Note: Descriptions are shown in the official language in which they were submitted.
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SECRETED PROTEINS AND POLYNUCLEOTIDES ENCODING THEM
This application is a continuation-in-part of Ser. No. 60/XXX,XXX (converted
to
a provisional application from non-provisional application Ser. No.
08/815,381), filed
March 11, 1997, which is 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
2 0 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
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
2 5 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
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
3 0 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
polynucleotides encoding them that the present invention is directed.
Si~TITUTE SHEET (RULE 26)
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SUMMARY OF THE INVENTION
In one embodiment, the present invention provides a composition comprising an
isolated palynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:1;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:1 from nucleotide 521 to nucleotide 1111;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:1 from nucleotide 536 to nucleotide 817;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone ax318 3 deposited under accession
number ATCC 98353;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone ax328 3 deposited under accession number ATCC 98353;
(f) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone ax318 3 deposited under accession number
ATCC 98353;
(g) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone ax318 3 deposited under accession number ATCC 98353;
2 0 (h) a poiynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:2;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:2 having biological activity, the fragment
comprising the amino acid sequence kom amino acid 93 to amino acid 102 of SEQ
2 5 ID N0:2;
(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
3 0 (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
NO:1 from nucleotide 521 to nucleotide 1111; the nucleotide sequence of SEQ ID
N0:1
from nucleotide 536 to nucleotide 817; the nucleotide sequence of the full-
length protein
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coding sequence of clone ax318_3 deposited under accession number ATCC 98353;
or the
nucleotide sequence of the mature protein coding sequence of clone ax318 3
deposited
under accession number ATCC 98353. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone ax318 3 deposited under accession number ATCC 98353. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:2 from amino acid 4 to amino
acid 99.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:1.
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 N0:2;
(b) the amino acid sequence of SEQ ID N0:2 from amino acid 4 to
2 5 amino acid 99;
(c) fragments of the amino acid sequence of SEQ ID N0:2 comprising
the amino acid sequence from amino acid 93 to amino acid 102 of SEQ ID N0:2;
and
(d) the amino acid sequence encoded by the cDNA insert of clone
2 0 ax318_3 deposited under accession number ATCC 98353;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:2 or the amino acid
sequence
of SEQ ID N0:2 from amino acid 4 to amino acid 99.
In one embodiment, the present invention provides a composition comprising an
2 5 isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3 from nucleotide 61 to nucleotide 864;
3 0 (c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3 from nucleotide 826 to nucleotide 1386;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone bg140 1 deposited under accession
number ATCC 98353;
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(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone bg140_1 deposited under accession number ATCC 98353;
(f) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone bg140_1 deposited under accession number
ATCC 98353;
(g) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone bg140_1 deposited under accession number ATCC 98353;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:4;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:4 having biological activity, the fragment
comprising the amino acid sequence from amino acid 129 to amino acid 138 of
SEQ ID N0:4;
(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 polynucleoHde capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(i).
2 0 Preferably, such polynucleotide comprises the nucleotide sequence of SEQ
ID
N0:3 from nucleotide 61 to nucleotide 864; the nucleotide sequence of SEQ ID
N0:3 from
nucleotide 826 to nucleotide 1386; the nucleotide sequence of the full-length
protein
coding sequence of clone bg140 1 deposited under accession number ATCC 98353;
or the
nucleotide sequence of the mature protein coding sequence of clone bg140_1
deposited
2 5 under accession number ATCC 98353. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone bg140_1 deposited under accession number ATCC 98353. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:31 from amino acid 148 to
amino acid
3 0 249.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:3.
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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 N0:4;
(b) the amino acid sequence of SEQ ID N0:31;
(c) the amino acid sequence of SEQ ID N0:31 from amino acid 148 to
amino acid 249;
(d) fragments of the amino acid sequence of SEQ ID N0:4 comprising
the amino acid sequence from amino acid 129 to amino acid 138 of SEQ ID N0:4;
(e) fragments of the amino acid sequence of SEQ ID N0:31comprising
the amino acid sequence from amino acid 163 to amino acid 172 of SEQ ID N0:31;
and
(f) the amino acid sequence encoded by the cDNA insert of clone
bg140 1 deposited under accession number ATCC 98353;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:4, the amino acid
sequence of
SEQ ID N0:31, or the amino acid sequence of SEQ ID N0:31 from amino acid 148
to
amino acid 249.
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
N0:5;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:5 from nucleotide 77 to nucleotide 1624;
2 5 (c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:5 from nucleotide 390 to nucleotide 789;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone bg465 2 deposited under accession
number ATCC 98353;
3 0 (e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone bg465 2 deposited under accession number ATCC 98353;
(f) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone bg465_2 deposited under accession number
ATCC 98353;
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(g) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone bg465_2 deposited under accession number ATCC 98353;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:6;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:6 having biological activity, the fragment
comprising the amino acid sequence from amino acid 253 to amino acid 262 of
SEQ ID N0:6;
(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
N0:5 from nucleotide 77 to nucleotide 1624; the nucleotide sequence of SEQ ID
N0:5
from nucleotide 390 to nucleotide 789; the nucleotide sequence of the full-
length protein
coding sequence of clone bg465 2 deposited under accession number ATCC 98353;
or the
nucleotide sequence of the mature protein coding sequence of clone bg465 2
deposited
2 0 under accession number ATCC 98353. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone bg465 2 deposited under accession number ATCC 98353. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:6 from amino acid 260 to amino
acid
2 5 343.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:5.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
3 0 consisting of:
(a) the amino acid sequence of SEQ ID N0:6;
(b) the amino acid sequence of SEQ iD N0:6 from amino acid 260 to
amino acid 343;
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(c) fragments of the amino acid sequence of SEQ ID N0:6 comprising
the amino acid sequence from amino acid 253 to amino acid 262 of SEQ ID N0:6;
and
(d) the amino acid sequence encoded by the cDNA insert of clone
bg465_2 deposited under accession number ATCC 98353;
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 260 to amino acid 343.
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 48 to nucleotide 1055;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:7 from nucleotide 216 to nucleotide 1055;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:7 from nucleotide 494 to nucleotide 958;
(e) a polynucleotide comprising the nucleotide sequence of the full
2 0 length protein coding sequence of clone bk291 3 deposited under accession
number ATCC 98353;
a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone bk291 3 deposited under accession number ATCC 98353;
(g) a polynucleotide comprising the nucleotide sequence of the mature
2 5 protein coding sequence of clone bk291 3 deposited under accession number
ATCC 98353;
(h) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone bk291 3 deposited under accession number ATCC 98353;
(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, the fragment
comprising the amino acid sequence from amino acid 85 to amino acid 94 of SEQ
ID N0:8;
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(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
N0:7 from nucleotide 48 to nucleotide 1055; the nucleotide sequence of SEQ ID
N0:7 from
nucleotide 216 to nucleotide 1055; the nucleotide sequence of SEQ ID N0:7 from
nucleotide 494 to nucleotide 958; the nucleotide sequence of the full-length
protein coding
sequence of clone bk291 3 deposited under accession number ATCC 98353; or the
nucleotide sequence of the mature protein coding sequence of clone bk291 3
deposited
under accession number ATCC 98353. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone bk291 3 deposited under accession number ATCC 98353. 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 188 to amino
acid
306.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
2 0 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
consisting of:
(a) the amino acid sequence of SEQ ID N0:8;
2 5 (b) the amino acid sequence of SEQ ID N0:8 from amino acid 188 to
amino acid 306;
(c) fragments of the amino acid sequence of SEQ ID N0:8 comprising
the amino acid sequence from amino acid 85 to amino acid 94 of SEQ ID N0:8;
and
(d) the amino acid sequence encoded by the cDNA insert of clone
3 0 bk291 3 deposited under accession number ATCC 98353;
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 188 to amino acid 306.
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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
N0:9;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:9 from nucleotide 64 to nucleotide 1197;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:9 from nucleotide 1 to nucleotide 828;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone bp537_4 deposited under accession
number ATCC 98353;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone bp537 4 deposited under accession number ATCC 98353;
a polynucleotide comprising the nucleotide sequence of the mature
25 protein coding sequence of clone bp537 4 deposited under accession number
ATCC 98353;
(g) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone bp537 4 deposited under accession number ATCC 98353;
(h) a polynucleotide encoding a protein comprising the amino acid
2 0 sequence of SEQ ID NO:10;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:10 having biological activity, the fragment
comprising the amino acid sequence from amino acid 184 to amino acid 193 of
SEQ ID N0:10;
2 5 (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
3 0 to any one of the polynucleotides specified in (a)-{i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:9 from nucleotide 64 to nucleotide 1197; the nucleotide sequence of SEQ ID
N0:9
from nucleotide 1 to nucleotide 828; the nucleotide sequence of the full-
length protein
coding sequence of clone bp537_4 deposited under accession number ATCC 98353;
or the
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nucleotide sequence of the mature protein coding sequence of clone bp537 4
deposited
under accession number ATCC 98353. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone bp537 4 deposited under accession number ATCC 98353. 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 1 to amino
acid
255.
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 NO:10;
(b) the amino acid sequence of SEQ ID NO:10 from amino acid 1 to
amino acid 255;
(c) fragments of the amino acid sequence of SEQ ID N0:10 comprising
the amino acid sequence from amino acid 184 to amino acid 193 of SEQ ID N0:10;
and
(d) the amino acid sequence encoded by the cDNA insert of clone
2 0 bp537 4 deposited under accession number ATCC 98353;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:10 or the amino acid
sequence
of SEQ ID NO:10 from amino acid 1 to amino acid 255.
In one embodiment, the present invention provides a composition comprising an
2 5 isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:I1;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:11 from nucleotide 581 to nucleotide 1534;
3 0 (c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:11 from nucleotide 928 to nucleotide 1510;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone cs431 2 deposited under accession
number ATCC 98353;
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(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone cs431 2 deposited under accession number ATCC 98353;
(f) a polynucleoHde comprising the nucleotide sequence of the mature
protein coding sequence of clone cs431 2 deposited under accession number
ATCC 98353;
(g) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone cs431 2 deposited under accession number ATCC 98353;
(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, the fragment
comprising the amino acid sequence from amino acid 154 to amino acid 163 of
SEQ ID N0:12;
(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).
2 0 Preferably, such polynucleotide comprises the nucleotide sequence of SEQ
ID
N0:11 from nucleotide 581 to nucleotide 1534; the nucleotide sequence of SEQ
ID N0:11
from nucleotide 928 to nucleotide 1510; the nucleotide sequence of the full-
length protein
coding sequence of clone cs431 2 deposited under accession number ATCC 98353;
or the
nucleotide sequence of the mature protein coding sequence of clone cs431 2
deposited
2 5 under accession number ATCC 98353. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone cs431 2 deposited under accession number ATCC 98353. 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 150 to
amino acid
3 0 310.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:11.
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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 N0:12;
(b) the amino acid sequence of SEQ ID N0:12 from amino acid 150 to
amino acid 310;
(c) fragments of the amino acid sequence of SEQ ID N0:12 comprising
the amino acid sequence from amino acid 154 to amino acid 163 of SEQ ID N0:12;
and
(d) the amino acid sequence encoded by the cDNA insert of clone
cs431 2 deposited under accession number ATCC 98353;
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 I50 to amino acid 310.
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
N0:13;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
2 0 N0:13 from nucleotide 29 to nucleotide 2227;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:13 from nucleotide 1334 to nucleotide 2227;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:13 from nucleotide 1 to nucleotide 746;
2 5 (e) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone cw976_1 deposited under accession
number ATCC 98353;
(f) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone cw976_1 deposited under accession number ATCC 98353;
3 0 (g) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone cw976_1 deposited under accession number
ATCC 98353;
(h) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone cw976_1 deposited under accession number ATCC 98353;
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(i) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID NO:14;
(j) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:14 having biological activity, the fragment
comprising the amino acid sequence from amino acid 361 to amino acid 370 of
SEQ ID N0:14;
(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
N0:13 from nucleotide 29 to nucleotide 2227; the nucleotide sequence of SEQ ID
N0:13
from nucleotide 1334 to nucleotide 2227; the nucleotide sequence of SEQ ID
N0:13 from
nucleotide 1 to nucleotide 746; the nucleotide sequence of the full-length
protein coding
sequence of clone cw976_1 deposited under accession number ATCC 98353; or the
nucleotide sequence of the mature protein coding sequence of clone cw976_1
deposited
under accession number ATCC 98353. In other preferred embodiments, the
2 0 polynucleotide encodes the full-length or mature protein encoded by the
cDNA insert of
clone cw976_1 deposited under accession number ATCC 98353. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:14 from amino acid 1 to amino
acid
239, or a polynucleotide encoding a protein comprising the amino acid sequence
of SEQ
2 5 ID N0:14 from amino acid 119 to amino acid 733.
Other embodiments provide the gene corresponding to the cDNA sequence of 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
3 0 consisting of:
(a) the amino acid sequence of SEQ ID N0:14;
(b) the amino acid sequence of SEQ ID N0:14 from amino acid 1 to
amino acid 239;
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(c) the amino acid sequence of SEQ ID N0:14 from amino acid 119 to
amino acid 733;
(d) fragments of the amino acid sequence of SEQ ID N0:14 comprising
the amino acid sequence from amino acid 361 to amino acid 370 of SEQ ID N0:14;
and
(e) the amino acid sequence encoded by the cDNA insert of clone
cw976_1 deposited under accession number ATCC 98353;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:14, the amino acid
sequence of
SEQ ID N0:14 from amino acid 1 to amino acid 239, or the amino acid sequence
of SEQ
ID N0:14 from amino acid 119 to amino acid 733.
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
N0:15;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:15 from nucleotide 364 to nucleotide 777;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:15 from nucleotide 1 to nucleotide 636;
2 0 (d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone cw1233 3 deposited under accession
number ATCC 98353;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone cw1233_3 deposited under accession number ATCC 98353;
2 5 (f) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone cw1233 3 deposited under accession number
ATCC 98353;
(g) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone cw1233 3 deposited under accession number ATCC 98353;
3 0 (h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:16;
(i} a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:16 having biological activity, the fragment
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comprising the amino acid sequence from amino acid 64 to amino acid 73 of SEQ
ID N0:16;
(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 polynucleoHde comprises the nucleotide sequence of SEQ ID
N0:15 from nucleotide 364 to nucleotide 777; the nucleotide sequence of SEQ ID
N0:15
from nucleotide 1 to nucleotide 636; the nucleotide sequence of the full-
length protein
coding sequence of clone cw1233_3 deposited under accession number ATCC 98353;
or
the nucleotide sequence of the mature protein coding sequence of clone
cw1233_3
deposited under accession number ATCC 98353. In other preferred embodiments,
the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone cw1233 3 deposited under accession number ATCC 98353. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:16 from amino acid 1 to amino
acid
91.
2 0 Other embodiments provide the gene corresponding to the cDNA sequence of
SEQ
ID N0:15.
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:
2 5 (a) the amino acid sequence of SEQ ID N0:16;
(b) the amino acid sequence of SEQ ID N0:16 from amino acid 1 to
amino acid 91;
(c) fragments of the amino acid sequence of SEQ ID N0:16 comprising
the amino acid sequence from amino acid 64 to amino acid 73 of SEQ ID N0:16;
3 0 and
(d) the amino acid sequence encoded by the cDNA insert of clone
cwi233_3 deposited under accession number ATCC 98353;
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the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:16 or the amino acid
sequence
of SEQ ID N0:16 from amino acid 1 to amino acid 91.
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
N0:17;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:17 from nucleotide 619 to nucleotide 1434;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:17 from nucleotide 520 to nucleotide 1323;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone dgl 1 deposited under accession number
ATCC 98353;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone dg1 1 deposited under accession number ATCC 98353;
(f) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone dg1 1 deposited under accession number ATCC
98353;
2 0 (g) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone dg1_1 deposited under accession number ATCC 98353;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:18;
(i) a polynucleotide encoding a protein comprising a fragment of the
2 5 amino acid sequence of SEQ ID N0:18 having~biological activity, the
fragment
comprising the amino acid sequence from amino acid 131 to amino acid 140 of
SEQ ID N0:18;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
3 0 (k) a polynucleotide which encodes a species homologue of the protein
of (h) or (i) above ; and
{l) a polynucleotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(i).
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Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:17 from nucleotide 619 to nucleotide 1434; the nucleotide sequence of SEQ
ID N0:17
from nucleotide 520 to nucleotide 1323; the nucleotide sequence of the full-
length protein
coding sequence of clone dgl_1 deposited under accession number ATCC 98353; or
the
nucleotide sequence of the mature protein coding sequence of clone dgl_1
deposited
under accession number ATCC 98353. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone dgl_1 deposited under accession number ATCC 98353. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:18 from amino acid 1 to amino
acid
235.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:17.
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 N0:18;
(b) the amino acid sequence of SEQ ID N0:18 from amino acid 1 to
amino acid 235;
2 0 (c) fragments of the amino acid sequence of SEQ ID N0:18 comprising
the amino acid sequence from amino acid 131 to amino acid 140 of SEQ ID N0:18;
and
(d) the amino acid sequence encoded by the cDNA insert of clone
dgl_1 deposited under accession number ATCC 98353;
2 5 the protein being substantially free from other mammalian proteins.
