CA2318303A1 - Secreted proteins and polynucleotides encoding them - Google Patents
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Abstract
Novel polynucleotides and the proteins encoded thereby are disclosed.
Description
SECRETED PROTEINS AND POLYNUCLEOTIDES ENCODING THEM
This application is a continuation-in-part of provisional application Ser. No.
60/072,134, filed January 22,1998, which is incorporated by reference herein.
FIELD OF THE INVENTION
The present invention provides novel polynucleotides and proteins encoded by such polynucleotides, along with therapeutic, diagnostic and research utilities for these polynucleotides and proteins.
BACKGROUND OF THE INVENTION
This application is a continuation-in-part of provisional application Ser. No.
60/072,134, filed January 22,1998, which is incorporated by reference herein.
FIELD OF THE INVENTION
The present invention provides novel polynucleotides and proteins encoded by such polynucleotides, along with therapeutic, diagnostic and research utilities for these polynucleotides and proteins.
BACKGROUND OF THE INVENTION
2 0 Technology aimed at the discovery of protein factors (including e.g., cytokines, such as lymphokines, interferons, CSFs and interleukins) has matured rapidly over the past decade. The now routine hybridization cloning and expression cloning techniques clone novel polynucleotides "directly" in the sense that they rely on information directly related to the discovered protein (i.e., partial DNA/amino acid sequence of the protein 2 5 in the case of hybridization cloning; activity of the protein in the case of expression cloning). More recent "indirect" cloning techniques such as signal sequence cloning, which isolates DNA sequences based on the presence of a now well-recognized secretory leader sequence motif, as well as various PCR-based or low stringency hybridization cloning techniques, have advanced the state of the art by making available large numbers of 3 0 DNA/amino acid sequences for proteins that are known to have biological activity by virtue of their secreted nature in the case of leader sequence cloning, or by virtue of the cell or tissue source in the case of PCR-based techniques. It is to these proteins and the polynucleotides encoding them that the present invention is directed.
WO 99/376'14 PCT/US99/01404 SUMMARY OF THE INVENTION
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:1;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:1 from nucleotide 734 to nucleotide 1873;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:1 from nucleotide 1403 to nucleotide 1873;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone cs756_2 deposited under accession number ATCC 98636;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone cs756 2 deposited under accession number ATCC 98636;
(f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone cs756_2 deposited under accession number ATCC 98636;
(g) a polynucleatide encoding a mature protein encoded by the cDNA
insert of clone cs756 2 deposited under accession number ATCC 98636;
2 0 (h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID N0:2;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:2 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID N0:2;
2 5 (j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and {l) a polynucleotide that hybridizes under stringent conditions to any 3 0 one of the polynucleotides specified in {a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
NO:1 from nucleotide 734 to nucleotide 1873; the nucleotide sequence of SEQ ID
NO:1 from nucleotide 1403 to nucleotide 1873; the nucleotide sequence of the full-length protein coding sequence of clone cs756_2 deposited under accession number ATCC
98636;
WO 99/376'14 PCT/US99/01404 SUMMARY OF THE INVENTION
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:1;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:1 from nucleotide 734 to nucleotide 1873;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:1 from nucleotide 1403 to nucleotide 1873;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone cs756_2 deposited under accession number ATCC 98636;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone cs756 2 deposited under accession number ATCC 98636;
(f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone cs756_2 deposited under accession number ATCC 98636;
(g) a polynucleatide encoding a mature protein encoded by the cDNA
insert of clone cs756 2 deposited under accession number ATCC 98636;
2 0 (h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID N0:2;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:2 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID N0:2;
2 5 (j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and {l) a polynucleotide that hybridizes under stringent conditions to any 3 0 one of the polynucleotides specified in {a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
NO:1 from nucleotide 734 to nucleotide 1873; the nucleotide sequence of SEQ ID
NO:1 from nucleotide 1403 to nucleotide 1873; the nucleotide sequence of the full-length protein coding sequence of clone cs756_2 deposited under accession number ATCC
98636;
or the nucleotide sequence of a mature protein coding sequence of clone cs756_2 deposited under accession number ATCC 98636. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert of clone cs756 2 deposited under accession number ATCC 98636. In further preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:2 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID N0:2, or a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:2 having biological activity, the fragment comprising the amino acid sequence from amino acid 185 to amino acid 194 of SEQ ID N0:2.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID NO:1.
Further embodiments of the invention provide isolated polynucleotides produced according to a process selected from the group consisting of:
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
2 0 (aa) SEQ ID N0:1, but excluding the poly(A) tail at the 3' end of SEQ ID NO:1; and (ab) the nucleotide sequence of the cDNA insert of clone cs756_2 deposited under accession number ATCC 98636;
(ii) hybridizing said probes) to human genomic DNA in 2 5 conditions at least as stringent as 4X SSC at 65 degrees C; and (iii) isolating the DNA polynucleotides detected with the probe(s);
and (b) a process comprising the steps of:
3 0 (i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(ba) SEQ ID NO:1, but excluding the poly(A) tail at the 3' end of SEQ ID NO:1; and (bb) the nucleotide sequence of the cDNA insert of clone cs756_2 deposited under accession number ATCC 98636;
(ii) hybridizing said primer{s) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C;
{iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii).
Preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:1, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID
NO:1 to a nucleotide sequence corresponding to the 3' end of SEQ ID NO:1 , but excluding the poly{A) tail at the 3' end of SEQ ID NO:1. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID NO:1 from nucleotide 734 to nucleotide 1873, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID N0:1 from nucleotide 734 to nucleotide 1873, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:1 from nucleotide 734 to nucleotide 1873. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:1 from nucleotide 1403 to nucleotide 1873, and extending contiguously from a 2 0 nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
NO:1 from nucleotide 1403 to nucleotide 1873, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:1 from nucleotide 1403 to nucleotide 1873.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group 2 5 consisting of:
(a) the amino acid sequence of SEQ ID N0:2;
(b) fragments of the amino acid sequence of SEQ ID N0:2, each fragment comprising eight consecutive amino acids of SEQ ID N0:2; and (c) the amino acid sequence encoded by the cDNA insert of clone 3 0 cs756 2 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID N0:2. In further preferred embodiments, the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID N0:2 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID N0:2, or a protein comprising a fragment of the amino acid sequence of SEQ ID N0:2 having biological activity, the fragment comprising the amino acid sequence from amino acid 185 to amino acid 194 of SEQ ID N0:2.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3 from nucleotide 26 to nucleotide 1738;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3 from nucleotide 140 to nucleotide 1738;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone ew150_1 deposited under accession number ATCC 98636;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone ew150_1 deposited under accession number ATCC 98636;
(f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone ew150_1 deposited under accession number 2 0 ATCC 98636;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone ew150_1 deposited under accession number ATCC 98636;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID N0:4;
2 5 (i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:4 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID N0:4;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above;
3 0 (k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and (1) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i).
insert of clone cs756 2 deposited under accession number ATCC 98636. In further preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:2 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID N0:2, or a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:2 having biological activity, the fragment comprising the amino acid sequence from amino acid 185 to amino acid 194 of SEQ ID N0:2.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID NO:1.
Further embodiments of the invention provide isolated polynucleotides produced according to a process selected from the group consisting of:
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
2 0 (aa) SEQ ID N0:1, but excluding the poly(A) tail at the 3' end of SEQ ID NO:1; and (ab) the nucleotide sequence of the cDNA insert of clone cs756_2 deposited under accession number ATCC 98636;
(ii) hybridizing said probes) to human genomic DNA in 2 5 conditions at least as stringent as 4X SSC at 65 degrees C; and (iii) isolating the DNA polynucleotides detected with the probe(s);
and (b) a process comprising the steps of:
3 0 (i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(ba) SEQ ID NO:1, but excluding the poly(A) tail at the 3' end of SEQ ID NO:1; and (bb) the nucleotide sequence of the cDNA insert of clone cs756_2 deposited under accession number ATCC 98636;
(ii) hybridizing said primer{s) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C;
{iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii).
Preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:1, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID
NO:1 to a nucleotide sequence corresponding to the 3' end of SEQ ID NO:1 , but excluding the poly{A) tail at the 3' end of SEQ ID NO:1. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID NO:1 from nucleotide 734 to nucleotide 1873, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID N0:1 from nucleotide 734 to nucleotide 1873, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:1 from nucleotide 734 to nucleotide 1873. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:1 from nucleotide 1403 to nucleotide 1873, and extending contiguously from a 2 0 nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
NO:1 from nucleotide 1403 to nucleotide 1873, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:1 from nucleotide 1403 to nucleotide 1873.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group 2 5 consisting of:
(a) the amino acid sequence of SEQ ID N0:2;
(b) fragments of the amino acid sequence of SEQ ID N0:2, each fragment comprising eight consecutive amino acids of SEQ ID N0:2; and (c) the amino acid sequence encoded by the cDNA insert of clone 3 0 cs756 2 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID N0:2. In further preferred embodiments, the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID N0:2 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID N0:2, or a protein comprising a fragment of the amino acid sequence of SEQ ID N0:2 having biological activity, the fragment comprising the amino acid sequence from amino acid 185 to amino acid 194 of SEQ ID N0:2.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3 from nucleotide 26 to nucleotide 1738;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:3 from nucleotide 140 to nucleotide 1738;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone ew150_1 deposited under accession number ATCC 98636;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone ew150_1 deposited under accession number ATCC 98636;
(f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone ew150_1 deposited under accession number 2 0 ATCC 98636;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone ew150_1 deposited under accession number ATCC 98636;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID N0:4;
2 5 (i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:4 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID N0:4;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above;
3 0 (k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and (1) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:3 from nucleotide 26 to nucleotide 1738; the nucleotide sequence of SEQ ID
N0:3 from nucleotide 140 to nucleotide 1738; the nucleotide sequence of the full-length protein coding sequence of clone ew150_1 deposited under accession number ATCC 98636;
or the nucleotide sequence of a mature protein coding sequence of clone ew150_1 deposited under accession number ATCC 98636. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert of clone ew150_1 deposited under accession number ATCC 98636. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID N0:4 from amino acid 108 to amino acid 166. In further preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID
N0:4 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID N0:4, or a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:4 having biological activity, the fragment comprising the amino acid sequence from amino acid 280 to amino acid 289 of SEQ ID N0:4.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:3.
2 0 Further embodiments of the invention provide isolated polynucleotides produced according to a process selected from the group consisting of:
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group 2 5 consisting of:
(aa) SEQ ID N0:3, but excluding the poly(A) tail at the 3' end of SEQ ID N0:3; and (ab) the nucleotide sequence of the cDNA insert of clone ew150_1 deposited under accession number ATCC 98636;
3 0 (ii) hybridizing said probes) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C; and (iii) isolating the DNA polynucleotides detected with the probe(s);
and (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(ba) SEQ ID N0:3, but excluding the poiy(A) tail at the 3' end of SEQ ID N0:3; and (bb) the nucleotide sequence of the cDNA insert of clone ew150_1 deposited under accession number ATCC 98636;
(ii) hybridizing said primer{s) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C;
(iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii).
Preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:3, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID
N0:3 to a nucleotide sequence corresponding to the 3' end of SEQ ID N0:3 , but excluding the poly(A) tail at the 3' end of SEQ ID N0:3. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:3 from nucleotide 26 to nucleotide 1738, and extending 2 0 contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID N0:3 from nucleotide 26 to nucleotide 1738, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID N0:3 from nucleotide 26 to nucleotide 1738. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
2 5 N0:3 from nucleotide 140 to nucleotide 1738, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
N0:3 from nucleotide 140 to nucleotide 1738, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID N0:3 from nucleotide 140 to nucleotide 1738.
