Note: Descriptions are shown in the official language in which they were submitted.
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10
SECRETED PROTEINS AND POLYNUCLEOTIDES ENCODING THEM
This is a continuation-in-part of Ser. No. 60/XXX,XXX, filed December 13, 1996
(converted to provisional application from non-provisional application Ser.
No.
08/766,263), which is incorporated by reference herein.
2 0 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.
2 5 BACKGROUND OF THE INVENTION
Technology aimed at the discovery of protein factors (including e.g.,
cytokines,
such as lymphokines, interferons, CSFs and interleukins) has matured rapidly
over the
past decade. The now routine hybridization cloning and expression cloning
techniques
clone novel polynucleotides "directly" in the sense that they rely on
information directly
3 0 related to the discovered protein (i.e., partial DNA / amino acid sequence
of the protein
in the case of hybridization cloning; activity of the protein in the case of
expression
cloning). More recent "indirect" cloning techniques such as signal sequence
cloning, which
isolates DNA sequences based on the presence of a now well-recognized
secretory leader
sequence motif, as well as various PCR-based or low stringency hybridization
cloning
3 5 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
NO:1 from nucleotide 22 to nucleotide 462;
(c) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone AJl 1 deposited under accession number
ATCC 98278;
(d) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone AJ1 1 deposited under accession number ATCC 98278;
(e) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone AJ1 1 deposited under accession number ATCC
98278;
(f) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone AJ1_1 deposited under accession number ATCC 98278;
(g) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:2;
2 0 (h) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:2 having biological activity;
{i) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(f) above;
(j) a polynucleotide which encodes a species homologue of the protein
2 5 of {g) or (h) above ; and
(k} a polynucleotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(h).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
NO:1 from nucleotide 22 to nucleotide 462; the nucleotide sequence of the full-
length
3 0 protein coding sequence of clone AJ1 1 deposited under accession number
ATCC 98278;
or the nucleotide sequence of the mature protein coding sequence of clone
AJl_1
deposited under accession number ATCC 98278. In other preferred embodiments,
the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone AJl 1 deposited under accession number ATCC 98278. In yet other
preferred
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embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:2 from amino acid 52 to amino
acid
147.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID NO:1 or SEQ ID N0:3.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:2;
(b) the amino acid sequence of SEQ ID N0:2 from amino acid 52 to
amino acid 147;
(c) fragments of the amino acid sequence of SEQ ID N0:2; and
(d) the amino acid sequence encoded by the cDNA insert of clone
AJ1_1 deposited under accession number ATCC 98278;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:2 or the amino acid
sequence
of SEQ ID N0:2 horn amino acid 52 to amino acid 147.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
2 0 (a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:4;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:4 from nucleotide 7 to nucleotide 1647;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
2 5 N0:4 from nucleotide 1 to nucleotide 305;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone AQ73_3 deposited under accession
number ATCC 98278;
(e) a polynucleotide encoding the full-length protein encoded by the
3 0 cDNA insert of clone AQ73 3 deposited under accession number ATCC 98278;
(f) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone AQ73_3 deposited under accession number
ATCC 98278;
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(g) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone AQ73_3 deposited under accession number ATCC 98278;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:5;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:5 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein
of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:4 from nucleotide 7 to nucleotide 1647; the nucleotide sequence of SEQ ID
N0:4 from
nucleotide 1 to nucleotide 305; the nucleotide sequence of the full-length
protein coding
sequence of clone AQ73 3 deposited under accession number ATCC 98278; or the
nucleotide sequence of the mature protein coding sequence of clone AQ73_3
deposited
under accession number ATCC 98278. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
2 0 clone AQ73 3 deposited under accession number ATCC 98278. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:5 from amino acid 1 to amino
acid 68.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:4.
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 N0:5;
{b) the amino acid sequence of SEQ ID N0:5 from amino acid 1 to
3 0 amino acid 68;
(c) fragments of the amino acid sequence of SEQ ID N0:5; and
(d) the amino acid sequence encoded by the cDNA insert of clone
AQ73 3 deposited under accession number ATCC 98278;
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the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:5 or the amino acid
sequence
of SEQ ID N0:5 from amino acid 1 to amino acid 68.
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
N0:6;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:6 from nucleotide 62 to nucleotide 757;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:6 from nucleotide 357 to nucleotide 703;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone BG142_1 deposited under accession
number ATCC 98278;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone BG142_1 deposited under accession number ATCC 98278;
(f) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone BG142_1 deposited under accession number
ATCC 98278;
2 0 (g) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone BG142_1 deposited under accession number ATCC 98278;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:7;
(i) a polynucleotide encoding a protein comprising a fragment of the
2 5 amino acid sequence of SEQ ID N0:7 having biological activity;
(j) a polynucleotide which is an allelic variant of a poiynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein
of (h) or (i) above ; and
3 0 (1) a polynucleotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:6 from nucleotide 62 to nucleotide 757; the nucleotide sequence of SEQ ID
N0:6 from
nucleotide 357 to nucleotide 703; the nucleotide sequence of the full-length
protein coding
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sequence of clone BG142_1 deposited under accession number ATCC 98278; or the
nucleotide sequence of the mature protein coding sequence of clone BG142_1
deposited
under accession number ATCC 98278. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone BG142_1 deposited under accession number ATCC 98278. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:7 from amino acid 184 to amino
acid
214.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:6.
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 N0:7;
(b) the amino acid sequence of SEQ ID N0:7 from amino acid 184 to
amino acid 214;
(c) fragments of the amino acid sequence of SEQ ID N0:7; and
(d) the amino acid sequence encoded by the cDNA insert of clone
BG142_1 deposited under accession number ATCC 98278;
2 0 the protein being substantially free from other mammalian proteins.
Preferably such
protein comprises the amino acid sequence of SEQ ID N0:7 or the amino acid
sequence
of SEQ ID N0:7 from amino acid 184 to amino acid 214.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
2 5 (a) a polynucleoHde comprising the nucleotide sequence of SEQ ID
N0:8;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:8 from nucleotide 404 to nucleotide 535;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
3 0 N0:8 from nucleotide 1 to nucleotide 666;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone BV66_1 deposited under accession
number ATCC 98278;
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(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone BV66_1 deposited under accession number ATCC 98278;
(f) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone BV66_1 deposited under accession number
ATCC 98278;
(g) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone BV66_1 deposited under accession number ATCC 98278;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:9;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:9 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein
of (h) or (i) above ; and
(1) a polynucleotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:8 from nucleotide 404 to nucleotide 535; the nucleotide sequence of SEQ ID
N0:8
2 0 from nucleotide 1 to nucleotide 666; the nucleotide sequence of the full-
length protein
coding sequence of clone BV66_1 deposited under accession number ATCC 98278;
or the
nucleotide sequence of the mature protein coding sequence of clone BV66_1
deposited
under accession number ATCC 98278. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
2 5 clone BV66_1 deposited under accession number ATCC 98278. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:9 from amino acid 1 to amino
acid 38.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:8.
3 0 In other embodiments, the present invention provides a composition
comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:9;
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(b) the amino acid sequence of SEQ ID N0:9 from amino acid 1 to
amino acid 38;
(c) fragments of the amino acid sequence of SEQ ID N0:9; and
(d) the amino acid sequence encoded by the cDNA insert of clone
BV66_1 deposited under accession number ATCC 98278;
the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:9 or the amino acid
sequence
of SEQ ID N0:9 from amino acid 1 to amino acid 38.
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
N0:10;
(b) a polynucleotide comprising the nucleotide sequence of SEQ iD
N0:10 from nucleotide 1204 to nucleotide 1389;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:10 from nucleotide 881 to nucleotide 1380;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone BV291 3 deposited under accession
number ATCC 98278;
2 0 (e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone BV291 3 deposited under accession number ATCC 98278;
(f) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone BV291 3 deposited under accession number
ATCC 98278;
2 5 (g) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone BV291 3 deposited under accession number ATCC 98278;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:11;
(i) a polynucleotide encoding a protein comprising a fragment of the
3 0 amino acid sequence of SEQ ID NO:11 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a}-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein
of (h) or (i) above ; and
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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
NO:10 from nucleotide 1204 to nucleotide 1389; the nucleotide sequence of SEQ
ID N0:10
' S from nucleotide 881 to nucleotide 1380; the nucleotide sequence of the
full-length protein
coding sequence of clone BV291 3 deposited under accession number ATCC 98278;
or the
nucleotide sequence of the mature protein coding sequence of clone BV291 3
deposited
under accession number ATCC 98278. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone BV291 3 deposited under accession number ATCC 98278. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID NO:11 from amino acid 1 to amino
acid
59.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID NO:10.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID NO:11;
2 0 (b) the amino acid sequence of SEQ ID N0:11 from amino acid 1 to
amino acid 59;
(c) fragments of the amino acid sequence of SEQ ID N0:11; and
{d) the amino acid sequence encoded by the cDNA insert of clone
BV291 3 deposited under accession number ATCC 98278;
2 5 the protein being substantially free from other mammalian proteins.
Preferably such
protein comprises the amino acid sequence of SEQ ID N0:11 or the amino acid
sequence
of SEQ ID N0:11 from amino acid 1 to amino acid 59.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleoHde selected from the group consisting of:
3 0 (a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:12;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:12 from nucleotide 189 to nucleotide 1115;
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(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:12 from nucleotide 1 to nucleotide 451;
{d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone CK201_1 deposited under accession
number ATCC 98278;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone CK201_1 deposited under accession number ATCC 98278;
(f) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone CK201 1 deposited under accession number
ATCC 98278;
(g) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone CK201 1 deposited under accession number ATCC 98278;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:13;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:13 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
{k) a polynucleotide which encodes a species homologue of the protein
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 polynucleotide comprises the nucleotide sequence of SEQ ID
N0:12 from nucleotide 189 to nucleotide 1115; the nucleotide sequence of SEQ
ID N0:12
2 5 from nucleotide 1 to nucleotide 451; the nucleotide sequence of the full-
length protein
coding sequence of clone CK201 1 deposited under accession number ATCC 98278;
or the
nucleotide sequence of the mature protein coding sequence of clone CK201 1
deposited
under accession number ATCC 98278. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
3 0 clone CK201 1 deposited under accession number ATCC 98278. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:13 from amino acid 1 to amino
acid
88.
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Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:12.
In other embodiments, the present invention provides a composition comprising
a protein, wherein said protein comprises an amino acid sequence selected kom
the group
consisting of:
{a) the amino acid sequence of SEQ ID N0:13;
(b) the amino acid sequence of SEQ ID N0:13 from amino acid 1 to
amino acid 88;
(c) fragments of the amino acid sequence of SEQ ID N0:13; and
(d) the amino acid sequence encoded by the cDNA insert of clone
CIC201 1 deposited under accession number ATCC 98278;
the protein being substantially kee from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:13 or the amino acid
sequence
of SEQ ID N0:13 from amino acid 1 to amino acid 88.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected kom 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
2 0 N0:14 from nucleotide 1I7 to nucleotide 923;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:14 from nucleotide 174 to nucleotide 923;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:14 from nucleotide 1 to nucleotide 316;
2 5 (e) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone CQ331 2 deposited under accession
number ATCC 9827$;
(f) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone CQ331 2 deposited under accession number ATCC 98278;
3 0 (g) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone CQ331 2 deposited under accession number
ATCC 98278;
(h) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone CQ331 2 deposited under accession number ATCC 98278;
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(i) a polynucleoHde encoding a protein comprising the amino acid
sequence of SEQ ID N0:15;
(j) a polynucleotide encoding a protein comprising a fragment of the~
amino acid sequence of SEQ ID N0:15 having biological activity;
(k) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(h) above;
(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
N0:14 from nucleotide 117 to nucleotide 923; the nucleotide sequence of SEQ ID
N0:14
from nucleotide 174 to nucleotide 923; the nucleotide sequence of SEQ ID N0:14
from
nucleotide 1 to nucleotide 316; the nucleotide sequence of the full-length
protein coding
sequence of clone CQ331 2 deposited under accession number ATCC 98278; or the
nucleotide sequence of the mature protein coding sequence of clone CQ331 2
deposited
under accession number ATCC 98278. in other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone CQ331 2 deposited under accession number ATCC 98278. In yet other
preferred
2 0 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
57.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:14.
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 N0:15;
(b) the amino acid sequence of SEQ ID N0:15 from amino acid 1 to
3 0 amino acid 57;
(c) fragments of the amino acid sequence of SEQ ID N0:15; and
(d) the amino acid sequence encoded by the cDNA insert of clone
CQ331 2 deposited under accession number ATCC 98278;
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the protein being substantially free from other mammalian proteins. Preferably
such
protein comprises the amino acid sequence of SEQ ID N0:15 or the amino acid
sequence
of SEQ ID N0:15 from amino acid 1 to amino acid 57.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:16;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:16 from nucleotide 223 to nucleotide 483;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:16 from nucleotide 22 to nucleotide 397;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone CT550_1 deposited under accession
number ATCC 98278;
(e) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone CT550_1 deposited under accession number ATCC 98278;
(f) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone CT550_1 deposited under accession number
ATCC 98278;
2 0 (g) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone CT550 1 deposited under accession number ATCC 98278;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:17;
(i) a polynucleotide encoding a protein comprising a fragment of the
2 5 amino acid sequence of SEQ ID N0:17 having biological activity;
(j) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein
of (h) or (i) above ; and
3 0 (1) a polynucleotide capable of hybridizing under stringent conditions
to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:16 from nucleotide 223 to nucleotide 483; the nucleotide sequence of SEQ ID
N0:16
from nucleotide 22 to nucleotide 397; the nucleotide sequence of the full-
length protein
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coding sequence of clone CT550_1 deposited under accession number ATCC 98278;
or the
nucleotide sequence of the mature protein coding sequence of clone CT550_1
deposited
under accession number ATCC 98278. In other preferred embodiments, the
polynudeotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone CT550_1 deposited under accession number ATCC 98278. 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 1 to amino
acid
58.
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 1 to
amino acid 58;
(c) fragments of the amino acid sequence of SEQ ID N0:17; and
(d) the amino acid sequence encoded by the cDNA insert of clone
CT550_1 deposited under accession number ATCC 98278;
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 1 to amino acid 58.
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 112 to nucleotide 969;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
3 0 N0:18 from nucleotide 154 to nucleotide 969;
(d) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:18 from nucleotide 1 to nucleotide 423;
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(e) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone CT585_1 deposited under accession
number ATCC 98278;
(f) a polynucleotide encoding the full-length protein encoded by the
cDNA insert of clone CT585_1 deposited under accession number ATCC 98278;
(g) a polynucleoHde comprising the nucleotide sequence of the mature
protein coding sequence of clone CT585_1 deposited under accession number
ATCC 98278;
{h) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone CT585_1 deposited under accession number ATCC 98278;
(i) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:19;
(j) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:19 having biological activity;
(k) a polynucleotide which is an allelic variant of a polynucleotide of
(a)-(h) above;
(1) a polynucleotide which encodes a species homologue of the protein
of (i) or (j) above ; and
(m) a polynucleotide capable of hybridizing under stringent conditions
2 0 to any one of the polynucleotides specified in (a)-(j).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:18 from nucleotide 112 to nucleotide 969; the nucleotide sequence of SEQ ID
N0:18
from nucleotide 154 to nucleotide 969; the nucleotide sequence of SEQ ID N0:18
from
nucleotide 1 to nucleotide 423; the nucleotide sequence of the full-length
protein coding
2 5 sequence of clone CT585_1 deposited under accession number ATCC 98278; or
the
nucleotide sequence of the mature protein coding sequence of clone CT585_1
deposited
under accession number ATCC 98278. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone CT585_1 deposited under accession number ATCC 98278. In yet other
preferred
3 0 embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:19 from amino acid 1 to amino
acid
104.
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 1 to
amino acid 104;
(c) fragments of the amino acid sequence of SEQ ID N0:19; and
(d) the amino acid sequence encoded by the cDNA insert of clone
CT585 1 deposited under accession number ATCC 98278;
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 1 to amino acid 104.
In one embodiment, the present invention provides a composition comprising an
isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:20;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:20 from nucleotide 37 to nucleotide 2766;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
2 0 N0:20 from nucleotide 243 to nucleotide 789;
(d) a polynucleotide comprising the nucleotide sequence of the full-
length protein coding sequence of clone CT797_3 deposited under accession
number ATCC 98278;
(e) a polynucleotide encoding the full-length protein encoded by the
2 5 cDNA insert of clone CT797_3 deposited under accession number ATCC 98278;
(f) a polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone CT797_3 deposited under accession number
ATCC 98278;
(g) a polynucleotide encoding the mature protein encoded by the
3 0 cDNA insert of clone CT797_3 deposited under accession number ATCC 98278;
(h) a polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID N0:21;
(i) a polynucleotide encoding a protein comprising a fragment of the
amino acid sequence of SEQ ID N0:21 having biological activity;
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(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
' S (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 37 to nucleotide 2766; the nucleotide sequence of SEQ ID
N0:20
from nucleotide 243 to nucleotide 789; the nucleotide sequence of the full-
length protein
coding sequence of clone CT797_3 deposited under accession number ATCC 98278;
or the
nucleotide sequence of the mature protein coding sequence of clone CT797_3
deposited
under accession number ATCC 98278. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by the cDNA
insert of
clone CT797_3 deposited under accession number ATCC 98278. In yet other
preferred
embodiments, the present invention provides a polynucleotide encoding a
protein
comprising the amino acid sequence of SEQ ID N0:21 from amino acid 75 to amino
acid
251.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:20.
2 0 In other embodiments, the present invention provides a composition
comprising
a protein, wherein said protein comprises an amino acid sequence selected from
the group
consisting of:
(a) the amino acid sequence of SEQ ID N0:21;
(b) the amino acid sequence of SEQ ID N0:21 from amino acid 75 to
2 5 amino acid 251;
{c) fragments of the amino acid sequence of SEQ ID N0:21; and
(d) the amino acid sequence encoded by the cDNA insert of clone
CT797 3 deposited under accession number ATCC 98278;
the protein being substantially free from other mammalian proteins. Preferably
such
3 0 protein comprises the amino acid sequence of SEQ ID N0:21 or the amino
acid sequence
of SEQ ID N0:21 from amino acid 75 to amino acid 251.
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.
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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.
Methods are also provided for preventing, treating or ameliorating a medical
condition which comprises administering to a mammalian subject a
therapeutically
effective amount of a composition comprising a protein of the present
invention and a
pharmaceutically acceptable carrier.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1A and 1B are schematic representations of the pED6 and pNOTs vectors,
respectively, used for deposit of clones disclosed herein.
2 0 DETAILED DESCRIPTION
ISOLATED PROTEINS AND POLYNUCLEOTIDES
Nucleotide and amino acid sequences, as presently determined, are reported
below for each clone and protein disclosed in the present application. The
nucleotide
sequence of each clone can readily be determined by sequencing of the
deposited clone
2 5 in accordance with known methods. The predicted amino acid sequence (both
full-length
and mature) can then be determined from such nucleotide sequence. The amino
acid
sequence of the protein encoded by a particular clone can also be determined
by
expression of the clone in a suitable host cell, collecting the protein and
determining its
sequence. For each disclosed protein applicants have identified what they have
3 0 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
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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 "AT1 1"
A polynucleotide of the present invention has been identified as clone "AJl
1".
AJl_1 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. AJ1_1 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
"AJ1 1 protein").
The nucleotide sequence of the 5' portion of AJ1_1 as presently determined is
reported in SEQ ID N0:1. What applicants presently believe is the proper
reading frame
for the coding region is indicated in SEQ ID N0:2. The predicted amino acid
sequence of
the AJl 1 protein corresponding to the foregoing nucleotide sequence is
reported in SEQ
ID N0:2. Additional nucleotide sequence from the 3' portion of AJ1 1,
including the
polyA tail, is reported in SEQ ID N0:3.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
2 0 AJ1_1 should be approximately 925 bp.
The predicted amino acid sequence disclosed herein for AJl_1 was searched
against the GenPept and GeneSeq amino acid sequence databases using the BLASTX
search protocol. The predicted AJ1 1 protein demonstrated at least some
similarity to
sequences identified as U39060 (GRIPl [Mus musculus]). Based upon sequence
similarity,
2 5 AJ1 1 proteins and each similar protein or peptide may share at least some
activity.
Clone "A073 3"
A polynucleotide of the present invention has been identified as clone "AQ73
3".
AQ73_3 was isolated from a human adult ovary (PA-1 teratocarcinoma, untreated
tissue
3 0 pooled with retinoic-acid-treated and activin-treated tissue) 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. AQ73_3 is a full-
length clone,
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including the entire coding sequence of a secreted protein (also referred to
herein as
"AQ73 3 protein').
The nucleotide sequence of AQ73 3 as presently determined is reported in SEQ
ID N0:4. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the AQ73_3 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:5.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
AQ73_3 should be approximately 2800 bp.
The nucleotide sequence disclosed herein for AQ73_3 was searched against the
2 0 GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. AQ73_3 demonstrated at least some similarity with
sequences
identified as AA514474 (nf57gOl.s1 NCI_CGAP Co3 Homo sapiens cDNA clone
924048),
T47520 (Human hepatoma-derived growth factor (HDGF-2) cDNA), W24708
(zb62e08.r1
Soares fetal lung NbHLI9W Homo sapiens cDNA clone 308198 5'), and W45513
(zc27g08.s1 Soares senescent fibroblasts NbHSF Homo sapiens cDNA clone 323582
3').
The predicted amino acid sequence disclosed herein for AQ73_3 was searched
against the
GenPept and GeneSeq amino acid sequence databases using the BLASTX search
protocol.
