Note: Descriptions are shown in the official language in which they were submitted.
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SECRETED PROTEINS AND POLYNUCLEOT1DES 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.
08/845,296), filed
April 25, 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
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 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
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.
SUBSTITUTE SHEET (RULE 26)
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SUMMARY OF THE INVENTION
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:1;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:1 from nucleotide 99 to nucleotide 902;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:1 from nucleotide 162 to nucleotide 902;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:1 from nucleotide 87 to nucleotide 219;
(e) a polynucleotide comprising the nucleotide sequence of the full
length protein coding sequence of clone ci25 4 deposited under accession
number
ATCC 98415;
(f) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone ci25 4 deposited under accession number ATCC 98415;
(g) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone ci25_4 deposited under accession number ATCC
2 0 98415;
(h) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone ci25 4 deposited under accession number ATCC 98415;
(i) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:2;
2 5 (j) 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 from amino acid 129 to amino acid 138 of
SEQ ID N0:2;
(k) a polynucleotide which is an allelic variant of a polynucleotide of
3 0 (a)-(h) above;
(1) a polynucleotide which encodes a species homologue of the protein
of {i) or (j) above ; and
(m) a polynucleotide that hybridizes under stringent conditions to any
one of the polynucleotides specified in (a)-(j).
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Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:1 from nucleotide 99 to nucleotide 902; the nucleotide sequence of SEQ ID
N0:1 from
nucleotide 162 to nucleotide 902; the nucleotide sequence of SEQ ID N0:1 from
nucleotide 87 to nucleotide 219; the nucleotide sequence of the full-length
protein coding
sequence of clone ci25 4 deposited under accession number ATCC 98415; or the
nucleotide sequence of a mature protein coding sequence of clone ci25_4
deposited under
accession number ATCC 98415. In other preferred embodiments, the
polynucleotide
encodes the full-length or a mature protein encoded by the cDNA insert of
clone ci25 4
deposited under accession number ATCC 98415.
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) fragments of the amino acid sequence of SEQ ID N0:2 comprising
the amino acid sequence from amino acid 129 to amino acid 138 of SEQ ID N0:2;
and
(c) the amino acid sequence encoded by the cDNA insert of clone
2 0 ci25_4 deposited under accession number ATCC 98415;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:2.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
2 5 (a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3 from nucleotide 283 to nucleotide 1158;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
3 0 N0:3 from nucleotide 1 to nucleotide 789;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone da228 6 deposited under accession
number ATCC 98415;
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(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone da228_6 deposited under accession number ATCC 98415;
(f) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone da228 6 deposited under accession number
ATCC 98415;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone da228_6 deposited under accession number ATCC 98415;
(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 141 to amino acid 150 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 polynucleotide that hybridizes 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 283 to nucleotide 1158; the nucleotide sequence of SEQ ID
N0:3
from nucleotide 1 to nucleotide 789; the nucleotide sequence of the full-
length protein
coding sequence of clone da228_6 deposited under accession number ATCC 98415;
or the
nucleotide sequence of a mature protein coding sequence of clone da228_6
deposited
2 5 under accession number ATCC 98415. In other preferred embodiments, the
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
of clone da228_6 deposited under accession number ATCC 98415. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:4 from amino acid 1 to amino
acid 169.
3 0 Other embodiments provide the gene corresponding to the cDNA sequence of
SEQ
ID N0:3.
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:
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(a) the amino acid sequence of SEQ ID N0:4;
(b) the amino acid sequence of SEQ ID N0:4 from amino acid 1 to
amino acid 169;
(c) fragments of the amino acid sequence of SEQ ID N0:4 comprising
' 5 the amino acid sequence from amino acid 141 to amino acid 150 of SEQ ID
N0:4;
and
(d) the amino acid sequence encoded by the cDNA insert of clone
da228_6 deposited under accession number ATCC 98415;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:4 or the amino acid
sequence
of SEQ ID N0:4 from amino acid 1 to amino acid 169.
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
NO:S;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:5 from nucleotide 152 to nucleotide 2182;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:5 from nucleotide 2 to nucleotide 931;
2 0 (d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone du410_5 deposited under accession
number ATCC 98415;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone du410 5 deposited under accession number ATCC 98415;
2 5 (f) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone du410 5 deposited under accession number
ATCC 98415;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone du410 5 deposited under accession number ATCC 98415;
30 (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
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comprising the amino acid sequence from amino acid 333 to amino acid 342 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 that hybridizes 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 152 to nucleotide 2182; the nucleotide sequence of SEQ ID
N0:5
from nucleotide 2 to nucleotide 931; the nucleotide sequence of the full-
length protein
coding sequence of clone du410 5 deposited under accession number ATCC 98415;
or the
nucleotide sequence of a mature protein coding sequence of clone du410 5
deposited
under accession number ATCC 98415. In other preferred embodiments, the
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
of clone du410_5 deposited under accession number ATCC 98415. 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 1 to amino
acid 260.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
2 0 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
consisting of:
(a) the amino acid sequence of SEQ ID N0:6;
2 5 (b) the amino acid sequence of SEQ ID N0:6 from amino acid 1 to
amino acid 260;
(c) fragments of the amino acid sequence of SEQ ID N0:6 comprising
the amino acid sequence from amino acid 333 to amino acid 342 of SEQ ID N0:6;
and
3 0 (d) the amino acid sequence encoded by the cDNA insert of clone
du410_5 deposited under accession number ATCC 98415;
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 1 to amino acid 260.
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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
N0:7;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:7 from nucleotide 51 to nucleotide 611;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:7 from nucleotide 1 to nucleotide 525;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone eh80_1 deposited under accession
number
ATCC 98415;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone eh80_1 deposited under accession number ATCC 98415;
(f) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone eh80_1 deposited under accession number ATCC
98415;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone eh80_1 deposited under accession number ATCC 98415;
(h) a polynucleotide encoding a protein comprising the amino acid
2 0 sequence of SEQ ID N0:8;
(i) 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 88 to amino acid 97 of SEQ
ID N0:8;
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 that hybridizes under stringent conditions to any
3 0 one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:7 from nucleotide 51 to nucleotide 611; the nucleotide sequence of SEQ ID
N0:7 from
nucleotide 1 to nucleotide 525; the nucleotide sequence of the full-length
protein coding
sequence of clone eh80_1 deposited under accession number ATCC 98415; or the
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nucleotide sequence of a mature protein coding sequence of clone eh80 1
deposited under
accession number ATCC 98415. In other preferred embodiments, the
polynucleotide
encodes the full-length or a mature protein encoded by the cDNA insert of
clone eh80_1
deposited under accession number ATCC 98415. In yet other preferred
embodiments,
the present invention provides a polynucleotide encoding a protein comprising
the amino
acid sequence of SEQ ID N0:8 from amino acid 1 to amino acid 158.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:7.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:8;
(b) the amino acid sequence of SEQ ID N0:8 from amino acid 1 to
amino acid 158;
(c) fragments of the amino acid sequence of SEQ ID N0:8 comprising
the amino acid sequence from amino acid 88 to amino acid 97 of SEQ ID N0:8;
and
(d) the amino acid sequence encoded by the cDNA insert of clone
eh80_1 deposited under accession number ATCC 98415;
the protein being substantially free from other mammalian proteins. Preferably
such
2 0 protein comprises the amino acid sequence of SEQ ID N0:8 or the amino acid
sequence
of SEQ ID N0:8 from amino acid 1 to amino acid 158.
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
2 5 N0:9;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:9 from nucleotide 431 to nucleotide 559;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:9 from nucleotide 518 to nucleotide 559;
3 0 (d) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:9 from nucleotide 190 to nucleotide 547;
(e) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone er369_1 deposited under accession
number ATCC 98415;
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(f) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone er369_1 deposited under accession number ATCC 98415;
(g) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone er369_1 deposited under accession number
' S ATCC 98415;
(h) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone er369_1 deposited under accession number ATCC 98415;
(i) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:10;
(j) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID NO:10 having biological activity, the fragment
comprising the amino acid sequence from amino acid 16 to amino acid 25 of SEQ
ID N0:10;
(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 that hybridizes under stringent conditions to any
one of the polynucleotides specified in (a)-(j).
2 0 Preferably, such polynucleotide comprises the nucleotide sequence of SEQ
ID
N0:9 from nucleotide 431 to nucleotide 559; the nucleotide sequence of SEQ ID
N0:9 from
nucleotide 518 to nucleotide 559; the nucleotide sequence of SEQ ID N0:9 from
nucleotide 190 to nucleotide 547; the nucleotide sequence of the full-length
protein coding
sequence of clone er369_1 deposited under accession number ATCC 98415; or the
2 5 nucleotide sequence of a mature protein coding sequence of clone er369_1
deposited
under accession number ATCC 98415. In other preferred embodiments, the
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
of clone er369_1 deposited under accession number ATCC 98415. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
3 0 comprising the amino acid sequence of SEQ ID N0:10 from amino acid 1 to
amino acid
39.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:9.
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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 N0:10;
(b) the amino acid sequence of SEQ ID NO:10 from amino acid 1 to
amino acid 39;
(c) fragments of the amino acid sequence of SEQ ID N0:10 comprising
the amino acid sequence from amino acid 16 to amino acid 25 of SEQ ID N0:10;
and
(d) the amino acid sequence encoded by the cDNA insert of clone
er369_1 deposited under accession number ATCC 98415;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID NO:10 or the amino acid
sequence
of SEQ ID N0:10 from amino acid 1 to amino acid 39.
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
N0:11;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
2 0 N0:11 from nucleotide 91 to nucleotide 2838;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:11 from nucleotide 2209 to nucleotide 2838;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:11 from nucleotide 839 to nucleotide 1197;
2 5 (e) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone fh123 5 deposited under accession
number ATCC 98415;
(f) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone fh123_5 deposited under accession number ATCC 98415;
3 0 (g) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone fh123 5 deposited under accession number
ATCC 98415;
{h) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone fh123_5 deposited under accession number ATCC 98415;
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(i) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:12;
(j) 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 453 to amino acid 462 of
SEQ ID N0:12;
(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 that hybridizes under stringent conditions to any
one of the polynucleotides specified in (a)-(j).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:11 from nucleotide 91 to nucleotide 2838; the nucleotide sequence of SEQ ID
N0:11
from nucleotide 2209 to nucleotide 2838; the nucleotide sequence of SEQ ID
N0:11 from
nucleotide 839 to nucleotide 1197; the nucleotide sequence of the full-length
protein
coding sequence of clone fh123 5 deposited under accession number ATCC 98415;
or the
nucleotide sequence of a mature protein coding sequence of clone fh123_5
deposited
under accession number ATCC 98415. In other preferred embodiments, the
2 0 polynucleotide encodes the full-length or a mature protein encoded by the
cDNA insert
of clone fh123 5 deposited under accession number ATCC 98415. 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 251 to
amino acid
369.
2 5 Other embodiments provide the gene corresponding to the cDNA sequence of
SEQ
ID NO:11.
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:
3 0 (a) the amino acid sequence of SEQ ID N0:12;
(b) the amino acid sequence of SEQ ID N0:12 from amino acid 251 to
amino acid 369;
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(c) fragments of the amino acid sequence of SEQ ID N0:12 comprising
the amino acid sequence from amino acid 453 to amino acid 462 of SEQ ID N0:12;
and
(d) the amino acid sequence encoded by the cDNA insert of clone
fh123 5 deposited under accession number ATCC 98415;
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 251 to amino acid 369.
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
NQ:13;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:13 from nucleotide 568 to nucleotide 978;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:13 from nucleotide 1084 to nucleotide 1854;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone fm60_1 deposited under accession
number ATCC 98415;
2 0 (e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone fm60_1 deposited under accession number ATCC 98415;
(f) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone fm60_1 deposited under accession number
ATCC 98415;
2 5 (g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone fm60_1 deposited under accession number ATCC 98415;
{h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:14;
(i) a polynucleotide encoding a protein comprising a fragment of the
3 0 amino acid sequence of SEQ ID N0:14 having biological activity, the
fragment
comprising the amino acid sequence from amino acid 63 to amino acid 72 of SEQ
ID N0:14;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
{a)-(g) above;
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(k) a polynucleotide which encodes a species homologue of the protein
of (h) or (i) above ; and
(1) a polynucleotide that hybridizes under stringent conditions to any
one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:13 from nucleotide 568 to nucleotide 978; the nucleotide sequence of SEQ ID
N0:13
from nucleotide 1084 to nucleotide 1854; the nucleotide sequence of the full-
length protein
coding sequence of clone fm60_1 deposited under accession number ATCC 98415;
or the
nucleotide sequence of a mature protein coding sequence of clone fm60_1
deposited under
accession number ATCC 98415. In other preferred embodiments, the
polynucleotide
encodes the full-length or a mature protein encoded by the cDNA insert of
clone fm60_1
deposited under accession number ATCC 98415.
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
consisting of:
(a) the amino acid sequence of SEQ ID N0:14;
(b) fragments of the amino acid sequence of SEQ ID N0:14 comprising
2 0 the amino acid sequence from amino acid 63 to amino acid 72 of SEQ ID
N0:14;
and
(c) the amino acid sequence encoded by the cDNA insert of clone
fm60_1 deposited under accession number ATCC 98415;
the protein being substantially free from other mammalian proteins. Preferably
such
2 5 protein comprises the amino acid sequence of SEQ ID N0:14.
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;
3 0 (b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:15 from nucleotide 16 to nucleotide 309;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:15 from nucleotide 127 to nucleotide 309;
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(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone fr473_2 deposited under accession
number ATCC 98415;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone fr473_2 deposited under accession number ATCC 98415;
(f) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone fr473_2 deposited under accession number
ATCC 98415;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone fr473_2 deposited under accession number ATCC 98415;
(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
comprising the amino acid sequence from amino acid 44 to amino acid 53 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
2 0 of (h) or (i) above ; and
(1) a polynucleotide that hybridizes under stringent conditions to any
one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:15 from nucleotide 16 to nucleotide 309; the nucleotide sequence of SEQ ID
N0:15
2 5 from nucleotide 127 to nucleotide 309; the nucleotide sequence of the full-
length protein
coding sequence of clone fr473_2 deposited under accession number ATCC 98415;
or the
nucleotide sequence of a mature protein coding sequence of clone fr473_2
deposited
under accession number ATCC 98415. In other preferred embodiments, the
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
3 0 of clone fr473_2 deposited under accession number ATCC 98415. 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
58.
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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:
(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 58;
{c) fragments of the amino acid sequence of SEQ ID N0:16 comprising
the amino acid sequence from amino acid 44 to amino acid 53 of SEQ ID N0:16;
and
(d) the amino acid sequence encoded by the cDNA insert of clone
fr473_2 deposited under accession number ATCC 98415;
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 58.
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.
2 0 Also provided by the present invention are organisms that have enhanced,
reduced, or
modified expression of the genes) corresponding to the polynucleotide
sequences
disclosed herein.
Processes are also provided for producing a protein, which comprise:
(a) growing a culture of the host cell transformed with such
2 5 polynucieotide compositions in a suitable culture medium; and
(b) purifying the protein from the culture.
The protein produced according to such methods is also provided by the present
invention. Preferred embodiments include those in which the protein produced
by such
process is a mature form of the protein.
3 0 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
CA 02288343 1999-10-15
WO 98/49302 PCT/US98/08336
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
in accordance with known methods. The predicted amino acid sequence (both full-
length
and mature forms) can then be determined from such nucleotide sequence. The
amino
acid sequence of the protein encoded by a particular clone can also be
determined by
expression of the clone in a suitable host cell, collecting the protein and
determining its
sequence. For each disclosed protein applicants have identified what they have
determined to be the reading frame best identifiable with sequence information
available
at the time of filing.
2 0 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
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
2 5 which are transported across the membrane of the endoplasmic reticulum.
Clone "ci25 4"
A polynucleotide of the present invention has been identified as clone
"ci25_4".
ci25_4 was isolated from a human adult brain cDNA library using methods which
are
3 0 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. ci25_4 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"ci25 4 protein").
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The nucleotide sequence of ci25 4 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 ci25 4 protein corresponding to the foregoing
nucleotide
sequence is reported in SEQ ID N0:2. Amino acids 9 to 21 are a predicted
leader/signal
sequence, with the predicted mature amino acid sequence beginning at amino
acid 22, or
are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
ci25_4 should be approximately 1700 bp.
The nucleotide sequence disclosed herein for ci25_4 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. ci25 4 demonstrated at least some similarity with
sequences
identified as AA243050 (zr24h03.r1 Stratagene NT2 neuronal precursor 937230
Homo
sapiens cDNA clone 664373 5'), AA316800 (EST188485 HCC cell line (matastasis
to liver
in mouse) II Homo sapiens cDNA 5' end}, AA340783 (EST46083 Fetal kidney II
Homo
sapiens cDNA 5' end), Q05686 (Islets of Langerhans cell clone ICA12.3 (ATCC
40703)),
812690 (yf40e07.s1 Homo Sapiens cDNA clone 129348 3'), 816432 (yf40e07.r1 Homo
sapiens cDNA clone), W81653 (zd84d12.r1 Soares fetal heart NbHHI9W Homo
sapiens
cDNA clone 347351 5'), and W81654 (zd84d12.s1 Soares fetal heart NbHHI9W Homo
sapiens cDNA clone 3473513'). Based upon sequence similarity, ci25 4 proteins
and each
2 0 similar protein or peptide may share at least some activity. The TopPredII
computer
program predicts five additional potential transmembrane domains within the
ci25_4
protein sequence, centered around amino acids 81,134,159,182, and 241 of SEQ
ID N0:2,
respectively.
2 5 Clone "da228 6"
A polynucleotide of the present invention has been identified as clone
"da228_6".
da228_6 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
3 0 analysis of the amino acid sequence of the encoded protein. da228 6 is a
full-length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"da228_6 protein").
The nucleotide sequence of da228_6 as presently determined is reported in SEQ
ID N0:3. What applicants presently believe to be the proper reading frame and
the
17
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WO 98/49302 PCTNS98/08336
predicted amino acid sequence of the da228 6 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:4.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
da228_6 should be approximately 1500 bp.