In other embodiments, the present invention provides a composition comprising 3 0 a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID N0:4;
(b) the amino acid sequence of SEQ ID N0:4 from amino acid 108 to amino acid 166;
N0:3 from nucleotide 26 to nucleotide 1738; the nucleotide sequence of SEQ ID
N0:3 from nucleotide 140 to nucleotide 1738; the nucleotide sequence of the full-length protein coding sequence of clone ew150_1 deposited under accession number ATCC 98636;
or the nucleotide sequence of a mature protein coding sequence of clone ew150_1 deposited under accession number ATCC 98636. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert of clone ew150_1 deposited under accession number ATCC 98636. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID N0:4 from amino acid 108 to amino acid 166. In further preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID
N0:4 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID N0:4, or a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:4 having biological activity, the fragment comprising the amino acid sequence from amino acid 280 to amino acid 289 of SEQ ID N0:4.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:3.
2 0 Further embodiments of the invention provide isolated polynucleotides produced according to a process selected from the group consisting of:
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group 2 5 consisting of:
(aa) SEQ ID N0:3, but excluding the poly(A) tail at the 3' end of SEQ ID N0:3; and (ab) the nucleotide sequence of the cDNA insert of clone ew150_1 deposited under accession number ATCC 98636;
3 0 (ii) hybridizing said probes) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C; and (iii) isolating the DNA polynucleotides detected with the probe(s);
and (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(ba) SEQ ID N0:3, but excluding the poiy(A) tail at the 3' end of SEQ ID N0:3; and (bb) the nucleotide sequence of the cDNA insert of clone ew150_1 deposited under accession number ATCC 98636;
(ii) hybridizing said primer{s) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C;
(iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii).
Preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:3, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID
N0:3 to a nucleotide sequence corresponding to the 3' end of SEQ ID N0:3 , but excluding the poly(A) tail at the 3' end of SEQ ID N0:3. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:3 from nucleotide 26 to nucleotide 1738, and extending 2 0 contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID N0:3 from nucleotide 26 to nucleotide 1738, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID N0:3 from nucleotide 26 to nucleotide 1738. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
2 5 N0:3 from nucleotide 140 to nucleotide 1738, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
N0:3 from nucleotide 140 to nucleotide 1738, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID N0:3 from nucleotide 140 to nucleotide 1738.
In other embodiments, the present invention provides a composition comprising 3 0 a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID N0:4;
(b) the amino acid sequence of SEQ ID N0:4 from amino acid 108 to amino acid 166;
(c) fragments of the amino acid sequence of SEQ ID N0:4, each fragment comprising eight consecutive amino acids of SEQ ID N0:4; and (d) the amino acid sequence encoded by the cDNA insert of clone ew150_1 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID N0:4 or the amino acid sequence of SEQ ID N0:4 from amino acid 108 to amino acid 166. In further preferred embodiments, the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID N0:4 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino ands of SEQ ID N0:4, or a protein comprising a fragment of the amino acid sequence of SEQ ID N0:4 having biological activity, the fragment comprising the amino acid sequence from amino acid 280 to amino acid 289 of SEQ ID N0:4.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:5;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:5 from nucleotide 1101 to nucleotide 1910;
2 0 (c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:5 from nucleotide 1260 to nucleotide 1920;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone gg894_13 deposited under accession number ATCC 98636;
2 5 (e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone gg894_13 deposited under accession number ATCC 98636;
(f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone gg894_13 deposited under accession number ATCC 98636;
3 0 {g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone gg894_13 deposited under accession number ATCC 98636;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID N0:6;
the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID N0:4 or the amino acid sequence of SEQ ID N0:4 from amino acid 108 to amino acid 166. In further preferred embodiments, the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID N0:4 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino ands of SEQ ID N0:4, or a protein comprising a fragment of the amino acid sequence of SEQ ID N0:4 having biological activity, the fragment comprising the amino acid sequence from amino acid 280 to amino acid 289 of SEQ ID N0:4.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:5;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:5 from nucleotide 1101 to nucleotide 1910;
2 0 (c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:5 from nucleotide 1260 to nucleotide 1920;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone gg894_13 deposited under accession number ATCC 98636;
2 5 (e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone gg894_13 deposited under accession number ATCC 98636;
(f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone gg894_13 deposited under accession number ATCC 98636;
3 0 {g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone gg894_13 deposited under accession number ATCC 98636;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID N0:6;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:6 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID N0:6;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and (1) a polynudeotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:5 from nucleotide 1101 to nucleotide 1910; the nucleotide sequence of SEQ
ID N0:5 from nucleotide 1260 to nucleotide 1910; the nucleotide sequence of the full-length protein coding sequence of clone gg894_13 deposited under accession number ATCC
98636; or the nucleotide sequence of a mature protein coding sequence of clone gg894_13 deposited under accession number ATCC 98636. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert of clone gg894_13 deposited under accession number ATCC 98636. In further preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:6 having biological 2 0 activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID N0:6, or a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:6 having biological activity, the fragment comprising the amino acid sequence from amino acid 130 to amino acid 139 of SEQ ID N0:6.
2 5 Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:5.
Further embodiments of the invention provide isolated polynucleotides produced according to a process selected from the group consisting of:
(a) a process comprising the steps of:
3 0 (i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(aa) SEQ ID N0:5, but excluding the poly(A) tail at the 3' end of SEQ ID N0:5; and (ab) the nucleotide sequence of the cDNA insert of clone gg894_13 deposited under accession number ATCC 98636;
(ii) hybridizing said probes) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C; and (iii) isolating the DNA polynucieotides detected with the probe(s);
and (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(ba) SEQ ID N0:5, but excluding the poly(A) tail at the 3' end of SEQ ID N0:5; and (bb) the nucleotide sequence of the cDNA insert of clone gg894_13 deposited under accession number ATCC 98636;
(ii) hybridizing said primers) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C;
(iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii).
2 0 Preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:5, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID
N0:5 to a nucleotide sequence corresponding to the 3' end of SEQ ID N0:5 , but excluding the poly(A) tail at the 3' end of SEQ ID N0:5. Also preferably the polynucleotide isolated 2 5 according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:5 from nucleotide 1101 to nucleotide 1910, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID N0:5 from nucleotide 1101 to nucleotide 1910, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID N0:5 from nucleotide 1101 to 3 0 nucleotide 1910. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
N0:5 from nucleotide 1260 to nucleotide 1910, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
N0:5 from nucleotide 1260 to nucleotide 1910, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID N0:5 from nucleotide 1260 to nucleotide 1910.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID N0:6;
(b) fragments of the amino acid sequence of SEQ ID N0:6, each fragment comprising eight consecutive amino acids of SEQ ID N0:6; and (c) the amino acid sequence encoded by the cDNA insert of clone gg894_13 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID N0:6. In further preferred embodiments, the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID N0:6 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino ands of SEQ ID N0:6, or a protein comprising a fragment of the amino acid sequence of SEQ ID N0:6 having biological activity, the fragment comprising the amino acid sequence from amino acid 130 to amino acid I39 of SEQ ID N0:6.
In one embodiment, the present invention provides a composition comprising an 2 0 isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:7;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:7 from nucleotide 452 to nucleotide 1102;
2 5 (c) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone it217 2 deposited under accession number ATCC 98636;
(d) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone it217_2 deposited under accession number ATCC 98636;
3 0 (e) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone it217_2 deposited under accession number ATCC
98636;
(f) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone it217_2 deposited under accession number ATCC 98636;
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID N0:8;
(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:8 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID N0:8;
(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 of (g) or (h) above ; and (k) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(h).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:7 from nucleotide 452 to nucleotide 1102; the nucleotide sequence of the full-length protein coding sequence of clone it217 2 deposited under accession number ATCC
98636;
or the nucleotide sequence of a mature protein coding sequence of clone it217_2 deposited under accession number ATCC 98636. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert of clone it217_2 deposited under accession number ATCC 98636. In further preferred embodiments, the present invention provides a polynucleotide encoding a protein 2 0 comprising a fragment of the amino acid sequence of SEQ ID N0:8 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID N0:8, or a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:8 having biological activity, the fragment comprising the amino acid sequence from amino and 103 2 5 to amino acid 112 of SEQ ID N0:8.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:7.
Further embodiments of the invention provide isolated polynucleotides produced according to a process selected from the group consisting of:
3 0 (a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(aa) SEQ ID N0:7, but excluding the poly(A) tail at the 3' end of SEQ ID N0:7; and (ab) the nucleotide sequence of the cDNA insert of clone it217_2 deposited under accession number ATCC 98636;
(ii) hybridizing said probes) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C; and (iii) isolating the DNA polynucleotides detected with the probe(s);
and (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(ba) SEQ ID N0:7, but excluding the poly(A) tail at the 3' end of SEQ ID N0:7; and (bb) the nucleotide sequence of the cDNA insert of done it217_2 deposited under accession number ATCC 98636;
(ii) hybridizing said primers) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C;
2 0 (iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii).
Preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:7, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID
N0:7 to 2 5 a nucleotide sequence corresponding to the 3' end of SEQ ID N0:7 , but excluding the poly(A) tail at the 3' end of SEQ ID N0:7. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:7 from nucleotide 452 to nucleotide 1102, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of 3 0 SEQ ID N0:7 from nucleotide 452 to nucleotide 1102, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID N0:7 from nucleotide 452 to nucleotide 1102.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID N0:8;
(b) fragments of the amino acid sequence of SEQ ID N0:8, each fragment comprising eight consecutive amino acids of SEQ ID N0:8; and (c) the amino acid sequence encoded by the cDNA insert of clone it217 2 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID N0:8. In further preferred embodiments, the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID N0:8 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino ands of SEQ ID N0:8, or a protein comprising a fragment of the amino acid sequence of SEQ ID N0:8 having biological activity, the fragment comprising the amino acid sequ~ce from amino acid 103 to amino acid 112 of SEQ ID N0:8.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
2 0 N0:9;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:9 from nucleotide 127 to nucleotide 387;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:9 from nucleotide 172 to nucleotide 387;
2 5 (d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone m1235_2 deposited under accession number ATCC 98636;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone m1235_2 deposited under accession number ATCC 98636;
3 0 (f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone m1235_2 deposited under accession number ATCC 98636;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone m1235 2 deposited under accession number ATCC 98636;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:10;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:10 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO:10;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and (1) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (ar(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:9 from nucleotide 127 to nucleotide 387; the nucleotide sequence of SEQ ID
N0:9 from nucleotide 172 to nucleotide 387; the nucleotide sequence of the full-length protein coding sequence of clone m1235_2 deposited under accession number ATCC 98636; or the nucleotide sequence of a mature protein coding sequence of clone m1235_2 deposited under accession number ATCC 98636. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert of clone m1235 2 deposited under accession number ATCC 98636. In further preferred 2 0 embodiments, the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:10 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO:20, or a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:10 having 2 5 biological activity, the fragment comprising the amino acid sequence from amino acid 38 to amino acid 47 of SEQ ID NO:10.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:9.
Further embodiments of the invention provide isolated polynucleotides produced 3 0 according to a process selected from the group consisting of:
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(aa) SEQ ID N0:9, but excluding the poly(A) tail at the 3' end of SEQ ID N0:9; and (ab) the nucleotide sequence of the cDNA insert of clone m1235 2 deposited under accession number A TCC 98636;
(ii) hybridizing said probes) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C; and (iii) isolating the DNA polynucleotides detected with the probe(s);
and (b) a process comprising the steps of:
(i) preparing one or more polynucleotfde primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(ba) SEQ ID N0:9, but excluding the poly(A) tail at the 3' end of SEQ ID N0:9; and (bb) the nucleotide sequence of the cDNA insert of clone m1235 2 deposited under accession number ATCC 98636;
(ii) hybridizing said primers) to human genomic DNA in conditions at least as stringent as 9:X SSC at 65 degrees C;
2 0 (iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii).
Preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:9, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID
N0:9 to 2 5 a nucleotide sequence corresponding to the 3' end of SEQ ID N0:9 , but excluding the poly(A) tail at the 3' end of SEQ ID N0:9. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:9 from nucleotide 127 to nucleotide 387, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of 3 0 SEQ ID N0:9 from nucleotide 127 to nucleotide 387, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID N0:9 from nucleotide 127 to nucleotide 387. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
N0:9 from nucleotide 172 to nucleotide 387, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
N0:9 from nucleotide 172 to nucleotide 387, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID N0:9 from nucleotide 172 to nucleotide 387.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:10;
(b) fragments of the amino acid sequence of SEQ ID NO:10, each fragment comprising eight consecutive amino acids of SEQ ID NO:10; and (c} the amino acid sequence encoded by the cDNA insert of clone m1235 2 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:10. In further preferred embodiments, the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID NO:10 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO:10, or a protein comprising a fragment of the amino acid sequence of SEQ ID N0:10 having biological activity, the fragment comprising the amino acid sequence from amino acid 38 to amino acid 47 of SEQ ID NO:10.
2 0 In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:11;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
2 5 N0:11 from nucleotide 147 to nucleotide 1163;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:11 from nucleotide 273 to nucleotide 1163;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone mt24 2 deposited under accession 3 0 number ATCC 98636;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone mt24_2 deposited under accession number ATCC 98636;
(f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone mt24_2 deposited under accession number ATCC 98636;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone mt24_2 deposited under accession number ATCC 98636;
(h) a polynudeotide encoding a protein comprising the amino acid sequence of SEQ ID N0:12;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:12 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID N0:12;
(j) a polynucleotide which is an allelic variant of a polynucleotfde of (ar(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and (1) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:11 from nucleotide 147 to nucleotide 1163; the nucleotide sequence of SEQ
ID N0:11 from nucleotide 273 to nucleotide 1163; the nucleotide sequence of the full-length protein 2 0 coding sequence of clone mt24 2 deposited under accession number ATCC
98636; or the nucleotide sequence of a mature protein coding sequence of clone mt24 2 deposited under accession number ATCC 98636. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone mt24 2 deposited under accession number ATCC 98636. In further preferred embodiments, the 2 5 present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:12 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID N0:12, or a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:12 having biological activity, the 3 0 fragment comprising the amino acid sequence from amino acid 164 to amino acid 173 of SEQ ID N0:12.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID NO:11.
Further embodiments of the invention provide isolated polynucleotides produced according to a process selected from the group consisting of:
(a) a process comprising the steps of:
{i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(aa) SEQ ID N0:11, but excluding the poly(A) tail at the 3' end of SEQ ID N0:11; and (ab) the nucleotide sequence of the cDNA insert of clone mt24 2 deposited under accession number ATCC 98636;
(ii} hybridizing said probes) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C; and {iii) isolating the DNA polynucleotides detected with the probe(s);
and (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
2 0 (ba) SEQ ID N0:11, but excluding the poly(A) tail at the 3' end of SEQ ID NO:11; and (bb) the nucleotide sequence of the cDNA insert of clone mt24 2 deposited under accession number ATCC 98636;
(ii) hybridizing said primers) to human genomic DNA in 2 5 conditions at least as stringent as 4X SSC at 65 degrees C;
(iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii).
Preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID NO:11, and 3 0 extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ
ID NO:11 to a nucleotide sequence corresponding to the 3' end of SEQ ID N0:11 , but excluding the poly(A) tail at the 3' end of SEQ ID NO:11. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:11 from nucleotide 147 to nucleotide 1163, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID NO:11 from nucleotide 147 to nucleotide 1163, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:11 from nucleotide 147 to nucleotide 1163. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:11 from nucleotide 273 to nucleotide 1163, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
NO:11 from nucleotide 273 to nucleotide 1163, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID N0:11 from nucleotide 273 to nucleotide 1163.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
{a) the amino acid sequence of SEQ ID N0:12;
(b) fragments of the amino acid sequence of SEQ ID N0:12, each fragment comprising eight consecutive amino acids of SEQ ID N0:12; and (c) the amino acid sequence encoded by the cDNA insert of clone mt24_2 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID N0:12. In further preferred 2 0 embodiments, the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID N0:12 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID N0:12, or a protein comprising a fragment of the amino acid sequence of SEQ ID N0:12 having biological activity, the fragment comprising the amino acid 2 5 sequence from amino acid 164 to amino acid 173 of SEQ ID N0:12.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:13;
3 0 (b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:13 from nucleotide 320 to nucleotide 1681;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:13 from nucleotide 437 to nucleotide 1681;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone pe584_2 deposited under accession number ATCC 98636;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone pe584_2 deposited under accession number ATCC 98636;
(f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone pe584 2 deposited under accession number ATCC 98636;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone pe584 2 deposited under accession number ATCC 98636;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID N0:14;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:14 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID N0:14;
(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 2 0 (1) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a~(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:13 from nucleotide 320 to nucleotide 1681; the nucleotide sequence of SEQ
ID N0:13 from nucleotide 437 to nucleotide 1681; the nucleotide sequence of the full-length protein 2 5 coding sequence of clone pe584_2 deposited under accession number ATCC
98636; or the nucleotide sequence of a mature protein coding sequence of clone pe584_2 deposited under accession number ATCC 98636. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert of clone pe584 2 deposited under accession number ATCC 98636. In further preferred 3 0 embodiments, the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:14 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID N0:14, or a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:14 having biological activity, the fragment comprising the amino acid sequence from amino acid 222 to amino acid 231 of SEQ ID N0:14.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:13.
Further embodiments of the invention provide isolated polynucleotides produced according to a process selected from the group consisting of:
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(aa) SEQ ID N0:13, but excluding the poly(A) tail at the 3' end of SEQ ID N0:13; and (ab) the nucleotide sequence of the cDNA insert of clone pe584 2 deposited under accession number ATCC 98636;
(ii) hybridizing said probes) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C; and (iii) isolating the DNA polynucleotides detected with the probe(s);
and 2 0 (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(ba) SEQ ID N0:13, but excluding the poly(A) tail at the 2 5 3' end of SEQ ID N0:13; and (bb) the nucleotide sequence of the cDNA insert of clone pe584 2 deposited under accession number ATCC 98636;
(ii) hybridizing said primers) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C;
3 0 (iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii).
Preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:13, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ
ID N0:13 to a nucleotide sequence corresponding to the 3' end of SEQ ID N0:13 , but excluding the poly(A) tail at the 3' end of SEQ ID N0:13. Also preferably .the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:13 from nucleotide 320 to nucleotide 1681, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID N0:13 from nucleotide 320 to nucleotide 1681, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID N0:13 from nucleotide 320 to nucleotide 1681. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
N0:13 from nucleotide 437 to nucleotide 1681, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
N0:13 from nucleotide 437 to nucleotide 1681, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID N0:13 from nucleotide 437 to nucleotide 1681.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID N0:14;
(b) fragments of the amino acid sequence of SEQ ID N0:14, each fragment comprising eight consecutive amino acids of SEQ ID N0:14; and 2 0 (c) the amino acid sequence encoded by the cDNA insert of clone pe584 2 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID N0:14. In further preferred embodiments, the present invention provides a protein comprising a fragment of the 2 5 amino acid sequence of SEQ ID N0:14 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID N0:14, or a protein comprising a fragment of the amino acid sequence of SEQ ID N0:14 having biological activity, the fragment comprising the amino acid sequence from amino acid 222 to amino acid 231 of SEQ ID N0:14.
3 0 In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:15;
i~VO 99/37674 PCT/US99/01404 (b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:15 from nucleotide 78 to nucleotide 1502;
- (c} a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:15 from nucleotide 564 to nucleotide 1502;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone pj323_2 deposited under accession number ATCC 98636;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone pj323 2 deposited under accession number ATCC 98636;
(f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone pj323 2 deposited under accession number ATCC 98636;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone pj323_2 deposited under accession number ATCC 98636;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID N0:16;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:16 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID N0:16;
2 0 (j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and a polynucleotide that hybridizes under stringent conditions to any 2 5 one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:15 from nucleotide 78 to nucleotide 1502; the nucleotide sequence of SEQ ID
N0:15 from nucleotide 564 to nucleotide 1502; the nucleotide sequence of the full-length protein coding sequence of clone pj323 2 deposited under accession number ATCC 98636;
or the 3 0 nucleotide sequence of a mature protein coding sequence of clone pj323_2 deposited under accession number ATCC 98636. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert of clone pj323 2 deposited under accession number ATCC 98636. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID N0:16 from amino acid 54 to amino acid 145. In further preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID
N0:16 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID N0:16, or a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:16 having biological activity, the fragment comprising the amino acid sequence from amino acid 232 to amino acid 241 of SEQ ID N0:16.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:15.
Further embodiments of the invention provide isolated polynucleotides produced according to a process selected from the group consisting of:
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(aa) SEQ ID N0:15, but excluding the poly(A) tail at the 3' end of SEQ ID N0:15; and (ab} the nucleotide sequence of the cDNA insert of clone 2 0 pj323_2 deposited under accession number ATCC 98636;
(ii) hybridizing said probes) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C; and (iii) isolating the DNA polynucleotides detected with the probe(s);
2 5 and (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
3 0 (ba) SEQ ID NO:IS, but excluding the poly(A) tail at the 3' end of SEQ ID N0:15; and (bb) the nucleotide sequence of the cDNA insert of clone pj323_2 deposited under accession number ATCC 98636;
(ii) hybridizing said primers) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C;
(iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii).
Preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:15, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ
ID N0:15 to a nucleotide sequence corresponding to the 3' end of SEQ ID N0:15 , but excluding the poly(A) tail at the 3' end of SEQ ID N0:15. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:15 from nucleotide 78 to nucleotide 1502, and extending contiguously from a nucleotide sequence corresponding to the 5' ~d of said sequence of SEQ ID N0:15 from nucleotide 78 to nucleotide 1502, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ TD N0:15 from nucleotide 78 to nucleotide 1502. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
N0:15 from nucleotide 564 to nucleotide 1502, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
N0:15 from nucleotide 564 to nucleotide 1502, to a nucleotide sequence corresponding to the 3' end 2 0 of said sequence of SEQ ID N0:15 from nucleotide 564 to nucleotide 1502.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID N0:16;
2 5 (b) the amino acid sequence of SEQ ID N0:16 from amino acid 54 to amino acid 145;
(c) fragments of the amino acid sequence of SEQ ID N0:16, each fragment comprising eight consecutive amino acids of SEQ ID N0:16; and (d) the amino acid sequence encoded by the cDNA insert of clone 3 0 pj323 2 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID N0:16 or the amino acid sequence of SEQ ID N0:16 from amino acid 54 to amino acid 145. In further preferred embodiments, the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID N0:16 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID N0:16, or a protein comprising a fragment of the amino acid sequence of SEQ ID N0:16 having biological activity, the fragment comprising the amino acid sequence from amino acid 232 to amino acid 241 of SEQ ID N0:16.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:17;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:17 from nucleotide 130 to nucleotide 294;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:17 from nucleotide 241 to nucleotide 294;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone yb24_1 deposited under accession number ATCC 98636;
{e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone yb24_1 deposited under accession number ATCC 98636;
(f) a polynucleotide comprising the nucleotide sequence of a mature 2 0 protein coding sequence of clone yb24_1 deposited under accession number ATCC
98636;
{g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone yb24_1 deposited under accession number ATCC 98636;
(h) a polynucleotide encoding a protein comprising the amino acid 2 5 sequence of SEQ ID N0:18;
(i) a polynucleofide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:18 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID N0:18;
(j) a polynucleotide which is an allelic variant of a polynucleotide of 3 0 (a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and (1) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (ar(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:17 from nucleotide 130 to nucleotide 294; the nucleotide sequence of SEQ ID
N0:17 from nucleotide 241 to nucleotide 294; the nucleotide sequence of the full-length protein coding sequence of clone yb24_1 deposited under accession number ATCC 98636;
or the nucleotide sequence of a mature protein coding sequence of clone yb24_1 deposited under accession number ATCC 98636. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone yb24_1 deposited under accession number ATCC 98636. In further preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:18 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID N0:18, or a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:18 having biological activity, the fragment comprising the amino acid sequence from amino acid 22 to amino acid 31 of SEQ
ID N0:18.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:17.