The predicted AQ73_3 protein demonstrated at least some similarity to
sequences
identified as D16431 (hepatoma-derived GF [Homo sapiens]), D63707 (mouse
hepatoma
2 0 derived growth factor (HDGF) [Mus musculus]), R66727 (Human hepatoma
derived
growth factor), U18997 (ORF_f299 (Escherichia coli]), U97193 (similar to S.
cerevisiae SIR2
(SP P06700) and mouse hepatoma derived growth factor HDGF (NID 8945418)
[Caenorhabditis elegans]), and W09404 (Human hepatoma-derived growth factor
(HDGF-2)). Based upon sequence similarity, AQ73_3 proteins and each similar
protein
2 5 or peptide may share at least some activity.
Clone "BG142 1"
A polynucleotide of the present invention has been identified as clone "BG
142_1 ".
BG142_1 was isolated from a human adult brain cDNA library using methods which
are
3 0 selective for cDNAs encoding secreted proteins (see U.S. Pat. No.
5,536,637), or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. BG142_1 is a full-
length
clone, including the entire coding sequence of a secreted protein (also
referred to herein
as "BG142_1 protein")
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The nucleotide sequence of BG142_1 as presently determined is reported in SEQ
ID N0:6. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the BG142_1 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:7.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
BG142_1 should be approximately 1100 bp.
The nucleotide sequence disclosed herein for BG 142_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. BG142_1 demonstrated at least some similarity with
sequences
identified as AA170261 (ms87h11.r1 Soares mouse 3NbMS Mus musculus cDNA clone
618597 5' similar to TR E245601 E245601 G-RICH BOX-BINDING PROTEIN), L04282
(Human CACCC box-binding protein mRNA, complete cds), N27696 (yx51h12.r1 Homo
Sapiens cDNA clone 265319 5'), W96110 {ze09a11.r1 Soares fetal heart NbHHI9W
Homo
sapiens cDNA clone 358460 5'), and W96111 (ze09a11.s1 Soares fetal heart
NbHHI9W
Homo sapiens cDNA clone 358460 3'). The predicted amino acid sequence
disclosed
herein for BG142_1 was searched against the GenPept and GeneSeq amino acid
sequence
databases using the BLASTX search protocol. The predicted BG142_1 protein
demonstrated at least some similarity to sequences identified as U80078
(transcription
factor BFCOL1 [Mus musculus]) and X98096 (G-rich box-binding protein [Mus
2 0 musculus]). Based upon sequence similarity, BG142_1 proteins and each
similar protein
or peptide may share at least some activity.
Clone "BV66 1"
A polynucleotide of the present invention has been identified as clone
"BV66_1".
2 5 BV66_1 was isolated from a human adult brain cDNA library using methods
which are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637),
or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. BV66_1 is a full-
length clone,
including the entire coding sequence of a secreted protein (also referred to
herein as
3 0 "BV66_1 protein").
The nucleotide sequence of BV66_1 as presently determined is reported in SEQ
ID
N0:8. What applicants presently believe to be the proper reading frame and the
predicted
amino acid sequence of the BV66_1 protein corresponding to the foregoing
nucleotide
sequence is reported in SEQ ID N0:9.
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The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
BV66_1 should be approximately 870 bp.
The nucleotide sequence disclosed herein for BV66_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN / BLASTX and
FASTA search protocols. No hits were found in the database. The nucleotide
sequence
of BV66_1 indicates that it may contain a TAAAl simple repeat element.
Clone "BV291 3"
A polynucleotide of the present invention has been identified as clone "BV291
3".
BV291 3 was isolated from a human adult brain cDNA library using methods which
are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637),
or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. BV291 3 is a full-
length
clone, including the entire coding sequence of a secreted protein (also
referred to herein
as "BV291 3 protein").
The nucleotide sequence of BV291 3 as presently determined is reported in SEQ
ID NO:10. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the BV291 3 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:11.
2 0 The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
BV291 3 should be approximately 2000 bp.
The nucleotide sequence disclosed herein for BV291 3 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. BV291 3 demonstrated at least some similarity with
sequences
2 5 identified as H10954 (ym06e09.r1 Homo sapiens cDNA clone 47034 5'), H10955
(ym06e09.s1 Homo sapiens cDNA clone 47034 3'), N25300 (yw52c10.s1 Homo sapiens
cDNA clone 255858 3'), T25940 (Human gene signature HUMGS08173), T68890
(yc30g11.s1 Homo Sapiens cDNA clone 82244 3'), T78286 (yc99a08.r1 Homo sapiens
cDNA
clone 24033 5'), 239987 (H. sapiens partial cDNA sequence; clone c-1oh05), and
247073
3 0 (Caenorhabditis elegans cosmid ZC506). The predicted amino acid sequence
disclosed
herein for BV292 3 was searched against the GenPept and GeneSeq amino acid
sequence
databases using the BLASTX search protocol. The predicted BV291 3 protein
demonstrated at least some similarity to sequences identified as X02155 (BTTGR
1
thyroglobulin [Bos taurus)). Based upon sequence similarity, BV291 3 proteins
and each
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similar protein or peptide may share at least some activity. The TopPredII
computer
program predicts a potential transmembrane domain within the BV291 3 protein
sequence centered around amino acid 48 of SEQ ID N0:11.
Clone "CK201 1"
A polynucleotide of the present invention has been identified as clone
"CK201_1".
CK201_1 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. CK201 1 is a full-
length
clone, including the entire coding sequence of a secreted protein (also
referred to herein
as "CK201 1 protein")
The nucleotide sequence of CK201_1 as presently determined is reported in SEQ
ID N0:12. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the CK201_1 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:13.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
CK201 1 should be approximately 1080 bp.
The nucleotide sequence disclosed herein for CK201_1 was searched against the
2 0 GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. CK201 1 demonstrated at least some similarity with
sequences
identified as AA229133 (zo09h12.s1 Stratagene neuroepithelium NT2RAMI 937234
Homo
Sapiens cDNA clone 567239 3' similar to contains Alu repetitive element),
D81444 (Human
fetal brain cDNA 5'-end GEN-164610), 836326 (yg69h09.r1 Homo sapiens cDNA
clone
2 5 38821 5'), T08553 (Oncogene R-ras mutant cDNA (exons 2-6)), T31595 (Probe
(BLUR13)
for Alu repeat sequence), X03273 (Human Alu-family cluster 5' of alpha(1)-acid
glycoprotein gene), and X69907 (H.sapiens gene for mitochondrial ATP synthase
c
subunit). The predicted amino acid sequence disclosed herein for CK201 1 was
searched
against the GenPept and GeneSeq amino acid sequence databases using the BLASTX
3 0 search protocol. The predicted CK201 1 protein demonstrated at least some
similarity to
sequences identified as D21827 (major surface glycoprotein [Pneumocystis
carinii]). Based
upon sequence similarity, CK201_1 proteins and each similar protein or peptide
may
share at least some activity. The nucleotide sequence of CK201_l indicates
that it may
contain an Alu repetitive element.
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Clone "CQ331 2"
A polynucleotide of the present invention has been identified as clone "CQ331
2".
CQ331 2 was isolated from a human adult heart 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. CQ331 2 is a full-
length
clone, including the entire coding sequence of a secreted protein (also
referred to herein
as "CQ331 2 protein'}.
The nucleotide sequence of CQ331 2 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 CQ331 2 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:15. Amino acids 7 to 19 are a
predicted
leader/signal sequence, with the predicted mature amino acid sequence
beginning at
amino acid 20, or are a transmembrane domain.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
CQ331 2 should be approximately 1600 bp.
The nucleotide sequence disclosed herein for CQ331 2 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. CQ331 2 demonstrated at least some similarity with
sequences
2 0 identified as J03766 (Canine cardiac calsequestrin mRNA, complete cds),
L29766 {Homo
sapiens epoxide hydrolase (EPHX) gene, complete cds), N8360I (KK1173F Homo
sapiens
cDNA clone KK1173 5' similar to CALSEQUESTRIN (CARDIAC)), T99646 (ye73f12.s1
Homo Sapiens cDNA clone 123407 3' similar to contains Alu repetitive
eiement;contains
PTR5 repetitive element), and W76326 (zd60d04.r1 Soares fetal heart NbHHI9W
Homo
2 5 Sapiens cDNA clone 3450315' similar to contains Alu repetitive element).
The predicted
amino acid sequence disclosed herein for CQ331 2 was searched against the
GenPept and
GeneSeq amino acid sequence databases using the BLASTX search protocol. The
predicted CQ331 2 protein demonstrated at least some similarity to sequences
identified
as J03766 (DOGCAL_1 Canine cardiac calsequestrin mRNA, complete cds [Cams
carus])
3 0 and X55040 (calsequestrin [Oryctolagus curuculus]). Based upon sequence
similarity,
CQ331 2 proteins and each similar protein or peptide may share at least some
activity.
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Clone "CT550 1"
A polynucleotide of the present invention has been identified as clone
"CT550_1".
CT550_l was isolated from a human adult brain cDNA library using methods which
are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637),
or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. CT550_1 is a full-
length
clone, including the entire coding sequence of a secreted protein (also
referred to herein
as "CT550_1 protein")
The nucleotide sequence of CT550_1 as presently determined is reported in SEQ
ID NO:16. What applicants presently believe to be the proper reading frame and
the
predicted amino acid sequence of the CT550_1 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:17.
The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
CT550_1 should be approximately 1070 bp.
The nucleotide sequence disclosed herein for CT550_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. No hits were found in the database. The TopPredII
computer
program predicts a potential transmembrane domain within the CT550_1 protein
sequence centered around amino acid 25 of SEQ ID N0:17.
Clone "CT585 1"
A polynucleotide of the present invention has been identified as clone
"CT585_1".
CT585_1 was isolated from a human adult brain cDNA library using methods which
are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637),
or was
2 5 identified as encoding a secreted or transmembrane protein on the basis of
computer
analysis of the amino acid sequence of the encoded protein. CT585 1 is a full-
length
clone, including the entire coding sequence of a secreted protein (also
referred to herein
as "CT585_1 protein").
The nucleotide sequence of CT585_1 as presently determined is reported in SEQ
3 0 ID N0:18. What applicants presently believe to be the proper reading frame
and the
predicted amino acid sequence of the CT585_1 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:19. Amino acids 2 to 14 are a
predicted
leader/signal sequence, with the predicted mature amino acid sequence
beginning at
amino acid 15, or are a transmembrane domain.
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The EcoRI/NotI restriction fragment obtainable from the deposit containing
clone
CT585_l should be approximately 2710 bp.
The nucleotide sequence disclosed herein for CT585_1 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. CT585_1 demonstrated at least some similarity with
sequences
identified as AA069442 (zf74b02.s1 Soares pineal gland N3HPG Homo Sapiens cDNA
clone 382635 3'), L38961 (Homo sapiens putative transmembrane protein (B5)
mRNA,
complete cds), N34932 (yy49b10.s1 Homo sapiens cDNA clone 276859 3'), N60101
(TgESTzy11f10.r1 Toxoplasma gondii cDNA clone tgzy11f10.r1 5'), and U13019
(Caenorhabditis elegans cosmid T12A2). The predicted amino acid sequence
disclosed
herein for CT585_1 was searched against the GenPept, GeneSeq, and SwissProt
amino
acid sequence databases using the BLASTX search protocol. The predicted
CT585_1
protein demonstrated at least some similarity to sequences identified as
L34260
(transmembrane protein [Mus musculus)), L38961 (transmembrane protein [Homo
Sapiens]}, P46975 (Caenorhabditis elegans oligosaccharyl transferase stt3
[Caenorhabditis
elegans]), and U13019 (Caenorhabditis elegans cosmid T12A2 [Caenorhabditis
elegans]).
Based upon sequence similarity, CT585_1 proteins and each similar protein or
peptide
may share at least some activity.
2 0 Clone "CT797 3"
A polynucleotide of the present invention has been identified as clone
"CT797_3".
CT797_3 was isolated from a human adult brain cDNA library using methods which
are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637),
or was
identified as encoding a secreted or transmembrane protein on the basis of
computer
2 5 analysis of the amino acid sequence of the encoded protein. CT797_3 is a
full-length
clone, including the entire coding sequence of a secreted protein (also
referred to herein
as "CT797_3 protein')
The nucleotide sequence of CT797_3 as presently determined is reported in SEQ
ID N0:20. What applicants presently believe to be the proper reading frame and
the
3 0 predicted amino acid sequence of the CT797_3 protein corresponding to the
foregoing
nucleotide sequence is reported in SEQ ID N0:21.
The EcoIZI/NotI restriction fragment obtainable from the deposit containing
clone
CT797 3 should be approximately 3300 bp.
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The nucleotide sequence disclosed herein for CT797_3 was searched against the
GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and
FASTA search protocols. CT797_3 demonstrated at least some similarity with
sequences
identified as AA573847 (nk08d06.s1 NCI_CGAP Co2 Homo sapiens cDNA clone
IMAGE:1012907). The predicted amino acid sequence disclosed herein for CT797_3
was
searched against the GenPept and GeneSeq amino acid sequence databases using
the
BLASTX search protocol. The predicted CT797_3 protein demonstrated at least
some
similarity to sequences identified as U18309 (chromokinesin [callus gallus])
and 282271
(T01G1.1 [Caenorhabditis elegans]). Based upon sequence similarity, CT797_3
proteins
and each similar protein or peptide may share at least some activity.
Deposit of Clones
Clones AJ1 1, AQ73_3, BG142_1, BV66_1, BV291 3, CK201 1, CQ331 2, CT550_l,
CT585 1 and CT797_3 were deposited on December 13, 1996 with the American Type
Culture Collection as an original deposit under the Budapest Treaty and were
given the
accession number ATCC 98278, from which each clone comprising a particular
polynucleotide is obtainable. All restrictions on the availability to the
public of the
deposited material will be irrevocably removed upon the granting of the
patent, except
for the requirements specified in 37 C.F.R. ~ 1.808(b).
2 0 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
2 5 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
3 0 orientation) in the deposited isolate. In such instances, the cDNA insert
can still be
isolated by digestion with EcoRI and NotI. However, NotI will then produce the
5' site
and EcoRI will produce the 3' site for placement of the cDNA in proper
orientation for
expression in a suitable vector. The cDNA may also be expressed from the
vectors in
which they were deposited.
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Bacterial cells containing a particular clone can be obtained from the
composite
deposit as follows:
An oligonucleotide probe or probes should be designed to the sequence that is
known for that particular clone. This sequence can be derived from the
sequences
provided herein, or from a combination of those sequences. The sequence of the
oligonucleotide probe that was used to isolate each full-length clone is
identified below,
and should be most reliable in isolating the clone of interest.
Clone Probe Sequence
AJ1 1 SEQ ID N0:22
AQ73 3 SEQ ID N0:23
BG142_1 SEQ ID N0:24
BV66_1 SEQ ID N0:25
BV291 3 SEQ ID N0:26
1 S CK201_1 SEQ ID N0:27
CQ331 2 SEQ ID N0:28
CT550_1 SEQ ID N0:29
CT585_1 SEQ ID N0:30, SEQ ID N0:32
CT797_3 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-
2 S 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;
3 0 (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
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label should preferably be removed by gel filtration chromatography or other
established
methods. The amount of radioactivity incorporated into the probe should be
quantitated
by measurement in a scintillation counter. Preferably, specific activity of
the resulting
probe should be approximately 4e+6 dpm/pmole.
The bacterial culture containing the pool of full-length clones should
preferably
be thawed and 100 ul of the stock used to inoculate a sterile culture flask
containing 25 ml
of sterile L-broth containing ampicillin at 100 ug/ml. The culture should
preferably be
grown to saturation at 37°C, and the saturated culture should
preferably be diluted in
fresh L-broth. Aliquots of these dilutions should preferably be plated to
determine the
dilution and volume which will yield approximately 5000 distinct and well-
separated
colonies on solid bacteriological media containing L-broth containing
ampicillin at 100
lzg/ml and agar at 1.5% in a 150 mm petri dish when grown overnight at
37°C. Other
known methods of obtaining distinct, well-separated colonies can also be
employed.
Standard colony hybridization procedures should then be used to transfer the
colonies to nitrocellulose filters and lyse, denature and bake them.
The filter is then preferably incubated at 65°C for 1 hour with gentle
agitation in
6X SSC (20X stock is 175.3 g NaCI/liter, 88.2 g Na citrate/liter, adjusted to
pH 7.0 with
NaOH) containing 0.5% SDS,100 ug/ml of yeast RNA, and 10 mM EDTA
(approximately
10 mL per 150 mm filter). Preferably, the probe is then added to the
hybridization mix at
2 0 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 0.1X SSC/0.5% SDS at 65°C for 30 minutes to 1 hour is
optional. The
2 5 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,
3 0 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.
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McDowell, et al., J. Amer. Chem. Soc.114, 9245-9253 (1992), both of which are
incorporated
herein by reference. Such fragments may be fused to carrier molecules such as
immunoglobulins for many purposes, including increasing the valency of protein
binding
sites. For example, fragments of the protein may be fused through "linker"
sequences to
the Fc portion of an immunoglobulin. For a bivalent form of the protein, such
a fusion
could be to the Fc portion of an IgG molecule. Other immunoglobulin isotypes
may also
be used to generate such fusions. For example, a protein - IgM fusion would
generate a
decavalent form of the protein of the invention.
The present invention also provides both full-length and mature forms of the
disclosed proteins. The full-length form of the such proteins is identified in
the sequence
listing by translation of the nucleotide sequence of each disclosed clone. The
mature form
of such protein may be obtained by expression of the disclosed full-length
polynucleotide
(preferably those deposited with ATCC) in a suitable mammalian cell or other
host cell.
The sequence of the mature form of the protein may also be determinable from
the amino
2 5 acid sequence of the full-length form.
The present invention also provides genes corresponding to the cDNA sequences
disclosed herein. "Corresponding genes" are the regions of the genome that are
transcribed to produce the mRNAs from which the cDNA sequences are derived and
may
include contiguous regions of the genome necessary for the regulated
expression of such
2 0 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 suppresser 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
25 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.
Where the protein of the present invention is membrane-bound (e.g., is a
receptor),
3 0 the present invention also provides for soluble forms of such protein. In
such forms part
or au 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.
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Proteins and protein fragments of the present invention include proteins with
amino acid sequence lengths that are at least 25%(more preferably at least
50%, and most
preferably at least 75%) of the length of a disclosed protein and have at
least 60% sequence
identity (more preferably, at least 75% identity; most preferably at least 90%
or 95%
identity) with that disclosed protein, where sequence identity is determined
by comparing
the amino acid sequences of the proteins when aligned so as to maximize
overlap and
identity while minimizing sequence gaps. Also included in the present
invention are
proteins and protein fragments that contain a segment preferably comprising 8
or more
(more preferably 20 or more, most preferably 30 or more) contiguous amino
acids that
shares at least 75% sequence identity (more preferably, at least 85% identity;
most
preferably at least 95% identity) with any such segment of any of the
disclosed proteins.
Species homologs of the disclosed polynucleotides and proteins are also
provided
by the present invention. As used herein, a "species homologue" is a protein
or
polynucleotide with a different species of origin from that of a given protein
or
polynucleotide, but with significant sequence similarity to the given protein
or
polynucleotide, as determined by those of skill in the art. Species homologs
may be
isolated and identified by making suitable probes or primers from the
sequences provided
herein and screening a suitable nucleic acid source from the desired species.
The invention also encompasses allelic variants of the disclosed
polynucleotides
2 0 or proteins; that is, naturally-occurring alternative forms of the
isolated polynucleotide
which also encode proteins which are identical, homologous, or related to that
encoded
by the polynucleotides .
The invention also includes polynucleotides with sequences complementary to
those of the polynucleotides disclosed herein.
2 5 The present invention also includes polynucleotides capable of hybridizing
under reduced stringency conditions, more preferably stringent conditions, and
most
preferably highly stringent conditions, to polynucleotides described herein.
Examples of
stringency conditions are shown in the table below: highly stringent
conditions are those
that are at least as stringent as, for example, conditions A-F; stringent
conditions are at
3 0 least as stringent as, for example, conditions G-L; and reduced stringency
conditions are
at least as stringent as, for example, conditions M-R.
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StringencyPolynucleotideHybridHybridization TemperatureWash
ConditionHybrid Lengthand Temperature
(bp)# Buffers and Buffers
A DNA:DNA s 50 65C; lxSSC -or- 65C; 0.3xSSC
42C; lxSSC, 50% formamide
B DNA:DNA <50 TB*; lxSSC TB*; lxSSC
C DNA:RNA z 50 67C; lxSSC -or- 67C; 0.3xSSC
45C; lxSSC, 50% formamide
D DNA:RNA <50 Tp*; lxSSC Tp*; lxSSC
E RNA:RNA z 50 70C; lxSSC -or- 70C; 0.3xSSC
50C; lxSSC, 50% formamide
F RNA:RNA <50 TF*; lxSSC TF*; IxSSC
G DNA:DNA Z 50 65C; 4xSSC -or- 65C; lxSSC
42C; 4xSSC, 50% formamide
H DNA:DNA <50 T,,*; 4xSSC TH*; 4xSSC
I DNA:RNA z 50 67C; 4xSSC -or- 67C; lxSSC
45C; 4xSSC, 50% formamide
J DNA:RNA <50 T~*; 4xSSC T~*; 4xSSC
K RNA:RNA z 50 70C; 4xSSC -or- 67C; lxSSC
50C; 4xSSC, 50% formamide
L RNA:RNA <50 T~*; 2xSSC TL*; 2xSSC
M DNA:DNA s 50 50C; 4xSSC -or- 50C; 2xSSC
40C; 6xSSC, 50% formamide
N DNA:DNA <50 TN*; 6xSSC TN*; 6xSSC
O DNA:RNA z 50 55C; 4xSSC -or- 55C; 2xSSC
42C; 6xSSC, 50% formamide
P DNA:RNA <50 TP*; 6xSSC TP*; 6xSSC
Q RNA:RNA s 50 60 C; 4xSSC -or- 60 C; 2xSSC
45C; 6xSSC, 50% formamide
2 R RNA:RNA <50 TR*; 4xSSC TR*; 4xSSC
0
$: The hybrid length is that anricipated for the hybridized regions) of the
hybridizing polynucleotides. When
hybridizing a polynucleoride to a target polynucleotide of unknown sequence,
the hybrid length is assumed
to be that of the hybridizing polynucleoHde. When polynucleotides of known
sequence are hybridized, the
2 5 hybrid length can be determined by aligning the sequences of the
polynucleorides and identifying the region
or regions of optimal sequence complementarity.