The nucleotide sequence disclosed herein for da228_6 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. da228_6 demonstrated at least some similarity with
sequences
identified as W57906 (zd17f1l.rl Soares fetal heart NbHHI9W Homo sapiens cDNA
clone
340941 5') and W57907 (zd17f11.s1 Soares fetal heart NbHHI9W Homo Sapiens cDNA
clone 340941 3'. Based upon sequence similarity, da228_6 proteins and each
similar
protein or peptide may share at least some activity.
Clone "du410 5"
A polynucleotide of the present invention has been identified as clone
"du410_5".
du410_5 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. du410_5 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
2 0 "du410_5 protein")
The nucleotide sequence of du410_5 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 du410_5 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:6.
2 5 The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
du410_5 should be approximately 2400 bp.
The nucleotide sequence disclosed herein for du410 5 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. du410 5 demonstrated at least some similarity with
sequences
3 0 identified as N44315 (EST51p19 WATMl Homo sapiens cDNA clone 51p19) and
N66980
(yz58d04.s1 Homo sapiens cDNA clone 287239 3'). The predicted amino acid
sequence
disclosed herein for du410 5 was searched against the GenPept and GeneSeq
amino acid
sequence databases using the BLASTX search protocol. The predicted du410 5
protein
demonstrated at least some similarity to sequences identified as U67604 (P115
protein
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WO 98/49302 PCT/US98/08336
[Methanococcus jannaschii]). Based upon sequence similarity, du410_5 proteins
and each
similar protein or peptide may share at least some activity.
Clone "eh80 1"
A polynucleotide of the present invention has been identified as clone
"eh80_1".
eh80_1 was isolated from a human adult blood (peripheral blood mononuclear
cells
treated with granulocyte-colony stimulating factor in vivo) 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. eh80_1 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"eh80_1 protein")
The nucleotide sequence of eh80_1 as presently determined is reported in SEQ
ID
N0:7. What applicants presently believe to be the proper reading frame and the
predicted
amino acid sequence of the eh80_1 protein corresponding to the foregoing
nucleotide
sequence is reported in SEQ ID N0:8. Another potential eh80_1 reading frame
and
predicted amino acid sequence is encoded by basepairs 41 to 1659 of SEQ ID
N0:7 and
is reported in SEQ ID N0:25. A frameshift in the nucleotide sequence of SEQ ID
N0:5
between about nucleotide 41 to about nucleotide 614 could join together
portions of the
2 0 overlapping reading frames of SEQ ID N0:8 and SEQ ID N0:25. -
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
eh80_1 should be approximately 2000 bp.
The nucleotide sequence disclosed herein for eh80_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
2 5 FASTA search protocols. eh80_1 demonstrated at least some similarity with
sequences
identified as AA012957 (ze27b03.r1 Soares retina N2b4HR Homo sapiens cDNA
clone
360173 5'), AA019878 (ze63b03.s1 Soares retina N2b4HR Homo Sapiens cDNA clone
363629 3'), AA505456 (nh84c07.s1 NCI CGAP_Brl.l Homo sapiens cDNA clone IMAGE
965196), Q60246 (Human brain Expressed Sequence Tag EST02242), 816603
(yf43c04.r1
3 0 Homo Sapiens cDNA clone 129606 5'), and T85469 (yd82f05.r1 Homo sapiens
cDNA clone
114753 5'). The predicted amino acid sequence disclosed herein for eh80_1 was
searched
against the GenPept and GeneSeq amino acid sequence databases using the BLASTX
search protocol. The predicted eh80_1 protein demonstrated at least some
similarity to
sequences identified as U40747 (FBP 11 [Mus musculus]). Based upon sequence
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WO 98/49302 PCT/IJS98/08336
similarity, eh80 1 proteins and each similar protein or peptide may share at
least some
activity. The TopPredII computer program predicts two potential transmembrane
domains within the amino acid sequence of SEQ ID N0:8, one centered around
amino
acid 107 and another around amino acid 131.
Clone "er369 1"
A polynucleotide of the present invention has been identified as clone
"er369_1".
er369_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. er369_1 is a full-
length clone,
including. the entire coding sequence of a secreted protein (also referred to
herein as
"er369_1 protein")
The nucleotide sequence of er369_1 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 er369_1 protein corresponding to the foregoing
nucleotide
sequence is reported in SEQ ID N0:10. Amino acids 17 to 29 are a predicted
leader/signal
sequence, with the predicted mature amino acid sequence beginning at amino
acid 30, or
are a transmembrane domain.
2 0 The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
er369_1 should be approximately 1500 bp.
The nucleotide sequence disclosed herein for er369_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. er369_1 demonstrated at least some similarity with
sequences
identified as H12227 (ym12g10.r1 Homo sapiens cDNA clone 47729 5'), H70978
(yr73g06.r1 Homo sapiens cDNA clone 210970 5'), M79179 (EST01327 Homo Sapiens
cDNA clone HHCP081), Q61324 (Human brain Expressed Sequence Tag EST01327), and
853554 (yg84e04.s1 Homo Sapiens cDNA clone 39854 3' similar to contains Alu
repetitive
element). Based upon sequence similarity, er369_1 proteins and each similar
protein or
3 0 peptide may share at least some activity. The nucleotide sequence of
er369_1 indicates
that it may contain an Alu repetitive element.
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Clone "fh123 5"
A polynucleotide of the present invention has been identified as clone
"fh123_5".
fh123 5 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
S identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. fh123_5 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"fh123_5 protein').
The nucleotide sequence of fh123 5 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 fh123 5 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:12. Amino acids 694 to 706 are a
predicted
leader/signal sequence, with the predicted mature amino acid sequence
beginning at
amino acid 707, or are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
fh123_5 should be approximately 2800 bp.
The nucleotide sequence disclosed herein for fh123 5 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. fh123 5 demonstrated at least some similarity with
sequences
2 0 identified as AA815253 {ai64d02.s1 Soares testis NHT Homo sapiens cDNA
clone 1375587
3'), AA855689 (vw71h04.r1 Stratagene mouse heart (#937316) Mus musculus cDNA
clone
1260439 5'), and W80785 (zd83d07.s1 Soares fetal heart NbHHI9W Homo sapiens
cDNA
clone 347245 3). The predicted amino acid sequence disclosed herein for fh123
5 was
searched against the GenPept and GeneSeq amino acid sequence databases using
the
2 5 BLASTX search protocol. The predicted fh123_5 protein demonstrated at
least some
similarity to sequences identified as D80005 (KIAA0183 [Homo sapiens]). Based
upon
sequence similarity, fh123 5 proteins and each similar protein or peptide may
share at
least some activity. The TopPredII computer program predicts five additional
possible
transmembrane domains within the fh123 5 protein sequence.
Clone "fm60 1"
A polynucleotide of the present invention has been identified as clone
"fm60_1".
fm60_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
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WO 98/49302 PCT/US98/08336
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. fm60 1 is a full-
length clone,
including the entire coding sequence of a secreted protein {also referred to
herein as
"fm60 1 protein").
The nucleotide sequence of fm60_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 fm60_1 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:14.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
fm~ 1 should be approximately 2200 bp.
The nucleotide sequence disclosed herein for fm60 1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. fm60_1 demonstrated at least some similarity with
sequences
identified as AA155574 (zo70a01.s1 Stratagene pancreas (#937208) Homo sapiens
cDNA
clone 592200 3'), AF015147 (Homo Sapiens clone HS19.1 Alu-Ya5 sequence),
N86095
(J6377F Fetal heart, Lambda ZAP Express Homo Sapiens cDNA clone J6377 5'
similar to
REPETITIVE ELEMENT ALU), U14567 (***ALU WARNING Human Alu-J subfamily
consensus sequence), and 282199 (Human DNA sequence from clone J316D5). Based
upon sequence similarity, fm60_1 proteins and each similar protein or peptide
may share
2 0 at least some activity. The TopPredII computer program predicts a
potential
transmembrane domain within the fm60_1 protein sequence centered around amino
acid
50 of SEQ ID N0:14. The nucleotide sequence of fm60_1 indicates that it may
contain one
or more of the following repetitive elements: Alu, L1.
2 5 Clone "fr473 2"
A polynucleotide of the present invention has been identified as clone "fr473
2".
fr473_2 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
3 0 analysis of the amino acid sequence of the encoded protein. fr473_2 is a
full-length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"fr473_2 protein"}.
The nucleotide sequence of fr473_2 as presently determined is reported in SEQ
ID
N0:15. What applicants presently believe to be the proper reading frame and
the
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WO 98/49302 PCT/US98/08336
predicted amino acid sequence of the fr473 2 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:16. Amino acids 25 to 37 are a
predicted
leader/signal sequence, with the predicted mature amino acid sequence
beginning at
amino acid 38, or are a transmembrane domain. Amino acids 62 to 74 are another
possible
leader/signal sequence, with the predicted mature amino acid sequence
beginning at
amino acid 75, or are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
fr473 2 should be approximately 605 bp.
The nucleotide sequence disclosed herein for fr473 2 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. fr473 2 demonstrated at least some similarity with
sequences
identified .as AA479559 (zu42a02.r1 Soares ovary tumor NbHOT Homo sapiens cDNA
clone 740618 5' similar to WP:F49C12.12 CE03372), H46855 (yo18g04.r1 Homo
sapiens
cDNA clone 178326 5'), T24372 (Human gene signature HUMGS06404), W31692
(zb93dOl.r1 Soares parathyroid tumor NbHPA Homo sapiens cDNA clone 320353 5'),
and
232877 (H. sapiens partial cDNA sequence; clone HEA41P; single read). The
predicted
amino acid sequence disclosed herein for fr473_2 was searched against the
GenPept and
GeneSeq amino acid sequence databases using the BLASTX search protocol. The
predicted fr473 2 protein demonstrated at least some similarity to sequences
identified
2 0 as 268227 (F49C12.12 [Caenorhabditis elegans]). Based upon sequence
similarity, fr473_2
proteins and each similar protein or peptide may share at least some activity.
Deposit of Clones
Clones ci25 4, da228 b, du410_5, eh80_l, er369_l, fh123_5, fm60_1, and fr473_2
2 5 were deposited on April 25, 1997 with the American Type Culture Collection
(10801
University Boulevard, Manassas, Virginia 20110-2209 U.S.A.) as an original
deposit under
the Budapest Treaty and were given the accession number ATCC 98415, from which
each
clone comprising a particular polynucleotide is obtainable. All restrictions
on the
availability to the public of the deposited material will be irrevocably
removed upon the
3 0 granting of the patent, except for the requirements specified in 37 C.F.R.
~ 1.808(b), and
the term of the deposit will comply with 37 C.F.R. ~ 1.806.
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
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WO 98/49302 PCT/US98/08336
appropriate fragment for such clone. Each clone was deposited in either the
pED6 or
pNOTs vector depicted in Figures lA and 1B, respectively. 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 an
oligonucleotide probe that was used to isolate or to sequence each full-length
clone is
2 0 identified below, and should be most reliable in isolating the clone of
interest.
Clone Probe Seauence
ci25_4 SEQ ID N0:17
da228_6 SEQ ID N0:18
2 5 du410_5 SEQ ID N0:19
eh80_1 SEQ ID N0:20
er369_1 SEQ ID N0:21
fh123 5 SEQ ID N0:22
~~_1 SEQ ID N0:23
3 0 fr473_2 SEQ ID N0:24
In the sequences listed above which include an N at position 2, that position
is occupied
in preferred probes/primers by a biotinylated phosphoaramidite residue rather
than a
nucleotide (such as , for example, that produced by use of biotin
phosphoramidite (1-
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WO 98/49302 PCT/US98/08336
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-32P 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 ul 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
grown to saturation at 37°C, and the saturated culture should
preferably be diluted in
2 0 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
pg/ml and agar at 1.5% in a 150 mm petri dish when grown overnight at
37°C. Other
known methods of obtaining distinct, well-separated colonies can also be
employed.
2 5 Standard colony hybridization procedures should then be used to transfer
the
colonies to nitrocellulose filters and lyse, denature and bake them.
The filter is then preferably incubated at 65°C for 1 hour with gentle
agitation in
6X SSC (20X stock is 175.3 g NaCI/liter, 88.2 g Na citrate/liter, adjusted to
pH 7.0 with
NaOH) containing 0.5% SDS,100 lzg/ml of yeast RNA, and 10 mM EDTA
(approximately
3 0 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
by 500 mL of 2X SSC/0.1% SDS at room temperature with gentle shaking for 15
minutes.
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A third wash with 0.1X 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, et al., Bio/Technology U 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
decavalent form of the protein of the invention.
2 0 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
forms) of such protein may be obtained by expression of the disclosed full-
length
polynucleotide (preferably those deposited with ATCC) in a suitable mammalian
cell or
2 5 other host cell. The sequences) of the mature forms) of the protein may
also be
determinable from the amino acid sequence of the full-length form.
The present invention also provides genes corresponding to the polynucleotide
sequences disclosed herein. "Corresponding genes" are the regions of the
genome that
are transcribed to produce the mRNAs from which cDNA polynucleotide sequences
are
3 0 derived and may include contiguous regions of the genome necessary for the
regulated
expression of such genes. Corresponding genes may therefore include but are
not limited
to coding sequences, 5' and 3' untranslated regions, alternatively spliced
exons, introns,
promoters, enhancers, and silencer or suppressor elements. The corresponding
genes can
be isolated in accordance with known methods using the sequence information
disclosed
26
CA 02288343 1999-10-15
WO 98/49302 PCT/US98/08336
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 mlZNA transcribed
from the
gene (Albert and Morns,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 B1, incorporated by reference
herein).
In addition, organisms are provided in which the genes) corresponding to the
2 0 polynucleotide sequences disclosed herein have been partially or
completely inactivated,
through insertion of extraneous sequences into the corresponding genes) or
through
deletion of au or part of the corresponding gene(s). Partial or complete gene
inactivation
can be accomplished through insertion, preferably followed by imprecise
excision, of
transposable elements (Plasterk,1992, Bioessays 14(9): 629-633; Zwaal et
al.,1993, Proc. Natl.
2 5 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;
5,616,491; and 5,679,523; all of which are incorporated by reference herein).
These
3 0 organisms with altered gene expression are preferably eukaryotes and more
preferably
are mammals. Such organisms are useful for the development of non-human models
for
the study of disorders involving the corresponding gene(s), and for the
development of
assay systems for the identification of molecules that interact with the
protein products)
of the corresponding gene(s).
27
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WO 98/49302 PCT/US98/08336
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.
Species homologues of the disclosed polynucleotides and proteins are also
provided by the present invention. As used herein, a "species homologue" is a
protein or
2 0 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. Preferably, polynucleotide species homologues have at least
60% sequence
identity (more preferably, at least 75% identity; most preferably at least 90%
identity) with
the given polynucleotide, and protein species homologues have at least 30%
sequence
2 5 identity (more preferably, at least 45% identity; most preferably at least
60% identity) with
the given protein, where sequence identity is determined by comparing the
nucleotide
sequences of the polynucleotides or the amino acid sequences of the proteins
when
aligned so as to maximize overlap and identity while minimizing sequence gaps.
Species
homologues may be isolated and identified by making suitable probes or primers
from
3 0 the sequences provided herein and screening a suitable nucleic acid source
from the
desired species. Preferably, species homologues are those isolated from
mammalian
species. Most preferably, species homologues are those isolated from certain
mammalian
species such as, for example, Pan troglodytes, Gorilla gorilla, Pongo
p~gmaeus, Hylobates
28
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WO 98/49302 PCTNS98/08336
coricolor, Macaca mulatta, Papio papio, Papio hamadryas, Cercopithecus
aethiops, Cebus capucinus,
Aotus trivirgatus, Sanguinus Oedipus, Microcebus murinus, Mus musculus, Rattus
norvegicus,
Cricetulus griseus, Felis catus, Mustela vison, Canis familiaris, Oryctolagus
cuniculus, Bos taurus,
Ovis aries, Sus scrofa, and Eguus caballus, for which genetic maps have been
created
allowing the identification of synteruc relationships between the genomic
organization of
genes in one species and the genomic organization of the related genes in
another species
{O'Brien and Seuanez, 1988, Ann. Rev. Genet. 22: 323-351; O'Brien et al.,
1993, Nature
Genetics 3:103-112; Johansson et al.,1995, Genomics 25: 682-690; Lyons et
al.,1997, Nature
Genetics 15: 47-56; O'Brien et al.,1997, Trends in Genetics 13(10): 393-399;
Carver and Stubbs,
1997, Genome Research 7:1123-1137; all of which are incorporated by reference
herein).
The invention also encompasses allelic variants of the disclosed
polynucleotides
or proteins; that is, naturally-occurnng alternative forms of the isolated
polynucleotides
which also encode proteins which are identical or have significantly similar
sequences to
those encoded by the disclosed polynucleotides. Preferably, allelic variants
have at least
60% sequence identity (more preferably, at least 75% identity; most preferably
at least 90%
identity) with the given polynucleotide, where sequence identity is determined
by
comparing the nucleotide sequences of the polynucleotides when aligned so as
to maximize
overlap and identity while minimizing sequence gaps. Allelic variants may be
isolated and
identified by making suitable probes or primers from the sequences provided
herein and
2 0 screening a suitable nucleic acid source from individuals of the
appropriate species.
The invention also includes polynucleotides with sequences complementary to
those of the polynucleotides disclosed herein.
The present invention also includes polynucleotides that hybridize under
reduced
stringency conditions, more preferably stringent conditions, and most
preferably highly
2 5 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.
