Note: Descriptions are shown in the official language in which they were submitted.
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SECRETED PROTEINS AND POLYNUCLEOTIDES ENCODING THEM
This application is a continuation-in-part of application Ser. No. 60/XXX,)OOC
(converted to a provisional application from non-provisional application Ser.
No.
08/823,330), filed March 28,1997, 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
fiechniques
clone novel polynucleotides "directly" in the sense that they rely on
information directly
related to the discovered protein (i.e., partial DNA/amino acid sequence of
the protein
2 0 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.
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SUMMARY OF THE INVENTION
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:1;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:1 from nucleotide 170 to nucleotide 322;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:1 from nucleotide 218 to nucleotide 322;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:1 from nucleotide 1814 to nucleotide 2355;
(e) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone b1209_10 deposited under accession
number ATCC 98379;
(f) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone b1209_10 deposited under accession number ATCC 98379;
(g) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone b1209_10 deposited under accession number
ATCC 98379;
2 0 (h) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone b1209_10 deposited under accession number ATCC 98379;
(i) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:2;
(j) a polynucleotide encoding a protein comprising a fragment of the
2 5 amino acid sequence of SEQ ID N0:2 having biological activity, the
fragment
comprising the amino acid sequence from amino acid 20 to amino acid 29 of SEQ
ID N0:2;
(k) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(h) above;
3 0 (1) a polynucleotide which encodes a species homologue of the protein
of (i) or (j) above ; and
(m) a polynucleotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(j).
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Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
NO:1 from nucleotide 170 to nucleotide 322; the nucleotide sequence of SEQ ID
NO:1 from
nucleotide 218 to nucleotide 322; the nucleotide sequence of SEQ ID N0:1 from
nucleotide 1814 to nucleotide 2355; the nucleotide sequence of the full-length
protein
coding sequence of clone b1209_10 deposited under accession number ATCC 98379;
or the
' nucleotide sequence of a mature protein coding sequence of clone b1209_10
deposited
under accession number ATCC 98379. In other preferred embodiments, the
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
of clone b1209_10 deposited under accession number ATCC 98379.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID NO: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 20 to amino acid 29 of SEQ ID N0:2;
and
(c) the amino acid sequence encoded by the cDNA insert of clone
b1209_10 deposited under accession number ATCC 98379;
2 0 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:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
2 5 N0:3;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3 from nucleotide 102 to nucleotide 1295;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3 from nucleotide 162 to nucleotide 1295;
3 0 (d) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3 from nucleotide 804 to nucleotide 1184;
(e) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone cr1162_25 deposited under accession
number ATCC 98379;
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(f) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone cr1162_25 deposited under accession number ATCC 98379;
(g) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone cr1162_25 deposited under accession number
ATCC 98379;
(h) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone cr1162_25 deposited under accession number ATCC 98379;
(i) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:4;
(j) 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 194 to amino acid 203 of
SEQ ID N0:4;
(k) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(h} above;
(1) a polynucleotide which encodes a species homologue of the protein
of (i) or (j) above ; and
(m) a polynucleotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(j).
2 0 Preferably, such polynucleotide comprises the nucleotide sequence of SEQ
ID
N0:3 from nucleotide 102 to nucleotide 1295; the nucleotide sequence of SEQ ID
N0:3
from nucleotide 162 to nucleotide 1295; the nucleotide sequence of SEQ ID N0:3
from
nucleotide 804 to nucleotide 1184; the nucleotide sequence of the full-length
protein
coding sequence of clone cr1162_25 deposited under accession number ATCC
98379; or
2 5 the nucleotide sequence of a mature protein coding sequence of clone
cr1162 25 deposited
under accession number ATCC 98379. In other preferred embodiments, the
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
of clone cr1162 25 deposited under accession number ATCC 98379. In yet other
preferred embodiments, the present invention provides a polynucleotide
encoding a
3 0 protein comprising the amino acid sequence of SEQ ID N0:4 from amino acid
236 to
amino acid 361.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:3.
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In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
. (a) the amino acid sequence of SEQ ID N0:4;
(b) the amino acid sequence of SEQ ID N0:4 from amino acid 236 to
~ amino acid 361;
(c) fragments of the amino acid sequence of SEQ ID N0:4 comprising
the amino acid sequence from amino acid 194 to amino acid 203 of SEQ ID N0:4;
and
(d) the amino acid sequence encoded by the cDNA insert of clone
cr1162_25 deposited under accession number ATCC 98379;
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 236 to amino acid 361.
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
N0:5;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
2 0 N0:5 from nucleotide 351 to nucleotide 842;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:5 from nucleotide 687 to nucleotide 842;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:5 from nucleotide 1 to nucleotide 689;
2 5 (e) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone dh40_3 deposited under accession
number ATCC 98379;
(f) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone dh40_3 deposited under accession number ATCC 98379;
3 0 (g) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone dh40_3 deposited under accession number
ATCC 98379;
(h) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone dh40 3 deposited under accession number ATCC 98379;
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(i) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:6;
(j) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:6 having biological activity, the fragment
comprising the amino acid sequence from amino acid 77 to amino acid 86 of SEQ
ID N0:6;
(k) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(h) above;
(1) a polynucleotide which encodes a species homologue of the protein
of (i) or (j) above ; and
(m) a polynucleotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(j).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:5 from nucleotide 351 to nucleotide 842; the nucleotide sequence of SEQ ID
N0:5 from
nucleotide 687 to nucleotide 842; the nucleotide sequence of SEQ ID N0:5 from
nucleotide 1 to nucleotide 689; the nucleotide sequence of the full-length
protein coding
sequence of clone dh40 3 deposited under accession number ATCC 98379; or the
nucleotide sequence of a mature protein coding sequence of clone dh40 3
deposited under
accession number ATCC 98379. In other preferred embodiments, the
polynucleotide
2 0 encodes the full-length or a mature protein encoded by the cDNA insert of
clone dh40 3
deposited under accession number ATCC 98379. 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 113.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
2 5 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;
3 0 (b) the amino acid sequence of SEQ ID N0:6 from amino acid 1 to
amino acid 113;
(c) fragments of the amino acid sequence of SEQ ID N0:6 comprising
the amino acid sequence from amino acid 77 to amino acid 86 of SEQ ID N0:6;
and
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(d) the amino acid sequence encoded by the cDNA insert of clone
dh40_3 deposited under accession number ATCC 98379; _
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 113.
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 2205 to nucleotide 2882;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:7 from nucleotide 2262 to nucleotide 2882;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:7 from nucleotide 2494 to nucleotide 3120;
(e) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone di39_9 deposited under accession
number
ATCC 98379;
(f) a polynucleotide encoding the full-length protein encoded by the
2 0 cDNA insert of clone di39_9 deposited under accession number ATCC 98379;
(g) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone di39_9 deposited under accession number ATCC
98379;
(h) a polynucleotide encoding a mature protein encoded by the cDNA
2 5 insert of clone di39_9 deposited under accession number ATCC 98379;
(i) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:8;
(j) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:8 having biological activity, the fragment
. 3 0 comprising the amino acid sequence from amino acid 108 to amino acid 117
of
SEQ ID N0:8;
(k) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(h) above;
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(1) a polynucleotide which encodes a species homologue of the protein
of (i) or (j) above ; and
(m) a polynucleotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a~(j).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:7 from nucleotide 2205 to nucleotide 2882; the nucleotide sequence of SEQ
ID N0:7
from nucleotide 2262 to nucleotide 2882; the nucleotide sequence of SEQ ID
N0:7 from
nucleotide 2494 to nucleotide 3120; the nucleotide sequence of the full-length
protein
coding sequence of clone di39_9 deposited under accession number ATCC 98379;
or the
nucleotide sequence of a mature protein coding sequence of clone di39_9
deposited under
accession number ATCC 98379. In other preferred embodiments, the
polynucleotide
encodes the full-length or a mature protein encoded by the cDNA insert of
clone di39 9
deposited under accession number ATCC 98379.
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;
2 0 (b) fragments of the amino acid sequence of SEQ ID N0:8 comprising
the amino acid sequence from amino acid 108 to amino acid 117 of SEQ ID N0:8;
and
(c) the amino acid sequence encoded by the cDNA insert of clone
di39 9 deposited under accession number ATCC 98379;
2 5 the protein being substantially free from other mammalian proteins.
Preferably such
protein comprises the amino acid sequence of SEQ ID N0:8.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
3 0 N0:9;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:9 from nucleotide 40 to nucleotide 1503;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:9 from nucleotide 863 to nucleotide 1377;
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(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone dt674 2 deposited under accession
number ATCC 98379;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone dt674_2 deposited under accession number ATCC 98379;
(f) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone dt674_2 deposited under accession number
ATCC 98379;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone dt674 2 deposited under accession number ATCC 98379;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:10;
(i) 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 238 to amino acid 247 of
SEQ ID N0:10;
(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 capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleoHde comprises the nucleotide sequence of SEQ ID
N0:9 from nucleotide 40 to nucleotide 1503; the nucleotide sequence of SEQ ID
N0:9
2 5 from nucleotide 863 to nucleotide 1377; the nucleotide sequence of the
full-length protein
coding sequence of clone dt674_2 deposited under accession number ATCC 98379;
or the
nucleotide sequence of a mature protein coding sequence of clone dt674_2
deposited
under accession number ATCC 98379. In other preferred embodiments, the
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
3 0 of clone dt674_2 deposited under accession number ATCC 98379. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID NO:10 from amino acid 277 to
amino acid
446.
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Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:9.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:10;
(b) the amino acid sequence of SEQ ID N0:10 from amino acid 277 to
amino acid 446;
(c) fragments of the amino acid sequence of SEQ ID N0:10 comprising
the amino acid sequence from amino acid 238 to amino acid 247 of SEQ ID NO:10;
and
(d) the amino acid sequence encoded by the cDNA insert of clone
dt674 2 deposited under accession number ATCC 98379;
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 277 to amino acid 446.
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 0 N0:11;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:11 from nucleotide 85 to nucleotide 450;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:11 from nucleotide 217 to nucleotide 450;
2 5 (d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone eh61 1 deposited under accession
number
ATCC 98379;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone eh61 1 deposited under accession number ATCC 98379;
3 0 (f) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone eh61 1 deposited under accession number ATCC
98379;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone eh61 1 deposited under accession number ATCC 98379;
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(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:12;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:12 having biological activity, the fragment
comprising the amino acid sequence from amino acid 55 to amino acid 64 of SEQ
ID N0:12;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a~(g) above;
(k) a polynucleotide which encodes a species homologue of the protein
of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotfde comprises the nucleotide sequence of SEQ ID
N0:11 from nucleotide 85 to nucleotide 450; the nucleotide sequence of SEQ ID
N0:11
from nucleotide 217 to nucleotide 450; the nucleotide sequence of the full-
length protein
coding sequence of clone eh61 1 deposited under accession number ATCC 98379;
or the
nucleotide sequence of a mature protein coding sequence of clone eh61 1
deposited under
accession number ATCC 98379. In other preferred embodiments, the
polynucleotide
encodes the full-length or a mature protein encoded by the cDNA insert of
clone eh61 1
2 0 deposited under accession number ATCC 98379. 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 9 to amino acid 94.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:11 or SEQ ID N0:13.
2 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 N0:12;
(b) the amino acid sequence of SEQ ID N0:12 from amino acid 9 to
3 0 amino acid 94;
(c) fragments of the amino acid sequence of SEQ ID N0:12 comprising
the amino acid sequence from amino acid 55 to amino acid 64 of SEQ ID N0:12;
and
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(d) the amino acid sequence encoded by the cDNA insert of clone
eh61_1 deposited under accession number ATCC 98379; _
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 9 to amino acid 94.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:14;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:14 from nucleotide 900 to nucleotide 1073;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:14 from nucleotide 544 to nucleotide 1022;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone fg265_1 deposited under accession
number ATCC 98379;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone fg265_1 deposited under accession number ATCC 98379;
(f) a polynucleotide comprising the nucleotide sequence of a mature
2 0 protein coding sequence of clone fg265_1 deposited under accession number
ATCC 98379;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone fg265_1 deposited under accession number ATCC 98379;
(h) a polynucleotide encoding a protein comprising the amino acid
2 5 sequence of SEQ ID N0:15;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:15 having biological activity, the fragment
comprising the amino acid sequence from amino acid 24 to amino acid 33 of SEQ
ID N0:15;
3 0 (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
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(1) a polynucleotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:14 from nucleotide 900 to nucleotide 1073; the nucleotide sequence of SEQ
ID N0:14
from nucleotide 544 to nucleotide 1022; the nucleotide sequence of the full-
length protein
coding sequence of clone fg265_1 deposited under accession number ATCC 98379;
or the
nucleotide sequence of a mature protein coding sequence of clone fg265_1
deposited
under accession number ATCC 98379. In other preferred embodiments, the
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
of clone fg265_1 deposited under accession number ATCC 98379. In yet other
vreferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:15 from amino acid 1 to amino
acid
41.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:14.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:15;
2 0 (b) the amino acid sequence of SEQ ID N0:15 from amino acid 1 to
amino acid 41;
(c) fragments of the amino acid sequence of SEQ ID N0:15 comprising
the amino acid sequence from amino acid 24 to amino acid 33 of SEQ ID N0:15;
and
2 5 (d) the amino acid sequence encoded by the cDNA insert of clone
fg265_1 deposited under accession number ATCC 98379;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:15 or the amino acid
sequence
of SEQ ID N0:15 from amino acid 1 to amino acid 41.
3 0 In one embodiment, the present invention provides a composition comprising
an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:16;
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(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:16 from nucleotide 119 to nucleotide 2440;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:16 from nucleotide 200 to nucleotide 2440;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:16 from nucleotide 460 to nucleotide 1153;
(e) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone fp273_10 deposited under accession
number ATCC 98379;
(f) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone fp273_10 deposited under accession number ATCC 98379;
(g) a polynucleoHde comprising the nucleotide sequence of a mature
protein coding sequence of clone fp273_10 deposited under accession number
ATCC 98379;
(h) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone fp273_10 deposited under accession number ATCC 98379;
(i) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:17;
(j) a polynucleotide encoding a protein comprising a fragment of the
2 0 amino acid sequence of SEQ ID N0:17 having biological activity, the
fragment
comprising the amino acid sequence from amino acid 382 to amino acid 391 of
SEQ ID N0:17;
(k} a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(h) above;
2 5 (1) a polynucleotide which encodes a species homologue of the protein
of (i) or (j) above ; and
(m) a polynucleotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(j).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
3 0 N0:16 from nucleotide 119 to nucleotide 2440; the nucleotide sequence of
SEQ ID N0:16
from nucleotide 200 to nucleotide 2440; the nucleotide sequence of SEQ ID
N0:16 from
nucleotide 460 to nucleotide 1153; the nucleotide sequence of the full-length
protein
coding sequence of clone fp273_10 deposited under accession number ATCC 98379;
or the
nucleotide sequence of a mature protein coding sequence of clone fp273_10
deposited
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under accession number ATCC 98379. In other preferred embodiments, the
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
of clone fp273_10 deposited under accession number ATCC 98379. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:17 from amino acid 115 to
amino acid
345.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:16.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:17;
(b) the amino acid sequence of SEQ ID N0:17 from amino acid 115 to
amino acid 345;
(c) fragments of the amino acid sequence of SEQ ID N0:17 comprising
the amino acid sequence from amino acid 382 to amino acid 391 of SEQ ID N0:17;
and
(d) the amino acid sequence encoded by the cDNA insert of clone
fp273_10 deposited under accession number ATCC 98379;
2 0 the protein being substantially free from other mammalian proteins.
Preferably such
protein comprises the amino acid sequence of SEQ ID N0:17 or the amino acid
sequence
of SEQ ID N0:17 from amino acid 115 to amino acid 345.
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
N0:18;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:18 from nucleotide 1187 to nucleotide 1804;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
3 0 N0:18 from nucleotide 674 to nucleotide 1014;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence , of clone fy243 8 deposited under accession
number ATCC 98379;
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(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone fy243_8 deposited under accession number ATCC 98379;
(f) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone fy243_8 deposited under accession number
ATCC 98379;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone fy243_8 deposited under accession number ATCC 98379;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:19;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:19 having biological activity, the fragment
comprising the amino acid sequence from amino acid 98 to amino acid 107 of SEQ
ID N0:19;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g} above;
(k) a polynucleotide which encodes a species homologue of the protein
of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(i).
2 0 Preferably, such polynucleotide comprises the nucleotide sequence of SEQ
ID
N0:18 from nucleotide 1187 to nucleotide 1804; the nucleotide sequence of SEQ
ID N0:18
from nucleotide 674 to nucleotide 1014; the nucleotide sequence of the full-
length protein
coding sequence of clone fy243_8 deposited under accession number ATCC 98379;
or the
nucleotide sequence of a mature protein coding sequence of clone fy243_8
deposited
2 5 under accession number ATCC 98379. In other preferred embodiments, the
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
of clone fy243_8 deposited under accession number ATCC 98379. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:19 from amino acid 21 to amino
acid
3 0 69.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:18.
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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 N0:19;
(b) the amino acid sequence of SEQ ID N0:19 from amino acid 21 to
amino acid 69;
(c) fragments of the amino acid sequence of SEQ ID N0:19 comprising
the amino acid sequence from amino acid 98 to amino acid 107 of SEQ ID N0:19;
and
(d) the amino acid sequence encoded by the cDNA insert of clone
fy243_8 deposited under accession number ATCC 98379;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:19 or the amino acid
sequence
of SEQ ID N0:19 from amino acid 21 to amino acid 69.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:20;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
2 0 N0:20 from nucleotide 99 to nucleotide 536;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:20 from nucleotide 1 to nucleotide 370;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone ga205 4 deposited under accession
2 5 number ATCC 98379;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone ga205_4 deposited under accession number ATCC 98379;
(f) a polynucleotide comprising the nucleotide sequence of a mature
protein coding sequence of clone ga205 4 deposited under accession number
3 0 ATCC 98379;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone ga205_4 deposited under accession number ATCC 98379;
(h) a polynucleotide encoding'a protein comprising the amino acid
sequence of SEQ ID N0:21;
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(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:21 having biological activity, the fragment
comprising the amino acid sequence from amino acid 68 to amino acid 77 of SEQ
ID N0:21;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotf de which encodes a species homologue of the protein
of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:20 from nucleotide 99 to nucleotide 536; the nucleotide sequence of SEQ ID
N0:20
from nucleotide 1 to nucleotide 370; the nucleotide sequence of the full-
length protein
coding sequence of clone ga205_4 deposited under accession number ATCC 98379;
or the
nucleotide sequence of a mature protein coding sequence of clone ga205 4
deposited
under accession number ATCC 98379. In other preferred embodiments, the
polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert
of clone ga205 4 deposited under accession number ATCC 98379. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
2 0 comprising the amino acid sequence of SEQ ID N0:21 from amino acid 1 to
amino acid
90.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:20.
In other embodiments, the present invention provides a composition comprising
2 5 a protein, wherein said protein comprises an amino acid sequence selected
from the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:21;
(b) the amino acid sequence of SEQ ID N0:21 from amino acid 1 to
amino acid 90;
3 0 (c) fragments of the amino acid sequence of SEQ ID N0:21 comprising
the amino acid sequence from amino acid 68 to amino acid 77 of SEQ ID N0:21;
and
(d) the amino acid sequence encoded by the cDNA insert of clone
ga205 4 deposited under accession number ATCC 98379;
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the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:21 or the_amino acid
sequence
of SEQ ID N0:21 from amino acid 1 to amino acid 90.
In certain preferred embodiments, the polynucleotide is operably linked to an
expression control sequence. The invention also provides a host cell,
including bacterial,
yeast, insect and mammalian cells, transformed with such polynucleotide
compositions.
Also provided by the present invention are organisms that have enhanced,
reduced, or
modified expression of the genes) corresponding to the polynucleotide
sequences
disclosed herein.
Processes are also provided for producing a protein, which comprise:
(a) growing a culture of the host cell transformed with such
polynucleotide compositions in a suitable culture medium; and
(b) purifying the protein from the culture.
The protein produced according to such methods is also provided by the present
invention. Preferred embodiments include those in which the protein produced
by such
process is a mature form of the protein.
Protein compositions of the present invention may further comprise a
pharmaceutically acceptable carrier. Compositions comprising an antibody which
specifically reacts with such protein are also provided by the present
invention.
2 0 Methods are also provided for preventing, treating or ameliorating a
medical
condition which comprises administering to a mammalian subject a
therapeutically
effective amount of a composition comprising a protein of the present
invention and a
pharmaceutically acceptable earner.
2 5 BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1A and 1B are schematic representations of the pED6 and pNOTs vectors,
respectively, used for deposit of clones disclosed herein.
DETAILED DESCRIPTION
3 0 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
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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.
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
which are transported across the membrane of the endoplasmic reticulum.
Clone "b1209 10"
A polynucleotide of the present invention has been identified as clone
"b1209_10".
b1209_10 was isolated from a human adult testes cDNA library using methods
which are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637),
or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. b1209_10 is a full-
length
2 0 clone, including the entire coding sequence of a secreted protein (also
referred to herein
as "b1209_10 protein').
The nucleotide sequence of b1209_10 as presently determined is reported in SEQ
ID NO:1. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the b1209_10 protein corresponding to the
foregoing
2 5 nucleotide sequence is reported in SEQ ID N0:2. Amino acids 4 to 16 are a
predicted
leader/signal sequence, with the predicted mature amino acid sequence
beginning at
amino acid 17, or are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
b1209_10 should be approximately 2400 bp.
3 0 The nucleotide sequence disclosed herein for b1209_10 was searched against
the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. b1209_10 demonstrated at least some similarity with
sequences
identified as AA522436 (ng30g05.s1 NCI CGAP_Co3 Homo sapiens cDNA clone IMAGE
936344), L06147 {Human (clone SY11) golgin-95 mRNA, complete cds), N29620
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(yw67d06.s1 Homo sapiens cDNA clone 2572913'), N41622 (yw67d06.r1 Homo Sapiens
cDNA clone 2572915'), N80172 (za65g07.s1 Homo sapiens cDNA clone 297468 3'),
and
U35022 (Rattus norvegicus cis-Golgi matrix protein GM130 mRNA, complete cds).
The
predicted amino acid sequence disclosed herein for b1209_10 was searched
against the
GenPept and GeneSeq amino acid sequence databases using the BLASTX search
protocol.
The predicted b1209_10 protein demonstrated at least some similarity to
sequences
identified as M34651 {immediate-early protein [Said herpesvirus]). Based upon
sequence
similarity, b1209_10 proteins and each similar protein or peptide may share at
least some
activity. [The TopPredII computer program predicts N potential transmembrane
domains
within the b1209_10 protein sequence, one around amino acid X and another
around
amino acid Y of SEQ ID N0:2.] (The nucleotide/amino acid sequence of b1209_10
indicates that it may contain an Alu repetitive element.]
Clone "cr1I62 25"
A polynucleotide of the present invention has been identified as clone "cr1162
25".
Secreted cDNA clones were first isolated from a human adult testes cDNA
library using
methods which are selective for cDNAs encoding secreted proteins (see U.S.
Pat. No.
5,53b,637), or were identified as encoding a secreted or transmembrane protein
on the
basis of computer analysis of the amino acid sequence of the encoded protein.