Preferably such
protein comprises the amino acid sequence of SEQ ID N0:18 or the amino acid
sequence
of SEQ ID N0:18 from amino acid 1 to amino acid 235.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
3 0 (a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:19;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:19 from nucleotide 2063 to nucleotide 2290;
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(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:19 from nucleotide 2276 to nucleotide 2290;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:19 from nucleotide 2037 to nucleotide 2405;
(e) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone ep234_1 deposited under accession
number ATCC 98353;
(f) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone ep234_1 deposited under accession number ATCC 98353;
(g) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone ep234_1 deposited under accession number
ATCC 98353;
(h) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone ep234_1 deposited under accession number ATCC 98353;
(i) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:20;
(j) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:20 having biological activity, the fragment
comprising the amino acid sequence from amino acid 33 to amino acid 42 of SEQ
2 0 ID N0:20;
{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 5 (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
N0:19 from nucleotide 2063 to nucleotide 2290; the nucleotide sequence of SEQ
ID N0:19
from nucleotide 2276 to nucleotide 2290; the nucleotide sequence of SEQ ID
N0:19 from
3 0 nucleotide 2037 to nucleotide 2405; the nucleotide sequence of the full-
length protein
coding sequence of clone ep234_1 deposited under accession number ATCC 98353;
or the
nucleotide sequence of the mature protein coding sequence of clone ep234_1
deposited
under accession number ATCC 98353. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
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clone ep234_1 deposited under accession number ATCC 98353. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:20 from amino acid 1 to amino
acid
69.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:19.
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 N0:20;
(b) the amino acid sequence of SEQ ID N0:20 from amino acid 1 to
amino acid 69;
(c) fragments of the amino acid sequence of SEQ ID N0:20 comprising
the amino acid sequence from amino acid 33 to amino acid 42 of SEQ ID N0:20;
and
(d) the amino acid sequence encoded by the cDNA insert of clone
ep234_1 deposited under accession number ATCC 98353;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:20 or the amino acid
sequence
2 0 of SEQ ID N0:20 from amino acid 1 to amino acid 69.
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
2 5 modified expression of the genes) corresponding to the polynucleotide
sequences
disclosed herein.
Processes are also provided for producing a protein, which comprise:
(a) growing a culture of the host cell transformed with such
polynucleotide compositions in a suitable culture medium; and
3 0 (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
process is a mature form of the protein.
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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
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.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures lA 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
2 0 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
2 S 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
3 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"ax318 3"
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A polynucleotide of the present invention has been identified as clone "ax318
3".
ax318 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
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. ax318_3 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"ax318_3 protein").
The nucleotide sequence of ax318 3 as presently determined is reported in SEQ
ID N0:1. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the ax318 3 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:2.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
ax318 3 should be approximately 1200 bp.
The nucleotide sequence disclosed herein for ax318 3 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. ax318 3 demonstrated at least some similarity with
sequences
identified as AA255872 (zs19a01.s1 NCI CGAP_GCB1 Homo Sapiens cDNA clone),
AA625610 (zv89h04.s1 Soares NhHMPu Sl Homo sapiens cDNA clone 766999 3'
similar
to WP:K10B2.1 CE02008 TRANSDUCIN BETA CHAIN), D86043 (Human mRNA for
2 0 SHPS-1, complete cds), H23217 (ym52f03.s1 Homo sapiens cDNA clone 51880
3'), U06701
(Human clone CCA53 mRNA containing CCA trinucleotide repeat), and W05822
(za90b02.r1 Soares fetal lung NbHLI9W Homo sapiens cDNA clone 299787 5'). The
predicted amino acid sequence disclosed herein for ax318_3 was searched
against the
GenPept and GeneSeq amino acid sequence databases using the BLASTX search
protocol.
2 5 The predicted ax318 3 protein demonstrated at least some similarity to
sequences
identified as D85183 (SHPS-1 [Rattus rattus]), D86043 (SHPS-1 [Homo Sapiens]),
R85852
(WD-40 domain-containing beta-transducin protein), Y10376 (SIRP-betal [Homo
sapiens]),
and 279757 (F55B12.3 [Caenorhabditis elegansJ). SHPS-1 is a membrane
glycoprotein that
binds the SH2-domain-containing protein tyrosine phosphatase SHP-2 in response
to
3 0 mitogens and cell adhesion (Fujioka et al., 1996, Mol. Cell. Biol. 16(12):
6887-6899,
incorporated by reference herein). Based upon sequence similarity, ax318_3
proteins and
each similar protein or peptide may share at least some activity. The
TopPredIi computer
program predicts a putative transmembrane domain within the ax318 3 protein
sequence,
centered around amino acid 28 of SEQ ID N0:2; amino acids 15 to 27 of SEQ ID
N0:2 are
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WO 98/40404 PG"T/US98/04601
also a possible leader/signal sequence, with the predicted mature amino acid
sequence
beginning at amino acid 28.
Clone "b lg 40 1"
A polynucleotide of the present invention has been identified as clone
"bg140_1".
bg140_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. bg140_1 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"bg140_1 protein").
The nucleotide sequence of bg140_1 as presently determined is reported in SEQ
ID N0:3. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the bg140 1 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:4. Another possible bg140_1
reading frame
and predicted amino acid sequence, encoded by base pairs 641 to 1648 of SEQ ID
N0:3,
is reported in SEQ ID N0:31. A frameshift in the nucleotide sequence of SEQ ID
N0:3,
caused, for example, by a single base pair deletion near position 825 of SEQ
ID N0:3,
could join the open reading frame encoding SEQ ID NO:4 with that encoding SEQ
ID
2 0 N0:31.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
bg140_1 should be approximately 2900 bp.
The nucleotide sequence disclosed herein for bg140_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
2 5 FASTA search protocols. bg140_1 demonstrated at least some similarity with
sequences
identified as AA075629 (zm89a04.s1 Stratagene ovarian cancer (#937219) Homo
Sapiens
cDNA clone 545070 3'), AA113989 (zn27h12.r1 Stratagene neuroepithelium NT2RAMI
937234 Homo Sapiens cDNA clone 548711 5'), N77135 (yz85hll.rl Homo sapiens
cDNA
clone 289893 5'), 811701 (yf49d02.r1 Homo Sapiens cDNA clone 25600 5'), 813178
3 0 (yf73e04.r1 Homo Sapiens cDNA clone 27855 5'), ail Y14946 {Homo sapiens
mRNA for
SPIN protein). The predicted amino acid sequence disclosed herein for bg140_1
was
searched against the GenPept and GeneSeq amino acid sequence databases using
the
BLASTX search protocol. The predicted bg140 1 protein demonstrated at least
some
similarity to sequences identified as AF038969 (general transcription factor 2-
I [Homo
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WO 98/40404 PCT/US98/04601
Sapiens]), U77948 (Bruton's tyrosine kinase-associated protein-135; BAP-135
[Homo
sapiens]), and Y14946 (SPIN protein [Homo sapiens]). Based upon sequence
similarity,
bg140_1 proteins and each similar protein or peptide may share at least some
activity.
Clone "b~465 2"
A polynucleotide of the present invention has been identified as clone "bg465_
2".
bg465 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 a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. bg465_2 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"bg465 2 protein").
The nucleotide sequence of bg465 2 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 bg465 2 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:6.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
bg465 2 should be approximately 2500 bp.
The nucleotide sequence disclosed herein for bg465_2 was searched against the
2 0 GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. bg465 2 demonstrated at least some similarity with
sequences
identified as AA133150 (zm25b07.r1 Stratagene pancreas (#937208) Homo sapiens
cDNA
clone 526645 5' similar to WP:F07F6.4 CE01896 ZINC FINGER PROTEIN), AA144842
(mr69hOl.r1 Stratagene mouse testis (#937308) Mus musculus cDNA clone 602737
5'),
2 5 AA484561 (nf06a12.s1 NCI CGAP_Lil Homo Sapiens cDNA clone), D16939 (Human
HepG2 3' region cDNA, clone hmd5d06), L24125 (Saccharomyces cerevisiae zinc
finger
protein (GCS1) gene, complete cds), 818422 (yg02f06.r1 Homo Sapiens cDNA clone
30950
5'), T32543 (EST50558 Homo Sapiens cDNA 5' end similar to None), W88569
(zh70b12.s1
Soares fetal liver spleen 1NFLS Sl Homo sapiens cDNA clone 417407 3'), 274274
3 0 (S.cerevisiae chromosome IV reading frame ORF YDL226c), and 282199 (Human
DNA
sequence *** SEQUENCING IN PROGRESS *** from clone 316D5; HTGS phase 1). The
predicted amino acid sequence disclosed herein for bg465 2 was searched
against the
GenPept and GeneSeq amino acid sequence databases using the BLASTX search
protocol.
The predicted bg465 2 protein demonstrated at least some similarity to
sequences
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identified as U23486 (similar to S. cerevisiae zinc finger protein GCSl (SP
GCSl YEAST)
[Caenorhabditis elegans]). Based upon sequence similarity, bg465_2 proteins
and each
similar protein or peptide may share at least some activity.
Clone "bk291 3"
A polynucleotide of the present invention has been identified as clone "bk291
3".
bk291 3 was isolated from a human adult retina 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. bk291 3 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"bk291 3 protein")
The nucleotide sequence of bk291 3 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 bk291 3 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:8. Amino acids 44 to 56 are a
predicted
leader/signal sequence, with the predicted mature amino acid sequence
beginning at
amino acid 57, or are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
2 0 bk291 3 should be approximately 1500 bp.
The nucleotide sequence disclosed herein for bk291 3 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. bk292 3 demonstrated at least some similarity with
sequences
identified as AA135915 (z119f04.r1 Soares pregnant uterus NbHPU Homo sapiens
cDNA
2 5 clone 502399 5'), AA156738 (z119f04.s1 Soares pregnant uterus NbHPU Homo
Sapiens
cDNA clone 502399 3' similar to WP:T05E11.5 CE06364 YEAST YKKO LIKE), AF017785
(Mus musculus MHC Class I Hl3a minor histocompatibility peptide (H13) mRNA,
partial
cds), N40134 (yw73a12.r1 Homo sapiens cDNA clone 257854 5' similar to
SW:YKKO_YEAST P34248 HYPOTHETICAL 67.5 KD PROTEIN IN APEl /LAP4-CWP1
3 0 INTERGENIC REGION), N42726 {yylldOl.r1 Homo Sapiens cDNA clone 270913 5'
similar
to SW:YKKO_YEAST P34248 HYPOTHETICAL 67.5 KD PROTEIN IN APEl /LAP4-CWP1
INTERGENIC REGION), 867144 (yi31h04.r1 Homo sapiens cDNA clone 140887 5'
similar
to SP:YKKO_YEAST P34248 HYPOTHETICAL 67.5 KD PROTEIN IN APE1/LAP4-MBR1
INTERGENIC), 878822 (yi90b05.r1 Homo sapiens cDNA clone 146481 5'), 879317
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(yi90b05.s1 Homo sapiens cDNA clone 146481 3'), T23944 (Human gene signature
HUMGS05889), and W04243 (za58h10.r1 Soares fetal liver spleen 1NFLS Homo
Sapiens
cDNA clone). The predicted amino acid sequence disclosed herein for bk291 3
was
searched against the GenPept and GeneSeq amino acid sequence databases using
the
BLASTX search protocol. The predicted bk291 3 protein demonstrated at least
some
similarity to sequences identified as X71133 (YKL450 (Saccharomyces
cerevisiae]), ~ 28100
(ORF YKL100c [Saccharomyces cerevisiae]), and 268751 (T05E11.5 (Caenorhabditis
elegans)). Based upon sequence similarity, bk291 3 proteins and each similar
protein or
peptide may share at least some activity. The TopPredII computer program
predicts six
additional potential transmembrane domains within the bk291 3 protein
sequence,
centered around amino acids 70, 125, 170, 220, 260, and 280 of SEQ ID N0:8.
Clone "b~537 4"
A polynucleotide of the present invention has been identified as clone
"bp537_4".
bp537_4 was isolated from a human fetal kidney 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. bp537 4 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
2 0 "bp537_4 protein").
The nucleotide sequence of bp537_4 as presently determined is reported in SEQ
ID N0:9. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the bp537_4 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:10.
2 5 The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
bp537_4 should be approximately 1500 bp.
The nucleotide sequence disclosed herein for bp537_4 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. bp537_4 demonstrated at least some similarity with
sequences
3 0 identified as AA007576 (zh99b06.r1 Soares fetal liver spleen 1NFLS S2 Homo
Sapiens
cDNA clone 429395 5'), AA287563 (zs52g02.r1 NCI CGAP_GCB1 Homo sapiens cDNA
5'), 809093 (yf21h09.r1 Homo sapiens cDNA clone 127553 5'), and T33088
(EST56631
Homo Sapiens cDNA 5' end similar to None). The predicted amino acid sequence
disclosed herein for bp537 4 was searched against the GenPept and GeneSeq
amino acid
CA 02283193 1999-09-02
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sequence databases using the BLASTX search protocol. The predicted bp537_4
protein
demonstrated at least some similarity to sequences identified as U34998 (Rad9
[Coprinus
cinereus]). Based upon sequence similarity, bp537_4 proteins and each similar
protein or
peptide may share at least some activity.
Clone "cs431 2"
A polynucleotide of the present invention has been identified as clone "cs431
2".
cs431 2 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. cs431 2 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"cs431 2 protein")
The nucleotide sequence of cs431 2 as presently determined is reported in SEQ
ID
N0:11. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the cs431 2 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:12.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
cs431 2 should be approximately 2300 bp.
2 0 The nucleotide sequence disclosed herein for cs431 2 was searched against
the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. cs431 2 demonstrated at least some similarity with
sequences
identified as AA021033 (ze67h12.s1 Soares retina N2b4HR Homo sapiens cDNA
clone
364103 3' similar to contains Alu repetitive element), H29349 (ym32c02.r1 Homo
sapiens
2 5 cDNA clone 49839 5' similar to SP:KYES_XIPHE P27447 PROTO-ONCOGENE
TYROSINE-PROTEIN KINASE YES), L14577 (Homo sapiens cystathionine beta-synthase
(CBS) mRNA, complete cds), Q87430 (Human cystathionine beta-synthase cDNA),
and
X92659 (H.sapiens intron 4 from p53 gene). The predicted amino acid sequence
disclosed
herein for cs431 2 was searched against the GenPept and GeneSeq amino acid
sequence
3 0 databases using the BLASTX search protocol. The predicted cs431 2 protein
demonstrated at least some similarity to sequences identified as L19501
(cystathionine
beta-synthase [Homo sapiens]), 871376 (Human cystathionine beta-synthase),
U61167
(SH3 domain-containing protein SH3P18 [Homo Sapiens]), and X82166
(cystathionine
beta-synthase [Homo sapiens]). Based upon sequence similarity, cs431 2
proteins and
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each similar protein or peptide may share at least some activity. The
TopPredII computer
program predicts a potential transmembrane domain within the cs431 2 protein
sequence
centered around amino acid 30 of SEQ ID N0:12. The nucleotide sequence of
cs431 2
indicates that it may contain an Alu repetitive element.
Clone "cw976 1"
A polynucleotide of the present invention has been identified as clone "cw976
1".
cw976_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. cw976_l is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"cw976_1 protein").
The nucleotide sequence of cw976_1 as presently determined is reported in SEQ
ID N0:13. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the cw976_1 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:14. Amino acids 423 to 435 are a
predicted
leader/signal sequence, with the predicted mature amino acid sequence
beginning at
amino acid 436, or are a transmembrane domain.
2 0 The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
cw976_1 should be approximately 3300 bp.
The nucleotide sequence disclosed herein for cw976_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. cw976_1 demonstrated at least some similarity with
sequences
2 5 identified as AA280747 (zs96a09.r1 NCI CGAP_GCB1 Homo Sapiens cDNA clone
IMAGE:711448 5'), AA323946 (EST26798 Cerebellum II Homo sapiens cDNA 5' end),
813665 (yf60g06.r1 Homo sapiens cDNA clone 26764 5'), 816892 (yf44d06.s2 Homo
sapiens cDNA clone 129707 3'), 885177 (yo43d07.r1 Homo sapiens cDNA clone
180685 5'),
and 889648 (ym97c02.r1 Homo sapiens cDNA clone 166850 5'). Based upon sequence
3 0 similarity, cw976_1 proteins and each similar protein or peptide may share
at least some
activity. The TopPredII computer program predicts a potential transrnembrane
domain
within the cw976_1 protein sequence at the amino terminus of SEQ ID N0:14.
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Clone "cw1233 3"
A polynucleotide of the present invention has been identified as clone
"cw1233_3".
cw1233_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 of
computer
analysis of the amino acid sequence of the encoded protein. cw1233_3 is a full-
length
clone, including the entire coding sequence of a secreted protein (also
referred to herein
as "cw1233_3 protein").
The nucleotide sequence of cw1233_3 as presently determined is reported in SEQ
ID N0:15. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the cw1233 3 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:16.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
cw1233_3 should be approximately 3000 bp.