': SSPE (lxSSPE is 0.15M NaCI, lOmM NaHzPOa, and 1.25mM EDTA, pH 7.4) can be
substituted for SSC
(lxSSC is 0.15M NaCI and lSmM sodium citrate) in the hybridization and wash
buffers; washes are
performed for 15 minutes after hybridization is complete.
3 0 *TB - TR: The hybridization temperature for hybrids anticipated to be less
than 50 base pairs in length should
be 5-10 ° C less than the melting temperature (Tm) of the hybrid, where
Tm is determined according to the
following equations. For hybrids less than 18 base pairs in length,
Tm(°C) = 2(# of A + T bases) + 4(# of G +
C bases). For hybrids between 18 and 49 base pairs in length, Tm(°C) =
81.5 + 16.6(log,°[Na']) + 0.41(%G+C)
(600/N), where N is the number of bases in the hybrid, and [Na'] is the
concentration of sodium ions in the
3 5 hybridization buffer ([Na'] for lxSSC = 0.165 M).
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Additional examples of stringency conditions for polynucleotide hybridization
are
provided in Sambrook, J., E.F. Fritsch, and T. Maniatis, 1989, Molecular
Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
NY,
chapters 9 and 11, and Current Protocols in Molecular Biology,1995, F.M.
Ausubel et al., eds.,
John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, incorporated herein by
reference.
Preferably, each such hybridizing polynucleotide has a length that is at least
25%(more preferably at least 50%, and most preferably at least 75%) of the
length of the
polynucleotide of the present invention to which it hybridizes, and has at
least 60%
sequence identity (more preferably, at least 75% identity; most preferably at
least 90% or
95% identity) with the polynucleotide of the present invention to which it
hybridizes,
where sequence identity is determined by comparing the sequences of the
hybridizing
polynucleotides when aligned so as to maximize overlap and identity while
minimizing
sequence gaps.
The isolated polynucleotide of the invention may be operably linked to an
expression control sequence such as the pMT2 or pED expression vectors
disclosed in
Kaufman et al., Nucleic Acids Res. 19, 4485-4490 (1991), in order to produce
the protein
recombinantly. Many suitable expression control sequences are known in the
art. General
methods of expressing recombinant proteins are also known and are exemplified
in R.
Kaufman, Methods in Enzymology 185, 537-566 (1990). As defined herein
"operably
2 0 linked" means that the isolated polynucleotide of the invention and an
expression control
sequence are situated within a vector or cell in such a way that the protein
is expressed
by a host cell which has been transformed (transfected) with the ligated
polynucleotide/expression control sequence.
A number of types of cells may act as suitable host cells for expression of
the
2 5 protein. Mammalian host cells include, for example, monkey COS cells,
Chinese Hamster
Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human
Co1o205
cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal
diploid cells, cell
strains derived from in vitro culture of primary tissue, primary explants,
HeLa cells,
mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.
3 0 Alternatively, it may be possible to produce the protein in lower
eukaryotes such
as yeast or in prokaryotes such as bacteria. Potentially suitable yeast
strains include
Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains,
Candida, or any
yeast strain capable of expressing heterologous proteins. Potentially suitable
bacterial
strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium,
or any bacterial
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strain capable of expressing heterologous proteins. If the protein is made in
yeast or
bacteria, it may be necessary to modify the protein produced therein, for
example by
phosphorylation or glycosylation of the appropriate sites, in order to obtain
the functional
protein. Such covalent attachments may be accomplished using known chemical or
enzymatic methods.
The protein may also be produced by operably linking the isolated
polynucleotide
of the invention to suitable control sequences in one or more insect
expression vectors,
and employing an insect expression system. Materials and methods for
baculovirus/insect cell expression systems are commercially available in kit
form from,
e.g., Invitrogen, San Diego, California, U.S.A. (the MaxBac~ kit), and such
methods are
well known in the art, as described in Summers and Smith, Texas Agricultural
Experiment
Station Bulletin No. 1555 (1987), incorporated herein by reference. As used
herein, an
insect cell capable of expressing a polynucleotide of the present invention is
"transformed."
T'he protein of the invention may be prepared by culturing transformed host
cells
under culture conditions suitable to express the recombinant protein. The
resulting
expressed protein may then be purified from such culture (i.e., from culture
medium or
cell extracts) using known purification processes, such as gel filtration and
ion exchange
chromatography. The purification of the protein may also include an affinity
column
2 0 containing agents which will bind to the protein; one or more column steps
over such
affinity resins as concanavalin A-agarose, heparin-toyopearl~ or Cibacrom blue
3GA
Sepharose~; one or more steps involving hydrophobic interaction chromatography
using
such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity
chromatography.
2 5 Alternatively, the protein of the invention may also be expressed in a
form which
will facilitate purification. For example, it may be expressed as a fusion
protein, such as
those of maltose binding protein (MBP}, glutathione-S-transferase (GST) or
thioredoxin
{TRX). Kits for expression and purification of such fusion proteins are
commercially
available from New England BioLab (Beverly, MA), Pharmacia (Piscataway, NJ)
and
3 0 InVitrogen, respectively. The protein can also be tagged with an epitope
and
subsequently purified by using a specific antibody directed to such epitope.
One such
epitope ("Flag") is commercially available from Kodak (New Haven, CT).
Finally, one or more reverse-phase high performance liquid chromatography (RP-
HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having
pendant
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CA 02274507 1999-06-11
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methyl or other aliphatic groups, can be employed to further purify the
protein. Some or
all of the foregoing purification steps, in various combinations, can also be
employed to
provide a substantially homogeneous isolated recombinant protein. The protein
thus
purified is substantially free of other mammalian proteins and is defined in
accordance
with the present invention as an "isolated protein."
The protein of the invention may also be expressed as a product of transgenic
animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or
sheep which
are characterized by somatic or germ cells containing a nucleotide sequence
encoding the
protein.
The protein may also be produced by known conventional chemical synthesis.
Methods for constructing the proteins of the present invention by synthetic
means are
known to those skilled in the art. The synthetically-constructed protein
sequences, by
virtue of sharing primary, secondary or tertiary structural and/or
conformational
characteristics with proteins may possess biological properties in common
therewith,
including protein activity. Thus, they may be employed as biologically active
or
immunological substitutes for natural, purified proteins in screening of
therapeutic
compounds and in immunological processes for the development of antibodies.
The proteins provided herein also include proteins characterized by amino acid
sequences similar to those of purified proteins but into which modification
are naturally
2 0 provided or deliberately engineered. For example, modifications in the
peptide or DNA
sequences can be made by those skilled in the art using known techniques.
Modifications
of interest in the protein sequences may include the alteration, substitution,
replacement,
insertion or deletion of a selected amino acid residue in the coding sequence.
For
example, one or more of the cysteine residues may be deleted or replaced with
another
2 5 amino acid to alter the conformation of the molecule. Techniques for such
alteration,
substitution, replacement, insertion or deletion are well known to those
skilled in the art
(see, e.g., U.S. Patent No. 4,518,584). Preferably, such alteration,
substitution, replacement,
insertion or deletion retains the desired activity of the protein.
Other fragments and derivatives of the sequences of proteins which would be
3 0 expected to retain protein activity in whole or in part and may thus be
useful for screening
or other immunological methodologies may also be easily made by those skilled
in the art
given the disclosures herein. Such modifications are believed to be
encompassed by the
presentinvention.
CA 02274507 1999-06-11
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USES AND BIOLOGICAL ACTIVITY
The polynucleotides and proteins of the present invention are expected to
exhibit
one or more of the uses or biological activities (including those associated
with assays
cited herein) identified below. Uses or activities described for proteins of
the present
invention may be provided by administration or use of such proteins or by
administration
or use of polynucleotides encoding such proteins (such as, for example, in
gene therapies
or vectors suitable for introduction of DNA).
Research Uses and Utilities
The polynucleotides provided by the present invention can be used by the
research
community for various purposes. The polynucleotides can be used to express
recombinant protein for analysis, characterization or therapeutic use; as
markers for
tissues in which the corresponding protein is preferentially expressed (either
constitutively or at a particular stage of tissue differentiation or
development or in disease
states); as molecular weight markers on Southern gels; as chromosome markers
or tags
(when labeled) to identify chromosomes or to map related gene positions; to
compare
with endogenous DNA sequences in patients to identify potential genetic
disorders; as
probes to hybridize and thus discover novel, related DNA sequences; as a
source of
information to derive PCR primers for genetic fingerprinting; as a probe to
"subtract-out"
2 0 known sequences in the process of discovering other novel polynucleotides;
for selecting
and making oligomers for attachment to a "gene chip" or other support,
including for
examination of expression patterns; to raise anti-protein antibodies using DNA
immunization techniques; and as an antigen to raise anti-DNA antibodies or
elicit another
immune response. Where the polynucleotide encodes a protein which binds or
potentially
2 5 binds to another protein (such as, for example, in a receptor-ligand
interaction), the
polynucleotide can also be used in interaction trap assays (such as, for
example, that
described in Gyuris et al., Cell 75:791-803 (1993)) to identify
polynucleotides encoding the
other protein with which binding occurs or to identify inhibitors of the
binding
interaction.
3 0 The proteins provided by the present invention can similarly be used in
assay to
determine biological activity, including in a panel of multiple proteins for
high-
throughput screening; to raise antibodies or to elicit another immune
response; as a
reagent (including the labeled reagent) in assays designed to quantitatively
determine
levels of the protein (or its receptor) in biological fluids; as markers for
tissues in which
36
CA 02274507 1999-06-11
WO 98/25962 PCT/US97/23224
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 correlatfve receptors or ligands. Where the protein binds
or potentially
binds to another protein (such as, for example, in a receptor-ligand
interaction), the
protein can be used to identify the other protein with which binding occurs or
to identify
inhibitors of the binding interaction. Proteins involved in these binding
interactions can
also be used to screen for peptide or small molecule inhibitors or agonists of
the binding
interaction.
Any or all of these research utilities are capable of being developed into
reagent
grade or kit format for commercialization as research products.
Methods for performing the uses listed above are well known to those skilled
in
the art. References disclosing such methods include without limitation
"Molecular
Cloning: A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory Press,
Sambrook,
J., E.F. Fritsch and T. Maniatis eds., 1989, and "Methods in Enzymology: Guide
to
Molecular Cloning Techniques", Academic Press, Bergen S.L. and A.R. Kimmel
eds.,1987.
Nutritional Uses
Polynucleotides and proteins of the present invention can also be used as
nutritional sources or supplements. Such uses include without limitation use
as a protein
2 0 or amino acid supplement, use as a carbon source, use as a nitrogen source
and use as a
source of carbohydrate. In such cases the protein or polynucleotide of the
invention can
be added to the feed of a particular organism or can be administered as a
separate solid
or liquid preparation, such as in the form of powder, pills, solutions,
suspensions or
capsules. In the case of microorganisms, the protein or polynucleotide of the
invention
2 5 can be added to the medium in or on which the microorganism is cultured.
Cytokine and Cell Proliferation/Differentiation Activity
A protein of the present invention may exhibit cytokine, cell proliferation
(either
3 0 inducing or inhibiting) or cell differentiation (either inducing or
inhibiting) activity or may
induce production of other cytokines in certain cell populations. Many protein
factors
discovered to date, including 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
37
CA 02274507 1999-06-11
WO 98125962 PCT/US97/23224
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, DAl, 123, T1165, HT2, CTLL2, TF-1, Mo7e and
CMK.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assays for T-cell or thymocyte proliferation include without limitation those
described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M.
Kruisbeek, D.H.
Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and
Wiley-
Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-
3.19; Chapter
7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500,
1986;
Bertagnolli et al., J. Immunol.145:1706-1712, 1990; Bertagnolli et al.,
Cellular Immunology
133:327-341, 1991; Bertagnolli, et al., J. Immunol. 149:3778-3783, 1992;
Bowman et al., J.
Immunol. 152: 1756-1761, 1994.
Assays for cytokine production and/or proliferation of spleen cells, lymph
node
cells or thymocytes include, without limitation, those described in:
Polyclonal T cell
stimulation, Kruisbeek, A.M. and Shevach, E.M. In Current Protocols in
Immunology. J.E.e.a.
Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and
Measurement of mouse and human Interferon y, Schreiber, R.D. In Current
Protocols in
2 0 Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley and
Sons, Toronto.1994.
Assays for proliferation and differentiation of hematopoietic and
lymphopoietic
cells include, without limitation, those described in: Measurement of Human
and Murine
Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L.S. and Lipsky, P.E. In
Current
Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John
Wiley and Sons,
2 5 Toronto. 1991; deVries et al., J. Exp. Med. 173:1205-1211, 1991; Moreau et
al., Nature
336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A. 80:2931-
2938, 1983;
Measurement of mouse and human interleukin 6 - Nordan, R. In Current Protocols
in
Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons,
Toronto.1991;
Smith et al., Proc. Natl. Acad. Sci. U.S.A. 83:1857-1861, 1986; Measurement of
human
3 0 Interleukin 11- Bennett, F., Giannotti, J., Clark, S.C. and Turner, K. J.
In 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.
38
CA 02274507 1999-06-11
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Assays for T-cell clone responses to antigens (which will identify, among
others,
proteins that affect APC-T cell interactions as well as direct T-cell effects
by measuring
proliferation and cytokine production) include, without limitation, those
described in:
Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H.
Margulies,
E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-
Interscience
(Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6,
Cytokines and
their cellular receptors; Chapter 7, Immunologic studies in Humans);
Weinberger et al.,
Proc. Natl. Acad. Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J.
Immun.
11:405-411, 1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al.,
J. Immunol.
140:508-512, 1988.
Immune Stimulatin, og r Suppressing Activity
A protein of the present invention may also exhibit immune stimulating or
immune suppressing activity, including without limitation the activities for
which assays
are described herein. A protein may be useful in the treatment of various
immune
deficiencies and disorders (including severe combined immunodeficiency
(SCID)), e.g.,
in regulating (up or down) growth and proliferation of T and/or B lymphocytes,
as well
as effecting the cytolytic activity of NK cells and other cell populations.
These immune
deficiencies may be genetic or be caused by viral (e.g., HIV) as well as
bacterial or fungal
2 0 infections, or may result from autoimmune disorders. More specifically,
infectious
diseases causes by viral, bacterial, fungal or other infection may be
treatable using a
protein of the present invention, including infections by HIV, hepatitis
viruses,
herpesviruses, mycobacteria, Leishmania spp., malaria spp. and various fungal
infections
such as candidiasis. Of course, in this regard, a protein of the present
invention may also
2 5 be useful where a boost to the immune system generally may be desirable,
i.e., in the
treatment of cancer.
Autoimmune disorders which may be treated using a protein of the present
invention include, for example, connective tissue disease, multiple sclerosis,
systemic
lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation,
3 0 Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent
diabetes mellitis,
myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye
disease.
Such a protein of the present invention may also to be useful in the treatment
of allergic
reactions and conditions, such as asthma (particularly allergic asthma) or
other respiratory
problems. Other conditions, in which immune suppression is desired (including,
for
39
CA 02274507 1999-06-11
WO 98/25962 PCT/US97/23224
example, organ transplantation), may also be treatable using a protein of the
present
invention.
Using the proteins of the invention it may also be possible to immune
responses,
in a number of ways. Down regulation may be in the form of inhibiting or
blocking an
immune response already in progress or may involve preventing the induction of
an
immune response. The functions of activated T cells may be inhibited by
suppressing T
cell responses or by inducing specific tolerance in T cells, or both.
Immunosuppression
of T cell responses is generally an active, non-antigen-specific, process
which requires
continuous exposure of the T cells to the suppressive agent. Tolerance, which
involves
inducing non-responsiveness . or anergy in T cells, is distinguishable from
immunosuppression in that it is generally antigen-specific and persists after
exposure to
the tolerizing agent has ceased. Operationally, tolerance can be demonstrated
by the lack
of a T cell response upon reexposure to specific antigen in the absence of the
tolerizing
agent.
Down regulating or preventing one or more antigen functions (including without
limitation B lymphocyte antigen functions (such as , for example, B7)), e.g.,
preventing
high level lymphokine synthesis by activated T cells, will be useful in
situations of tissue,
skin and organ transplantation and in graft-versus-host disease (GVHD). For
example,
blockage of T cell function should result in reduced tissue destruction in
tissue
2 0 transplantation. Typically, in tissue transplants, rejection of the
transplant is initiated
through its recognition as foreign by T cells, followed by an immune reaction
that destroys
the transplant. The administration of a molecule which inhibits or blocks
interaction of
a B7 lymphocyte antigen with its natural ligand(s) on immune cells (such as a
soluble,
monomeric form of a peptide having B7-2 activity alone or in conjunction with
a
2 5 monomeric form of a peptide having an activity of another B lymphocyte
antigen (e.g., B7-
1, B7-3) or blocking antibody), prior to transplantation can lead to the
binding of the
molecule to the natural ligand(s) on the immune cells without transmitting the
corresponding costimulatory signal. Blocking B lymphocyte antigen function in
this
matter prevents cytokine synthesis by immune cells, such as T cells, and thus
acts as an
3 0 immunosuppressant. Moreover, the lack of costimulation may also be
sufficient to
anergize the T cells, thereby inducing tolerance in a subject. Induction of
long-term
tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of
repeated
administration of these blocking reagents. To achieve sufficient
immunosuppression or
CA 02274507 1999-06-11
WO 98/25962 PCT/IJS97/23224
tolerance in a subject, it may also be necessary to block the function of a
combination of
B lymphocyte antigens.
The efficacy of particular blocking reagents in preventing organ transplant
rejection or GVHD can be assessed using animal models that are predictive of
efficacy in
humans. Examples of appropriate systems which can be used include allogeneic
cardiac
grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of
which have been
used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in
vivo as
described in Lenschow et al., Science 257:789-792 (1992) and Turka et al.,
Proc. Natl. Acad.
Sci USA, 89:11102-11105 (1992). In addition, marine models of GVHD (see Paul
ed.,
Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847} can be used
to
determine the effect of blocking B lymphocyte antigen function in vivo on the
development
of that disease.
Blocking antigen function may also be therapeutically useful for treating
autoimmune diseases. Many autoimmune disorders are the result of inappropriate
activation of T cells that are reactive against self tissue and which promote
the production
of cytokines and autoantibodies involved in the pathology of the diseases.
Preventing the
activation of autoreactive T cells may reduce or eliminate disease symptoms.
Administration of reagents which block costimulation of T cells by disrupting
receptor:ligand interactions of B lymphocyte antigens can be used to inhibit T
cell
2 0 activation and prevent production of autoantibodies or T cell-derived
cytokines which
may be involved in the disease process. Additionally, blocking reagents may
induce
antigen-specific tolerance of autoreactive T cells which could lead to long-
term relief from
the disease. The efficacy of blocking reagents in preventing or alleviating
autoimmune
disorders can be determined using a number of well-characterized animal models
of
2 5 human autoimmune diseases. Examples include marine experimental autoimmune
encephalitis, systemic lupus erythmatosis in MRL/lpr/lpr mice or NZB hybrid
mice,
marine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB
rats, and
marine experimental myasthenia gravis (see Paul ed., Fundamental Immunology,
Raven
Press, New York, 1989, pp. 840-856).
3 0 Upregulation of an antigen function (preferably a B lymphocyte antigen
function),
as a means of up regulating immune responses, may also be useful in therapy.
Upregulation of immune responses may be in the form of enhancing an existing
immune
response or eliciting an initial immune response. For example, enhancing an
immune
response through stimulating B lymphocyte antigen function may be useful in
cases of
41
CA 02274507 1999-06-11
WO 98/25962 PCT/1JS97/23224
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, cosHmulating the T cells in vitro with
viral antigen-
pulsed APCs either expressing a peptide of the present invention or together
with a
stimulatory form of a soluble peptide of the present invention and
reintroducing the in
vitro activated T cells into the patient. Another method of enhancing anti-
viral immune
responses would be to isolate infected cells from a patient, transfect them
with a nucleic
acid encoding a protein of the present invention as described herein such that
the cells
express all or a portion of the protein on their surface, and reintroduce the
transfected
cells into the patient. The infected cells would now be capable of delivering
a
costimulatory signal to, and thereby activate, T cells in vivo.
In another application, up regulation or enhancement of antigen function
(preferably B lymphocyte antigen function) may be useful in the induction of
tumor
immunity. Tumor cells (e.g., sarcoma, melanoma, lymphoma, leukemia,
neuroblastoma,
carcinoma) transfected with a nucleic acid encoding at least one peptide of
the present
invention can be administered to a subject to overcome tumor-specific
tolerance in the
subject. If desired, the tumor cell can be transfected to express a
combination of peptides.