Further embodiments of the invention provide isolated polynucleotides produced according to a process selected from the group consisting of:
2 0 {a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(aa) SEQ ID N0:17, but excluding the poly(A) tail at the 2 5 3' end of SEQ ID N0:17; and (ab) the nucleotide sequence of the cDNA insert of clone yb24_1 deposited under accession number ATCC 98636;
(ii) hybridizing said probes) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C; and 3 0 (iii) isolating the DNA polynucleotides detected with the probe(s);
and (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected_from the group consisting of:
(ba) SEQ ID N0:17, but excluding the poly(A) tail at the 3' end of SEQ ID N0:17; and (bb) the nucleotide sequence of the cDNA insert of clone yb24_1 deposited under accession number ATCC 98636;
(ii) hybridizing said primers) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C;
(iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii).
Preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:17, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ
ID N0:17 to a nucleotide sequence corresponding to the 3' end of SEQ ID N0:17 , but excluding the poly(A) tail at the 3' end of SEQ ID N0:17. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:17 from nucleotide 130 to nucleotide 294, and extending contiguously from a nucleotide sequence corresponding to the 5' end 2 0 of said sequence of SEQ ID N0:17 from nucleotide 130 to nucleotide 294, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID N0:17 from nucleotide 130 to nucleotide 294. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
N0:17 from nucleotide 241 to nucleotide 294, and extending contiguously from a 2 5 nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
N0:17 from nucleotide 241 to nucleotide 294, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID N0:17 from nucleotide 241 to nucleotide 294.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group 3 0 consisting of:
(a) the amino acid sequence of SEQ ID N0:18;
(b) fragments of the amino acid sequence of SEQ ID N0:18, each fragment comprising eight consecutive amino acids of SEQ ID N0:18; and (c) the amino acid sequence encoded by the cDNA insert of clone yb24_1 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID N0:18. In further preferred embodiments, the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID N0:18 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID N0:18, or a protein comprising a fragment of the amino acid sequence of SEQ ID N0:18 having biological activity, the fragment comprising the amino acid sequence from amino acid 22 to amino acid 31 of SEQ ID N0:18.
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
N0:19;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:19 from nucleotide 514 to nucleotide 1707;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:19 from nucleotide 580 to nucleotide 1707;
(d) a polynucleotide comprising the nucleotide sequence of the full-2 0 length protein coding sequence of clone yb44_1 deposited under accession number ATCC 98636;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone yb44_1 deposited under accession number ATCC 98636;
(f) a polynucleotide comprising the nucleotide sequence of a mature 2 5 protein coding sequence of clone yb44_1 deposited under accession number ATCC
98636;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone yb44_1 deposited under accession number ATCC 98636;
(h) a polynucleotide encoding a protein comprising the amino acid 3 0 sequence of SEQ ID N0:20;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:20 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID N0:20;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and (1) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:19 from nucleotide 514 to nucleotide 1707; the nucleotide sequence of SEQ
ID N0:19 from nucleotide 580 to nucleotide 1707; the nucleotide sequence of the full-length protein coding sequence of clone yb44_1 deposited under accession number ATCC 98636;
or the nucleotide sequence of a mature protein coding sequence of clone yb44_1 deposited under accession number ATCC 98636. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone yb44_1 deposited under accession number ATCC 98636. In further preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:20 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID N0:20, or a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:20 having biological activity, the 2 0 fragment comprising the amino acid sequence from amino acid 194 to amino acid 203 of SEQ ID N0:20.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:19.
Further embodiments of the invention provide isolated polynucleotides produced 2 5 according to a process selected from the group consisting of:
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
3 0 (aa) SEQ ID N0:19, but excluding the poly(A) tail at the 3' end of SEQ ID N0:19; and (ab) the nucleotide sequence of the cDNA insert of clone yb44_1 deposited under accession number ATCC 98636;
(ii) hybridizing said probes) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C; and (iii) isolating the DNA polynucleotides detected with the probe(s);
and (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(ba) SEQ ID N0:19, but excluding the poly(A) tail at the 3' end of SEQ ID N0:19; and (bb) the nucleotide sequence of the cDNA insert of clone yb44_1 deposited under accession number ATCC 98636;
(ii) hybridizing said primers) to human genomic DNA in conditions at least as stringent as 4X SSC at b5 degrees C;
(iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii.).
Preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:19, and 2 0 extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ
ID N0:19 to a nucleotide sequence corresponding to the 3' end of SEQ ID N0:19 , but excluding the poly(A) tail at the 3' end of SEQ ID N0:19. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:19 from nucleotide 514 to nucleotide 2 5 1707, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID N0:19 from nucleotide 514 to nucleotide 1707, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID N0:19 from nucleotide 514 to nucleotide 1707. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
3 0 N0:19 from nucleotide 580 to nucleotide 1707, and extending contiguously from a nucleotide sequence corresponding to the.5' end of said sequence of SEQ ID
N0:19 from nucleotide 580 to nucleotide 1707, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID N0:19 from nucleotide 580 to nucleotide 1707.
'WO 99/37674 PCTNS99/01404 In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID N0:20;
(b) fragments of the amino acid sequence of SEQ ID N0:20, each fragment comprising eight consecutive amino acids of SEQ ID N0:20; and (c) the amino acid sequence encoded by the cDNA insert of clone yb44_1 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID N0:20. In further preferred embodiments, the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID N0:20 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID N0:20, or a protein comprising a fragment of the amino acid sequence of SEQ ID N0:20 having biological activity, the fragment comprising the amino acid sequence from amino acid 194 to amino acid 203 of SEQ ID N0:20.
In certain preferred embodiments, the polynucleotide is operably linked to an expression control sequence. The invention also provides a host cell, including bacterial, yeast, insect and mammalian cells, transformed with such polynucleotide compositions.
2 0 Also provided by the present invention are organisms that have enhanced, reduced, or modified expression of the genes) corresponding to the polynucleotide sequences disclosed herein.
Processes are also provided for producing a protein, which comprise:
(a) growing a culture of the host cell transformed with such 2 5 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.
Protein compositions of the present invention may further comprise a 3 0 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 WO 99/37674 PC"T/US99/01404 effective amount of a composition comprising a protein of the present invention and a pharmaceutically acceptable carrier.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures lA and 1B are schematic representations of the pED6 and pNOTs vectors, respectively, used for deposit of clones disclosed herein.
DETAILED DESCRIPTION
ISOLATED PROTEINS AND POLYNUCLEOTIDES
Nucleotide and amino acid sequences, as presently determined, are reported below for each clone and protein disclosed in the present application. The nucleotide sequence of each clone can readily be determined by sequencing of the deposited clone in accordance with known methods. The predicted amino acid sequence (both full-length and mature forms) can then be determined from such nucleotide sequence. The amino acid sequence of the protein encoded by a particular clone can also be determined by expression of the clone in a suitable host cell, collecting the protein and determining its sequence. For each disclosed protein applicants have identified what they have determined to be the reading frame best identifiable with sequence information available at the time of filing.
2 0 As used herein a "seae~ed" protein is one which, when expressed in a suitable host cell, is transported across or through a membrane, including transport as a result of signal sequences in its amino acid sequence. "Secreted" proteins include without limitation proteins secreted wholly (e.g., soluble proteins) or partially (e.g. , receptors) from the cell in which they are expressed. "Secreted" proteins also include without limitation proteins 2 5 which are transported across the membrane of the endoplasmic reticulum.
Clone "cs756 2"
A polynucleotide of the present invention has been identified as clone "cs756_2".
cs756_2 was isolated from a human fetal 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. cs756_2 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "cs756 2 protein").
VIrO 99/37674 PCTNS99/01404 The nucleotide sequence of cs756_2 as presently determined is reported in SEQ
ID
NOa, and includes a poly(A) tail. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the cs756 2 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID N0:2.
Amino acids 211 to 223 of SEQ ID N0:2 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 224. Due to the hydrophobic nature of the predicted leader/signal sequence, it is likely to act as a transmembrane domain should the predicted leader/signal sequence not be separated from the remainder of the cs756 2 protein. The TopPredII computer program predicts a potential transmembrane domain within the cs756_2 protein sequence of SEQ ID
N0:2, centered around amino acid 15 of SEQ ID N0:2; amino acids 2 to 14 of SEQ ID
N0:2 are also a possible leader/signal sequence, with the predicted mature amino acid sequence in that case beginning at amino acid 15.
Another possible cs756 2 reading frame and predicted amino acid sequence, encoded by base pairs 385 to 825 of SEQ ID NO:1, is reported in SEQ ID N0:30;
the TopPredII computer program predicts a potential transmembrane domain centered around amino acid 100 of SEQ ID N0:30. The open reading frames corresponding to SEQ
ID N0:30 and SEQ ID N0:2 could be joined if a frameshift were introduced into the nucleotide sequence of SEQ ID N0:1.
2 0 The EcoRI/NotI restriction fragment obtainable from the deposit containing clone cs756_2 should be approximately 3000 bp.
The nucleotide sequence disclosed herein for cs756 2 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. cs756_2 demonstrated at least some similarity with sequences 2 5 identified as AA398077 (zt58c03.s1 Soares testis NHT Homo Sapiens cDNA
clone 726532 3'), AA541286 (nf97e03.s1 NCI_CGAP_Co3 Homo Sapiens cDNA clone 1MAGE:927868), W28620 (49c2 Human retina cDNA randomly primed sublibrary Homo Sapiens cDNA), and W47601 (zc35g08.r1 Soares senescent fibroblasts NbHSF Homo sapiens cDNA
clone 324350 5'). The predicted amino acid sequence disclosed herein for SEQ ID
N0:30 was 3 0 searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted SEQ ID N0:30 protein demonstrated at least some similarity to sequences identified as L76938 (Werner syndrome gene, complete cds [Homo sapiensJ). "Werner's syndrome (WS) is an inherited disease with clinical symptoms resembling premature aging ... [the] predicted protein is 1432 amino acids in WO 99/376'14 PCTNS99/01404 length and shows significant similarity to DNA helicases" (Yu et al., 1996, Science 272(5259):258-262, which is incorporated by reference herein). Based upon sequence similarity, cs756 2 proteins and each similar protein or peptide may share at least some activity. The nucleotide sequence of cs756 2 indicates that it may contain one or more of the following repetitive elements: MIR, MER.
Clone "ew150 1"
A polynucleotide of the present invention has been identified as clone "ew150_1".
ew150_1 was isolated from a human adult placenta cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No.