These
2 0 cDNA clones were then used to isolate cr1162_25, a full-length human cDNA
clone which
includes the entire coding sequence of a secreted protein (also referred to
herein as
"cr1162_25 protein'), from a human fetal brain cDNA lbrary.
The nucleotide sequence of cr1162_25 as presently determined is reported in
SEQ
ID N0:3. What applicants presently believe to be the proper reading frame and
the
2 5 predicted amino acid sequence of the cr1162_25 protein corresponding to
the foregoing
nucleotide sequence is reported in SEQ ID N0:4. Amino acids 8 to 20 are a
predicted
leader/signal sequence, with the predicted mature amino acid sequence
beginning at
amino acid 21, or are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
3 0 cr1162 25 should be approximately 3700 bp.
The nucleotide sequence disclosed herein for cr1162_25 was searched against
the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. cr1162_25 demonstrated at least some similarity with
sequences
identified as H14720 (ym24b05.r1 Homo sapiens cDNA clone 48883 5'), H15268
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(ym30d11.r1 Homo sapiens cDNA clone 49904 5'), and N45514 (yy59g07.r1 Homo
sapiens
cDNA clone 277884 5'). The predicted amino acid sequence disclosed herein for
cr1162 25
was searched against the GenPept, GeneSeq, and SwissProt amino acid sequence
databases using the BLASTX search protocol. The predicted cr1162 25 protein
demonstrated at least some similarity to sequences identified as D12612
(poliovirus
receptor gene [Cercopithecus aethiops]), D26156 (hSNF2b; transcriptional
activator [Homo
sapiens], L12589 (B-lymphocyte activation antigen 7 [Mus musculus]), P51532
(POSSIBLE
GLOBAL TRANSCRIPTION ACTIVATOR SNF2L3 ( OR SNF2-BETA OR BRG-1) (Homo
sapiens]), R07130 (H20B receptor), U29175 (transcriptional activator (BRG1~))
[Homo
sapiens]), X57516 (poliovirus receptor alpha [Homo sapiens]), X60958 (B
lymphocyte
activation antigen [Mus musculus]), X64116 (poliovirus receptor alpha [Homo
sapiens]),
and X68274 (TAG-1/axonin-1 [Homo sapiens]). Based upon sequence similarity,
cr1162 25 proteins and each similar protein or peptide may share at least some
activity.
The TopPredII computer program predicts an additional potential transmembrane
domain at the carboxy terminus of the cr1162_25 protein sequence, centered
around
amino acid 342 of SEQ ID N0:4.
Clone "dh40 3"
A polynucleotide of the present invention has been identified as clone "dh40
3".
2 0 dh40_3 was isolated from a human fetal brain cDNA library using methods
which are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637),
or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. dh40 3 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"dh40_3 protein')
The nucleotide sequence of dh40_3 as presently determined is reported in SEQ
ID
N0:5. What applicants presently believe to be the proper reading frame and the
predicted
amino acid sequence of the dh40 3 protein corresponding to the foregoing
nucleotide
sequence is reported in SEQ ID N0:6. Amino acids 100 to I12 are a predicted
3 0 leader/signal sequence, with the predicted mature amino acid sequence
beginning at
amino acid 113, or are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
dh40 3 should be approximately 3000 bp.
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The nucleotide sequence disclosed herein for dh40_3 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. dh40_3 demonstrated at least some similarity with
sequences
identified as AG005063 (Homo sapiens genomic DNA, 21q region, clone
T1957SpN11),
267586 (H.sapiens DNA segment containing (CA) repeat), and 274023 (Human DNA
sequence from cosmid LUCA3 on chromosome 3p21.3. contains ESTs). Based upon
sequence similarity, dh40_3 proteins and each similar protein or peptide may
share at
least some activity. The TopPredII computer program predicts an additional
potential
transmembrane domain within the dh40_3 protein sequence at the extreme carboxy
terminus of SEQ ID N0:6.
Clone "di39 9"
A polynucleotide of the present invention has been identified as clone "di39
9".
di39_9 was isolated from a human adult testes cDNA library using methods which
are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637),
or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. di39 9 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"di39 9 protein").
2 0 The nucleotide sequence of di39_9 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 di39 9 protein corresponding to the foregoing
nucleotide
sequence is reported in SEQ ID N0:8. Amino acids 7 to 19 are a predicted
leader/signal
sequence, with the predicted mature amino acid sequence beginning at amino
acid 20, or
2 5 are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
di39 9 should be approximately 3000 bp.
The nucleotide sequence disclosed herein for di39_9 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
3 0 FASTA search protocols. di39 9 demonstrated at least some similarity with
sequences
identified as AA249116 (hfe0042.seq.F Human fetal heart, Lambda ZAP Express
Homo
Sapiens cDNA 5'), AA598667 (ae40a05.s1 Gessler Wilms tumor Homo Sapiens cDNA
clone
898256 3'), N53166 (yv56e11.s1 Homo sapiens cDNA clone 246764 3'}, N80292
(za96h08.s1
Homo sapiens cDNA clone 300447 3'), T86182 QTV1 coding sequence), U24169
(Human
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JT'V-1 (JTV-1) mRNA, complete cds), U38964 (Human PMS2 related (hPMSR2) gene,
complete cds), and W24630 (zb62g08.r1 Soares fetal lung NbHLI9W Homo sapiens
cDNA
clone 308222 5'). The predicted amino acid sequence disclosed herein for
di39_9 was
searched against the GenPept and GeneSeq amino acid sequence databases using
the
BLASTX search protocol. The predicted di39_9 protein demonstrated at least
some
similarity to sequences identified as U24169 (JTV-1 (Homo sapiens]), U38964
(hPMSR2
[Homo sapiens]), and W25776 QTVl protein}. The positioning of the regions of
similarity
to hPMSR2 and JTV-1 relative to each other in the di39_9 sequence is quite
similar to that
of the JTV-1 and PMS2 sequences in the human genome. Based upon sequence
similarity,
di39_9 proteins and each similar protein or peptide may share at least some
activity. The
TopPredII computer program predicts two additional potential transmembrane
domains
within the di39_9 protein sequence, one centered around amino acid 160 and
another
around amino acid 200 of SEQ ID N0:8.
Clone "dt674 2"
A polynucleotide of the present invention has been identified as clone "dt674
2".
dt674_2 was isolated from a human adult brain cDNA library using methods which
are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5;536,637),
or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
2 0 analysis of the amino acid sequence of the encoded protein. dt674_2 is a
full-length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"dt674 2 protein').
The nucleotide sequence of dt674 2 as presently determined is reported in SEQ
ID
N0:9. What applicants presently believe to be the proper reading frame and the
predicted
2 5 amino acid sequence of the dt674 2 protein corresponding to the foregoing
nucleotide
sequence is reported in SEQ ID NO:10.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
dt674 2 should be approximately 3500 bp.
The nucleotide sequence disclosed herein for dt674_2 was searched against the
3 0 GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. dt674 2 demonstrated at least some similarity with
sequences
identified as T06736 (EST04625 Homo sapiens cDNA clone HFBDX78). The predicted
amino acid sequence disclosed herein for dt674_2 was searched against the
GenPept and
GeneSeq amino acid sequence databases using the BLASTX search protocol. The
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predicted dt674 2 protein demonstrated at least some similarity to sequences
identified
as 272807 (ORF YGR023w [Saccharomyces cerevisiae]). Based upon_sequence
similarity,
dt674_2 proteins and each similar protein or peptide may share at least some
activity. The
nucleotide sequence of dt674_2 indicates that it may contain at least one copy
of one or
more repetitive elements.
Clone "eh61 1"
A polynucleotide of the present invention has been identified as clone "eh61
1".
eh61 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. eh61 1 is a full-
length clone,
including the entire coding sequence of a secreted protein {also referred to
herein as
"eh61 1 protein").
The nucleotide sequence of the 5' portion of eh61_1 as presently determined is
reported in SEQ ID NO:11. What applicants presently believe is the proper
reading frame
for the coding region is indicated in SEQ ID N0:12. The predicted amino acid
sequence
of the eh61 1 protein corresponding to the foregoing nucleotide sequence is
reported in
2 0 SEQ ID N0:12. Amino acids 32 to 44 are a predicted leader/signal sequence,
with the
predicted mature amino acid sequence beginning at amino acid 45, or are a
transmembrane domain. Additional nucleotide sequence from the 3' portion of
eh61 1,
including the polyA tail, is reported in SEQ ID N0:13.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
2 5 eh61 1 should be approximately 2200 bp.
The nucleotide sequence disclosed herein for eh61 1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. eh61 1 demonstrated at least some similarity with
sequences
identified as AA114131 (zn75g05.s1 Stratagene NT2 neuronal precursor 937230
Homo
3 0 sapiens cDNA clone 564056 3' similar to contains Alu repetitive
element;contains element
TAR1 repetitive element), H53674 (yu38e03.r1 Homo sapiens cDNA clone 236092
5'),
L24093 (Gorilla gorilla ADP-ribosyltransferase (NAD+) pseudogene, repeat
region),
N38129 (19356 Arabidopsis thaliana cDNA clone 219I8T7), T04321 (368
Arabidopsis
thaliana cDNA clone), U45981 (Schizosaccharomyces pombe Ste20-related protein
kinase
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(shk2) gene, complete cds), and X97774 (A.thaliana mRNA for light represssible
receptor
protein kinase). The predicted amino acid sequence disclosed herein for eh61 1
was
searched against the GenPept and GeneSeq amino acid sequence databases using
the
BLASTX search protocol. The predicted eh61 1 protein demonstrated at least
some
similarity to sequences identified as D10152 (protein tyrosine-serine-
threonine kinase
[Arabidopsis thaliana]), L24521 (transformation-related protein [Homo
sapiens]), and
L76191 (interleukin-1 receptor-associated kinase [Homo Sapiens]). Based upon
sequence
similarity, eh61 1 proteins and each similar protein or peptide may share at
least some
activity. The nucleotide sequence of eh61 1 indicates that it may contain an
Alu repetitive
element.
Clone "fg2651"
A polynucleotide of the present invention has been identified as clone
"fg265_1".
fg265_1 was isolated from a human adult brain cDNA library using methods which
are
2 5 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. fg265_1 is a full-
length clone,
including the entire coding sequence of a secreted protein {also referred to
herein as
"fg265_1 protein')
2 0 The nucleotide sequence of fg265_1 as presently determined is reported in
SEQ ID
N0:14. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the fg265_1 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:15.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
2 5 fg265_1 should be approximately 3100 bp.
The nucleotide sequence disclosed herein for fg265_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
PASTA search protocols. fg265_1 demonstrated at least some similarity with
sequences
identified as AA076592 (zm91h10.r1 Stratagene ovarian cancer (#937219) Homo
Sapiens
3 0 cDNA clone 545347 5'), AA482600 (zt34a12.s1 Soares ovary tumor NbHOT Homo
Sapiens
cDNA), N23393 (yx83d12.s1 Homo sapiens cDNA clone 268343 3'), 810011
(yf34g05.r1
Homo Sapiens cDNA clone 128792 5'), 841186 (yf84c08.s1 Homo sapiens cDNA clone
29313 3'), and W87844 (zh68a05.r1 Soares fetal liver spleen 1NFLS Sl Homo
Sapiens cDNA
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clone 417200 5'). Based upon sequence similarity, fg265_1 proteins and each
similar
protein or peptide may share at least some activity. .
Clone "fp273_10"
A polynucleotide of the present invention has been identified as clone
"fp273_10".
fp273_10 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
analysis of the amino acid sequence of the encoded protein. fp273_10 is a full-
length
clone, including the entire coding sequence of a secreted protein (also
referred to herein
as "fp273_10 protein').
The nucleotide sequence of fp273_10 as presently determined is reported in SEQ
ID N0:16. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the fp273_10 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:17. Amino acids 15 to 27 are a
predicted
leader/signal sequence, with the predicted mature amino acid sequence
beginning at
amino acid 28, or are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
fp273_10 should be approximately 3800 bp.
2 0 The nucleotide sequence disclosed herein for fp273_10 was searched against
the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. fp273_10 demonstrated at least some similarity with
sequences
identified as 816387 (yf9lgOl.r1 Homo Sapiens cDNA clone 29825 5'), 817806
(yg09b06.r1
Homo sapiens cDNA clone 31763 5'), and T65784 (yc11f10.s1 Homo sapiens cDNA
clone
2 5 80395 3' similar to contains L1 repetitive element). Based upon sequence
similarity,
fp273_10 proteins and each similar protein or peptide may share at least some
activity.
The TopPredII computer program predicts four additional potential
transmembrane
domains within the fp273_10 protein sequence, centered around amino acids 140,
530, 560,
and 720 of SEQ ID N0:17, respectively. At amino acid 449 of SEQ ID N0:17, the
fp273_10
3 0 protein has a C-5 cytosine-specific DNA methylase motif.
Clone ", 243 8"
A polynucleotide of the present invention has been identified as clone
"fy243_8".
fy243 8 was isolated from a human adult placenta cDNA library using methods
which are
27
CA 02285447 1999-09-23
WO 98/44113 PCT/US98/06176
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. fy243_8 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"fy243_8 protein').
The nucleotide sequence of fy243_8 as presently determined is reported in SEQ
ID
N0:18. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the fy243_8 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:19. Additional open reading
frames for
fy243 8 are predicted at basepairs 297 to 635, at basepairs 826 to 1014, and
at basepairs
1102 to 1248 of SEQ ID N0:18; the predicted amino acid sequences corresponding
to the
foregoing nucleotide sequences are reported in SEQ ID N0:32, SEQ ID N0:33, and
SEQ
ID N0:34, respectively. The open reading frame for SEQ ID N0:19 could be
joined to
those for SEQ ID N0:32, SEQ ID N0:33, and SEQ ID N0:34 if the intervening
nucleotide
sequences of SEQ ID N0:18 were removed.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
fy243_8 should be approximately 2200 bp.
The nucleotide sequence disclosed herein for fy243_8 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
2 0 FASTA search protocols. fy243_8 demonstrated at least some similarity with
sequences
identified as AA121177 (z188h03.s1 Stratagene colon (#937204) Homo sapiens
cDNA clone
511733 3'), AA121218 (z188h03.r1 Stratagene colon (#937204) Homo Sapiens cDNA
clone
511733 5' similar to WP F44B9.5 CE00552), AA126582 (zn86g12.s1 Stratagene lung
carcinoma 937218 Homo Sapiens cDNA clone 565126 3'), 873372 (y110g08.r1 Homo
2 5 Sapiens cDNA clone 157886 5' similar to SP F44B9.5 CE00552), T27033
(NIBT173E09R
Infant brain, LLNL array of Dr. M. Soares 1NIB Homo Sapiens cDNA clone
LLAB173E09
5'end), and U41736 (Mus musculus ancient ubiquitous 46 kDa protein AUP1
precursor
(Aupl) mRNA, complete cds). The predicted amino acid sequence disclosed herein
for
fy243_8 was searched against the GenPept and GeneSeq amino acid sequence
databases
3 0 using the BLASTX search protocol. The predicted fy243_8 protein
demonstrated at least
some similarity to sequences identified as U41736 (ancient ubiquitous 46 kDa
protein
AUP46 precursor (Mus musculus]). Based upon sequence similarity, fy243 8
proteins and
each similar protein or peptide may share at least some activity.
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WO 98/44113 PCTNS98/06176
Clone '~a205 4"
A polynucleotide of the present invention has been identified as clone "ga205
4".
ga205 4 was isolated from a human adult testes cDNA library using methods
which are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637),
or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. ga205_4 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"ga205 4 protein').
The nucleotide sequence of ga205_4 as presently determined is reported in SEQ
ID N0:20. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the ga205 4 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:21.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
ga205 4 should be approximately 1000 bp.
The nucleotide sequence disclosed herein for ga205 4 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. ga205_4 demonstrated at least some similarity with
sequences
identified as AA075247 (zm86e01.r1 Stratagene ovarian cancer (#937219) Homo
sapiens
cDNA clone 544824 5'), AA081273 (zn33e12.s1 Stratagene endothelial cell 937223
Homo
2 0 sapiens cDNA clone 549262 3'), AA203476 (zx55e01.r1 Soares fetal liver
spleen 1NFLS S1
Homo Sapiens cDNA clone 446424 5' similar to contains element Ll repetitive
element),
T21011 (Human gene signature HUMGS02293), and U73030 (Rattus norvegicus
pituitary
tumor-specific transforming factor mRNA, complete cds). The predicted amino
acid
sequence disclosed herein for ga205_4 was searched against the GenPept and
GeneSeq
2 5 amino acid sequence databases using the BLASTX search protocol. The
predicted ga205 4
protein demonstrated at least some similarity to sequences identified as
U73030 (PTTG
gene product [Rattus norvegicus]). Based upon sequence similarity, ga205 4
proteins and
each similar protein or peptide may share at least some activity.
3 0 Deposit of Clones
Clones b1209_10, cr1162 25, dh40 3, di39_9, dt674_2, eh61 1, fg265_l,
fp273_10,
fy243 8, and ga205 4 were deposited on March 28,1997 with the American Type
Culture
Collection as an original deposit under the Budapest Treaty and were given the
accession
number ATCC 98379, from which each clone comprising a particular
polynucleotide is
29
CA 02285447 1999-09-23
WO 98/44113 PCT/US98/06176
obtainable. All restrictions on the availability to the public of the
deposited material will
be irrevocably removed upon the granting of the patent, except for the
requirements
specified in 37 C.F.R. ~ 1.808(b), 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
appropriate fragment for such clone. Each clone was deposited in either the
pED6 or
pNOTs vector depicted in Fig. 1. The pED6dpc2 vector ("pED6") was derived from
pED6dpc1 by insertion of a new polylinker to facilitate cDNA cloning (Kaufman
et al.,
1991, Nucleic Acids Res. 19: 4485-4490); the pNOTs vector was derived from
pMT2
(Kaufman et al., 1989, Mol. Cell. Biol. 9: 946-958) by deletion of the DHFR
sequences,
insertion of a new polylinker, and insertion of the M13 origin of replication
in the CIaI site.
In some instances, the deposited clone can become "flipped" (i.e., in the
reverse
orientation) in the deposited isolate. In such instances, the cDNA insert can
stiD 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.
2 0 Bacterial cells containing a particular clone can be obtained from the
composite
deposit as follows:
An oligonucleotide probe or probes should be designed to the sequence that is
known for that particular clone. This sequence can be derived from the
sequences
provided herein, or from a combination of those sequences. The sequence of the
2 5 oligonucleotide probe that was used to isolate each full-length clone is
identified below,
and should be most reliable in isolating the clone of interest.
Clone Probe Sequence
b1209_10 SEQ ID N0:22
3 0 cr1162_25 SEQ ID N0:23
dh40 3 SEQ ID N0:24
di39 9 SEQ ID N0:25
dt674_2 SEQ ID N0:26
eh61 1 SEQ ID N0:27
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WO 98/44113 PCT/US98/06176
fg265_1 SEQ ID N0:28
fp273_10 SEQ ID N0:29_
fy243_8 SEQ ID N0:30
ga205_4 SEQ ID N0:31
In the sequences listed above which include an N at position 2, that position
is occupied
in preferred probes/primers by a biotinylated phosphoaramidite residue rather
than a
nucleotide (such as , for example, that produced by use of biotin
phosphoramidite (1-
dimethoxytrityloxy-2-(N-biotinyl-4-aminobutyl)-propyl-3-O-(2-cyanoethyl)-(N,N-
diisopropyl)-phosphoramadite) (Glen Research, cat. no.10-1953)).
The design of the oligonucleotide probe should preferably follow these
parameters:
{a) It should be designed to an area of the sequence which has the fewest
ambiguous bases ("N's'"), if any;
I5 (b) It should be designed to have a Tm of approx. 80 ° C {assuming
2° for each
A or T and 4 degrees for each G or C).
The oligonucleotide should preferably be labeled with g 3zP ATP (specific
activity 6000
Ci/mmole) and T4 polynucleotide kinase using commonly employed techniques for
labeling oligonucleotides. Other labeling techniques can also be used.
Unincorporated
2 0 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
2 5 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
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
3 0 colonies on solid bacteriological media containing L-broth containing
ampicillin at 100
ug/ml and agar at 1.5% in a 150 mm petri dish when grown overnight at
37°C. Other
known methods of obtaining distinct, well-separated colonies can also be
employed.
Standard colony hybridization procedures should then be used to transfer the
colonies to nitrocellulose filters and lyse, denature and bake them.
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CA 02285447 1999-09-23
WO 98/44113 PCT/US98/06176
The filter is then preferably incubated at 65°C for 1 hour with gentle
agitation in
6X SSC (20X stock is 175.3 g NaCI/liter, 88.2 g Na citrate/liter, adjusted to
pH 7.0 with
NaOH) containing 0.5% SDS,100 ug/ml of yeast RNA, and 10 mM EDTA
(approximately
mL per 150 mm filter). Preferably, the probe is then added to the
hybridization mix at
5 a concentration greater than or equal to 1e+6 dpm/mL. The filter is then
preferably
incubated at 65°C with gentle agitation overnight. The filter is then
preferably washed in
500 mL of 2X SSC/0.5% SDS at room temperature without agitation, preferably
followed
by 500 mL of 2X SSC/0.1% SDS at room temperature with gentle shaking for 15
minutes.
A third wash with O.1X SSC/0.5% SDS at 65°C for 30 minutes to 1 hour is
optional. The
10 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 10, 773-778 (1992) and
in R.S.
2 0 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
2 5 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.
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
3 0 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
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.
32
CA 02285447 1999-09-23
WO 98/44113 PCT/US98/06176
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 mIZNAs from which cDNA polynucleotide sequences
are
derived and may include contiguous regions of the genome necessary for the
regulated
expression of such genes. Corresponding genes may therefore include but are
not limited
to coding sequences, 5' and 3' untranslated regions, alternatively spliced
exons, introns,
promoters, enhancers, and silencer or suppressor elements. The corresponding
genes can
be isolated in accordance with known methods using the sequence information
disclosed
herein. Such methods include the preparation of probes or primers from the
disclosed
sequence information for identification and/or amplification of genes in
appropriate
genomic libraries or other sources of genomic materials. An "isolated gene" is
a gene that
has been separated from the adjacent coding sequences, if any, present in the
genome of
the organism from which the gene was isolated.
Organisms that have enhanced, reduced, or modified expression of the genes)
corresponding to the polynucleodde 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 mIZNA 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-
2 0 39; all of which are incorporated by reference herein). Transgenic animals
that have
multiple copies of the genes) corresponding to the polynucleotfde 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
2 5 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
polynucleotide sequences disclosed herein have been partially or completely
inactivated,
through insertion of extraneous sequences into the corresponding genes) or
through
3 0 deletion of all or part of the corresponding gene(s). Partial or complete
gene inactivation
can be accomplished through insertion, preferably followed by imprecise
excision, of
transposable elements (Plasterk,1992, Bioessays 14(9): 629-633; Zwaal et
al.,1993, Proc. Natl.
Acad. Sci. LISA 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,
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CA 02285447 1999-09-23
WO 98/44113 PCT/US98/06176
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
organisms with altered gene expression are preferably eukaryotes and more
preferably
are mammals. Such organisms are useful for the development of non-human models
for
the study of disorders involving the corresponding gene(s), and for the
development of
assay systems for the identification of molecules that interact with the
protein products)
of the corresponding gene(s).