The nucleotide sequence disclosed herein for cw1233 3 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. cw1233_3 demonstrated at least some similarity with
sequences
identified as 861600 (yh16f08.r1 Homo Sapiens cDNA clone 37807 5'), 883929
(yp06h11.s1
Homo sapiens cDNA clone 186693 3' similar to contains Alu repetitive
element;contains
2 0 TART repetitive element), 887877 (yo45h05.r1 Homo sapiens cDNA clone
180921 5'),
U14568 (***ALU WARNING Human Alu-Sb subfamily consensus sequence). The
predicted amino acid sequence disclosed herein for cw1233_3 was searched
against the
GenPept and GeneSeq amino acid sequence databases using the BLASTX search
protocol.
The predicted cw1233_3 protein demonstrated at least some similarity to
sequences
2 5 identified as AB002317 (KIAA0319 [Homo sapiens]). Based upon sequence
similarity,
cw1233_3 proteins and each similar protein or peptide may share at least some
activity.
The nucleotide sequence of cw1233_3 indicates that it may contain an Alu
repetitive
element.
3 0 Clone "dg1 1"
A polynucleotide of the present invention has been identified as clone "dg1
1".
dgl_1 was isolated from a human adult placenta 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
28
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WO 98/40404 PCT/US98/04601
analysis of the amino acid sequence of the encoded protein. dgl_1 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"dg1_1 protein").
The nucleotide sequence of dg1_1 as presently determined is reported in SEQ ID
N0:17. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the dg1 1 protein corresponding to the
fore;oing
nucleotide sequence is reported in SEQ ID N0:18.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
dg1_1 should be approximately 1900 bp.
The nucleotide sequence disclosed herein for dg1_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. dg1_1 demonstrated at least some similarity with
sequences
identified as AA73128i (nw57f05.s1 NCI CGAP_GCB1 Homo Sapiens cDNA clone
IMAGE 1250721 similar to gb Y00345 cdsl POLYADENYLATE-BINDING PROTEIN
(HUMAN)). The predicted amino acid sequence disclosed herein for dgl 1 was
searched
against the GenPept and GeneSeq amino acid sequence databases using the BLASTX
search protocol. The predicted dg1_1 protein demonstrated at least some
similarity to
sequences identified as M81878 (hyaluronidase [Clostridium perfringens]) and
U28742
(similar to hyaluronoglucosaminidase (SPNAGH_CLOPE, P26831) [Caenorhabditis
2 0 elegans]). Hyaluronoglucosaminidase is a secreted protein found, for
example, in bee
venom. Based upon sequence similarity, dg1_1 proteins and each similar protein
or
peptide may share at least some activity. The TopPredII computer program
predicts a
potential transmembrane domain within the dgl_1 protein sequence centered
around
amino acid 150 of SEQ ID N0:18.
Clone "ep234 1"
A polynucleotide of the present invention has been identified as clone
"ep234_1".
ep234_1 was isolated from a human adult placenta 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. ep234_1 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"ep234_1 protein").
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The nucleotide sequence of ep234_1 as presently determined is reported in SEQ
ID N0:19. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the ep234_1 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:20. Amino acids 59 to 71 are a
predicted
leader/signal sequence, with the predicted mature amino acid sequence
beginning at
amino acid 72, or are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
ep234_1 should be approximately 2500 bp.
The nucleotide sequence disclosed herein for ep234_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. ep234_1 demonstrated at least some similarity with
sequences
identified as AA044714 (zf54d09.r1 Soares retina N2b4HR Homo sapiens cDNA
clone
380753 5'), AA454840 (zx79d09.s1 Soares ovary tumor NbHOT Homo Sapiens cDNA
clone
809969 3'), AA470137 (zu11e01.s1 Soares testis NHT Homo sapiens cDNA clone
731544
3'), H24852 (y142c11.r1 Homo Sapiens cDNA clone 160916 5'), N64648 (yz87b06.s1
Homo
Sapiens cDNA clone 290003 3'), 845788 (Ha662-f Homo sapiens cDNA clone a662-
f),
T70546 (yd15b07.s1 Homo Sapiens cDNA clone 108277 3'), W73481 (zd54e01.s1
Soares
fetal heart NbHHI9W Homo sapiens cDNA clone 344472 3'), and W73553 (zd54e01.r1
Soares fetal heart). Based upon sequence similarity, ep234_1 proteins and each
similar
2 0 protein or peptide may share at least some activity. The TopPredII
computer program
predicts at least two potential transmembrane domains within the ep234_1
protein
sequence of SEQ ID N0:20.
Deposit of Clones
2 5 Clones ax318 3, bg140_l, bg465_2, bk291 3, bp537_4, cs431 2, cw976_1,
cw1233_3, dg1 1, and ep234_1 were deposited on March 11,1997 with the American
Type
Culture Collection as an original deposit under the Budapest Treaty and were
given the
accession number ATCC 98353, from which each clone comprising a particular
polynucleotide is obtainable. All restrictions on the availability to the
public of the
3 0 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
CA 02283193 1999-09-02
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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 al., 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.
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
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
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.
Clone Probe Sequence
ax318_3 SEQ ID N0:21
bg140_1 SEQ ID N0:22
bg465 2 SEQ ID N0:23, SEQ ID N0:32
2 5 bk291 3 SEQ ID N0:24
bp537_4 SEQ ID N0:25
cs431 2 SEQ ID N0:26
cw976_1 S EQ ID N0:27
cw1233 3 SEQ ID N0:28
3 0 dgl_1 SEQ ID N0:29
ep234_1 SEQ ID N0:30
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
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CA 02283193 1999-09-02
WO 98/40404 PCT/US98/04601
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:
(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 3zP ATP (specific
activity 6000
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
probe should be approximately 4e+6 dpm/pmole.
The bacterial culture containing the pool of full-length clones should
preferably
be thawed and 100 pl of the stock used to inoculate a sterile culture flask
containing 25 ml
of sterile L-broth containing ampicillin at 100 ug/ml. The culture should
preferably be
2 0 grown to saturation at 37°C, and the saturated culture should
preferably be diluted in
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
2 5 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
3 0 NaOH) containing 0.5% SDS,100 Ng/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 le+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
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CA 02283193 1999-09-02
WO 98/40404 PCT/LTS98/04601
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.
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
protein may be in linear form or they may be cyclized using known methods, for
example,
as described in H.U. Saragovi, ef 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
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
2 0 decavalent form of the protein of the invention.
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
2 5 (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
3 0 are transcribed to produce the mRNAs 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
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CA 02283193 1999-09-02
WO 98/40404 PC"T/US98/04601
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
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
gene (Albert and Morris, 1994, Trends Pharmacol. Sci.15(7): 250-254; Lavarosky
et al., 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
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).
2 0 In addition, organisms are provided in which the genes) corresponding to
the
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
2 5 transposable elements (Plasterk,1992, Bioessays 14(9): 629-633; Zwaal et
al.,1993, Proc. Natl.
Acad. Sci. USA 90(16): 7431-7435; Clark et al.,1994, Proc. Natl. Acad. Sci.
USA 91(2): 719-722;
all of which are incorporated by reference herein), or through homologous
recombination,
preferably detected by positive/negative 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;
3 0 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
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CA 02283193 1999-09-02
WO 98/40404 PCT/US98/04601
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
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
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
(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.
2 0 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
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
2 5 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.
Preferably,
species homologs are those isolated from mammalian species.
The invention also encompasses allelic variants of the disclosed
polynucleotides
or proteins; that is, naturally-occurring alternative forms of the isolated
polynucleotide
3 0 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.
CA 02283193 1999-09-02
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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
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.
StringencyPolynucleotideHybridHybridization TemperatureWash
ConditionHybrid Lengthand Temperature
(bp)$ Buffers 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 Tp*; lxSSC Tp*; lxSSC
E RNA:RNA s 50 70C; lxSSC -or- 70C; 0.3xSSC
50C; lxSSC, 50% formamide
F RNA:RNA <50 TF*; lxSSC TF*; lxSSC
G DNA:DNA z 50 65C; 4xSSC -or- 65C; lxSSC
42C; 4xSSC, 50% formamide
H DNA:DNA <50 TH*; 4xSSC T"*; 4xSSC
I DNA:RNA ~ 50 67C; 4xSSC -or- 67C; lxSSC
45C; 4xSSC, 50% formamide
J DNA:RNA <50 T~*; 4xSSC T~*; 4xSSC
2 K RNA:RNA s 50 70C; 4xSSC -or- 67C; lxSSC
0 50C; 4xSSC, 50% formamide
L RNA:RNA <50 T~*; 2xSSC T~*; 2xSSC
M DNA:DNA z 50 50C; 4xSSC -or- 50C; 2xSSC
40C; 6xSSC, 50% formamide
N DNA:DNA <50 TN*; 6xSSC TN*; 6xSSC
O DNA:RNA z 50 55C; 4xSSC -or- 55C; 2xSSC
42C; 6xSSC, 50% formamide
2 P DNA:RNA <50 TP*; 6xSSC TP*; 6xSSC
5
Q RNA:RNA s 50 60C; 4xSSC -or- 60C; 2xSSC
45C; 6xSSC, 50% formamide
R RNA:RNA <50 TR*; 4xSSC TR*; 4xSSC
$: The hybrid length is that anticipated for the hybridized regions) of the
hybridizing polynucleotides. When
3 0 hybridizing a polynucleoHde to a target polynucleotide of unknown
sequence, the hybrid length is assumed
36
CA 02283193 1999-09-02
WO 98140404 PCT/US98/04601
to be that of the hybridizing polynucleotide. When polynucleotides of known
sequence are hybridized, the
hybrid length can be determined by aligning the sequences of the
polynucleotides and identifying the region
or regions of optimal sequence complementarity.
i: SSPE (lxSSPE is 0.15M NaCI, lOmM NaHzPOa, and I.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.
*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 (T",) 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 ion;, in the
hybridization buffer ([Na'] for lxSSC = 0.165 M).
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 Biologt~,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
2 0 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
2 5 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
3 0 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
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
3 5 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
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
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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.
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, Bacilhts sitbtilis, Salmonella typhimurium,
or any bacterial
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
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,
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
2 0 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."
The protein of the invention may be prepared by culturing transformed host
cells
under culture conditions suitable to express the recombinant protein. The
resulting
2 5 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
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
3 0 Sepharose~; one or more steps involving hydrophobic interaction
chromatography using
such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity
chromatography.
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
38
CA 02283193 1999-09-02
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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
InVitrogen, respectively. The protein can also be tagged with an epitope and
' S 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
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
2 0 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
2 5 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
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
3 0 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
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
39
CA 02283193 1999-09-02
WO 98/40404 PCT/US98/04601
(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
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
present invention.
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
2 0 recombinant protein for analysis, characterization or therapeutic use; as
markers for
tissues in which the corresponding protein is preferentially expressed (either
constitutively 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 map related gene positions; to
compare
2 5 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"
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
3 0 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
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
CA 02283193 1999-09-02
WO 98/40404 PCT/US98/04601
described in Gyuris et al., Cell 75:791-803 (1993)) to identify
polynucleotides encoding the
other protein with which binding occurs or to identify inhibitors of the
binding
interaction.
The proteins provided by the present invention can similarly be used in assay
to
' S 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
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
2 0 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.
2 5 Nutritional Uses
PolynucleoHdes and proteins of the present invention can also be used as
nutritional sources or supplements. Such uses include without limitation use
as a protein
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
3 0 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
can be added to the medium in or on which the microorganism is cultured. '
41
CA 02283193 1999-09-02
WO 98!40404 PCT/US98104601
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
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
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 3.1-
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
2 0 133:327-341, 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 Current Protocols in
Immunology. J.E.e.a.
2 5 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.
Assays for proliferation and differentiation of hematopoietic and
lymphopoietic
cells include, without limitation, those described in: Measurement of Human
and Murine
3 0 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
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
42
CA 02283193 1999-09-02
WO 98/40404 PCT/US98/04601
Immunolog~~. 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 - Bennett, F., Giannotti, J., Clark, S.C. and Turner, K. J. In
Current Protocols
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. Vol
1 pp. 6.13.1,
John Wiley and Sons, Toronto. 1991.
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
2 0 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
(SLID)), e.g.,
in regulating (up or down) growth and proliferation of T and/or B lymphocytes,
as well
2 5 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
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,
3 0 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
be useful where a boost to the immune system generally may be desirable, i.e.,
in the
treatment of cancer.
43
CA 02283193 1999-09-02
WO 98/40404 PCT/US98/04601
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,
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
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
which 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
2 0 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.g.,
preventing
2 5 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
example,
blockage of T cell function should result in reduced tissue destruction in
tissue
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
3 0 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
monomeric form of a peptide having an activity of another B lymphocyte antigen
(e.g., B7-
1, B7-3) or blocking antibody), prior to transplantation can lead to the
binding of the
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CA 02283193 1999-09-02
WO 98/40404 PCT/IJS98/04601
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
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
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 al.,
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.
2 0 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.
2 5 Administration of reagents which block costimulaHon of T cells by
disrupting
receptor:ligand interactions of B lymphocyte antigens can be used to inhibit T
cell
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
3 0 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
human autoimmune diseases. Examples include murine experimental autoimmune
encephalitis, systemic lupus erythmatosis in MRL/Ipr/lpr mice or NZB hybrid
mice,
murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB
rats, and
CA 02283193 1999-09-02
WO 98/40404 PCT/US98/04601
murine experimental myasthenia gravis (see Paul ed., Fundamental Immunology,
Raven
Press, New York,1989, pp. 840-856).
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
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
2 0 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
2 5 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.
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
3 0 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
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
46
CA 02283193 1999-09-02
WO 98/40404 PCT/US98/04601
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
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 ~
chain
protein to thereby express MHC class I or MHC class II proteins on the cell
surface.
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
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
2 0 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.,
2 5 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,
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;
Bertagnolli et al.,
3 0 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
(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
47
CA 02283193 1999-09-02
WO 98/40404 PCT/US98/04601
antibody production, Mond, J.J. and Brunswick, M. In Current Protocols in
Immunolog~~.
J.E.e.a. Coligan eds. Vol 1 pp. 3.8.1- 3.8.16, John Wiley and Sons, Toronto.
1994.
Mixed lymphocyte reaction (MLR) assays (which will identify, among others,
proteins that generate predominantly Thl 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
7, Immunologic studies in Humans); Takai et al., J. Immunol. 237:3494-3500,
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. Immunoi. 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,
2 0 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;
2 5 Gorczyca et al., International Journal of Oncology 1:639-648,1992.
Assays for proteins that influence early steps of T-cell commitment and
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 Regulating Activity
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
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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,
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
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 thrombocytoperua, 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-vivo or
ex-vivv (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.
2 0 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 hematopoietic
lines
are cited above.
Assays for embryonic stem cell differentiation (which will identify, among
others,
2 5 proteins that influence embryonic differentiation hematopoiesis) include,
without
limitation, those described in: Johansson et al. Cellular Biology 15:141-151,
1995; Kelley et
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
3 0 others, proteins that regulate lympho-hematopoiesis) include, without
limitation, those
described in: Methylcellulose colony forming assays, Freshney, M.G. In Culture
of
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
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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,
1994; Cobblestone area forming cell assay, Ploemacher, R.E. In Culture of
Hematopoietic
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,
H.J. In Culture of Hematopoietic Cells. R.I. Freshney, et al. 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
2 0 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
2 5 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
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
3 0 destruction (collagenase activity, osteoclast activity, etc.) mediated by
inflammatory
processes.
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
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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
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
formarion induced by a composition of the present invention contributes to the
repair of
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
2 0 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,
2 5 Parkinson s disease, Huntington's disease, amyotrophic lateral sclerosis,
and Shy-Drager
syndrome. Further conditions which may be treated in accordance with the
present
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
3 0 invention.
Proteins of the invention may also be useful to promote better or faster
closure of
non-healing wounds, including without limitation pressure ulcers, ulcers
associated with
vascular insufficiency, surgical and traumatic wounds, and the like.
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It is expected that a protein of the present invention 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)
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.
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 ).
2 0 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).
Activin/Inhibin Activity
A protein of the present invention may also exhibit activin- or inhibin-
related
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
3 0 invention, alone or in heterodimers with a member of the inhibin a family,
rnay be useful
as a contraceptive based on the ability of inhibins to decrease fertility in
female mammals
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-
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~i 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,
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:
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
2 0 treatment of localized infections. For example, attraction of lymphocytes,
monocytes or
neutrophils to tumors or sites of infection may result in improved immune
responses
against the tumor or infecting agent.
A protein or peptide has chemotactic activity for a particular cell population
if it
can stimulate, directly or indirectly, the directed orientation or movement of
such cell
2 5 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
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
3 0 by the following methods:
Assays for chemotactic activity (which will identify proteins that induce or
prevent
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
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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 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 al 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.
Hemostatic and Thrombolytic 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 hemophiliac) 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
2 0 described in: Linet et al., J. Clin. Pharmacol. 26:131-i40, 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
2 5 A protein of the present invention may also demonstrate activity as
receptors,
receptor ligands or inhibitors or agonists of receptor/ligand interactions.
Examples of
such receptors and ligands include, without limitation, cytokine receptors and
their
ligands, receptor kinases and their ligands, receptor phosphatases and their
ligands,
receptors involved in cell-cell interactions and their ligands (including
without limitation,
3 0 cellular adhesion molecules (such as selectins, integrins and their
ligands) and
receptor/ligand pairs involved in antigen presentation, antigen recognition
and
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
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limitation, fragments of receptors and ligands) may themselves be useful as
inhibitors of
receptor/ligand interactions.