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WO 98/49302 PCT/US98/08336
StringencyPolynucleotideHybridHybridization TemperatureWash
ConditionHybrid Lengthand Temperature
(bp)t Buffer' and Buffer'
A DNA:DNA z 50 65C; lxSSC -or- 65C; 0.3xSSC
42C; lxSSC, 50% formamide
B DNA:DNA <50 TB*; lxSSC TB*; lxSSC
C DNA:RNA Z 50 67C; lxSSC -or- 67C; 0.3xSSC
45C; lxSSC, 50% formamide
D DNA:RNA <50 Tp*; lxSSC Tp*; lxSSC
E RNA:RNA z 50 70C; lxSSC -or- 70C; 0.3xSSC
50C; lxSSC, 50% formamide
F RNA:RNA <50 TF*; lxSSC TF*; lxSSC
G DNA:DNA a 50 65C; 4xSSC -or- 65C; lxSSC
42C; 4xSSC, 50% formamide
H DNA:DNA <50 T"*; 4xSSC TH*; 4xSSC
I DNA:RNA s 50 67C; 4xSSC -or- 67C; lxSSC
45C; 4xSSC, 50% formamide
J DNA:RNA <50 T~*; 4xSSC T~*; 4xSSC
K RNA:RNA z 50 70C; 4xSSC -or- 67C; lxSSC
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
P DNA:RNA <50 Tr*; 6xSSC T,,*; 6xSSC
Q RNA:RNA s 50 60C; 4xSSC -or- 60C; 2xSSC
45C; 6xSSC, 50% formamide
2 R RNA:RNA <50 TR*; 4xSSC Ta*; 4xSSC
0
3: The hybrid length is that anticipated for the hybridized regions) of the
hybridizing polynucleoHdes. When
hybridizing a polynucleotide to a target polynucleotide of unknown sequence,
the hybrid length is assumed
to be that of the hybridizing polynucleotide. When polynucleotides of known
sequence are hybridized, the
2 5 hybrid length can be determined by aligning the sequences of the
polynucleotides and identifying the region
or regions of optimal sequence compiementarity.
': SSPE (lxSSPE is 0.15M NaCI, lOmM NaHzPO,, and 1.25mM EDTA, pH 7.4) can be
substituted for SSC
(lxSSC is 0.15M NaCI and l5mM sodium citrate) in the hybridization and wash
buffers; washes are
performed for 15 minutes after hybridization is complete.
3 0 "TB - TR: The hybridization temperature for hybrids anticipated to be less
than 50 base pairs in length should
be 5-10°C less than the melting temperature (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, T,"(°C) =
81.5 + 16.6{log,o[Na']) + 0.41(%G+C)
(600/N), where N is the number of bases in the hybrid, and [Na'] is the
concentration of sodium ions in the
3 5 hybridization buffer ([Na"] for lxSSC = 0.165 M).
CA 02288343 1999-10-15
WO 98/49302 PCT/US98/08336
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 Biology,1995, F.M.
Ausubel et al., eds.,
John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, incorporated herein by
reference.
Preferably, each such hybridizing polynucleotide has a length that is at least
25%(more preferably at least 50%, and most preferably at least 75%) of the
length of the
polynucleotide of the present invention to which it hybridizes, and has at
least 60%
sequence identity (more preferably, at least 75% identity; most preferably at
least 90% or
95% identity) with the polynucleotide of the present invention to which it
hybridizes,
where sequence identity is determined by comparing the sequences of the
hybridizing
polynucleotides when aligned so as to maximize overlap and identity while
minimizing
sequence gaps.
The isolated polynucleotide of the invention may be operably linked to an
expression control sequence such as the pMT2 or pED expression vectors
disclosed in
Kaufman et al., Nucleic Acids Res. 19, 4485-4490 (1991), in order to produce
the protein
recombinantly. Many suitable expression control sequences are known in the
art. General
methods of expressing recombinant proteins are also known and are exemplified
in R.
Kaufman, Methods in Enzymology 185, 537-566 (1990). As defined herein
"operably
2 0 linked" means that the isolated polynucleotide of the invention and an
expression control
sequence are situated within a vector or cell in such a way that the protein
is expressed
by a host cell which has been transformed (transfected) with the ligated
polynucleotide/expression control sequence.
A number of types of cells may act as suitable host cells for expression of
the
2 5 protein. Mammalian host cells include, for example, monkey COS cells,
Chinese Hamster
Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human
Co1o205
cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal
diploid cells, cell
strains derived from in vitro culture of primary tissue, primary explants,
HeLa cells,
mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.
3 0 Alternatively, it may be possible to produce the protein in lower
eukaryotes such
as yeast or in prokaryotes such as bacteria. Potentially suitable yeast
strains include
Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains,
Candida, or any
yeast strain capable of expressing heterologous proteins. Potentially suitable
bacterial
strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium,
or any bacterial
31
CA 02288343 1999-10-15
WO 98/49302 PCT/US98/08336
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
phosphoryiation 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
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
expressed protein may then be purified from such culture (i.e., from culture
medium or
cell extracts) using known purification processes, such as gel filtration and
ion exchange
chromatography. The purification of the protein may also include an affinity
column
2 0 containing agents which will bind to the protein; one or more column steps
over such
affinity resins as concanavalin A-agarose, heparin-toyopearl~ or Cibacrom blue
3GA
Sepharose~; one or more steps involving hydrophobic interaction chromatography
using
such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity
chromatography.
2 5 Alternatively, the protein of the invention may also be expressed in a
form which
will facilitate purification. For example, it may be expressed as a fusion
protein, such as
those of maltose binding protein (MBP), glutathione-S-transferase (GST) or
thioredoxin
(TRX). Kits for expression and purification of such fusion proteins are
commercially
available from New England BioLab (Beverly, MA), Pharmacia (Piscataway, NJ)
and
3 0 InVitrogen, respectively. The protein can also be tagged with an epitope
and
subsequently purified by using a specific antibody directed to such epitope.
One such
epitope ("Flag") is commercially available from Kodak (New Haven, CT).
Finally, one or more reverse-phase high performance liquid chromatography (IZP-
HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having
pendant
32
CA 02288343 1999-10-15
WO 98/49302 PCT/US98/08336
methyl or other aliphatic groups, can be employed to further purify the
protein. Some or
ail of the foregoing purification steps, in various combinations, can also be
employed to
provide a substantially homogeneous isolated recombinant protein. The protein
thus
purified is substantially free of other mammalian proteins and is defined in
accordance
with the present invention as an "isolated protein."
The protein of the invention may also be expressed as a product of transgenic
animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or
sheep which
are characterized by somatic or germ cells containing a nucleotide sequence
encoding the
protein.
The protein may also be produced by known conventional chemical synthesis.
Methods for constructing the proteins of the present invention by synthetic
means are
known to those skilled in the art. The synthetically-constructed protein
sequences, by
virtue of sharing primary, secondary or tertiary structural and/or
conformational
characteristics with proteins may possess biological properties in common
therewith,
including protein activity. Thus, they may be employed as biologically active
or
immunological substitutes for natural, purified proteins in screening of
therapeutic
compounds and in immunological processes for the development of antibodies.
The proteins provided herein also include proteins characterized by amino acid
sequences similar to those of purified proteins but into which modification
are naturally
2 0 provided or deliberately engineered. For example, modifications in the
peptide or DNA
sequences can be made by those skilled in the art using known techniques.
Modifications
of interest in the protein sequences may include the alteration, substitution,
replacement,
insertion or deletion of a selected amino acid residue in the coding sequence.
For
example, one or more of the cysteine residues may be deleted or replaced with
another
2 5 amino acid to alter the conformation of the molecule. Techniques for such
alteration,
substitution, replacement, insertion or deletion are well known to those
skilled in the art
(see, e.g., U.S. Patent No. 4,518,584). Preferably, such alteration,
substitution, replacement,
insertion or deletion retains the desired activity of the protein.
Other fragments and derivatives of the sequences of proteins which would be
3 0 expected to retain protein activity in whole or in part and may thus be
useful for screening
or other immunological methodologies may also be easily made by those skilled
in the art
given the disclosures herein. Such modifications are believed to be
encompassed by the
present invention.
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CA 02288343 1999-10-15
WO 98/49302 PCT/US98/08336
USES AND BIOLOGICAL ACTIVITY
The polynucleotides and proteins of the present invention are expected to
exhibit
one or more of the uses or biological activities (including those associated
with assays
cited herein) identified below. Uses or activities described for proteins of
the present
invention may be provided by administration or use of such proteins or by
administration
or use of polynucleotides encoding such proteins (such as, for example, in
gene therapies
or vectors suitable for introduction of DNA).
Research Uses and Utilities
The polynucleotides provided by the present invention can be used by the
research
community for various purposes. The polynucleotides can be used to express
recombinant protein for analysis, characterization or therapeutic use; as
markers for
tissues in which the corresponding protein is preferentially expressed (either
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
with endogenous DNA sequences in patients to identify potential genetic
disorders; as
probes to hybridize and thus discover novel, related DNA sequences; as a
source of
information to derive PCR primers for genetic fingerprinting; as a probe to
"subtract-out"
2 0 known sequences in the process of discovering other novel polynucleotides;
for selecting
and making oligomers for attachment to a "gene chip" or other support,
including for
examination of expression patterns; to raise anti-protein antibodies using DNA
immunization techniques; and as an antigen to raise anti-DNA antibodies or
elicit another
immune response. Where the polynucleotide encodes a protein which binds or
potentially
2 5 binds to another protein (such as, for example, in a receptor-ligand
interaction), the
polynucleotide can also be used in interaction trap assays (such as, for
example, those
described in Gyuris et al., 1993, Cell 75: 791-803 and in Rossi et al., 1997,
Proc. Natl. Acad.
Sci. USA 94: 8405-8410, all of which are incorporated by reference herein) to
identify
polynucleotides encoding the other protein with which binding occurs or to
identify
3 0 inhibitors of the binding interaction.
The proteins provided by the present invention can similarly be used in assay
to
determine biological activity, including in a panel of multiple proteins for
high-
throughput screening; to raise antibodies or to elicit another immune
response; as a
reagent (including the labeled reagent) in assays designed to quantitatively
determine
34
CA 02288343 1999-10-15
WO 98/49302 PCT/US98/08336
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
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, Berger, S.L. and A.R. Kimmel
eds.,1987.
Nutritional Uses
Poiynucleotides and proteins of the present invention can also be used as
2 0 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
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
2 5 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.
Cytokine and Cell Proliferation/Differentiation ActivitX
A protein of the present invention may exhibit cytokine, cell proliferation
(either
3 0 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 au 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
CA 02288343 1999-10-15
WO 98/49302 PCT/US98/08336
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
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.
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
2 0 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
iymphopoietic
cells include, without limitation, those described in: Measurement of Human
and Murine
Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L.S. and Lipsky, P.E. In
Current
Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John
Wiley and Sons,
2 5 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
Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons,
Toronto.1991;
Smith et al., Proc. Natl. Acad. Sci. U.S.A. 83:1857-1861, 1986; Measurement of
human
3 0 Interleukin 11- Bennett, F., Giannotti, J., Clark, S.C. and Turner, K. J.
In Casrrent 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.
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CA 02288343 1999-10-15
WO 98/49302 PCT/US98/08336
Assays for T-cell clone responses to antigens (which will identify, among
others,
proteins that affect APC-T cell interactions as well as direct T-cell effects
by measuring
proliferation and cytokine production) include, without limitation, those
described in:
Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H.
Margulies,
E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-
Interscience
(Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6,
Cytokines and
their cellular receptors; Chapter 7, Immunologic studies in Humans);
Weinberger et al.,
Proc. Natl. Acad. Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J.
Immun.
11:405-411, 1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al.,
J. Immunol.
140:508-512, 1988.
Immune Stimulating or Suppressing Activity
A protein of the present invention may also exhibit immune stimulating or
immune suppressing activity, including without limitation the activities for
which assays
are described herein. A protein may be useful in the treatment of various
immune
deficiencies and disorders (including severe combined immunodeficiency
(SLID)), e.g.,
in regulating (up or down) growth and proliferation of T and/or B lymphocytes,
as well
as effecting the cytolytic activity of NK cells and other cell populations.
These immune
deficiencies may be genetic or be caused by viral (e.g., HIV) as well as
bacterial or fungal
2 0 infections, or may result from autoimmune disorders. More specifically,
infectious
diseases causes by viral, bacterial, fungal or other infection may be
treatable using a
protein of the present invention, including infections by HIV, hepatitis
viruses,
herpesviruses, mycobacteria, Leishmania spp., malaria spp. and various fungal
infections
such as candidiasis. Of course, in this regard, a protein of the present
invention may also
2 5 be useful where a boost to the immune system generally may be desirable,
i.e., in the
treatment of cancer.
Autoimmune disorders which may be treated using a protein of the present
invention include, for example, connective tissue disease, multiple sclerosis,
systemic
lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation,
3 0 Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent
diabetes mellitis,
myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye
disease.
Such a protein of the present invention may also to be useful in the treatment
of allergic
reactions and conditions, such as asthma (particularly allergic asthma) or
other respiratory
problems. Other conditions, in which immune suppression is desired (including,
for
37
CA 02288343 1999-10-15
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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
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
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
2 0 transplantation. Typically, in tissue transplants, rejection of the
transplant is initiated
through its recognition as foreign by T cells, followed by an immune reaction
that destroys
the transplant. The administration of a molecule which inhibits or blocks
interaction of
a B7 lymphocyte antigen with its natural ligand(s) on immune cells (such as a
soluble,
monomeric form of a peptide having B7-2 activity alone or in conjunction with
a
2 5 monomeric form of a peptide having an activity of another B lymphocyte
antigen (e.g., B7-
1, B7-3) or blocking antibody), prior to transplantation can lead to the
binding of the
molecule to the natural ligand(s) on the immune cells without transmitting the
corresponding costimulatory signal. Blocking B lymphocyte antigen function in
this
matter prevents cytokine synthesis by immune cells, such as T cells, and thus
acts as an
3 0 immunosuppressant. Moreover, the lack of costimulation may also be
sufficient to
anergize the T cells, thereby inducing tolerance in a subject. Induction of
long-term
tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of
repeated
administration of these blocking reagents. To achieve sufficient
immunosuppression or
38
CA 02288343 1999-10-15
WO 98/49302 PCT/US98/08336
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, marine models of GVHD (see Paul
ed.,
Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847) can be used
to
determine the effect of blocking B lymphocyte antigen function in vivo on the
development
of that disease.
Blocking antigen function may also be therapeutically useful for treating
autoimmune diseases. Many autoimmune disorders are the result of inappropriate
activation of T cells that are reactive against self tissue and which promote
the production
of cytokines and autoantibodies involved in the pathology of the diseases.
Preventing the
activation of autoreactive T cells may reduce or eliminate disease symptoms.
Administration of reagents which block costimulation of T cells by disrupting
receptor:ligand interactions of B lymphocyte antigens can be used to inhibit T
cell
2 0 activation and prevent production of autoantibodies or T cell-derived
cytokines which
may be involved in the disease process. Additionally, blocking reagents may
induce
antigen-specific tolerance of autoreactive T cells which could lead to long-
term relief from
the disease. The efficacy of blocking reagents in preventing or alleviating
autoimmune
disorders can be determined using a number of well-characterized animal models
of
2 5 human autoimmune diseases. Examples include marine experimental autoimmune
encephalitis, systemic lupus erythmatosis in MRL/Ipr/lpr mice or NZB hybrid
mice,
marine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB
rats, and
marine experimental myasthenia gravis (see Paul ed., Fundamental Immunology,
Raven
Press, New York,1989, pp. 840-856).
3 0 Upregulation of an antigen function (preferably a B lymphocyte antigen
function),
as a means of up regulating immune responses, may also be useful in therapy.
Upregulation of immune responses may be in the form of enhancing an existing
immune
response or eliciting an initial immune response. For example, enhancing an
immune
response through stimulating B lymphocyte antigen function may be useful in
cases of
39
CA 02288343 1999-10-15
WO 98/49302 PCT/IJS98/08336
vital infection. In addition, systemic viral diseases such as influenza, the
common cold,
and encephalitis might be alleviated by the administration of stimulatory
forms of B
lymphocyte antigens systemically.
Alternatively, anti-viral immune responses may be enhanced in an infected
patient
by removing T cells from the patient, costimulating the T cells in vitro with
viral antigen-
pulsed APCs either expressing a peptide of the present invention or together
with a
stimulatory form of a soluble peptide of the present invention and
reintroducing the in
vitro activated T cells into the patient. Another method of enhancing anti-
viral immune
responses would be to isolate infected cells from a patient, transfect them
with a nucleic
acid encoding a protein of the present invention as described herein such that
the cells
express all or a portion of the protein on their surface, and reintroduce the
transfected
cells into the patient. The infected cells would now be capable of delivering
a
costimulatory signal to, and thereby activate, T cells in vivo.
In another application, up regulation or enhancement of antigen function
(preferably B lymphocyte antigen function) may be useful in the induction of
tumor
immunity. Tumor cells (e.g., sarcoma, melanoma, lymphoma, leukemia,
neuroblastoma,
carcinoma) transfected with a nucleic acid encoding at least one peptide of
the present
invention can be administered to a subject to overcome tumor-specific
tolerance in the
subject. If desired, the tumor cell can be transfected to express a
combination of peptides.
2 0 For example, tumor cells obtained from a patient can be transfected ex
vivo with an
expression vector directing the expression of a peptide having B7-2-like
activity alone, or
in conjunction with a peptide having B7-1-like activity and/or B7-3-like
activity. The
transfected tumor cells are returned to the patient to result in expression of
the peptides
on the surface of the transfected cell. Alternatively, gene therapy techniques
can be used
2 5 to target a tumor cell for transfection in vivo.
The presence of the peptide of the present invention having the activity of a
B
lymphocyte antigens) on the surface of the tumor cell provides the necessary
costimulation signal to T cells to induce a T cell mediated immune response
against the
transfected tumor cells. In addition, tumor cells which lack MHC class I or
MHC class II
3 0 molecules, or which fail to reexpress sufficient amounts of MHC class I or
MHC class II
molecules, can be transfected with nucleic acid encoding all or a portion of
{e.g., a
cytoplasmic-domain truncated portion) of an MHC class I a chain protein and pz
microglobulin protein or an MHC class II a chain protein and an MHC class II
~3 chain
protein to thereby express MHC class I or MHC class II proteins on the cell
surface.