Where the protein of the present invention is membrane-bound (e.g., is a
receptor),
the present invention also provides for soluble forms of such protein. In such
forms part
or all of the intracellular and transmembrane domains of the protein are
deleted such that
the protein is fully secreted from the cell in which it is expressed. The
intracellular and
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
2 0 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
2 5 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
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
3 0 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
identity {more preferably, at least 45% identity; most preferably at least 60%
identity) with
34
CA 02285447 1999-09-23
WO 98/44113 PCT/US98/06176
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
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
pygmaeus, Hylobates
concolor, 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 Equus caballus, for which genetic maps have been
created
allowing the identification of syntenic 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
2 0 or proteins; that is, naturally-occurring 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
2 5 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
screening a suitable nucleic acid source from individuals of the appropriate
species.
The invention also includes polynucleotides with sequences complementary to
3 0 those of the polynucleotides disclosed herein.
The present invention also includes polynucleotides capable of hybridizing
under
reduced stringency conditions, more preferably stringent conditions, and most
preferably
highly stringent conditions, to polynucleotides described herein. Examples of
stringency
CA 02285447 1999-09-23
WO 98/44113 PCT/US98/06176
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.
S StringencyPolynucleotideHybrid Hybridization TemperatureWash
ConditionHybrid Length and Temperature
(bp)x Buffers 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 s 50 67C; lxSSC -or- 67C; 0.3xSSC
45C; lxSSC, 50% formamide
D DNA:RNA <50 TD*; lxSSC Tp*; lxSSC
E RNA:RNA s 50 70C; lxSSC -or- 70C; 0.3xSSC
50C; lxSSC, 50% formamide
F RNA:RNA <50 Tr*; lxSSC TF*; lxSSC
G DNA:DNA z 50 65C; 4xSSC -or- 65C; lxSSC
42C;4xSSC,50% formamide
H DNA:DNA <50 T"*; 4xSSC T"*; 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 TL*; 2xSSC T~*; 2xSSC
M DNA:DNA z 50 50C; 4xSSC -or- 50C; 2xSSC
40C; 6xSSC, 50% formamide
2 N DNA:DNA <50 TN*; 6xSSC TN*; 6xSSC
0
O DNA:RNA Z 50 55C; 4xSSC -or- 55C; 2xSSC
42C; 6xSSC, 50% formamide
P DNA:RNA <50 T,~*; 6xSSC T,.*; 6xSSC
Q RNA:RNA z 50 60C; 4xSSC -or- 60C; 2xSSC
45C;6xSSC,50% fonmamide
R RNA:RNA <50 Ta*; 4xSSC TR*; 4xSSC
_: The hybrid length is that anticipated for the hybridized regions) of the
hybridizing polynucleotides. 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
hybrid length can be determined by aligning the sequences of the
polynucleoHdes and identifying the region
3 0 or regions of optimal sequence complementarity.
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CA 02285447 1999-09-23
WO 98/44113 PCT/US98/06176
': 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. _
'~TB - TR: The hybridization temperature for hybrids anticipated to be less
than 50 base pairs in length should
be 5-10°C less than the melting temperature (Tm) of the hybrid, where
Tm is determined according to the
following equations. For hybrids less than 18 base pairs in length,
T,~(°C) = 2(# of A + T bases) + 4(# of G +
C bases). For hybrids between 18 and 49 base pairs in length, Tm(°C) =
81.5 + 16.6(log,o[Na+]) + 0.41 (%G+C)
(600/N), where N is the number of bases in the hybrid, and [Na'] is the
concentration of sodium ions in the
hybridization buffer ([Na'] for lxSSC = 0.165 M).
Additional examples of stringency conditions for polynucleotide hybridization
are
provided in Sambrook, J., E.F. Fritsch, and T. Maniatis, 1989, Molecular
Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
NY,
chapters 9 and 11, and Current Protocols in Molecular Biology,1995, F.M.
Ausubel et al., eds.,
John Wiley & Sons, Inc., sections 2.10 and 6.3-b.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
2 0 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
2 5 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
3 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
3 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
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strains derived from in vitro culture of primary tissue, primary explants,
HeLa cells,
mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells. _
Alternatively, it may be possible to produce the protein in lower eukaryotes
such
as yeast or in prokaryotes such as bacteria. Potentially suitable yeast
strains include
Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains,
Candida, or any
yeast strain capable of expressing heterologous proteins. Potentially suitable
bacterial
strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium,
or any bacterial
strain capable of expressing heterologous proteins. If the protein is made in
yeast or
bacteria, it may be necessary to modify the protein produced therein, for
example by
phosphorylation or glycosylation of the appropriate sites, in order to obtain
the functional
protein. Such covalent attachments may be accomplished using known chemical or
enzymatic methods.
The protein may also be produced by operably linking the isolated
polynucleotide
of the invention to suitable control sequences in one or more insect
expression vectors,
and employing an insect expression system. Materials and methods for
baculovirus/insect cell expression systems are commercially available in kit
form from,
e.g., Invitrogen, San Diego, California, U.S.A. (the MaxBacGR' 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
2 0 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
2 5 cell extracts) using known purification processes, such as gel filtration
and ion exchange
chromatography. The purification of the protein may also include an affinity
column
containing agents which will bind to the protein; one or more column steps
over such
affinity resins as concanavalin A-agarose, heparin-toyopearl~ or Cibacrom blue
3GA
Sepharose~; one or more steps involving hydrophobic interaction chromatography
using
3 0 such resins as phenyl ether, butyl ether, or propyl ether; or
immunoaffinity
chromatography.
Alternatively, the protein of the invention may also be expressed in a form
which
will facilitate purification. For example, it may be expressed as a fusion
protein, such as
those of maltose binding protein (MBP), glutathione-S-transferase (GST} or
thioredoxin
38
CA 02285447 1999-09-23
WO 98/44113 PCT/US98/06176
(TRX). Kits for expression and purification of such fusion proteins are
commercially
available from New England BioLab (Beverly, MA), Pharmacia (Piscataway, NJ)
and
InVitrogen, respectively. The protein can also be tagged with an epitope and
subsequently purified by using a specific antibody directed to such epitope.
One such
epitope ("Flag") is commercially available from Kodak (New Haven, CT).
Finally, one or more reverse-phase high performance liquid chromatography {RP-
HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having
pendant
methyl or other aliphatic groups, can be employed to further purify the
protein. Some or
all of the foregoing purification steps, in various combinations, can also be
employed to
provide a substantially homogeneous isolated recombinant protein. The protein
thus
purified is substantially free of other mammalian proteins and is defined in
accordance
with the present invention as an "isolated protein."
The protein of the invention may also be expressed as a product of transgenic
animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or
sheep which
are characterized by somatic or germ cells containing a nucleotide sequence
encoding the
protein.
The protein may also be produced by known conventional chemical synthesis.
Methods for constructing the proteins of the present invention by synthetic
means are
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.
2 5 The proteins provided herein also include proteins characterized by amino
acid
sequences similar to those of purified proteins but into which modification
are naturally
provided or deliberately engineered. For example, modifications in the peptide
or DNA
sequences can be made by those skilled in the art using known techniques.
Modifications
of interest in the protein sequences may include the alteration, substitution,
replacement,
3 0 insertion or deletion of a selected amino acid residue in the coding
sequence. For
example, one or more of the cysteine residues may be deleted or replaced with
another
amino acid to alter the conformation of the molecule. Techniques for such
alteration,
substitution, replacement, insertion or deletion are well known to those
skilled in the art
39
CA 02285447 1999-09-23
WO 98/44113 PCT/US98/06176
(see, e.g., U.S. Patent No. 4,518,584). Preferably, such alteration,
substitution, replacement,
insertion or deletion retains the desired activity of the protein. _
Other fragments and derivatives of the sequences of proteins which would be
expected to retain protein activity in whole or in part and may thus be useful
for screening
or other immunological methodologies may also be easily made by those skilled
in the art
given the disclosures herein. Such modifications are believed to be
encompassed by the
present invention.
USES AND BIOLOGICAL ACTIVITY
The polynucleotides and proteins of the present invention are expected to
exhibit
one or more of the uses or biological activities (including those associated
with assays
cited herein) identified below. Uses or activities described for proteins of
the present
invention may be provided by administration or use of such proteins or by
administration
or use of polynucleotides encoding such proteins (such as, for example, in
gene therapies
or vectors suitable for introduction of DNA).
Research Uses and Utilities
The polynucleotides provided by the present invention can be used by the
research
community for various purposes. The polynucleotides can be used to express
2 0 recombinant protein for analysis, characterization or therapeutic use; as
markers for
tissues in which the corresponding protein is preferentially expressed {either
constitutively or at a particular stage of tissue differentiation or
development or in disease
states); as molecular weight markers on Southern gels; as chromosome markers
or tags
(when labeled) to identify chromosomes or to map related gene positions; to
compare
2 5 with endogenous DNA sequences in patients to identify potential genetic
disorders; as
probes to hybridize and thus discover novel, related DNA sequences; as a
source of
information to derive PCR primers for genetic fingerprinting; as a probe to
"subtract-out"
known sequences in the process of discovering other novel polynucleotides; for
selecting
and making oligomers for attachment to a "gene chip" or other support,
including for
3 0 examination of expression patterns; to raise anti-protein antibodies using
DNA
immunization techniques; and as an antigen to raise anti-DNA antibodies or
elicit another
immune response. Where the polynucleotide encodes a protein which binds or
potentially
binds to another protein {such as, for example, in a receptor-ligand
interaction), the
polynucleotide can also be used in interaction trap assays (such as, for
example, those
CA 02285447 1999-09-23
WO 98/44113 PCT/US98/06176
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
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
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.
2 0 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
Polynucleotides and proteins of the present invention can also be used as
nutritional sources or supplements. Such uses include without limitation use
as a protein
or amino acid supplement, use as a carbon source, use as a nitrogen source and
use as a
3 0 source of carbohydrate. In such cases the protein or polynucleotide of the
invention can
be added to the feed of a particular organism or can be administered as a
separate solid
or liquid preparation, such as in the form of powder, pills, solutions,
suspensions or
capsules. In the case of microorganisms, the protein or polynucleotide of the
invention
can be added to the medium in or on which the microorganism is cultured.
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CA 02285447 1999-09-23
WO 98/44113 PCT/US98/06176
Cytokine and Cell Proliferation/Differentiation Activity
A protein of the present invention may exhibit cytokine, cell_proliferation
(either
inducing or inhibiting) or cell differentiation (either inducing or
inhibiting) activity or may
induce production of other cytokines in certain cell populations. Many protein
factors
discovered to date, including all known cytokines, have exhibited activity in
one or more
factor dependent cell proliferation assays, and hence the assays serve as a
convenient
confirmation of cytokine activity. The activity of a protein of the present
invention is
evidenced by any one of a number of routine factor dependent cell
proliferation assays
for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11,
BaF3,
MC9/G, M+ (preB M+), 2E8, RBS, DA1,123, T1165, HT2, CTLL2, TF-1, Mo7e and CMK.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assays for T-cell or thymocyte proliferation include without limitation those
described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M.
Kruisbeek, D.H.
Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and
Wiley-
Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-
3.19; Chapter
7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500,
1986;
Bertagnolli et al., J. Immunol.145:1706-1712, 1990; Beitagnolli et al.,
Cellular Immunology
2 0 133:327-341,1991; Bertagnolli, et al., J. Immunol. 149:3778-3783,1992;
Bowman et al., J.
Immunol. 152: 1756-1761, 1994.
Assays for cytokine production and/or proliferation of spleen cells, lymph
node
cells or thymocytes include, without limitation, those described in:
Polyclonal T cell
stimulation, Kruisbeek, A.M. and Shevach, E.M. In Current Protocols in
Immunology. J.E.e.a.
2 5 Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994;
and
Measurement of mouse and human Interferon y, Schreiber, R.D. In Current
Protocols in
Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley and Sons,
Toronto. 1994.
Assays for proliferation and differentiation of hematopoietic and
lymphopoietic
cells include, without limitation, those described in: Measurement of Human
and Murine
3 0 Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L.S. and Lipsky,
P.E. In Current
Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John
Wiley and Sons,
Toronto. 1991; deVries et al., J. Exp. Med. 173:1205-1211, 1991; Moreau et
al., Nature
336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A. 80:2931-
2938, 1983;
Measurement of mouse and human interleukin 6 - Nordan, R. In Current Protocols
in
42
CA 02285447 1999-09-23
WO 98/44113 PCT/US98/06176
Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons,
Toronto.1991;
Smith et al., Proc. Natl. Acad. Sci. U.S.A. 83:1857-1861, 1986; Measurement of
human
Interleukin 11- Bennett, F., Giannotti, J., Clark, S.C. and Turner, K. j. In
Current Protocols
in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.15.1 John Wiley and Sons,
Toronto. 1991;
Measurement of mouse and human Interleukin 9 - Ciarletta, A., Giannotti, J.,
Clark, S.C.
and Turner, K.J. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol
1 pp. 6.13.1,
John Wiley and Sons, Toronto. 1991.
Assays for T-cell clone responses to antigens (which will identify, among
others,
proteins that affect APC-T cell interactions as well as direct T-cell effects
by measuring
proliferation and cytokine production) include, without limitation, those
described in:
Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H.
Margulies,
E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-
Interscience
(Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6,
Cytokines and
their cellular receptors; Chapter 7, Immunologic studies in Humans);
Weinberger et al.,
Proc. Natl. Acad. Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J.
Immun.
11:405-411, 1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al.,
J. Immunol.
140:508-512, 1988.
Immune Stimulating or Su~uressing Activity
2 0 A protein of the present invention may also exhibit immune stimulating or
immune suppressing activity, including without limitation the activities for
which assays
are described herein. A protein may be useful in the treatment of various
immune
deficiencies and disorders (including severe combined immunodeficiency
(SCID)), e.g.,
in regulating (up or down) growth and proliferation of T and/or B lymphocytes,
as well
2 5 as effecting the cytolytic activity of NK cells and other cell
populations. These immune
deficiencies may be genetic or be caused by viral (e.g., HIV) as well as
bacterial or fungal
infections, or may result from autoimmune disorders. More specifically,
infectious
diseases causes by viral, bacterial, fungal or other infection may be
treatable using a
protein of the present invention, including infections by HIV, hepatitis
viruses,
3 0 herpesviruses, mycobacteria, Leishmarua spp., malaria spp. and various
fungal infections
such as candidiasis. Of course, in this regard, a protein of the present
invention may also
be useful where a boost to the immune system generally may be desirable, i.e.,
in the
treatment of cancer.
43
CA 02285447 1999-09-23
WO 98/44113 PCT/US98/06176
Autoimmune disorders which may be treated using a protein of the present
invention include, for example, connective tissue disease, multiple sclerosis,
systemic
lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation,
Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes
mellitis,
myasthenia gravis, graft-versus-host disease and autoimmufte inflammatory eye
disease.
Such a protein of the present invention may also to be useful in the treatment
of allergic
reactions and conditions, such as asthma (particularly allergic asthma) or
other respiratory
problems. Other conditions, in which immune suppression is desired (including,
for
example, organ transplantation), may also be treatable using a protein of the
present
invention.
Using the proteins of the invention it may also be possible to immune
responses,
in a number of ways. Down regulation may be in the form of inhibiting or
blocking an
immune response already in progress or may involve preventing the induction of
an
immune response. The functions of activated T cells may be inhibited by
suppressing T
cell responses or by inducing specific tolerance in T cells, or both.
Immunosuppression
of T cell responses is generally an active, non-antigen-specific, process
which requires
continuous exposure of the T cells to the suppressive agent. Tolerance, which
involves
inducing non-responsiveness or anergy in T cells, is distinguishable from
immunosuppression in that it is generally antigen-specific and persists after
exposure to
2 0 the tolerizing agent has ceased. Operationally, tolerance can be
demonstrated by the lack
of a T cell response upon reexposure to specific antigen in the absence of the
tolerizing
agent.
Down regulating or preventing one or more antigen functions (including without
limitation B lymphocyte antigen functions (such as , for example, B7)), e.g.,
preventing
2 5 high level lymphokine synthesis by activated T cells, will be useful in
situations of tissue,
skin and organ transplantation and in graft-versus-host disease (GVHD). For
example,
blockage of T cell function should result in reduced tissue destruction in
tissue
transplantation. Typically, in tissue transplants, rejection of the transplant
is initiated
through its recognition as foreign by T cells, followed by an immune reaction
that destroys
3 0 the transplant. The administration of a molecule which inhibits or blocks
interaction of
a B7 lymphocyte antigen with its natural ligand(s) on immune cells (such as a
soluble,
monomeric form of a peptide having B7-2 activity alone or in conjunction with
a
monomeric form of a peptide having an activity of another B lymphocyte antigen
(e.g., B7-
I, B7-3) or blocking antibody), prior to transplantation can lead to the
binding of the
44
CA 02285447 1999-09-23
WO 98/44113 PCT/US98/06176
molecule to the natural ligand(s) on the immune cells without transmitting the
corresponding costimulatory signal. Blocking B lymphocyte antigen function in
this
matter prevents cytokine synthesis by immune cells, such as T cells, and thus
acts as an
immunosuppressant. Moreover, the lack of costimulation may also be sufficient
to
anergize the T cells, thereby inducing tolerance in a subject. Induction of
long-term
tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of
repeated
administration of these blocking reagents. To achieve sufficient
immunosuppression or
tolerance in a subject, it may also be necessary to block the function of a
combination of
B lymphocyte antigens.
The efficacy of particular blocking reagents in preventing organ transplant
rejection or GVHD can be assessed using animal models that are predictive of
efficacy in
humans. Examples of appropriate systems which can be used include allogeneic
cardiac
grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of
which have been
used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in
vivo as
described in Lenschow et al., Science 257:789-792 (1992) and Turka et al.,
Proc. Natl. Acad.
Sci USA, 89:11102-11105 (1992). In addition, murine models of GVHD (see Paul
ed.,
Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847) can be used
to
determine the effect of blocking B lymphocyte antigen function in vivo on the
development
of that disease.
2 0 Blocking antigen function may also be therapeutically useful for treating
autoimmune diseases. Many autoimmune disorders are the result of inappropriate
activation of T cells that are reactive against self tissue and which promote
the production
of cytokines and autoantibodies involved in the pathology of the diseases.
Preventing the
activation of autoreactive T cells may reduce or eliminate disease symptoms.
2 5 Administration of reagents which block costimulation of T cells by
disrupting
receptor:ligand interactions of B lymphocyte antigens can be used to inhibit T
cell
activation and prevent production of autoantibodies or T cell-derived
cytokines which
may be involved in the disease process. Additionally, blocking reagents may
induce
antigen-specific tolerance of autoreactive T cells which could lead to long-
term relief from
3 0 the disease. The efficacy of blocking reagents in preventing or
alleviating autoimmune
disorders can be determined using a number of well-characterized animal models
of
human autoimmune diseases. Examples include murine experimental autoimmune
encephalitis, systemic lupus erythmatosis in MRL/Ipr/Ipr mice or NZB hybrid
mice,
murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB
rats, and
CA 02285447 1999-09-23
WO 98/44113 PCT/US98/06176
marine experimental myasthenia gravis (see Paul ed., Fundamental Immunology,
Raven
Press, New York,1989, pp. 840-856). _
Upregulation of an antigen function (preferably a B lymphocyte antigen
function),
as a means of up regulating immune responses, may also be useful in therapy.
Upregulation of immune responses may be in the form of enhancing an existing
immune
response or eliciting an initial immune response. For example, enhancing an
immune
response through stimulating B lymphocyte antigen function may be useful in
cases of
viral infection. In addition, systemic viral diseases such as influenza, the
common cold,
and encephalitis might be alleviated by the administration of stimulatory
forms of B
lymphocyte antigens systemically.
Alternatively, anti-viral immune responses may be enhanced in an infected
patient
by removing T cells from the patient, costimulating the T cells in vitro with
viral antigen-
pulsed APCs either expressing a peptide of the present invention or together
with a
stimulatory form of a soluble peptide of the present invention and
reintroducing the in
vitro activated T cells into the patient. Another method of enhancing anti-
viral immune
responses would be to isolate infected cells from a patient, transfect them
with a nucleic
acid encoding a protein of the present invention as described herein such that
the cells
express all or a portion of the protein on their surface, and reintroduce the
transfected
cells into the patient. The infected cells would now be capable of delivering
a
2 0 costimulatory signal to, and thereby activate, T cells in vivo.
In another application, up regulation or enhancement of antigen function
(preferably B lymphocyte antigen function) may be useful in the induction of
tumor
immunity. Tumor cells (e.g., sarcoma, melanoma, lymphoma, leukemia,
neuroblastoma,
carcinoma) transfected with a nucleic acid encoding at least one peptide of
the present
2 5 invention can be administered to a subject to overcome tumor-specific
tolerance in the
subject. If desired, the tumor cell can be transfected to express a
combination of peptides.
For example, tumor cells obtained from a patient can be transfected ex vivo
with an
expression vector directing the expression of a peptide having B7-2-like
activity alone, or
in conjunction with a peptide having B7-1-like activity and/or B7-3-like
activity. The
3 0 transfected tumor cells are returned to the patient to result in
expression of the peptides
on the surface of the transfected cell. Alternatively, gene therapy techniques
can be used
to target a tumor cell for transfection in vivo.
The presence of the peptide of the present invention having the activity of a
B
lymphocyte antigen{s) on the surface of the tumor cell provides the necessary
46
CA 02285447 1999-09-23
WO 98/44113 PCT/US98/06176
costimulation signal to T cells to induce a T cell mediated immune response
against the
transfected tumor cells. In addition, tumor cells which lack MHC class I or
MHC class II
molecules, or which fail to reexpress sufficient amounts of MHC class I or MHC
class II
molecules, can be transfected with nucleic acid encoding all or a portion of
(e.g., a
cytoplasmic-domain truncated portion) of an MHC class I a chain protein and
~3z
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.
Expression of the appropriate class I or class II MHC in conjunction with a
peptide having
the activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a T
cell mediated
immune response against the transfected tumor cell. Optionally, a gene
encoding an
antisense construct which blocks expression of an MHC class II associated
protein, such
as the invariant chain, can also be cotransfected with a DNA encoding a
peptide having
the activity of a B lymphocyte antigen to promote presentation of tumor
associated
antigens and induce tumor specific immunity. Thus, the induction of a T cell
mediated
immune response in a human subject may be sufficient to overcome tumor-
specific
tolerance in the subject.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Suitable assays for thymocyte or splenocyte cytotoxicity include, without
2 0 limitation, those described in: Current Protocols in Immunology, Ed by J.
E. Coligan, A.M.
Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing
Associates
and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte
Function 3.1-
3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl.
Acad. Sci.
USA 78:2488-2492,1981; Hernnann et al., J. Immunol. 128:1968-1974,1982; Handa
et al.,
2 S J. Immunol.135:1564-1572,1985; Takai et al., J. Immunol.137:3494-
3500,1986; Takai et al.,
J. Immunol.140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA
78:2488-2492,
1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J.
Immunol.
135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Bowmanet
al., J.
Virology 61:1992-1998; Takai et aL, J. Immunol. 140:508-512, 1988; Bertagnolli
et al.,
3 0 Cellular Immunology 133:327-341, 1991; Brown et al., J. Immunol. 153:3079-
3092, 1994.