2 0 For example, tumor cells obtained from a patient can be transfected ex
vivo with an
expression vector directing the expression of a peptide having B7-2-like
activity alone, or
in conjunction with a peptide having B7-1-like activity and/or B7-3-like
activity. The
transfected tumor cells are returned to the patient to result in expression of
the peptides
on the surface of the transfected cell. Alternatively, gene therapy techniques
can be used
2 5 to target a tumor cell for transfection in vivo.
The presence of the peptide of the present invention having the activity of a
B
lymphocyte antigens) on the surface of the tumor cell provides the necessary
costimulation signal to T cells to induce a T cell mediated immune response
against the
transfected tumor cells. In addition, tumor cells which lack MHC class I or
MHC class II
3 0 molecules, or which fail to reexpress sufficient amounts of MHC class I or
MHC class II
molecules, can be transfected with nucleic acid encoding all or a portion of
(e.g., a
cytoplasmic-domain truncated portion) of an MHC class I a chain protein and
X32
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.
42
CA 02274507 1999-06-11
WO 98/25962 PCT/US97/23224
Expression of the appropriate class I or class II MHC in conjunction with a
peptide having
the activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a T
cell mediated
immune response against the transfected tumor cell. Optionally, a gene
encoding an
antisense construct which blocks expression of an MHC class II associated
protein, such
as the invariant chain, can also be cotransfected with a DNA encoding a
peptide having
the activity of a B lymphocyte antigen to promote presentation of tumor
associated
antigens and induce tumor specific immunity. Thus, the induction of a T cell
mediated
immune response in a human subject may be sufficient to overcome tumor-
specific
tolerance in the subject.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Suitable assays for thymocyte or splenocyte cytotoxicity include, without
limitation, those described in: Current Protocols in Immunology, Ed by J. E.
Coligan, A.M.
Kruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing
Associates
and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte
Function 3.1-
3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl.
Acad. Sci.
USA 78:2488-2492,1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa
et al.,
J. Immunol. 135:1564-1572,1985; Takai et al., J. Immunol. 137:3494-3500, 1986;
Takai et al.,
J: Immunol. 140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA
78:2488-2492,
2 0 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J.
Immunol.
135:1564-1572, 1985; Takai et al., J. Immunol. 137:3494-3500, 1986; Bowmanet
al., J.
Virology 61:1992-1998; Takai et al., J. Immunol. 140:508-512, 1988;
Bertagnolli et al.,
Cellular Immunology 133:327-341, 1991; Brown et al., J. Immunol. 153:3079-
3092, 1994.
Assays for T-cell-dependent immunoglobulin responses and isotype switching
2 5 (which will identify, among others, proteins that modulate T-cell
dependent antibody
responses and that affect Th1 /Th2 profiles) include, without limitation,
those described
in: Maliszewski, J. Immunol. 144:3028-3033, 1990; and Assays for B cell
function: In vitro
antibody production, Mond, J.J. and Brunswick, M. In Current Protocols in
Immunology.
j.E.e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto.
1994.
3 0 Mixed lymphocyte reaction (MLR) assays (which will identify, among others,
proteins that generate predominantly Thl and CTL responses) include, without
limitation,
those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M.
Kruisbeek,
D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and
Wiley-
Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-
3.19; Chapter
43
CA 02274507 1999-06-11
WO 98/25962 PCT/LTS97/23224
7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500,
1986; Takai
et al., J. Immunol. 140:508-512, 1988; Bertagnolli et al., J. Immunol.
149:3778-3783, 1992.
Dendritic cell-dependent assays (which will identify, among others, proteins
expressed by dendritic cells that activate naive T-cells) include, without
limitation, those
described in: Guery et al., J. Immunol. 134:536-544, 1995; Inaba et al.,
Journal of
Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal of
Immunology
154:5071-5079,1995; Porgador et al., Journal of Experimental Medicine 182:255-
260, 1995;
Nair et al., journal of Virology 67:4062-4069, 1993; Huang et al., Science
264:961-965,
1994; Macatonia et al., Journal of Experimental Medicine 169:1255-1264, 1989;
Bhardwaj
et al., Journal of Clinical Investigation 94:797-807, 1994; and Inaba et al.,
Journal of
Experimental Medicine 172:631-640, 1990.
Assays for lymphocyte survival/apoptosis (which will identify, among others,
proteins that prevent apoptosis after superantigen induction and proteins that
regulate
lymphocyte homeostasis) include, without limitation, those described in:
Darzynkiewicz
et al., Cytometry 13:795-808,1992; Gorczyca et al., Leukemia 7:659-670, 1993;
Gorczyca et
al., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991;
Zacharchuk,
Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry 14:891-897,
1993;
Gorczyca et al., International Journal of Oncology 1:639-648, 1992.
Assays for proteins that influence early steps of T-cell commitment and
2 0 development include, without limitation, those described in: Antica et
al., Blood
84:111-117, 1994; Fine et al., Cellular Immunology 155:111-122, 1994; Galy et
al., Blood
85:2770-2778,1995; Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551, 1991.
Hematopoiesis Re ulatin A~ ctivity
2 5 A protein of the present invention may be useful in regulation of
hematopoiesis
and, consequently, in the treatment of myeloid or lymphoid cell deficiencies.
Even
marginal biological activity in support of colony forming cells or of factor-
dependent cell
lines indicates involvement in regulating hematopoiesis, e.g. in supporting
the growth and
proliferation of erythroid progenitor cells alone or in combination with other
cytokines,
3 0 thereby indicating utility, for example, in treating various anemias or
for use in
conjunction with irradiation/chemotherapy to stimulate the production of
erythroid
precursors and / or erythroid cells; irt 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
44
CA 02274507 1999-06-11
WO 98/25962 PCT/US97/23224
myelo-suppression; in supporting the growth and proliferation of
megakaryocytes and
consequently of platelets thereby allowing prevention or treatment of various
platelet
disorders such as thrombocytopenia, and generally for use in place of or
complimentary
to platelet transfusions; and/or in supporting the growth and proliferation of
hematopoietic stem cells which are capable of maturing to any and all of the
above-
mentioned hematopoietic cells and therefore find therapeutic utility in
various stem cell
disorders (such as those usually treated with transplantation, including,
without
limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well
as in
repopulating the stem cell compartment post irradiation/chemotherapy, either
in-vivo or
ex-vivo (i.e., in conjunction with bone marrow transplantation or with
peripheral
progenitor cell transplantation (homologous or heterologous)) as normal cells
or
genetically manipulated for gene therapy.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Suitable assays for proliferation and differentiation of various hematopoietic
lines
are cited above.
Assays for embryonic stem cell differentiation (which will identify, among
others,
proteins that influence embryonic differentiation hematopoiesis) include,
without
limitation, those described in: Johansson et al. Cellular Biology 15:141-151,
1995; Keller et
2 0 al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al.,
Blood
81:2903-2915, 1993.
Assays for stem cell survival and differentiation (which will identify, among
others, proteins that regulate lympho-hematopoiesis) include, without
limitation, those
described in: Methylcellulose colony forming assays, Freshney, M.G. In
Culticre of
2 5 Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 265-268, Wiley-
Liss, Inc., New York,
NY. 1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992;
Primitive
hematopoietic colony forming cells with high proliferative potential, McNiece,
LK. and
Briddell, R.A. In Culture of Hematopoietic Cells. R.I. Freshney, et aI. eds.
VoI pp. 23-39,
Wiley-Liss, Inc., New York, NY. 1994; Neben et al., Experimental Hematology
22:353-359,
3 0 1994; Cobblestone area forming cell assay, Ploemacher, R.E. In Culture of
Hematopoietic
Cells. R.I. Freshney, et al. eds. Vol pp. 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,
CA 02274507 1999-06-11
WO 98/25962 PCTIUS97I23224
H.J. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 139-
162, Wiley-Liss,
Inc., New York, NY. 1994.
Tissue Growth Activity
A protein of the present invention also may have utility in compositions used
for
bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration,
as well as
for wound healing and tissue repair and replacement, and in the treatment of
burns,
incisions and ulcers.
A protein of the present invention, which induces cartilage and / or bone
growth
in circumstances where bone is not normally formed, has application in the
healing of
bone fractures and cartilage damage or defects in humans and other animals.
Such a
preparation employing a protein of the invention may have prophylactic use in
closed as
well as open fracture reduction and also in the improved fixation of
artificial joints. De
novo bone formation induced by an osteogenic agent contributes to the repair
of
congenital, trauma induced, or oncologic resection induced craniofacial
defects, and also
is useful in cosmetic plastic surgery.
A protein of this invention may also be used in the treatment of periodontal
disease, and in other tooth repair processes. Such agents may provide an
environment
to attract bone-forming cells, stimulate growth of bone-forming cells or
induce
2 0 differentiation of progenitors of bone-forming cells. A protein of the
invention may also
be useful in the treatment of osteoporosis or osteoarthritis, such as through
stimulation
of bone and / or cartilage repair or by blocking inflammation or processes of
tissue
destruction (collagenase activity, osteoclast activity, etc.) mediated by
inflammatory
processes.
2 5 Another category of tissue regeneration activity that may be attributable
to the
protein of the present invention is tendon/ligament formation. A protein of
the present
invention, which induces tendon/ligament-like tissue or other tissue formation
in
circumstances where such tissue is not normally formed, has application in the
healing of
tendon or ligament tears, deformities and other tendon or ligament defects in
humans and
3 0 other animals. Such a preparation employing a tendon/ligament-like tissue
inducing
protein may have prophylactic use in preventing damage to tendon or ligament
tissue, as
well as use in the improved fixation of tendon or ligament to bone or other
tissues, and
in repairing defects to tendon or ligament tissue. De novo tendon/ligament-
like tissue
formation induced by a composition of the present invention contributes to the
repair of
46
CA 02274507 1999-06-11
WO 98/25962 PCT/US97/23224
congenital, trauma induced, or other tendon or ligament defects of other
origin, and is also
useful in cosmetic plastic surgery for attachment or repair of tendons or
ligaments. The
compositions of the present invention may provide an environment to attract
tendon- or
ligament-forming cells, stimulate growth of tendon- or ligament-forming cells,
induce
differentiation of progenitors of tendon- or ligament-forming cells, or induce
growth of
tendon/ligament cells or progenitors ex vivo for return in vivo to effect
tissue repair. The
compositions of the invention may also be useful in the treatment of
tendinitis, carpal
tunnel syndrome and other tendon or ligament defects. The compositions may
also
include an appropriate matrix and/or sequestering agent as a carrier as is
well known in
the art.
The protein of the present invention may also be useful for proliferation of
neural
cells and for regeneration of nerve and brain tissue, i.e. for the treatment
of central and
peripheral nervous system diseases and neuropathies, as well as mechanical and
traumatic disorders, which involve degeneration, death or trauma to neural
cells or nerve
tissue. More specifically, a protein may be used in the treatment of diseases
of the
peripheral nervous system, such as peripheral nerve injuries, peripheral
neuropathy and
localized neuropathies, and central nervous system diseases, such as
Alzheimer's,
Parkinson's disease, Huntingtori s disease, amyotrophic lateral sclerosis, and
Shy-Drager
syndrome. Further conditions which may be treated in accordance with the
present
2 0 invention include mechanical and traumatic disorders, such as spinal cord
disorders, head
trauma and cerebrovascular diseases such as stroke. Peripheral neuropathies
resulting
from chemotherapy or other medical therapies may also be treatable using a
protein of the
invention.
Proteins of the invention may also be useful to promote better or faster
closure of
2 5 non-healing wounds, including without limitation pressure ulcers, ulcers
associated with
vascular insufficiency, surgical and traumatic wounds, and the like.
It is expected that a protein of the present invention may also exhibit
activity for
generation or regeneration of other tissues, such as organs {including, for
example,
pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth,
skeletal or cardiac)
3 0 and vascular (including vascular endothelium) tissue, or for promoting the
growth of cells
comprising such tissues. Part of the desired effects may be by inhibition or
modulation
of fibrotic scarring to allow normal tissue to regenerate. A protein of the
invention may
also exhibit angiogenic activity.
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CA 02274507 1999-06-11
WO 98/25962 PCT/US97/23224
A protein of the present invention may also be useful for gut protection or
regeneration and treatment of lung or liver fibrosis, reperfusion injury in
various tissues,
and conditions resulting from systemic cytokine damage.
A protein of the present invention may also be useful for promoting or
inhibiting
differentiation of tissues described above from precursor tissues or cells; or
for inhibiting
the growth of tissues described above.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assays for tissue generation activity include, without limitation, those
described
in: International Patent Publication No. W095/16035 (bone, cartilage, tendon);
International Patent Publication No. W095/05846 (nerve, neuronal);
International Patent
Publication No. W091/07491 (skin, endothelium ).
Assays for wound healing activity include, without limitation, those described
in:
Winter, Epidermal Wound Healing, pps. 71-112 (Maibach, HI and Rovee, DT,
eds.), Year
Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J.
Invest.
Dermatol 71:382-84 (1978).
Activin/Inhibin Activity
A protein of the present invention may also exhibit activin- or inhibin-
related
2 0 activities. Inhibins are characterized by their ability to inhibit the
release of follicle
stimulating hormone (FSH), while activins and are characterized by their
ability to
stimulate the release of follicle stimulating hormone (FSH). Thus, a protein
of the present
invention, alone or in heterodimers with a member of the inhibin a family, may
be useful
as a contraceptive based on the ability of inhibins to decrease fertility in
female mammals
2 5 and decrease spermatogenesis in male mammals. Administration of sufficient
amounts
of other inhibins can induce infertility in these mammals. Alternatively, the
protein of the
invention, as a homodimer or as a heterodimer with other protein suburuts of
the inhibin-
~3 group, may be useful as a fertility inducing therapeutic, based upon the
ability of activin
molecules in stimulating FSH release from cells of the anterior pituitary.
See, for example,
3 0 United States Patent 4,798,885. A protein of the invention may also be
useful for
advancement of the onset of fertility in sexually immature mammals, so as to
increase the
lifetime reproductive performance of domestic animals such as cows, sheep and
pigs.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
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CA 02274507 1999-06-11
WO 98/25962 PCT/US97/23224
Assays for activin/inhibin activity include, without limitation, those
described in:
Vale et al., Endocrinology 91:562-572, 1972; Ling et al., Nature 321:779-782,
1986; Vale et
al., Nature 321:776-779,1986; Mason et al., Nature 318:659-663, 1985; Forage
et al., Proc.
Natl. Acad. Sci. USA 83:3091-3095, 1986.
Chemotactic/Chemokinetic Active
A protein of the present invention may have chemotactic or chemokinetic
activity
(e.g., act as a chemokine) for mammalian cells, including, for example,
monocytes,
fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and /
or endothelial cells.
Chemotactic and chemokinetic proteins can be used to mobilize or attract a
desired cell
population to a desired site of action. Chemotactic or chemokinetic proteins
provide
particular advantages in treatment of wounds and other trauma to tissues, as
well as in
treatment of localized infections. For example, attraction of lymphocytes,
monocytes or
neutrophils to tumors or sites of infection may result in improved immune
responses
against the tumor or infecting agent.
A protein or peptide has chemotactic activity for a particular cell population
if it
can stimulate, directly or indirectly, the directed orientation or movement of
such cell
population. Preferably, the protein or peptide has the ability to directly
stimulate directed
movement of cells. Whether a particular protein has chemotactic activity for a
population
2 0 of cells can be readily determined by employing such protein or peptide in
any known
assay for cell chemotaxis.
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assays for chemotactic activity (which will identify proteins that induce or
prevent
2 5 chemotaxis) consist of assays that measure the ability of a protein to
induce the migration
of cells across a membrane as well as the ability of a protein to induce the
adhesion of one
cell population to another cell population. Suitable assays for movement and
adhesion
include, without limitation, those described in: Current Protocols in
Immunology, Ed by
J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, W.Strober, Pub.
Greene
3 0 Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of
alpha and
beta Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:1370-
1376,1995; Lind et al.
APMIS 103:140-146, 1995; Muller et al Eur. J. Immunol. 25: 1744-1748; Gruber
et al. j. of
Immunol. 152:5860-5867, 1994; Johnston et al. J. of Immunol. 153: 1762-1768,
1994.
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Hemostatic and ThrombolYtic Activity
A protein of the invention may also exhibit hemostatic or thrombolytic
activity.
As a result, such a protein is expected to be useful in treatment of various
coagulation
disorders (including hereditary disorders, such as hemophilias) or to enhance
coagulation
and other hemostatic events in treating wounds resulting from trauma, surgery
or other
causes. A protein of the invention may also be useful for dissolving or
inhibiting
formation of thromboses and for treatment and prevention of conditions
resulting
therefrom (such as, for example, infarction of cardiac and central nervous
system vessels
(e.g., stroke).
The activity of a protein of the invention may, among other means, be measured
by the following methods:
Assay for hemostatic and thrombolytic activity include, without limitation,
those
described in: Linet et al., J. Clin. Pharmacol. 26:131-140, 1986; Burdick et
al., Thrombosis
Res. 45:413-419,1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub,
Prostaglandins
35:467-474, 1988.
Receptor/Li~and ActivitX
A protein of the present invention may also demonstrate activity as receptors,
receptor ligands or inhibitors or agonists of receptor/ligand interactions.
Examples of
2 0 such receptors and ligands include, without limitation, cytokine receptors
and their
ligands, receptor kinases and their ligands, receptor phosphatases and their
ligands,
receptors involved in cell-cell interactions and their ligands (including
without limitation,
cellular adhesion molecules (such as selectins, integrins and their ligands)
and
receptor/ligand pairs involved in antigen presentation, antigen recognition
and
2 5 development of cellular and humoral immune responses}. Receptors and
ligands are also
useful for screening of potential peptide or small molecule inhibitors of the
relevant
receptor/ligand interaction. A protein of the present invention (including,
without
limitation, fragments of receptors and ligands) may themselves be useful as
inhibitors of
receptor/ligand interactions.
3 0 The activity of a protein of the invention may, among other means, be
measured
by the following methods:
Suitable assays for receptor-ligand activity include without limitation those
described in:Current Protocols in Immunology, Ed by J.E. Coligan, A.M.
Kruisbeek, D.H.
Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and
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Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under
static
conditions 7.28.1-7.28.22), Takai et al., Proc. Natl. Acad. Sci. USA 84:6864-
6868, 1987;
Bierer et al., J. Exp. Med. 168:1145-1156, 1988; Rosenstein et al., J. Exp.
Med. 169:149-160
1989; Stoltenborg et al., J. Immunol. Methods 175:59-68,1994; Stitt et al.,
Cell 80:661-670,
1995.
Anti-Inflammatory Activity
Proteins of the present invention may also exhibit anti-inflammatory activity.
The
anti-inflammatory activity may be achieved by providing a stimulus to cells
involved in
the inflammatory response, by inhibiting or promoting cell-cell interactions
(such as, for
example, cell adhesion), by inhibiting or promoting chemotaxis of cells
involved in the
inflammatory process, inhibiting or promoting cell extravasation, or by
stimulating or
suppressing production of other factors which more directly inhibit or promote
an
inflammatory response. Proteins exhibiting such activities can be used to
treat
inflammatory conditions including chronic or acute conditions), including
without
limitation inflammation associated with infection (such as septic shock,
sepsis or systemic
inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin
lethality, arthritis, complement-mediated hyperacute rejection, nephritis,
cytokine or
chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or
resulting
2 0 from over production of cytokines such as TNF or IL-1. Proteins of the
invention may also
be useful to treat anaphylaxis and hypersensitivity to an antigenic substance
or material.
Cadherin/Tumor Invasion Suppressor ActivitX
Cadherins are calcium-dependent adhesion molecules that appear to play major
2 5 roles during development, particularly in defining specific cell types.
Loss or alteration
of normal cadherin expression can lead to changes in cell adhesion properties
linked to
tumor growth and metastasis. Cadherin malfunction is also implicated in other
human
diseases, such as pemphigus vulgaris and pemphigus foliaceus (auto-immune
blistering
skin diseases), Crohn's disease, and some developmental abnormalities.
3 0 The cadherin superfamily includes well over forty members, each with a
distinct
pattern of expression. All members of the superfamily have in common conserved
extracellular repeats (cadherin domains), but structural differences are found
in other
parts of the molecule. The cadherin domains bind calcium to form their
tertiary structure
and thus calcium is required to mediate their adhesion. Only a few amino acids
in the
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first cadherin domain provide the basis for hemophilic adhesion; modification
of this
recognition site can change the specificity of a cadherin so that instead of
recognizing only
itself, the mutant molecule can now also bind to a different cadherin. In
addition, some
cadherins engage in heterophilic adhesion with other cadherins.
E-cadherin, one member of the cadherin superfamily, is expressed in epithelial
cell
types. Pathologically, if E-cadherin expression is lost in a tumor, the
malignant cells
become invasive and the cancer metastasizes. Transfection of cancer cell lines
with
polynucleotides expressing E-cadherin has reversed cancer-associated changes
by
returning altered cell shapes to normal, restoring cells' adhesiveness to each
other and to
their substrate, decreasing the cell growth rate, and drastically reducing
anchorage-
independent cell growth. Thus, reintroducing E-cadherin expression reverts
carcinomas
to a less advanced stage. It is likely that other cadherins have the same
invasion
suppresser role in carcinomas derived from other tissue types. Therefore,
proteins of the
present invention with cadherin activity, and polynucleotides of the present
invention
encoding such proteins, can be used to treat cancer. Introducing such proteins
or
polynucleotides into cancer cells can reduce or eliminate the cancerous
changes observed
in these cells by providing normal cadherin expression.