CA 02288343 1999-10-15
WO 98/49302 PCT/IIS98/08336
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
limitation, those described in: Current Protocols in Immunology, Ed by J. E.
Coligan, A.M.
Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing
Associates
and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte
Function 3.1-
3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl.
Acad. Sci.
USA 78:2488-2492,1981; Herrmann et al., J. Immunol. 128:1968-1974,1982; Handa
et al.,
J. Immunol.135:1564-1572,1985; Takai et al., J. Immunol. 137:3494-3500,1986;
Takai et al.,
J. Immunol.140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA
78:2488-2492,
2 0 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J.
Immunol.
135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Bowmanet
al., J.
Virology 61:1992-1998; Takai et al., J. Immunol. 140:508-512, 1988;
Bertagnolli et al.,
Cellular Immunology 133:327-341,1991; Brown et al., J. Immunol. 153:3079-3092,
1994.
Assays for T-cell-dependent immunoglobulin responses and isotype switching
2 5 (which will identify, among others, proteins that modulate T-cell
dependent antibody
responses and that affect Thl /Th2 profiles) include, without limitation,
those described
in: Maliszewski, J. Immunol. 144:3028-3033,1990; and Assays for B cell
function: In vitro
antibody production, Mond, J.J. and Brunswick, M. In Current Protocols in
Immunology.
J.E.e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto.
1994.
3 0 Mixed lymphocyte reaction (MLR) assays (which will identify, among others,
proteins that generate predominantly Th1 and CTL responses) include, without
limitation,
those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M.
Kruisbeek,
D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and
Wiley-
Interscience (Chapter 3, In Vitro assays fox Mouse Lymphocyte Function 3.1-
3.19; Chapter
41
CA 02288343 1999-10-15
WO 98/49302 PCT/US98/08336
7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500,
1986; Takai
et al., J. Immunol. 140:508-512, 1988; Bertagnolli et al., J. Immunol.
149:3778-3783, 1992.
Dendritic cell-dependent assays (which will identify, among others, proteins
expressed by dendritic cells that activate naive T-cells) include, without
limitation, those
described in: Guery et aL, J. Immunol. 134:536-544, 1995; Inaba et al.,
Journal of
Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal of
Immunology
154:5071-5079,1995; Porgador et al., Journal of Experimental Medicine 282:255-
260, 1995;
Nair et al., Journal of Virology 67:4062-4069, 1993; Huang et al., Science
264:961-965,
1994; Macatonia et al., Journal of Experimental Medicine 169:1255-1264,1989;
Bhardwaj
et al., Journal of Clinical Investigation 94:797-807, 1994; and Inaba et al.,
Journal of
Experimental Medicine 172:631-640,1990.
Assays for lymphocyte survival/apoptosis (which will identify, among others,
proteins that prevent apoptosis after superantigen induction and proteins that
regulate
lymphocyte homeostasis) include, without limitation, those described in:
Darzynkiewicz
et al., Cytometry 13:795-808,1992; Gorczyca et al., Leukemia 7:659-670,1993;
Gorczyca et
al., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991;
Zacharchuk,
Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry 14:891-897,
1993;
Gorczyca et al., International Journal of Oncology 1:639-648, 1992.
Assays for proteins that influence early steps of T-cell commitment and
2 0 development include, without limitation, those described in: Antica et
al., Blood
84:111-117, 1994; Fine et al., Cellular Immunology 155:111-122, 1994; Galy et
al., Blood
85:2770-2778, 1995; Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551, 1991.
Hematopoiesis Re u-g lating Activity
2 5 A protein of the present invention may be useful in regulation of
hematopoiesis
and, consequently, in the treatment of myeloid or lymphoid cell deficiencies.
Even
marginal biological activity in support of colony forming cells or of factor-
dependent cell
lines indicates involvement in regulating hematopoiesis, e.g. in supporting
the growth and
proliferation of erythroid progenitor cells alone or in combination with other
cytokines,
3 0 thereby indicating utility, for example, in treating various anemias or
for use in
conjunction with irradiation/chemotherapy to stimulate the production of
erythroid
precursors and/or erythroid cells; in supporting the growth and proliferation
of myeloid
cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF
activity)
useful, for example, in conjunction with chemotherapy to prevent or treat
consequent
42
CA 02288343 1999-10-15
WO 98/49302 PCT/US98/08336
myelo-suppression; in supporting the growth and proliferation of
megakaryocytes and
consequently of platelets thereby allowing prevention or treatment of various
platelet
disorders such as thrombocytopenia, and generally for use in place of or
complimentary
to platelet transfusions; and/or in supporting the growth and proliferation of
hematopoietic stem cells which are capable of maturing to any and all of the
above-
mentioned hematopoietic cells and therefore find therapeutic utility in
various stem cell
disorders (such as those usually treated with transplantation, including,
without
limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well
as in
repopulating the stem cell compartment post irradiation/chemotherapy, either
in-vivo or
ex-vivo (i.e., in conjunction with bone marrow transplantation or with
peripheral
progenitor cell transplantation (homologous or heterologous)) as normal cells
or
genetically manipulated for gene therapy.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Suitable assays for proliferation and differentiation of various hematopoietic
lines
are cited above.
Assays for embryonic stem cell differentiation (which will identify, among
others,
proteins that influence embryonic differentiation hematopoiesis) include,
without
limitation, those described in: Johansson et al. Cellular Biology 15:141-
151,1995; Keller et
2 0 al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al.,
Blood
81:2903-2915, 1993.
Assays for stem cell survival and differentiation (which will identify, among
others, proteins that regulate lympho-hematopoiesis) include, without
limitation, those
described in: Methylcellulose colony forming assays, Freshney, M.G. In Culture
of
2 5 Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 265-268, Wiley-
Liss, Inc., New York,
NY. 1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992;
Primitive
hematopoietic colony forming cells with high proliferative potential, McNiece,
LK. and
Briddell, R.A. In Culture of Hemafopoietic Cells. R.I. Freshney, et al. eds.
Vol pp. 23-39,
Wiley-Liss, Inc., New York, NY.1994; Neben et al., Experimental Hematology
22:353-359,
3 0 1994; Cobblestone area forming cell assay, Ploemacher, R.E. In Culture of
Hematopoietic
Cells. R.I. Freshney, et al. eds. Vol pp. l-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,
43
CA 02288343 1999-10-15
WO 98/49302 PCT/US98/08336
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
well as open fracture reduction and also in the improved fixation of
artificial joints. De
novo bone formation induced by an osteogenic agent contributes to the repair
of
congenital, trauma induced, or oncologic resection induced craniofacial
defects, and also
is useful in cosmetic plastic surgery.
A protein of this invention may also be used in the treatment of periodontal
disease, and in other tooth repair processes. Such agents may provide an
environment
to attract bone-forming cells, stimulate growth of bone-forming cells or
induce
2 0 differentiation of progenitors of bone-forming cells. A protein of the
invention may also
be useful in the treatment of osteoporosis or osteoarthritis, such as through
stimulation
of bone and/or cartilage repair or by blocking inflammation or processes of
tissue
destruction (collagenase activity, osteoclast activity, etc.) mediated by
inflammatory
processes.
2 5 Another category of tissue regeneration activity that may be attributable
to the
protein of the present invention is tendon/ligament formation. A protein of
the present
invention, which induces tendon/ligament-like tissue or other tissue formation
in
circumstances where such tissue is not normally formed, has application in the
healing of
tendon or ligament tears, deformities and other tendon or ligament defects in
humans and
3 0 other animals. Such a preparation employing a tendon/ligament-like tissue
inducing
protein may have prophylactic use in preventing damage to tendon or ligament
tissue, as
well as use in the improved fixation of tendon or ligament to bone or other
tissues, and
in repairing defects to tendon or ligament tissue. De novo tendon/ligament-
like tissue
formation induced by a composition of the present invention contributes to the
repair of
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CA 02288343 1999-10-15
WO 98/49302 PCT/US98/08336
congenital, trauma induced, or other tendon or ligament defects of other
origin, and is also
useful in cosmetic plastic surgery for attachment or repair of tendons or
ligaments. The
compositions of the present invention may provide an environment to attract
tendon- or
ligament-forming cells, stimulate growth of tendon- or ligament-forming cells,
induce
differentiation of progenitors of tendon- or ligament-forming cells, or induce
growth of
tendon/ligament cells or progenitors ex vivo for return in vivo to effect
tissue repair. The
compositions of the invention may also be useful in the treatment of
tendinitis, carpal
tunnel syndrome and other tendon or ligament defects. The compositions may
also
include an appropriate matrix and/or sequestering agent as a carrier as is
well known in
the art.
The protein of the present invention may also be useful for proliferation of
neural
cells and for regeneration of nerve and brain tissue, i.e. for the treatment
of central and
peripheral nervous system diseases and neuropathies, as well as mechanical and
traumatic disorders, which involve degeneration, death or trauma to neural
cells or nerve
tissue. More specifically, a protein may be used in the treatment of diseases
of the
peripheral nervous system, such as peripheral nerve injuries, peripheral
neuropathy and
localized neuropathies, and central nervous system diseases, such as
Alzheimer's,
Parkinson s disease, Huntingtori s disease, amyotrophic lateral sclerosis, and
Shy-Drager
syndrome. Further conditions which may be treated in accordance with the
present
2 0 invention include mechanical and traumatic disorders, such as spinal cord
disorders, head
trauma and cerebrovascular diseases such as stroke. Peripheral neuropathies
resulting
from chemotherapy or other medical therapies may also be treatable using a
protein of the
invention.
Proteins of the invention may also be useful to promote better or faster
closure of
2 5 non-healing wounds, including without limitation pressure ulcers, ulcers
associated with
vascular insufficiency, surgical and traumatic wounds, and the like.
It is expected that a protein of the present 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)
3 0 and vascular (including vascular endothelium) tissue, or for promoting the
growth of cells
comprising such tissues. Part of the desired effects may be by inhibition or
modulation
of fibrotic scarring to allow normal tissue to regenerate. A protein of the
invention may
also exhibit angiogenic activity.
CA 02288343 1999-10-15
WO 98/49302 PCT/US98/08336
A protein of the present invention may also be useful for gut protection or
regeneration and treatment of lung or liver fibrosis, reperfusion injury in
various tissues,
and conditions resulting from systemic cytokine damage.
A protein of the present invention may also be useful for promoting or
inhibiting
differentiation of tissues described above from precursor tissues or cells; or
for inhibiting
the growth of tissues described above.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assays for tissue generation activity include, without limitation, those
described
in: International Patent Publication No. W095/16035 (bone, cartilage, tendon);
International Patent Publication No. W095/05846 (nerve, neuronal);
International Patent
Publication No. W091/07491 (skin, endothelium ).
Assays for wound healing activity include, without limitation, those described
in:
Winter, Epidermal Wound Healing, pps. 71-112 (Maibach, HI and Rovee, DT,
eds.), Year
Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J.
Invest.
Dermatol 71:382-84 (1978).
Activin/Inhibin ActivitX
A protein of the present invention may also exhibit activin- or inhibin-
related
2 0 activities. Inhibins are characterized by their ability to inhibit the
release of follicle
stimulating hormone (FSH), while activins and are characterized by their
ability to
stimulate the release of follicle stimulating hormone (FSH). Thus, a protein
of the present
invention, alone or in heterodimers with a member of the inhibin a family, may
be useful
as a contraceptive based on the ability of inhibins to decrease fertility in
female mammals
2 5 and decrease spermatogenesis in male mammals. Administration of sufficient
amounts
of other inhibins can induce infertility in these mammals. Alternatively, the
protein of the
invention, as a homodimer or as a heterodimer with other protein subunits of
the inhibin-
~3 group, may be useful as a fertility inducing therapeutic, based upon the
ability of activin
molecules in stimulating FSH release from cells of the anterior pituitary.
See, for example,
3 0 United States Patent 4,798,885. A protein of the invention may also be
useful for
advancement of the onset of fertility in sexually immature mammals, so as to
increase the
lifetime reproductive performance of domestic animals such as cows, sheep and
pigs.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
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CA 02288343 1999-10-15
WO 98/49302 PCT/CTS98/08336
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.
Chemotacti~Chemokinetic Actfvity
A protein of the present invention may have chemotactic or chemokinetic
activity
(e.g., act as a chemokine) for mammalian cells, including, for example,
monocytes,
fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or
endothelial cells.
Chemotactic and chemokinetic proteins can be used to mobilize or attract a
desired cell
population to a desired site of action. Chemotactic or chemokinetic proteins
provide
particular advantages in treatment of wounds and other trauma to tissues, as
well as in
treatment of localized infections. For example, attraction of lymphocytes,
monocytes or
neutrophils to tumors or sites of infection may result in improved immune
responses
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
population. Preferably, the protein or peptide has the ability to directly
stimulate directed
movement of cells. Whether a particular protein has chemotactic activity for a
population
2 0 of cells can be readily determined by employing such protein or peptide in
any known
assay for cell chemotaxis.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assays for chemotactic activity (which will identify proteins that induce or
prevent
2 5 chemotaxis) consist of assays that measure the ability of a protein to
induce the migration
of cells across a membrane as well as the ability of a protein to induce the
adhesion of one
cell population to another cell population. Suitable assays for movement and
adhesion
include, without limitation, those described in: Current Protocols in
Immunology, Ed by
J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W.Strober, Pub.
Greene
3 0 Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of
alpha and
beta Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:1370-
1376,1995; Lind et al.
APMIS 103:140-146, 1995; Muller et 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.
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CA 02288343 1999-10-15
WO 98/49302 PCT/US98/08336
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 hemophilias) or to enhance
coagulation
and other hemostatic events in treating wounds resulting from trauma, surgery
or other
causes. A protein of the invention may also be useful for dissolving or
inhibiting
formation of thromboses and for treatment and prevention of conditions
resulting
therefrom (such as, for example, infarction of cardiac and central nervous
system vessels
(e.g., stroke).
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assay for hemostatic and thrombolytic activity include, without limitation,
those
described in: Linet et al., J. Clin. Pharmacol. 26:131-140,1986; Burdick et
al., Thrombosis
Res. 45:413-419,1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub,
Prostaglandins
35:467-474, 1988.
Receptor/Ligand Activity
A protein of the present invention may also demonstrate activity as receptors,
receptor ligands or inhibitors or agonists of receptor/ligand interactions.
Examples of
2 0 such receptors and ligands include, without limitation, cytokine receptors
and their
ligands, receptor kinases and their ligands, receptor phosphatases and their
ligands,
receptors involved in cell-cell interactions and their ligands (including
without limitation,
cellular adhesion molecules (such as selectins, integrins and their ligands)
and
receptor/ligand pairs involved in antigen presentation, antigen recognition
and
2 5 development of cellular and humoral immune responses). Receptors and
ligands are also
useful for screening of potential peptide or small molecule inhibitors of the
relevant
receptor/ligand interaction. A protein of the present invention (including,
without
limitation, fragments of receptors and ligands) may themselves be useful as
inhibitors of
receptor/ligand interactions.
3 0 The activity of a protein of the invention may, among other means, be
measured
by the following methods:
Suitable assays for receptor-ligand activity include without limitation those
described in:Current Protocols in Immunology, Ed by J.E. Coligan, A.M.
Kruisbeek, D.H.
Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and
48
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WO 98/49302 PCT/US98/08336
Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under
static
conditions 7.28.1-7.28.22), Takai et al., Proc. Natl. Acad. Sci. USA 84:6864-
6868, 1987;
Bierer et al., J. Exp. Med.168:1145-1156, 1988; Rosenstein et al., J. Exp.
Med. 169:149-160
1989; Stoltenborg et al., J. Immunol. Methods 175:59-68,1994; Stitt et al.,
Cell 80:661-670,
1995.
Anti-Inflammatory Activity
Proteins of the present invention may also exhibit anti-inflammatory activity.
The
anti-inflammatory activity may be achieved by providing a stimulus to cells
involved in
the inflammatory response, by inhibiting or promoting cell-cell interactions
(such as, for
example, cell adhesion), by inhibiting or promoting chemotaxis of cells
involved in the
inflammatory process, inhibiting or promoting cell extravasation, or by
stimulating or
suppressing production of other factors which more directly inhibit or promote
an
inflammatory response. Proteins exhibiting such activities can be used to
treat
inflammatory conditions including chronic or acute conditions), including
without
limitation inflammation associated with infection (such as septic shock,
sepsis or systemic
inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin
lethality, arthritis, complement-mediated hyperacute rejection, nephritis,
cytokine or
chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or
resulting
2 0 from over production of cytokines such as TNF or IL-1. Proteins of the
invention may also
be useful to treat anaphylaxis and hypersensitivity to an antigenic substance
or material.
Cadherin/Tumor Invasion Suppressor Activity
Cadherins are calcium-dependent adhesion molecules that appear to play major
2 5 roles during development, particularly in defining specific cell types.
Loss or alteration
of normal cadherin expression can lead to changes in cell adhesion properties
linked to
tumor growth and metastasis. Cadherin malfunction is also implicated in other
human
diseases, such as pemphigus vulgaris and pemphigus foliaceus (auto-immune
blistering
skin diseases), Crohn's.disease, and some developmental abnormalities.
3 0 The cadherin superfamily includes well over forty members, each with a
distinct
pattern of expression. All members of the superfamily have in common conserved
extracellular repeats (cadherin domains), but structural differences are found
in other
parts of the molecule. The cadherin domains bind calcium to form their
tertiary structure
and thus calcium is required to mediate their adhesion. Only a few amino acids
in the
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first cadherin domain provide the basis for homophilic adhesion; modification
of this
recognition site can change the specificity of a cadherin so that instead of
recognizing only
itself, the mutant molecule can now also bind to a different cadherin. In
addition, some
cadherins engage in heterophilic adhesion with other cadherins.