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
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
2 0 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
2 5 lethality, arthritis, complement-mediated hyperacute rejection, nephritis,
cytokine or
chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or
resulting
from over production of cytokines 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.
3 0 Cadherin/Tumor Invasion Suppressor Activit-y
Cadherins are calcium-dependent adhesion molecules that appear to play major
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
CA 02283193 1999-09-02
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diseases, such as pemphigus vulgaris and pemphigus foliaceus (auto-immune
blistering
skin diseases), Crohn's disease, and some developmental abnormalities.
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
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
polynucleoHdes 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
2 0 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.
2 5 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
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
3 0 reducing or eliminating the tendency of the cells to metastasize.
Additionally, proteins of the present invention with cadherin activity, and
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
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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
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
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: 1-Iortsch 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.
2 0 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
2 5 or inhibiting factors, agents or cell types which promote tumor growth.
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
3 0 agents, including, without limitation, bacteria, viruses, fungi and other
parasites; effecting
(suppressing or enhancing) bodily characteristics, including, without
limitation, height,
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;
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effecting the fertility of male or female subjects; effecting the metabolism,
catabolism,
anabolism, processing, utilization, storage or elimination of dietary fat,
lipid, protein,
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
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,
2 0 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
2 5 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-I0, IL-11,
IL-12, IL-13, IL-14, IL-15, IFN, TNFO, TNFl, TNF2, G-CSF, Meg-CSF,
thrombopoietin, stem
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
3 0 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
in formulations of the particular cytokine, lymphokine, other hematopoietic
factor,
thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize
side effects
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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
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
and/or peptide antigen will deliver a stimulatory signal to both B and T
lymphocytes. B
lymphocytes will respond to antigen through their surface immunogiobulin
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
immunolgobuiin
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.
2 0 The pharmaceutical composition of the invention may 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
iamellar layers
in aqueous solution. Suitable lipids for liposomal formulation include,
without limitation,
2 5 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; U.S.
Patent No. 4,837,028; and U.S. Patent No. 4,737,323, all of which are
incorporated herein
by reference.
3 0 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
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
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WO 98/40404 PCT/US98104601
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.
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
or other hematopoietic factors. When co-administered with one or more
cytokines,
lymphokines 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
2 0 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
2 5 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.
When administered in liquid form, a liquid carrier such as water, petroleum,
oils of animal
or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil,
or synthetic oils
3 0 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
form, the pharmaceutical composition contains from about 0.5 to 90% by weight
of protein
CA 02283193 1999-09-02
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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
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
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
2 0 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 lZg
to about 100
mg (preferably about O.lng to about 10 mg, more preferably about 0.1 ug to
about 1 mg)
2 5 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
contemplated that the duration of each application of the protein of the
present invention
3 0 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
polyclonal and monoclonal antibodies which specifically react with the
protein. Such
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CA 02283193 1999-09-02
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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
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
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
2 0 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
2 5 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
applications.
3 0 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
sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid, polyglycolic
acid and
62
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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
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.
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, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-
methylcellulose, and carboxymethylcellulose, the most preferred being cationic
salts of
carboxymethylcellulose (CMC). Other preferred sequestering agents include
hyaluronic
2 0 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
2 5 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 with other
agents beneficial to the treatment of the bone and/or cartilage defect, wound,
or tissue in
question. These agents include various growth factors such as epidermal growth
factor
3 0 (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
humans, are desired patients for such treatment with proteins of the present
invention.
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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
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
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
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.
2 0 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.
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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
(ii) TITLE OF INVENTION: SECRETED PROTEINS AND POLYNUCLEOTIDES
ENCODING THEM
(iii) NUMBER OF SEQUENCES: 32
(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 N0:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1328 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
i
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(ii) MOLECULE TYPE: eDNA
(xi) SEQUENCE
DESCRIPTION:
SEQ ID
N0:1:
CGTTAACAAGTGGAATGGAACTCAAAGACA ATATTCTTGTCTCTGGGAAT GCAGATTCTA60
CAGTTAAAATCTGGGATATCAAAACAGGAC AGTGTTTACAAACATTGCAA GGTCCCAACA120
AGCATCAGAGTGCTGTGACCTGTTTACAGT TCAACAAGAACTTTGTAATT ACCAGCTCAG180
ATGATGGAACTGTAAAACTATGGGACTTGA AAACGGGTGAATTTATTCGA AACCTAGTCA240
CATTGGAGAGTGGGGGGAGTGGGGGAGTTG TGTGGCGGATCACAGCCTCA AACACAAAGC300
TGGTGTGTGCAGTTGGGAGTCGGAATGGGA CTGAAGAAACCAAGCTGCTG GTGCTGGACT360
TTGATGTGGACATGAAGTGAAGAGCAGAAA AGATGAATTTGTCCAATTGT GTAGACGATA420
TACTCCCTGCCCTTCCCCCTGCAAAAAGAA P,~~i~AAAAAAAA,~~AAAAAAAA AAAAAAAAAA480
P~1~~AAAAAAAAAAAAAGCGCAAGCTCCAGG TTTCACCACAATGCCCATCC CTGCCTCCCC540
ACTCCACCCACCTCTGCCTTCCTTACTGCT GTATCTGCTGCTTGAACTGG CAGGAGTCAC600
ACATGTGTTCCATGTGCAACAAACGGAGAT GTCACAGACTGTATCAACTG GGGAGTCAAT660
CATCTTGAGTTGCAGCGTACCCGATACCTT ACCAAATGGACCTGTCTTGT GGTTCAAGGG720
AACAGGGCCAAACCGGAAATTAATCTACAA TTTCAAACAAGGTAACTTTC CCAGAGTAAA780
AGAGATTGGAGACACCACCAAGCCTGGCAA CACAGACTTTTCCACCCGCA TCCGTGAAAT840
CTCTCTTGCTGATGCTGGCACCTATTACTG CGTGAAGTTCATAAAAGGAA GAGCTATCAA900
GGAGTACCAATCAGGTCGGGGCACTCAGGT GTTTGTTACTGAGCAGAATC CAAGACCTCC960
CAAGAACAGACCTGCAGGCAGAGCAGGCTC CAGGGCCCACCATGATGCCC ATACCTGCCT1020
CTCGGCCCTGCCTGAGAGAAACAGCACAAA CTATTTCGTCCAACCCTGCT GCTGCCTCCG1080
GCTGCTGGGACTCACAGGCTTGCTGTCAAA ATAATCCAAACAGGGAAGGA ACGTACAAGT1140
AAATAACAAAAGCCCCCATACTCTTCTGAC TCCCTGGAGACAGCTACTTT TTAGGAGTTT1200
CATTTGCCTTCTTCAAGAGAGCTTTCTTCC ACTGACATAAAATGCCAGCT TGATCGTACA1260
ATAAATCTGTCTATTTACCTGGGTCCAAAA F,~~i~AAAP.AAAP~~~AAAAAAA P~~;AAAAAAAA13
2
0
AAAAAAAA 1328
(2) INFORMATION
FOR SEQ
ID N0:2:
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 197 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met Pro Ile Pro Ala Ser Pro Leu His Pro Pro Leu Pro Ser Leu Leu
1 5 10 15
Leu Tyr Leu Leu Leu Glu Leu Ala Gly Val Thr His Val Phe His Val
20 25 30
Gln Gln Thr Glu Met Ser Gln Thr Val Ser Thr Gly Glu Ser Ile Ile
35 40 45
Leu Ser Cys Ser Val Pro Asp Thr Leu Pro Asn Gly Pro Val Leu Trp
50 55 60
Phe Lys Gly Thr Gly Pro Asn Arg Lys Leu Ile Tyr Asn Phe Lys Gln
65 70 75 80
Gly Asn Phe Pro Arg Val Lys Glu Ile Gly Asp Thr Thr Lys Pro Gly
85 90 95
Asn Thr Asp Phe Ser Thr Arg Ile Arg Glu Ile Ser Leu Ala Asp Ala
100 105 110
Gly Thr Tyr Tyr Cys Val Lys Phe Ile Lys Gly Arg Ala Ile Lys Glu
115 120 125
Tyr Gln Ser Gly Arg Gly Thr Gln Val Phe Val Thr Glu Gln Asn Pro
130 135 140
Arg Pro Pro Lys Asn Arg Pro Ala Gly Arg Ala Gly Ser Arg Ala His
145 150 155 160-
His Asp Ala His Thr Cys Leu Ser Ala Leu Pro Glu Arg Asn Ser Thr
165 170 175
Asn Tyr Phe Val Gln Pro Cys Cys Cys Leu Arg Leu Leu Gly Leu Thr
180 185 190
Gly Leu Leu Ser Lys
195
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
67
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(A) LENGTH: 2835 base pairs
(B) TYPE. nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
{xi) SEQUENCE
DESCRIPTION:
SEQ ID
N0:3:
GCTTGATAAGCCAGCTTCAGGAGTAAAGGAAGAATGGTATGCCAGAATCACTAAATTAAG60
ATGGTGGATCAGCTTTTCTGCAAAAAATTTGCGGAAGCCTTGGGGAGCACTGAAGCCAAG120
GCTGTACCGTACCAAAAATTTGAGGCACACCCGAATGATCTGTACGTGGAAGGACTGCCA180
GAAAACATTCCTTTCCGAAGTCCCTCATGGTATGGAATCCCAAGGCTGGAAAAAATCATT240
CAAGTGGGCAATCGAATTAAATTTGTTATTAAAAGACCAGAACTTCTGACTCACAGTACC300
ACTGAAGTTACTCAGCCAAGAACGAATACACCAGTCAAAGAAGATTGGAATGTCAGAATT360
ACCAAGCTACGGAAGCAAGTGGAAGAGATTTTTAATTTGAAATTTGCTCAAGCTCTTGGA420