5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. evV150_1 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "ew150_1 protein').
The nucleotide sequence of ew150_1 as presently determined is reported in SEQ
ID N0:3, and includes a poly(A) tail. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the ew150_1 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID N0:4.
Amino acids 26 to 38 of SEQ ID N0:4 are a predicted leader/signal sequence, with the predicted 2 0 mature amino acid sequence beginning at amino acid 39. Due to the hydrophobic nature of the predicted leader/signal sequence, it is likely to act as a transmembrane domain should the predicted leader/signal sequence not be separated from the remainder of the ew150_1 protein.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone 2 5 ew150_1 should be approximately 2000 bp.
The nucleotide sequence disclosed herein for ew150_1 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. ew150_1 demonstrated at least some similarity with sequences identified as AA563938 (nk23b12.s1 NCI_CGAP Col1 Homo sapiens cDNA clone IMAGE
3 0 1014335), D63209 (Human placenta cDNA 5'-end GEN-506F01), M90423 (Bacteriphage US3 lytic-enzyme), W23461 (zb33cOl.r1 Soares parathyroid tumor NbHPA Homo sapiens cDNA clone 305376 5'), and 256916 (H.sapiens CpG DNA, clone 153b7, forward read cpg153b7.ftla). In the region around position 1514 of SEQ ID N0:3, ew150_1 also demonstrated at least some similarity with sequences encoding a mitochondria) energy-transfer proteins signature motif which is found in mitochondrial and other membrane proteins. Based upon sequence similarity, ew150_1 proteins and each similar protein or peptide may share at least some activity. The TopPredII computer program predicts ten potential transmembrane domains within the ew150_1 protein sequence, which are centered around amino acids 70,106,133, 200, 314, 349, 387, 457, 504, and 527 of SEQ ID
N0:4, respectively.
lone "g~894 1~"
A polynucleotide of the present invention has been identified as clone "gg894_13".
gg894_13 was isolated from a human fetal kidney cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. gg894_13 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "gg894_13 protein").
The nucleotide sequence of gg894_13 as presently determined is reported in SEQ
ID N0:5, and includes a poly(A) tail. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the gg894_13 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID N0:6.
Amino 2 0 acids 41 to 53 of SEQ ID N0:6 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 54. Due to the hydrophobic nature of the predicted leader/signal sequence, it is likely to act as a transmembrane domain should the predicted leader/signal sequence not be separated from the remainder of the gg894_13 protein. Another possible gg894_13 reading frame and predicted amino acid 2 5 sequence, encoded by base pairs 602 to 1129 of SEQ ID N0:5, is reported in SEQ ID
N0:31. The open reading frames corresponding to SEQ ID N0:31 and SEQ ID N0:6 could be joined if a frameshift were introduced into the nucleotide sequence of SEQ
ID N0:5.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone gg894_13 should be approximately 2400 bp.
3 0 The nucleotide sequence disclosed herein for gg894_13 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. gg894_13 demonstrated at least some similarity with sequences identified as H57424 (yr13a10.s1 Homo sapiens cDNA clone 205146 3'), T23885 (Human gene signature HUMGS05820), and W80358 (zh49a07.s1 Soares fetal liver spleen WO 99!37674 PCT/US99/01404 Sl Homo sapiens cDNA clone 415380 3'). Based upon sequence similarity, gg894_13 proteins and each similar protein or peptide may share at least some activity.
The TopPredII computer program predicts a potential transmembrane domain within the gg894_13 protein sequence centered around amino acid 115 of SEQ ID N0:6. The nucleotide sequence of gg894_13 indicates that it may contain a RBMI
repetitive element.
Clone "it217 2"
A polynucleotide of the present invention has been identified as clone "it217_2".
it217 2 was isolated from a human adult brain (thalamus) 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. it217 2 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "it217_2 protein").
The nucleotide sequence of it217 2 as presently determined is reported in SEQ
ID
N0:7, and includes a poly(A) tail. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the it217_2 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID N0:8.
Another possible it217 2 reading frame and predicted amino acid sequence, encoded by base pairs 2 0 45 to 311 of SEQ ID N0:7, is reported in SEQ ID N0:32. Amino acids 36 to 48 of SEQ ID
N0:32 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 49. Due to the hydrophobic nature of the predicted leader/signal sequence, it is likely to act as a transmembrane domain should the predicted leader/signal sequence not be separated from the remainder of the it217_2 protein. The 2 5 open reading frames corresponding to SEQ ID N0:32 and SEQ ID N0:8 could be joined if at least one frameshift were introduced into the nucleotide sequence of SEQ
ID N0:7.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone it217_2 should be approximately 2250 bp.
The nucleotide sequence disclosed herein for it217 2 was searched against the 3 0 GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. it217_2 demonstrated at least some similarity with sequences identified as AA242969 (zr65h09.r1 Soares NhHMPu S1 Homo sapiens cDNA clone 668321 5' similar to SW SCC2_HUMAN P48594 SQUAMOUS CELL CARCINOMA
ANTIGEN 2 ;contains Alu repetitive element), B44876 (HS-1060-Al-G06-MR.abi CTT
i~NO 99/37674 PGT/US99/01404 Human Genomic Sperm Library C Homo sapien genomic clone Plate CT 782 Col 11 Row M), H82168 (yv78d08.r1 Homo sapiens cDNA clone), 566896 (squamous cell carcinoma antigen), U19556 (Human squamous cell carcinoma antigen 1 (SCCAl) mRNA, complete cds), U19557 (Human squamous cell carcinoma antigen 2 (SCCA2) mRNA, complete cds), S and U35459 (Human bomapin mRNA, complete cds). The predicted amino acid sequence disclosed herein for it217_2 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted it217_2 protein demonstrated at least some similarity to sequences identified as L40377 (cytoplasmic antiproteinase 2 [Homo sapiens]), M34352 (ovalbumin (callus gallus]), M91161 (serpin [Equus caballus]), 825276 (SCC antigen), 848379 (Human megakaryocyte differentiation factor), 566896 (squamous cell carcinoma antigen, SCC antigen serine protease inhibitor [human, Peptide, 390 aa] [Homo sapiens]), U19568 (squamous cell carcinoma antigen [Homo sapiens]), and U19576 (squamous cell carcinoma antigen [Homo sapiens]).
Human bomapin may play a role in the regulation of protease activities during hematopoiesis (Riewald et al.,1995, J. Biol. Chem. 270: 26754, which is incorporated by reference herein). Serpins are SERine Proteinase IlVhibitors and are considered extracellular in localization. Human squamous cell carcinoma antigen (SSCA) is a member of the serpin family of proteinase inhibitors, purified from sera of cancer patients.
Based upon sequence similarity, it217 2 proteins and each similar protein or peptide may 2 0 share at least some activity.
Clong "m1235 2"
A polynucleotide of the present invention has been identified as clone "m1235 2".
m1235_2 was isolated from a human adult brain (caudate nucleus) cDNA library using 2 5 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. m1235 2 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "m1235 2 protein').
3 0 The nucleotide sequence of m1235 2 as presently determined is reported in SEQ
ID N0:9, and includes a poly(A) tail. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the m1235 2 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID
N0:10. Amino acids 3 to 15 of SEQ ID NO:10 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 16. Due to the hydrophobic nature of the predicted leader/signal sequence, it is likely to act as a transmembrane domain should the predicted leader/signal sequence not be separated from the remainder of the m1235 2 protein.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone m1235 2 should be approximately 1400 bp.
The nucleotide sequence disclosed herein for m1235 2 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. m1235_2 demonstrated at least some similarity with sequences identified as AA160887 (zo79b05.s1 Stratagene pancreas (#937208} Homo sapiens cDNA
clone 593073 3'), 814349 (yf79f12.r1 Homo sapiens cDNA clone 28451 5'), and (yg74f07.r1 Homo sapiens cDNA clone 39059 5'). Based upon sequence similarity, m1235 2 proteins and each similar protein or peptide may share at least some activity.
The TopPredII computer program predicts a potential transmembrane domain within the m1235_2 protein sequence centered around amino acid 25 of SEQ ID N0:10.
Clone "mt24 2"
A polynudeotide of the present invention has been identified as clone "mt24 2".
mt24_2 was isolated from a human adult testes cDNA library using methods which are 2 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. mt24 2 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "mt24 2 protein').
2 5 The nucleotide sequence of mt24 2 as presently determined is reported in SEQ ID
N0:11, and includes a poly(A) tail. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the mt24 2 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID
N0:12. Amino acids 30 to 42 of SEQ ID N0:12 are a predicted leader/signal sequence, with the predicted 3 0 mature amino acid sequence beginning at amino acid 43. Due to the hydrophobic nature of the predicted leader/signal sequence, it is likely to act as a transmembrane domain should the predicted leader/signal sequence not be separated from the remainder of the mt24 2 protein.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone mt24 2 should be approximately 1400 bp.
The nucleotide sequence disclosed herein for mt24_2 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. mt24 2 demonstrated at least some similarity with sequences identified as AA062589 (zf68f04.r1 Soares pineal gland N3HPG Homo sapiens cDNA
clone 3821115') and T19332 (b08016t Testis 1 Homo sapiens cDNA clone b08016 5' end).
Based upon sequence similarity, mt24 2 proteins and each similar protein or peptide may share at least some activity. The TopPredII computer program predicts four potential transmembrane domains within the mt24_2 protein sequence centered around amino acids 38,153,167, and 232 of SEQ ID N0:12, respectively.
Clone"pe584 2"
A polynucleotide of the present invention has been identified as clone "pe584_2".
pe584_2 was isolated from a human adult blood (chronic myelogenous leukemia K5) 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. pe584 2 is a full-length clone, including the entire coding sequence of a secreted 2 0 protein (also referred to herein as "pe584 2 protein").
The nucleotide sequence of pe584 2 as presently determined is reported in SEQ
ID N0:13, and includes a poly(A) tail. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the pe584_2 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID
N0:14. Amino 2 5 acids 27 to 39 of SEQ ID N0:14 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 40. Due to the hydrophobic nature of the predicted leader/signal sequence, it is likely to act as a transmembrane domain should the predicted leader/signal sequence not be separated from the remainder of the pe584_2 protein.
3 0 The EcoRI/NotI restriction fragment obtainable from the deposit containing clone pe584 2 should be approximately 3000 bp.
The nucleotide sequence disclosed herein for pe584_2 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. pe584 2 demonstrated at least some similarity with sequences identified as AA303149 (EST13039 Uterus tumor I), AA405004 (zt06e03.s1 NCI_CGAP_GCBl Homo Sapiens cDNA clone IMAGE 712348 3'), AA481230 (aa34gOl.r1 NCI_CGAP GCB1 Homo sapiens cDNA clone 815184 5' similar to SW TCR2_ECOLI
P02981 TETRACYCLINE RESISTANCE PROTEIN), D88315 (Mouse mRNA for tetracycline transporter-like protein, complete cds), and T10077 (seq1295 Homo sapiens cDNA clone b4HB3MA-COT8-HAP-Ft109 5'}. The predicted amino acid sequence disclosed herein for pe584 2 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted pe584_2 protein demonstrated at least some similarity to sequences identified as D88315 (tetracycline transporter-like protein [Mus musculus]). Mouse tetracycline transporter-like protein is a sugar transporter (Matsuo et al.,1997, Biochem. Biophys. Res. Comm. 238: 126-192, which is incorporated by reference herein). Based upon sequence similarity, pe584 2 proteins and each similar protein or peptide may share at least some activity. The TopPredII
computer program predicts eleven potential transmembrane domains within the pe584 2 protein sequence, which are centered around amino acids 32, 55, 78, 114,142, 196, 235, 264, 287, 332, and 375 of SEQ ID N0:14, respectively.