E-cadherin, one member of the cadherin superfamily, is expressed in epithelial
cell
types. Pathologically, if E-cadherin expression is lost in a tumor, the
malignant cells
become invasive and the cancer metastasizes. Transfection of cancer cell lines
with
polynucleotides expressing E-cadherin has reversed cancer-associated changes
by
returning altered cell shapes to normal, restoring cells' adhesiveness to each
other and to
their substrate, decreasing the cell growth rate, and drastically reducing
anchorage-
independent cell growth. Thus, reintroducing E-cadherin expression reverts
carcinomas
to a less advanced stage. It is likely that other cadherins have the same
invasion
suppressor role in carcinomas derived from other tissue types. Therefore,
proteins of the
present invention with cadherin activity, and polynucleotides of the present
invention
encoding such proteins, can be used to treat cancer. Introducing such proteins
or
polynucleotides into cancer cells can reduce or eliminate the cancerous
changes observed
in these cells by providing normal cadherin expression.
Cancer cells have also been shown to express cadherins of a different tissue
type
than their origin, thus allowing these cells to invade and metastasize in a
different tissue
2 0 in the body. Proteins of the present invention with cadherin activity, and
polynucleotides
of the present invention encoding such proteins, can be substituted in these
cells for the
inappropriately expressed cadherins, restoring normal cell adhesive properties
and
reducing or eliminating the tendency of the cells to metastasize.
Additionally, proteins of the present invention with cadherin activity, and
2 5 polynucleotides of the present invention encoding such proteins, can used
to generate
antibodies recognizing and binding to cadherins. Such antibodies can be used
to block
the adhesion of inappropriately expressed tumor-cell cadherins, preventing the
cells from
forming a tumor elsewhere. Such an anti-cadherin antibody can also be used as
a marker
for the grade, pathological type, and prognosis of a cancer, i.e. the more
progressed the
3 0 cancer, the less cadherin expression there will be, and this decrease in
cadherin expression
can be detected by the use of a cadherin-binding antibody.
Fragments of proteins of the present invention with cadherin activity,
preferably
a polypeptide comprising a decapeptide of the cadherin recognition site, and
poly-
nucleotides of the present invention encoding such protein fragments, can also
be used
CA 02288343 1999-10-15
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to block cadherin function by binding to cadherins and preventing them from
binding in
ways that produce undesirable effects. Additionally, fragments of proteins of
the present
invention with cadherin activity, preferably truncated soluble cadherin
fragments which
have been found to be stable in the circulation of cancer patients, and
polynucleotides
encoding such protein fragments, can be used to disturb proper cell-cell
adhesion.
Assays for cadherin adhesive and invasive suppressor activity include, without
limitation, those described in: Hortsch et al. J Biol Chem 270 (32): 18809-
18817, 1995;
Miyaki et al. Oncogene 11: 2547-2552,1995; Ozawa et al. Cell 63: 1033-
1038,1990.
Tumor Inhibition Activity
In addition to the activities described above for immunological treatment or
prevention of tumors, a protein of the invention may exhibit other anti-tumor
activities.
A protein may inhibit tumor growth directly or indirectly (such as, for
example, via
ADCC). A protein may exhibit its tumor inhibitory activity by acting on tumor
tissue or
tumor precursor tissue, by inhibiting formation of tissues necessary to
support tumor
growth (such as, for example, by inhibiting angiogenesis), by causing
production of other
factors, agents or cell types which inhibit tumor growth, or by suppressing,
eliminating
or inhibiting factors, agents or cell types which promote tumor growth.
2 0 Other Activities
A protein of the invention may also exhibit one or more of the following
additional
activities or effects: inhibiting the growth, infection or function of, or
killing, infectious
agents, including, without limitation, bacteria, viruses, fungi and other
parasites; effecting
(suppressing or enhancing) bodily characteristics, including, without
limitation, height,
2 5 weight, hair color, eye color, skin, fat to lean ratio or other tissue
pigmentation, or organ
or body part size or shape (such as, for example, breast augmentation or
diminution,
change in bone form or shape); effecting biorhythms or caricadic cycles or
rhythms;
effecting the fertility of male or female subjects; effecting the metabolism,
catabolism,
anabolism, processing, utilization, storage or elimination of dietary fat,
lipid, protein,
3 0 carbohydrate, vitamins, minerals, cofactors or other nutritional factors
or component(s);
effecting behavioral characteristics, including, without limitation, appetite,
libido, stress,
cognition (including cognitive disorders), depression (including depressive
disorders) and
violent behaviors; providing analgesic effects or other pain reducing effects;
promoting
differentiation and growth of embryonic stem cells in lineages other than
hematopoietic
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lineages; hormonal or endocrine activity; in the case of enzymes, correcting
deficiencies
of the enzyme and treating deficiency-related diseases; treatment of
hyperproliferative
disorders (such as, for example, psoriasis); immunoglobulin-like activity
(such as, for
example, the ability to bind antigens or complement); and the ability to act
as an antigen
in a vaccine composition to raise an immune response against such protein or
another
material or entity which is cross-reactive with such protein.
ADMINISTRATION AND DOSING
A protein of the present invention {from whatever source derived, including
without limitation from recombinant and non-recombinant sources) may be used
in a
pharmaceutical composition when combined with a pharmaceutically acceptable
carrier.
Such a composition may also contain (in addition to protein and a carrier)
diluents, fillers,
salts, buffers, stabilizers, solubilizers, and other materials well known in
the art. The term
"pharmaceutically acceptable" means a non-toxic material that does not
interfere with the
effectiveness of the biological activity of the active ingredient(s). The
characteristics of the
carrier will depend on the route of administration. The pharmaceutical
composition of
the invention may also contain cytokines, lymphokines, or other hematopoietic
factors
such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-
9, IL-10, IL-11,
2 0 IL-12, IL-13, IL-14, IL-15, IFN, TNFO, TNF1, 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
pharmaceutical composition to produce a synergistic effect with protein of the
invention,
2 5 or to minimize side effects. Conversely, protein of the present invention
may be included
in formulations of the particular cytokine, lymphokine, other hematopoietic
factor,
thromboiytic or anti-thrombotic factor, or anti-inflammatory agent to minimize
side effects
of the cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-
thrombotic
factor, or anti-inflammatory agent.
3 0 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.
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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 immunoglobulin
receptor. T
lymphocytes will respond to antigen through the T cell receptor (TCR)
following
presentation of the antigen by MHC proteins. MHC and structurally related
proteins
including those encoded by class I and class II MHC genes on host cells will
serve to
present the peptide antigens) to T lymphocytes. The antigen components could
also be
supplied as purified MHC-peptide complexes alone or with co-stimulatory
molecules that
can directly signal T cells. Alternatively antibodies able to bind surface
immunolgobulin
and other molecules on B cells as well as antibodies able to bind the TCR and
other
molecules on T cells can be combined with the pharmaceutical composition of
the
invention.
The pharmaceutical composition of the invention may be in the form of a
liposome
in which protein of the present invention is combined, in addition to other
pharmaceutically acceptable Garners, with amphipathic agents such as lipids
which exist
in aggregated form as micelles, insoluble monolayers, liquid crystals, or
lamellar layers
in aqueous solution. Suitable lipids for liposomal formulation include,
without limitation,
monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids,
saponin, bile acids,
2 0 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.
As used herein, the term "therapeutically effective amount" means the total
2 5 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
ingredient, administered alone, the term refers to that ingredient alone. When
applied to
3 0 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
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CA 02288343 1999-10-15
WO 98/49302 PCTIUS98/08336
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
cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection.
Intravenous administration to the patient is preferred.
When a therapeutically effective amount of protein of the present invention is
administered orally, protein of the present invention will be in the form of a
tablet,
capsule, powder, solution or elixir. When administered in tablet form, the
pharmaceutical
composition of the invention may additionally contain a solid carrier such as
a gelatin or
2 0 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
may be added. The liquid form of the pharmaceutical composition may further
contain
2 5 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
of the present invention, and preferably from about 1 to 50% protein of the
present
invention.
3 0 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
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CA 02288343 1999-10-15
WO 98/49302 PCT/US98/08336
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
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 pg
to about 100
mg (preferably about 0.lng to about 10 mg, more preferably about 0.1 lzg to
about 1 mg)
of protein of the present invention per kg body weight.
The duration of intravenous therapy using the pharmaceutical composition of
the
2 0 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
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
2 5 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
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
3 0 carboxyl terminus, and are conjugated to a hapten such as keyhole limpet
hemocyanin
(KLFi). 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
CA 02288343 1999-10-15
WO 98/49302 PCT/US98/08336
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
tissue damage. Topical administration may be suitable for wound healing and
tissue
repair. Therapeutically useful agents other than a protein of the invention
which may also
optionally be included in the composition as described above, may
alternatively or
additionally, be administered simultaneously or sequentially with the
composition in the
methods of the invention. Preferably for bone and/or cartilage formation, the
composition would include a matrix capable of delivering the protein-
containing
composition to the site of bone and/or cartilage damage, providing a structure
for the
2 0 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.
The choice of matrix material is based on biocompatibility, biodegradability,
mechanical properties, cosmetic appearance and interface properties. The
particular
2 5 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
polyanhydrides. Other potential materials are biodegradable and biologically
well-
defined, such as bone or dermal collagen. Further matrices are comprised of
pure proteins
3 0 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-
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CA 02288343 1999-10-15
WO 98/49302 PCT/US98/08336
alurriinate-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
acid, sodium alginate, polyethylene glycol), polyoxyethylene oxide,
carboxyvinyI
polymer and polyvinyl alcohol). The amount of sequestering agent useful herein