CTCACCGAGGCAGTAAAAGTACCATATCCTGTGTTTGAATCAAACCCGGAGTTCTTGTAT480
GTGGAAGGCTTGCCAGAGGGGATTCCCTTCCGAAGCCCTACCTGGTTTGGAATTCCACGA540
CTTGAAAGGATCGTCCACGGGAGTAATAAAATCAAGTTCGTTGTTAAAAAACCTGAACTA600
GTTATTTCCTACTTGCCTCCTGGGATGGCTAGTAAAATAAACACTAAAGCTTTGCAGTCC660
CCCAAAAGACCACGAAGTCCTGGGAGTAATTCAAAGGTTCCTGAAATTGAGGTCACCGTG720
GAAGGCCYTAATAACAACAATCCTCAAACCTCAGCTGTTCGAACCCCGACCCAGACTAAC780
GGTTCTAACGTTCCCTTCAAGCCACGAGGGAGAGAGTTTTCCTTTGGCCTGGAATGCCAA840
AATCACGGACCTAAAACAGAAAGTTGAAAATCTCTTCAATGAGAAATGTGGGGAAGCTCT900
TGGCCTTAAACAAGCTGTGAAGGTGCCGTTCGCGTTATTTGAGTCTTTCCCGGAAGACTT960
TTATGTGGAAGGCTTACCTGAGGGTGTGCCATTCCGAAGACCATCGACTTTTGGCATTCC1020
GAGGCTGGAGAAGATACTCAGAAACAAAGCCAAAATTAAGTTCATCATTAAAAAGCCCGA1080
AATGTTTGAGACGGCGATTAAGGAGAGCACCTCCTCTAAGAGCCCTCCCAGAAAAATAAA1140
TTCATCACCCAATGTTAATACTACTGCATCAGGTGTTGAAGACCTTAACATCATTCAGGT1200
GACAATTCCAGATGATGATAATGAAAGACTCTCGAAAGTTGAAAAAGCTAGACAGCTAAG1260
AGAACAAGTGAATGACCTCTTTAGTCGGAAATTTGGTGAAGCTATTGGTATGGGTTTTCC1320
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TGTGAAAGTT CCCTACAGGA AAATCACAAT TAACCCTGGC TGTGTGGTGG TTGATGGCAT 1380
GCCCCCGGGG GTGTCCTTCA AAGCCCCCAG CTACCTGGAA ATCAGCTCCA TGAGAAGGAT 1440
CTTAGACTCT GCCGAGTTTA TCAAATTCAC GGTCATTAGA CCATTTCCAG GACTTGTGAT 1500
TAATAACCAG CTGGTTGATC AGAGTGAGTC AAAAGGCCCC GTGATACAAG AATCAGCTGA 1560
ACCAAGCCAG TTGGAAGTTC CAGCCACAGA AGAAATAAAA GAGACTGATG GAAGCTCTCA 1620
GATCAAGCAA GAACCAGACC CCACGTGGTA GACCTCTTCC CTCCTAGGCT TAAAGTATCA 1680
GTGGTTGAGA AGAGCTTTTC GGACCTGTTA CTACCCCAAG CTGTGTAATA TACTTGTATA 1740
ACAGAAATAC CTTCTATACA AACCTTTTTT TCTACTTTTA GATAGAAATG TCTACTTTTT 1800
CAGCAGTTCT GTGAATTAAA GAGCAGAGTG ACTGTGGGTC TGGAATGGCT GGTGTACTTG 1860
GGAATGTACT ATCAGGATTT TACAGCAATG CTGGGAAATG ACAGGGAAAA TGACAGGAAT 1920
GAATCTCACC AGATTTTTTA TGTACTCAGC AGAGCCTTGA GTTACGGTGT TTATTTTCCA 1980
ATCAAGTGAA GATATCTCCT ACTTCTCCTA CTGGAACATC TCAGCTTCTG CAGTGAAGAA 2040
AAATTCCTGT GATAGTTCAG TTCTTTAGTT TTTCTATTTG AAAAAP.P,AAA ATCATTTAAA 2100
TGATCCTTTG TTCACGGCTC TCCTTAATGA CTGAGTGAAC AGTTCCTATC TGTATATTTG 2160
ACTAAACCTT TTCCTAAGCT ATCTCTCATG GTTCCTATGT TTTTTTATCA TAATTAAAAG 2220
CAAAACCATT TGGATCACCT AACAGTCAGA GGTCAGTATC TCAGCGTGTG AATTATAGAG 2280
GAAATACAGA GAGAACCTCT TCCACTTTTA CTTTTCGTCC AAATAAAATG CATGGTGTAC 2340
CAGAAGTTGA AGATCGGGTT GAGGATTGGG GCTAGCTCGA TGACACTAAG GCCCCAACAT 2400
CGCGGGACCT GCTGTGGCGC GGATTCTTAG GAACGCTGTT CTAGCCGGCC CCCTCTCCAG 2460
GGGTCGCCGT GGCCGGCATT ATTTCCTAGT TCTTCTTGTA ACCCTGAGGT GCCAGCGCGG 2520
GGAGTGAGGA GGGGTCAGGG GGCTAAGGAT GCAACCTCTG ACGTTCTGCG CCTTCCTAGG 2580
AGAGTCTTAC ATGTGTTGAG ATTTCACAAG CAATGCGAGT TGTAAAATAC CAGCTCTACA 2640
AGAAGCTAGG CTCTGTGACG GCATAGTTTT CAGTAGCTTT ATCACAATAT TCACAATGGA 2700
GAATTATATG ACATGGTAGC AGAAATAGGC CCTTTTATGT GTTGCTTCTA TTTTACCTCA 2760
AATTGTAGAT ATAGGGTAAT CAATAAAATC CATCCATGCC TTTCAAAAAA AAAAAAAAAA 2820
AA ~~AAA~1AA Ap p,Ap' 2 8 3 5
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
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(A) LENGTH: 268 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Met Val Asp Gln Leu Phe Cys Lys Lys Phe Ala Glu Ala Leu Gly Ser
1 5 10 15
Thr Glu Ala Lys Ala Val Pro Tyr Gln Lys Phe Glu Ala His Pro Asn
20 25 30
Asp Leu Tyr Val Glu Gly Leu Pro Glu Asn Ile Pro Phe Arg Ser Pro
35 40 45
Ser Trp Tyr Gly Ile Pro Arg Leu Glu Lys Ile Ile Gln Val Gly Asn
50 55 60
Arg Ile Lys Phe Val Ile Lys Arg Pro Glu Leu Leu Thr His Ser Thr
65 70 75 80
Thr Glu Val Thr Gln Pro Arg Thr Asn Thr Pro Val Lys Glu Asp Trp
85 90 95
Asn Val Arg Ile Thr Lys Leu Arg Lys Gln Val Glu Glu Ile Phe Asn
100 105 110
Leu Lys Phe Ala Gln Ala Leu Gly Leu Thr Glu Ala Val Lys Val Pro
115 120 125
Tyr Pro Val Phe Glu Ser Asn Pro Glu Phe Leu Tyr Val Glu Gly Leu
130 135 140
Pro Glu Gly Ile Pro Phe Arg Ser Pro Thr Trp Phe Gly Ile Pro Arg
145 150 155 160
Leu Glu Arg Ile Val His Gly Ser Asn Lys Ile Lys Phe Val Val Lys
165 170 175
Lys Pro Glu Leu Val Ile Ser Tyr Leu Pro Pro Gly Met Ala Ser Lys
180 185 190
Ile Asn Thr Lys Ala Leu Gln Ser Pro Lys Arg Pro Arg Ser Pro Gly
195 200 205
Ser Asn Ser Lys Val Pro Glu Ile Glu Val Thr Val Glu Gly Xaa Asn
210 215 220
Asn Asn Asn Pro Gln Thr Ser Ala Val Arg Thr Pro Thr Gln Thr Asn
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225 230 235 240
Gly Ser Asn Val Pro Phe Lys Pro Arg Gly Arg Glu Phe Ser Phe Gly
245 250 255
Leu Glu Cys Gln Asn His Gly Pro Lys Thr Glu Ser
260 265
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2705 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
TAGGCCATGA AGGCCGCGCTTTTCGTCGACTCTTACCGGT TGGCTGGGCCAGCTGCGCCG 60
CGGCTCACAG CTGACGATGGGGGACCCCAGCAAGCAGGAC ATCTTGACCATCTTCAAGCG 120
CCTCCGCTCG GTGCCCACTAACAAGGTGTGTTTTGATTGT GGTGCCAAAAATCCCAGCTG 180
GGCAAGCATA ACCTATGGAGTGTTCCTTTGCATTGATTGC TCAGGGTCCCACCGGTCACT 240
TGGTGTTCAC TTGAGTTTTATTCGATCTACAGAGTTGGAT TCCAACTGGTCATGGTTTCA 300
GTTGCGATGC ATGCAAGTCGGAGGAAACGCTAGTGCATCT TCCTTTTTTCATCAACATGG 360
GTGTTCCACC AATGACACCAATGCCAAGTACAACAGTCGT GCTGCTCAGCTCTATAGGGA 420
GAAAATCAAA TCGCTCGCCTCTCAAGCAACACGGAAGCAT GGCACTGATCTGTGGCTTGA 480
TAGTTGTGTG GTTCCACCTTTGTCCCCTCCACCAAAGGAG GAAGATTTTTTTGCCTCTCA 540
CGTTTCTCCT GAGGTGAGTGACACAGCGTGGGCATCAGCA ATAGCAGAACCATCTTCTTT 600
AACATCAAGG CCTGTGGAAACCACTTTGGAAAATAATGAA GGTGGACAAGAGCAAGGACC 660
AAGTGTGGAA GGTCTTAATGTACCAACAAAGGCTACTTTA GAGGTATCCTCTATCATAAA 720
AAAGAAACCA AATCAAGCTAAAAAAGGCCTTGGGGCCAAA AAAGGAAGTTTGGGAGCTCA 780
GAAACTGGCA AACACATGCTTTAATGAAATTGAAAAACAA GCTCAAGCTGCGGATAAAAT 840
GAAGGAGCAG AAAGACCTGGCCAAGGTGGTATCTAAAGAA GAATCAATTGTTTCATCATT 900
ACGATTAGCC TATAAGGATCTTGAAATTCAAATGAAGAAA GACGAAAAGATGAACATTAG 960
71
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TGGCAAAAAA CAGACAGACTCGGCATGGGATTTGGAAATTGCAGAAGTGT 1020
AATGTTGACT
TATTTCACATTCAGTGACTTCAGATATGCAGACCATAGAGCAGGAATCACCCATTATGGC 1080
AAAACCAAGAAAAAAGTATAATGATGACAGTGACGATTCATATTTTACTTCCAGCTCAAG 1140
TTACTTTGACGAGCCAGTGGAGTTAAGGAGCAGTTCTTTCTCTAGCTGGGATGACAGTTC 1200
AGATTCCTATTGGAAAAAAGAGACCAGCAAAGATACTGAAACAGTTCTGAAAACCACAGG 1260
CTATTCAGACAGACCTACTGCTCGCCGCAAGCCAGATTATGAGCCAGTTGAAAATACAGA 1320
TGAGGCCCAGAAGAAGTTTGGCAATGTCAAGGCCATTTCATCAGATATGTATTTTGGAAG 1380
ACAATCCCAGGCTGATTATGAGACCAGGGCCCGCCTAGAGAGGCTGTCGGCAAGTTCCTC 1440
CATAAGCTCGGCTGATCTGTTCGAGGAGCCGAGGAAGCAGCCAGCAGGGAACTACAGCCT 1500
GTCCAGTGTGCTGCCCAACGCCCCCGACATGGCGCAGTTCAAGCAGGGAGTGAGATCGGT 1560
TGCTGGAAAACTCTCCGTCTTTGCTAATGGAGTCGTGACTTCAATTCAGGATCGCTACGG 1620
TTCTTAATACTGAAGTCATGATGTGTATTTCCTGGAGAAATTCCTCTTTAAATGAACAAG 1680
TAACCACATCTCAGGCGGCAGTGAAGTCCAGATAGTTTTGCAGATTGTTTTGCTACTTTT 1740
TCATATGGTATATGTTTCTGATTTTTAATATTTCTTTTGAGAAATTCTGAGTTCTGATGT 1800
AGGAGCTTTCCTGTGATTTCTGTTTCACGTTCCTTCCTGTCACACCCTCCTTTGGCGTCT 1860
CTGTGTATATCCTTGCTTTATTTTCTTGGAACCTTTGATTTCAACACTGAGGGCCTGGAG 1920
ACCTCGGCTCCTCCTGCTCCTGAACCAGGAGGCTTCATGTGGGGGAGGAGGAGAGGTCTC 1980
CATGTGACACATGGGCTCAGGGCTGCCAGAATCAGCGGATGCTGGATGGGCCTGCAGAAA 2040
CAACACTCACCACACACACTTCCTTCAAAAGACCAAAAGTGACTGGTGTCTCGTGTGACA 2100
GATTGCTTCATTTATGTTTCTACATAGTAAGGTGACTGCCAAATAATATTTGAAGTCATC 2160
TGTCTCTTTGTAAATTATTTTATATGACCTATAAATTTAAAAATGTTTTTCAGTGAGTGC 2220
TTTTAACAAACTTAAGCTTCTGCCCTGCCAAGGGAATTAATGTTATCTTGTGAAAGGTGT 2280
TGCTGTTTGAATTGATGAGAAATGGAAGATGAGAACTCCCTAAGAGTTCTCATAATAAAT 2340
CATCTCATCACAAATCAATACGGTATACAGAGTTAAAGTGGAATGAGGTAAGAAGATACA 2400
GCTACAGAAAATAGTTGCGTGTATGGGAGAACAGTCATTGTAATTGGGTAGTTTTGTTAA 2460
TAAATATTTTTAAATCTTGCTTTTCAGAAATTACCGAATGTGTATAAACAAATAAAGAAA 2520
AATAATTTAGCTGTGTTTTAGACAGCATTAGAATATATTGTTCAGCACAGTAAAATATAT 2580
TTGAAATTTGATAAGCCAAAAATGTGGTTTTGAATGAATATTTTGTGAATCTTTCTTAAA 2640
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AGCTCAAATT TGTAGACTTC TAAATAGAAT AAACACTTGC AGCAGATGGA AAAAAAAAAA 2700
AAAAA
2705
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 516 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Met Gly Asp Pro Ser Lys Gln Asp Ile Leu Thr Ile Phe Lys Arg Leu
1 5 10 15
Arg Ser Val Pro Thr Asn Lys Val Cys Phe Asp Cys Gly Ala Lys Asn
20 25 30
Pro Ser Trp Ala Ser Ile Thr Tyr Gly Val Phe Leu Cys Ile Asp Cys
35 40 45
Ser Gly Ser His Arg Ser Leu Gly Val His Leu Ser Phe Ile Arg Ser
50 55 60
Thr Glu Leu Asp Ser Asn Trp Ser Trp Phe Gln Leu Arg Cys Met Gln
65 70 75 80
Val Gly Gly Asn Ala Ser Ala Ser Ser Phe Phe His Gln His Gly Cys
85 90 95
Ser Thr Asn Asp Thr Asn Ala Lys Tyr Asn Ser Arg Ala Ala Gln Leu
100 105 110
Tyr Arg Glu Lys Ile Lys Ser Leu Ala Ser Gln Ala Thr Arg Lys His
115 120 125
Gly Thr Asp Leu Trp Leu Asp Ser Cys Val Val Pro Pro Leu Ser Pro
130 135 140
Pro Pro Lys Glu Glu Asp Phe Phe Ala Ser His Val Ser Pro Glu Val
145 150 155
160
Ser Asp Thr Ala Trp Ala Ser Ala Ile Ala Glu Pro Ser Ser Leu Thr
165 170 175
Ser Arg Pro Val Glu Thr Thr Leu Glu Asn Asn Glu Gly Gly Gln Glu
180 185 190
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Gln Gly Pro Ser Val Glu Gly Leu Asn Val Pro Thr Lys Ala Thr Leu
195 200 205
Glu Val Ser Ser Ile Ile Lys Lys Lys Pro Asn Gln Ala Lys Lys Gly
210 215 220
Leu Gly Ala Lys Lys Gly Ser Leu Gly Ala Gln Lys Leu Ala Asn Thr
225 230 235 240
Cys Phe Asn Glu Ile Glu Lys Gln Ala Gln Ala Ala Asp Lys Met Lys
245 250 255
Glu Gln Lys Asp Leu Ala Lys Val Val Ser Lys Glu Glu Ser Ile Val
260 265 270
Ser Ser Leu Arg Leu Ala Tyr Lys Asp Leu Glu Ile Gln Met Lys Lys
275 280 285
Asp Glu Lys Met Asn Ile Ser Gly Lys Lys Asn Val Asp Ser Asp Arg
290 295 300
Leu Gly Met Gly Phe Gly Asn Cys Arg Ser Val Ile Ser His Ser Val
305 310 315 320
Thr Ser Asp Met Gln Thr Ile Glu Gln Glu Ser Pro Ile Met Ala Lys
325 330 335
Pro Arg Lys Lys Tyr Asn Asp Asp Ser Asp Asp Ser Tyr Phe Thr Ser
340 345 350
Ser Ser Ser Tyr Phe Asp Glu Pro Val Glu Leu Arg Ser Ser Ser Phe
355 360 365
Ser Ser Trp Asp Asp Ser Ser Asp Ser Tyr Trp Lys Lys Glu Thr Ser
370 375 380
Lys Asp Thr Glu Thr Val Leu Lys Thr Thr Gly Tyr Ser Asp Arg Pro
385 390 395 400
Thr Ala Arg Arg Lys Pro Asp Tyr Glu Pro Val Glu Asn Thr Asp Glu
405 410 415
Ala Gln Lys Lys Phe Gly Asn Val Lys Ala Ile Ser Ser Asp Met Tyr
420 425 430
Phe Gly Arg Gln Ser Gln Ala Asp Tyr Glu Thr Arg Ala Arg Leu Glu
435 440 445
Arg Leu Ser Ala Ser Ser Ser Ile Ser Ser Ala Asp Leu Phe Glu Glu
450 455 460
Pro Arg Lys Gln Pro Ala Gly Asn Tyr Ser Leu Ser Ser Val Leu Pro
465 470 475 480
Asn Ala Pro Asp Met Ala Gln Phe Lys Gln Gly Val Arg Ser Val Ala
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485 490 495
Gly Lys Leu Ser Val Phe Ala Asn Gly Val Val Thr Ser Ile Gln Asp
500 505 510
Arg Tyr Gly Ser
515
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1414 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
{ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE SEQ ID
DESCRIPTION: N0:7:
CTTCCACGCCCGAGGGCATCGCGCTGGCCT CCTGCTCATG 60
ACGGCAGCCT GCGCTGCTGC
CCATCTTCTTCGGCGCCCTGCGCTCCGTACGCTGCGCCCGCGGCAAGAATGCTTCAGACA 120
TGCCTGAAACAATCACCAGCCGGGATGCCGCCCGCTTCCCCATCATCGCCAGCTGCACAC 180
TCTTGGGGCTCTACCTCTTTTTCAAAATATTCTCCCAGGAGTACATCAACCTCCTGCTGT 240
CCATGTATTTCTTCGTGCTGGGAATCCTGGCCCTGTCCCACACCATCAGCCCCTTCATGA 300
ATAAGTTTTTTCCAGCCAGCTTTCCAAATCGACAGTACCAGCTGCTCTTCACACAGGGTT 360
CTGGGGAAAACAAGGAAGAGATCATCAATTATGAATTTGACACCAAGGACCTGGTGTGCC 420