Assays for T-cell-dependent immunoglobulin responses and isotype switching
(which will identify, among others, proteins that modulate T-cell dependent
antibody
responses and that affect Thl/Th2 profiles) include, without limitation, those
described
in: Maliszewski, J. Immunol. 144:3028-3033, 1990; and Assays for B cell
function: In vitro
47
CA 02285447 1999-09-23
WO 98/44113 PCT/US98/06176
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.
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.
ICruisbeek,
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. lmmunol.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. l.mmunol. 134:536-544, 1995; Inaba et al.,
Journal of
Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal of
Immunology
154:5071-5079,1995; Porgador et al., Journal of Experimental Medicine 182:255-
260, 1995;
Nair et al., Journal of Virology 67:4062-4069, 1993; Huang et al., Science
264:961-965,
1994; Macatonia et al., Journal of Experimental Medicine 169:1255-1264,1989;
Bhardwaj
et al., Journal of Clinical Investigation 94:797-807, 1994; and Inaba et aL,
Journal of
Experimental Medicine 172:631-640,1990.
Assays for lymphocyte survival/apoptosis (which will identify, among others,
2 0 proteins that prevent apoptosis after superantigen induction and proteins
that regulate
lymphocyte homeostasis) include, without limitation, those described in:
Darzynkiewicz
et al., Cytometry 13:795-808,1992; Gorczyca et al., Leukemia 7:659-670,1993;
Gorczyca et
al., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991;
Zacharchuk,
Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry 14:891-897,
1993;
2 5 Gorczyca et al., International Journal of Oncology 1:639-648,1992.
Assays for proteins that influence early steps of T-cell commitment and
development include, without limitation, those described in: Antica et al.,
Blood
84:111-117, 1994; Fine et al., Cellular Immunology 155:111-122, 1994; Galy et
al., Blood
85:2770-2778,1995; Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551,1991.
Hematopoiesis Regulatin "~ Activity
A protein of the present invention may be useful in regulation of
hematopoiesis
and, consequently, in the treatment of myeloid or lymphoid cell deficiencies.
Even
marginal biological activity in support of colony forming cells or of factor-
dependent cell
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lines indicates involvement in regulating hematopoiesis, e.g. in supporting
the growth and
proliferation of erythroid progenitor cells alone or in combination with other
cytokines,
thereby indicating utility, for example, in treating various anemias or for
use in
conjunction with irradiation/chemotherapy to stimulate the production of
erythroid
precursors and/or erythroid cells; in supporting the growth and proliferation
of myeloid
cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF
activity)
useful, for example, in conjunction with chemotherapy to prevent or treat
consequent
myelo-suppression; in supporting the growth and proliferation of
megakaryocytes and
consequently of platelets thereby allowing prevention or treatment of various
platelet
disorders such as 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.
2 0 The activity of a protein of the invention may, among other means, be
measured
by the following methods:
Suitable assays for proliferation and differentiation of various hematopoietic
lines
are cited above.
Assays for embryonic stem cell differentiation (which will identify, among
others,
2 5 proteins that influence embryonic differentiation hematopoiesis) include,
without
limitation, those described in: johansson et al. Cellular Biology 15:141-
151,1995; Keller et
al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al., Blood
81:2903-2915,1993.
Assays for stem cell survival and differentiation (which will identify, among
3 0 others, proteins that regulate lympho-hematopoiesis) include, without
limitation, those
described in: Methylcellulose colony forming assays, Freshney, M.G. In Culture
of
Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss,
Inc., New York,
NY. 1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992;
Primitive
hematopoietic colony forming cells with high proliferative potential, McNiece,
LK. and
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Briddell, R.A. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds.
Vol pp. 23-39,
Wiley-Liss, Inc., New York, NY.1994; Neben et al., Experimental Hematology
22:353-359,
1994; Cobblestone area forming cell assay, Ploemacher, R.E. In Culture of
Hematopoietic
Cells. R.I. Freshney, et al. eds. Vol pp. 1-21, Wiley-Liss, Inc.., New York,
NY. 1994; Long
term bone marrow cultures in the presence of stromal cells, Spooncer, E.,
Dexter, M. and
Allen, T. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol
pp. 163-179,
Wiley-Liss, Inc., New York, NY.1994; Long term culture initiating cell assay,
Sutherland,
H.J. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp.139-
162, Wiley-Liss,
Inc., New York, NY.1994.
Tissue Growth Activity
A protein of the present invention also may have utility in compositions used
for
bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration,
as well as
for wound healing and tissue repair and replacement, and in the treatment of
burns,
incisions and ulcers.
A protein of the present invention, which induces cartilage and/or bone growth
in circumstances where bone is not normally formed, has application in the
healing of
bone fractures and cartilage damage or defects in humans and other animals.
Such a
preparation employing a protein of the invention may have prophylactic use in
closed as
2 0 well as open fracture reduction and also in the improved fixation of
artificial faints. De
novo bone formation induced by an osteogenic agent contributes to the repair
of
congenital, trauma induced, or oncologic resection induced craniofacial
defects, and also
is useful in cosmetic plastic surgery.
A protein of this invention may also be used in the treatment of periodontal
2 5 disease, and in other tooth repair processes. Such agents may provide an
environment
to attract bone-forming cells, stimulate growth of bone-forming cells or
induce
differentiation of progenitors of bone-forming cells. A protein of the
invention may also
be useful in the treatment of osteoporosis or osteoarthritis, such as through
stimulation
of bone and/or cartilage repair or by blocking inflammation or processes of
tissue
3 0 destruction (collagenase activity, osteoclast activity, etc.) mediated by
inflammatory
processes.
Another category of tissue regeneration activity that may be attributable to
the
protein of the present invention is tendon/ligameitt formation. A protein of
the present
invention, which induces tendon/ligament-like tissue or other tissue formation
in
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circumstances where such tissue is not normally formed, has application in the
healing of
tendon or ligament tears, deformities and other tendon or ligament defects in
humans and
other animals. Such a preparation employing a tendon/ligament-like tissue
inducing
protein may have prophylactic use in preventing damage to tendon or ligament
tissue, as
well as use in the improved fixation of tendon or ligament to bone or other
tissues, and
in repairing defects to tendon or ligament tissue. De novo tendon/ligament-
like tissue
formation induced by a composition of the present invention contributes to the
repair of
congenital, trauma induced, or other tendon or ligament defects of other
origin, and is also
useful in cosmetic plastic surgery for attachment or repair of tendons or
ligaments. The
compositions of the present invention may provide an environment to attract
tendon- or
ligament-forming cells, stimulate growth of tendon- or ligament-forming cells,
induce
differentiation of progenitors of tendon- or ligament-forming cells, or induce
growth of
tendon/ligament cells or progenitors ex vivo for return in vivo to effect
tissue repair. The
compositions of the invention may also be useful in the treatment of
tendinitis, carpal
tunnel syndrome and other tendon or ligament defects. The compositions may
also
include an appropriate matrix and/or sequestering agent as a carrier as is
well known in
the art.
The protein of the present invention may also be useful for proliferation of
neural
cells and for regeneration of nerve and brain tissue, i.e. for the treatment
of central and
2 0 peripheral nervous system diseases and neuropathies, as well as mechanical
and
traumatic disorders, which involve degeneration, death or trauma to neural
cells or nerve
tissue. More specifically, a protein may be used in the treatment of diseases
of the
peripheral nervous system, such as peripheral nerve injuries, peripheral
neuropathy and
localized neuropathies, and central nervous system diseases, such as
Alzheimer's,
2 5 Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis,
and Shy-Drager
syndrome. Further conditions which may be treated in accordance with the
present
invention include mechanical and traumatic disorders, such as spinal cord
disorders, head
trauma and cerebrovascular diseases such as stroke. Peripheral neuropathies
resulting
from chemotherapy or other medical therapies may also be treatable using a
protein of the
3 0 invention.
Proteins of the invention may also be useful to promote better or faster
closure of
non-healing wounds, including without limitation pressure ulcers, ulcers
associated with
vascular insufficiency, surgical and traumatic wounds, and the like.
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It is expected that a protein of the present invention may also exhibit
activity for
generation or regeneration of other tissues, such as organs (including, for
example,
pancreas, liver, intestine, kidney, skin, endothelium), muscle {smooth,
skeletal or cardiac)
and vascular (including vascular endothelium) tissue, or for promoting the
growth of cells
comprising such tissues. Part of the desired effects may be by inhibition or
modulation
of fibrotic scarring to allow normal tissue to regenerate. A protein of the
invention may
also exhibit angiogenic activity.
A protein of the present invention may also be useful for gut protection or
regeneration and treatment of lung or liver fibrosis, reperfusion injury in
various tissues,
and conditions resulting from systemic cytokine damage.
A protein of the present invention may also be useful for promoting or
inhibiting
differentiation of tissues described above from precursor tissues or cells; or
for inhibiting
the growth of tissues described above.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assays for tissue generation activity include, without limitation, those
described
in: International Patent Publication No. W095/16035 (bone, cartilage, tendon);
International Patent Publication No. W095/05846 (nerve, neuronal);
International Patent
Publication No. W091/07491 (skin, endothelium ).
2 0 Assays for wound healing activity include, without limitation, those
described in:
Winter, Epidermal Wound Healing, pps. 71-112 (Maibach, HI and Rovee, DT,
eds.), Year
Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J.
Invest.
Dermatol 71:382-84 (1978).
2 5 Activin / Inhibin Activity
A protein of the present invention may also exhibit activin- or inhibin-
related
activities. Inhibins are characterized by their ability to inhibit the release
of follicle
stimulating hormone (FSH), while activins and are characterized by their
ability to
stimulate the release of follicle stimulating hormone (FSH). Thus, a protein
of the present
3 0 invention, alone or in heterodimers with a member of the inhibin a family,
may be useful
as a contraceptive based on the ability of inhibins to decrease fertility in
female mammals
and decrease spermatogenesis in male mammals. Administration of sufficient
amounts
of other inhibins can induce infertility in these mammals. Alternatively, the
protein of the
invention, as a homodimer or as a heterodimer with other protein subunits of
the inhibin-
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p group, may be useful as a fertility inducing therapeutic, based upon the
ability of activin
molecules in stimulating FSH release from cells of the anterior pituitary.
See, for example,
United States Patent 4,798,885. A protein of the invention may also be useful
for
advancement of the onset of fertility in sexually immature mammals, so as to
increase the
lifetime reproductive performance of domestic animals such as cows, sheep and
pigs.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assays for activin/inhibin activity include, without limitation, those
described in:
Vale et al., Endocrinology 91:562-572,1972; Ling et al., Nature 321:779-782,
1986; Vale et
al., Nature 321:776-779,1986; Mason et al., Nature 318:659-663,1985; Forage et
al., Proc.
Natl. Acad. Sci. USA 83:3091-3095,19$6.
Chemotactic/Chemokinetic Activit~r
A protein of the present invention may have chemotactic or chemokinetic
activity
(e.g., act as a chemokine) for mammalian cells, including, for example,
monocytes,
fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or
endothelial cells.
Chemotactic and chemokinetic proteins can be used to mobilize or attract a
desired cell
population to a desired site of action. Chemotactic or chemokinetic proteins
provide
particular advantages in treatment of wounds and other trauma to tissues, as
well as in
2 0 treatment of localized infections. For example, attraction of lymphocytes,
monocytes or
neutrophils to tumors or sites of infection may result in improved immune
responses
against the tumor or infecting agent.
A protein or peptide has chemotactic activity for a particular cell population
if it
can stimulate, directly or indirectly, the directed orientation or movement of
such cell
2 5 population. Preferably, the protein or peptide has the ability to directly
stimulate directed
movement of cells. Whether a particular protein has chemotactic activity for a
population
of cells can be readily determined by employing such protein or peptide in any
known
assay for cell chemotaxis.
The activity of a protein of the invention may, among other means, be measured
3 0 by the following methods:
Assays for chemotactic activity (which will identify proteins that induce or
prevent
chemotaxis) consist of assays that measure the ability of a protein to induce
the migration
of cells across a membrane as well as the ability of a protein to induce the
adhesion of one
cell population to another cell population. Suitable assays for movement and
adhesion
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include, without limitation, those described in: Current Protocols in
Immunology, Ed by
J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W.Strober, Pub.
Greene
Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of
alpha and
beta Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:1370-
1376,1995; Lind et al.
APMIS 103:140-146,1995; Muller et al Eur. J. Imrnunol. 25:1744-1748; Gruber et
al. j. of
Immunol. 152:5860-5867,1994; Johnston et al. J. of Immunol. 153:1762-
1768,1994.
Hemostatic and Thrombolytic Activity
A protein of the invention may also exhibit hemostatic or thrombolytic
activity.
As a result, such a protein is expected to be useful in treatment of various
coagulation
disorders (including hereditary disorders, such as hemophiliac) or to enhance
coagulation
and other hemostatic events in treating wounds resulting from trauma, surgery
or other
causes. A protein of the invention may also be useful for dissolving or
inhibiting
formation of thromboses and for treatment and prevention of conditions
resulting
therefrom (such as, for example, infarction of cardiac and central nervous
system vessels
(e.g., stroke).
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assay for hemostatic and thrombolytic activity include, without (imitation,
those
2 0 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/Li~and ActivitX
2 5 A protein of the present invention may also demonstrate activity as
receptors,
receptor ligands or inhibitors or agonists of receptor/ligand interactions.
Examples of
such receptors and ligands include, without limitation, cytokine receptors and
their
ligands, receptor kinases and their ligands, receptor phosphatases and their
ligands,
receptors involved in cell-cell interactions and their ligands (including
without limitation,
3 0 cellular adhesion molecules (such as selectins, integrins and their
ligands) and
receptor/ligand pairs involved in antigen presentation, antigen recognition
and
development of cellular and humoral immune responses). Receptors and ligands
are also
useful for screening of potential peptide or small molecule inhibitors of the
relevant
receptor/ligand interaction. A protein of the present invention (including,
without
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limitation, fragments of receptors and ligands) may themselves be useful as
inhibitors of
receptor/ligand interactions. _
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Suitable assays for receptor-ligand activity include without limitation those
described in:Current Protocols in Immunology, Ed by J.E. Coligan, A.M.
Kruisbeek, D.H.
Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and
Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under
static
conditions 7.28.1-7.28.22), Takai et al., Proc. Natl. Acad. Sci. USA 84:6864-
6868, 1987;
Bierer et al., J. Exp. Med. 168:1145-1156, 1988; Rosenstein et al., j. Exp.
Med. 169:149-160
1989; Stoltenborg et al:, J. Immunol. Methods 175:59-68,1994; Stitt et al.,
Cell 80:661-670,
1995.
Anti-Inflammatory Activity
Proteins of the present invention may also exhibit anti-inflammatory activity.
The
anti-inflammatory activity may be achieved by providing a stimulus to cells
involved in
the inflammatory response, by inhibiting or promoting cell-cell interactions
(such as, for
example, cell adhesion), by inhibiting or promoting chemotaxis of cells
involved in the
inflammatory process, inhibiting or promoting cell extravasation, or by
stimulating or
2 0 suppressing production of other factors which more directly inhibit or
promote an
inflammatory response. Proteins exhibiting such activities can be used to
treat
inflammatory conditions including chronic or acute conditions), including
without
limitation inflammation associated with infection (such as septic shock,
sepsis or systemic
inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin
2 5 lethality, arthritis, complement-mediated hyperacute rejection, nephritis,
cytokine or
chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or
resulting
from over production of cytokines such as TNF or IL-1. Proteins of the
invention may also
be useful to treat anaphylaxis and hypersensitivity to an antigenic substance
or material.
3 0 Cadherin/Tumor Invasion Suppressor Activity
Cadherins are calcium-dependent adhesion molecules that appear to play major
roles during development, particularly in defining specific cell types. Loss
or alteration
of normal cadherin expression can lead to change's in cell adhesion properties
linked to
tumor growth and metastasis. Cadherin malfunction is also implicated in other
human
CA 02285447 1999-09-23
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diseases, such as pemphigus vulgaris and pemphigus foiiaceus (auto-immune
blistering
skin diseases), Crohn's disease, and some developmental abnormalities.
The cadherin superfamily includes well over forty members, each with a
distinct
pattern of expression. All members of the superfamily have in common conserved
extracellular repeats (cadherin domains), but structural differences are found
in other
parts of the molecule. The cadherin domains bind calcium to form their
tertiary structure
and thus calcium is required to mediate their adhesion. Only a few amino acids
in the
first cadherin domain provide the basis for homophilic adhesion; modification
of this
recognition site can change the specificity of a cadherin so that instead of
recognizing only
itself, the mutant molecule can now also bind to a different cadherin. In
addition, some
cadherins engage in heterophilic adhesion with other cadherins.
E-cadherin, one member of the cadherin superfamily, is expressed in epithelial
cell
types. Pathologically, if E-cadherin expression is lost in a tumor, the
malignant cells
become invasive and the cancer metastasizes. Transfection of cancel 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
2 0 suppressor role in carcinomas derived from other tissue types. Therefore,
proteins of the
present invention with cadherin activity, and polynucleotides of the present
invention
encoding such proteins, can be used to treat cancer. Introducing such proteins
or
polynucleotides into cancer cells can reduce or eliminate the cancerous
changes observed
in these cells by providing normal cadherin expression.
2 5 Cancer cells have also been shown to express cadherins of a different
tissue type
than their origin, thus allowing these cells to invade and metastasize in a
different tissue
in the body. Proteins of the present invention with cadherin activity, and
polynucleotides
of the present invention encoding such proteins, can be substituted in these
cells for the
inappropriately expressed cadherins, restoring normal cell adhesive properties
and
3 0 reducing or eliminating the tendency of the cells to metastasize.
Additionally, proteins of the present invention with cadherin activity, and
polynucleotides of the present invention encoding such proteins, can used to
generate
antibodies recognizing and binding to cadherins. Such antibodies can be used
to block
the adhesion of inappropriately expressed tumor-cell cadherins, preventing the
cells from
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forming a tumor elsewhere. Such an anti-cadherin antibody can also be used as
a marker
for the grade, pathological type, and prognosis of a cancer, i.e. the more
progressed the
cancer, the less cadherin expression there will be, and this decrease in
cadherin expression
can be detected by the use of a cadherin-binding antibody.
Fragments of proteins of the present invention with cadherin activity,
preferably
a polypeptide comprising a decapeptide of the cadherin recognition site, and
poly-
nucleotides of the present invention encoding such protein fragments, can also
be used
to block cadherin function by binding to cadherins and preventing them from
binding in
ways that produce undesirable effects. Additionally, fragments of proteins of
the present
invention with cadherin activity, preferably truncated soluble cadherin
fragments which
have been found to be stable in the circulation of cancer patients, and
polynucleotides
encoding such protein fragments, can be used to disturb proper cell-cell
adhesion.
Assays for cadherin adhesive and invasive suppressor activity include, without
limitation, those described in: 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.
2 0 A protein may inhibit tumor growth directly or indirectly (such as, for
example, via
ADCC). A protein may exhibit its tumor inhibitory activity by acting on tumor
tissue or
tumor precursor tissue, by inhibiting formation of tissues necessary to
support tumor
growth (such as, for example, by inhibiting angiogenesis), by causing
production of other
factors, agents or cell types which inhibit tumor growth, or by suppressing,
eliminating
2 5 or inhibiting factors, agents or cell types which promote tumor growth.
Other Activities
A protein of the invention may also exhibit one or more of the following
additional
activities or effects: inhibiting the growth, infection or function of, or
killing, infectious
3 0 agents, including, without limitation, bacteria, viruses, fungi and other
parasites; effecting
(suppressing or enhancing) bodily.characteristics, including, without
limitation, height,
weight, hair color, eye color, skin, fat to lean ratio or other tissue
pigmentation, or organ
or body part size or shape (such as, for example, breast augmentation or
diminution,
change in bone form or shape); effecting biorhythms or caricadic cycles or
rhythms;
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effecting the fertility of male or female subjects; effecting the metabolism,
catabolism,
anabolism, processing, utilization, storage or elimination of dieta _ry fat,
lipid, protein,
carbohydrate, vitamins, minerals, cofactors or other nutritional factors or
component(s);
effecting behavioral characteristics, including, without limitation, appetite,
libido, stress,
cognition (including cognitive disorders), depression (including depressive
disorders) and
violent behaviors; providing analgesic effects or other pain reducing effects;
promoting
differentiation and growth of embryonic stem cells in lineages other than
hematopoietic
lineages; hormonal or endocrine activity; in the case of enzymes, correcting
deficiencies
of the enzyme and treating deficiency-related diseases; treatment of
hyperproliferative
disorders (such as, for example, psoriasis); immunoglobulin-like activity
(such as, for
example, the ability to bind antigens or complement); and the ability to act
as an antigen
in a vaccine composition to raise an immune response against such protein or
another
material or entity which is cross-reactive with such protein.
ADMINISTRATION AND DOSING
A protein of the present invention (from whatever source derived, including
without limitation from recombinant and non-recombinant sources) may be used
in a
pharmaceutical composition when combined with a pharmaceutically acceptable
carrier.
2 0 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
2 5 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,
ILrl2, IL-13, IL-14, IL-15, IFN, TNFO, TNFl, TNF2, G-CSF, Meg-CSF,
thrombopoietin, stem
cell factor, and erythropoietin. The pharmaceutical composition may further
contain other
agents which either enhance the activity of the protein or compliment its
activity or use
3 0 in treatment. Such additional factors and /or agents may be included in
the
pharmaceutical composition to produce a synergistic effect with protein of the
invention,
or to minimize side effects. Conversely, protein of the present invention may
be included
in formulations of the particular cytokine, lymphokine, other hematopoietic
factor,
thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize
side effects
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of the cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-
thrombotic
factor, or anti-inflammatory agent. _
A protein of the present invention may be active in multimers (e.g.,
heterodimers
or homodimers) or complexes with itself or other proteins. As a result,
pharmaceutical
compositions of the invention may comprise a protein of the invention in such
multimeric
or complexed form.
The pharmaceutical composition of the invention may be in the form of a
complex
of the proteins) of present invention along with protein or peptide antigens.
The protein
and/or peptide antigen will deliver a stimulatory signal to both B and T
lymphocytes. B
lymphocytes will respond to antigen through their surface 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.
2 0 The pharmaceutical composition of the invention may be in the form of a
liposome
in which protein of the present invention is combined, in addition to other
pharmaceutically acceptable earners, 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,
2 5 monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids,
saponin, bile acids,
and the like. Preparation of such liposomal formulations is within the level
of skill in the
art, as disclosed, for example, in U.S. Patent No. 4,235,871; U.S. Patent No.
4,501,728; U.S.
Patent No. 4,837,028; and U.S. Patent No. 4,737,323, all of which are
incorporated herein
by reference.
3 0 As used herein, the term "therapeutically effective amount" means the
total
amount of each active component of the pharmaceutical composition or method
that is
sufficient to show a meaningful patient benefit, i.e., treatment, healing,
prevention or
amelioration of the relevant medical condition, or an increase in rate of
treatment, healing,
prevention or amelioration of such conditions. When applied to an individual
active
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ingredient, administered alone, the term refers to that ingredient alone. When
applied to
a combination, the term refers to combined amounts of the active ingredients
that result
in the therapeutic effect, whether administered in combination, serially or
simultaneously.