is 0.5-20
wt%, preferably 1-10 wt% based on total formulation weight, which represents
the
amount necessary to prevent desorbtion of the protein from the polymer matrix
and to
provide appropriate handling of the composition, yet not so much that the
progenitor cells
are prevented from infiltrating the matrix, thereby providing the protein the
opportunity
to assist the osteogenic activity of the progenitor cells.
2 0 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
(EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF-
a and
TGF-(3), and insulin-like growth factor (IGF).
2 5 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.
The dosage regimen of a protein-containing pharmaceutical composition to be
used in tissue regeneration will be determined by the attending physician
considering
3 0 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
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CA 02288343 1999-10-15
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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.
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 J.
{ii) TITLE OF INVENTION: SECRETED PROTEINS AND POLYNUCLEOTIDES
ENCODING THEM
(iii) NUMBER OF SEQUENCES: 25
(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: 1480 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
59
CA 02288343 1999-10-15
WO 98/49302 PCT/US98/08336
{ii) MOLECULE TYPE: cDNA
(xi)
SEQUENCE
DESCRIPTION:
SEQ
ID NO:1:
AGGCGCCCTCCCTTCCTGAGGAGCTGTTGGCCTGGGTGGGCAGGAACTGCAGTATGGCCA 60
TGGGCTGAGCAGGCTGAGCACCTCAGCCTTTAGGGCTTATGGCCAGGGGACACTGTATGA 120
CTCTCCTCTCCTGCAGGTGTCTATCCACCTGGGGTATGGCATCTACCGACCTGTCTCCCT 180
GGGGTCACATGCTTTGTTTCCATTCTTGTCCTGGCTGGACCAGCCACTGTGGGACCAACA 240
CCCCTCCCACACTCCCCCAGACTGCTCGTCTATCACCAGGATCGCTTTGTACTTTGTGCA 300
AAAGGGTCTGGCTGTCCCTTGCTGTTTTCATCTCTGCCAAGCCTATTGTGCCTCTGGCTG 360
CTGTATGTGTGCGCGTGCACGTGTGTGTGTTTCATCTGTTCATTCACTGCACAAGATATT 420
TATTGAGTGCCCACTACGTGCCAGGCACTGTTGCTGAGTTCCTGTGGGTGTGTCTCTCGA 480
TGCCACTCCTGCTTCTCTGGGGGCCTCTTTCTGTGCTTCTCTTTGTCCCCAAATTGCTAC 540
CTCTTTGTCAGTCTGGGTGTCTCAGGTTCTGTGTGTCCTTGTGTGCATTTCTGTCTCTCT 600
CTGTCCTCGTCTCTCTGCAAGGCCCTCTATTTCTCTCTTTCTTGGTGTCTGTCCTTTGCC 660
CCCTGTGCCCTCTGGATTCTCTGGGTCTATGTAGGCCCCTGGTCTGCCCTGGGCTCATCA 720
GCCTTCCTGACCTCCTCCTGCCCTCCCCTTCACTCCCTCCCTGGCTCTGCCAGTCGGTTC 780
CCACGGAGCCATTTTTAGCTCTGATCAGCATGGGAATGTGCCTCGGCCTCCAAGGGGCTT 840
TGTCCTGGTGCCCCCGCCCCTGGTCCCAACCTGATCCCACGAGGGAGTTGGGACAGGAGG 900
ATTGATGGTGCTCCCCTTCCTGCCAGCGTCAGAGGCCCTGGAGAGGGGCTGTCCATGGCA 960
GCTGGTCTTTATTCCTCCCTCATGAGCACAGGGTCGGGGGGTCCCCATTCTTGGAAGAGG 1020
TTGAGAAGACTCCTGGGCTTCAGCCTCTCCCACCCAGCCCTGCCCCTCACCTGCCTGCCC 1080
TCCCCTCCCCCACTCTATACTAGGGACTGGATCTCAGCCTCTGATCAGTTTCACAAAGTT 1140
TGTTCCCTAAGGAAATCAAATCCCATTGTCACCTAACTCTGAAGATCTAAATAGCCCTTG 1200
GATCAGTACGGGAACCCCAAATCCCACAGGGCCAGATGTGGAGTCTGTGTCTGCCCCCGT 1260
CTTCTCTCCATCCTCAAAGCCCCCACTTCTCTCCAGGCTGTTTCTTTTTTTATGACTGTA 1320
AACATAGATAGTGCTTTATTTTGTTAATAATAAGATAATGATGAGTAACTTAACCAGCAC 1380
ATTTCTCCTGTTTACACTCGGGGGATTTTTTTGTTTTCTGATGACATAATAAAGACAGAT 1440
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CATTTCAGAA P.~~e~AAAAAAA P,~~~AAAAAAA AAAAAAAAAA 14 8 0
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 268 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi)SEQUENCE
DESCRIPTION:
SEQ
ID
N0:2:
Met AlaArgGly HisCysMet ThrLeuLeu SerCys ArgCysLeu Ser
1 5 10 15
Thr TrpGlyMet AlaSerThr AspLeuSer ProTrp GlyHisMet Leu
20 25 30
Cys PheHisSer CysProGly TrpThrSer HisCys GlyThrAsn Thr
35 40 45
Pro ProThrLeu ProGlnThr AlaArgLeu SerPro GlySerLeu Cys
50 55 60
Thr LeuCysLys ArgValTrp LeuSerLeu AlaVal PheIleSer Ala
65 70 75 80
Lys ProIleVal ProLeuAla AlaValCys ValArg ValHisVal Cys
85 90 95
Val PheHisLeu PheIleHis CysThrArg TyrLeu LeuSerAla His
100 105 110
Tyr ValProGly ThrValAla GluPheLeu TrpVal CysLeuSer Met
115 120 125
Pro LeuLeuLeu LeuTrpGly ProLeuSer ValLeu LeuPheVal Pro
130 135 140
Lys Leu Leu Pro Leu Cys Gln Ser Gly Cys Leu Arg Phe Cys Val Ser
145 150 155 160
Leu Cys Ala Phe Leu Ser Leu Ser Val Leu Val Ser Leu Gln Gly Pro
165 170 175
Leu Phe Leu Ser Phe Leu Val Ser Val Leu Cys Pro Leu Cys Pro Leu
180 185 190
Asp Ser Leu Gly Leu Cys Arg Pro Leu Val Cys Pro Gly Leu Ile Ser
195 200 205
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Leu Pro Asp Leu Leu Leu Pro Ser Pro Ser Leu Pro Pro Trp Leu Cys
210 215 220
Gln Ser Val Pro Thr Glu Pro Phe Leu Ala Leu Ile Ser Met Gly Met
225 230 235 240
Cys Leu Gly Leu Gln Gly Ala Leu Ser Trp Cys Pro Arg Pro Trp Ser
245 250 255
Gln Pro Asp Pro Thr Arg Glu Leu Gly Gln Glu Asp
260 265
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1436 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi)
SEQUENCE
DESCRIPTION:
SEQ
ID N0:3:
CCCGGCGGCTCCTGGAACCC CGGTTCGCGGCGATGCCAGCCACCCCAGCG AAGCCGCCGC60
AGTTCAGTGCTTGGATAATT TGAAAGTACAATAGTTGGTTTCCCTGTCCA CCCGCCCCAC120
TTCGCTTGCCATCACAGCAC GCCTATCGGATGTGAGAGGAGAAGTCCCGC TGCTCGGGCA180
CTGTCTATATACGCCTAACA CCTACATATATTTTAAAAACATTAAATATA ATTAACAATC240
AAAAGAAAGAGGAGAAAGGA AGGGAAGCATTACTGGGTTACTATGCACTT GCGACTGATT300
TCTTGGCTTTTTATCATTTT GAACTTTATGGAATACATCGGCAGCCAAAA CGCCTCCCGG360
GGAAGGCGCCAGCGAAGAAT GCATCCTAACGTTAGTCAAGGCTGCCAAGG AGGCTGTGCA420
ACATGCTCAGATTACAATGG ATGTTTGTCATGTAAGCCCAGACTATTTTT TGCTCTGGAA480
AGAATTGGCATGAAGCAGAT TGGAGTATGTCTCTCTTCATGTCCAAGTGG ATATTATGGA540
ACTCGATATCCAGATATAAA TAAGTGTACAAAATGCAAAGCTGACTGTGA TACCTGTTTC600
AACAAAAATTTCTGCACAAA ATGTAAAAGTGGATTTTACTTACACCTTGG AAAGTGCCTT660
GACAATTGCCCAGAAGGGTT GGAAGCCAACAACCATACTATGGAGTGTGT CAGTATTGTG720
CACTGTGAGGTCAGTGAATG GAATCCTTGGAGTCCATGCACGAAGAAGGG AAAAACATGT780
GGCTTCAAAAGAGGGACTGA AACACGGGTCCGAGAAATAATACAGCATCC TTCAGCAAAG840
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GGTAACCTGT GTCCCCCAAC AAATGAGACA AGAAAGTGTACAGTGCAAAG GAAGAAGTGT900
CAGAAGGGAG AACGAGGAAA AAAAGGAAGG GAGAGGAAAAGAAAAAAACC TAATAAAGGA960
GAAAGTAAAG AAGCAATACC TGACAGCAAA AGTCTGGAATCCAGCAAAGA AATCCCAGAG1020
CAACGAGAAA ACAAACAGCA GCAGAAGAAG CGAAAAGTCCAAGATAAACA GAAATCGGGG1080
ATTGAAGTCA CCCTAGCTGA AGGCCTCACC AGTGTTTCACAGAGGACACA GCCCACCCCT1140
TGCAGGAGGA GGTATCTCTG AGTGTGCAGC ACAGAATCGCATGACCCACC TTAACCTTCC1200
TGTTGTCATG GAAGGATGCA CGGCTGCTCT GTCCACTGTGATTCCTAGCC CTCTCAAGAT1260
CACTGCTTTC TGAAGAATTT GCAATGACTC TGGCTTCTGGCTGCTTATCT CTGGACACCC1320
GTTCTCCACC AGTTGTACAG TTCATGTAAT CTACTTGGCTTAATTGATTT TCCACTTCTC1380
TCTTCCTCTT CTAAGATATA AACATTTTAA ATGATTTAAAAP,~~P.AAAP.AA AAAAAA143
6
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 292 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Met His Leu Arg Leu Ile Ser Trp Leu Phe Ile Ile Leu Asn Phe Met
1 5 10 15
Glu Tyr Ile Gly Ser Gln Asn Ala Ser Arg Gly Arg Arg Gln Arg Arg
20 25 30
Met His Pro Asn Val Ser Gln Gly Cys Gln Gly Gly Cys Ala Thr Cys
35 40 45
Ser Asp Tyr Asn Gly Cys Leu Ser Cys Lys Pro Arg Leu Phe Phe Ala
50 55 60
Leu Glu Arg Ile Gly Met Lys Gln Ile Gly Val Cys Leu Ser Ser Cys
65 70 75 80
Pro Ser Gly Tyr Tyr Gly Thr Arg Tyr Pro Asp Ile Asn Lys Cys Thr
85 90 95
Lys Cys Lys Ala Asp Cys Asp Thr Cys Phe Asn Lys Asn Phe Cys Thr
100 105 110
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Lys Cys Lys Ser Gly Phe Tyr Leu His Leu Gly Lys Cys Leu Asp Asn
115 120 125
Cys Pro Glu Gly Leu Glu Ala Asn Asn His Thr Met Glu Cys Val Ser
130 135 140
Ile Val His Cys Glu Val Ser Glu Trp Asn Pro Trp Ser Pro Cys Thr
145 150 155 160
Lys Lys Gly Lys Thr Cys Gly Phe Lys Arg Gly Thr Glu Thr Arg Val
165 170 175
Arg Glu Ile Ile Gln His Pro Ser Ala Lys Gly Asn Leu Cys Pro Pro
180 185 190
Thr Asn Glu Thr Arg Lys Cys Thr Val Gln Arg Lys Lys Cys Gln Lys
195 200 205
Gly Glu Arg Gly Lys Lys Gly Arg Glu Arg Lys Arg Lys Lys Pro Asn
210 215 220
Lys Gly Glu Ser Lys Glu Ala Ile Pro Asp Ser Lys Ser Leu Glu Ser
225 230 235 240
Ser Lys Glu Ile Pro Glu Gln Arg Glu Asn Lys Gln Gln Gln Lys Lys
245 250 255
Arg Lys Val Gln Asp Lys Gln Lys Ser Gly Ile Glu Val Thr Leu Ala
260 265 270
Glu Gly Leu Thr Ser Val Ser Gln Arg Thr Gln Pro Thr Pro Cys Arg
275 280 285
Arg Arg Tyr Leu
290
(2) INFORMATION FOR SEQ ID N0:5:
{i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2322 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
GGTTAAGAGC AGATTAGAAC AGAAATCAGG AGAACTTGGG AAGAAGATCA CTGAGTTAAC 60
ATTGAAAAAT CAGACACTAC AAAAGGAAAT TGAAAAAGTT TATTTGGATA ATAAGCTCCT 120
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CAAGGAGCAA GCACATAACT TAACAATTGA AATGAAAAAT CATTATGTTC CTTTAAAAGT180
AAGTGAAGAC ATGAAAAAGT CACATGATGC AATTATTGAT GATCTTAATA GAAAGCTTTT240
AGATGTAACA CAAAAATATA CAGAAAAGAA GTTGGAAATG GAGAAATTGC TACTGGAAAA300
TGACAGCTTA AGTAAGGATG TAAGCCGCCT AGAAACTGTG TTTGTACCTC CTGAGAAACA360
TGAAAAAGAG ATAATAGCTC TGAAATCCAA TATTGTTGAA CTTAAGAAAC AGCTGTCTGA420
ACTTAAGAAA AAATGTGGTG AAGACCAGGA GAAAATACAC GCTCTCACAT CTGAAAACAC480
TAACTTGAAG AAGATGATGA GTAATCAGTA TGTGCCAGTT AAAACCCATG AAGAGGTTAA540
AATGACACTG AATGACACGT TAGCCAAAAC TAACAGAGAA TTATTAGATG TGAAGAAAAA600
ATTTGAAGAT ATAAATCAGG AATTTGTAAA AATAAAAGAT AAGAATGAAA TATTAAAAAG660
AAACCTGGAA AACACTCAGA ACCAAATAAA AGCTGAGTAC ATCAGCCTGG CAGAGCACGA720
GGCAAAGATG AGCTCGCTAA GTCAGAGCAT GAGAAAGGTG CAGGATAGTA ATGCTGAAAT780
CTTGGCCAAC TACAGAAAAG GCCAAGAAGA GATTGTGACA CTGCATGCCG AAATTAAAGC840
CCAGAAGAAG GAGCTCGACA CAATACAAGA ATGCATTAAG GTAAAATATG CCCCAATTGT900
CAGCTTTGAG GAGTGCGAGA GAAAATTTAA AGCAACAGAG AAAGAACTAA AAGACCAGTT960
ATCAGAGCAG ACACAAAAGT ATAGTGTCAG TGAAGAAGAA GTCAAGAAAA ACAAGCAAGA1020
GAATGACAAG TTAAAGAAGG AGATTTTTAC CCTTCAGAAA GATTTGAGAG ATAAGACAGT1080
TCTCATTGAG AAGTCTCATG AAATGGAAAG AGCATTAAGC AGAAAAACAG ACGAGCTAAA1140
CAAACAGTTA AAAGACTTGT CACAGAAATA CACGGAAGTA AAGAATGTGA AAGAGAAGCT1200
AGTAGAAGAA AATGCCAAAC AGACTTCTGA GATACTTGCA GTGCAAAATC TTTTGCAAAA1260
ACAACATGTT CCATTGGAAC AGGTTGAGGC TCTGAAAP.AA TCTCTTAATG GCACAATTGA1320
AAATCTAAAG GAAGAACTGA AGAGTATGCA AAGGTGTTAC GAGAAAGAGC AGCAGACAGT1380
GACCAAACTG CATCAATTGT TGGAGAATCA AAAGAACTCT TCTGTACCCC TGGCAGAGCA1440
TTTGCAGATT AAAGAAGCAT TTGAGAAAGA AGTTGGAATC ATAAAAGCCA GCTTGAGAGA1500
AAAGGAAGAA GAAAGCCAAA ACAAAATGGA AGAAGTCTCC AAACTTCAGT CGGAGGTTCA1560
GAATACTAAA CAAGCATTAA AAAAATTAGA GACTAGAGAG GTAGTTGACT TGTCTAAATA1620
TAAAGCAACA AAAAGTGATT TGGAGACACA GATTTCTAGC TTAAATGAAA AATTGGCCAA1680
TCTGAATAGA AAGTATGAGG AAGTATGTGA GGAAGTTTTG CATGCCAAAA AGAAGGAAAT1740
ATCTGCAAAA GATGAGAAGG AATTACTGCA TTTCAGCATT GAGCAAGAAA TTAAGGATCA1800
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GAAGGAACGA TGTGATAAGT CCTTAACAAC TTACAAAGAA GAATACAAGA1860
AATCACAGAG
ATCTGCTAAA CAAATAGAAG CAAAAGATAATAAGATAACTGAACTGCTTA ATGATGTGGA1920
AAGATTAAAA CAGGCACTCA ATGGCCTTTCCCAACTCACCTACACAAGTG GGAACCCCAC1980
CAAGAGGCAG AGCCAGCTGA TTGACACTCTGCAGCACCAAGTGAAATCTC TGGAGCAACA2040
GCTGGCCGAT GCTGACAGAC AGCACCAAGAAGTAATTGCAATTTATCGGA CACACCTTCT2100
TAGTGCTGCA CAGGGTCACA TGGATGAAGATGTTCAGGAGGCTCTGCTCC AGATCATACA2160
AATGCGGCAG GGGCTTGTGT GCTAGCCGTTAGCACTGACTGCCAGTATCT GTTTTATCTT2220
GCTGGTGCTG AACATTCTTT GTGCAACTCCATGGTCTTTCTGGGCCTTAC TGTGCTGGTA2280
TAATTAAAAT AAAATATATT TTGTTCTAAAP,~~e~AAAP.AAAAA 2
3
2
2
(2) INFORMATION FOR SEQ
ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 677 amino acids
(B) TYPE: amino acid
(Cj STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Met Lys Asn His Tyr Val Pro Leu Lys Val Ser Glu Asp Met Lys Lys
1 5 10 15
Ser His Asp Ala Ile Ile Asp Asp Leu Asn Arg Lys Leu Leu Asp Val
20 25 30
Thr Gln Lys Tyr Thr Glu Lys Lys Leu Glu Met Glu Lys Leu Leu Leu
35 40 45
Glu Asn Asp Ser Leu Ser Lys Asp Val Ser Arg Leu Glu Thr Val Phe
50 55 60
Val Pro Pro Glu Lys His Glu Lys Glu Ile Ile Ala Leu Lys Ser Asn
65 70 75 80
Ile Val Glu Leu Lys Lys Gln Leu Ser Glu Leu Lys Lys Lys Cys Gly
85 90 95
Glu Asp Gln Glu Lys Ile His Ala Leu Thr Ser Glu Asn Thr Asn Leu
100 105 110
Lys Lys Met Met Ser Asn Gln Tyr Val Pro Val Lys Thr His Glu Glu
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115 120 125
Val Lys Met Thr Leu Asn Asp Thr Leu Ala Lys Thr Asn Arg Glu Leu
130 135 140
Leu Asp Val Lys Lys Lys Phe Glu Asp Ile Asn Gln Glu Phe Val Lys
145 150 155 160
Ile Lys Asp Lys Asn Glu Ile Leu Lys Arg Asn Leu Glu Asn Thr Gln
165 170 175
Asn Gln Ile Lys Ala Glu Tyr Ile Ser Leu Ala Glu His Glu Ala Lys
180 185 190
Met Ser Ser Leu Ser Gln Ser Met Arg Lys Val Gln Asp Ser Asn Ala
195 200 205
Glu Ile Leu Ala Asn Tyr Arg Lys Gly Gln Glu Glu Ile Val Thr Leu
210 215 220
His Ala Glu Ile Lys Ala Gln Lys Lys Glu Leu Asp Thr Ile Gln Glu
225 230 235 240
Cys Ile Lys Val Lys Tyr Ala Pro Ile Val Ser Phe Glu Glu Cys Glu
245 250 255
Arg Lys Phe Lys Ala Thr Glu Lys Glu Leu Lys Asp Gln Leu Ser Glu
260 265 270