Cancer cells have also been shown to express cadherins of a different tissue
type
than their origin, thus allowing these cells to invade and metastasize in a
different tissue
2 0 in the body. Proteins of the present invention with cadherin activity, and
polynucleotides
of the present invention encoding such proteins, can be substituted in these
cells for the
inappropriately expressed cadherins, restoring normal cell adhesive properties
and
reducing or eliminating the tendency of the cells to metastasize.
Additionally, proteins of the present invention with cadherin activity, and
2 5 polynucleotides of the present invention encoding such proteins, can used
to generate
antibodies recognizing and binding to cadherins. Such antibodies can be used
to block
the adhesion of inappropriately expressed tumor-cell cadherins, preventing the
cells from
forming a tumor elsewhere. Such an anti-cadherin antibody can also be used as
a marker
for the grade, pathological type, and prognosis of a cancer, i.e. the more
progressed the
3 0 cancer, the less cadherin expression there will be, and this decrease in
cadherin expression
can be detected by the use of a cadherin-binding antibody.
Fragments of proteins of the present invention with cadherin activity,
preferably
a polypeptide comprising a decapeptide of the cadherin recognition site, and
poly-
nucleotides of the present invention encoding such protein fragments, can also
be used
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to block cadherin function by binding to cadherins and preventing them from
binding in
ways that produce undesirable effects. Additionally, fragments of proteins of
the present
invention with cadherin activity, preferably truncated soluble cadherin
fragments which
have been found to be stable in the circulation of cancer patients, and
polynucleotides
encoding such protein fragments, can be used to disturb proper cell-cell
adhesion.
Assays for cadherin adhesive and invasive suppressor activity include, without
limitation, those described in: Hortsch et al. J Biol Chem 270 (32): 18809-
18817, 1995;
Miyaki et al. Oncogene 11: 2547-2552, 1995; Ozawa et al. Cell 63: 1033-1038,
1990.
Tumor Inhibition Activity
In addition to the activities described above for immunological treatment or
prevention of tumors, a protein of the invention may exhibit other anti-tumor
activities.
A protein may inhibit tumor growth directly or indirectly (such as, for
example, via
ADCC). A protein may exhibit its tumor inhibitory activity by acting on tumor
tissue or
tumor precursor tissue, by inhibiting formation of tissues necessary to
support tumor
growth (such as, for example, by inhibiting angiogenesis), by causing
production of other
factors, agents or cell types which inhibit tumor growth, or by suppressing,
eliminating
or inhibiting factors, agents or cell types which promote tumor growth.
2 0 Other Activities
A protein of the invention may also exhibit one or more of the following
additional
activities or effects: inhibiting the growth, infection or function of, or
killing, infectious
agents, including, without limitation, bacteria, viruses, fungi and other
parasites; effecting
(suppressing or enhancing) bodily characteristics, including, without
limitation, height,
2 5 weight, hair color, eye color, skin, fat to lean ratio or other tissue
pigmentation, or organ
or body part size or shape (such as, for example, breast augmentation or
diminution,
change in bone form or shape); effecting biorhythms or caricadic cycles or
rhythms;
effecting the fertility of male or female subjects; effecting the metabolism,
catabolism,
anabolism, processing, utilization, storage or elimination of dietary fat,
lipid, protein,
3 0 carbohydrate, vitamins, minerals, cofactors or other nutritional factors
or component(s);
effecting behavioral characteristics, including, without limitation, appetite,
libido, stress,
cognition {including cognitive disorders), depression (including depressive
disorders) and
violent behaviors; providing analgesic effects or other pain reducing effects;
promoting
differentiation and growth of embryonic stem cells in lineages other than
hematopoietic
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CA 02274507 1999-06-11
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Iineages; hormonal or endocrine activity; in the case of enzymes, correcting
deficiencies
of the enzyme and treating deficiency-related diseases; treatment of
hyperproliferative
disorders (such as, for example, psoriasis); immunoglobulin-like activity
{such as, for
example, the ability to bind antigens or complement); and the ability to act
as an antigen
in a vaccine composition to raise an immune response against such protein or
another
material or entity which is cross-reactive with such protein.
ADMINISTRATION AND DOSING
A protein of the present invention (from whatever source derived, including
without limitation from recombinant and non-recombinant sources) may be used
in a
pharmaceutical composition when combined with a pharmaceutically acceptable
carrier.
Such a composition may also contain (in addition to protein and a carrier)
diluents, fillers,
salts, buffers, stabilizers, solubilizers, and other materials well known in
the art. The term
"pharmaceutically acceptable" means a non-toxic material that does not
interfere with the
effectiveness of the biological activity of the active ingredient(s). The
characteristics of the
carrier will depend on the route of administration. The pharmaceutical
composition of
the invention may also contain cytokines, lymphokines, or other hematopoietic
factors
such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-
9, IL-10, IL-11,
1L-12, IL-13, IL-14, IL-15,1FN, TNFO, TNF1, TNF2, GCSF, Meg-CSF,
thrombopoietin, stem
2 0 cell factor, and erythropoietin. The pharmaceutical composition may
further contain other
agents which either enhance the activity of the protein or compliment its
activity or use
in treatment. Such additional factors and/or agents may be included in the
pharmaceutical composition to produce a synergistic effect with protein of the
invention,
or to minimize side effects. Conversely, protein of the present invention may
be included
2 5 in formulations of the particular cytokine, lymphokine, other
hematopoietic factor,
thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize
side effects
of the cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-
thrombotic
factor, or anti-inflammatory agent.
A protein of the present invention may be active in muitimers (e.g.,
heterodimers
3 0 or homodimers) or complexes with itself or other proteins. As a result,
pharmaceutical
compositions of the invention may comprise a protein of the invention in such
multimeric
or complexed form.
The pharmaceutical composition of the invention may be in the form of a
complex
of the proteins) of present invention along with protein or peptide antigens.
The protein
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and/or peptide antigen will deliver a stimulatory signal to both B and T
lymphocytes. B
lymphocytes will respond to antigen through their surface immunoglobulin
receptor. T
lymphocytes will respond to antigen through the T cell receptor (TCR)
following
presentation of the antigen by MHC proteins. MHC and structurally related
proteins
including those encoded by class I and class II MHC genes on host cells will
serve to
present the peptide antigens) to T lymphocytes. The antigen components could
also be
supplied as purified MHC-peptide complexes alone or with co-stimulatory
molecules that
can directly signal T cells. Alternatively antibodies able to bind surface
immunolgobulin
and other molecules on B cells as well as antibodies able to bind the TCR and
other
molecules on T cells can be combined with the pharmaceutical composition of
the
invention.
The pharmaceutical composition of the invention may be in the form of a
liposome
in which protein of the present invention is combined, in addition to other
pharmaceutically acceptable 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,
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.
2 0 Patent No. 4,837,028; and U.S. Patent No. 4,737,323, all of which are
incorporated herein
by reference.
As used herein, the term "therapeutically effective amount" means the total
amount of each active component of the pharmaceutical composition or method
that is
sufficient to show a meaningful patient benefit, i.e., treatment, healing,
prevention or
2 5 amelioration of the relevant medical condition, or an increase in rate of
treatment, healing,
prevention or amelioration of such conditions. When applied to an individual
active
ingredient, administered alone, the term refers to that ingredient alone. When
applied to
a combination, the term refers to combined amounts of the active ingredients
that result
in the therapeutic effect, whether administered in combination, serially or
simultaneously.
3 0 In practicing the method of treatment or use of the present invention, a
therapeutically effective amount of protein of the present invention is
administered to a
mammal having a condition to be treated. Protein of the present invention may
be
administered in accordance with the method of the invention either alone or in
combination with other therapies such as treatments employing cytokines,
lymphokines
CA 02274507 1999-06-11
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or other hematopoietic factors. When co-administered with one or more
cytokines,
lymphokines or other hematopoietic factors, protein of the present invention
may be
administered either simultaneously with the cytokine(s), lymphokine(s), other
hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or
sequentially. If
administered sequentially, the attending physician will decide on the
appropriate
sequence of administering protein of the present invention in combination with
cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or
anti-thrombotic
factors.
Administration of protein of the present invention used in the pharmaceutical
composition or to practice the method of the present invention can be carried
out in a
variety of conventional ways, such as oral ingestion, inhalation, topical
application or
cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection.
Intravenous administration to the patient is preferred.
When a therapeutically effective amount of protein of the present invention is
administered orally, protein of the present invention will be in the form of a
tablet,
capsule, powder, solution or elixir. When administered in tablet form, the
pharmaceutical
composition of the invention may additionally contain a solid carrier such as
a gelatin or
an adjuvant. The tablet, capsule, and powder contain from about 5 to 95%
protein of the
present invention, and preferably from about 25 to 90% protein of the present
invention.
2 0 When administered in liquid form, a liquid earner such as water,
petroleum, oils of animal
or plant origin such as peanut oil, mineral oil, soybean oii, or sesame oil,
or synthetic oils
may be added. The liquid form of the pharmaceutical composition may further
contain
physiological saline solution, dextrose or other saccharide solution, or
glycols such as
ethylene glycol, propylene glycol or polyethylene glycol. When administered in
liquid
2 5 form, the pharmaceutical composition contains from about 0.5 to 90% by
weight of protein
of the present invention, and preferably from about 1 to 50% protein of the
present
invention.
When a therapeutically effective amount of protein of the present invention is
administered by intravenous, cutaneous or subcutaneous injection, protein of
the present
3 0 invention will be in the form of a pyrogen-free, parenterally acceptable
aqueous solution.
The preparation of such parenterally acceptable protein solutions, having due
regard to
pH, isotonicity, stability, and the like, is within the skill in the art. A
preferred
pharmaceutical composition for intravenous, cutaneous, or subcutaneous
injection should
contain, in addition to protein of the present invention, an isotonic vehicle
such as Sodium
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Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and
Sodium Chloride
Injection, Lactated Ringer's Injection, or other vehicle as known in the art.
The
pharmaceutical composition of the present invention may also contain
stabilizers,
preservatives, buffers, antioxidants, or other additives known to those of
skill in the art.
The amount of protein of the present invention in the pharmaceutical
composition
of the present invention will depend upon the nature and severity of the
condition being
treated, and on the nature of prior treatments which the patient has
undergone.
Ultimately, the attending physician will decide the amount of protein of the
present
invention with which to treat each individual patient. Initially, the
attending physician
will administer low doses of protein of the present invention and observe the
patient's
response. Larger doses of protein of the present invention may be administered
until the
optimal therapeutic effect is obtained for the patient, and at that point the
dosage is not
increased further. It is contemplated that the various pharmaceutical
compositions used
to practice the method of the present invention should contain about 0.01 ug
to about 100
mg (preferably about 0.lng to about 10 mg, more preferably about 0.1 ~g to
about 1 mg)
of protein of the present invention per kg body weight.
The duration of intravenous therapy using the pharmaceutical composition of
the
present invention will vary, depending on the severity of the disease being
treated and
the condition and potential idiosyncratic response of each individual patient.
It is
2 0 contemplated that the duration of each application of the protein of the
present invention
will be in the range of 12 to 24 hours of continuous intravenous
administration.
Ultimately the attending physician will decide on the appropriate duration of
intravenous
therapy using the pharmaceutical composition of the present invention.
Protein of the invention may also be used to immunize animals to obtain
2 5 polyclonal and monoclonal antibodies which specifically react with the
protein. Such
antibodies may be obtained using either the entire protein or fragments
thereof as an
immunogen. The peptide immunogens additionally may contain a cysteine residue
at the
carboxyl terminus, and are conjugated to a hapten such as keyhole limpet
hemocyanin
(KLH). Methods for synthesizing such peptides are known in the art, for
example, as in
3 0 R.P. Merrifield, J. Amer.Chem.Soc. 85, 2149-2154 (1963); J.L. Krstenansky,
et al., FEBS Lett.
211, 10 {1987). Monoclonal antibodies binding to the protein of the invention
may be
useful diagnostic agents for the immunodetection of the protein. Neutralizing
monoclonal
antibodies binding to the protein may also be useful therapeutics for both
conditions
associated with the protein and also in the treatment of some forms of cancer
where
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abnormal expression of the protein is involved. In the case of cancerous cells
or leukemic
cells, neutralizing monoclonal antibodies against the protein may be useful in
detecting
and preventing the metastatic spread of the cancerous cells, which may be
mediated by
the protein.
For compositions of the present invention which are useful for bone,
cartilage,
tendon or ligament regeneration, the therapeutic method includes administering
the
composition topically, systematically, or locally as an implant or device.
When
administered, the therapeutic composition for use in this invention is, of
course, in a
pyrogen-free, physiologically acceptable form. Further, the composition may
desirably
be encapsulated or injected in a viscous form for delivery to the site of
bone, cartilage or
tissue damage. Topical administration may be suitable for wound healing and
tissue
repair. Therapeutically useful agents other than a protein of the invention
which may also
optionally be included in the composition as described above, may
alternatively or
additionally, be administered simultaneously or sequentially with the
composition in the
methods of the invention. Preferably for bone and/or cartilage formation, the
composition would include a matrix capable of delivering the protein-
containing
composition to the site of bone and / or cartilage damage, providing a
structure for the
developing bone and cartilage and optimally capable of being resorbed into the
body.
Such matrices may be formed of materials presently in use for other implanted
medical
2 0 applications.
The choice of matrix material is based on biocompatibility, biodegradability,
mechanical properties, cosmetic appearance and interface properties. The
particular
application of the compositions will define the appropriate formulation.
Potential
matrices for the compositions may be biodegradable and chemically defined
calcium
2 5 sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid,
polyglycolic acid and
polyanhydrides. Other potential materials are biodegradable and biologically
well-
defined, such as bone or dermal collagen. Further matrices are comprised of
pure proteins
or extracellular matrix components. Other potential matrices are
nonbiodegradable and
chemically defined, such as sintered hydroxapatite, bioglass, aluminates, or
other
3 0 ceramics. Matrices may be comprised of combinations of any of the above
mentioned
types of material, such as polylactic acid and hydroxyapatite or collagen and
tricalciumphosphate. The bioceramics may be altered in composition, such as in
calcium-
aluminate-phosphate and processing to alter pore size, particle size, particle
shape, and
biodegradability.
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Presently preferred is a 50:50 (mole weight) copolymer of lactic acid and
glycolic
acid in the form of porous particles having diameters ranging from 150 to 800
microns.
In some applications, it will be useful to utilize a sequestering agent, such
as
carboxymethyl cellulose or autologous blood clot, to prevent the protein
compositions
from disassociating from the matrix.
A preferred family of sequestering agents is cellulosic materials such as
alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose,
ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-
methylcellulose, and carboxymethylcellulose, the most preferred being cationic
salts of
carboxymethylcellulose (CMC). Other preferred sequestering agents include
hyaluronic
acid, sodium alginate, polyethylene glycol), polyoxyethylene oxide,
carboxyvinyl
polymer and polyvinyl alcohol). The amount of sequestering agent useful herein
is 0.5-20
wt%, preferably 1-10 wt% based on total formulation weight, which represents
the
amount necessary to prevent desorbtion of the protein from the polymer matrix
and to
provide appropriate handling of the composition, yet not so much that the
progenitor cells
are prevented from infiltrating the matrix, thereby providing the protein the
opportunity
to assist the osteogenic activity of the progenitor cells.
In further compositions, proteins of the invention may be combined with other
agents beneficial to the treatment of the bone and / or cartilage defect,
wound, or tissue in
2 0 question. These agents include various growth factors such as epidermal
growth factor
(EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF-
a and
TGF-(3), and insulin-like growth factor (IGF).
The therapeutic compositions are also presently valuable for veterinary
applications. Particularly domestic animals and thoroughbred horses, in
addition to
2 5 humans, are desired patients for such treatment with proteins of the
present invention.
The dosage regimen of a protein-containing pharmaceutical composition to be
used in tissue regeneration will be determined by the attending physician
considering
various factors which modify the action of the proteins, e.g., amount of
tissue weight
desired to be formed, the site of damage, the condition of the damaged tissue,
the size of
3 0 a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and
diet, the severity
of any infection, time of administration and other clinical factors. The
dosage may vary
with the type of matrix used in the reconstitution and with inclusion of other
proteins in
the pharmaceutical composition. For example, the addition of other known
growth
factors, such as IGF I (insulin like growth factor I), to the final
composition, may also effect
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the dosage. Progress can be monitored by periodic assessment of tissue/bone
growth
and/or repair, for example, X-rays, histomorphometric determinations and
tetracycline
labeling.
Polynucleotides of the present invention can also be used for gene therapy.
Such
polynucleotides can be introduced either in vivo or ex vivo into cells for
expression in a
mammalian subject. Polynucleotides of the invention may also be administered
by other
known methods for introduction of nucleic acid into a cell or organism
(including, without
limitation, in the form of viral vectors or naked DNA).
Cells may also be cultured ex vivo in the presence of proteins of the present
invention in order to proliferate or to produce a desired effect on or
activity in such cells.
Treated cells can then be introduced in vivo for therapeutic purposes.
Patent and literature references cited herein are incorporated by reference as
if
fully set forth.
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Jacobs, Kenneth
McCoy, John M.
LaVallie, Edward R.
Racie, Lisa A.
Merberg, David
Treacy, Maurice
Spaulding, Vikki
Agostino, Michael J.
(ii) TITLE OF INVENTION: SECRETED PROTEINS AND POLYNUCLEOTIDES
ENCODING THEM
(iii) NUMBER OF SEQUENCES: 32
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Genetics Institute, Inc.
(B) STREET: 87 CambridgePark Drive
(C) CITY: Cambridge
(D) STATE: MA
(E) COUNTRY: U.S.A.
(F) ZIP: 02140
(v) COMPUTER READABLE FORM:
{A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Sprunger, Suzanne A.