Clony '~i3,~_' A polynucleotide of the present invention has been identified as clone "pj323 2".
2 0 pj323 2 was isolated from a human fetal carcinoma (NTD2 cells treated with retinoic acid for 23 days) 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. pj323 2 is a full-length clone, including the entire coding sequence 2 5 of a secreted protein (also referred to herein as "pj323 2 protein").
The nucleotide sequence of pj323_2 as presently determined is reported in SEQ
ID
N0:15, and includes a poly(A) tail. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the pj323 2 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID
N0:16. Amino 3 0 acids 150 to 162 of SEQ ID N0:16 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 163. Due to the hydrophobic nature of the predicted leader/signal sequence, it is likely to act as a transmembrane domain should the predicted leader/signal sequence not be separated from the remainder of the pj323_2 protein.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone pj323_2 should be approximately 2500 bp.
The nucleotide sequence disclosed herein for pj323_2 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. pj323 2 demonstrated at least some similarity with sequences identified as AA160454 (zo74g05.r1 Stratagene pancreas (#937208) Homo sapiens cDNA
clone 592664 5'), AA398257 (zt60a08.s1 Scares testis NHT Homo Sapiens cDNA
clone 726710 3'), and T47284 (yb64g11.s1 Homo Sapiens cDNA clone 76004 3'). The predicted amino acid sequence disclosed herein for pj323 2 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted pj323 2 protein demonstrated at least some similarity to human integral nuclear envelope protein, lamin B receptors from several species, and sterol reductases from several species. Lamin B receptors have hydrophobic carboxy terminal portions and hydrophilic amino terminal portions. Antibodies to lamin B receptors have been found in patients with primary biliary cirrhosis. Sterol reductases demonstrate sequence similarity to the hydrophobic portions of lamin B receptors. Based upon sequence similarity, pj323_2 proteins and each similar protein or peptide may share at least some activity. The TopPredII computer program predicts six potential transmembrane domains within the pj323 2 protein sequence, which are centered around amino acids 47,106,164, 2 0 187, 341, and 432 of SEQ ID N0:16, respectively.
pj323 2 protein was expressed in a COS cell expression system, and an expressed protein band of approximately 46 kDa was detected in membrane fractions using SDS
polyacrylamide gel electrophoresis.
2 S Clone "vb24 1"
A polynucleotide of the present invention has been identified as clone "yb24_1".
yb24_1 was isolated from a human fetal brain cDNA library and was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. yb24_1 is a full-length clone, including the 3 0 entire coding sequence of a secreted protein (also referred to herein as "yb24_1 protein").
The nucleotide sequence of yb24_1 as presently determined is reported in SEQ
ID
N0:17, and includes a poly(A) tail. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the yb24_1 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID
N0:18. Amino acids 25 to 37 of SEQ ID N0:18 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 38. Due to the hydrophobic nature of the predicted leader/signal sequence, it is likely to act as a transmembrane domain should the predicted leader/signal sequence not be separated from the remainder of the yb24_1 protein.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone yb24_1 should be approximately 1700 bp.
The nucleotide sequence disclosed herein for yb24_1 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. yb24_1 demonstrated at least some similarity with sequences identified as AA149807 (z147c09.s1 Soares pregnant uterus NbHPU Homo sapiens cDNA
clone 505072 3') and AB003515 (Rat mRNA for GEF-2, complete cds). Based upon sequence similarity, yb24_1 proteins and each similar protein or peptide may share at least some activity.
A polynucleotide of the present invention has been identified as clone "yb44_1".
yb44_1 was isolated from a human fetal brain cDNA library and was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the 2 0 amino acid sequence of the encoded protein. yb44_1 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "yb44_1 protein").
The nucleotide sequence of yb44_1 as presently determined is reported in SEQ
ID
N0:19, and includes a poly(A) tail. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the yb44_1 protein 2 5 corresponding to the foregoing nucleotide sequence is reported in SEQ ID
N0:20. Amino acids 10 to 22 of SEQ ID N0:20 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 23. Due to the hydrophobic nature of the predicted leader/signal sequence, it is likely to act as a transmembrane domain should the predicted leader/signal sequence not be separated from the remainder of the 3 0 yb44_1 protein.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone yb44_1 should be approximately 2000 bp.
The nucleotide sequence disclosed herein for yb44_1 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. yb44_1 demonstrated at least some similarity with sequences identified as AC000016 {*** SEQUENCING IN PROGRESS *** EPMl /APECED region of chromosome 21, BAC clone B4P3; HTGS phase 1,10 unordered pieces). The predicted amino acid sequence disclosed herein for yb44_1 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted yb44_1 protein demonstrated at least some similarity to sequences identified as 872377 (Human auxiliary cytochrome P450 species 2D6 variant 2 protein) and (cytochrome P450 [Drosophila melanogaster]). Based upon sequence similarity, yb44_1 proteins and each similar protein or peptide may share at least some activity.
The TopPredII computer program predicts three additional potential transmembrane domains within the yb44_1 protein sequence, which are centered around amino acids 82,128, and 361 of SEQ ID N0:20, respectively. The nucleotide sequence of yb44_1 indicates that it may contain one or more of the following repetitive elements: Alu, AT, TATACA, MER44A, TACA.
Den osit of ~,lones Clones cs756_2, ew150_l, gg894_13, it217_2, m1235_2, mt24_2, pe584 2, pj323 2, yb24_l, and yb44_1 were deposited on January 22,1998 with the American Type Culture Collection (10801 University Boulevard, Manassas, Virginia 20110-2209 U.S.A.) as an 2 0 original deposit under the Budapest Treaty and were given the accession number ATCC
98636, 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), and the term of the deposit will comply with 37 C.F.R. ~
1.806.
2 5 Each clone has been transfected into separate bacterial cells (E. coh~ 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 pEDb or pNOTs vector depicted in Figures lA and 1B, respectively. The pED6dpc2 vector 3 0 ("pED6") was derived from pED6dpc1 by insertion of a new polylinker to facilitate cDNA cloning (Kaufman et al.,1991, Nucleic Acids Res.19: 4485-4490); the pNOTs vector was derived from pMT2 (Kaufman et al.,1989, Mol. Cell. Biol. 9: 946-958) by deletion of the DHFR sequences, insertion of a new polylinker, and insertion of the M13 origin of replication in the CIaI site. In some instances, the deposited clone can become "flipped"
'WO 99/37674 PCT/US99/01404 (i.e., in the reverse orientation) in the deposited isolate. In such instances, the cDNA insert can still be isolated by digestion with EcoRI and NotI. However, NotI will then produce the 5' site and EcoRI will produce the 3' site for placement of the cDNA in proper orientation for expression in a suitable vector. The cDNA may also be expressed from the vectors in which they were deposited.
Bacterial cells containing a particular clone can be obtained from the composite deposit as follows:
An oligonucleotide probe or probes should be designed to the sequence that is known for that particular clone. This sequence can be derived from the sequences provided herein, or from a combination of those sequences. The sequence of an oligonudeotide probe that was used to isolate or to sequence each full-length clone is identified below, and should be most reliable in isolating the clone of interest.
Clone Probe Sequence cs756_2 SEQ ID N0:21 ew150_1 SEQ ID N0:22 gg894_13 SEQ ID N0:23 it217_2 SEQ ID N0:24 m1235 2 SEQ ID N0:25 2 0 mt24_2 SEQ ID N0:26 pe584_2 ~ SEQ ID N0:27 pj323_2 SEQ ID N0:28 yb24_1 SEQ ID N0:29 2 5 In the sequences listed above which include an N at position 2, that position is occupied in preferred probes/primers by a biotinylated phosphoaramidite residue rather than a nucleotide (such as, for example, that produced by use of biotin phosphoramidite (1-dimethoxytrityloxy-2-(N-biotinyl-4-aminobutyl)-propyl-3-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramadite) (Glen Research, cat. no.10-1953)).
3 0 The design of the oligonucleotide probe should preferably follow these parameters:
(a) It should be designed to an area of the sequence which has the fewest ambiguous bases ("N's"), if any;
(b) It should be designed to have a Tm of approx. 80 ° C (assuming 2° for each A or T and 4 degrees for each G or C).
The oligonucleotide should preferably be labeled with y 32P ATP (specific activity 6000 Ci/mmole) and T4 polynucleotide kinase using commonly employed techniques for labeling oligonucleotides. Other labeling techniques can also be used.
Unincorporated label should preferably be removed by gel filtration chromatography or other established methods. The amount of radioactivity incorporated into the probe should be quantitated by measurement in a scintillation counter. Preferably, specific activity of the resulting probe should be approximately 4e+6 dpm/pmole.
The bacterial culture containing the pool of full-length clones should preferably be thawed and 100 Ixl 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 ug/ml and agar at 1.5% in a 150 mm petri dish when grown overnight at 37°C. Other known methods of obtaining distinct, well-separated colonies can also be employed.
Standard colony hybridization procedures should then be used to transfer the 2 0 colonies to nitrocellulose filters and lyre, 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 ~g/ml of yeast RNA, and 10 mM EDTA
(approximately 10 mL per 150 mm filter). Preferably, the probe is then added to the hybridization mix at a concentration greater than or equal to 1e+6 dpm/mL. The filter is then preferably incubated at 65°C with gentle agitation overnight. The filter is then preferably washed in 500 mL of 2X SSC/0.5% SDS at room temperature without agitation, preferably followed by 500 mL of 2X SSC/0.1% SDS at room temperature with gentle shaking for 15 minutes.
A third wash with O.1X SSC/0.5% SDS at 65°C for 30 minutes to 1 hour is optional. The 3 0 filter is then preferably dried and subjected to autoradiography for sufficient time to visualize the positives on the X-ray film. Other known hybridization methods can also be employed.
The positive colonies are picked, grown in culture, and plasmid DNA isolated using standard procedures. The clones can then be verified by restriction analysis, hybridization analysis, or DNA sequencing.
Fragments of the proteins of the present invention which are capable of exhibiting biological activity are also encompassed by the present invention. Fragments of the protein may be in linear form or they may be cyclized using known methods, for example, as described in H.U. Saragovi, et al., Bio/Technology 1~0 773-778 (1992) and in R.S.
McDowell, et al., J. Amer. Chem. Soc. ~~, 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 forms) of such protein may be obtained by expression of the disclosed full-length 2 0 polynucleotide (preferably those deposited with ATCC) in a suitable mammalian cell or other host cell. The sequences) of the mature forms) of the protein may also be determinable from the amino acid sequence of the full-length form.
The present invention also provides genes corresponding to the polynucleotide sequences disclosed herein. "Corresponding genes" are the regions of the genome that 2 5 are transcribed to produce the mRNAs from which cDNA polynucleotide sequences are derived and may include contiguous regions of the genome necessary for the regulated expression of such genes. Corresponding genes may therefore include but are not limited to coding sequences, 5' and 3' untranslated regions, alternatively spliced exons, introns, promoters, enhancers, and silencer or suppressor elements. The corresponding genes can 3 0 be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include the preparation of probes or primers from the disclosed sequence information for identification and/or amplification of genes in appropriate genomic libraries or other sources of genomic materials. An "isolated gene" is a gene that has been separated from the adjacent coding sequences, if any, present in the genome of the organism from which the gene was isolated.
The chromosomal location corresponding to the polynucleotide sequences disclosed herein may also be determined, for example by hybridizing appropriately labeled polynucleotides of the present invention to chromosomes in situ. It may also be possible to determine the corresponding chromosomal location for a disclosed polynucleotide by identifying significantly similar nucleotide sequences in public databases, such as expressed sequence tags (ESTs), that have already been mapped to particular chromosomal locations. For at least some of the polynucleotide sequences disclosed herein, public database sequences having at least some similarity to the polynucleotide of the present invention have been listed by database accession number.