Gln Thr Gln Lys Tyr Ser Val Ser Glu Glu Glu Val Lys Lys Asn Lys
275 280 285
Gln Glu Asn Asp Lys Leu Lys Lys Glu Ile Phe Thr Leu Gln Lys Asp
290 295 300
Leu Arg Asp Lys Thr Val Leu Ile Glu Lys Ser His Glu Met Glu Arg
305 310 315 320
Ala Leu Ser Arg Lys Thr Asp Glu Leu Asn Lys Gln Leu Lys Asp Leu
325 330 335
Ser Gln Lys Tyr Thr Glu Val Lys Asn Val Lys Glu Lys Leu Val Glu
340 345 350
Glu Asn Ala Lys Gln Thr Ser Glu Ile Leu Ala Val Gln Asn Leu Leu
355 360 365
Gln Lys Gln His Val Pro Leu Glu Gln Val Glu Ala Leu Lys Lys Ser
370 375 380
Leu Asn Gly Thr Ile Glu Asn Leu Lys Glu Glu Leu Lys Ser Met Gln
385 390 395 400
Arg Cys Tyr Glu Lys Glu Gln Gln Thr Val Thr Lys Leu His Gln Leu
405 410 415
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Leu Glu Asn Gln Lys Asn Ser Ser Val Pro Leu Ala Glu His Leu Gln
420 425 430
Ile Lys Glu Ala Phe Glu Lys Glu Val Gly Ile Ile Lys Ala Ser Leu
435 440 445
Arg Glu Lys Glu Glu Glu Ser Gln Asn Lys Met Glu Glu Val Ser Lys
450 455 460
Leu Gln Ser Glu Val Gln Asn Thr Lys Gln Ala Leu Lys Lys Leu Glu
465 4?0 475 480
Thr Arg Glu Val Val Asp Leu Ser Lys Tyr Lys Ala Thr Lys Ser Asp
485 490 495
Leu Glu Thr Gln Ile Ser Ser Leu Asn Glu Lys Leu Ala Asn Leu Asn
500 505 510
Arg Lys Tyr Glu Glu Val Cys Glu Glu Val Leu His Ala Lys Lys Lys
515 520 525
Glu Ile Ser Ala Lys Asp Glu Lys Glu Leu Leu His Phe Ser Ile Glu
530 535 540
Gln Glu Ile Lys Asp Gln Lys Glu Arg Cys Asp Lys Ser Leu Thr Thr
545 550 555 560
Ile Thr Glu Leu Gln Arg Arg Ile Gln Glu Ser Ala Lys Gln Ile Glu
565 570 575
Ala Lys Asp Asn Lys Ile Thr Glu Leu Leu Asn Asp Val Glu Arg Leu
580 585 590
Lys Gln Ala Leu Asn Gly Leu Ser Gln Leu Thr Tyr Thr Ser Gly Asn
595 600 605
Pro Thr Lys Arg Gln Ser Gln Leu Ile Asp Thr Leu Gln His Gln Val
610 615 620
Lys Ser Leu Glu Gln Gln Leu Ala Asp Ala Asp Arg Gln His Gln Glu
625 630 635 640
Val Ile Ala Ile Tyr Arg Thr His Leu Leu Ser Ala Ala Gln Gly His
645 650 655
Met Asp Glu Asp Val Gln Glu Ala Leu Leu Gln Ile Ile Gln Met Arg
660 665 670
Gln Gly Leu Val Cys
675
(2} INFORMATION FOR SEQ ID N0:7:
(i} SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2041 base pairs
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(B) TYPE: nucleic acid
(C} STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi)
SEQUENCE
DESCRIPTION:
SEQ
ID N0:7:
TCTCCCCCCTCCCCGACACACACTCACAGG CCGGGCATTGATGGTAATGT ATGCGAGGAA60
ACAGCAGAGACTCAGTGATGGCTGTCACGA CCGGAGGGGGGACTCGCAGC CTTACCAGGC120
ACTTAAGTATTCATCGAAGAGTCACCCCAG TAGCGGTGATCACAGACATG AAAAGATGCG180
AGACGCCGGAGATCCTTCACCACCAAATAA AATGTTGCGGAGATCTGATA GTCCTGAAAA240
CAAATACAGTGACAGCACAGGTCACAGTAA GGCCAAAAATGTGCATACTC ACAGAGTTAG300
AGAGAGGGATGGTGGGACCAGTTACTCTCC ACAAGAAAATTCACACAACC ACAGTGCTCT360
TCATAGTTCAAATTCACATTCTTCTAATCC AAGCAATAACCCAAGCAAAA CTTCAGATGC420
ACCTTATGATTCTGCAGATGACTGGTCTGA GCATATTAGCTCTTCTGGGA AAAAGTACTA480
CTACAATTGTCGAACAGAAGTTTCACAATG GGAAAAACCAAAAGAGTGGC TTGAAAGAGA540
ACAGAGACAAAAAGAAGCAAACAAGATGGC AGTCAACAGCTTCCCAAAAG ATAGGGATTA600
CAGAAGAGAGGTGATGCAAGCAACAGCCAC TAGTGGGTTTGCCAGTGGAA AATCTACATC660
AGGAGACAAACCCGTATCACATTCTTGCAC AACTCCTTCCACGTCTTCTG CCTCTGGACT720
GAACCCCACATCTGCACCTCCAACATCTGC TTCAGCGGTCCCTGTTTCTC CTGTTCCACA780
GTCGCCAATACCTCCCTTACTTCAGGACCC AAATCTTCTTAGACAATTGC TTCCTGCTTT840
GCAAGCCACGCTGCAGCTTAATAATTCTAA TGTGGACATATCTAAAATAA ATGAAGTTCT900
TACAGCAGCTGTGACACAAGCCTCACTGCA GTCTATAATTCATAAGTTTC TTACTGCTGG960
ACCATCTGCTTTCAACATAACGTCTCTGAT TTCTCAAGCTGCTCAGCTCT CTACACAAGC1020
CCAGCCATCTAATCAGTCTCCGATGTCTTT AACATCTGATGCGTCATCCC CAAGATCATA1080
TGTTTCTCCAAGAATAAGCACACCTCAAAC TAACACAGTCCCTATCAAAC CTTTGATCAG1140
TACTCCTCCTGTTTCATCACAGCCAAAGGT TAGTACTCCAGTAGTTAAGC AAGGACCAGT1200
GTCACAGTCAGCCACACAGCAGCCTGTAAC TGCTGACAAGCAGCAAGGTC ATGAACCTGT1260
CTCTCCTCGAAGTCTTCAGCGCTCAAGCCA GAGAAGTCCATCACCTGGTC CCAATCATAC1320
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TTCTAATAGTAGTAATGCAT CAAATGCAACAGTTGTACCA CAGAATTCTTCTGCCCGATC1380
CACGTGTTCATTAACGCCTG CACTAGCAGCACACTTCAGT GAAAATCTCATAAAACACGT1440
TCAAGGATGGCCTGCAGATC ATGCAGAGAAGCAGGCATCA AGATTACGCGAAGAAGCGCA1500
TAACATGGGAACTATTCACA TGTCCGAAATTTGTACTGAA TTAAAAAATTTAAGATCTTT1560
AGTCCGAGTATGTGAAATTC AAGCAACTTTGCGAGAGCAA AGGATACTATTTTTGAGACA1620
ACAAATTAAGGAACTTGAAA AGCTAAAAAATCAGAATTCC TTCATGGTGTGAAGATGTGA1680
ATAATTGCACATGGTTTTGA GAACAGGAACTGTAAATCTG TTGCCCAATCTTAACATTTT1740
TGAGCTGCATTTAAGTAGAC TTTGGACCGTTAAGCTGGGC AAAGGAAATGACAAGGGGAC1800
GGGGTCTGTGAGAGTCAATT CAGGGGAAAGATACAAGATT GATTTGTAAAACCCTTGAAA1860
TGTAGATTTCTTGTAGATGT ATCCTTCACGTTGTAAATAT GTTTTGTAGAGTGAAGCCAT1920
GGGAAGCCATGTGTAACAGA GCTTAGACATCCAAAACTAA TCAATGCTGAGGTGGCTAAA1980
TACCTAGCCTTTTACATGTA AACCTGTCTGCAAAATTAGC TTTTTTAAAAP~~~AAAAAAA2040
A 2041
(2) INFORMATION
FOR
SEQ
ID N0:8:
(i) SEQUENCE
CHARACTERISTICS:
(A) LENGTH: 187 acids
amino
(B} TYPE: amino
acid
(C} STRANDEDNESS:
(D) TOPOLOGY: linear
(ii)
MOLECULE
TYPE:
protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
Met Arg Gly Asn Ser Arg Asp Ser Val Met Ala Val Thr Thr Gly Gly
1 5 10 15
Gly Thr Arg Ser Leu Thr Arg His Leu Ser Ile His Arg Arg Val Thr
20 25 30
Pro Val Ala Val Ile Thr Asp Met Lys Arg Cys Glu Thr Pro Glu Ile
35 40 45
Leu His His Gln IIe Lys Cys Cys Gly Asp Leu Ile Val Leu Lys Thr
50 55 60
Asn Thr Val Thr Ala Gln Val Thr Val Arg Pro Lys Met Cys Ile Leu
65 70 75 80
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Thr Glu Leu Glu Arg Gly Met Val Gly Pro Val Thr Leu His Lys Lys
85 90 95
Ile His Thr Thr Thr Val Leu Phe Ile Val Gln Ile His Ile Leu Leu
100 105 110
Ile Gln Ala Ile Thr Gln Ala Lys Leu Gln Met His Leu Met Ile Leu
115 120 125
Gln Met Thr Gly Leu Ser Ile Leu Ala Leu Leu Gly Lys Ser Thr Thr
130 135 140
Thr Ile Val Glu Gln Lys Phe His Asn Gly Lys Asn Gln Lys Ser Gly
145 150 155 160
Leu Lys Glu Asn Arg Asp Lys Lys Lys Gln Thr Arg Trp Gln Ser Thr
165 170 175
Ala Ser Gln Lys Ile Gly Ile Thr Glu Glu Arg
180 185
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1163 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
GCCCTATCCA CTTAATAGAT GCCAATTCAA AGAGGTTAAA TGATTAGACT AAGGCACCTA 60
ACTTATGTGA GTGTCAGGCT TCAATGCCTG TGTTAGAGCT ACTCCTTCAC ACAAAATAGT 120
TCAGAACATA GAGAAGGACC AAGGTTAATA AATGATTTTC ATCCCAAACA CTAAACATGA 180
TTGATGGGTA GAGGCTGCCC GAAGTACTGT GTAAAGATGG AATCTGAGAT AGAAGAATGC 240
TGTGGTCAAT TAGTAATTCT TGCCCATGGA GGGATTAGTG ACACATGCCT TGTATATTTG 300
TCATCTGTGG CCTAAACTCT GCCCCTGAAG GTTTGTTTTC TAATTCAGAG GTTTAAATTA 360
ATCTAGCCCA CTTAATAAAA CCAGAGATCC TATGGGAAAT TTAGCCTAAG ACAGTGCTGG 420
AAATTGCCAT ATGTTGATAC AAAGAAGTGT TTGGCCACAT TACAGGTCTC AGACTCAACT 480
GCTATGTGTG ACTGCCGCTC TGTGCCTATG TCTTGCTTTT TTGCTGAGTT CCCTATTTCC 540
ATATCTCCAG GTGAATCCAT GAGAAGCGAG AGGGTGGCTG AGAGGCCTGG GCCTCTGGGA 600
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TTCCACCTTGCTATCTCTGC TCTTCAACCATTGTTTTAGACTCTGAACAC CAGATCCTCA660
TATCTGAAAGTGATTTGGAG ACCTGGGCATCAAGTGCTCTTTTAAGAAGG GGCTATCCCA720
GAGGACTGTTCAAAAGTCTC ATTCAATAGAGATGTTGGAGTCCCAGAACA AAGTTAGGGA780
GCAAACCAGTAACCTATGCT GGTSGTAACAGAGGATCCTACAATTACGTT TGTTTTTAAG840
ACAGGATTTTGCTGTGTTGC CCAGACTGGTCTCAAACTCCTGGGTTCAAG AGATCCATCC900
TCCCACCTCAGTCTCCTGAA AGCTGGGATGACAGGCACATGCCACCACAC CTAGCTCCTT960
ACAACCATTTATTTTAACTT ATTTCATTTATAACTGGTATCTTTCATTTG TATGTGGCAG1020.
CTAGAGATTTATATAGGATG GAAGTAATTTATTTTTAATTTAAATATTTC ATGTTGAACT1080
GTTTGCCTTGTATGGAACAT TTTACTTGGCCAATTCAAATAAAAATAAAG TCAGCTTTGT1140
TTGTGACAAAAAAAAAAAAA AAA 1163
(2) INFORMATION
FOR
SEQ
ID N0:10:
(i) S EQUENCE CHARACTERISTICS:
(A) LENGTH: 43
amino acids
(B) TYPE: amino
acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii)
MOLECULE
TYPE:
protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:10:
Met Leu Ile Gln Arg Ser Val Trp Pro His Tyr Arg Ser Gln Thr Gln
1 5 10 15
Leu Leu Cys Val Thr Ala Ala Leu Cys Leu Cys Leu Ala Phe Leu Leu
20 25 30
Ser Ser Leu Phe Pro Tyr Leu Gln Val Asn Pro
35 40
(2) INFORMATION FOR SEQ ID N0:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3067 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
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(xi) SEQUENCE DESCRIPTION:
SEQ ID N0:11:
GCGGTGGCTG AGGCGGCTGGGCCTAGGGTG CAGCGGGCGC GTCTGCGGCTGGTGTTGGCG60
CATCTCTAGA TCCTTTCCCGGAGTTCAGTT ATGGGTGTGA GAGGTTTGCAAGGATTTGTG120
GGAAGTACCT GCCCACATATATGTACAGTA GTAAATTTCA AAGAACTGGCAGAGCACCAC180
CGAAGCAAGT ATCCTGGATGTACCCCTACC ATTGTGGTTG ATGCCATGTGTTGTCTCAGA240
TATTGGTATA CTCCAGAATCTTGGATCTGC GGTGGCCAGT GGCGAGAATACTTTTCTGCT300
TTGCGAGATT TTGTTAAAACTTTTACGGCA GCTGGGATCA AGTTGATATTCTTCTTTGAT360
GGCATGGTGG AGCAGGATAAGAGAGATGAA TGGGTGAAAC GAAGGCTCAAGAACAACAGG420
GAGATATCCA GGATTTTTCATTACATCAAG TCACACAAGG AGCAGCCAGGCAGAAATATG480
TTCTTCATCC CCTCAGGGCTAGCTGTGTTT ACACGATTTG CTCTAAAGACACTGGGCCAG540
GAAACTTTGT GTTCTTTGCAGGAAGCAGAT TATGAGGTAG CTTCCTATGGCCTCCAGCAT600
AACTGTCTTG GGATTCTGGGGGAAGACACT GATTACCTAA TCTATGACACTTGTCCCTAC660
TTTTCAATTA GCGAGCTCTGCCTAGAGAGC CTGGACACCG TCATGCTCTGCAGAGAGAAG720
CTCTGTGAGA GTCTGGGCCTCTGTGTGGCC GACCTTCCTC TTCTGGCCTGCCTCCTTGGC780
GACGACATAA TCCCAGAGGGCATGTTTGAA AGCTTTAGGT ACAAATGCTTATCGTCCTAC840
ACCTCTGTAA AAGAGAACTTTGACAAAAAA GGTAACATCA TATTAGCTGTGTCAGACCAT900
ATATCGAAAG TTCTTTACTTGTATCAAGGT GAGAAAAAAT TAGAAGAGATATTACCTCTG960
GGACCAAACA AAGCTCTTTTTTATAAAGGA ATGGCATCAT ATCTTTTACCAGGACAAAAA1020
TCTCCATGGT TTTTCCAAAAACCCAAAGGT GTAATAACTT TGGACAAACAAGTAATATCC1080
ACGAGTTCAG ACGCCGAATCCAGGGAAGAA GTTCCCATGT GTTCAGATGCTGAATCCAGG1140
CAAGAAGTTC CCATGTGTACAGGCCCTGAA TCCAGGCGAG AAGTTCCCGTGTATACAGAT1200
TCTGAACCCA GGCAAGAAGTTCCCATGTGT TCAGACCCTG AACCCAGGCAAGAAGTTCCC1260
ACATGTACAG GCCCTGAATCCAGGCGAGAA GTTCCCATGT GTTCAGACCCTGAACCCAGG1320
CAAGAAGTTC CCATGTGTACAGGCCCTGAA GCCAGGCAAG AAGTTCCCATGTATACAGAC1380
TCTGAACCCA GGCAAGAAGTTCCCATGTAT ACAGACTCTG AACCCAGGCAAGAAGTTCCC1440
ATGTATACAG GCTCTGAACCCAGGCAAGAA GTTCCCATGT ATACAGGCCCTGAATCCAGG1500
CAAGAAGTTC CCATGTATACAGGCCCTGAA TCCAGGCAAG AAGTTTTAATACGGACAGAC1560
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CCTGAATCTA GGCAAGAAATTATGTGTACAGGCCATGAATCCAAACAGGA AGTTCCCATA1620
TGTACAGATC CTATATCCAAGCAAGAAGACTCCATGTGTACACACGCTGA AATCAATCAA1680
AAATTACCTG TAGCAACAGATTTTGAATTTAAGCTAGAAGCTCTCATGTG TACAAACCCT1740
GAAATTAAAC AAGAAGACCCCACAAATGTGGGGCCTGAAGTAAAGCAACA AGTAACCATG1800
GTTTCAGACA CTGAAATCTTAAAGGTTGCTAGAACACATCACGTCCAAGC AGAAAGCTAC1860
CTGGTGTACA ACATCATGAGCAGTGGAGAGATTGAATGCAGCAACACCCT AGAAGATGAG1920
CTTGACCAGG CCTTACCCAGCCAGGCCTTCATTTACCGTCCCATTCGACA GCGGGTCTAC1980
TCACTCTTAC TGGAGGACTGTCAAGATGTCACCAGCACCTGCCTAGCTGT CAAGGAGTGG2040
TTTGTGTATC CTGGGAACCCACTGAGGCACCCGGACCTCGTCAGGCCGCT GCAGATGACC2100
ATTCCAGGGG GAACGCCTAGTTTGAAAATATTATGGCTGAACCAAGAGCC AGAAATACAG2160
GTTCGGCGCT TGGACACACTCCTAGCCTGTTTCAATCTTTCCTCCTCAAG AGAAGAGCTG2220
CAGGCTGTCG AAAGCCCATTTCAAGCTTTGTGCTGCCTCTTGATCTACCT CTTTGTCCAG2280
GTGGACACGC TTTGCCTGGAGGATTTGCATGCGTTTATTGCGCAGGCCTT GTGCCTCCAA2340
GGAAAATCCA CCTCGCAGCTTGTAAATCTACAGCCTGATTACATCAACCC CAGAGCCGTG2400
CAGCTGGGCT CCCTTCTCGTCCGCGGCCTCACCACTCTGGTTTTAGTCAA CAGCGCATGT2460
GGCTTCCCCT GGAAGACGAGTGATTTCATGCCCTGGAATGTATTTGACGG GAAGCTTTTT2520
CATCAGAAGT ACTTGCAATCTGAAAAGGGTTATGCTGTGGAGGTTCTTTT AGAACAAAAT2580
GGAGGTGGGG AAGACAGGGCTCCAGCTACCACAGGACGGGCTCTGGGTAT AGCCGTTCCA2640
GTCAGGGACA GCCGTGGAGAGACCAGGGACCAGGAAGCAGACAGTATGAG CATGACCAGT2700
GGAGAAGGTA CTAGTCAACCTCCAGAAAGAGTATGGAGAGAAAAAGAGGC ACACCTGGAC2760
GCAGAGCCCT GCCAGCGCCCTCCTCTGCTGTTGCAGCTGCAAGGAGACCA TGCCTGTGGG2820
AGCCAGGCCT CGCTTGCATGAAGAAGGAACGATGCCTTTTTCAATGGTGT CTCCCTCCCA2880
TTGTGCAGAA GAGCTTTTGTTGGCTTCTCTCCCGAGCTTGTGCCTGATTC TGTGGCCCAA2940
AACAATCATT GTTAACATCTTCATGTGTTTCATTCTGATCTTTCATTCAT ATATATGATG3000
CCTAGCTAAT TTCATTTTAAAATAAATGGGAATCTGTTGTA,~~1AAAAAAA P,~~AAAAAAAA3
0
6
0
3067
(2) INFORMATION EQ ID
FOR S N0:12:
(i) SEQUENCE RACTERISTICS:
CHA
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(A) LENGTH: 916 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
Met Gly Val Arg Gly Leu Gln Gly Phe Val Gly Ser Thr Cys Pro His
1 5 10 15
Ile Cys Thr Val Val Asn Phe Lys Glu Leu Ala Glu His His Arg Ser
20 25 30
Lys Tyr Pro Gly Cys Thr Pro Thr Ile Val Val Asp Ala Met Cys Cys
35 40 45
Leu Arg Tyr Trp Tyr fihr Pro Glu Ser Trp Ile Cys Gly Gly Gln Trp
50 55 60
Arg Glu Tyr Phe Ser Ala Leu Arg Asp Phe Val Lys Thr Phe Thr Ala
65 70 75 80
Ala Gly Ile Lys Leu Ile Phe Phe Phe Asp Gly Met Val Glu Gln Asp
85 90 95
Lys Arg Asp Glu Trp Val Lys Arg Arg Leu Lys Asn Asn Arg Glu Ile
100 105 110
Ser Arg Ile Phe His Tyr Ile Lys Ser His Lys Glu Gln Pro Gly Arg
115 120 125
Asn Met Phe Phe Ile Pro Ser Gly Leu Ala Val Phe Thr Arg Phe Ala
130 135 140
Leu Lys Thr Leu Gly Gln Glu Thr Leu Cys Ser Leu Gln Glu Ala Asp
145 150 155 160