In practicing the method of treatment or use of the present invention, a
therapeutically effective amount of protein of the present invention is
administered to a
mammal having a condition to be treated. Protein of the present invention may
be
administered in accordance with the method of the invention either alone or in
combination with other therapies such as treatments employing cytokines,
lympholcines
or other hematopoietic factors. When co-administered with one or more
cytolcines,
lymphokines or other hematopoietic factors, protein of the present invention
may be
administered either simultaneously with the cytolcine(s), lympholcine{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
cytolcine(s), lympholcine(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 carned
out in a
variety of conventional ways, such as oral ingestion, inhalation, topical
application or
2 0 cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous
injection.
Intravenous administration to the patient is preferred.
When a therapeutically effective amount of protein of the present invention is
administered orally, protein of the present invention will be in the form of a
tablet,
capsule, powder, solution or elixir. When administered in tablet form, the
pharmaceutical
2 5 composition of the invention may additionally contain a solid carrier such
as a gelatin or
an adjuvant. The tablet, capsule, and powder contain from about 5 to 95%
protein of the
present invention, and preferably from about 25 to 90% protein of the present
invention.
When administered in liquid form, a liquid carrier such as water, petroleum,
oils of animal
or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil,
or synthetic oils
3 0 may be added. The liquid form of the pharmaceutical composition may
further contain
physiological saline solution, dextrose or other saccharide solution, or
glycols such as
ethylene glycol, propylene glycol or polyethylene glycol. When administered in
liquid
form, the pharmaceutical composition contains from about 0.5 to 90% by weight
of protein
CA 02285447 1999-09-23
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of the present invention, and preferably from about 1 to 50% protein of the
present
invention.
When a therapeutically effective amount of protein of the present invention is
administered by intravenous, cutaneous or subcutaneous injection, protein of
the present
invention will be in the form of a pyrogen-free, parenterally acceptable
aqueous solution.
The preparation of such parenterally acceptable protein solutions, having due
regard to
pH, isotonicity, stability, and the like, is within the skill in the art. A
preferred
pharmaceutical composition for intravenous, cutaneous, or subcutaneous
injection should
contain, in addition to protein of the present invention, an isotonic vehicle
such as Sodium
Chloride Injection, Ringer s Injection, Dextrose Injection, Dextrose and
Sodium Chloride
Injection, Lactated Ringer's Injection, or other vehicle as known in the art.
The
pharmaceutical composition of the present invention may also contain
stabilizers,
preservatives, buffers, antioxidants, or other additives known to those of
skill in the art.
The amount of protein of the present invention in the pharmaceutical
composition
of the present invention will depend upon the nature and severity of the
condition being
treated, and on the nature of prior treatments which the patient has
undergone.
Ultimately, the attending physician will decide the amount of protein of the
present
invention with which to treat each individual patient. Initially, the
attending physician
will administer low doses of protein of the present invention and observe the
patient's
2 0 response. Larger doses of protein of the present invention may be
administered until the
optimal therapeutic effect is obtained for the patient, and at that point the
dosage is not
increased further. It is contemplated that the various pharmaceutical
compositions used
to practice the method of the present invention should contain about 0.01 Ilg
to about 100
mg (preferably about 0.lng to about 10 mg, more preferably about 0.1 pg to
about 1 mg)
2 5 of protein of the present invention per kg body weight.
The duration of intravenous therapy using the pharmaceutical composition of
the
present invention will vary, depending on the severity of the disease being
treated and
the condition and potential idiosyncratic response of each individual patient.
It is
contemplated that the duration of each application of the protein of the
present invention
3 0 will be in the range of 12 to 24 hours of continuous intravenous
administration.
Ultimately the attending physician will decide on the appropriate duration of
intravenous
therapy using the pharmaceutical composition of the present invention.
Protein of the invention may also be used to immunize animals to obtain
polyclonal and monoclonal antibodies which specifically react with the
protein. Such
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antibodies may be obtained using either the entire protein or fragments
thereof as an
immunogen. The peptide immunogens additionally may contain a cysteine residue
at the
carboxyl terminus, and are conjugated to a hapten such as keyhole limpet
hemocyanin
(KLH). Methods for synthesizing such peptides are known in the art, for
example, as in
R.P. Merrifield, J. Amer.Chem.Soc. 5 2149-2154 (1963); J.L. ICrstenansky, et
al., FEBS Lett.
211, 10 (1987). Monoclonal antibodies binding to the protein of the invention
may be
useful diagnostic agents for the immunodetection of the protein. Neutralizing
monoclonal
antibodies binding to the protein may also be useful therapeutics for both
conditions
associated with the protein and also in the treatment of some forms of cancer
where
abnormal expression of the protein is involved. In the case of cancerous cells
or leukemic
cells, neutralizing monoclonal antibodies against the protein may be useful in
detecting
and preventing the metastatic spread of the cancerous cells, which may be
mediated by
the protein.
For compositions of the present invention which are useful for bone,
cartilage,
tendon or ligament regeneration, the therapeutic method includes administering
the
composition topically, systematically, or locally as an implant or device.
When
administered, the therapeutic composition for use in this invention is, of
course, in a
pyrogen-free, physiologically acceptable form. Further, the composition may
desirably
be encapsulated or injected in a viscous form for delivery to the site of
bone, cartilage or
2 0 tissue damage. Topical administration may be suitable for wound healing
and tissue
repair. Therapeutically useful agents other than a protein of the invention
which may also
optionally be included in the composition as described above, may
alternatively or
additionally, be administered simultaneously or sequentially with the
composition in the
methods of the invention. Preferably for bone and/or cartilage formation, the
2 5 composition would include a matrix capable of delivering the protein-
containing
composition to the site of bone and/or cartilage damage, providing a structure
for the
developing bone and cartilage and optimally capable of being resorbed into the
body.
Such matrices may be formed of materials presently in use for other implanted
medical
applications.
3 0 The choice of matrix material is based on biocompatibility,
biodegradability,
mechanical properties, cosmetic appearance and interface properties. The
particular
application of the compositions will define the appropriate formulation.
Potential
matrices for the compositions may be biodegradable and chemically defined
calcium
sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid, polyglycolic
acid and
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polyanhydrides. Other potential materials are biodegradable and biologically
well-
defined, such as bone or dermal collagen.. Further matrices are comprised of
pure proteins
or extracellular matrix components. Other potential matrices are
nonbiodegradable and
chemically defined, such as sintered hydroxapatite, bioglass, aluminates, or
other
ceramics. Matrices may be comprised of combinations of any of the above
mentioned
types of material, such as polylactic acid and hydroxyapatite or collagen and
tricalciumphosphate. The bioceramics may be altered in composition, such as in
calcium-
aluminate-phosphate and processing to alter pore size, particle size, particle
shape, and
biodegradability.
Presently preferred is a 50:50 (mole weight) copolymer of lactic acid and
glycolic
acid in the form of porous particles having diameters ranging from 150 to 800
microns.
In some applications, it will be useful to utilize a sequestering agent, such
as
carboxymethyl cellulose or autologous blood clot, to prevent the protein
compositions
from disassociating from the matrix.
A preferred family of sequestering agents is cellulosic materials such as
alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose,
ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-
methylcellulose, and carboxymethylcellulose, the most preferred being cationic
salts of
carboxymethylcellulose (CMC). Other preferred sequestering agents include
hyaluronic
2 0 acid, sodium alginate, polyethylene glycol), polyoxyethylene oxide,
carboxyvinyl
polymer and polyvinyl alcohol). The amount of sequestering agent useful herein
is 0.5-20
wt%, preferably 1-10 wt% based on total formulation weight, which represents
the
amount necessary to prevent desorbtion of the protein from the polymer matrix
and to
provide appropriate handling of the composition, yet not so much that the
progenitor cells
2 5 are prevented from infiltrating the matrix, thereby providing the protein
the opportunity
to assist the osteogenic activity of the progenitor cells.
In further compositions, proteins of the invention may be combined with other
agents beneficial to the treatment of the bone and/or cartilage defect, wound,
or tissue in
question. These agents include various growth factors such as epidermal growth
factor
3 0 (EGF), platelet derived growth factor (PDGF), transforming growth factors
(TGF-a and
TGF-Vii), and insulin-like growth factor (IGF).
The therapeutic compositions are also presently valuable for veterinary
applications. Particularly domestic animals and thoroughbred horses, in
addition to
humans, are desired patients for such treatment with proteins of the present
invention.
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The dosage regimen of a protein-containing pharmaceutical composition to be
used in tissue regeneration will be determined by the attending physician
considering
various factors which modify the action of the proteins, e.g., amount of
tissue weight
desired to be formed, the site of damage, the condition of the damaged tissue,
the size of
a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and
diet, the severity
of any infection, time of administration and other clinical factors. The
dosage may vary
with the type of matrix used in the reconstitution and with inclusion of other
proteins in
the pharmaceutical composition. For example, the addition of other known
growth
factors, such as IGF I (insulin like growth factor I), to the final
composition, may also effect
the dosage. Progress can be monitored by periodic assessment of tissue/bone
growth
and/or repair, for example, X-rays, histomorphometric determinations and
tetracycline
labeling.
Polynucleotides of the present invention can also be used for gene therapy.
Such
polynucleotides can be introduced either in vivo or ex vivo into cells for
expression in a
mammalian subject. Polynucleotides of the invention may also be administered
by other
known methods for introduction of nucleic acid into a cell or organism
(including, without
limitation, in the form of viral vectors or naked DNA).
Cells may also be cultured ex vivo in the presence of proteins of the present
invention in order to proliferate or to produce a desired effect on or
activity in such cells.
2 0 Treated cells can then be introduced in vivo for therapeutic purposes.
Patent and literature references cited herein are incorporated by reference as
if
fully set forth.
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SEQUENCE LISTING
{1) GENERAL INFORMATION:
(i) APPLICANT: Jacobs, Kenneth
McCoy, John M.
LaVallie, Edward R.
Racie, Lisa A.
Merberg, David
Treacy, Maurice
Spaulding, Vikki
Agostino, Michael J.
(ii) TITLE OF INVENTION: SECRETED PROTEINS AND POLYNUCLEOTIDES
ENCODING THEM
(iii) NUMBER OF SEQUENCES: 34
(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: 2355 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
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(ii) MOLECULE TYPE: cDNA
(xi)
SEQUENCE
DESCRIPTION:
SEQ
ID N0:1:
CGCTTTTTTTTTTTTTTTTTTTCAGAAGGAGGAAGCTCATTATGTTTGGATCACCCACAG60
CTATAGATTCTAAAAATATTTTGGCTTTTTTTGAGGTGCTTTAGTAAAATATAACCCCAA120
ATGATTCACTTGGACAAGTGGTCTTAACAGCAAGGAAAACAAACACTTTATGAAAACAGC180
TATAAGCCTTCTGTCTTTTATCTTTACTATTTTCTCCGAGTCTGGCATGAAACAGATACA240
CAGCAGCCTCCACAGGGGGTTAAGTARAGAACCATCCAAGCATCACAGAGTGTCATCCAG300
AATTCTGATGACTTCCATTCGTTGACTCTGATGCACAATATGCCTGGCTTGGGATGCAGC360
GACCATGATGCCCCTCCCAGAACAGACACTTGCAGAGTGTTCCAGGAACAGCAGCTCCCT420
CCAGCCCCCAGCACAAGATGCACACATCTCAGAACAAGCCTCCATCCTTTTCCTAGAGAA480
CTGAGCATAAATAACTTGTTCTATATCTGGCTCCAAGTCCATTTCTGTTCTGTCTTGGAG540
TAGAGTCTTAGCTCCCAGTTTGTTTTAGGTCAACTTTCAGCACCTACTTCAGCTCACTTG600
TTTGATTTACTAAGCTCTTGCTTCTGTATATTATCAAATGTAGGGATGTAGGGAGAATAA660
AAGGATCTAGATACTTGCTTTTAGGAGAGATTAGAACAAAGCTGAAGGTGGAGGCATTAG720
TTCCTAGGTCTTCAGATCTCAGAGCAAAGGACCCACTCTGGAGCCTAAATTCTATGAGAG780
ACCACAGAGCAGCCTGAAATCCAAAGGAGTTTTACACAGGAAAAAAAAAATACTGTGAGG840
ACTTACACTAAATAATAATGTTGTTTTGAATGGGGTTGTGGGTAATTCCTATATTCTTCT900
TTATAACTTTTGTACTTTTCAAATTCCCTAATGTGAACTCACTACTTAGTAGGTCTGTAA960
GCTTAAACATTACTATGGCTTGGAATCTCATTTCP~P.AAAATCTTTAAAATGGGGACAAGA1020
GTAAAAATTTCTTAGCTTCTATGGAAGAATAAAATGAAATTATAATGATACAGTGCCTGG1080
CATGTTGTGGTCGCTCAATAAACACTGCTTTCCTCCCCATTGTCCTCCTCTTTATTCTGT1140
TTCATTACAAGGTCAGCAGATTGAATCAGGACCAGCTGGGAGGGCTACTTCTATGAGAGA1200
AGATCTGTCCACAGTCATGGTTTTCAATGTTTAGTGCACCAGAATCACCTTGAGGGTTTG1260
TTAAAACAGACTGCTGAACATAACACATCTATGAGAATGGCCAAAATCCAGAACACCAAA1320
TGCTGGTGAGGATGTGGAGCAATAAAAACTCTCATTTATTGCTGATGGCAATGCAAAATG1380
GTACAGCCACTTTGGAAGACAATTTGCCAAATTTTTACAAAACTAAGTGTACTCTTACCA1440
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TACAATCTAGCAATCATGCT CCCTGGTATT AGTTAAAAAC TTATGTCTAG1500
TACCTAAAGG
ACAGAAACCTGCATATGAAT GTTTATAGCAGTTTTTTTCATAATTGCTAA ACTTTGGAAG1560
TAACCAAGATGCCCTTCAGC AGGTGAATGGACAAATAAACTGCAGTAGAT GCAGACAGTG1620
GAATATCATTCTAGGCCATG AAGGCCGAATTCGGCCTTCATGGCCTAATT AAAGAAAGTC1680
AGGATAAAAATTTTAAAAAG CAGGCCACTGTCAGCAAAGCCTGGAGAAGT GGGGCCGGAG1740
GYTCCGCCCCCATCATGTGC CTGCCACCCCTTCCCAGTCATCCCTTTAYT CTTACAGTAG1800
CAAATAAGACCCCTGTCTAA TGGGGGGAGACAAATGTGTAGACCCTTAGC CACCTTGGCC1860