Searches using the GenBank accession numbers of these public database sequences can then be performed at an Internet site provided by the National Center for Biotechnology Information having the address http://www.ncbi.nlm.nih.gov/UniGene/, in order to identify "UniGene clusters" of overlapping sequences. Many of the "UniGene clusters"
so identified will already have been mapped to particular chromosomal sites.
Organisms that have enhanced, reduced, or modified expression of the genes) corresponding to the polynucleotide sequences disclosed herein are provided.
The desired change in gene expression can be achieved through the use of aniisense 2 0 polynucleotides or ribozymes that bind and/or cleave the mRNA transcribed from the gene (Albert and Morris,1994, Trends Phurmacol. Sci.15(~: 250-254; Lavarosky et al.,1997, Biochem. Mol. Med. 62(1):11-22; and Hampel,1998, Prog. Nucleic Acid Res. Mol.
Biol. 58:1-39; all of which are incorporated by reference herein). Transgenic animals that have multiple copies of the genes) corresponding to the polynucleotide sequences disclosed 2 5 herein, preferably produced by transformation of cells with genetic constructs that are stably maintained within the transformed cells and their progeny, are provided.
Transgenic animals that have modified genetic control regions that increase or reduce gene expression levels, or that change temporal or spatial patterns of gene expression, are also provided (see European Patent No. 0 649 464 B1, incorporated by reference herein).
3 0 In addition, organisms are provided in which the genes) corresponding to the polynucleotide sequences disclosed herein have been partially or completely inactivated, through insertion of extraneous sequences into the corresponding genes) or through deletion of all or part of the corresponding gene(s). Partial or complete gene inactivation can be accomplished through insertion, preferably followed by imprecise excision, of transposable elements (Plasterk,1992, Bioessays 14(9): 629-633; Zwaal et al.,1993, Proc. Natl.
Acad. Sci. LISA 90(16): 7431-7435; Clark et al.,1994, Proc. Natl. Acad. Sci.
LISA 91(2): 719-722;
all of which are incorporated by reference herein), or through homologous recombination, preferably detected by positive/negative genetic selection strategies (Mansour et al.,1988, Nature 336: 348-352; U.S. Patent Nos. 5,464,764; 5,487,992; 5,627,059;
5,631,153; 5,614, 396;
5,616,491; and 5,679,523; all of which are incorporated by reference herein).
These organisms with altered gene expression are preferably eukaryotes and more preferably are mammals. Such organisms are useful for the development of non-human models for the study of disorders involving the corresponding gene(s), and for the development of assay systems for the identification of molecules that interact with the protein products) of the corresponding gene(s).
Where the protein of the present inv~tion is membrane-bound (e.g., is a receptor), the present invention also provides for soluble forms of such protein. In such forms, part or all of the intracellular and transmembrane domains of the protein are deleted such that the protein is fully secreted from the cell in which it is expressed. The intracellular and transmembrane domains of proteins of the invention can be identified in accordance with known techniques for determination of such domains from sequence information.
For example, the TopPredII computer program can be used to predict the location of transmembrane domains in an amino acid sequence, domains which are described by the 2 0 location of the center of the transmsmbrane domain, with at least ten transmembrane amino acids on each side of the reported central residue(s).
Proteins and protein fragments of the present invention include proteins with amino acid sequence lengths that are at least 25%(more preferably at least 50%, and most preferably at least 75%) of the length of a disclosed protein and have at least 60% sequence 2 5 identity (more preferably, at least 75% identity; most preferably at least 90% or 95%
identity) with that disclosed protein, where sequence identity is determined by comparing the amino acid sequences of the proteins when aligned so as to maximize overlap and identity while mi~aimizing sequence gaps. Also included in the present invention are proteins and protein fragments that contain a segment preferably comprising 8 or more 3 0 (more preferably 20 or more, most preferably 30 or more) contiguous amino acids that shares at least 75% sequence identity (more preferably, at least 85% identity;
most preferably at least 95% identity) with any such segment of any of the disclosed proteins.
In particular, sequence identity may be determined using WU-BLAST
(Washington University BLAST) version 2.0 software, which builds upon WU-BLAST
version 1.4, which in turn is based on the public domain NCBI-BLAST version 1.4 (Altschul and Gish, 1996, Local alignment statistics, Doolittle ed., Methods in Enzymology 266: 460-480; Altschul et al., 1990, Basic local alignment search tool, Journal of Molecular Biology 2I5: 403-410; Gish and States, 1993, Identification of protein coding regions by database similarity search, Nature Genetics 3: 266-272; Karlin and Altschul, 1993, Applications and statistics for multiple high-scoring segments in molecular sequences, Proc. Natl. Acad. Sci. USA 90: 5873-5877; all of which are incorporated by reference herein). WU-BLAST version 2.0 executable programs for several UNIX
platforms can be downloaded from ftp://blast.wustl.edu/blast/executables. The complete suite of search programs (BLASTP, BLASTN, BLASTX, TBLASTN, and TBLASTX) is provided at that site, in addition to several support programs. WU-BLAST 2.0 is copyrighted and may not be sold or redistributed in any form or manner without the express written consent of the author; but the posted executables may otherwise be freely used for commercial, nonprofit, or academic purposes. In all search programs in the suite -- BLASTP, BLASTN, BLASTX, TBLASTN and TBLASTX -- the gapped alignment routines are integral to the database search itself, and thus yield much better sensitivity and selectivity while producing the more easily interpreted output. Gapping can optionally be fumed off in all of these programs, if desired. The default penalty (Q) for a gap of length one is Q=9 for proteins and BLASTP, and Q=10 for BLASTN, but may be changed to any 2 0 integer value including zero, one through eight, nine, ten, eleven, twelve through twenty, twenty-one through fifty, fifty-one through one hundred, etc. The default per-residue penalty for extending a gap (R) is R=2 for proteins and BLASTP, and R=10 for BLASTN, but may be changed to any integer value including zero, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve through twenty, twenty-one through fifty, fifty-one 2 5 through one hundred, etc. Any combination of values for Q and R can be used in order to align sequences so as to maximize overlap and identity while minimizing sequence gaps.
The default amino acid comparison matrix is BLOSUM62, but other amino acid comparison matrices such as PAM can be utilized.
Species homologues of the disclosed polynucleotides and proteins are also 3 0 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. Preferably, polynucleotide species homologues have at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90%
identity) with the given polynudeotide, and protein species homologues have at least 30%
sequence identity (more preferably, at least 45% identity; most preferably at least 60%
identity) with the given protein, where sequence identity is determined by comparing the nucleotide sequences of the polynucleotides or the amino acid sequences of the proteins when aligned so as to maximize overlap and identity while minimizing sequence gaps.
Species homologues may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from the desired species. Preferably, species homologues are those isolated from mammalian species. Most preferably, species homologues are those isolated from certain mammalian species such as, for example, Pan troglodytes, Gorilla gorilla, Pongo pygmaeus, Hylobates concolor, Macaca mulatta, Papio papio, Papio hamadryas, Cercopithecus aethiops, Cebus capucinus, Aotus trivirgatus, Sanguinus Oedipus, Microcebus murinus, Mus musculus, Rattus norvegicus, CricetuIus griseus, Fells catus, Mustela visor, Canis familiaris, O~yctolagus cuniculus, Bos taurus, Ovis aries, Sus scrota, and Equus caballus, for which genetic maps have been created allowing the identification of syntenic relationships between the genomic organization of genes in one species and the genomic organization of the related genes in another species (O'Brien and Seuanez, 1988, Ann. Rev. Genet. 22: 323-351; OBrien et al., 1993, Nature 2 0 Genetics 3:103-112; Johansson et al.,1995, Genomics 25: 682-690; Lyons et al.,1997, Nature Genetics 15: 47-56; O'Brien et al.,1997, Trends in Genetics 13(10): 393-399;
Carver and Stubbs, 1997, Genome Research 7:1123-1137; all of which are incorporated by reference herein).
The invention also encompasses allelic variants of the disclosed polynucleotides or proteins; that is, naturally-occurring alternative forms of the isolated polynucleotides 2 5 which also encode proteins which are identical or have significantly similar sequences to those encoded by the disclosed polynucleotides. Preferably, allelic variants have at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90%
identity} with the given polynudeotide, where sequence identity is determined by comparing the nucleotide sequences of the polynucleotides when aligned so as to maximize 3 0 overlap and identity while minimizing sequence gaps. Allelic variants may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from individuals of the appropriate species.
VlrO 99/37674 PCTNS99/01404 The invention also includes polynucleotides with sequences complementary to those of the polynucleotides disclosed herein.
The present invention also includes polynucleotides that hybridize under reduced stringency conditions, more preferably stringent conditions, and most preferably highly stringent conditions, to polynucleotides described herein. Examples of stringency conditions are shown in the table below: highiy stringent conditions are those that are at least as stringent as, for example, conditions A-F; stringent conditions are at least as stringent as, for example, conditions G-L; and reduced stringency conditions are at least as stringent as, for example, conditions M-R.
StringencyPolynucleotideHybridHybridization TemperatureWash ConditionHybrid Lengthand Temperature (bp)= Buffer' and Buffers _ A DNA:DNA z 50 65C; lxSSC -or- 65C; 0.3xSSC
42C; lxSSC, 50% formamide B DNA:DNA <50 TB*; lxSSC Te*; 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, SO% formamide F RNA:RNA <50 TF*; lxSSC TF*; ixSSC
G DNA:DNA 2 SO 65C; 4xSSC -or- 65C; lxSSC
42C; 4xSSC, 50% formamide 1 H DNA:DNA <50 T"*; 4xSSC TH'; 4xSSC
O
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 2 50 70C; 4xSSC -or- 67C; lxSSC
50C; 4xSSC, 50% formamide L RNA:RNA <50 T~'; 2xSSC T~'; 2xSSC
M DNA:DNA z 50 50C; 4xSSC -or- 50C; 2xSSC
40C; 6xSSC, 50% formamide N DNA:DNA <50 TN*; 6xSSC TN*; 6xSSC
O DNA:RNA a 50 55C; 4xSSC -or- 55C; 2xSSC
42C; 6xSSC, 509 formamide P DNA:RNA <50 TP*; 6xSSC TP'; 6xSSC
Q RNA:RNA a 50 60C; 4xSSC -or- 60C; 2xSSC
45C; 6xSSC, 50~ formamide 2 R RNA:RNA <50 TR'; 4xSSC TR*; 4xSSC
~: The hybrid length is that anticipated for the hybridized regions) of the hybridizing polynudeotides. When hybridizing a polynucleotide to a target polynucleotide of unknown sequence, the hybrid length is assumed to be that of the hybridizing polynucleotide. When polynucleotides of known sequence are hybridized, the 2 5 hybrid length can be determined by aligning the sequences of the polynudeotides and identifying the region or regions of optimai sequence complementarily.
t: SSPE (lxSSPE is 0.15M NaCI, IOmM NaH2P0" and 1.25mM EDTA, pH 7.4) can be substituted for SSC
(lxSSC is O.15M NaCI and lSmM sodium citrate) in the hybridization and wash buffers; washes are performed for 15 minutes after hybridization is complete.
3 0 "Ts - 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 aaording 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'j) + 0.41(~°G+C) (600/N), where N is the number of bases in the hybrid, and [Na'j is the concentration of sodium ions in the 3 5 hybridization buffer ([Na') for lxSSC = 0.165 M).