Tyr Glu Val Ala Ser Tyr Gly Leu Gln His Asn Cys Leu Gly Ile Leu
265 170 175
Gly Glu Asp Thr Asp Tyr Leu Ile Tyr Asp Thr Cys Pro Tyr Phe Ser
180 185 190
Ile Ser Glu Leu Cys Leu Glu Ser Leu Asp Thr Val Met Leu Cys Arg
195 200 205
Glu Lys Leu Cys Glu Ser Leu Gly Leu Cys Val Ala Asp Leu Pro Leu
210 215 220
Leu Ala Cys Leu Leu Gly Asp Asp Ile Ile Pro Glu Gly Met Phe Glu
7$
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225 230 235 240
Ser Phe Arg Tyr Lys Cys Leu S2r Ser Tyr Thr Ser Val Lys Glu Asn
245 250 ~ 255
Phe Asp Lys Lys Gly Asn Ile Ile Leu Ala Val Ser Asp His Ile Ser
260 265 270
Lys Val Leu Tyr Leu Tyr Gln Gly Glu Lys Lys Leu Glu Glu Ile Leu
275 280 285
Pro Leu Gly Pro Asn Lys Ala Leu Phe Tyr Lys Gly Met Ala Ser Tyr
290 295 300
Leu Leu Pro Gly Gln Lys Ser Pro Trp Phe Phe Gln Lys Pro Lys Gly
305 310 315 320
Val Ile Thr Leu Asp Lys Gln Val Ile Ser Thr Ser Ser Asp Ala Glu
325 330 335
Ser Arg Glu Glu Val Pro Met Cys Ser Asp Ala Glu Ser Arg Gln Glu
340 345 350
Val Pro Met Cys Thr Gly Pro Glu Ser Arg Arg Glu Val Pro Val Tyr
355 360 365
Thr Asp Ser Glu Pro Arg Gln Glu Val Pro Met Cys Ser Asp Pro Glu
370 375 380
Pro Arg Gln Glu Val Pro Thr Cys Thr Gly Pro Glu Ser Arg Arg Glu
385 390 395 400
Val Pro Met Cys Ser Asp Pro Glu Pro Arg Gln Glu Val Pro Met Cys
405 410 415
Thr Gly Pro Glu Ala Arg Gln Glu Val Pro Met Tyr Thr Asp Ser Glu
420 425 430
Pro Arg Gln Glu Val Pro Met Tyr Thr Asp Ser Glu Pro Arg Gln Glu
435 440 445
Val Pro Met Tyr Thr Gly Ser Glu Pro Arg Gln Glu Val Pro Met Tyr
450 455 460
Thr Gly Pro Glu Ser Arg Gln Glu Val Pro Met Tyr Thr Gly Pro Glu
465 470 475 480
Ser Arg Gln Glu Val Leu Ile Arg Thr Asp Pro Glu Ser Arg Gln Glu
485 490 495
Ile Met Cys Thr Gly His Glu Ser Lys Gln Glu Val Pro Ile Cys Thr
500 505 510
Asp Pro Ile Ser Lys Gln Glu Asp Ser Met Cys Thr His Ala Glu Ile
515 520 525
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Asn Gln Lys Leu Pro Val Ala Thr Asp Phe Glu Phe Lys Leu Glu Ala
530 535 540
Leu Met Cys Thr Asn Pro Glu Ile Lys Gln Glu Asp Pro Thr Asn Val
545 550 555 560
Gly Pro Glu Val Lys Gln Gln Val Thr Met Val Ser Asp Thr Glu Ile
565 570 575
Leu Lys Val Ala Arg Thr His His Val Gln Ala Glu Ser Tyr Leu Val
580 585 590
Tyr Asn Ile Met Ser Ser Gly Glu Ile Glu Cys Ser Asn Thr Leu Glu
595 600 605
Asp Glu Leu Asp Gln Ala Leu Pro Ser Gln Ala Phe Ile Tyr Arg Pro
610 615 620
Ile Arg Gln Arg Val Tyr Ser Leu Leu Leu Glu Asp Cys Gln Asp Val
625 630 635 640
Thr Ser Thr Cys Leu Ala Val Lys Glu Trp Phe Val Tyr Pro Gly Asn
645 650 655
Pro Leu Arg His Pro Asp Leu Val Arg Pro Leu Gln Met Thr Ile Pro
660 665 670
Gly Gly Thr Pro Ser Leu Lys Ile Leu Trp Leu Asn Gln Glu Pro Glu
675 680 685
Ile Gln Val Arg Arg Leu Asp Thr Leu Leu Ala Cys Phe Asn Leu Ser
690 695 700
Ser Ser Arg Glu Glu Leu Gln Ala Val Glu Ser Pro Phe Gln Ala Leu
705 710 715 720
Cys Cys Leu Leu Ile Tyr Leu Phe Val Gln Val Asp Thr Leu Cys Leu
725 730 735
Glu Asp Leu His Ala Phe Ile Ala Gln Ala Leu Cys Leu Gln Gly Lys
740 745 750
Ser Thr Ser Gln Leu Val Asn Leu Gln Pro Asp Tyr Ile Asn Pro Arg
?55 760 765
Ala Val Gln Leu Gly Ser Leu Leu Val Arg Gly Leu Thr Thr Leu Val
770 775 780
Leu Val Asn Ser Ala Cys Gly Phe Pro Trp Lys Thr Ser Asp Phe Met
785 790 795 800
Pro Trp Asn Val Phe Asp Gly Lys Leu Phe His Gln Lys Tyr Leu Gln
805 810 815
Ser Glu Lys Gly Tyr Ala Val Glu Val Leu Leu Glu Gln Asn Gly Gly
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820 825 830
Gly Glu Asp Arg Ala Pro Ala Thr Thr Gly Arg Ala Leu Gly Ile Ala
835 840 845
Val Pro Val Arg Asp Ser Arg Gly Glu Thr Arg Asp Gln Glu Ala Asp
850 855 860
Ser Met Ser Met Thr Ser Gly Glu Gly Thr Ser Gln Pro Pro Glu Arg
865 870 875 880
Val Trp Arg Glu Lys Glu Ala His Leu Asp Ala Glu Pro Cys Gln Arg
885 890 895
Pro Pro Leu Leu Leu Gln Leu Gln Gly Asp His Ala Cys Gly Ser Gln
900 905 910
Ala Ser Leu Ala
915
(2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1914 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
- (xi) SEQUENCE
DESCRIPTION:
SEQ ID N0:13:
AGCTGTCTGC TCTCCTGGCAGGAATCGCTGAGGGAGGGAAACGCGGCTCTGAATCAGCCC60
AGAACGAGCC TTCGGGAAGCTCACCCTCCGATCTCGGTGTGATTGTTGTGATTGTTGTGA120
TTTCCTGTCT CGTTTGCCTTGACCGCCATGTGAAAGAATCTGTTCCCCAGCTAGGTGGGG180
AAAATTCACA GGTGGGCTGTCTGTAGAGAGAACTGGCTGATTAAAGGCTTCTCGTCCCGA240
TTTTGTGATA GCCAAGTGCTTGGCCTGGTCGACGGTCTTTGCTCCTTTACAAATAAAGTG300
TTCTGTTTCA GTTCGTCCCAAGTTTTCCATGAAGGGCAGTGGTTCCCTGACCTCCCAGGT360
GCCTGGGCTT CCCCAGGTTCCTGATCTGGGGCTTGGGGCCCTGTGTTTGGGGATCGTGGC420
ACTGTGTGCA CCAGCCTGGAAGCACTGGGCCAGTCTTGGCCAAGCTTTCCATCAGGGATG480
ATTTGATCTT GGTGCTACAGGTCTGTGGTACGACCATTGTTCCACACCACATGTCATTAA540
TAATGCTTCC CATGCTTCTGCTTGCAAATGACCAGCCTTCCAAACAGCCAGAGCTGTTTC600
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GAGGTGTTTC TGCAGGCAGGTGCAGGCGTGCCCTCAAATAAGCTTTGCCA ATGGAGTCTC660
AGCAAGAGCA AAACCTGGTCAGGAAAGACAAAGCCTGGGAATCCACCCCC ATGCCCTGCA720
GGTTGGCTGG CCCTGGAGCCATTTATTATAGTGCTAATCATGTTTCTAGG CAGGTGCAGA780
TGGCAAGGGC AGTGTCTTGGTGAGCTTTTTAGCACGAAGAGCCAGGTCTG TCGAAGCCTT840
TGTGAGAGCT GGAAACGCAGGTGTGCTGGGCATGCGCAGTATGGGGTTTC GGGCTCAGGG900
CTTGCCCTTT GGCATCAGACAGACCTGGCTTCGCATCCTGGATTTGCTTC TGACGTGCAC960
CCTTCCCTTT GGGTCTCGTGATGTGAAATGGAGATGTTGTCATTTGTGAG GGCTCCATGA1020
AGTTTCGTTG AAATGACAAATACTAATTTCTTCATCTGTGAAATGGAGAT AATAGTGCTG1080
ACCTCAGAAC AGCTGAGAGGACTAAATGAAATGATGTTGGATGTAGCCAT AAAGAACGAA1140
GTCAGGCACT GGTGCACGCCTGGAATCCCAGCTCTTGGGAGACCGAGACA GGTGGATTGC1200
TTGAGCTCAG GAGTTTGAGACCAGCCTGAGCAACATAGGGAGGTCCAGTC TCTACAAAAA1260
ATATGAAAAG TAGCTGGGCGTGGTGGCGCATGCCTGTAGTCCCACTACTT GGAAGGCTTC1320
GTTGGGAGGA TCACTTGAGCCCAGAAGATTGAGGCTGCAGTAAGCCGTGA TCGTGCCACT1380
GCATTCCAGC CTGGGCAACAGAGCGAGACACTGTCTCAAATAAAAP,AGAT GGGAATAGTA1440
GACACTGGGG GCTCCAGAAGGAGGGAGGGAGGGAGGAAGGGGAGGAAGGG CTGAAATGCT1500
TTCTATTGGA TACTATCTGGGCATATTACTTCCTGTGGTTCACTGTCTGG GTGACAGGAT1560
TCATAGAAGC CCAAACTTTAGCACCACGCAGCATACCCTTGTAACAAAGC CGCACACGTA1620
CGCCCTCAAG CTAAAACAAAAGTGGACCGGGAGGCCGAGGTCGGGGGATC ATGAGGTCAG1680
GAGTTTGAGA CCAGCCTGGCAGATAACGGTGAAACCCCGTCTCTACTAAA AATACCAAAA1740
AAAGTTAGCC GGACATGGTGGCAGGTGCCTGTAGTCCCAGCTACTTGGGA GGCTGGGGCA1800
GAAGAATCGC TTGAACCCAGGAGGCGGAGGTTGCAGTGAGCCGAGATTGC GCCACTGCAC1860
TCCAGCCTGT GCGACAGAGTGAGACTCCGTCTCAAAAAAAAAAAAAAAAA AAAA 1914
(2) INFORMATION EQ ID N0:14:
FOR S
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 137 amino acids
(B) TYPE: amino
acid
(C) STRANDEDNESS:
(D) TOPOLOG Y: linear
(ii) MOLECULE E: protein
TYP
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
Met Thr Ser Leu Pro Asn Ser Gln Ser Cys Phe Glu Val Phe Leu Gln
1 5 10 15
Ala Gly Ala Gly Val Pro Ser Asn Lys Leu Cys Gln Trp Ser Leu Ser
20 25 30
Lys Ser Lys Thr Trp Ser Gly Lys Thr Lys Pro Gly Asn Pro Pro Pro
35 40 45
Cys Pro Ala Gly Trp Leu Ala Leu Glu Pro Phe Ile Ile Val Leu Ile
50 55 60
Met Phe Leu Gly Arg Cys Arg Trp Gln Gly Gln Cys Leu Gly Glu Leu
65 70 75 80
Phe Ser Thr Lys Ser Gln Val Cys Arg Ser Leu Cys Glu Ser Trp Lys
85 90 95
Arg Arg Cys Ala Gly His Ala Gln Tyr Gly Val Ser Gly Ser Gly Leu
100 105 110
Ala Leu Trp His Gln Thr Asp Leu Ala Ser His Pro Gly Phe Ala Ser
115 120 125
Asp Val His Pro Ser Leu Trp Val Ser
130 135
(2) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 575 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
CCGACTCCCT TCTTTATGGC GTCGCTCCTG TGCTGTGGGC CGAAGCTGGC CGCCTGCGGC 60
ATCGTCCTCA GCGCCTGGGG AGTGATCATG TTGATAATGC TCGGAATATT TTTCAATGTC 120
CATTCCGCTG TGTTGATTGA GGACGTTCCC TTCACGGAGA AAGATTTTGA GAATGGCCCC 180
CAGAACATAT ACAACCTTTA CGAGCAAGTC AGCTACAACT GTTTCATCGC TGCAGGCCTT 240
TACCTCCTCC TCGGAGGCTT CTCTTTCTGC CAAGTTCGGC TCAATAAGCG CAAGGAATAC 300
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ATGGTGCGCT AGGGCCCCGG CGCGTTTCCC CGCTCCAGCC CCTCCTCTAT TTAAAGACTC 360
CCTGCACCGT GTCACCCAGG TCGCGTCCCA CCCTTGCCGG CGCCCTCTGT GGGACTGGGT 420
TTCCCGGGCG AGAGACTGAA TCCCTTCTCC CATCTCTGGC ATCCGGCCCC CGTGGAGAGG 480
GCTGAGGCTG GGGGGCTGTT CCGTCTCTCC ACCCTTCGCT GTGTCCCGTA TCTCAATAAA 540
GAGAATCTGC TCTCTTCAAA AAAAAAAAAA AAAAA 575
(2) INFORMATION FOR SEQ ID N0:16:
{i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 98 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:
Met Ala Ser Leu Leu Cys Cys Gly Pro Lys Leu Ala Ala Cys Gly Ile
1 5 10 15
Val Leu Ser Ala Trp Gly Val Ile Met Leu Ile Met Leu Gly Ile Phe
20 25 30
Phe Asn Val His Ser Ala Val Leu Ile Glu Asp Val Pro Phe Thr Glu
35 40 45
Lys Asp Phe Glu Asn Gly Pro Gln Asn Ile Tyr Asn Leu Tyr Glu Gln
50 55 60
Val Ser Tyr Asn Cys Phe Ile Ala Ala Gly Leu Tyr Leu Leu Leu Gly
65 70 75 80
Gly Phe Ser Phe Cys Gln Val Arg Leu Asn Lys Arg Lys Glu Tyr Met
85 90 95
Val Arg
(2) INFORMATION FOR SEQ ID N0:17:
(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"
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
GNAGCCCAGGA GTCTTCTCAA CCTCTTCC 29
(2) INFORMATION FOR SEQ ID N0:18:
(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 N0:18:
ANCAGTCGCAA GTGCATAGTA ACCCAGTA 29
(2) INFORMATION FOR SEQ ID N0:19:
(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 N0:19:
TNCTCAGCTTT TATTTGGTTC TGAGTGTT 29
(2) INFORMATION FOR SEQ ID N0:20:
(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"
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:20:
TNTGCTCAGAC CAGTCATCTG CAGAATCA 29
(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 NO:21:
TNCAGCACTGT CTTAGGCTAA ATTTCCCA 29
(2) INFORMATION FOR SEQ ID N0:22:
(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 N0:22:
GNATTCGGCGT CTGAACTCGT GGATATTA . 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"
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:
ANATGCCCAGA TAGTATCCAA TAGAAAGC 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:
CNACAGCACAG GAGCGACGCC ATAAAGAA 29
(2) INFORMATION FOR SEQ ID N0:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 543 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:
Met Val Met Tyr Ala Arg Lys Gln Gln Arg Leu Ser Asp Gly Cys His
1 5 10 15
Asp Arg Arg Giy Asp Ser Gln Pro Tyr Gln Ala Leu Lys Tyr Ser Ser
20 25 30
Lys Ser His Pro Ser Ser Gly Asp His Arg His Glu Lys Met Arg Asp
35 40 45
Ala Gly Asp Pro Ser Pro Pro Asn Lys Met Leu Arg Arg Ser Asp Ser
50 55 60
Pro Glu Asn Lys Tyr Ser Asp Ser Thr Gly His Ser Lys Ala Lys Asn
65 70 75 80
Val His Thr His Arg Val Arg Glu Arg Asp Gly Gly Thr Ser Tyr Ser
85 90 95
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Pro Gln Glu Asn Ser His Asn His Ser Ala Leu His Ser Ser Asn Ser
100 105 110
His Ser Ser Asn Pro Ser Asn Asn Pro Ser Lys Thr Ser Asp Ala Pro
115 120 125
Tyr Asp Ser Ala Asp Asp Trp Ser Glu His Ile Ser Ser Ser Gly Lys
130 135 140
Lys Tyr Tyr Tyr Asn Cys Arg Thr Glu Val Ser Gln Trp Glu Lys Pro
145 150 155 160
Lys Glu Trp Leu Glu Arg Glu Gln Arg Gln Lys Glu Ala Asn Lys Met
165 170 175
Ala Val Asn Ser Phe Pro Lys Asp Arg Asp Tyr Arg Arg Glu Val Met
180 185 190
Gln Ala Thr Ala Thr Ser Gly Phe Ala Ser Gly Lys Ser Thr Ser Gly
195 200 205
Asp Lys Pro Val Ser His Ser Cys Thr Thr Pro Ser Thr Ser Ser Ala
210 215 220
Ser Gly Leu Asn Pro Thr Ser Ala Pro Pro Thr Ser Ala Ser Ala Val
225 230 235 240
Pro Val Ser Pro Val Pro Gln Ser Pro Ile Pro Pro Leu Leu Gln Asp
245 250 255
Pro Asn Leu Leu Arg Gln Leu Leu Pro Ala Leu Gln Ala Thr Leu Gln
260 265 270
Leu Asn Asn Ser Asn Val Asp Ile Ser Lys Ile Asn Glu Val Leu Thr
275 280 285
Ala Ala Val Thr Gln Ala Ser Leu Gln Ser Ile Ile His Lys Phe Leu
290 295 300
Thr Ala Gly Pro Ser Ala Phe Asn Ile Thr Ser Leu Ile Ser Gln Ala
305 310 315 320
Ala Gln Leu Ser Thr Gln Ala Gln Pro Ser Asn Gln Ser Pro Met Ser
325 330 335
Leu Thr Ser Asp Ala Ser Ser Pro Arg Ser Tyr Val Ser Pro Arg Ile
340 345 350
Ser Thr Pro Gln Thr Asn Thr Val Pro Ile Lys Pro Leu Ile Ser Thr
355 360 365
Pro Pro Val Ser Ser Gln Pro Lys Val Ser Thr Pro Val Val Lys Gln
370 375 380
Gly Pro Val Ser Gln Ser Ala Thr Gln Gln Pro Val Thr Ala Asp Lys
g5
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385 390 395 400
Gln Gln Gly His Glu Pro Val Ser Pro Arg Ser Leu Gln Arg Ser Ser
405 410 415
Gln Arg Ser Pro Ser Pro Gly Pro Asn His Thr Ser Asn Ser Ser Asn
420 425 430
Ala Ser Asn Ala Thr Val Val Pro Gln Asn Ser Ser Ala Arg Ser Thr
435 440 445
Cys Ser Leu Thr Pro Ala Leu Ala Ala His Phe Ser Glu Asn Leu Ile
450 455 460
Lys His Val Gln Gly Trp Pro Ala Asp His Ala Glu Lys Gln Ala Ser
465 470 475 480
Arg Leu Arg Glu Glu Ala His Asn Met Gly Thr Ile His Met Ser Glu
485 490 495
Ile Cys Thr Glu Leu Lys Asn Leu Arg Ser Leu Val Arg Val Cys Glu
500 505 510
Ile Gln Ala Thr Leu Arg Glu Gln Arg Ile Leu Phe Leu Arg Gln Gln
515 520 525
Ile Lys Glu Leu Glu Lys Leu Lys Asn Gln Asn Ser Phe Met Val
530 535 540
86