TGGGCCTGAGCAGCATCGTTGGCGTCTGGTACCTGCTGAGGAAGCACTGGATTGCCAACA 480
ACCTTTTTGGCCTGGCCTTCTCCCTTAATGGAGTAGAGCTCCTGCACCTCAACAATGTCA 540
GCACTGGCTGCATCCTGCTGGGCGGACTCTTCATCTACGATGTCTTCTGGGTATTTGGCA 600
CCAATGTGATGGTGACAGTGGCCAAGTCCTTCGAGGCACCAATAAAATTGGTGTTTCCCC 660
AGGATCTGCTGGAGAAAGGCCTCGAAGCAAACAACTTTGCCATGCTGGGACTTGGAGATG 720
TCGTCATTCCAGGGATCTTCATTGCCTTGCTGCTGCGCTTTGACATCAGCTTGAAGAAGA 780
ATACCCACACCTACTTCTACACCAGCTTTGCAGCCTACATCTTCGGCCTGGGCCTTACCA 840
TCTTCATCATGCACATCTTCAAGCATGCTCAGCCTGCCCTCCTATACCTGGTCCCCGCCT 900
GCATCGGTTTTCCTGTCCTGGTGGCGCTGGCCAAGGGAGAAGTGACAGAGATGTTCAGTT 960
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ATGAGGAGTC AAATCCTAAG GATCCAGCGGCAGTGACAGAATCCAAAGAG GGAACAGAGG1020
CATCAGCATC GAAGGGGCTG GAGAAGAAAGAGAAATGATGCAGCTGGTGC CCGAGCCTCT1080
CAGGGCCAGA CCAGACAGAT GGGGGCTGGGCCCACACAGGCGTGCACCGG TAGAGGGCAC1140
AGGAGGCCAA GGGCAGCTCC AGGACAGGGCAGGGGGCAGCAGGATACCTC CAGCCAGGCC1200
TCTGTGGCCT CTGTTTCCTT CTCCCTTTCTTGGCCCTCCTCTGCTCCTCC CCACACCCTG1260
CAGGCAAAAG AAACCCCCAG CTTCCCCCCTCCCCGGGAGCCAGGTGGGAA AAGTGGGTGT1320
GATTTTTAGA TTTTGTATTG TGGACTGATTTTGCCTCACATTAAAAACTC ATCCCATGGC1380
CAGGGCGGGC CACTGTAAAA AAA.AAAAAAAAAAA 1414
{2) INFORMATION FOR SEQ
ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 336 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
{xi) SEQUENCE DESCRIPTION: SEQ ID NO: B:
Met Ala Leu Leu Pro Ile Phe Phe Gly Ala Leu Arg Ser Val Arg Cys
1 5 10 15
Ala Arg Gly Lys Asn Ala Ser Asp Met Pro Glu Thr Ile Thr Ser Arg
20 25 30
Asp Ala Ala Arg Phe Pro Ile Ile Ala Ser Cys Thr Leu Leu Gly Leu
35 40 45
Tyr Leu Phe Phe Lys Ile Phe Ser Gln Glu Tyr Ile Asn Leu Leu Leu
50 55 60
Ser Met Tyr Phe Phe Val Leu Gly Ile Leu Ala Leu Ser His Thr Ile
65 70 75 80
Ser Pro Phe Met Asn Lys Phe Phe Pro Ala Ser Phe Pro Asn Arg Gln
85 90 95
Tyr Gln Leu Leu Phe Thr Gln Gly Ser Gly Glu Asn Lys Glu Glu Ile
100 105 110
Ile Asn Tyr Glu Phe Asp Thr Lys Asp Leu Val Cys Leu Gly Leu Ser
115 120 125
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Ser Ile Val Gly Val Trp Tyr Leu Leu Arg Lys His Trp Ile Ala Asn
130 135 140
Asn Leu Phe Gly Leu Ala Phe Ser Leu Asn Gly Val Glu Leu Leu His
145 150 155 160
Leu Asn Asn Val Ser Thr Gly Cys Ile Leu Leu Gly Gly Leu Phe Ile
165 170 175
Tyr Asp Val Phe Trp Val Phe Gly Thr Asn Val Met Val Thr Val Ala
180 185 190
Lys Ser Phe Glu Ala Pro Ile Lys Leu Val Phe Pro Gln Asp Leu Leu
195 200 205
Glu Lys Gly Leu Glu Ala Asn Asn Phe Ala Met Leu Gly Leu Gly Asp
210 215 220
Val Val Ile Pro Gly Ile Phe Ile Ala Leu Leu Leu Arg Phe Asp Ile
225 230 235 240
Ser Leu Lys Lys Asn Thr His Thr Tyr Phe Tyr Thr Ser Phe Ala Ala
245 250 255
Tyr Ile Phe Gly Leu Gly Leu Thr Ile Phe Ile Met His Ile Phe Lys
260 265 270
His Ala Gln Pro Ala Leu Leu Tyr Leu Val Pro Ala Cys Ile Gly Phe
275 280 285
Pro Val Leu Val Ala Leu Ala Lys Gly Glu Val Thr Glu Met Phe Ser
290 295 300
Tyr Glu Glu Ser Asn Pro Lys Asp Pro Ala Ala Val Thr Glu Ser Lys
305 310 315 320
Glu Gly Thr Glu Ala Ser Ala Ser Lys Gly Leu Glu Lys Lys Glu Lys
325 330 335
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1583 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
77
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AGACGAGCCTTTCTTATTTCTTTACTCAACCTCTTTGATGACACAGCAAA 60
AACAGACGTG
ACTATGCTCTTGTATATAGCAGACAATCTAGCCTGTTTTCCATACCAGACACAGGAAGAG 120
CCGTTGTTTATAATGCATCATATAGACATTACACTCTCAGTTTCTGGTAGTAACCTACTG 180
CAGTCATTCAAGGAGTCTATGGTAAAGGACAAAAGGAAAGAGAGAAAATCATCACCTAGT 240
AAGGAAAATGAGTCAAGCGACAGTGAAGAAGAAGTTTCCAGGCCTCGGAAGTCACGGAAA 300
CGTGTAGATTCAGATTCAGATTCAGATTCAGAAGACGATATAAATTCAGTGATGAAATGT 360
TTGCCAGAAAATTCAGCTCCTTTAATCGAATTTGCAAATGTGTCCCAGGGTATTTTATTA 420
CTTCTCATGTTAAAACAACATTTGAAGAATCTTTGTGGATTTTCTGATAGTAAAATTCAG 480
AAGTACTCTCCATCTGAATCTGCAAAAGTATATGATAAAGCGATAAACCGAAAAACAGGA 540
GTTCATTTTCATCCAAAACAAACACTGGACTTCCTGCGGAGTGACATGGCTAATTCCAAA 600
ATCACAGAAGAGGTGAAAAGGAGTATAGTAAAACAGTATCTAGATTTCAAACTTCTCATG 660
GAACATCTGGACCCTGATGAAGAAGAAGAAGAAGGGGAGGTTTCAGCTAGCACAAATGCT 720
CGGAACAAAGCAATTACCTCACTGCTTGGAGGAGGCAGCCCTAAAAATAATACAGCAGCA 780
GAGACAGAAGATGATGAAAGTGATGGGGAGGATAGAGGAGGAGGCACTTCAGGGTCATTG 840
AGAAGGTCAAAACGAAATTCAGACTCTACGGAGTTGGCAGCACAGATGAATGAAAGTGTT 900
GACGTCATGGATGTCATCGCTATTTGCTGTCCAAAGTACAAAGATCGACCACAAATTGCA 960
AGAGTAGTGCAGAAAACCAGCAGTGGCTTCAGTGTTCAGTGGATGGCAGGCTCCTACAGT 1020
GGCTCCTGGACTGAGGCTAAGCGCCGTGATGGCCGCAAACTGGTGCCTTGGGTAGACACT 1080
ATTAAAGAGTCAGACATTATTTACAAAAAAATTGCTCTAACGAGTGCTAATAAGCTGACT 1140
AATAAAGTTGTTCAGACTTTACGATCCCTGTATGCCGCCAAGGATGGGACTTCCAGCTAA 1200
TGAATTTGTACATGCAGCCAAATTTACAGGAATTTTTTTAAAAGGCAGAAAAACTTGAAA 1260
TACCAACATTCTGGCAAAAAAAAATCAGTTTTATGAAGAGTAAGTGGAACCTGGGATGCA 1320
GGAACAAAAGAAGGAAATGTTGGGCAAACATTTTTGTGGGAGCTCCCTTCGCTGTTGTGC 1380
AGCAGAAACAGATTCTCAGTTCATTTTTACTCCCACTGTATTATAGTTTAACAAAAATTG 1440
TTTATATCTTGGAAAAAAAAACTTTCTGTTTAAAAAAAATAAACAAGTGAATGTTGGAAA 1500
TTAGTCTGTTAATGTTCTTAATAAAGTGTTCTTGGAGTTTTp,AAAAAAAAP~~iAAAAAAAA15
6
0
p,~~~AAAAAAAP,2~~AAAAAAAAAA 15
8
3
(2) INFORMATION
FOR
SEQ
ID NO:10:
7g
CA 02283193 1999-09-02
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 378 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi)SEQUENCE
DESCRIPTION:
SEQ
ID
N0:10:
MetLeu LeuTyrIle AlaAspAsn LeuAlaCys PhePro TyrGlnThr
1 5 10 15
GlnGlu GluProLeu PheIleMet HisHisIle AspIle ThrLeuSer
20 25 30
ValSer GlySerAsn LeuLeuGln SerPheLys GluSer MetValLys
35 40 45
AspLys ArgLysGlu ArgLysSer SerProSer LysGlu AsnGluSer
50 55 60
SerAsp SerGluGlu GluValSer ArgProArg LysSer ArgLysArg
65 70 75 80
ValAsp SerAspSer AspSerAsp SerGluAsp AspIle AsnSerVal
85 90 95
MetLys CysLeuPro GluAsnSer AlaProLeu IleGlu PheAlaAsn
100 105 110
ValSer GlnGlyIle LeuLeuLeu LeuMetLeu LysGln HisLeuLys
115 120 125
AsnLeu CysGlyPhe SerAspSer LysIleGln LysTyr SerProSer
130 135 140
GluSer AlaLysVal TyrAsp.Lys AlaIleAsn ArgLys ThrGlyVal
145 150 155 160
HisPhe HisProLys GlnThrLeu AspPheLeu ArgSer AspMetAla
165 170 175
AsnSer LysIleThr GluGluVal LysArgSer IleVal LysGlnTyr
180 185 190
LeuAsp PheLysLeu LeuMetGlu HisLeuAsp ProAsp GluGluGlu
195 200 205
GluGlu GlyGluVal SerAlaSer ThrAsnAla ArgAsn LysAlaIle
210 215 220
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Thr Ser Leu Leu Gly Gly Gly Ser Pro Lys Asn Asn Thr Ala Ala Glu
225 230 235 240
Thr Glu Asp Asp Glu Ser Asp Gly Glu Asp Arg Gly Gly Gly Thr Ser
245 250 255
Gly Ser Leu Arg Arg Ser Lys Arg Asn Ser Asp Ser Thr Glu Leu Ala
260 265 270
Ala Gln Met Asn Glu Ser Val Asp Val Met Asp Val Ile Ala Ile Cys
275 280 285
Cys Pro Lys Tyr Lys Asp Arg Pro Gln Ile Ala Arg Val Val Gln Lys
290 295 300
Thr Ser Ser Gly Phe Ser Val Gln Trp Met Ala Gly Ser Tyr Ser Gly
305 310 315 320
Ser Trp Thr Glu Ala Lys Arg Arg Asp Gly Arg Lys Leu Val Pro Trp
325 330 335
Val Asp Thr Ile Lys Glu Ser Asp Ile Ile Tyr Lys Lys Ile Ala Leu
340 345 350
Thr Ser Ala Asn Lys Leu Thr Asn Lys Val Val Gln Thr Leu Arg Ser
355 360 365
Leu Tyr Ala Ala Lys Asp Gly Thr Ser Ser
370 375
(2) INFORMATION FOR SEQ ID N0:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2353 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
TCCCGTCGCG CTCAGCATCC TCAATCATCC GCAGGCTGAT GCGGTCCTTC ACGCTCCCGC 60
CCGCGTTGAA GAACTCACAC TTGGCCAAGA GCTCACACTT CAGGCCGAAC TTCTTCCCAA 120
TCTTGTTGAT TCTGACCATA GGGGTGTCCC CGATTTTCTT CAGAATATCT GGCAAGATTT 180
TTGGAGATTT TGCCGGGGCA GTGTGGTGAT GTGGGGACTC GGAGGCAGGC CGGCCCAGCT 240
GCCAGGTGCA CCTGCTCGGA GCATCGGGCC GGATCCACAG GGGCTCCTTG GCTTCCTTAT 300
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CCTCTGGGGA CCCCTTCTCC AGGCTCCCCTTCGCCGAGTG TGGCCCTGAGCGGTGGGGGC360
AGCCTGTGGG CCCCACTTCT GCCTGGGGGGTCTCAGAAGG CATGCTGGGACCGGACAGAT420
GGTGCTGACA GTTCAGCACC GCTCAAACTCTGTAAGGAGA CGTGACAACAGAATCTGTCA480
TGGGATCCTG GAATAGAAAA GGGCAGTGAGGAGAAAACTC TGCAAATCTGACTCAAGCGC540
GGACTTTGGT TAGGCCTCTT TGGACAGTACTTTGAGAATT ATGGATCTCCGGATATTAGT600
AGCAAGGAAG TTTGTAGGAG CAGCAAATTATCGGGAGAAG ATTCATAGTACTTTGACTCC660
ATGTGGGACT TTTCTGTTTG CTGGAAGTGAGGATGGTATA GTGTATGTTTGGAACCCAGA720
AACAGGAGAA CAAGTAGCCA TGTATTCTGACTTGCCATTC AAGTCACCCATTCGAGACAT780
TTCTTATCAT CCATTTGAAA ATATGGTTGCATTCTGTGCA TTTGGGCAAAATGAGCCAAT840
TCTTCTGTAT ATTTACGATT TCCATGTTGCCCAGCAGGAG GCTGAAATGTTCAAACGCTA900
CAATGGAACA TTTCCATTAC CTGGAATACACCAAAGTCAA GATGCCCTATGTACCTGTCC960
AAAACTACCC CATCAAGGCT CTTTTCAGATTGATGAATTT GTCCACACTGAAAGTTCTTC1020
AACGAAGATG CAGCTAGTAA AACAGAGGCTTGAAACTGTC ACAGAGGTGATACGTTCCTG1080
TGCTGCAAAA GTCAACAAAA ATCTCTCATTTACTTCACCA CCAGCAGTTTCCTCACAACA1140
GTCTAAGTTA AAGCAGTCAA ACATGCTGACCGCTCAAGAG ATTCTACATCAGTTTGGTTT1200
CACTCAGACC GGGATTATCA GCATAGAAAGAAAGCCTTGT AACCATCAGGTAGATACAGC1260
ACCAACGGTA GTGGCTCTTT ATGACTACACAGCGAATCGA TCAGATGAACTAACCATCCA1320
TCGCGGAGAC ATTATCCGAG TGTTTTTCAAAGATAATGAA GACTGGTGGTATGGCAGCAT1380
AGGAAAGGGA CAGGAAGGTT ATTTTCCAGCTAATCATGTG GCTAGTGAAACACTGTATCA1440
AGAACTGCCT CCTGAGATAA AGGAGCGATCCCCTCCTTTA AGCCCTGAGGAAAAAACTAA1500
AATAGAAAAA TCTCCAGCTC CTCAAAAGGTAAAATAAAAC AAAACAGCTCACAGAGACAC1560
CTCCTTCTTC CCCCTGCTTC TGCCTCCATGAGTAACTACC TATATGACGTGCTGCTGCTG1620
AGACCAAGGA ATGAGTGAGT AAAGGTGTTTGGAAGTCAAA TATTGGTCCTAGTTAAGTAA1680
GTGTCCTTTT CAAGTAAGTG GTAAACTTTGTAATGTGGAC CCCCTTCTAACTTAGGATTC1740
ATATAATTTG AACACAGGCT AAGCTGCCTCTGCATCATTA AAAAGGATTTGGGTATGCCA1800
GTGATAGGAA TACACAGTAG AAAAGATGGCAGGGACCATG TTTAACATACCTAAGACAAA1860
CTTTCTGAGG ATATCACAGC ATCCATTTCACATGAACAGA CTTAGCAGAATTGGCAAGAC1920
CTGCACATGG CAACATATTA TTCATTTTTC AAAGTAACAT1980
AGGATAACAA AAAATAATTA
gl
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TGGCTTAACA CTGGTATATA GTTCAGTATGACTTCTCTCCCCTCAACCTC TGGAATAGGT2040
TCAAATTGAT GAGAATTTCT GATTAGAGCCCTTAATGTTGAGTTTTTTGA AAAGTTTTAT2100
CAAGTTTCAT ATATACCTAT ATTGATGGTAAGTTGCTGGTCTTGCCATGG GCAAAGAGAG2160
AAAAATGATA YTGAGACCTT GTAAAGAATAGYTGGACATGGAGGCGCACA CCTGTAATCC2220
CAGCAAYTCA GAAGGCTGAG GCGGGAGGATTGCTTGAGCCCAGGAGTTCA AGGCTGCAGT2280
GAGCTACGAT CATGCCACTG TACTCCAGCCTGAGCAACAAAGCATGACCC AATCTCTTAA2340
p,~~iAAAAi~AAA AAA 2
3
5
3
(2) INFORMATION FOR SEQ
ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 318 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
Met Asp Leu Arg Ile Leu Val Ala Arg Lys Phe Val Gly Ala Ala Asn
1 5 10 15
Tyr Arg Glu Lys Ile His Ser Thr Leu Thr Pro Cys Gly Thr Phe Leu
20 25 30
Phe Ala Gly Ser Glu Asp Gly Ile Val Tyr Val Trp Asn Pro Glu Thr
35 40 45
Gly Glu Gln Val Ala Met Tyr Ser Asp Leu Pro Phe Lys Ser Pro Ile
50 55 60
Arg Asp Ile Ser Tyr His Pro Phe Glu Asn Met Val Ala Phe Cys Ala-
65 70 75 80
Phe Gly Gln Asn Glu Pro Ile Leu Leu Tyr Ile Tyr Asp Phe His Val
85 90 95
Ala Gln Gln Glu Ala Glu Met Phe Lys Arg Tyr Asn Gly Thr Phe Pro
100 105 110
Leu Pro Gly Ile His Gln Ser Gln Asp Ala Leu Cys Thr Cys Pro Lys
115 120 125
Leu Pro His Gln Gly Ser Phe Gln Ile Asp Glu Phe Val His Thr Glu
130 135 140
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Ser Ser Ser Thr Lys Met Gln Leu Val Lys Gln Arg Leu Glu Thr Val
145 150 155 160
Thr Glu Val Ile Arg Ser Cys Ala Ala Lys Val Asn Lys Asn Leu Ser
165 170 175
Phe Thr Ser Pro Pro Ala Val Ser Ser Gln Gln Ser Lys Leu Lys Gln
180 185 190
Ser Asn Met Leu Thr Ala Gln Glu Ile Leu His Gln Phe Gly Phe Thr
195 200 205