AGGGCTGACTCCTTAAATTT CTGGATGATGATGATTGTTATTTAATAGCC AGAGGCTCAT1920
ATAATTGGCCTCTTTGGAAG AGGCCTCATGGCCTCCTTACTCTCACCAAA GCAATTTTTC1980
CCTCAGGGGGGCTCCCATCT TCTTACACAGAGAGGCAGCTGAGGCAGGAC AGTGGGGCTA2040
ACTGTAGACCAGGCGAGGGC ACGGGCTGCTGGGGTGGCCCTGCTTCCCCA GTGTACATAT2100
TGTATCTGTGTAACATTTTG TATATTCCAGGGGTAGGGCCGCCCCCTGTA TCATACCTAG2160
CAGAGGTTGGAGCTGGCACA TGGGGAGGAGGTTCTAATAATTATTTGGGG CTGGGAAACT2220
TATTTATTGATAGCATAGGA CAGAGGAAGGAGGCGGGGATGGGGTCGTGG CGCCCTGGTG2280
ATGCGACTCCTGTTTATTTT GCTTTTTATTTCGGAATAAATGGATTTAGC CATAAAAAAA2340
~ AAAAA 2
3
5
5
(2) INFORMATION
FOR SEQ
ID N0:2:
(i) S EQUENCE CHARACTERISTICS:
(A) LENGTH: 51
amino acids
(B) TYPE: amino
acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE
TYPE:
protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met Lys Thr Ala Ile Ser Leu Leu Ser Phe Ile Phe Thr Ile Phe Ser
1 5 10 15
Glu Ser Gly Met Lys Gln Ile His Ser Ser Leu His Arg Gly Leu Ser
20 25 30
Xaa Glu Pro Ser Lys His His Arg Val Ser Ser Arg Ile Leu Met Thr
35 40 45
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Ser Ile Arg
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2496 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE
DESCRIPTION:
SEQ ID
N0:3:
GCGCCCTTTCGGTCAACATCGTAGTCCACCCCCTCCCCATCCCCAGCCCCCGGGGATTCA 60
GGCTCGCCAGCGCCCAGCCAGGGAGCCGGCCGGGAAGCGCGATGGGGGCCCCAGCCGCCT 120
CGCTCCTGCTCCTGCTCCTGCTGTTCGCCTGCTGCTGGGCGCCCGGCGGGGCCAACCTCT 180
CCCAGGACGACAGCCAGCCCTGGACATCTGATGAAACAGTGGTGGCTGGTGGCACCGTGG 240
TGCTCAAGTGCCAAGTGAAAGATCACGAGGACTCATCCCTGCAATGGTCTAACCCTGCTC 300
AGCAGACTCTCTACTTTGGGGAGAAGAGAGCCCTTCGAGATAATCGAATTCAGCTGGTTA 360
CCTCTACGCCCCACGAGCTCAGCATCAGCATCAGCAATGTGGCCCTGGCAGACGAGGGCG 420
AGTACACCTGCTCAATCTTCACTATGCCTGTGCGAACTGCCAAGTCCCTCGTCACTGTGC 480
TAGGAATTCCACAGAAGCCCATCATCACTG.GTTATAAATCTTCATTACGGGAAAAAGACA 540
CAGCCACCCTAAACTGTCAGTCTTCTGGGAGCAAGCCTGCAGCCCGGCTCACCTGGAGAA 600
AGGGTGACCAAGAACTCCACGGAGAACCAACCCGCATACAGGAAGATCCCAATGGTAAAA 660
CCTTCACTGTCAGCAGCTCGGTGACATTCCAGGTTACCCGGGAGGATGATGGGGCGAGCA 720
TCGTGTGCTCTGTGAACCATGAATCTCTAAAGGGAGCTGACAGATCCACCTCTCAACGCA 780
TTGAAGTTTTATACACACCAACTGCGATGATTAGGCCAGACCCTCCCCATCCTCGTGAGG 840
GCCAGAAGCTGTTGCTACACTGTGAGGGTCGCGGCAATCCAGTCCCCCAGCAGTACCTAT 900
GGGAGAAGGAGGGCAGTGTGCCACCCCTGAAGATGACCCAGGAGAGTGCCCTGATCTTCC 960
CTTTCCTCAACAAGAGTGACAGTGGCACCTACGGCTGCACAGCCACCAGCAACATGGGCA 1020
GCTACAAGGCCTACTACACCCTCAATGTTAATGACCCCAGTCCGGTGCCCTCCTCCTCCA 1080
GCACCTACCACGCCATCATCGGTGGGATCGTGGCTTTCATTGTCTTCCTGCTGCTCATCA 1140
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TGCTCATCTT CCTCGGCCAC TACTTGATCCGGCACAAAGG 1200
AACCTACCTG
ACACATGAGG
CAAAAGGCTC CGACGATGCT CCAGACGCGGACACGGCCATCATCAATGCA GAAGGCGGGC1260
AGTCAGGAGG GGACGACAAG AAGGAATATTTCATCTAGAGGCGCCTGCCC ACTTCCTGCG1320
CCCCCCAGGG GCCCTGTGGG GACTGCTGGGGCCGTCACCAACCCGGACTT GTACAGAGCA1380
ACCGCAGGGC CGCCCCTCCC GCTTGCTCCCCAGCCCACCCACCCCCCTGT ACAGAATGTC1440
TGCTTTGGGT GCGGTTTTGT ACTCGGTTTGGAATGGGGAGGGAGGAGGGC GGGGGGAGGG1500
GAGGGTTGCC CTCAGCCCTT TCCGTGGCTTCTCTGCATTTGGGTTATTAT TATTTTTGTA1560
ACAATCCCAA ATCAAATCTG TCTCCAGGCTGGAGAGGCAGGAGCCCTGGG GTGAGAAAAG1620
CP.AAAAACAA ACAAAAAACA AAACCCTGGAGTGTTAGGAGGAGAGTGAAG GTAGAGGGGT1680
GAGGAAGGGT AAGGGGCAGG GCTGGTTTCAGCTGGGGGCTCTCACCAGCC CTCCTTTCAG1740
CCTCTACAAC AGAGCAGCTT CCCAGACTTCTCCAGGAACCCAGAAACGGG ATGGTTGTCG1800
GCAAAGGTTG GGAGTGGCTT TTCCTCTGGTAGCCACACACCTGAGCACTA CGGACAGGGA1860
GGCAGGTGCC ACCTTGACAC CTCTCTTCCATAGCAATGGGAAAGTGATGA GTGCGGGAGT1920
CCTGAGGAGA TGTGGCCTGC AGACAACATGCAGCCATGCAGGGACCCAGG ACTGTAACCT1980
GGGGAGGACG CGGGTCCCTG CAAGGAAGAGTAGATTTGGAGAGGAAGGAT GGAGGTGGAC2040
TCTCACCCCA TTCCCCCCGG AAATGAACAAAGCCGGGCCCTTTCCATAGG AACTGCCCTT2100
GGAGATAGCA GAGTGTGGCT GCCCCTCCTTGCTCCAGCAGCAGTGGGAGA GGCACTGCTC2160
TGGGGCCTGA ACTGCCTCTG CTTCCCCCCCTGAGGGGCCCCTCACTCTTA CCCAAGACTC2220
TGGATTGTTG CACGGCAACC ACTCCTCCCATGGCATTGCTCAGCAACTAC TTCTCCCTTC2280
CCGGCCACCC TGTGCCCCCT TCCTGGTCCCAACGCCAGCCCTTCATCCTT CCTCCCTCAG2340
CAGCCAGGCA GACATAACAA CAAAACTACTAAAAGGAGCTTCP,AAAAAAA
AAAAAAAAAA2400
AAAAAAAAAA P~~~AAAAAAA AAA,AAAAAAAAAAAAAAAAAP,~~~AAAAAAA
AAAAAAAAAA2
4
6
0
AAAAAAAAAA p~~iA,AAAAAAA AAAAAAAAAAAAAAAA 2
4
9
6
(2) INFORMATION FOR
SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 398 acids
amino
(B) TYPE: amino
acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE:
protein
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
Met Gly Ala Pro Ala Ala Ser Leu Leu Leu Leu Leu Leu Leu Phe Ala
1 5 10 15
Cys Cys Trp Ala Pro Gly Gly Ala Asn Leu Ser Gln Asp Asp Ser Gln
20 25 30
Pro Trp Thr Ser Asp Glu Thr Val Val Ala Gly Gly Thr Val Val Leu
35 40 45
Lys Cys Gln Val Lys Asp His Glu Asp Ser Ser Leu Gln Trp Ser Asn
50 55 60
Pro Ala Gln Gln Thr Leu Tyr Phe Gly Glu Lys Arg Ala Leu Arg Asp
65 70 75 BO
Asn Arg Ile Gln Leu Val Thr Ser Thr Pro His Glu Leu Ser Ile Ser
85 90 95
Ile Ser Asn Val Ala Leu Ala Asp Glu Gly Glu Tyr Thr Cys Ser Ile
100 105 110
Phe Thr Met Pro Val Arg Thr Ala Lys Ser Leu Val Thr Val Leu Gly
115 120 125
Ile Pro Gln Lys Pro Ile Ile Thr Gly Tyr Lys Ser Ser Leu Arg Glu
130 135 140
Lys Asp Thr Ala Thr Leu Asn Cys Gln Ser Ser Gly Ser Lys Pro Ala
145 150 155 160
Ala Arg Leu Thr Trp Arg Lys Gly Asp Gln Glu Leu His Gly Glu Pro
165 170 175
Thr Arg Ile Gln Glu Asp Pro Asn Gly Lys Thr Phe Thr Val Ser Ser
180 185 190
Ser Val Thr Phe Gln Val Thr Arg Glu Asp Asp Gly Ala Ser Ile Val
195 200 205
Cys~Ser Val Asn His Glu Ser Leu Lys Gly Ala Asp Arg Ser Thr Ser
210 215 220
Gln Arg Ile Glu Val Leu Tyr Thr Pro Thr Ala Met Ile Arg Pro Asp
225 230 235 240
Pro Pro His Pro Arg Glu Gly Gln Lys Leu Leu Leu His Cys Glu Gly
245 250 255
Arg Gly Asn Pro Val Pro Gln Gln Tyr Leu Trp Glu Lys Glu Gly Ser
260 265 270
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Val Pro Pro Leu Lys Met Thr Gln Glu Ser Ala Leu Ile Phe Pro Phe
275 280 285
Leu Asn Lys Ser Asp Ser Gly Thr Tyr Gly Cys Thr Ala Thr Ser Asn
290 295 300
Met Gly Ser Tyr Lys Ala Tyr Tyr Thr Leu Asn Val Asn Asp Pro Ser
305 310 315 320
Pro Val Pro Ser Ser Ser Ser Thr Tyr His Ala Ile Ile Gly Gly Ile
325 330 335
Val Ala Phe Ile Val Phe Leu Leu Leu Ile Met Leu Ile Phe Leu Gly
340 345 350
His Tyr Leu Ile Arg His Lys Gly Thr Tyr Leu Thr His Glu Ala Lys
355 360 365
Gly Ser Asp Asp Ala Pro Asp Ala Asp Thr Ala Ile Ile Asn Ala Glu
370 375 380
Gly Gly Gln Ser Gly Gly Asp Asp Lys Lys Glu Tyr Phe Ile
385 390 395
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2764 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE
DESCRIPTION:
SEQ ID
N0:5:
GGGCCAAAGAGGCCTACCAGCTGCTGTTGACCGCTGGACTCACAAACCTTTCTTTCTACT 60
CTTGTTTTTCATTCACTTTGGGTCATTTTTCAGTGTTGATGGGGACGTAATAAAGCACGG 120
TAAGAAAATCCGTGAATTCCGTCAGAGCAGTCGTCCAGAGGGAAGGCGCGCCCGGCGTAG 180
GGAGGTCAGAGCTCATGTTAGCTATGAACACAGGTCACAGGGGCGTACGGCGATGGGAAA 240
CACTGAGATGCTCAATATATTGATTATTTAATAGTGTTTAGCAAAATGGTCTTTTTTTAT 300
TCCTTAAATCAACTGAAACTCACTTCACGTCTCTTTCCTTGTAGAGCATCATGCTTATTT 360
CTGGCTCACTCACATCTTTGTCTCGGGAGTTCTCTGCCGAGCCATTGCCCCCTACAGCAG 420
AGAGCACAGCTGGCTGCACTAGTGCTGAAGGAGCCAGCCCCAGAGCAGGGCATTTCCAGG 480
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GGCTCTTGTCCCAGAGCGGCAGGCGTTGTG GCCCCTCCCACGCAGCACAG 540
TGCAGAGAAC
AGAACGCGGGGTGGGTGTGTGGCTCCGGGCCTGTGGGGCTTAGGCTGCCTGAACCACCGC 600
CGACTGGCACCATGACTCGGCATTCCTGGAAGTGCCTTACCAAGTTGTTGTTGTTGTTTfi660
GTTGTTTTTTAAGAGACGGGCTTGCTCTATCATCCAGGCTCGAGTGCAATGGCACAGTCA 720
CAGCTCACTGCAGCCTTGAACTCGTGGGCTCAAGCCATCCTCCTGTGTCAGCCTCCCCAG 780
TACCTGGGACTGTGGGCATGAGCACTGCGCCTGGCAGCTGTATCAGTGTTGACTCCACAT 840
TTTAATAGTTGCTTCTTGAAATTAAAATGCTTTGATTCAGCCTTCAAGCCATCAGGAAAG 900
TTTGCCCCTCTGAGTCACACCTGGTGGTCTCCAGGGTTCCTGCCCCTCCCTCCTGAGCCA 960
GCTCCTCAGAGCGGATAGAGGCAGGACCCCCACCCAGGTCTTGAGACCCCCCTGCCCCGC 1020
ACTCCCCCGGAGACGGGCTACCCCTGCAGATGCAGATAGTCAAAGCTCAGGTTTCTTCCA 1080
AAGCTTTTAAAAAGATATTGTACCTTGAGCACTTTAAAAATGTCTTAAAATTGCCATACA 1140
GGCTCTTAAAAGCTTATACGTTTAAACTGTTGATAGATGGGCCTTTACTAAAATGCATTC 1200
ATTTATTTTCCTAATCCCTTGGTTGTTAAATAATTCTGGGGAAGGGCCCCGAGCACGACA 1260
GCCGCAGTCTCCACCCAGAACCAGAGAGTCCCCCCCAACCCGGGATGTACCCTCTGGCCA 1320
CACCAGGGACCCTGCCAGAGGCCGCAGACTGGCAGCAGCAGCCTCCCCACACAGTGGGGG 1380
AAGGTCAGTGTGATGCCTTCAGGCCCCGTCTCCTGCCAGGGCTCTCCCTCCAGCCTACAT 1440
AGGGCCTCAGAGAAATGCATTTTTAGTTCTGGCTTTGGCCCAGCCCAGGGCAAGGCAGGA 1500
AACTCTCCAGCGTGAGTCCGTGAGGGCCAAGAAGTCCCGCCCTGTTCTGGGGGAGGACCT 1560
GGCTTTTCTGGTGTCTCTGGTGCCCGAGAGCCCGGTGCTGCCATCTTTAGTGAAAGAGTA 1620
AATGGTGGCCGAGGGCTCCTTTTGTGAGGGATGTGCCTTGGTGAAGAAGGCATGTTCCCT 1680
GCCGTGAAGATACTTGGAAGCTCTGGGTGGAGAGGGAAAAGGGATACCCCTGGTGCTCCC 1740
TGGGCCTGGCGGAAGGCTAGGAGGAAGGACAGCTGAGGTGAGGACTGAGTGGGGCAGGTA 1800
TCACCCTGACAAACAGTTTGGGAAGATCAGGAAAGGCAGGTGAGACCTGGTGCAGAATCC 1860
AGGTTGGGTAATAGATACATCGTCGAAGATGTAGCAAGCAAAGTAATATACTCAACTCTG 1920
GAACATTGCACAGAAGCTTTTAAAGCACTCTGTGACACTTTTTGTAATGAGGGATCTGAA 1980
GGAAACGGCCCCAGAGTCACCCATCCCCACGGGTCTGGTTGGCGGGGCTGGTGCCTTTCT 2040
TCTGCACTCAGTCACCATGGCTCCGTCTGTCAAACTCAACTCTTTTTTTTTTTTTTTTTC 2100
TTCTCTTGGTGTGGTAATTTGTTTGAAGAGCCACTCCATCCCCAAATTCAAGATTAGAAA 2160
72
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GATCCCTGAC TGCTTCTCAA GATCCAGAAC ATTCCTTGACAGAGTATATT CACCATTTAG2220
AAGTGATCCA GCAAAGATTG GGAGGGGTAC TACCAGATTCTACTTCAAAG A~1ATCCTGCC2280
ACCCGATGAT TAAACAGTGA ATAAAATGTC ATGGCTCTTTCCTGCGACAA TTCTATTTGA2340
GGAAAAGATT TGTTTTTCCC TTTTCCCAAG GAAGCTCGTGGGACAGCATG GGCACTACTC2400
TTCATGTGCG GTGACACCAG CCCCCAGATG CCTTGAATTAAGTGTCCTCA CCTTTATGCA2460
TGACTGCAAA GCCAGCTGGA GCATTTTCTA TGGAGCCTCCGTATGTTTTA GGCCCATGAC2520
CTTCGTGAGG TGATGGGCAC TCACTCCCAT GAGCCCTGGCTGTGTGCTGT TGTGTGCCTA2580
TCGGCAGATC CATCCTTCCT GCCTCCAAGG AGGATACACAGAGAATGGCT TCCTGTTGTT2640
TTGTTTATTT TCTTAACGTG TACAGATGGA AACTTCATTTAAAAATAAAA ACAAAACAAY2700
TCNAAAAAAA AAAAAAAAAA AAAAAi~AAAA A,AAAAAAAAAp,~~ AAAAAAAAAA 2
7
6
0
2764
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 164 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
Met Leu Ile Ser Gly Ser Leu Thr Ser Leu Ser Arg Glu Phe Ser Ala
1 5 20 15
Glu Pro Leu Pro Pro Thr Ala Glu Ser Thr Ala Gly Cys Thr Ser Ala
20 25 30
Glu Gly Ala Ser Pro Arg Ala Gly His Phe Gln Gly Leu Leu Ser Gln
35 40 45
Ser Gly Arg Arg Cys Val Gln Arg Thr Pro Leu Pro Arg Ser Thr Glu
50 55 60
Asn Ala Gly Trp Val Cys Gly Ser Gly Pro Val Gly Leu Arg Leu Pro
65 70 75 80
Glu Pro Pro Pro Thr Gly Thr Met Thr Arg His Ser Trp Lys Cys Leu
85 ~90 95
73
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Thr Lys Leu Leu Leu Leu Phe Cys Cys Phe Leu Arg Asp Gly Leu Ala
100 105 110
Leu Ser Ser Arg Leu Glu Cys Asn Gly Thr Val Thr Ala His Cys Ser
115 120 125
Leu Glu Leu Val Gly Ser Ser His Pro Pro Val Ser Ala Ser Pro Val
130 135 140
Pro Gly Thr Val Gly Met Ser Thr Ala Pro Gly Ser Cys Ile Ser Val
145 ~ 150 155 160
Asp Ser Thr Phe
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3367 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi)
SEQUENCE
DESCRIPTION:
SEQ
ID N0:7:
CAGAAGGGAGGTAGTCGCCCTCCGTCGTGGCCTGGCGTGGATTCCGAGCGTTGGTGTCTG60
GCGGTTTCCGACCGTTGGTGTCTGGCACGCGCCACCCCGATGTACCAGGTAAAGCCCTAT120
CACGGGGTCGGCGCCCCTCTCCGTGTGGAGCCCACCTGCATGTACTGGCTCCCCAACATG180
CACGGCAGGAGCGGCGGCCCAGCACTCGGCACTGGCCACTTGCAGACAAGAAGACAAGAA240
AATGATTTGAGGACAGCTTCAATCGCGGTGTGAAGAAGAAAGCAACAAAACGACCACTGA300
AAACAATGCCGGTGGCAAAACATCCAAAGAAAGGGTCCCAAGCGGTACATCGTCATAGCT360
GGAAACAGTCAGAGCCACCAGCCAATGATCTTTTCAATGCTGCGAAAGCTGCCAAAAGTG420
ACATGCAGTGTGGCCATGAGGTCTGCCGGAAGTGACTTGTTGGTGTTATCTCCTGAGTTA480
AAATGTGAAGGGATTTTTTTTTTTCAGATTACTGAGAGTCTTCTGTTACTAGTTTGTCTT540
TCCTAGATCCAGACACGGGGACTGCAGAGAAAGGCTGTGTGCATCCGCTGTCTACTCCAC600
TGTCTCCTCTGCAGAGGCGGATTTCCCTGACTGAAGACCATGTTGCAGGCCCACAGCTGC660
CTACAGAACCGTCCCAAAATATGGCAAAGAAACCTATTCTGAGGGTCTCACCATGTTGCC720
CAGGCTGGTCTTGAACTCCTGGACTCATCCTAAAGTGCTGGCCTCTCATTCCCTGTCTGT780
74
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GCACACCTCA CGGCAAGGGCCAGCCTGTTTCCTCCCGGTCACCTCCAAATCTTGCTGCTT840
TTAATTCAAC TCAGAGGCCTAGCCAGGGTTGAGTTCTCACCCACCTGTGCCGCCCTGCCT900
TGTTACCTGG AAGCACAGCCTTGGGGACTGAGCAGGCCCTCACTGTCACTTTAAGAAGGG960
AATCAGCCAC TTTGTGCTCACCACCTCTGGGGAAGGTGTGAGAGGAGAGAAGGAAGTGGC1020
TGTTTGGCTG CTGACAACATGAAGACTTCCTGCGATGAGAACAGAGGCACAGGTGCCGGC1080
CCTGCAGCCC CCAGAACCCGGACTGGAGGGGGCCATGGGGCGCCGGACCCTGGCCCTGCC1140
CTGGGTGCTG CTGACCCTGCGTGTCACTGCAGGGACCCCGGAGGTGTGAGTACAAGTTCG1200
GATGGAGGCC ACCGAGCTCTCGTCCTTCACCATCCGTTGTGGGTTCCTGGAGTCTGGCTC1260
CATCTCCCTG GTGACTGTGAGCTGGGGGGGCCCCGATGGTGCTGGGGGGACCACGCTGGC1320
TGTGTTGCAC CCGGAACTTGGCATCCAGCAATGGGCCCCTGCTCGCCAGGCCCGCTGGGA1380
AACCCAGAGC AGCGTCTCTCTTGCCCTGGAAGTCTCTGGGGCCAGCAGCCCCTGCACCAA1440
CACCACCTTC TGCTGCAAGTTTGCGTCCTTCCCTGAGGGCTCCTGGGAGGCCTCTGGGAG1500
CCTCCCGCCC AGCTCAGACCCAGGGCTCTCTGTCCCGCCGACTCCTGCCCCCATTCTGCG1560
GGCAGACCTG GCCGGGATCTTGGGGGTCTCAGGAGTCCTTCTCTTTGACTGTGGCTACCT1620
CCTTCATCTG CTGTGCCGACAGAAGCACCGCCCTGCCCCTAGGCTCCAGCCATCCCACAC1680
CAGCTCCTAG GCACTGAGAGCACGAGCATGGGCACCCAGCCAGGCCTCCCAGGCTGCTCT1740
CCACGTCCCT TATGCCACTATCAACACCAGCTGCTGCCCAGCTACTTTGGACACAGCTCA1800
CCCCCGACAG GGGGCCGTCCTGTCGTTTCCTGCTGTGACTAAGTCAGCAACACAGTTCCT1860
CTGACATGGG CCTTGGCTGTGCTTCTTTGGGGGTGAAGAGATTGGGGAGGAAGTCTCCAC1920
CCCTGGGAGG CAGAAGCCAGGCATAGCGCGCTGGCTAGGACTCCAGTACCGTGAAGGGAG1980
GCAGTGAGAG CAGACATCTGTGTCTCATTCCTGATCTCAAGGGGAAAGCAAGAACAAGGG2040
AGGCTTCCTC AGGATCTCAAACCTGCGGAAGGAGGACCAGTCTGTGTACTTCTGCCAAGT2100
CCAGCTGGAC ATACAGATCAGCCCTCAGGCAGCCCCTCCACAGGACCCCTCTCCTGCCTG2160
GACAGCTCTG CTGGTCTCCCCGTCCCCTGGAGAAGAACAAGGCCATGGGTCGGCCCCTGC2220
TGCTGCCCCT GCTGCTCCTGCTGCAGCCGCCAGCATTTCTGCAGCCTGGTGGCTCCACAG2280
GATCTGGTCC AAGCTACCTTTATGGGGTCACTCAACCAAAACACCTCTCAGCCTCCATGG2340
GTGGCTCTGT GGAAATCCCCTTCTCCTTCTATTACCCCTGGGAGTTAGCCACAGCTCCCG2400
ACGTGAGAAT ATCCTGGAGACGGGGCCACTTCCACGGGCAGTCCTTCTACAGCACAAGGC2460
7$
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CGCCTTCCAT TCACAAGGAT TATGTGAACC GAACTGGACA GAGGGTCAGG2520
GGCTCTTTCT
AGAGCGGCTT CCTCAGGATC TCAAACCTGC CCAGTCTGTG TATTTCTGCC2580
GGAAGGAGGA
GAGTCGAGCT GGACACCCGG AGATCAGGGA GCAGTCCATC AAGGGGACCA2640
GGCAGCAGTT
AACTCACCAT CACCCAGGCT GTCACAACCA GACGCCCAGC AGCACAACCA2700
CCACCACCTG
CCATAGCCGG CCTCAGGGTC ACAGAAAGCA AGAATCATGG CACCTAAGTC2760
AAGGGCACTC
TGGACACTGC CATCAGGGTT GCATTGGCTG CAAAACTGTC ATTTTGGGAC2820
TCGCTGTGCT
TGCTGTGCCT CCTCCTGTGG TGGAGGAGAA CAGGGCGCCA AGCAGTGACT2880
GGAAAGGTAG
TCTGACCAAC AGAGTGTGGG GAGAAGGGAT CCCGGAGGAC GTGATGTGAG2940
GTGTATTAGC
ACCCGCTTGT GAGTCCTCCA CACTCGTTCC GATACATGGA GAGCACCCTG3000
CCATTGGCAA
AGGACCTTTA AAAGGCAAAG CCGCAAGGCA GGGTCCCTGA ATCACCGACT3060
GAAGGAGGCT
GGAGGAGAGT TACCTACAAG AGCCTTCATC CACACTGCAA TGATATAGGA3120
CAGGAGCATC
WTGAGGTCTG AACTCCACTG AATTAAACCA GGGGCTGTTC ATTATAGCAG3180
CTGGCATTTG
TGCAAAGAGT TCCTTTATCC TCCCCAAGGA AATTTATTTT GCTTACCATA3240
TGGAAAATAC
CACCCCTTTT CTCTTCGTCC ACATTTTCCA TGGCTGTCTT CTATGGCAGA3300
ATCTGTATGG
AGGTTTTGGG GAATAAATAG CGTGAAATGC AAAAAAAAAA P,~~~AAAAAAA3360
TAAAAAAAAA
AAAAAAA 3367
(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 226 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
Met Gly Arg Pro Leu Leu Leu Pro Leu Leu Leu Leu Leu Gln Pro Pro
1 5 10 15
Ala Phe Leu Gln Pro Gly Gly Ser Thr Gly Ser Gly Pro Ser Tyr Leu
20 25 30
Tyr Gly Val Thr Gln Pro Lys His Leu, Ser Ala Ser Met Gly Gly Ser
35 40 45
76
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Val Glu Ile Pro Phe Ser Phe Tyr Tyr Pro Trp Glu Leu Ala Thr Ala