(B) REGISTRATION NUMBER: 41,323
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: {617) 498-8284
(B) TELEFAX: (617) 876-5851
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 462 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
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(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
GTGGAAGGAG TGGATAATAA AATGAGTCAG TGCACCAGCT TAGCTCAAGT 60
CCACCATTCC
CAAGAGAAAG ACCCTAAAAT TAAGACAGAG ACAAGTGAAG AGACTTGGAT 120
AGGGATCTGG
AATCTAGATG CTATTCTTGG TGATCTGACT AGTTCTGACT TTCCATATCC 180
TTTACAATAA
TCAAATGGTA GTCATCTGGG GACTAAGCAA CAGGTGTTTC TTCTCTGGGT 240
AAGGAACTAA
TTGAAAAGTT CACAGTCTGT GCAGTCTATT CGTCCTCCAT AGTGTCTCTG 300
ATAACCGAGC
GATAGCCCTG TTTCTGTTGG CTCAAGTCCT CCAGTAAAAA TTTCCCCATG 360
ATATCAGTGC
TTACCAAAGC AACCCATGTT GGGTGGGAAT CCAAGAATGA RGAAAATTAT 420
TGGATAGTCA
GGCTCAAGTA TGGGAGACTG GGGCTTACCA AACTCAAAGG 462
CC
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 147 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi)SEQUENCE
DESCRIPTION:
SEQ
ID
N0:2:
Met SerGln CysThrSer SerThrIle ProSer SerSerGln GluLys
1 5 10 15
Asp ProLys IleLysThr GluThrSer GluGlu GlySerGly AspLeu
20 25 30
Asp AsnLeu AspAlaIle LeuGlyAsp LeuThr SerSerAsp PheTyr
35 40 45
Asn AsnSer IleSerSer AsnGlySer HisLeu GlyThrLys GlnGln
50 55 60
Val PheGln GlyThrAsn SerLeuGly LeuLys SerSerGln SerVal
65 70 75 gp
Gln SerIle ArgProPro TyrAsnArg AlaVal SerLeuAsp SerPro
85 90 95
62
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Val Ser Val Gly Ser Ser Pro Pro Val Lys Asn Ile Ser Ala Phe Pro
100 105 110
Met Leu Pro Lys Gln Pro Met Leu Gly Gly Asn Pro Arg Met Met Asp
115 120 125
Ser Gln Glu Asn Tyr Gly Ser Ser Met Gly Asp Trp Gly Leu Pro Asn
130 135 140
Ser Lys Ala
145
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 119 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
p~~~.AAAAAAA F,~~4AAAAAAA P,~~P.AAAAAAA P~~?~AAAAAAA AAAAAAAAAA AAAAAAAAAA 6 0
AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA F~~.AAAAAAA 119
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3316 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE
DESCRIPTION:
SEQ ID
N0:4:
GGTAAGATGGCGGCTGTGAG TCTGCGGCTCGGCGACTTGG TGTGGGGGAA ACTCGGCCGA60
TATCCTCCTTGGCCAGGAAA GATTGTTAATCCACCAAAGG ACTTGAAGAA ACCTCGCGGA120
AAGAAATGCTTCTTTGTGAA ATTTTTTGGAACAGAAGATC ATGCCTGGAT CAAAGTGGAA180
CAGCTGAAGCCATATCATGC TCATAAAGAGGAAATGATAA AAATTAACAA GGGTAAACGA240
TTCCAGCAAGCGGTAGATGC TGTCGAAGAGTTCCTCAGGA GAGCCAAAGG GAAAGACCAG300
63
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ACGTCATCCCACAATTCTTCTGATGACAAG AATCGACGTAATTCCAGTGAGGAGAGAAGT360
AGGCCAAACTCAGGTGATGAGAAGCGCAAA CTTAGCCTGTCTGAAGGGAAGGTGAAGAAG420
AACATGGGAGAAGGAAAGAAGAGGGTGTCT TCAGGCTCTTCAGAGAGAGGCTCCAAATCC480
CCTCTGAAAAGAGCCCAAGAGCAAAGTCCC CGGAAGCGGGGTCGGCCCCCAAAGGATGAG540
AAGGATCTCACCATCCCGGAGTCTAGTACC GTGAAGGGGATGATGGCCGGACCGATGGCC600
GCGTTTAAATGGCAGCCAACCGCAAGCGAG CCTGTTAAAGATGCAGATCCTCATTTCCAT660
CATTTCCTGCTAAGCCAAACAGAGAAGCCA GCTGTCTGTTACCAGGCAATCACGAAGAAG720
TTGAAAATATGTGAAGAGGAAACTGGCTCC ACCTCCATCCAGGCAGCTGACAGCACAGCC780
GTGAATGGCAGCATCACACCCACAGACAAA AAGATAGGATTTTTGGGCCTTGGTCTCATG840
GGAAGTGGAATCGTCTCCAACTTGCTAAAA ATGGGTCACACAGTGACTGTCTGGAACCGC900
ACTGCAGAGAAAGAGGGGGCCCGTCTGGGA AGAACCCCCGCTGAAGTCGTCTCAACCTGC960
GACATCACTTTCGCCTGCGTGTCGGATCCC AAGGCGGCCAAGGACCTGGTGCTGGGCCCC1020
AGTGGTGTGCTGCAAGGGATCCGCCCTGGG AAGTGCTACGTGGACATGTCAACAGTGGAC1080
GCTGACACCGTCACTGAGCTGGCCCAGGTG ATTGTGTCCAGGGGGGGGCGCTTTCTGGAA1140
GCCCCCGTCTCAGGGAATCAGCAGCTGTCT AATGACGGGATGTTGGTGATCTTAGCGGCT1200
GGAGACAGGGGCTTATATGAGGACTGCAGC AGCTGCTTCCAGGCGATGGGGAAGACCTCC1260
TTCTTCCTAGGTGAAGTGGGCAATGCAGCC AAGATGATGCTGATCGTGAACATGGTCCAA1320
GGGAGCTTCATGGCCACTATTGCCGAGGGG CTGACCCTGGCCCAGGTGACAGGCCAGTCC1380
CAGCAGACACTCTTGGACATCCTCAATCAG GGACAGTTGGCCAGCATCTTCCTGGACCAG1440
AAGTGCCAAAATATCCTGCAAGGAAACTTT AAGCCTGATTTCTACCTGAAATACATTCAG1500
AAGGATCTCCGCTTAGCCATTGCGCTGGGT GATGCGGTCAACCATCCGACTCCCATGGCA1560
GCTGCAGCAAATGAGGTGTACAAAAGAGCC AAGGCGCTGGACCAGTCTGACAACGATATG1620
TCCGCCGTGTACCGAGCCTACATACACTAA GCTGTCGACACCCCGCCCTCACCCCTCCAA1680
TCCCCCCTCTGACCCCCTCTTCCTCACATG GGGTCGGGGGCCTGGGAGTTCATTCTGGAC1740
CAGCCCACCTATCTCCATTTCCTTTTATAC AGACTTTGAGACTTGCCATCAGCACAGCAC1800
ACAGCAGCACCCTTCCCCTGAGGCCGGTGG GGAGGGGACAAGTGTCAGCAGGATTGGCGT1860
GTGGGAAAGCTCTTGAGCTGGGCACTGGCC CCCCGGACGAGGTGGCTGTGTGTTCACACA1920
CACACACACACACACACACACACAGGCTCT CGCCCCAGGATAGAAGCTGCCCAGAAACTG1980
64
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CTGCCTGGCT TTTTTTCTTCCGAGCTTGTCTTATCTCAAA 2040
CCCCTTCCAG
TCAAGGAACT
AGAATCAGCA ACGAGAGTTGGAAGCCTTCCCACAGCTTCCCCCAGAGCGA AGAGGCTGTA2100
GTCATGTCCC CATCCCCCACTGGATTCCCTACAAGGAGAGGCCTTGGGCC CAGATGAGCC2160
AGTACAGACT CCAGACAGAGGGGCCCTTGGGGCCCTCCAACCTCAGGTGA TGAGCTGAGA2220
AAGATGTTCA CGTCTAAGCGTCCAGTGTGCACCCAGCGCTCCATAGACGC CTTTGTGAAC2280
TGAAAAGAGA CTGGCAGAGTCCCGAGAAGATGGGGCCCTGGCTTTCCAGG GAGTGCAGCA2340
AGCAGCCGGC CTGCAGACCCAGCCTGACCAACGATGAGCATTTCTTAGGC TCAGCTCTTG2400
ATACGGAAAC GAGTGTCTTCACTCCAGCCAGCATCATGGTCTTCGGTGCT TCCCGGGCCC2460
GGGGTCTGTC GGGAGGGAAGAGAACTGGGCCTGACCTACCTGAACTGACT GGCCCTCCGA2520
GGTGGGTCTG GGACATCCTAGAGGCCCTACATTTGTCCTTGGATAGGGGA CCGGGGGGGG2580
CTTGGAATGT TGCAAAAAAAAAAGTTACCCAAGGGATGTCAGTTTTTTAT CCCTCTGCAT2640
GGGTTGGATT TTCCAAAATCATAATTTGCAGAAGGAAGGCCAGCATTTAC GATGCAATAT2700
GTAATTATAT ATAGGGTGGCCACACTAGGGCGGGGTCCTTCCCCCCTCAC AGCTTTGGCC2760
CCTTTCAGAG ATTAGAAACTGGGTTAGAGGATTGCAGAAGACGAGTGGGG GGAGGGCAGG2820
GAAGATGCCT GTCGGGTTTTTAGCACAGTTCATTTCACTGGGATTTTGAA GCATTTCTGT2880
CTGGACACAA AGCCTGTTCTAGTCCTGGCGGAACACACTGGGGGTGGGGG CGGGGGAAGA2940
TGCGGTAATG AAACCGGTTAGTCAATTTTGTCTTAATATTGTTGACAATT CTGTAAAGTT3000
CCTTTTTATG AATATTTCTGTTTAAGCTATTTCACCTTTCTTTTGAAATC CTTCCCTTTT3060
AAGGAGAAAA TGTGACACTTGTGAAAAAGCTTGTAAGAAAGCCCCTCCCT TTTTTCTTTA3120
AACCTTTAAA TGACAAATCTAGGTAATTAAGGTTGTGAATTTTTATTTTT GCTTTGTTTT3180
TAATGAACAT TTGTCTTTCAGAATAGGATTGTGTGATAATGTTTAAATGG CAAAAACAAA3240
ACATGATTTT GTGCAATTAACAAAGCTACTGCAAGAAAAATAAAACACTT CTTGGTAACA3300
Cpl ~~ 3
316
(2) INFORMATION EQ ID
FOR S N0:5:
(i) SEQUENCE CHARACTERISTIC S:
(A) LENGTH: 547 aminoacids
(B) TYPE: amino
acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE:
protein
CA 02274507 1999-06-11
WO 98/25962 PCT/US97/23224
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
Met Ala Ala Val Ser Leu Arg Leu Gly Asp Leu Val Trp Gly Lys Leu
1 5 10 15
Gly Arg Tyr Pro Pro Trp Pro Gly Lys Ile Val Asn Pro Pro Lys Asp
20 25 30
Leu Lys Lys Pro Arg Gly Lys Lys Cys Phe Phe Val Lys Phe Phe Gly
35 40 45
Thr Glu Asp His Ala Trp Ile Lys Val Glu Gln Leu Lys Pro Tyr His
50 55 60
Ala His Lys Glu Glu Met Ile Lys Ile Asn Lys Gly Lys Arg Phe Gln
65 70 75 80
Gln Ala Val Asp Ala Val Glu Glu Phe Leu Arg Arg Ala Lys Gly Lys
85 90 95
Asp Gln Thr Ser Ser His Asn Ser Ser Asp Asp Lys Asn Arg Arg Asn
100 105 110
Ser Ser Glu Glu Arg Ser Arg Pro Asn Ser Gly Asp Glu Lys Arg Lys
115 120 125
Leu Ser Leu Ser Glu Gly Lys Val Lys Lys Asn Met Gly Glu Gly Lys
130 135 140
Lys Arg Val Ser Ser Gly Ser Ser Glu Arg Gly Ser Lys Ser Pro Leu
145 150 155 160
Lys Arg Ala Gln Glu Gln Ser Pro Arg Lys Arg Gly Arg Pro Pro Lys
165 170 175
Asp Glu Lys Asp Leu Thr Ile Pro Glu Ser Ser Thr Val Lys Gly Met
180 185 190
Met Ala Gly Pro Met Ala Ala Phe Lys Trp Gln Pro Thr Ala Ser Glu
195 200 205
Pro Val Lys Asp Ala Asp Pro His Phe His His Phe Leu Leu Ser Gln
210 215 220
Thr Glu Lys Pro Ala Val Cys Tyr Gln Ala Ile Thr Lys Lys Leu Lys
225 230 235 240
Ile Cys Glu Glu Glu Thr Gly Ser Thr Ser Ile Gln Ala Ala Asp Ser
245 250 255
Thr Ala Val Asn Gly Ser Ile Thr Pro Thr Asp Lys Lys Ile Gly Phe
260 265 270
66
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Leu Gly Leu Gly Leu Met Gly Ser Gly Ile Val Ser Asn Leu Leu Lys
275 280 285
Met Gly His Thr Val Thr Val Trp Asn Arg Thr Ala Glu Lys Glu Gly
290 295 300
Ala Arg Leu Gly Arg Thr Pro Ala Glu Val Val Ser Thr Cys Asp Ile
305 310 315 320
Thr Phe Ala Cys Val Ser Asp Pro Lys Ala Ala Lys Asp Leu Val Leu
325 330 335
Gly Pro Ser Gly Val Leu Gln Gly Ile Arg Pro Gly Lys Cys Tyr Val
340 345 350
Asp Met Ser Thr Val Asp Ala Asp Thr Val Thr Glu Leu Ala Gln Val
355 360 365
Ile Val Ser Arg Gly Gly Arg Phe Leu Glu Ala Pro Val Ser Gly Asn
370 375 380
Gln Gln Leu Ser Asn Asp Gly Met Leu Val Ile Leu Ala Ala Gly Asp
385 390 395 400
Arg Gly Leu Tyr Glu Asp Cys Ser Ser Cys Phe Gln Ala Met Gly Lys
405 410 415
Thr Ser Phe Phe Leu Gly Glu Val Gly Asn Ala Ala Lys Met Met Leu
420 425 430
Ile Val Asn Met Val Gln Gly Ser Phe Met Ala Thr Ile Ala Glu Gly
435 440 445
Leu Thr Leu Ala Gln Val Thr Gly Gln Ser Gln Gln Thr Leu Leu Asp
450 455 460
Ile Leu Asn Gin Gly Gln Leu Ala Ser Ile Phe Leu Asp Gln Lys Cys
465 470 475 480
Gln Asn Ile Leu Gln Gly Asn Phe Lys Pro Asp Phe Tyr Leu Lys Tyr
485 490 495
Ile Gln Lys Asp Leu Arg Leu Ala Ile Ala Leu Gly Asp Ala Val Asn
500 505 510
His Pro Thr Pro Met Ala Ala Ala Ala Asn Glu Val Tyr Lys Arg Ala
515 520 525
Lys Ala Leu Asp Gln Ser Asp Asn Asp Met Ser Ala Val Tyr Arg Ala
530 535 540
Tyr Ile His
. 545
(2) INFORMATION FOR SEQ ID N0:6:
67
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1097 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION:
SEQ ID N0:6:
ATTGGTTCAA GAAGAAAATTTGAGCCCAGGCACCCAAACACCTTCAAATG ATAAAGCAAG60
TATGTTGCAA GAATACTCCAAATACCTCCAACAGGCTTTTGAAAAATCCA CTAATGCAAG120
TTTTACTCTT GGACACGGTTTCCAATTTGTCAGTTTGTCTTCACCTCTCC ACAACCACAC180
TTTGTTTCCA GAAAAACAAATATACACTACGTCTCCTTTGGAGTGTGGTT TCGGCCAATC240
TGTTACCTCA GTGTTGCCATCTTCATTGCCAAAGCCTCCTTTTGGGATGT TGTTTGGATC300
TCAGCCAGGT CTTTATTTGTCTGCTTTGGATGCTACACATCAGCAGTTGA CACCTTCCCA360
GGAGCTGGAT GATCTGATAGATTCTCAGAAGAACTTAGAGACTTCATCAG CCTTCCAGTC420
CTCATCTCAG AAATTGACTAGCCAGAAGGAACAGAAAAACTTAGAGTCTT CAACAGGCTT480
TCAGATTCCA TCTCAGGAGTTAGCTAGCCAGATAGATCCTCAGAAAGACA TAGAGCCTAG540
AACAACGTAT CAGATTGAGAACTTTGCACAAGCGTTTGGTTCTCAGTTTA AGTCGGGCAG600
CAGGGTGCCA ATGACCTTTATCACTAACTCTAATGGAGAAGTGGACCATA GAGTAAGGAC660
TTCAGTGTCA GATTTCTCAGGGTATACAAATATGATGTCTGATGTAAGTG AGCCATGTAG720
TACAAGAGTA AAGACACCCACCAGCCAGAGTTACAGGTAAGGTCCCAAAA GTGGCCAGGC780
TGGAGGTTTT TTAATGTAATTTTGTTTTATTTTGAGAACACTGCCATTGG AATGTTTTTA840
CACGATCCTA TTAAGAATAATGTGATGCCCTTTCAATGCAACTTTTCATA TTTAGTTTAT900
TTTGTTAGCG TGATTTTAGCTCTGTTTGTATTATGATTTTTAATCAAAAT CAATAGATTA960
AAAATAGTTT GACATTCAAAGTGACAATGTTTAGCAATCAAATTTACATG TATAGATTGT1020
CAGGGAATAG CCCAAATGTTTTAAACGCAAP,~~AAAAAAAAF,~~~A AAAAAAAAAA 10
8
0
p,~~AAAAAAAA AAAAAAA 10
9
7
(2) INFORMATION EQ ID
FOR S N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 232 aminoacids
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(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
Met Leu Gln Glu Tyr Ser Lys Tyr Leu Gin Gln Ala Phe Glu Lys Ser
1 5 10 15
Thr Asn Ala Ser Phe Thr Leu Gly His Gly Phe Gln Phe Val Ser Leu
20 25 30
Ser Ser Pro Leu His Asn His Thr Leu Phe Pro Glu Lys Gln Ile Tyr
35 40 45
Thr Thr Ser Pro Leu Glu Cys Gly Phe Gly Gln Ser Val Thr Ser Val
50 55 60
Leu Pro Ser Ser Leu Pro Lys Pro Pro Phe Gly Met Leu Phe Gly Ser
65 70 75 80
Gln Pro Gly Leu Tyr Leu Ser Ala Leu Asp Ala Thr His Gln Gln Leu
85 90 95
Thr Pro Ser Gln Glu Leu Asp Asp Leu Ile Asp Ser Gln Lys Asn Leu
100 105 110
Glu Thr Ser Ser Ala Phe Gln Ser Ser Ser Gln Lys Leu Thr Ser Gln
115 120 125
Lys Glu Gln Lys Asn Leu Glu Ser Ser Thr Gly Phe Gln Ile Pro Ser
130 135 140
Gln Glu Leu Ala Ser Gln Ile Asp Pro Gln Lys Asp Ile Glu Pro Arg
145 150 155 160
Thr Thr Tyr Gln Ile Glu Asn Phe Ala Gln Ala Phe Gly Ser Gln Phe
165 170 175
Lys Ser Gly Ser Arg Val Pro Met Thr Phe Ile Thr Asn Ser Asn Gly
180 185 190
Glu Val Asp His Arg Val Arg Thr Ser Val Ser Asp Phe Ser Gly Tyr
195 200 205
Thr Asn Met Met Ser Asp Val Ser Glu Pro Cys Ser Thr Arg Val Lys
210 215 220
Thr Pro Thr Ser Gln Ser Tyr Arg
225 230
69
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(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 775 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID
N0:8:
GTGTCACATA CCACTCTTGT AGGTGTCCTC TTTTCCCACA AAATACACAG60
AATAATCCCC
GGTGTATTAT CTTTCTCTTT ATTCACCCCC ACTGAAGTTA ATTACATAGC120
ACTTTGCTGA
CTTTCTTCTA ACCTCCTTAG TAATGAACCT TGTATTTACA GCGTCTGTGG180
TCACATAAAG
TAGCCAGCCC TTCCTCCTCT ACTTTCTAGG CAATAACTAG GAATTTAATG240
AGGGGATAGC
ACAGATTTTT TTTTTCTTTG AAATAAATGG TCCATTTTAG AATTTTGTTG300
CCAGAGTTTC
TCCTCCTTAA TCATCTGCTT ACCTAGTCAT GCAGAAACTT CATAAAGGAA360
TACTCAATCT
AAGTGCTGCA TTGTTTTTAC AAATAACAGT AATATGACAA ACCTCAACTA420
TTGTAGGGAA
TGGGAGTTGT CCACAATACA AAATTTTGAA ATAGTGATAA TATCATACTT480
AAAACATTAC
GGTTGTTAGG CTTGTTGCTT CCCCACATCA ATGATTTATC TTTTGTAATT540
GAGGCATCTA
GCTGTGAACT TTTTTAAATA AGCCATTTAG TCATGTATCA AATGGCTATT600
TGTGAAATTG
GGAAATGGAC TTTACTCAAT TTTAATTCCA GCCGGAGTGA CAGAGTAAGA660
CTGCACTCTA
CTCTGTCTCA AAAATAAATA AATAAATAAA AATAAATAAA TAAATAAAAA720
TAAATAAATA
ATAATAATAC AAGTTTTCAT AAGGAAAAAA P,~~,AAAAAAAA AAAAA 7
F~~P.AAAAAAA 7
5
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
{A) LENGTH: 44 amino acids
{B) TYPE: amino acid
(C) STRANDEDNESS:
{D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
7
CA 02274507 1999-06-11
WO 98/25962 PCT/US97123224
Met Thr Asn Leu Asn Tyr Gly Ser Cys Pro Gln Tyr Lys Ile Leu Lys
1 5 10 15
Lys His Tyr Ile Val Ile Ile Ser Tyr Leu Val Val Arg Leu Val Ala
20 25 30
Ser Pro His Gln Arg His Leu Met Ile Tyr Leu Leu
35 40
(2) INFORMATION FOR SEQ ID N0:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2060 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE
DESCRIPTION:
SEQ ID
N0:10:
GAAAAAGAAGACAAAGCTCACCTTCAGGCGGAGGTGCAGCACCTGCGAGAGGACAACCTG 60
AGGCTACAGGAGGAGTCCCAGAACGCCTCGGACAAGCTGAAGAAGTTCACAGAATGGGTC 120
TTCAACACCATAGACATGAGCTAGGGAAGGCTGAGGAGGACAGGAGAAGGGCCCAGACAC 180
TCCCTCCAGTGAGTGTCCTGCAGCCCTTATTCCCTCCATAGAAAGCATCCTCAGAGCACC 240