Gln Thr Gly Ile Ile Ser Ile Glu Arg Lys Pro Cys Asn His Gln Val
210 215 220
Asp Thr Ala Pro Thr Val Val Ala Leu Tyr Asp Tyr Thr Ala Asn Arg
225 230 235 240
Ser Asp Glu Leu Thr Ile His Arg Gly Asp Ile Ile Arg Val Phe Phe
245 250 255
Lys Asp Asn Glu Asp Trp Trp Tyr Gly Ser Ile Gly Lys Gly Gln Glu
260 265 270
Gly Tyr Phe Pro Ala Asn His Val Ala Ser Glu Thr Leu Tyr Gln Glu
275 280 285
Leu Pro Pro Glu Ile Lys Glu Arg Ser Pro Pro Leu Ser Pro Glu Glu
290 295 300
Lys Thr Lys Ile Glu Lys Ser Pro Ala Pro Gln Lys Val Lys
305 310 315
(2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2567 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
AGCCCACCTA CACACCCAGC TCCCACCCAT GGAGAGCACC CCAGTCCTGT TCCTCCTGCC 60
CTGGACCCTG GCCAACTCTG CCACAAGCTC TACCCTCGGT ACAACAGGCT CTGTCCCCAC 120
ATCTACAGAC CCTGCCCCAT CTGCACACCT AGACTCAGTT CATAAGTCCA CAGACTCTGG 180
CCCTTCAGAA CTGCCAGGCC CCACTCACAC CACTACAGGC TCTACCTATA GTGCCATTAC 240
83
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CACTACCCACAGTGCTCCAA TCACACTACTACAGGCTCCACCCACAAGCC300
GCCCCCTCAC
CATAATCTCTACCCTTACTACTACAGGCCCTACCCTCAATATCATAGGCCCAGTCCAGAC360
TACCACAAGCCCCACCCACACTATGCCAAGCCCTACCCATACCACAGCAAGCCCCACTCA420
TACTTCCACAAGCCCCACCCATACCCCCACAAGTCCCACCCACAAAACCAGTATGTCACC480
TCCCACCACTACAAGTCCTACCCCCAGTGGTATGGGCCTAGTCCAGACTGCCACAAGTCC540
CACCCATCCTACCACAAGCCCCACCCATCCCACCACAAGCCCCATCCTTATAAATGTAAG600
CCCTTCCACTTCTCTAGAACTTGCTACCCTCTCCAGCCCCTCCAAACACTCAGACCCCAC660
CCTCCCAGGCACTGACTCCCTTCCCTGTAGTCCCCCAGTCTCCAATTCCTACACTCAGGC720
AGACCCTATGGCCCCCAGAACTCCCCACCCAAGTCCTGCCCATTCCAGTAGGAAACCCCT780
CACAAGCCCTGCCCCAGATCCCTCAGAGTCTACGGTTCAGAGTCTAAGCCCCACTCCCTC840
ACCCCCAACCCCTGCACCCCAGCATTCAGACCTTTGCCTGGCCATGGCTGTCCAGACCCC900
AGTCCCAACGGCAGCCGGAGGGTCTGGGGACAGGAGCCTGGAGGAGGCACTGGGGGCCCT960
AATGGCTGCCCTGGATGACTACCGTGGCCAGTTTCCTGAGCTGCAGGGCCTGGAGCAGGA1020
GGTGACCCGCCTAGAAAGTCTGCTCATGCAGAGACAAGGTCTGACTCGCAGCCGGGCCTC1080
CAGTCTCAGCATCACTGTGGAGCATGCCTTGGAGAGCTTCAGCTTCCTCAATGAAAACGA1140
AGATGAAGACAATGATGTTCCTGGGGACAGGCCTCCAAGCAGCCCGGAGGCTGGGGCTGA1200
GGACAGCATAGACTCACCCAGTGCCCGCCCCCTCAGCACGGGGTGTCCAGCTCTGGATGC1260
TGCCTTGGTCCGGCACCTGTACCACTGCAGTCGCCTCCTGCTGAAACTGGGCACATTTGG1320
GCCCCTGCGCTGCCAGGAGGCATGGGCCCTGGAGCGGCTGCTGCGGGAAGCCCGAGTACT1380
GGAGGCAGTATGCGAGTTCAGCAGGCGGTGGGAGATCCCGGCCAGCTCTGCCCAGGAAGT1440
GGTGCAGTTCTCGGCCTCTCGGCCTGGCTTCCTGACCTTCTGGGACCAGTGCACAGAGAG1500
ACTCAGCTGCTTCCTCTGCCCGGTGGAGCGGGTGCTTCTCACCTTCTGCAACCAGTATGG1560
TGCCCGCCTCTCCCTGCGCCAGCCAGGCTTGGCTGAGGCTGTGTGTGTGAAGTTCCTGGA1620
GGATGCCCTGGGGCAGAAGCTGCCCAGAAGGCCCCAGCCAGGGCCTGGAGAGCAGCTCAC1680
AGTCTTCCAGTTCTGGAGTTTTGTGGAAACCTTGGACAGCCCCACCATGGAGGCCTACGT1740
GACTGAGACCGCTGAGGAGGTGCTACTGGTGCGGAATCTGAACTCGGATGATCAGGCTGT1800
TGTGCTGAAGGCCCTGAGATTGGCGCCCGAGGGGCGTCTGCGAAGGGACGGGCTGCGGGC1860
CCTCAGCTCCCTGCTCGTCCATGGCAACAACAAGGTCATGGCTGCTGTCAGCACCCAGCT1920
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CCGGAGCCTG TCACTGGGCC CTACCTTCCGGGAGAGGGCCCTCCTGTGCT TCCTGGACCA1980
GCTGGAGGAT GAGGACGTGC AGACTCGAGTGGCTGGCTGCCTGGCCCTAG GCTGCATCAA2040
GGCTCCCGAG GGCATTGAGC CCCTGGTGTACCTCTGCCAAACTGACACAG AAGCTGTGAG2100
GGAAGCTGCC CGGCAAAGCC TACAGCAGTGTGGAGAAGAGGGACAGTCTG CCCATCGACG2160
GCTGGAGGAG TCCCTGGACG CCCTGCCCCGCATCTTTGGGCCTGGCAGCA TGGCCAGCAC2220
AGCATTCTAA ACTATTCACC CATGGGTTCCTGGTGCCCCTTTCCCCCCAC TTTCAGGGCT2280
CACCAGGCAC TGGCAGGGAG GGTAAGGGCTGGCTCCAGATACCCCTCCCC CACAGATTCC2340
TAGCAATGAA AATCTAATAT ATTCTTCTGTTGCCCCTGGGGTTGGAGAGT CAGTGCCTGC2400
AGTCAAGTGC CTCCCAGCCT CGGCTCAGCACATCCCTTGCCACAAATCAG TGTCTGGGGC2460
TTGGCCACCC TGCCGCTGCC CAGCCACATCCCTTGGTTTTGTATTTTATT TACAGAGTTT2520
TACAGAAAAT AAAAAAGCAA AATGTCTTTCCTAAAAAAAAAAAAAAA 2567
(2) INFORMATION FOR SEQ :
ID N0:14
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 733 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi)SEQUENCE
DESCRIPTION:
SEQ
ID
N0:14:
MetGlu SerThr ProValLeu PheLeu LeuProTrp ThrLeuAla Asn
1 5 10 15
SerAla ThrSer SerThrLeu GlyThr ThrGlySer ValProThr Ser
20 25 30
ThrAsp ProAla ProSerAla HisLeu AspSerVal HisLysSer Thr
35 40 45
AspSer GlyPro SerGluLeu ProGly ProThrHis ThrThrThr Gly
50 55 60
SerThr TyrSer AlaIleThr ThrThr HisSerAla ProSerPro Leu
65 70 75
80
ThrHis ThrThr ThrGlySer ThrHis LysProIle IleSerThr Leu
85 90 95
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Thr Thr Thr Gly Pro Thr Leu Asn Ile Ile Gly Pro Val Gln Thr Thr
100 105 110
Thr Ser Pro Thr His Thr Met Pro Ser Pro Thr His Thr Thr Ala Ser
115 120 125
Pro Thr His Thr Ser Thr Ser Pro Thr His Thr Pro Thr Ser Pro Thr
130 135 140
His Lys Thr Ser Met Ser Pro Pro Thr Thr Thr Ser Pro Thr Pro Ser
145 150 155 160
Gly Met Gly Leu Val Gln Thr Ala Thr Ser Pro Thr His Pro Thr Thr
165 170 175
Ser Pro Thr His Pro Thr Thr Ser Pro Ile Leu Ile Asn Val Ser Pro
180 185 190
Ser Thr Ser Leu Glu Leu Ala Thr Leu Ser Ser Pro Ser Lys His Ser
195 200 205
Asp Pro Thr Leu Pro Gly Thr Asp Ser Leu Pro Cys Ser Pro Pro Val
210 215 220
Ser Asn Ser Tyr Thr Gln Ala Asp Pro Met Ala Pro Arg Thr Pro His
225 230 235 240
Pro 5er Pro Ala His Ser Ser Arg Lys Pro Leu Thr Ser Pro Ala Pro
245 250 255
Asp Pro Ser Glu Ser Thr Val Gln Ser Leu Ser Pro Thr Pro Ser Pro
260 265 270
Pro Thr Pro Ala Pro Gln His Ser Asp Leu Cys Leu Ala Met Ala Val
275 280 285
Gln Thr Pro Val Pro Thr Ala Ala Gly Gly Ser Gly Asp Arg Ser Leu
290 295 300
Glu Glu Ala Leu Gly Ala Leu Met Ala Ala Leu Asp Asp Tyr Arg Gly
305 310 315 320
Gln Phe Pro Glu Leu Gln Gly Leu Glu Gln Glu Val Thr Arg Leu Glu
325 330 335
Ser Leu Leu Met Gln Arg Gln Gly Leu Thr Arg Ser Arg Ala Ser Ser
340 345 350
Leu Ser Ile Thr Val Glu His Ala Leu Glu Ser Phe Ser Phe Leu Asn
355 360 365
Glu Asn Glu Asp Glu Asp Asn Asp Val Pro Gly Asp Arg Pro Pro Ser
370 375 380
Ser Pro Glu Ala Gly Ala Glu Asp Ser Ile Asp Ser Pro Ser Ala Arg
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385 390 395 400
Pro Leu Ser Thr Gly Cys Pro Ala Leu Asp Ala Ala Leu Val Arg His
405 410 415
Leu Tyr His Cys Ser Arg Leu Leu Leu Lys Leu Gly Thr Phe Gly Pro
420 425 430
Leu Arg Cys Gln Glu Ala Trp Ala Leu Glu Arg Leu Leu Arg Glu Ala
435 440 445
Arg Val Leu Glu Ala Val Cys Glu Phe Ser Arg Arg Trp Glu Ile Pro
450 455 460
Ala Ser Ser Ala Gln Glu Val Val Gln Phe Ser Ala Ser Arg Pro Gly
465 470 475 480
Phe Leu Thr Phe Trp Asp Gln Cys Thr Glu Arg Leu Ser Cys Phe Leu
485 490 495
Cys Pro Val Glu Arg Val Leu Leu Thr Phe Cys Asn Gln Tyr Gly Ala
500 505 510
Arg Leu Ser Leu Arg Gln Pro Gly Leu Ala Glu Ala Val Cys Val Lys
515 520 525
Phe Leu Glu Asp Ala Leu Gly Gln Lys Leu Pro Arg Arg Pro Gln Pro
530 535 540
Gly Pro Gly Glu Gln Leu Thr Val Phe Gln Phe Trp Ser Phe Val Glu
545 550 555 560
Thr Leu Asp Ser Pro Thr Met Glu Ala Tyr Val Thr Glu Thr Ala Glu
565 570 575
Glu Val Leu Leu Val Arg Asn Leu Asn Ser Asp Asp Gln Ala Val Val
580 585 590
Leu Lys Ala Leu Arg Leu Ala Pro Glu Gly Arg Leu Arg Arg Asp Gly
595 600 605
Leu Arg Ala Leu Ser Ser Leu Leu Val His Gly Asn Asn Lys Val Met-
610 615 620
Ala Ala Val Ser Thr Gln Leu Arg Ser Leu Ser Leu Gly Pro Thr Phe
625 630 635 640
Arg Glu Arg Ala Leu Leu Cys Phe Leu Asp Gln Leu Glu Asp Glu Asp
645 650 655
Val Gln Thr Arg Val Ala Gly Cys Leu Ala Leu Gly Cys Ile Lys Ala
660 665 670
Pro Glu Gly Ile Glu Pro Leu Val Tyr Leu Cys Gln Thr Asp Thr Glu
67s 68o ss5
87
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Ala Val Arg Glu Ala Ala Arg Gln Ser Leu Gln Gln Cys Gly Glu Glu
690 695 700
Gly Gln Ser Ala His Arg Arg Leu Glu Glu Ser Leu Asp Ala Leu Pro
705 710 715 720
Arg Ile Phe Gly Pro Gly Ser Met Ala Ser Thr Ala Phe
725 730
{2) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2501 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE
DESCRIPTION:
SEQ ID
N0:15:
GGCAGTCAAAGCACTGATGA TGATAAAATCGTTCAGTACCATTGGGAAGAACTTAAGGGG60
CCTCTAAGAGAAGAGAAGAT TTCTGAAGATACAGCCATATTAAAACTAAGTAAACTCGTC120
CCTGGGAACTACACTTTCAG CTTGACTGTAGTAGACTCTGATGGAGCTACCAACTCTACT180
ACTGCAAACCTGACAGTGAA CAAAGCTGTGGATTACCCCCCTGTGGCCAACGCAGGCCCC240
AACCAAGTGATCACCCTGCC CCAAAACTCCATCACCCTCTTTGGGAACCAGAGCACTGAT300
GATCATGGCATCACCAGCTA TGAGTGGTCACTCAGCCCAAGCAGCAAAGGGAAAGTGGTG360
GAGATGCAGGGTGTTAGAAC ACCAACCTTACAGCTCTCTGCGATGCAAGAAGGAGACTAC420
ACTTACCAGCTCACAGTGAC TGACACAATAGGACAGCAGGCCACTGCTCAAGTGACTGTT480
ATTGTGCAACCTGAAAACAA TAAGCCTCCTCAGGCAGATGCAGGCCCAGATAAAGAGCTG540
ACCCTTCCTGTGGATAGCAC AACCCTGGATGGCAGCAAGAGCTCAGATGATCAGAAAATT600
ATCTCATATCTCTGGGAAAA AACACAGGGACCTGATGGGGTGCAGCTCGAGAATGCTAAC660
AGCAGTGTTGCTACTGTGAC TGGGCTGCAAGTGGGGACCTATGTGTTCACCTTGACTGTC720
AAAGATGAGAGGAACCTGCA AAGCCAGAGCTCTGTGAATGTCATTGTCAAAGAAGAATAA780
ACAAACCACCTATAGCCAAG ATAACTGGGAATGTGGTGATTACCCTACCCACGAGCACAG840
CAGAGCTGGATGGCTCTAAG TCCTCAGATGACAAGGGAATAGTCAGCTACCTCTGGACTC900
GAGATGAGGGGAGCCCAGCA GCAGGGGAGGTGTTAAATCACTCTGACCATCACCCTATCC960
gg
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TTTTTCTTTC AAACCTGGTT GAGGGAACCT ACACTTTTCA CCTGAAAGTG ACCGATGCAA 1020
AGGGTGAGAG TGACACAGAC CGGACCACTG TGGAGGTGAA ACCTGATCCC AGGAAAAACA 1080
ACCTGGTGGA GATCATCTTG GATATCAACG TCAGTCAGCT AACTGAGAGG CTGAAGGGGA 1140
TGTTCATCCG CCAGATTGGG GTCCTCCTGG GGGTGCTGGA TTCCGACATC ATTGTGCAAA 1200
AGATTCAGCC GTACACGGAG CAGAGCACCA AAATGGTATT TTTTGTTCAA AACGAGCCTC 1260
CCCACCAGAT CTTCAAAGGC CATGAGGTGG CAGCGATGCT CAAGAGTGAG CTGCGGAAGC 1320
AAAAGGCAGA CTTTTTGATA TTCAGAGCCT TGGAAGTCAA CACTGTCACA TGTCAGCTGA 1380
ACTGTTCCGA CCATGGCTAC TGTGACTCGT TCACCAAACG CTGTATCTGT GACCCTTTTT 1440
GGATGGAGAA TTTCATCAAG GTGCAGCTGA GGGATGGAGA CAGCAACTGT GAGTGGAGCG 1500
TGTTATATGT TATCATTGCT ACCTTTGTCA TTGTTGTTGC CTTGGGAATC CTGTCTTGGA 1560
CTGTGATCTG TTGTTGTAAG AGGCAAAAAG GAAAACCCAA GAGGAAAAGC AAGTACAAGA 1620
TCTGGATGCC ACGGATCAGG AAAGCTTGGA GCTGAAGCCA ACCTCCCGAG CAGGCATCAA 1680
ACAGAAAGGC CTTTTGCTAA GTAGCAGCCT GATGCACTCC GAGTCAGAGC TGGACAGCGA 1740
TGATGCCATC TTTACATGGC CAGACCGAGA GAAGGGCAAA CTCCTGCATG GTCAGAATGG 1800
CTCTGTACCC AACGGGCAGA CCCCTCTGAA GGCCAGGAGC CCGCGGGAGG AGATCCTGTA 1860
GCCACCTGGT CTGTCTCCTC AGGGCAGGGC CCAGCACACT GCCCGGCCAG TCCTCCTACC 1920
TCCCGAGTCT GCGGGCAGCT GCTGTCCCAG CATCTGCTGG TCATTTCGCC CTGACAGTCC 1980
CAACCAGAAC CCCTGGGACT TGAATCCAGA GACGTCCTTC AGGAACCCCT CAACGAAGCT 2040
GTGAATGAAG AGGTTTCCTC TTTAAACCTG TCTGGTGGGC CCCCAGATAT CCTCACCTCA 2100
GGGCCTCCTT TTTTTGCAAA CTCCTCCCCT CCCCCGAGGG CAGACCCAGC CAGCTGCTAA 2160
GCTCTGCAGC TCCCCAGTGG ACAGTGTCAT TGTGCCCAGA GTGCTGCAAG GTGAGGCCTG 2220
CTGTGCTGCC CGCACACCTG AGTGCAAAAC CAAGCACTGT GGGCATGGTG TTTCCCTCTC 2280
TGGGGTAGAG TACGCCCTCT CGCTGGGCAA AGAGGAAGTG GCACCCCTCC CCTCACCACA 2340
GATGCTGAGA TGGTAGCATA GAAATGATGG CCGGGCGCGG TGGCTMACGC CTGTAATCCC 2400
AGCACTTTGG GAGGCCGAGG CGGGCGGATC ATGAGGTCAG GAGATCAAGA CCACCCTGGC 2460
TAACACGGTG AAACCCCATC TCTACTAAAA h,F~~AAAAAAp, A 2 5 O 1
(2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
89
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(A) LENGTH: 138 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:
Met Gln Gly Val Arg Thr Pro Thr Leu Gln Leu Ser Ala Met Gln Glu
1 5 10 15
Gly Asp Tyr Thr Tyr Gln Leu Thr Val Thr Asp Thr Ile Gly Gln Gln
20 25 30
Ala Thr Ala Gln Val Thr Val Ile Val Gln Pro Glu Asn Asn Lys Pro
35 40 45
Pro Gln Ala Asp Ala Gly Pro Asp Lys Glu Leu Thr Leu Pro Val Asp