50 55 60
Pro Asp Val Arg Ile Ser Trp Arg Arg Gly His Phe His Gly Gln Ser
65 70 75 gp
Phe Tyr Ser Thr Arg Pro Pro Ser Ile His Lys Asp Tyr Val Asn Arg
85 90 95
Leu Phe Leu Asn Trp Thr Glu Gly Gln Glu Ser Gly Phe Leu Arg Ile
100 105 110
Ser Asn Leu Arg Lys Glu Asp Gln Ser Val Tyr Phe Cys Arg Val Glu
115 120 125
Leu Asp Thr Arg Arg Ser Gly Arg Gln Gln Leu Gln Ser Ile Lys Gly
130 135 140
Thr Lys Leu Thr Ile Thr Gln Ala Val Thr Thr Thr Thr Thr Trp Thr
145 150 155 160
Pro Ser Ser Thr Thr Thr Ile Ala Gly Leu Arg Val Thr Glu Ser Lys
165 170 175
Gly His Ser Glu Ser Trp His Leu Ser Leu Asp Thr Ala Ile Arg Val
180 185 190
Ala Leu Ala Val Ala Val Leu Lys Thr Val Ile Leu Gly Leu Leu Cys
195 200 205
Leu Leu Leu Trp Trp Arg Arg Arg Lys Gly Ser Arg Ala Pro Ser Ser
210 215 220
Asp Phe
225
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3899 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
GGGAAGAGAT GGTGACTGAG GCAGAAGCTA ATAGGGAAGA TGATAGGAAA GAAATTTTAC 60
CCAAGGGAAT TAGATTTAGC AAGAGAGCGA AGGAAAGCTG AGAGGCCAAA AACATCTCTG 120
77
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AGGAAAACTGACTCTGAGAGAGAAGAGGTG 180
ACAAGGGCAA
ATGCACTCAA
GGATGAAGAT
GCTTTTAAAGAAGAGCAAAAACTTAAAGCGGAAGAAGGGGAAACAGAGACAGAAGTWAGA 240
GCTGAGGAAGAGACAAAAGCTCCCCCAAATGAAATGGGATCTGATGCTGARAACGAASCA 300
CCTGTGGAGGCTTCTGAGTTGTCTGACAATCCAGGGCTTCTAGGAGAARATTCACTAAAA 360
GAGACAGTGGTTCCCATATTTGAAGCAACGCCTGGATTTGAAAAGTCGCTGGAAAACATA 420
ACAGCTCTGAGGAAAGAAGGAGGAGGGGAAAGACTGAGTGAAGCCAGAGACACAGAGCAC 480
AAAGACAGAGAAGAGCTGTCCAGCAGGGAGAATAGGGCCCTGAAGGAAGGGCACCGCCAA 540
GATGGAGAGGGGGCCTTAGCAGCTCCTGAAGCTGAGCCAGCAGGAAAGGTGCAGGCCCCT 600
GAGGGGCTGATCCCAGCCACAGGCCAGGCAGAGGAGCTAGCAGCCAAAGATCACGACTCC 660
TGCGCAGGACTGGAGGGGAGAGCTGAAGGGCAAGGAGGAGTGGATGTCGTGCTAAGGACC 720
CAGGAAGCTGTTGCTGAGGAAGATCCCATA4JTGGCAGAAAAGTTCAGGGAGGAAGCGGTG 780
GATGAGGACCCAGAGGAGGAAGAGGACAAAGAGTGCAYTCTGGAGACAGAAGCGATGCAG 840
GACAGGAACTCGGAAGGGGACGGGGACATGGAAGGAGAAGGAAACACACAAAAGAATGAG 900
GGCATGGGAGGAGGAAGGGTTGTGGCTGTGGAAGTTCTACACGGAGGTGGTGAAACGGCA 960
GAAACAGCCGCAGAGGAGAGGGAGGTGTTGGCAGGTTCGGAGACAGCCGAGGAGAAAACA 1020
ATAGCAAATAAAGCCTCCTCCTTTTCAGATGTTGCTGAGGAAGAAACCTGGCACCAACAG 1080
GATGAGTTAGTAGGAAAAACAGCAGCTGCAGGGAAGGTGGTGGTAGAGGAATTAGCACGG 1140
AGTGGGGAGGAAGTGCCAGCAGCAGAGGAGATGACAGTGACATATACAACAGAGGCTGGG 1200
GTGGGCACTCCAGGAGCCCTGGAGCGGAAGACCTCAGGGCTAGGACAGGAGCAAGAGGAA 1260
GGGTCAGAGGGCCAGGAGGCAGCCACTGGGAGTGGCGATGGGAGGCAGGAGACAGGAGCA 1320
GCTGAAAAATTCCGATTAGGATTATCACGGGAGGGAGAGAGGGAATTGAGTCCGGAGAGT 1380
CTACAGGCGATGGCAACACTTCCAGTGAAGCCTGATTTCACTGAAACCCGAGAGAAGCAA 1440
CAGCATATGGTGCAAGGAGAAAGCGAGACTGCAGATGTTTCCCCCAACAACATGCAGGTC 1500
TAGGAGACTTGCTGGCAGACGGATAATTTAAAGATGTCTTCTGAAGATGTAAAGAGTGGA 1560
GAAAGATTCACGCAAGCATCTCACCAGGATTCTTGATTTTCTCTCTCTCCTCTTTAGTTG 1620
CTGGTTGCGCTTGTCTGAGATGATTCCCAATCTGTCAGCCCTGGTCAGTAGCTCAGTAAG 1680
CACCTTGAGAATAGCTCAAGTAGATCTGTAGGACCCTTCTTAGAAGCAGTGGTTCCTCAT 1740
GGAGAAACTTGTGAGGCTGTTACACATTCTACACACCTAACATTATTTTCAAACAAAAAT 1800
7g
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GATAATTTTC AGATGCTTGACTTTTACCAA CCTAATGTTA1860
AGATCACTGG
AAGGCCCAGT
GGGGTTTGTT TAAAGTCCTTTTTATTTTACAATACAGAGCCCCAGTCAATTCCACAATCT1920
CAATTTCATA CATGGGAATTTTATTTAAAAATCTGTGGTTTGGGGCTTTAATGAATTGGC1980
CTGTGAAAAT GAGCTCTAAATTTCCTCCCACGTACACTCAAAACTCAAGATTGCTCCAAA2040
TCTCTAAGTT CTTCCAGCAAAAGATTTCTTGGCATGTATATTCACTTATACTTAGAAATA2100
TTCATTCTTT TAATTTATGCCAGAATAACAAAGTGGAAATCTTATTTCAAAATGCTCTTT2160
GTTTTTTTGT GTGTGTTTCTGTAGTTCTGCTTTCTGGGGTAGACTAGTAAAATGGTAGCT2220
TCCAGCATTT TGTCCCTGGGGCCTTCTTTATAGGGCCACTCAAATTTAAATAAAAGTAGT2280
AAATAATTTA GCTAAGTGGAATAAGTATAATAATTATAGTGGTAAGCATAGCACATCAGC2340
ATTATGCCAA CATTCTAGACTCTTTAGTTGATGTCATTAAATGGAAAAGAAACTTGGATT2400
AAATGAGTGT GCTGCTCACCTTCCCAAGTTCTGTTATTTCAAACCTGTGAACTAACCTTG2460
CAGTTCATTA TAAATCAACAGTAACAACTGCATTCTAAATTACTCCCTGATATTATTTTC2520
TAGTTGTGTA TCAGCCTGTCTCCTAGGGGTTTTCATTTCCCTGAAGACATACAAGTGCCC2580
CAGAGCGCAT GTATATGTCTACCATTTCTCTATATGAGAAGGTAAAAAAAATTTCCTTAA2640
GCAGTGATTT TCCAGCCAGAATATACATTAGATTTTCATGGGACGCTTTTATAAATGACT2700
CAACCCTTTT CCCCACCCCAGAGATTCAGACTTAATTCGTTTTAGATGGATCTACACATC2760
AGTATATATA TATTTTTAACTTTTCACTTGATTCTTCTCTGTAGCCAAGGTTGAGAACCG2820
CTGTTCTAAA TCATCATATAATCCATGCTGGCCACATTACACTCAAGGTCCCTAGGGACC2880
AGGCATATTA TCATAGTAGGTATCTTCCATTTTAATGTGTAATGGAGCCATTCAATGATC2940
AAAAATACAC TGGACCAGATAGTAGACTGGTCCCTTGATCAGAAGCATCAGCACATCAGC3000
ATCACCTGGA AATTGTTCCCAGCCTTTGTCTCCTACCTACTAAATTAGAAACTCTTGGTG3060
GGTTCCAGTA ATCCATAGCTTAACAAGCCCTGCAGTTAATACTGATGTACACTGATGTCC3120
AAAAACTGCT GTCATGGACTATTGATTGTATTGAGGATTAGTCTCAGTTGGAAAGCCAAC3180
TACAGAGGCA TTTTGAACTTTCTTTCTTTGCCTCTCTATGTCTCTCTGTCTTTTCCTGTC3240
TTCTGATTTA TCTGTCTTTCTTTCTCTAGTAAATGGCACTCAATATAAAAGTGGTGGAGT3300
CAATCTTAAA CTTATTTTTATTATGATTGTATTGATACATGCACGAAGTCCCTCTGCCCT3360
ACTCCCTATT CAAGGATATTACTCACTGCACATCATAAATCTCCATCATCTGTCTTAAAG3420
TTTTATGAGT AGATTTCATCTACATTATATTCAAGTTCATTTATTACTGAGCTGTATTAC3480
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TGTGGAGCTC TAACAGTATT TGTTTCCTGA TTTCAAACTC AATGCTACAG AGCACTTTGA 3540
ATACATCACA CCTTATAGGA AAGATAGTAA ATGTATTAAT CCCATTGAAA AATTAGTTTT 3600
GTACAATGTG CTAAATAGTA TTGCATTGGA TTACTTTTAT ATTTAACACA CTCCATCAAA 3660
ACATCCCATA ACATAATTTT ACAATCTGCA TGTGAATTTA ACTGTGAAAT TCAGTATTGT 3720
GATATTTTGA ATAAGTGAAT TCTTTCTCTG CAAATACTAT GTTGATAAAA TTACTTGTAT 3780
GTTCCCCTGA 1?~~iAAAAAAAA AP~~AAAAAAA AAAAAAAAAA P,~~~AAAAAAA P~~~AAAAAAA 3840
p.,~~i~P.AAAAAA p~~i4AAAAAAA p~~i~AAAAAAA AAAAAAAAAA P,F~~AAAAAAA AAAAAAAGA 3
8 9 9
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 487 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:10:
Met Ile Gly Lys Lys Phe Tyr Pro Arg Glu Leu Asp Leu Ala Arg Glu
1 5 10 15
Arg Arg Lys Ala Glu Arg Pro Lys Thr Ser Leu Arg Lys Thr Asp Ser
20 25 30
Glu Arg Glu Glu Val Thr Arg Ala Asn Ala Leu Lys Asp Glu Asp Ala
35 40 45
Phe Lys Glu Glu Gln Lys Leu Lys Ala Glu Glu Gly Glu Thr Glu Thr
50 55 60
Glu Val Arg Ala Glu Glu Glu Thr Lys Ala Pro Pro Asn Glu Met Gly
65 70 75 80
Ser Asp Ala Glu Asn Glu Xaa Pro Val Glu Ala Ser Glu Leu Ser Asp
85 90 95
Asn Pro Gly Leu Leu Gly Glu Xaa Ser Leu Lys Glu Thr Val Val Pro
100 105 110
Ile Phe Glu Ala Thr Pro Gly Phe Glu Lys Ser Leu Glu Asn Ile Thr
115 120 125
Ala Leu Arg Lys Glu Gly Gly Gly Glu Arg Leu Ser Glu Ala Arg Asp
130 135 140
g0
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Thr Glu His Lys Asp Arg Glu Glu Leu Ser Ser Arg Glu Asn Arg Ala
145 150 155 160
Leu Lys Glu Gly His Arg Gln Asp Gly Glu Gly Ala Leu AIa Ala Pro
165 170 175
Glu Ala Glu Pro Ala Gly Lys Val Gln Ala Pro Glu Gly Leu Ile Pro
180 185 190
Ala Thr Gly Gln Ala Glu Glu Leu Ala Ala Lys Asp His Asp Ser Cys
195 200 205
Ala Gly Leu Glu Gly Arg Ala Glu Gly Gln Gly Gly Val Asp Val Val
210 215 220
Leu Arg Thr Gln Glu Ala Val Ala Glu Glu Asp Pro Ile Xaa Ala Glu
225 230 235 240
Lys Phe Arg Glu Glu Ala Val Asp Glu Asp Pro Glu Glu Glu Glu Asp
245 250 255
Lys Glu Cys Xaa Leu Glu Thr Glu Ala Met Gln Asp Arg Asn Ser Glu
260 265 270
Gly Asp Gly Asp Met Glu Gly Glu Gly Asn Thr Gln Lys Asn Glu Gly
275 280 285
Met Gly Gly Gly Arg Val Val Ala Val Glu Val Leu His Gly Gly Gly
290 295 300
Glu Thr Ala Glu Thr Ala Ala Glu Glu Arg Glu Val Leu Ala Gly Ser
305 310 315 320
Glu Thr Ala Glu Glu Lys Thr Ile Ala Asn Lys Ala Ser Ser Phe Ser
325 330 335
Asp Val Ala Glu Glu Glu Thr Trp His Gln Gln Asp Glu Leu Val Gly
340 345 350
Lys Thr Ala Ala Ala Gly Lys Val Val Val Glu Glu Leu Ala Arg Ser
355 360 365
Gly Glu Glu Val Pro Ala Ala Glu Glu Met Thr Val Thr Tyr Thr Thr
370 375 380
Glu Ala Gly Val Gly Thr Pro Gly Ala Leu Glu Arg Lys Thr Ser Gly
385 390 395 400
Leu Gly Gln Glu Gln Glu Glu Gly Ser Glu Gly Gln Glu Ala Ala Thr
405 410 415
Gly Ser Gly Asp Gly Arg Gln Glu Thr Gly Ala Ala Glu Lys Phe Arg
420 425 430
Leu Gly Leu Ser Arg Glu Gly Glu Arg Glu Leu Ser Pro Glu Ser Leu
81
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435 440 445
Gln Ala Met Ala Thr Leu Pro Val Lys Pro Asp Phe Thr Glu Thr Arg
450 455 460
Glu Lys Gln Gln His Met Val Gln Gly Glu Ser Glu Thr Ala Asp Val
465 470 475 480
Ser Pro Asn Asn Met Gln Val
485
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 483 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:11:
CATTGCTAGA CAGACTCTCT TGCTTGGATG GTACTCCACC CACATGAGAT 60
ACTTTCTTGG
GCAAGATTGC TCAGGGTGCA GCTAATGGCA TCAATTTTCT CATCATATTC 120
ACATGAAAAT
ATAGAGATAT TAAAAGTGCA AATATCTTAC TGGATGAAGC AAAATATCTG 180
TTTTACTGCT
ACTTTGGCCT TGCACGGGCT TCTGAGAAGT TTTGCCCAGA TAGCAGAATT 240
CAGTCATGAC
GTGGGAACAA CAGCTTATAT GGCACCAGAA GCTTTGCGTG ACCCAAATCT 300
GAGAAATAAC
GATATTTACA GCTTTGGTGT GGTTTTACTA GAAATAATAA AGCTGTGGAT 360
CTGGACTTCC
GAACACCGTG AACCTCAGTT ATTGCTAGAT ATTAAAGAAG TGAAGAAAAG 420
AAATTGAAGA
ACATTGAAGA TTATATTGAT AAAAAGATGA ATGATGCTGA GTTGAAGCTA 480
TTCCACTTCA
TGT 483
(2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 121 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
82
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
Met Ala Ser Ile Phe Tyr Met Lys Ile Ile Ile Phe Ile Glu Ile Leu
1 5 10 15
Lys Val Gln Ile Ser Tyr Trp Met Lys Leu Leu Leu Leu Lys Tyr Leu
20 25 30
Thr Leu Ala Leu His Gly Leu Leu Arg Ser Phe Ala Gln Thr Val Met
35 40 45
Thr Ser Arg Ile Val Gly Thr Thr Ala Tyr Met Ala Pro Glu Ala Leu
50 55 60
Arg Gly Glu Ile Thr Pro Lys Ser Asp Ile Tyr Ser Phe Gly Val Val
65 70 75 80
Leu Leu Glu Ile Ile Thr Gly Leu Pro Ala Val Asp Glu His Arg Glu
85 90 95
Pro Gln Leu Leu Leu Asp Ile Lys Glu Glu Ile Glu Asp Glu Glu Lys
100 105 110
Thr Leu Lys Ile Ile Leu Ile Lys Arg
115 120
(2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 493 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
AATCTGAGTC AGCTTAGAAG ATANTCCAAG CTTCAGATGA TAACCACAGC CTGGGCTGAC 60
ACCTGGATTT CAGCTTTGCA TGATCCTCAG TATGAGAATC TATCTGTTCT GTGCTGGACT 120
TCTAATATAT AGAACTGTGA GATAATGGGT CACATTGGCT GGATGTGGTG GCTCATACCT 180
GTAAATCCCA GCACTTTGGG AGGCCGAGGC AGGCAGATCA CCTGAGGTCA GGAGTTCAAG 240
ACCGGCCTGG CCAGCATGGT GAAGCCCCGT CTTTACTAGA AATACAAAAA TTAGACGAGC 300
GTGGTGGTGG ACACCTGTGT TCCCAGCTAC TTGGGAGGCT GAGGCAGGAG ACTGGCTGGA 360
ACCAGGGAGG TAGAGGTTGC AGTGAGCTGA GATCGTGCCA CTGCACTCCA GCCTGGGTGA 420
83
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CAGAGTGAGA CTCCATCATA AATAAATAAA TAAATAAATG GGTCACATTA AGCCTTTAAA 480
1~,F~AAAPu'~AAA AAA - 493
{2) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
{A) LENGTH: 2682 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE
DESCRIPTION:
SEQ ID N0:14:
GGTTCCCAGA AGAGTTTGCGACGTGGTAAAGAAATAAGGCGAGTACACAA GCGAAGACTT60
TCCAGCTCAG AGAGTGAAGAGAGCTATTTGTCCAAGAACTCTGAAGATGA TGAGCTAGCT120
AAAGAATCAA AGCGGTCAGTTCGAAAGCGGGGCCGAAGCACAGACGAGTA TTCAGAAGCA180
GATGAGGAGG AGGAGGAAGARGAAGGCAAACCATCCCGCAAACGGCTACA CCGGATTGAG240
ACGGATGAGG ARGAGAGTTGTGACAATGCTCATGGAGATGCAAATCAGCC TGCCCGTGAC300
AGCCAGCCTA GGGTCCTGCCCTCAGAACAAGAGAGCACCAAGAAGCCCTA CCGGATAGAA360
AGTGATGAGG AAGAGGACTTTGAAAATGTAGGCAAAGTGGGGAGCCCATT GGACTATAGC420
TTAGTGGACT TACCTTCAACCAATGGACAGAGCCCTGGCAAAGCCATTGA GAACTTGATT480
GGCAAGCCTA CTGAGAAGTCTCAGACCCCCAAGGACAACAGCACAGCCAG TGCAAGCCTA540
GCYTCCCAAT GGGACAAGTGGTGGGCAGGAGGCAGGAGCACCAGAAGAGG AGGAAGATGA600
GCTTTTGAGA GTGACTGACCTTGTTGATTATGTCTGTAACAGTGAACAGT TATAAGACTT660
TTTTTCCATT TTTGTGCTAATTTATTCCACGGTAGCTCTCACACCAGCGG GCCAGTTATT720
AAAAGCTGTT TAATTTTTCCTAGAAAACTCCACTACAGAATGACTTTTAG AAGAAAAATT780
TCAACAAATC CTGAAGTCTTTCTGTGAAGTGACCAGTTCTGAACTTTGAA GATAAATAAT840
TGCTGTAAAT TCCTTTTGATTTTCTTTTTCCAGGTTCATGGTCCTTGGTA ATTTCATTCA900
TGGAAAAAAA TCTTATTATAATAACAACAAAGATTTGTATATTTTTGACT TTATATTTCC960
TGAGCTCTCC TGACTTTGTGAAAAAGGGTGATGAAAATGCATTCCGAATC TGTGAGGGCC1020
CAAAACAGAA TTTAGGGGTGGGTGAAAGCACTTGTGCTTTAGCTTTTTCA TATTAAATAT1080
84
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ATATTATATT TAAACATTCA TGGCATAGAT GATGATTTAC AGACAATTTA 1140
AAAGTTCAAG
TCTGTACTGT TACAGTTTGA GAATTGTAGA TAACATCATA CATAAGTCAT 1200
TTAGTAACAG
CCTTTGTGAA ATGAACTTGT TTACTATTGG AGATAACCAC ACTTAATAAA 1260
GAAGAGACAG
TGAAAGTACC ATCATAATTA ACCTAAATTT TTGTTATAGC AGAGTTTCTT 1320
GTTTAAAAAA
AAAWAAAAWG CRKCYGMAAA GCATTTGTAC AGTAAAATGT ATAATGAAGC 1380
TTTGCCAACC
AGACTGTGCT AGCAACAAAT TTTTTTAAAT AAGCTTTATG CAGTGGTAAT 1440
AAGGTGGCCT
CAAATATATT GTGTCTGATG GAGAGTTATT AGTGAAATGA ATGTGGTCTT 1500
TCTTAAGGCC
TGGGTGGACT GTAAACTTTG CCAATAGTAT AACTCTTGTC TTCTGGCCAC 1560
TTGATGTTTA
AATATCTGAA ATATCATTTT GAAAAAAATA CATCTATATA TAACATACAT 1620
GAAGAGATGC
TAAGCTGACA GTGATATTTT AGCACATTTG AAGACTGGGA AGAGATTTTC 1680
AGGTGAATTT
TAACTGGTCT ATTCTTGCCC TTAGTATCTA CTTCAAATTG AAGTCTACAA 1740
ACAAAGCAGT
TCCTTTGGGA GGTTTTTAGT TTGAGTTTTA GCGTGTGTGT GTGTTTGTGT 1800
GTGTGCGTGT
GCGTGTGTGT GTGTGTTGGA ATTTCCTATC TGCCTGGATA TATTAGCAGA 1860
GTTTGAATGT
AGTTTTGGCC TTTGGCCATT AGACTTCTAT TAAAATTCAT TAATAGTCAT 1920
ACAACCAACA
TAGAGTTGAA TGAGAACTGC CGATGTAATT AATAGGCATG ACATCCATTT 1980
CAAACATCTC
AACACTTTAA AGAAAAGCCC TTTGTTTCAA GAAAAAAGGG TTTGTAACTA 2040
ACTAAATACC
TAACATGTAA TTGACACTAA AATATGAACT TTGTCTTATT TAGTTTCTGT 2100
TATAGCTGTA
AAATTTCAGG CAGAGCCATA ACATTGTACA GAGTGTAGCA CTTGTGATTA 2160
AACCTAGCCT
GTTAAATCCT GAAACCTTCA ACCATTACTT CTGTGAATAC TTTAGCCCTG 2220
GGATTTGGGT
TTTTCTGTTC CGGTGTTGTG TCTGTTGCCG GCAATGGACA CACCATATCT 2280
GCTGCTGGCC
CAAGGAACGT CATTAATTTT TCTTTCCAAA TTAAGTATTA TGTGCTAGTC 2340
AGTGTATAGT
AAAGCACTTC TCTTTTTTAT TACTAAAAAG CTGGCATTAG ATTTGCATTA 2400
TAAATACCTC
TCTAGGAACT TTATACTCCT TTTCCTTCTT CAACAGGTAT TGCCCTTAAA 2460
TCTTATCTTT
TGGCCTTGAA AGTTTATAGC TATTGTTTTT CAGTTGTTCG TTGTTTTGTT 2520
TTGTTTCACT
TTAGTTCTGT AGTACCTGCC CATTAATATT TTTGCTTTGA TTCTAGCAAT 2580
GTGTATGTAT
CTGTATAAAA AATAAAATAA TGAAAGCAAC CTAAAAATAG GATGCACCAA 2640
TTAAAAAAAA
~~~~AAAAAAA AAAAAAAAAA P,~~iAAAAAAAA AAAAAAAAAA AA 2
6
8
2
(2) INFORMATION FOR SEQ ID N0:15:
g$
CA 02285447 1999-09-23
WO 98/44113 PCT/US98/06176
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 58 amino acids
(B) TYPE: amino acid -
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
Met Glu Lys Asn Leu Ile Ile Ile Thr Thr Lys Ile Cys Ile Phe Leu
1 5 10 15
Thr Leu Tyr Phe Leu Ser Ser Pro Asp Phe Val Lys Lys Gly Asp Glu
20 25 30
Asn Ala Phe Arg Ile Cys Glu Gly Pro Lys Gln Asn Leu Gly Val Gly
35 40 45
Glu Ser Thr Cys Ala Leu Ala Phe Ser Tyr
50 55
(2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2522 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION:
SEQ ID N0:16:
GCCGAGCGCC CGCGCCGCCGCTGCCTCTGTCCTCCGCGCG CTGCTCAGCTGAAGGCGCAC 60
AGGATTCAAT TACTGGACTTGTCAACTCTGCCAGTGTACG TGCCATTTCTCTTCCACTAT 120
GAGAGGACCG ATTGTATTGCACATTTGTCTGGCTTTCTGT AGCCTTCTGCTTTTCAGCGT 180
TGCCACACAA TGTCTGGCCTTCCCCAAAATAGAAAGGAGG AGGGAGATAGCACATGTTCA 240
TGCGGAAAAA GGGCAGTCCGATAAGATGAACACCGATGAC CTAGAAAATAGCTCTGTTAC 300
CTCAAAGCAG ACTCCCCAACTGGTGGTCTCTGAAGATCCA ATGATGATGTCAGCAGTACC 360
ATCGGCAACA TCATTAAATAAAGCATTCTCGATTAACAAA GAAACCCAGCCTGGACAAGC 420
TGGGCTCATG CAAACAGAACGCCCTGGTGTTTCCACACYT ACTGAGTCAGGTGTCCCCTC 480
86
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AGCTGAAGAA GTATTTGGTT AGAGAGAATATCTCCTGAAA 540
CCAGCCAGCC GTGGACTTGC
CAAGGCCATG TTAACCATTG CTATCACTGCGACTCCTTCTCTGACTGTTGATGAAAAGGA 600
GGAACTCCTT ACAAGCACTA ACTTTCAGCCCATTGTAGAAGAGATCACAGAAACCACAAA 660
AGGTTTTCTG AAGTATATGG ATAATCAATCATTTGCAACTGAAAGTCAGGAAGGAGTTGG 720