TTCCCTGGCTTCCTACTCTGCCCCCTTTCGGGGAGTGCACAACACAATAGTTGCAGATCA 300
ACAATCATCACCTGCCTTTTGTAGAAAAGAAAAACAAAAAAAGTAAATAAAAATTTTAAA 360
CAGTAAAATAAAAGTTTAACTGCTAAAATGTGAATGTCTTTATTTTTTTGCACAATATCT 420
TTATCTGTTATGTATTTAAGAAGAAACTGGGCCTTGGACCAGGGCGCCCCCTGGCCCATC 480
CGCCTCTATTCCCATCAGCTTTCTTATCAACTTCAGGTAACCCAAGCTTTCCCTTGTTAT 540
TCTAACAAATATCATTATTCCTAGAAAAAGAATGTTTTTATAACTTGTTTGGGGAGTAGA 600
GAGGGATATTTCCTTACCTTCTTCCCTAAAATGCCTGGAGAGGGAGTTGCTTTGAGAAAA 660
TGCCTACCTCCCTTGAATGACTCGTGCATGAGCTAGTGCTGTCTGTACCTGTCCTCCAGA 720
GATCAGCAGGACCGGAGTTAAATATTTAACAGCAAGTCTGTAAACCAGAGCAGCTCTGAC 780
AGTGCCTGCAGGCCACACCCCTTCTCAGTCCTGCATTGTGAGGTCATTTCCTGCTTCTCC 840
CTTTCCCCAGGAAGATGGTCCCACTTGTGCTGCAAGACTCTTTTTTGTTTGGCTTAATTG 900
AGCCCCACTAAATTGGAATCAATCTCTCTTACAGCTTCCTGGCTCCAAACATTAATTGAT 960
71
r
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TTCAGAATTC CCCCAAACTA AAACCTTATC TTGAATGCAT TTTGGTCAAA1020
TGTCTGCATT
AGTATACGTT TTAAAGATTT TTAAAGATAA CAACTGGTTT TTTTAGCTTG1080
AAATGTGGCA
CTGAAAATGA CCATATCTCT AAATTAATCT AGCAAGACTT CACCAGTATT1140
TTCTCTCCAG
TGTAACTAGG AGAAGCTAAG TGAATGTTTA TTAATCATTG CTTGTTAGGA1200
ATTGTGAATT
ATAATGACTG TGATACTAGA ATGGGCTTTT TGTCCCAGTG TGAAATTTCA1260
GAAACCTGCA
GCACGGCATT TTCTGCATCC TTTCATGGCC TTCCGCTGCA GAAATTATTG1320
ATCCAAAGGA
ATGTGCTATT TTTGCTGTCT TGTGATGCAG GCCCCTGGGT CACTCTTCCA1380
GCTGCTTTGG
AGGCTGCTGT AGAGCACAGA GACATGGGGC TGACTGACCT GAGGAGACCC1440
TGGCCAGTGT
CTTTGTTTGT TGCCTTCATA ACTGTCACTA TCTGCCCTTT CAGTGGCAAC1500
AACCGACCCC
TCTGGTCTAA GGGAACATTC AGCACTCTAG TTGGAAGTTC CCTCACCCAA1560
CGGCATCTGA
GTAATCTCAA TTCCTTCCTC TCTCCATCCC AGGATGGATT TTCCTCTCTT1620
TGAAAGAAAC
CTCCCTGCTA CATTCACTAC CAGATTTTTA TCATTCTTGA TTGTGATTTC1680
TGCTACAGTT
TCCATGGAAT TTTTTTTTTC TGGTGACATT AAATAGGAAG ATTTCGGAGT1740
TCTATCATGG
GCTTTGTGAA GATTTCAATT GTCTGTCTCT GACTTGTATG AAGGAGATTG1800
TTCTCTCTTT
TACATTGCCT GATATCTCTT TGTAAATGAG AACATCCAAG CATTCTGAAG1860
AAATATTGCT
TCTTGCTTAT CCTTCTGAGT TTAGTTCTCA CATTTTGTTT GGGGACTTGG1920
TTTTGTTTTA
GGCAAGCTAT TTATTAGAGT TTTGCAACAG TGAAGCCTCT AAAGACTACC1980
AGTTCTTGTT
TGTAAAATTC AAAGAATAAA ATTCATTTTA TAAAAP.AAAA F~~?~AAAAAAP,2040
AACGCTCTTT
p,~~~AA.AAAAA p~~i~AAAAAAA 2
0
6
0
(2) INFORMATION FOR SEQ ID N0:11:
{i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 62 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D} TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:11:
Met Thr Val Ile Leu Glu Trp Ala Phe Glu Thr Cys Met Ser Gln Cys
1 5 10 15
72
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WO 98/25962 PCT/US97/23224
Glu Ile Ser Ala Arg His Phe Leu His Pro Phe Met Ala Ile Gln Arg
20 25 30
Ile Pro Leu Gln Lys Leu Leu Met Cys Tyr Phe Cys Cys Leu Val Met
35 40 45
Gln Ala Ala Leu Gly Pro Trp Val Thr Leu Pro Arg Leu Leu
50 55 60
(2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1160 base pairs
(B) TYPE: nucleic acid
{C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE
DESCRIPTION:
SEQ ID
N0:12:
GATTATTTTCAGTAGGCAGACATCTAATCG GAATCTTGCTCTTGTTGCCCAGGCTGGAGT 60
GTAATGGCACAATCTCGGCTTACTGCAACC TCTGCCTCCTGGATTCAAGTGATTCTCCTG 120
CCTCAGCCTCCCAAGTAGCTGGGATTACAG CCCTGAAAACCACTCGCTTGCAGAGCGCTG 180
GATCAGCAATGCCTACTAGTTCTTCATTCA AACACCGGATTAAAGAGCAGGAAGACTACA 240
TCCGAGATTGGACTGCTCATCGAGAAGAGA TAGCCAGGATCAGCCAAGATCTTGCTCTCA 300
TTGCTCGGGAGATCAACGATGTAGCAGGAG AGATAGATTCAGTGACTTCATCAGGCACTG 360
CCCCTAGTACCACAGTAAGCACTGCTGCCA CCACCCCTGGCTCTGCCATAGACACTAGAG 420
AAGAGTTGGTTGATCGTGTTTTTGATGAAA GCCTCAACTTCCAAAAGATTCCTCCATTAG 480
TTCATTCCAAAACACCAGAAGGAAACAACG GTCGATCTGGTGATCCAAGACCTCAAGCAG 540
CAGAGCCTCCCGATCACTTAACAATTACAA GGCGGAGAACCTGGAGCAGGGATGAAGTCA 600
TGGGAGATAATCTGCTGCTGTCATCCGTCT TTCAGTTCTCTARGAAGATAAGACAATCTA 660
TAGATAAGACAGCTGGAAAGATCAGAATAT TATTTAAAGACAAAGATCGGAATTGGGATG 720
ACATAGAAAGCAAATTAAGAGCCGAAAGTG AAGTCCCTATTGTGAAAACCTCGAGCATGG 780
AGATTTCTTCTATCTTACAGGAACTGAAAA GAGTAGAAAAGCAGCTACAAGCAATCAATG 840
CTATGATTGATCCTGATGGAACTTTGGAGG CTCTGAACAACATGGGATTTCCCAGTGCTA 900
TGTTGCCATCTCCACCGAAACAGAAGTCCA GCCCTGTGAATAACCACCACAGCCCGGGTC 960
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AGACACCAAC ACTTGGCCAA CCAGAAGCTA GGGCTCTTCA TCCTGCTGCT GTTTCAGCCG 1020
CAGCTGAATT TGAGAATGCT GAATCTGAGG CTGATTTCAG TATACATTTC AATAGAGTCA 1080
ACCCTGATGG GGAAGAGGAA GATGTTACAG TAACATAAAT GACTTTCTCT TGATTGTTGA 1140
p~~~AAAAAAA P~~~,?~AAAAAA 116 0
(2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 309 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
Met Pro Thr Ser Ser Ser Phe Lys His Arg Ile Lys Glu Gln Glu Asp
1 5 10 15
Tyr Ile Arg Asp Trp Thr Ala His Arg Glu Glu Ile Ala Arg Ile Ser
20 25 30
Gln Asp Leu Ala Leu Ile Ala Arg Glu Ile Asn Asp Val Ala Gly Glu
35 40 45
Ile Asp Ser Val Thr Ser Ser Gly Thr Ala Pro Ser Thr Thr Val Ser
50 55 60
Thr Ala Ala Thr Thr Pro Gly Ser Ala Ile Asp Thr Arg Glu Glu Leu
65 70 75 80
Val Asp Arg Val Phe Asp Glu Ser Leu Asn Phe Gln Lys Ile Pro Pro
85 90 95
Leu Val His Ser Lys Thr Pro Glu Gly Asn Asn Gly Arg Ser Gly Asp
100 105 110
Pro Arg Pro Gln Ala Ala Glu Pro Pro Asp His Leu Thr Ile Thr Arg
115 120 125
Arg Arg Thr Trp Ser Arg Asp Glu Val Met Gly Asp Asn Leu Leu Leu
130 135 140
Ser Ser Val Phe Gln Phe Ser Xaa Lys Ile Arg Gln Ser Ile Asp Lys
145 150 155 160
Thr Ala Gly Lys Ile Arg Ile Leu Phe Lys Asp Lys Asp Arg Asn Trp
165 170 175
74
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Asp Asp Ile Glu Ser Lys Leu Arg Ala Glu Ser Glu Val Pro Ile Val
180 185 190
Lys Thr Ser Ser Met Glu Ile Ser Ser Ile Leu Gln Glu Leu Lys Arg
195 200 205
Val Glu Lys Gln Leu Gln Ala Ile Asn Ala Met Ile Asp Pro Asp Gly
210 215 220
Thr Leu Glu Ala Leu Asn Asn Met Gly Phe Pro Ser Ala Met Leu Pro
225 230 235 240
Ser Pro Pro Lys Gln Lys Ser Ser Pro Val Asn Asn His His Ser Pro
245 250 255
Gly Gln Thr Pro Thr Leu Gly Gln Pro Glu Ala Arg Ala Leu His Pro
260 265 270
Ala Ala Val Ser Ala Ala Ala Glu Phe Glu Asn Ala Glu Ser Glu Ala
275 280 285
Asp Phe Ser Ile His Phe Asn Arg Val Asn Pro Asp Gly Glu Glu Glu
290 295 300
Asp Val Thr Val Thr
305
(2) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1536 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
GAAGAGAGAA AATCAGCCTG TCTGCTCTCT CCTTGGCTCA ACAAGGCCTC TAACAGTCTT 60
CTGTCCTCTA TTCTGCACAC GGCATATTTG GGAACGAGAA ACAAAAGTTT TCCCAAATGA 120
AGAGAACTCA CTTGTTTATT GTGGGGATTT ATTTTCTGTC CTCTTGCAGG GCAGAAGAGG 180
GGCTTAATTT CCCCACATAT GATGGGAAGG ACCGAGTGGT AAGTCTTTCC GAGAAGAACT 240
TCAAGCAGGT TTTAAAGAAA TATGACTTGC TTTGCCTCTA CTACCATGAG CCGGTGTCTT 300
CAGATAAGGT CACGCANAAA CAGTTCCAAC TGAAAGAAAT CGTGCTTGAG CTTGTGGCCC 360
ACGTCCTTGA ACATAAAGCT ATAGGCTTTG TGATGGTGGA TGCCAAGAAA GAAGCCAAGC 420
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TTGCCAAGAA ACTGGGTTTTGATGAAGAAGGAAGCCTGTATATTCTTAAG GGTGATCGCA480
CAATAGAGTT TGATGGCGAGTTTGCAGCTGATGTCTTGGTGGAGTTCCTC TTGGATCTAA540
TTGAAGACCC AGTGGAGATCATCAGCAGCAAACTGGAAGTCCAAGCCTTC GAACGCATTG600
AAGACTACAT CAAACTCATTGGCTTTTTCAAGAGTGAGGACTCAGAATAC TACAAGGCTT660
TTGAAGAAGC AGCTGAACACTTCCAGCCTTACATCAAATTCTTTGCCACC TTTGACAAAG720
GGGTTGCAAA GAAATTATCTTTGAAGATGAATGAGGTTGACTTCTATGAG CCATTTATGG780
ATGAGCCCAT TGCCATCCCCAACAAACCTTACACAGAAGAGGAGCTGGTG GAGTTTGTGA840
AGGAACACCA AAGGTGCCTGAGATGGCATGTGGGGGCTGGGGGCCTGGGG TCTGGGGAAT900
GGAGAGGAGC CTCTCTGTGCTAACATTTCAGACCTGCCAAGAGCAACAAC CTAGTTAGTA960
CCCCAGCAGT ACAGAACTCAGTAGTATGGCTTTGTTGATCAGTAATGACT AGCAGGGATG1020
TTATTACTTC TGAATCTAAGTCTGCACCTGCAAGCAGAGTTTGATAAATC CCTCAGTCAG1080
CAAATCCCCT CAAAGCCAGGGCAAGATATAAATAAAATTCTATACTAGGA ATGAGAGCAA1140
TTTAGTGAAA GTTCCCATATACCAATAACCATGCCCAGTGCTTTAGGGAA ACTATTTTAT1200
CTAATCTCCA ACCTTAGGGAGTAATTATTATTATCCCAATTTTACAGATC AAGGAATTGG1260
ACTCAATAGT TAAGTAACTTAGCCAAGGATGAACACTCTATGCATAGAAC TTCTGGGAGA1320
GAAATGCTTG ATACCACTTAGTGTAGCTCCAGCATGGATCAGCAAACTTT TTCTGTAAAG1380
AACAAAATGG TAAATATTTCAGGTTCTGTGGGCCAGATGGCGTCTGTAGC AACTACTTAA1440
CTGCGGCTGT GGCATGAAAGCAGCCATGGATCATGTATAAACAAATGGGT GTGGCTGTGT1500
ACCAGTAAAA GTTTATTTAGGp~AAAAAAAAAAAAAA 1536
(2) INFORMATION EQ ID
FOR S N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 268 aminoacids
(B) TYPE: a mino acid
(C) STRANDE DNESS:
(D) TOPOLOG Y: linear
(ii) MOLECULE TYP E: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
Met Lys Arg Thr His Leu Phe Ile Val Gly Ile Tyr Phe Leu Ser Ser
1 5 10 15
76
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Cys Arg Ala Glu Glu Gly Leu Asn Phe Pro Thr Tyr Asp Gly Lys Asp
20 25 30
Arg Val Val Ser Leu Ser Glu Lys Asn Phe Lys Gln Val Leu Lys Lys
35 40 45
Tyr Asp Leu Leu Cys Leu Tyr Tyr His Glu Pro Val Ser Ser Asp Lys
50 55 60
Val Thr Xaa Lys Gln Phe Gln Leu Lys Glu Ile Val Leu Glu Leu Val
65 70 75 80
Ala His Val Leu Glu His Lys Ala Ile Gly Phe Val Met Val Asp Ala
85 90 95
Lys Lys Glu Ala Lys Leu Ala Lys Lys Leu Gly Phe Asp Glu Glu Gly
100 105 110
Ser Leu Tyr Ile Leu Lys Gly Asp Arg Thr Ile Glu Phe Asp Gly Glu
115 120 125
Phe Ala Ala Asp Val Leu Val Glu Phe Leu Leu Asp Leu Ile Glu Asp
130 135 140
Pro Val Glu Ile Ile Ser Ser Lys Leu Glu Val Gln Ala Phe Glu Arg
145 150 155 160
Ile Glu Asp Tyr Ile Lys Leu Ile Gly Phe Phe Lys Ser Glu Asp Ser
165 170 175
Glu Tyr Tyr Lys Ala Phe Glu Glu Ala Ala Glu His Phe Gln Pro Tyr
180 185 190
Ile Lys Phe Phe Ala Thr Phe Asp Lys Gly Val Ala Lys Lys Leu Ser
195 200 205
Leu Lys Met Asn Glu Val Asp Phe Tyr Glu Pro Phe Met Asp Glu Pro
210 215 220
Ile Ala Ile Pro Asn Lys Pro Tyr Thr Glu Glu Glu Leu Val Glu Phe
225 230 235 240
Val Lys Glu His Gln Arg Cys Leu Arg Trp His Val Gly Ala Gly Gly
245 250 255
Leu Gly Ser Gly Glu Trp Arg Gly Ala Ser Leu Cys
260 265
(2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1009 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
7
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(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID
N0:16:
GAATTCGGCC TTCATGCGCC TGCAGGAAAG CATCACACTG TGTTTTCCTT60
AATCTGACAT
AACTTGACAG GAAGTCAACT TCAAGCAGAT CGGGATCTCA TTTAGGAAGC120
TGACTTGAAA
ATAAGTGTCC AATCAAAAAC TGTGTATTTT GAAAATACTC AAGTTCCAGT180
TTTAAATTTG
TGCTTATCAT TCTCCTTCAC TTTCTGAAAA CCATGTGGAC TTCTGGTAGA240
CCTGGCAATC
ATGAGCAATG CAAAGAACTG GCTTGGACTT TGTACTTCTG GGGGCTGATG300
GGCATGTCCT
GACCTTACGA CCACCGTTCT CTCGGACACC AAGGTGAATT AGAAGCACTC360
CCAACACCAC
CTGTCAGACA AGCCACAGTC ACATCAGCGG GCTGGGTTTG GAACCAGTTT420
ACCAAGARGA
TTCGTTCTGG AAGAGTACAC TGGGACCGAC TCGGCAAGGT AAGAAATGCC480
CCTTTGTATG
AAGTAGAAAT GACCCGGGTA GTGGATATTG TGAATTGAGT ATCAAAGTTG540
AAATTGAATA
ACCTAGCCTT TATYTGAGAC CTGAGAAAAA TGGTACGTTA CTTGACACCT600
CTAGAACAAG
AGCTAAAATG TAACTTCTGC TTTGTCAGAG AAGGAAAGAT TTATTTCCTT660
ACCAGTCTGA
GTCCATGTCT CTGGTATGAA TGGGAAAAAG GATTTGGAGG AAAAGGCTCA720
TGGGAATTGG
GACCCTGCAA GAGCTATTCA AGTCCTAAAA CAGCTGTCTG GGAATGACAG780
GAGGCAGCAG
AATGGGGGAG AGGGAAACTT GGAAATACAA GAGTTTTTTG CTTTGTGTTT840
GAAGAGTACA
TTATGGGGTT TTTTTCAGAG CATTTCCAGA GAGGTGCAAA TTTAATGAAA900
GGTATTGCCT
AAAATAAAAA TAAAACATTT TTCATTTCAG CATGTGCTTT AGGATAGTTG960
AAGATCTTAG
GAGACAATAA ATATATTTAT AAATGTTAAA P~AAAAAAAA 10
P~~?~AAAAAAA 0
9
(2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 87 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
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Met Trp Thr Ser Gly Arg Met Ser Asn Ala Lys Asn Trp Leu Gly Leu
1 5 10 15
Gly Met Ser Leu Tyr Phe Trp Gly Leu Met Asp Leu Thr Thr Thr Val
20 25 30
Leu Ser Asp Thr Pro Thr Pro Gln Gly Glu Leu Glu Ala Leu Leu Ser
35 40 45
Asp Lys Pro Gln Ser His Gln Arg Thr Lys Xaa Ser Trp Val Trp Asn
50 55 60
Gln Phe Phe Val Leu Glu Glu Tyr Thr Gly Thr Asp Pro Leu Tyr Val
65 70 75 80
Gly Lys Val Arg Asn Ala Lys
(2) INFORMATION FOR SEQ ID N0:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2546 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION:
SEQ ID N0:18:
AAAAGAAACC AAGGAAATTTGTATGATAAG GCAGGTAAAGTGAGGAAACATGCAACTGAA60
CAGGAAAAAA CTGAAGAGGGATTAGGCCCT AATATAAAAAGCATTGTCACCATGTTGATG120
CTGATGCTAT TGATGATGTTTGCTGTCCAC TGTACCTGGGTCACAAGCAATGCCTACTCT180
AGTCCAAGTG TAGTCCTGGCCTCATACAAT CATGATGGCACCAGGAATATCTTAGATGAT240
TTTAGAGAAG CTTACTTTTGGCTAAGGCAA AATACAGATGAACATGCACGAGTAATGTCT300
TGGTGGGATT ATGGCTATCAGATAGCTGGA ATGGCTAATAGAACTACGTTGGTGGATAAT360
AACACCTGGA ATAACAGCCACATAGCACTG GTGGGAAAAGCTATGTCTTCTAATGAAACA420
GCAGCCTATA AAATCATGAGGACTCTAGAT GTAGATTATGTTTTGGTTATTTTTGGAGGG480
GTTATTGGCT ATTCTGGTGATGATATCAAC AAATTTCTCTGGATGGTTAGGATAGCTGAA540
GGAGAACATC CCAAAGACATTCGGGAAAGT GACTATTTTACCCCACAGGGAGAATTCCGT600
GTAGACAAAG CAGGATCCCCTACTTTGTTG AATTGCCTTATGTATAAAATGTCATACTAC660
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AGATTTGGAG GGATTTTCGTACACCCCCAGGTTTTGACCG AACACGTAAT720
AAATGCAGCT
GCTGAGATTGGAAATAAGGACATTAAATTCAAACATTTGGAAGAAGCCTT TACATCAGAA780
CACTGGCTTGTTAGGATATATAAAGTAAAAGCACCTGATAACAGGGAGAC ATTAGATCAC840
AAACCTCGAGTCACCAACATTTTCCCAAAACAGAAGTATTTGTCAAAGAA GACTACCAAA900
AGGAAGCGTGGCTACATTAAAAATAAGCTGGTTTTTAAGAAAGGCAAGAA AATATCTAAG960
AAGACTGTTTAAATGCACTGTTCTGGTTCCTAACTTGAAGCAGTTGTCCT TGTGAGAACC1020
GGTCTTTGCCTTTAGCTCATGTCGTGTTTCACAGCAAAGAGGGTACAGAA CCATCACTGG1080
TCCAGGTTAATGTACAAAATTTTCTGGCAATGCCTGATTAAAAAAATAAA ATTGGCTTGT1140
TGAGAACAGCTGTTTTCGATTTCTAATGTGAAGCAAGACAGAGCACTGCT GTAAATGTCT1200
AGCAGCAGATTTTTTTTTTATTGGTACATATTATCCTTCAAATCTGAGAA TTTGGACTAA1260
CTGCACCAAAGAACCCTCTAATTTGGTCCCTGGCACATGCATACTTGTCA ATGTTTTTAT1320
TCTCTTACAAGACCTGCATTTTATTTGAATTACCCGAATAGCAATATGTA AAATACAAGT1380
GACAAAATGTGATGAGAGCTTCTTGAACCGGTAAACTAGTACAGGTCTGA GAAAGACATA1440
TTAGAAGAATCATTATACTTCCCTGAATTATATTTATTTTCATGTTTCTC TAATGCAAAG1500
AATGTTTCATCAAATGTATATTTTCTGTTGCTTACTGTTTGCTCTGAGAA GAAGCTGCTG1560
TTTCAAAGATGGACCTCTGAGTAGCTAATTGATTCAAGTAGTTTTTTTAT GTTGACACAT1620
TATTACTGCTGTTAGCAGTCGTTTTCACCAGGTACTTACAGAGCAGATTT CATACATCAT1680
TCATTCAAGGGCTAAATTTATATTTTTTGGAAATCATGGCAACTACACAG GATGTTGCTT1740
ACCAGGACGGAGTTTTGGTATCTTAGTACTGAAGTTAGCACTATGTTTAC ATGCAAAAGA1800