50 55 60
Ser Thr Thr Leu Asp Gly Ser Lys Ser Ser Asp Asp Gln Lys Ile Ile
65 70 75 80
Ser Tyr Leu Trp Glu Lys Thr Gln Gly Pro Asp Gly Val Gln Leu Glu
85 90 95
Asn Ala Asn Ser Ser Val Ala Thr Val Thr Gly Leu Gln Val Gly Thr
100 105 110
Tyr Val Phe Thr Leu Thr Val Lys Asp Glu Arg Asn Leu Gln Ser Gln
115 120 125
Ser Ser Val Asn Val Ile Val Lys Glu Glu
130 135
(2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1820 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
GGCAGCAGCA GAGGGAGAGC TCGGGGCTTG GAGGGGAAAC AGCGGAAGAC CTAAGATTAT 60
CA 02283193 1999-09-02
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CGGGAGGGCA GCAGAGGCAG AGAACGAGGA CAGGACCCTT GGCCGTCTTC 120
TTCCAGGGAA
CGAGAGGTCA CAGCCTCGCT CTCCGCTTAG GCTTCTGGCG CCCCAGCTTA 180
AAGCCGAGGC
TGCGGCTGAC AAAGGGCTCG CGCCGGTGCC GCCGCCCTTC TCATCCGGGC 240
ATTCGGGTCC
CTGCGGAGAG GGAGGGGGAA GGGCAGAGGG GGAGGGGAAG GAGCCSGAGG 300
GGCSCACACT
TGGAGCTGAA GCCCTCTCCA GGGCTCCGGG CCGGTGCCCC AACGGACAGA 360
GGTCGAGGAG
GACCCGCAGA GGTGGCAGCG GCCGGGGGCA GGAGGATGGT GCAGAAGGAG 420
AGTCAAGCGA
CGTTGGAGGA GCGGGAGAGC GAGCTCAGTT CCAACCCTGC CGCCTCTGCG 480
GGGGCATCGC
TGGAGCCGCC GGCAGCTCCG GCACCCGGAG AAGACAACCC CGCCGGGGCT 540
GGGGGAGCGG
CGGTGGCCGG GGCTGCAGGA GGGGCTCGGC GGTTTCCTCT GCGGTGTGGT 600
GGAAGGATTT
TATGGAAGAC CTTGGGTTAT GGAACAGAGA AAAGAACTCT TTAGAAGGCT 660
CCAGAAATGG
GAATTAAATA CATACTTGTA TGCCCCAAAA GATGACTACA AACATAGGAT 720
GTTTTGGCGA
GAGATGTATT CAGTGGAGGA AGCTGAGCAA CTTATGACTC TCATCTCTGC 780
TGCACGAGAA
TATGAGATAG AGTTCATCTA TGCGATCTCA CCTGGATTGG ATATCACTTT 840
TTCTAACCCC
AAGGAAGTAT CCACATTGAA ACGTAAATTG GACCAGGTTT CTCAGTTTGG 900
GTGCAGATCA
TTTGCTTTGC TTTTTGATGA TATAGACCAT AATATGTGTG CAGCAGACAA 960
AGAGGTATTC
AGTTCTTTTG CTCATGCCCA AGTCTCCATC ACAAATGAAA TCTATCAGTA 1020
CCTAGGAGAG
CCAGAAACTT TCCTCTTCTG TCCCACAGAA TACTGTGGCA CTTTCTGTTA 1080
TCCAAATGTG
TCTCAGTCTC CATATTTAAG GACTGTGGGT GAAAAGCTTC TACCTGGAAT 1140
TGAAGTGCTT
TGGACAGGTC CCAAAGTTGT TTCTAAAGAA ATTCCAGTAG AGTCCATCGA 1200
AGAGGTTTCT
AAGATTATTA AGAGAGCTCC AGTAATCTGG GATAACATTC ATGCTAATGA 1260
TTATGATCAG
AAGAGACTGT TTCTGGGCCC GTACAAAGGA AGATCCACAG AACTCATCCC 1320
ACGGTTAAAA
GGAGTCCTCA CTAATCCAAA TTGTGAATTT GAAGCCAACT ACGTTGCTAT 1380
CCACACCCTT
GCCACCTGGT ACAAATCAAA CATGAATGGG AGTGAGAAAA GATGTAGTGA 1440
TGACTGACAG
TGAAGATAGT ACTGTGTCCA TCCAGATAAA ATTAGAAAAT GAAGGCAGTG 1500
ATGAAGATAT
TGAAACTGAT GTACTCTATA GTCCACAGAT GGCTCTAAAG CTAGCATTAA 1560
CAGAATGGTT
GCAAGAGTTT GGTGTGCYTC ATCAATACAG CAGTAGGCAA GTTGCACACA 1620
GTGGAGCTAA
AGCAAGTGTA GTTGATGGGA CTCCTTTAGT TGCAGCACCC TCTTTAAATG 1680
CCACAACCGT
AGTAACAACA GTTTATCAGG AGCCCATTAT GAGCCAGGGA GCAGCCTTGA 1740
GTGGTGAGCC
91
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TACTACTCTG ACCAAGGAAG AAGAAAAGAA ACAGCCTGAT GAAGAACCCA TGGACATGGT 1800
GGTGGAAAAA P~~AAAAAAAP. 1820
(2) INFORMATION FOR SEQ ID N0:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 272 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:
Met Glu Gln Arg Lys Glu Leu Phe Arg Arg Leu Gln Lys Trp Glu Leu
1 5 10 15
Asn Thr Tyr Leu Tyr Ala Pro Lys Asp Asp Tyr Lys His Arg Met Phe
20 25 30
Trp Arg Glu Met Tyr Ser Val Glu Glu Ala Glu Gln Leu Met Thr Leu
35 40 45
Ile Ser Ala Ala Arg Glu Tyr Glu Ile Glu Phe Ile Tyr Ala Ile Ser
50 55 60
Pro Gly Leu Asp Ile Thr Phe Ser Asn Pro Lys Glu Val Ser Thr Leu
65 70 75 80
Lys Arg Lys Leu Asp Gln Val Ser Gln Phe Gly Cys Arg Ser Phe Ala
85 90 95
Leu Leu Phe Asp Asp Ile Asp His Asn Met Cys Ala Ala Asp Lys Glu
100 105 110
Val Phe Ser Ser Phe Ala His Ala Gln Val Ser Ile Thr Asn Glu Ile
115 120 125
Tyr Gln Tyr Leu Gly Glu Pro Glu Thr Phe Leu Phe Cys Pro Thr Glu
130 135 140
Tyr Cys Gly Thr Phe Cys Tyr Pro Asn Val Ser Gln Ser Pro Tyr Leu
145 150 155 160
Arg Thr Val Gly Glu Lys Leu Leu Pro Gly Ile Glu Val Leu Trp Thr
165 170 175
Gly Pro Lys Val Val Ser Lys Glu Ile Pro Val Glu Ser Ile Glu Glu ,
180 185 190
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Val Ser Lys Ile Ile Lys Arg Ala Pro Val Ile Trp Asp Asn Ile His
195 200 205
Ala Asn Asp Tyr Asp Gln Lys Arg Leu Phe Leu Gly Pro Tyr Lys Gly
210 215 220
Arg Ser Thr Glu Leu Ile Pro Arg Leu Lys Gly Val Leu Thr Asn Pro
225 230 235 240
Asn Cys Glu Phe Glu Ala Asn Tyr Val Ala Ile His Thr Leu Ala Thr
245 250 255
Trp Tyr Lys Ser Asn Met Asn Gly Ser Glu Lys Arg Cys Ser Asp Asp
260 265 270
(2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2405 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION:
SEQ ID N0:19:
TATCCATTAC GTCGACTAATACGTACATAA GAATTCAATCGGGAGACAATGTAATAACCC 60
AAACACTGGG TATTCATATTTGACACATGG GCAAACTTGCCAGTGGAATGGAATTGTGAC 120
CTGACAGAGA AGGGAAGGCAGGCTGACGAA GGTGATCGAATGGGAGAACAGTGCTGTGGT 180
GATCATGAGA ATGAGGCTTTTCTGTAGCAT GTAAACCAAACCGGACCCTTGGCAGTTCGT 240
CGTCCCTCAG TTCTCCAGATGCTATTTTTT GCAGGTTCTACCAAGTGCTTGTTGATTACC 300
CTAGTTGTAA TTATCTAGGGAAGAGATGAA TGTAAGTGAGAGTGCAGAGCACTGGGGAGG 360
GTGACAGTGA AATGCAATTAGAGGCAGCAA GAGAGTCCTAGTCTGTTCTCACATAACCGG 420
ACTTGAACGC TCCAGTGCGAGCAGAGTGCT GGGGGTGGATTCCACTGCCGAACCACGGCA 480
GCTTTGCTTT ACTCTTCAGCATGGGGGTGG TAACTAGCTGCACAGCAAGTTATGAAATGG 540
AAAGCAAGCT TAACAGCTGTAATCTCATCG GATACCCTGGAGCAAATGCCTTGGGATTGC 600
CTGAAGTGAA GTGTTTAGCATCCACCAAAT AGTTGAGTTTCTAAGATGGGCCATGCGGGA 660
TCCCTGCCAC ACGGGTGTGGGGGCAGCGCG CTCCTCCCTGCCTCGGCTGCTGTGTGTCTT 720
93
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AGTCTTCATGTTTCCCCTTGGCGTCACCTCTGGTCCTGTCCATCTTGTGCACATGTGTCC780
CTGAGGGCGAGTCACACCTGTGTCTTGAGTCTGCTGTAAGGTGTTCAGTCTAC_CTCAAGG840
GGTCATCATGGTAAGCTCTATCCCACGGCATCCTCCGTCCATCACCACACACACTGGAAT900
GTATCCCCCCCGCCCTGCCCCTTCGATAACCTCCTGACCTTGTCTTTCCTATAATCACCA960
CAAACTCCTAGTGCAAATTGGATGCTGCTTTAAATGTGAAAACAATTGTTCAAAAGCTAT1020
AAAACCTGAATGAAAGCTGAAGCTGAATTTATAAGCCTTGTTGCATATGAGCCAAAAGTG1080
CAAAAAGCTCTATATAATGAACTAACCTGCCACTCGTATAAATATAAATATATATAAATA1140
TATTAAAATCAGACTGTTCTACAAAATTGTGTATTTGTATTTTTGTGTACGTACAATTTT1200
CTGCTAAGCAGAAGGAGGATGTAGTATAGGATGCTGTGAGAAGAGATTTGGTTTAATCCT1260
ATTTCTTTGTTACTTATTTTTGTTAACATCAGATGCCTGTCTTTGTCTTATATGTGTATG1320
ACACTGTTTGGAAAGCTGCAGTATCTCTCTTCCTGAGGTCCAGAGGCCATTAAAGAATAA1380
ACATGGGGCTTAGAGGTGTCTTGGCCACCAAACACAGGTAAGCCAACAGAGTTCGGCTCC1440
CGTACGTGGTGTGGTGGACCTGGGGCCTGGAGCCACTTGTACTCCTGGGGTGGGACTGAG1500
GGAGGGAAAGGGCTCCCCCACTTCAGGCTGGTCACTGTATGTTGACTTCCCTCTGGGAAG1560
CAAAATTCCACTTGTAAAAATCAGCTTCCAAGACAAAAGATGATCCGATGTCACTTCTGG1620
GTCACCAGGAAGGGACACAGGATCTCTCTGAACTTTGGAACAGACCCTCATATTCTGGGG1680
CCAGAAGTTTGCCCAGAAAGCAGCAGGTGGCTCTGCCTGGCTGTAGAGCCCAGCTCATTG1740
GCTGTCCCTGGGCTTGGTCTCCCTCTTCCGAGTAGTTGCTGCCTTTCTTCAGATCAGGTT1800
ACCACAATGCCTCCCCGCTGCTGACGCTTCATCCCCCACACCTCCAGCCCCAGTTACCTG1860
GAGCTTCTCAGAACCCACTTTGCCGGTGCTAAAACACAAGAGGGGGTGAAAGTGGCTGCC1920
AGTAATGGCCAGAAACCAACCACCAGAGGCCAGGCTGAAAGACAAGCTCCGGGTGTCCAG1980
GGGCTGACGGGCCAACCATGTGGCAGGTCCCAGGCCCCACCCACTGCGCCATCCGCCTCT2040
GAGCTCCACAGTGGTCCCACTAATGGGAACCTCCTCTAGGGAGAGTGATACTGCACCTTC2100
ACCCGTAGGACTCATATTTATAACAATGTGTAATGGCTGTAGCAAAAAGCCCTTGTTTCT2160
AGATGTAAATGGTCAAAGAAACAAGCGCTCTATTGTTTTGAATAAAATAGTTCAAATGAG2220
TCCTGTATCATTGTATCTCCTATTCTGGATTAGTGCCTTTTGGACAGTAGACTGTTCTGT2280
AATTAAAATGTAGTATACTGCTTTTTTGTACAGTTTTGTTTTAATAAAACTTTTTTTTAA2340
TTTGTGTTTATTTTAGTATTGTACCTATTAGAGAATAAAATGTATAACTGP~~P.AAAAA:4A2400
94
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2405
(2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 76 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi)SEQUENCE
DESCRIPTION:
SEQ
ID
N0:20:
MetGlyThr SerSerArg GluSer AspThrAla ProSer ProValGly
5 10 15
LeuIlePhe IleThrMet CysAsn GlyCysSer LysLys ProLeuPhe
20 25 30
LeuAspVal AsnGlyGln ArgAsn LysArgSer IleVal LeuAsnLys
35 40 45
IleValGln MetSerPro ValSer LeuTyrLeu LeuPhe TrpIleSer
50 55 60
AlaPheTrp ThrValAsp CysSer ValIleLys Met
65 70 75
(2) INFORMATION FOR SEQ ID N0:21:
(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 N0:21:
CNAGCAGCAGA TACAGCAGTA AGGAAGGC 29
(2) INFORMATION FOR SEQ ID N0:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
i
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(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
GNGCTCTCCTT AATCGCCGTC TCAAACAT 29
(2) INFORMATION FOR SEQ ID N0:23:
(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) SEQ.UENCE DESCRIPTION: SEQ ID N0:23:
CNTTGGCCAGG TCTTCCTGCT CCTTCATT 29
(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: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:
ANGCTGCAAAG CTGGTGTAGA AGTAGGTG 29
(2) INFORMATION FOR SEQ ID N0:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
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(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:
ANTTAGCCATG TCACTCCGCA GGAAGTCC 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:
TNTAGTCATAA AGAGCCACTA CCGTTGGT 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:
TNGTATGGGTA GGGCTTGGCA TAGTGTGG 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
97
i
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(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:
TNTCAGGTTTG CAGTAGTAGA GTTGGTAG 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:
CNTCTTCGATG GACTCTACTG GAATTTCT 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:
TNAGCCTGGCC TCTGGTGGTT GGTTTCTG 29
(2) INFORMATION FOR SEQ ID N0:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 336 amino acids
(B) TYPE: amino acid
(C} STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
98
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:31:
Thr Leu Lys Leu Cys Ser Pro Pro Lys Asp His Glu Val Leu Gly Val
1 5 10 15
Ile Gln Arg Phe Leu Lys Leu Arg Ser Pro Trp Lys Ala Leu Ile Thr
20 25 30
Thr Pro Phe Arg Arg Thr
Ile Gln Glu Pro Leu Val
Leu Leu Pro 45
Lys 40
35
Leu Phe Ser HisGlu Gly Ser PhePro LeuAlaTrp
Thr Pro Ser 55 Glu 60
50
Asn Lys IleThrAsp LeuLys GlnLysVal GluAsn LeuPheAsn
Ala 70 75 80
65
GluLysCys GlyGluAla LeuGly LeuLysGln AlaVal LysValPro
85 90 95
PheAlaLeu PheGluSer PhePro GluAspPhe TyrVal GluGlyLeu
100 105 110
ProGluGly ValProPhe ArgArg ProSerThr PheGly IleProArg
115 120 125
LeuGluLys IleLeuArg AsnLys AlaLysIle LysPhe IleIleLys
130 135 140
LysProGlu MetPheGlu ThrAla IleLysGlu SerThr SerSerLys
145 150 155
160
SerProPro ArgLysIle AsnSer SerProAsn ValAsn ThrThrAla
165 170 175
SerGlyVal GluAspLeu AsnIle IleGlnVal ThrIle ProAspAsp
180 185 190
AspAsnGlu ArgLeuSer LysVal GluLysAla ArgGln LeuArgGlu
195 200 205
GlnValAsn AspLeuPhe SerArg LysPheGly GluAla IleGlyMet
210 215 220
GlyPhePro ValLysVal ProTyr ArgLysIle ThrIle AsnProGly
225 230 235
240
CysValVal Val Gly MetPro ProGlyVal SerPhe LysAlaPro
Asp 250 255
245
SerTyrLeu GluIleSer Ser ArgArgIle Leu Ser Glu
260 Met 265 Asp Ala
270
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Phe Ile Lys Phe Thr Val Ile Arg Pro Phe Pro Gly Leu Val Ile Asn
275 280 285
Asn Gln Leu Val Asp Gln Ser Glu Ser Lys Gly Pro Val Ile Gln Glu
290 295 300
Ser Ala Glu Pro Ser Gln Leu Glu Val Pro Ala Thr Glu Glu Ile Lys
305 310 315 320
Glu Thr Asp Gly Ser Ser Gln Ile Lys Gln Glu Pro Asp Pro Thr Trp
325 330 335
(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:
ANCTTGGCCA GGTCTTCCTG CTCCTTCAT 29
100