TTTGGGACAT TCACCTTCAT CCTATGTGAATACTAAGGAAATGCTAACCACCAATCCAAA 780
GACTGAGAAA TTTGAAGCAG ACACAGACCACAGGACAACTTCTTTTCCTGGTGCTGAGTC 840
CACAGCAGGC AGTGAGCCTG GAAGCCTCACCCCTGATAAGGAGAAGCCTTCGCAGATGAC 900
AGCTGATAAC ACCCAGGCTG CTGCCACCAAGCAACCACTCGAAACTTCCGAGTACACCCT 960
GAGTGTTGAG CCAGAAACTG ATAGTCTGCTGGGAGCCCCAGAAGTCACAGTGAGTGTCAG 1020
CACAGCTGTT CCAGCTGCCT CTGCCTTAAGTGATGAGTGGGATGACACCAAATTAGAGAG 1080
TGTAAGCCGG ATAAGGACCC CCAAGCTTGGAGACAATGAAGAGACTCAGGTGAGAACGGA 1140
GATGTCTCAG ACAGCACAAG TAAGCCATGAGGGTATGGAAGGAGGCCAGCCTTGGACAGA 1200
GGCTGCACAG GTGGCTCTGG GGCTGCCTGAAGGGGAAACACACACGGGCACAGCCCTGCT 1260
AATAGCGCAT GGGAATGAGA GATCACCTGCTTTCACTGATCAAAGTTCCTTTACCCCCAC 1320
AAGTCTGATG GAAGACATGA AAGTTTCCATTGTGAACTTGCTCCAAAGTACGGGAGACTT 1380
CACGGAATCC ACCAAGGAAA ACGATGCCCTGTTTTTCTTAGAAACCACTGTTTCTGTCTC 1440
TGTATATGAG TCTGAGGCAG ACCAACTGTTGGGAAATACAATGAAAGACATCATCACTCA 1500
AGAGATGACA ACAGCTGTTC AAGAGCCAGATGCCACTTTATCCATGGTGACACAAGAGCA 1560
GGTTGCTACC CTCGAGCTTA TCAGAGACAGTGGCAAGACTGAGGAAGAAAAGGAGGACCC 1620
CTCTCCTGTG TCTGACGTTC CTGGTGTTACTCAGCTGTCAAGAAGATGGGAGCCTCTGGC 1680
CACTACAATT TCAACTACAG TCGTCCCTTTGTCTTTTGAAGTTACTCCCACTGTGGAAGA 1740
ACAAATGGAC ACAGTCACAG GGCCAAATGAGGAGTTCACACCAGTTCTGGGATCTCCAGT 1800
GACACCTCCT GGAATAATGG TGGGGGAACCCAGCATTTCCCCTGCACTTCCTGCTTTGGA 1860
GGCATCCTCT GAGAGAAGAA CTGTTGTTCCATCTATTACTCGTGTTAATACAGCTGCCTC 1920
ATATGGCCTG GACCAACTTG AATCTGAAGAGGGACAAGAAGATGAGGATGAAGAGGATGA 1980
AGAAGATGAA GATGAAGAAG AGGAAGATGAGGAAGAAGATGAGGAAGATAAAGATGCAGA 2040
CTCGCTGGAT GAGGGCTTGG ATGGTGACACTGAGCTGCCAGGTTTTACCCTCCCTGGTAT 2100
CACATCCCAG GAACCAGGCT TAGAGGAGGGAAACATGGACCTGTTGGAGGGAGCTACCTA 2160
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CCAGGTGCCA GATGCCYTCG AGTGGGAACA 2220
GCAGAATCAA GGCCTGGTGA GAAGCTGGAT
GGAAAAATTM AAAGACAAGG CTGGTTACAT CTGGTGCCTG TAGGGGTTGG2280
GTCTGGGATG
GATAGCTGGA GCCTTGTTCA TCTTGGGAGC ATTAAGGTTA TGAATCGCCG2340
CCTCTACAGC
AAGGAGAAAT GGCTTCAAAA GGCATAAAAG GAATTCAACA GCATGCAAGA2400
AAAGCAGAGA
TCGAGTAATG CTCTTAGCCG ACAGCTCTGA TGAATTGGAC TGGGTTTTAA2460
AGATGAATTT
TTGGGATATT CAACGATGCT ACTATTCTAA GGAGCAGAAA P.~F~i~AAAAAAA2520
TTTTTATTTT
AA 2522
(2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 774 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
Met Arg Gly Pro Ile Val Leu His Ile Cys Leu Ala Phe Cys Ser Leu
1 5 10 15
Leu Leu Phe Ser Val Ala Thr Gln Cys Leu Ala Phe Pro Lys Ile Glu
20 25 30
Arg Arg Arg Glu Ile Ala His Val His Ala Glu Lys Gly Gln Ser Asp
35 40 45
Lys Met Asn Thr Asp Asp Leu Glu Asn Ser Ser Val Thr Ser Lys Gln
50 55 60
Thr Pro Gln Leu Val Val Ser Glu Asp Pro Met Met Met Ser Ala Val
65 70 75 80
Pro Ser Ala Thr Ser Leu Asn Lys Ala Phe Ser Ile Asn Lys Glu Thr
85 90 95
Gln Pro Giy Gln Ala Gly Leu Met Gln Thr Glu Arg Pro Gly Val Ser
100 105 110
Thr Xaa Thr Glu Ser Gly Val Pro Ser Ala Glu Glu Val Phe Gly Ser
115 120 125
Ser Gln Pro Glu Arg Ile Ser Pro Glu~Ser Gly Leu Ala Lys Ala Met
130 135 140
88
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Leu Thr Ile Ala Ile Thr Ala Thr Pro Ser Leu Thr Val Asp Glu Lys
145 150 155 160
Glu Glu Leu Leu Thr Ser Thr Asn Phe Gln Pro Ile Val Glu Glu Ile
165 170 175
Thr Glu Thr Thr Lys G1y Phe Leu Lys Tyr Met Asp Asn Gln Ser Phe
180 185 190
Ala Thr Glu Ser Gln Glu Gly Val Gly Leu Gly His Ser Pro Ser Ser
195 200 205
Tyr Val Asn Thr Lys Glu Met Leu Thr Thr Asn Pro Lys Thr Glu Lys
210 215 220
Phe Glu Ala Asp Thr Asp His Arg Thr Thr Ser Phe Pro Gly Ala Glu
225 230 235 240
Ser Thr Ala Gly Ser Glu Pro Gly Ser Leu Thr Pro Asp Lys Glu Lys
245 250 255
Pro Ser Gln Met Thr Ala Asp Asn Thr Gln Ala Ala Ala Thr Lys Gln
260 265 270
Pro Leu Glu Thr Ser Glu Tyr Thr Leu Ser Val Glu Pro Glu Thr Asp
275 280 285
Ser Leu Leu Gly Ala Pro Glu Val Thr Val Ser Val Ser Thr Ala Val
290 295 300
Pro Ala Ala Ser Ala Leu Ser Asp Glu Trp Asp Asp Thr Lys Leu Glu
305 310 315 320
Ser Val Ser Arg Ile Arg Thr Pro Lys Leu Gly Asp Asn Glu Glu Thr
325 330 335
Gln Val Arg Thr Glu Met Ser Gln Thr Ala Gln Val Ser His Glu Gly
340 345 350
Met Glu Gly Gly Gln Pro Trp Thr Glu Ala Ala Gln Val Ala Leu Gly
355 360 365
Leu Pro Glu Gly Glu Thr His Thr Gly Thr Ala Leu Leu Ile Ala His
370 375 380
Gly Asn Glu Arg Ser Pro Ala Phe Thr Asp Gln Ser Ser Phe Thr Pro
385 390 395 400
Thr Ser Leu Met Glu Asp Met Lys Val Ser Ile Val Asn Leu Leu Gln
405 410 415
Ser Thr Gly Asp Phe Thr Glu Ser Thr Lys Glu Asn Asp Ala Leu Phe
420 ~ 425 430
Phe Leu Glu Thr Thr Val Ser Val Ser Val Tyr Glu Ser Glu Ala Asp
89
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435 440 445
Gln Leu Leu Gly Asn Thr Met Lys Asp Ile Ile Thr Gln Glu Met Thr
450 455 460
Thr Ala Val Gln Glu Pro Asp Ala Thr Leu Ser Met Val Thr Gln Glu
465 470 475 480
Gln Val Ala Thr Leu Glu Leu Ile Arg Asp Ser Gly Lys Thr Glu Glu
485 490 495
Glu Lys Glu Asp Pro Ser Pro Val Ser Asp Val Pro Gly Val Thr Gln
500 505 510
Leu Ser Arg Arg Trp Glu Pro Leu Ala Thr Thr Ile Ser Thr Thr Val
515 520 525
Val Pro Leu Ser Phe Glu Val Thr Pro Thr Val Glu Glu Gln Met Asp
530 535 540
Thr Val Thr Gly Pro Asn Glu Glu Phe Thr Pro Val Leu Gly Ser Pro
545 550 555 560
Val Thr Pro Pro Gly Ile Met Val Gly Glu Pro Ser Ile Ser Pro Ala
565 570 575
Leu Pro Ala Leu Glu Ala Ser Ser Glu Arg Arg Thr Val Val Pro Ser
580 585 590
Ile Thr Arg Val Asn Thr Ala Ala Ser Tyr Gly Leu Asp Gln Leu Glu
595 600 605
Ser Glu Glu Gly Gln Glu Asp Glu Asp Glu Glu Asp Glu Glu Asp Glu
610 625 620
Asp Glu Glu Glu Glu Asp Glu Glu Glu Asp Glu Glu Asp Lys Asp Ala
625 630 635 640
Asp Ser Leu Asp Glu Gly Leu Asp Gly Asp Thr Glu Leu Pro Gly Phe
645 650 655
Thr Leu Pro Gly Ile Thr Ser Gln Glu Pro Gly Leu Glu Glu Gly Asn
660 665 670
Met Asp Leu Leu Glu Gly Ala Thr Tyr Gln Val Pro Asp Ala Xaa Glu
675 680 685
Trp Glu Gln Gln Asn Gln Gly Leu Val Arg Ser Trp Met Glu Lys Xaa
690 695 700
Lys Asp Lys Ala Gly Tyr Met Ser Gly Met Leu Val Pro Val Gly Val
705 710 715 720
Gly Ile Ala Gly Ala Leu Phe Ile Leu Gly Ala Leu Tyr Ser Ile Lys
725 730 735
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Val Met Asn Arg Arg Arg Arg Asn Gly Phe Lys Arg His Lys Arg Lys
740 745 750
Gln Arg Glu Phe Asn Ser Met Gln Asp Arg Val Met Leu Leu Ala Asp
755 760 765
Ser Ser Glu Asp Glu Phe
770
(2) INFORMATION FOR SEQ ID N0:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2002 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE
DESCRIPTION:
SEQ ID
N0:18:
GGCACGCCGGTACCTGAAGTCCTTCAGAAGTGCACGCCGGGACCAGGATTCCGGGAGGCC 60
GACTCCTCCCTGCCCCACGAATGCCGGGAATTGTGGTCTCCGCCGGACGCGAGTTGTGAG 120
ACGGCCCAAGGGGCCGCGGGGTATGCTGGGACCGCTAGCCCTTCCGGCGCGCCTCAGGAC 180
TTCGGGTCCCCTCACCCCGGGCGGATGCCCAAAGACTCCGCCTTCCCAAGAGCCCCTGCG 240
GCCGGGCGCGAAAATGGCGGCGGCGGCGACGGCCGGGCGCTCCTGAAGCAGCAGTTATGG 300
AGCTTCCCTCAGGGCCGGGGCCGGAGCGGCTCTTTGACTCGCACCGGCTTCCGGGTGACT 360
GCTTCCTACTGCTCGTGCTGCTGCTCTACGCGCCAGTCGGGTTCTGCCTCCTCGTCCTGC 420
GCCTGTTTCTCGGGATCCACGTCTTCCTGGTCAGCTGCGCGCTGCCAGACAGCGTCCTTC 480
GCAGATTCGTAGTGCGGACCATGTGTGCGGTGCTAGGGCTCGTGGCCCGGCAGGAGGACT 540
CCGGACTCCGGGATCACAGTGTCAGGGTCCTCATTTCCAACCATGTGACACCTTTCGACC 600
ACAACATAGTCAATTTGCTTACCACCTGTAGCACCGTGAGTGAGAGCGAGGCCGAGAGCG 660
CCACGGGGCGGTTCCCTGGGGCCCAGCTGAAGGCCCCCCTGTCCCCACTCGCGTTCCCCA 720
TGGAGGATACTGAGCCTTACCCCTAACCCCGATCCTCTACCCAACATGTCAGTTTTTTTT 780
TTCATTTTCCTCAATATTTTTCTTCTTGCTTTCTCTTCTCCTGGTTCCCAGCCTCTACTC 840
AATAGTCCCCCCAGCTTTGTGTGCTGGTCTCGGGGCTTCATGGAGATGAATGGGCGGGGG 900
GAGTTGGTGGAGTCACTCAAGAGATTCTGTGCTTCCACGAGGCTTCCCCCCACTCCTCTG 960
91
CA 02285447 1999-09-23
WO 98144113 PCT/US98/06I76
CTGCTATTCC CTGAGGAAGAGGCCACCAATGGCCGGGAGGGGCTCCTGCG CTTCAGAGTT1020
TGACAGTTGC CTGTTATAAGGCAGGTGTGAGCTGCTGACTAGGCTGGCTG GATTCCCATC1080
CTACTTTCTC CTTCCTCTTCTAGTTCCTGGCCATTTTCTATCCAAGATGT GGTACAACCT1140
CTTACCCTGC AAGTTCAGAGACCCCTGGTCTCTGTGACGGTGTCAGATGC CTCCTGGGTC1200
TCAGAACTGC TGTGGTCACTTTTCGTCCCTTTCACGGTGTATCAAGTGGC TTCGTCCTGT1260
TCATCGCCAA CTAGGGGAAGCGAATGAGGAGTTTGCACTCCGTGTACAAC AGCTGGTGGC1320
CAAGGAATTG GGCCAGACAGGGACACGGCTCACTCCAGCTGACAAAGCAG AGCACATGAA1380
GCGACAAAGA CACCCCAGATTGCGCCCCCAGTCAGCCCAGTCTTCTTTCC CTCCCTCCCC1440
TGGTCCTTCT CCTGATGTGCAACTGGCAACTCTGGCTCAGAGAGTCAAGG AAGTTTTGCC1500
CCATGTGCCA TTTGGTGTCATCCAGAGAGACCTGGCCAAGACTGGCTGTG TAGACTTGAC1560
TATCACTAAT CTGCTTGAGGGGGCCGTAGCTTTCATGCCTGAAGACATCA CCAAGGGAAC1620
TCAGTCCCTA CCCACAGCCTCTGCCTCCAAGTTTCCCAGCTCTGGCCCGG TGACCCCTCA1680
GCCAACAGCC CTAACATTTGCCAAGTCTTCCTGGGCCCGGCAGGAGAGCC TGCAGGAGCG1740
CAAGCAAGCA CTATATGAATACGCAAGAAGGAGATTCACAGAGAGACGAG CCCAGGAGGC1800
TGACTGAGCT CAAAGGAACAGGATGGCACCCAGAGCCGCAGGACGGAGAC TGGGGGCAGC1860
CCTCACCCAA CTCACAACAGGCTGGATGGGTGGGTGGTAAAAAGGGAAGG ATGAGGCTCC1920
CCCAATGTCA CATTAAATTCATGGTTTTCATTCAAGGVAAP,2~e~AAAAAAA AAAAAAAAAA1980
1?~~iAAAAAAAA 1?,~~~AAAAAAAAA 2
0
0
2
(2) INFORMATION
FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 206 aminoacids
(B) TYPE: amino
acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE:
protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:19:
Met Pro Pro Gly Ser Gln Asn Cys Cys Gly His Phe Ser Ser Leu Ser
1 5 . 10 15
Arg Cys Ile Lys Trp Leu Arg Pro Val His Arg Gln Leu Gly Glu Ala
92
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20 25 30
Asn Glu Glu Phe Ala Leu Arg Val Gln Gln Leu Val Ala Lys Glu Leu
35 40 45
Gly Gln Thr Gly Thr Arg Leu Thr Pro Ala Asp Lys Ala Glu His Met
50 55 60
Lys Arg Gln Arg His Pro Arg Leu Arg Pro Gln Ser Ala Gln Ser Ser
65 70 75 g0
Phe Pro Pro Ser Pro Gly Pro Ser Pro Asp Val Gln Leu Ala Thr Leu
85 90 95
Ala Gln Arg Val Lys Glu Val Leu Pro His Val Pro Phe Gly Val Ile
100 I05 110
Gln Arg Asp Leu Ala Lys Thr Gly Cys Val Asp Leu Thr Ile Thr Asn
115 120 125
Leu Leu Glu Gly Ala Val Ala Phe Met Pro Glu Asp Ile Thr Lys Gly
130 135 140
Thr Gln Ser Leu Pro Thr Ala Ser Ala Ser Lys Phe Pro Ser Ser Gly
145 150 155 160
Pro Val Thr Pro Gln Pro Thr Ala Leu Thr Phe Ala Lys Ser Ser Trp
165 170 175
Ala Arg Gln Glu Ser Leu Gln Glu Arg Lys Gln Ala Leu Tyr Glu Tyr
180 185 190
Ala Arg Arg Arg Phe Thr Glu Arg Arg Ala Gln Glu Ala Asp
195 200 205
(2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 819 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:20:
CAATTGGGCC GCGAGTTGTG GTTTAAACCA GGAGTGCGCC GCGTCCGTTC ACCGCGGCCT 60
CAGATGAATG CGGCTGTTAA GACCTGCAAT AATCCAGAAT GGCTACTCTG ATCTATGTTG 120
ATAAGGAAAA TGGAGAACCA GGCACCCGTG TGGTTGCTAA GGATGGGCTG AAGCTGGGGT 180
93
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CTGGACCTTC AATCAAAGCC TTAGATGGGAGATCTCAAGT TTCAACACCACGTTTTGGCA240
AAACGTTCGA TGCCCCACCA GCCTTACCTAAAGCTACTAG AAAGGCTTTGGGAACTGTCA300
ACAGAGCTAC AGAAAAGTCT GTAAAGACCAAGGGACCCCT CAAACAAAAACAGCCAAGCT360
TTTCTGCCAA AAAGATGACT GAGAAGACTGTTAAAGCAAA AAGCTCTGTTCCTGCCTCAG420
ATGATGCCTA TCCAGAAATA GAAAAATTCTTTCCCTTCAA TCCTCTAGACTTTGAGAGTT480
TTGACCTGCC TGAAGAGCAC CAGATTGCGCACCTCCCCTT GAGTGGAGTGCCTCTCWTGA540
TCCTTGACGA GGAGAGAGAG CTTGAAAAGCTGTTTCAGCT GGGCCCCCCTTCACCTGTGA600
AGATGCCCTC TCCACCATGG GAATCCAATCTGTTGCAGTC TCCTTCAAGCATTCTGTCGA660
CCCTGGATGT TGAATTGCCA CCTGTTTGCTGTGACATAGA TATTTAAATTTCTTAGTGCT720
TCAGAGTTTG TGTGTATTTG TATTAATAAAGCATTCTTTA ACAGAAAAAA~1,F~~.AAAAAAA780
1?,~~i~AAAAAAA AAAAAAAAAA p~~AAAAAAA 819
AP.AAAAF1AAA
(2) INFORMATION FOR SEQ
ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 146 amino acids
(B) TYPE: amino acid
(C} STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:
Met Ala Thr Leu Ile Tyr Val Asp Lys Glu Asn Gly Glu Pro Gly Thr
1 5 10 15
Arg Val Val Ala Lys Asp Gly Leu Lys Leu Gly Ser Gly Pro Ser Ile
20 25 30
Lys Ala Leu Asp Gly Arg Ser Gln Val Ser Thr Pro Arg Phe Gly Lys
35 40 45
Thr Phe Asp Ala Pro Pro Ala Leu Pro Lys Ala Thr Arg Lys Ala Leu
50 55 60
Gly Thr Val Asn Arg Ala Thr Glu Lys Ser Val Lys Thr Lys Gly Pro
65 70 75 80
Leu Lys Gln Lys Gln Pro Ser Phe Ser Ala Lys Lys Met Thr Glu Lys
85 90 95
94
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Thr Val Lys Ala Lys Ser Ser Val Pro Ala Ser Asp Asp Ala Tyr Pro
100 105 110
Glu Ile Glu Lys Phe Phe Pro Phe Asn Pro Leu Asp Phe Glu Ser Phe
115 120 125
Asp Leu Pro Glu Glu His Gln Ile Ala His Leu Pro Leu Ser Gly Val
130 135 140
Pro Leu
145
(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:
TNTCCTGCCTC AGCTGCCTCT CTGTGTAA 29
(2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:
CNCACTGCCCT CCTTCTCCCA TAGGTACT 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
CA 02285447 1999-09-23
WO 98/44113 PCT/US98/06176
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:
GNAATAAGCAT GATGCTCTAC AAGGAAAG 29
(2) INFORMATION FOR SEQ ID N0:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:
TNGGTGCCATG ATTCTGAGTG CCCTTTGC 29
(2) INFORMATION FOR SEQ ID N0:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C} STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi} SEQUENCE DESCRIPTION: SEQ ID N0:26:
GNATATGTCAC TGTCATCTCC TCTGCTGC 29
(2) INFORMATION FOR SEQ ID N0:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
{C) STRANDEDNESS: single
{D) TOPOLOGY: linear
{ii) MOLECULE TYPE: other nucleic acid
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(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:27:
ANAAGCTTCAT CCAGTAAGAT ATTTGCAC 29
(2) INFORMATION FOR SEQ ID N0:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:
ANTTCAGAACT GGTCACTTCA CAGAAAGA 29
(2) INFORMATION FOR SEQ ID N0:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:
GNATTCACATA GGATGAAGGT GAATGTCC 29
(2) INFORMATION FOR SEQ ID N0:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:30:
ANTAGAGGCTG GGAACCAGGA GAAGAGAA 29
(2) INFORMATION FOR SEQ ID N0:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:31:
TNTTGCAGGTC TTAACAGCCG CATTCATC 29
(2) INFORMATION FOR SEQ ID N0:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 113 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:32:
Met Glu Leu Pro Ser Gly Pro Gly Pro Glu Arg Leu Phe Asp Ser His
1 5 10 15
Arg Leu Pro Gly Asp Cys Phe Leu Leu Leu Val Leu Leu Leu Tyr Ala
20 25 30
Pro Val Gly Phe Cys Leu Leu Val Leu Arg Leu Phe Leu Gly Ile His
35 40 45
Val Phe Leu Val Ser Cys Ala Leu Pro Asp Ser Val Leu Arg Arg Phe
50 55 60
Val Val Arg Thr Met Cys Ala Val Leu Gly Leu Val Ala Arg Gln Glu
65 70 75 80
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Asp Ser Gly Leu Arg Asp His Ser Val Arg Val Leu Ile Ser Asn His
85 90 95
Val Thr Pro Phe Asp His Asn Ile Val Asn Leu Leu Thr Thr Cys Ser
100 105 110
Thr
(2) INFORMATION FOR SEQ ID N0:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 63 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:33:
Ser Gln Pro Leu Leu Asn Ser Pro Pro Ser Phe Val Cys Trp Ser Arg
1 5 10 15
Gly Phe Met Glu Met Asn Gly Arg Gly Glu Leu Val Glu Ser Leu Lys
20 25 30
Arg Phe Cys Ala Ser Thr Arg Leu Pro Pro Thr Pro Leu Leu Leu Phe
35 40 45
Pro Glu Glu Glu Ala Thr Asn Gly Arg Glu Gly Leu Leu Arg Phe
50 55 60
(2) INFORMATION FOR SEQ ID N0:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 49 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:34:
Ser Ser Trp Pro Phe Ser Ile Gln Asp Val Val Gln Pro Leu Thr Leu
1 5 10 15
Gln Val Gln Arg Pro Leu Val Ser Val Thr Val Ser Asp Ala Ser Trp
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20 25 30
Val Ser Glu Leu Leu Trp Ser Leu Phe Val Pro Phe Thr Val Tyr Gln
35 40 45
Val
100