TTAAGGAAAAAACCCTTAAAGTGGACAGGTATCCAAAGTTCATTTTCTGT GACTCATCAA1860
AGTGACAAAAGACTTGTAACAACTTTGCCTGGACTTTTTTCATTTTACAA CAGTTCATCC1920
ATTCACAATGATTTTGTTCTCTGCTCCATATTTTTTAATCCCTTAAGCAT TTGATGAAAC1980
ACTCTTTAGTGCTATATGCATTTTCTTACTTTTGTTAAAAATGTGACAAT TGTCAAAAAA2040
TGCACTAAAATGTAAATGGAGATTGAACAAGTTCACTTTCCAGCTTATAG GCAACTTTAT2100
ACAGACTTGAACATTTTCTCCAGTTGTTTAGTAAAAGTGAAAGAGAAAGG GTTTTTCCTG2160
CCACAGGATATAACTTTTTTTTATATAACAAGCATAACACACCACTGCTT TTGGTGGAAA2220
AGTGCAGAATAGTATGTACCTTTTATGAAGAAAAATGTAATTTACAATAT TCAGTGAGAA2280
TGTTACTGCTGATTTTCTTTTCCAAGGTGTAGAATATTCTTTGATTTATA GAATTCATTT2340
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TTGACCCAGA TGATGGTTCC TTTACAGAAC AATAAAATGG CTGAACATTT TCACAAATAG 2400
AGTGTAACGA AGTCTGGATT TCTGATACCT TGTCATTTGG GGGATTTTAT TTTACTTTGT 2460
TGCTTTAAAA TTCAATGCAG AGAAGTTGTT GACTGTAGGG GAAATAAAGT TAATTCAAAT 2520
TTTGAAAAAA F,~~AAAAAAAA AAAAAA 2 5 4 6
(2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 286 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:19:
Met Leu Met Leu Met Leu Leu Met Met Phe Ala Val His Cys Thr Trp
1 5 10 15
Val Thr Ser Asn Ala Tyr Ser Ser Pro Ser Val Val Leu Ala Ser Tyr
20 25 30
Asn His Asp Gly Thr Arg Asn Ile Leu Asp Asp Phe Arg Glu Ala Tyr
35 40 45
Phe Trp Leu Arg Gln Asn Thr Asp Glu His Ala Arg Val Met Ser Trp
50 55 60
Trp Asp Tyr Gly Tyr Gln Ile Ala Gly Met Ala Asn Arg Thr Thr Leu
65 70 75 80
Val Asp Asn Asn Thr Trp Asn Asn Ser His Ile Ala Leu Val Gly Lys
85 90 95
Ala Met Ser Ser Asn Glu Thr Ala Ala Tyr Lys Ile Met Arg Thr Leu
100 105 110
Asp Val Asp Tyr Val Leu Val Ile Phe Gly Gly Val Ile Gly Tyr Ser
115 120 125
Gly Asp Asp Ile Asn Lys Phe Leu Trp Met Val Arg Ile Ala Glu Gly
130 135 140
Glu His Pro Lys Asp Ile Arg Glu Ser Asp Tyr Phe Thr Pro Gln Gly
145 150 155 160
Glu Phe Arg Val Asp Lys Ala Gly Ser Pro Thr Leu Leu Asn Cys Leu
165 170 175
81
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Met Tyr Lys Met Ser Tyr Tyr Arg Phe Gly Glu Met Gln Leu Asp Phe
180 185 190
Arg Thr Pro Pro Gly Phe Asp Arg Thr Arg Asn Ala Glu Ile Gly Asn
195 200 205
Lys Asp Ile Lys Phe Lys His Leu Glu Glu Ala Phe Thr Ser Glu His
210 215 220
Trp Leu Val Arg Ile Tyr Lys Val Lys Ala Pro Asp Asn Arg Glu Thr
225 230 235 240
Leu Asp His Lys Pro Arg Val Thr Asn Ile Phe Pro Lys Gln Lys Tyr
245 250 255
Leu Ser Lys Lys Thr Thr Lys Arg Lys Arg Gly Tyr Ile Lys Asn Lys
260 265 270
Leu Val Phe Lys Lys Gly Lys Lys Ile Ser Lys Lys Thr Val
275 280 285
(2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4061 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION:
SEQ ID N0:20:
AAGCAATTGA AGAAATTGCAGCAGGATGTG ATGGAAATGA AAAAAACAAA 60
GGTTCGCCTA
ATGAAACAAA TGAAAGAAGAACAAGAGAAA GCCAGACTGA CTGAGTCTAGAAGAAACAGA 120
GAGATTGCTC AGTTGAAAAAGGATCAACGT AAAAGAGATC ATCMACTTAGACTTCTGGAA 180
GCCCAAAAAA GAAACCAAGAAGTGGTTCTA CGTCGCAAAA CTGAAGAGGTTACGGCTCTT 240
CGTCGGCAAG TAAGACCCATGTCAGATAAA GTGGCTGGGA AAGTTACTCGGAAGCTGAGT 300
TCATCTGATG CACCTGCTCAGGACACAGGT TCCAGTGCAG CTGCTGTCGAAACAGATGCA 360
TCAAGGACAG GAGCCCAGCAGAAAATGAGA ATTCCTGTGG CGAGAGTCCAGGCCTTACCA 420
ACGCCGGCAA CAAATGGAAACAGGAAAAAA TATCAGAGGA AAGGATTGACTGGCCGAGTG 480
TTTATTTCCA AGACAGCTCGCATGAAGTGG CAGCTCCTTG AGCGCAGGGTCACAGACATC 540
ATCATGCAGA AGATGACCATTTCCAACATG GAGGCAGATA TGAATAGACTCCTCAAGCAA 600
82
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CGGGAGGAACTCACAAAAAGACGAGAGAAA 660
CTTTCAAAAA
GAAGGGAGAA
GATAGTCAAG
GAGAATGGAGAGGGAGATAAAAATGTGGCTAATATCAATGAAGAGATGGA GTCACTGACT720
GCTAATATCGATTACATCAATGACAGTATTTCTGATTGTCAGGCCAACAT AATGCAGATG780
GAAGAAGCAAAGGAAGAAGGTGAGACATTGGATGTTACTGCAGTCATTAA TGCCTGCACC840
CTTACAGAAGCCCGATACCTGCTAGATCACTTCCTGTCAATGGGCATCAA TAAGGGTCTT900
CAGGCTGCCCAGAAAGAGGCTCAAATTAAAGTACTGGAAGGTCGACTCAA ACAAACAGAA960
ATAACCAGTGCTACCCAAAACCAGCTCTTATTCCATATGTTGAAAGAGAA GGCAGAATTA1020
AATCCTGAGCTAGATGCTTTACTAGGCCATGCTTTACAAGATCTAGATAG CGTACCATTA1080
GAAAATGTAGAGGATAGTACTGATGAGGATGCTCCTTTAAACAGCCCAGG ATCAGAAGGA1140
AGCACGCTGTCTTCAGATCTCATGAAGCTTTGTGGTGAAGTGAAACCTAA GAACAAGGCC1200
CGAAGGAGAACCACCACTCAGATGGAATTGCTGTATGCAGATAGCAGTGA ACTAGCTTCA1260
GACACTAGTACAGGAGATGCCTCCTTGCCTGGCCCTCTCACACCTGTTGC AGAAGGGCAA1320
GAGATTGGAATGAATACAGAGACAAGTGGTACTTCTGCTAGGGAAAAAGA GCTCTCTCCC1380
CCACCTGGCTTACCTTCTAAGATAGGCAGCATTTCCAGGCAGTCATCTCT ATCAGAAAAA1440
AAAATTCCAGAGCCTTCTCCTGTAACAAGGAGAAAGGCATATGAGAAAGC AGAAAAATCA1500
AAGGCCAAGGAACAAAAGCACTCAGATTCTGGAACTTCAGAGGCTAGTCT TTCACCTCCT1560
TCTTCCCCACCAAGCCGGCCCCGTAATGAACTGAATGTTTTTAATCGTCT TACTGTTTCT1620
CAGGGAAACACATCAGTTCAGCAGGATAAGTCTGATGAAAGTGACTCCTC TCTCTCGGAG1680
GTACACAGCAGATCCTCCAGAAGGGGCATAATCAACCCATTTCCTGCTTC AAAAGGAATC1740
AGAGCTTTTCCACTTCAGTGTATTCACATAGCTGAAGGGCATACAAAAGC TGTGCTCTGT1800
GTGGATTCTACTGATGATCTCCTCTTCACTGGATCAAAAGATCGTACTTG TAAAGTATGG1860
AATCTGGTGACTGGGCAGGAAATAATGTCACTGGGGGGTCATCCCAACAA TGTCGTGTCT2920
GTAAAATACTGTAATTATACCAGTTTGGTCTTCACTGTATCAACATCTTA TATTAAGGTG1980
TGGGATATCAGAGATTCAGCAAAGTGCATTCGAACACTAACGTCTTCAGG TCAAGTTACT2040
CTTGGAGATGCTTGTTCTGCAAGTACCAGTCGAACAGTAGCTATTCCTTC TGGAGAGAAC2100
CAGATCAATCAAATTGCCCTAAACCCAACTGGCACCTTCCTCTATGCTGC TTCTGGAAAT2160
GCTGTCAGGATGTGGGATCTTAAAAGGTTTCAGTCTACAGGAAAGTTAAC AGGACACCTA2220
GGCCCTGTTATGTGCCTTACTGTGGATCAGATTTCCAGTGGACAAGATCT AATCATCACT2280
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GGCTCCAAGGATCATTACATCAAAATGTTTGATGTTACAG TGGGACTGTG 2340
AAGGAGCTCT
AGTCCCACCCACAATTTTGAACCCCCTCATTATGATGGCATAGAAGCACTAACCATTCAA 2400
GGGGATAACCTATTTAGTGGGTCTAGAGATAATGGAATCAAGAAATGGGACTTAACTCAA 2460
AAAGACCTTCTTCAGCAAGTTCCAAATGCACATAAGGATTGGGTCTGTGCCCTGGGAGTG 2520
GTGCCAGACCACCCAGTTTTGCTCAGTGGCTGCAGAGGGGGCATTTTGAAAGTCTGGAAC 2580
ATGGATACTTTTATGCCAGTGGGAGAGATGAAGGGTCATGATAGTCCTATCAATGCCATA 2640
TGTGTTAATTCCACCCACATTTTTACTGCAGCTGATGATCGAACTGTGAGAATTTGGAAG 2700
GCTCGCAATTTGCAAGATGGTCAGATCTCTGACACAGGAGATCTGGGGGAAGATATTGCC 2760
AGTAATTAAACATGAATGAAGATAGGTTGTAAACTGAATGCTGTGATAATACTCTGTATT 2820
CTTTATGGAAAATGTTGTCCTGTACTTACTAGGCAAAACGTATGAATCGGATTAACTGGA 2880
AAATATATCTGAATTCAACTGCTGACTATAAATGGTATTCTAATAAAATTGTGTACTATC 2940
CTGTGTGCTTAGTTTTAAGATCAACCAATAGATATATATCCTACAATTGATATATTGCTT 3000
TATTCACACTTTTATTGTGGCTGAATTTTTGTGCCTATCTATAAAACACACTTTCAAATT 3060
ATTTGAATTACCAAGACGTCTGCTTTTGTGACAGTCAGAAAACACACCTGGAATACGATG 3120
CAGCCCACCATTAACTCATTCATGTAGTTTATTCAAGTGATTTATGTATTTAAACTAAAT 3180
ATTGAAAATGTTAGTCAAATTGTGGTTTGCTTGTCAGGTATTTATATCAGTCTGTAGTGG 3240
ATTCCCAAATTTCAAAGCTCTTTTAATGTAATGGACAAAAATAAGATATGAGAATATTAT 3300
TGATGAATTTTCATAAGGTGGAATTGATCTTAATCTACTAACAGAGAAGGGTAGACAGTT 3360
TGTGTTAAATGTTGGCATTTACTTGTATTGACCAAAGTTTTGCAGCTCTACTATATTCTG 3420
TGCTCAGGACTAAAATGCTGTTAATTTTTTTTTTTTTTTTTCCAGTGCTGTGCATATATT 3480
CTGTGATGGGAAACATTGTTGATGTCCTAACAGAAATATATTTTGATCTATTTTCCTATG 3540
GAGTTGTTTCTATTATGACCATTTAATTTTGTTTTTATTTAATAGTAGTATTTCCTTCCC 3600
TTTTATCTAATTTTTTATATGCTGCTAAATATATTTTAAATATACTATGTTTGCGAACCT 3660
TGGTAGCTATGATGAGAGCTATTATCATCTGTGGTGGGAAAAGCTATGTAAATAGGTAGA 3720
TTGTATAGAGAGACTATCTTGTGTTGTGCCTGTATGAATTTTTAAAAGTTGTTGACTGGA 3780
TTTTGCAAAAGGATGTATAATATTTCTGTCTGCTCAGAATATTAATTTGTAAATTCTGCA 3840
AGTTTAATTTTTATGTAGATGGTATAACATTTGAAAATATTGTCTTATGTGATTTTTTCC 3900
CCTGAAAATATTTGCTTGTAAATGAAAACTTAGCTAGGGCTTAAATAAACATGTTGCTAT 3960
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GA~:ATKRAAA P~~~AAAAAAA AAAARAAAAA F~1AAAAAAAA p~~tIAAAAAAA p~~i~AAAAAAA 4 0 2
0
A .FtAAP.AAAA P~~~AAAAAAA p~~~.AAAAAAA F,F~AAAAAAAA A 4 0 61
,2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 910 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi)SEQUENCE
DESCRIPTION:
SEQ
ID
N0:21:
Met LysLysThr Lys Arg LeuMetLys GlnMet LysGluGlu Gln
Val
1 5 10 15
Glu LysAlaArg Leu Glu SerArgArg AsnArg GluIleAla Gln
Thr
20 25 30
Leu LysLysAsp Gln Lys ArgAspHis XaaLeu ArgLeuLeu Glu
Arg
35 40 45
Ala GlnLysArg Asn Glu ValValLeu ArgArg LysThrGlu Glu
Gln
50 55 60
Val Thr Ala Leu Arg Arg Gln Val Arg Pro Met Ser Asp Lys Val Ala
65 70 75 80
Gly Lys Val Thr Arg Lys Leu Ser Ser Ser Asp Ala Pro Ala Gln Asp
85 90 95
Thr Gly Ser Ser Ala Ala Ala Val Glu Thr Asp Ala Ser Arg Thr Gly
100 105 110
Ala Gln Gln Lys Met Arg Ile Pro Val Ala Arg Val Gln Ala Leu Pro
115 220 125
Thr Pro Ala Thr Asn Gly Asn Arg Lys Lys Tyr Gln Arg Lys Gly Leu
130 135 140
Thr Gly Arg Val Phe Ile Ser Lys Thr Ala Arg Met Lys Trp Gln Leu
145 150 155 160
Leu Glu Arg Arg Val Thr Asp Ile Ile Met Gln Lys Met Thr Ile Ser
165 170 175
Asn Met Glu Ala Asp Met Asn Arg Leu Leu Lys Gln Arg Glu Glu Leu
180 185 190
g$
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Thr Lys Arg Arg Glu Lys Leu Ser Lys Arg Arg Glu Lys Ile Val Lys
195 200 205
Glu Asn Gly Glu Gly Asp Lys Asn Val Ala Asn Ile Asn Glu Glu Met
210 215 220
Glu Ser Leu Thr Ala Asn Ile Asp Tyr Ile Asn Asp Ser Ile Ser Asp
225 230 235 240
Cys Gln Ala Asn Ile Met Gln Met Glu Glu Ala Lys Glu Glu Gly Glu
245 250 255
Thr Leu Asp Val Thr Ala Val Ile Asn Ala Cys Thr Leu Thr Glu Ala
260 265 270
Arg Tyr Leu Leu Asp His Phe Leu Ser Met Gly Ile Asn Lys Gly Leu
275 280 285
Gln Ala Ala Gln Lys Glu Ala Gln Ile Lys Val Leu Glu Gly Arg Leu
290 295 300
Lys Gln Thr Glu Ile Thr Ser Ala Thr Gln Asn Gln Leu Leu Phe His
305 310 315 320
Met Leu Lys Glu Lys Ala Glu Leu Asn Pro Glu Leu Asp Ala Leu Leu
325 330 335
Gly His Ala Leu Gln Asp Leu Asp Ser Val Pro Leu Glu Asn Val Glu
340 345 350
Asp Ser Thr Asp Glu Asp Ala Pro Leu Asn Ser Pro Gly Ser Glu Gly
355 360 365
Ser Thr Leu Ser Ser Asp Leu Met Lys Leu Cys Gly Glu Val Lys Pro
370 375 380
Lys Asn Lys Ala Arg Arg Arg Thr Thr Thr Gln Met Glu Leu Leu Tyr
385 390 395 400
Ala Asp Ser Ser Glu Leu Ala Ser Asp Thr Ser Thr Gly Asp Ala Ser
405 410 415
Leu Pro Gly Pro Leu Thr Pro Val Ala Glu Gly Gln Glu Ile Gly Met
420 425 430
Asn Thr Glu Thr Ser Gly Thr Ser Ala Arg Glu Lys Glu Leu Ser Pro
435 440 445
Pro Pro Gly Leu Pro Ser Lys Ile Gly Ser Ile Ser Arg Gln Ser Ser
450 455 460
Leu Ser Glu Lys Lys Ile Pro Glu Pro Ser Pro Val Thr Arg Arg Lys
465 470 475 480
Ala Tyr Glu Lys Ala Glu Lys Ser Lys Ala Lys Glu Gln Lys His Ser
86
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485 490 495
Asp Ser Gly Thr Ser Glu Ala Ser Leu Ser Pro Pro Ser Ser Pro Pro
500 505 510
Ser Arg Pro Arg Asn Glu Leu Asn Val Phe Asn Arg Leu Thr Val Ser
515 520 525
Gln Gly Asn Thr Ser Val Gln Gln Asp Lys Ser Asp Glu Ser Asp Ser
530 535 540
Ser Leu Ser Glu Val His Ser Arg Ser Ser Arg Arg Gly Ile Ile Asn
545 550 555 560
Pro Phe Pro Ala Ser Lys Gly Ile Arg Ala Phe Pro Leu Gln Cys Ile
565 570 575
His Ile Ala Glu Gly His Thr Lys Ala Val Leu Cys Val Asp Ser Thr
580 585 590
Asp Asp Leu Leu Phe Thr Gly Ser Lys Asp Arg Thr Cys Lys Val Trp
595 600 605
Asn Leu Val Thr Gly Gln Glu Ile Met Ser Leu Gly Gly His Pro Asn
610 615 620
Asn Val Val Ser Val Lys Tyr Cys Asn Tyr Thr Ser Leu Val Phe Thr
625 630 635 640
Val Ser Thr Ser Tyr Ile Lys Val Trp Asp Ile Arg Asp Ser Ala Lys
645 65C 655
Cys Ile Arg Thr Leu Thr Ser Ser Gly Gln Val Thr Leu Gly Asp Ala
660 665 670
Cys Ser Ala Ser Thr Ser Arg Thr Val Ala Ile Pro Ser Gly Glu Asn
675 680 685
Gln Ile Asn Gln Ile Ala Leu Asn Pro Thr Gly Thr Phe Leu Tyr Ala
690 695 700
Ala Ser Gly Asn Ala Val Arg Met Trp Asp Leu Lys Arg Phe Gln Ser
705 710 715 720
Thr Gly Lys Leu Thr Gly His Leu Gly Pro Val Met Cys Leu Thr Val
725 730 735
Asp Gln Ile Ser Ser Gly Gln Asp Leu Ile Ile Thr Gly Ser Lys Asp
740 745 750
His Tyr Ile Lys Met Phe Asp Val Thr Glu Gly Ala Leu Gly Thr Val
755 760 765
Ser Pro Thr His Asn Phe Glu Pro Pro His Tyr Asp Gly Ile Glu Ala
770 775 780
87
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Leu Thr Ile Gln Gly Asp Asn Leu Phe Ser Gly Ser Arg Asp Asn Gly
785 790 795 800
Ile Lys Lys Trp Asp Leu Thr Gln Lys Asp Leu Leu Gln Gln Val Pro
805 810 815
Asn Ala His Lys Asp Trp Val Cys Ala Leu Gly Val Val Pro Asp His
820 825 830
Pro Val Leu Leu Ser Gly Cys Arg Gly Gly Ile Leu Lys Val Trp Asn
835 840 845
Met Asp Thr Phe Met Pro Val Gly Glu Met Lys Gly His Asp Ser Pro
850 855 860
Ile Asn Ala Ile Cys Val Asn Ser Thr His Ile Phe Thr Ala Ala Asp
865 870 875 880
Asp Arg Thr Val Arg Ile Trp Lys Ala Arg Asn Leu Gln Asp Gly Gln
885 890 895
Ile Ser Asp Thr Gly Asp Leu Gly Glu Asp Ile Ala Ser Asn
900 905 910
(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 = "oigonucleotide"
{xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
TNCTCGGTTAT ATGGAGGACG AATAGACT 29
(2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
gg
CA 02274507 1999-06-11
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:
CTCTTTATGA GCATGATATG GCTTCAG 27
(2) INFORMATION FOR SEQ ID N0:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:
CNTGCTGCCCG ACTTAAACTG AGAACCAA 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:
GNGGACAACTC CCATAGTTGA GGTTTGTC 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:
89
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TNGGAAGAGTG ACCCAGGGGC CCAAAGCA 29
(2) INFORMATION FOR SEQ ID N0:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:27:
CNCTGCAAGCG AGTGGTTTTC AGGGCTGT 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:
TNGAGCCAAGG AGAGAGCAGA CAGGCTGA 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:
ANCCCCCAGAA GTACAAGGAC ATGCCAAG 29
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(2) INFORMATION FOR SEQ ID N0:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
{B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:30:
CNATAGCATCA GCATCAACAT GGTGACAA 2g
(2) INFORMATION FOR SEQ ID N0:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 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:
CTCCATTCTC CTTGACTATC TTCTCCC 27
(2) INFORMATION FOR SEQ ID N0:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "oligonucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:32:
TNCCTGGTGCC ATCATGATTG TATGAGGC 2g
91