Additional examples of stringency conditions for polynucleotide hybridization are provided in Sambrook, J., E.F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, chapters 9 and 11, and Current Protocols in Molecular Biology,1995, F.M.
Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, 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 ICaufman et al., Nucleic Acids Res. 12, 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.
ICaufman, Methods in Enzymology ~$~, 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 i~ 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 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 Ae~ricultural Experiment Station Bulletin No. 1555 (198, incorporated herein by reference. As used herein, an insect cell capable of expressing a polynucleotide of the present invention is "transformed."
The protein of the invention may be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant protein. The resulting expressed protein may then be purified from such culture (i.e., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography. The purification of the protein may also include an affinity column 2 0 containing agents which will bind to the protein; one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearl~ or Cibacrom blue 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 BioLabs (Beverly, MA), Pharmacia (Piscataway, NJ) and 3 0 Invitrogen Corporation (Carlsbad, CA), 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 the Eastman Kodak Company (New Haven, CT).
Finally, one or more reverse-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify the protein. Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant protein. The protein thus purified is substantially free of other mammalian proteins and is defined in accordance with the present invention as an "isolated protein."
The protein of the invention may also be expressed as a product of transgeruc 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 struchlral and/or conformational characteristics with proteins may possess biological properties in common therewith, including protein activity. Thus, they may be employed as biologically active or immunological substitutes for natural, purified proteins in screening of therapeutic compounds and in immunological processes for the development of antibodies.
2 0 The proteins provided herein also include proteins characterized by amino acid sequences similar to those of purified proteins but into which modification are naturally provided or deliberately engineered. For example, modifications in the peptide or DNA
sequences can be made by those skilled in the art using known techniques.
Modifications of interest in the protein sequences may include the alteration, substitution, replacement, 2 5 insertion or deletion of a selected amino acid residue in the coding sequence. For example, one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule. Techniques for such alteration, substitution, replacement, insertion or deletion are well known to those skilled in the art (see, e.g., U.S. Patent No. 4,518,584). Preferably, such alteration, substitution, replacement, 3 0 insertion or deletion retains the desired activity of the protein.
Other fragments and derivatives of the sequences of proteins which would be expected to retain profiein activity in whole or in part and may thus be useful for screening or other immunological methodologies may also be easily made by those skilled in the art given the disclosures herein. Such modifications are believed to be encompassed by the present invention.
USES AND BIOLOGICAL ACTIVL~
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 2 0 (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"
known sequences in the process of discovering other novel polynucleotides; for selecting 2 5 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 binds to another protein (such as, for example, in a receptor-ligand interaction), the 3 0 polynucleotide can also be used in interaction trap assays (such as, for example, those described in Gyuris et al.,1993, Cell 75: 791-803 and in Rossi et al.,1997, Proc. Natl. Acad.
Sci. LISA 94: 8405-8410, all of which are incorporated by reference herein) to identify polynucleotides encoding the other protein with which binding occurs or to identify inhibitors of the binding interaction.
The proteins provided by the present invention can similarly be used in assay to determine biological activity, including in a panel of multiple proteins for high-throughput screening; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological fluids; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state); and, of course, to isolate correlative receptors or ligands. Where the protein binds or potentially binds to another protein {such as, for example, in a receptor-ligand interaction), the protein can be used to identify the other protein with which binding occurs or to identify inhibitors of the binding interaction. Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.
Any or alI 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 2 0 Molecular Cloning Techniques", Academic Press, Berger, S.L. and A.R.
ICimmel 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 5 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 3 0 can be added to the medium in or on which the microorganism is cultured.
~,tr,k;np and Cell Proliferation/Differentiation Activity A protein of the present invention may exhibit cytokine, cell proliferation (either inducing or intu'biting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations. Many protein factors discovered to date, including all known cytokines, have exhibited activity in one or more factor-dependent cell proliferation assays, and hence the assays serve as a convenient confirmation of cytokine activity. The activity of a protein of the present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+ (preB M+), 2E8, RBS, DA1,123, T1165, HT2, CTLL2, TF-1, Mo7e and CMK.
The activity of a protein of the invention may, among other means, be measured by the following methods:
Assays for T-cell or thymocyte proliferation include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M.
Kruisbeek, D.H.
Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986;
Bertagnolli et al., J. Immunol.145:1706-1712, 1990; Bertagnolli et al., Cellular Immunology 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 2 0 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 Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley and Sons, Toronto.1994.
2 5 Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those described in: Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L.S. and Lipsky, P.E. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto. 1991; deVries et al., J. Exp. Med. 173:1205-1211, 1991; Moreau et al., Nature 3 0 336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A.
80:2931-2938, 1983;
Measurement of mouse and human interleukin 6 - Nordan, R. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.6.1-6.6.5, john Wiley and Sons, Toronto.1991;
Smith et al., Proc. Natl. Acad. Sci. U.S.A. 83:1857-1861, 1986; Measurement of human Interleukin 11- Bennett, F., Giannotti, J., Clark, S.C. and Turner, K. J. In Current Protocols WO 99/3?6?4 PCT/US99/01404 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. Coiigan eds. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto.1991.
Assays for T-cell clone responses to antigens (which will identify, among others, proteins that affect APC-T cell interactions as well as direct T-cell effects by measuring proliferation and cytokine production) include, without limitation, those described in:
Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H.
Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their cellular receptors; Chapter 7, Immunologic studies in Humans);
Weinberger et al., Proc. Natl. Acad. Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J.
Immun.
(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 polynudeotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:5 from nucleotide 1101 to nucleotide 1910; the nucleotide sequence of SEQ
ID N0:5 from nucleotide 1260 to nucleotide 1910; the nucleotide sequence of the full-length protein coding sequence of clone gg894_13 deposited under accession number ATCC
98636; or the nucleotide sequence of a mature protein coding sequence of clone gg894_13 deposited under accession number ATCC 98636. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert of clone gg894_13 deposited under accession number ATCC 98636. In further preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:6 having biological 2 0 activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID N0:6, or a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:6 having biological activity, the fragment comprising the amino acid sequence from amino acid 130 to amino acid 139 of SEQ ID N0:6.
2 5 Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:5.
Further embodiments of the invention provide isolated polynucleotides produced according to a process selected from the group consisting of:
(a) a process comprising the steps of:
3 0 (i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(aa) SEQ ID N0:5, but excluding the poly(A) tail at the 3' end of SEQ ID N0:5; and (ab) the nucleotide sequence of the cDNA insert of clone gg894_13 deposited under accession number ATCC 98636;
(ii) hybridizing said probes) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C; and (iii) isolating the DNA polynucieotides detected with the probe(s);
and (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(ba) SEQ ID N0:5, but excluding the poly(A) tail at the 3' end of SEQ ID N0:5; and (bb) the nucleotide sequence of the cDNA insert of clone gg894_13 deposited under accession number ATCC 98636;
(ii) hybridizing said primers) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C;
(iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii).
2 0 Preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:5, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID
N0:5 to a nucleotide sequence corresponding to the 3' end of SEQ ID N0:5 , but excluding the poly(A) tail at the 3' end of SEQ ID N0:5. Also preferably the polynucleotide isolated 2 5 according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:5 from nucleotide 1101 to nucleotide 1910, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID N0:5 from nucleotide 1101 to nucleotide 1910, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID N0:5 from nucleotide 1101 to 3 0 nucleotide 1910. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
N0:5 from nucleotide 1260 to nucleotide 1910, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
N0:5 from nucleotide 1260 to nucleotide 1910, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID N0:5 from nucleotide 1260 to nucleotide 1910.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID N0:6;
(b) fragments of the amino acid sequence of SEQ ID N0:6, each fragment comprising eight consecutive amino acids of SEQ ID N0:6; and (c) the amino acid sequence encoded by the cDNA insert of clone gg894_13 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID N0:6. In further preferred embodiments, the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID N0:6 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino ands of SEQ ID N0:6, or a protein comprising a fragment of the amino acid sequence of SEQ ID N0:6 having biological activity, the fragment comprising the amino acid sequence from amino acid 130 to amino acid I39 of SEQ ID N0:6.
In one embodiment, the present invention provides a composition comprising an 2 0 isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:7;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:7 from nucleotide 452 to nucleotide 1102;
2 5 (c) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone it217 2 deposited under accession number ATCC 98636;
(d) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone it217_2 deposited under accession number ATCC 98636;
3 0 (e) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone it217_2 deposited under accession number ATCC
98636;
(f) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone it217_2 deposited under accession number ATCC 98636;
(g) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID N0:8;
(h) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:8 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID N0:8;
(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 of (g) or (h) above ; and (k) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(h).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:7 from nucleotide 452 to nucleotide 1102; the nucleotide sequence of the full-length protein coding sequence of clone it217 2 deposited under accession number ATCC
98636;
or the nucleotide sequence of a mature protein coding sequence of clone it217_2 deposited under accession number ATCC 98636. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert of clone it217_2 deposited under accession number ATCC 98636. In further preferred embodiments, the present invention provides a polynucleotide encoding a protein 2 0 comprising a fragment of the amino acid sequence of SEQ ID N0:8 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID N0:8, or a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:8 having biological activity, the fragment comprising the amino acid sequence from amino and 103 2 5 to amino acid 112 of SEQ ID N0:8.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:7.
Further embodiments of the invention provide isolated polynucleotides produced according to a process selected from the group consisting of:
3 0 (a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(aa) SEQ ID N0:7, but excluding the poly(A) tail at the 3' end of SEQ ID N0:7; and (ab) the nucleotide sequence of the cDNA insert of clone it217_2 deposited under accession number ATCC 98636;
(ii) hybridizing said probes) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C; and (iii) isolating the DNA polynucleotides detected with the probe(s);
and (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(ba) SEQ ID N0:7, but excluding the poly(A) tail at the 3' end of SEQ ID N0:7; and (bb) the nucleotide sequence of the cDNA insert of done it217_2 deposited under accession number ATCC 98636;
(ii) hybridizing said primers) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C;
2 0 (iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii).
Preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:7, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID
N0:7 to 2 5 a nucleotide sequence corresponding to the 3' end of SEQ ID N0:7 , but excluding the poly(A) tail at the 3' end of SEQ ID N0:7. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:7 from nucleotide 452 to nucleotide 1102, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of 3 0 SEQ ID N0:7 from nucleotide 452 to nucleotide 1102, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID N0:7 from nucleotide 452 to nucleotide 1102.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID N0:8;
(b) fragments of the amino acid sequence of SEQ ID N0:8, each fragment comprising eight consecutive amino acids of SEQ ID N0:8; and (c) the amino acid sequence encoded by the cDNA insert of clone it217 2 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID N0:8. In further preferred embodiments, the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID N0:8 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino ands of SEQ ID N0:8, or a protein comprising a fragment of the amino acid sequence of SEQ ID N0:8 having biological activity, the fragment comprising the amino acid sequ~ce from amino acid 103 to amino acid 112 of SEQ ID N0:8.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
2 0 N0:9;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:9 from nucleotide 127 to nucleotide 387;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:9 from nucleotide 172 to nucleotide 387;
2 5 (d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone m1235_2 deposited under accession number ATCC 98636;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone m1235_2 deposited under accession number ATCC 98636;
3 0 (f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone m1235_2 deposited under accession number ATCC 98636;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone m1235 2 deposited under accession number ATCC 98636;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:10;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:10 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO:10;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and (1) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (ar(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:9 from nucleotide 127 to nucleotide 387; the nucleotide sequence of SEQ ID
N0:9 from nucleotide 172 to nucleotide 387; the nucleotide sequence of the full-length protein coding sequence of clone m1235_2 deposited under accession number ATCC 98636; or the nucleotide sequence of a mature protein coding sequence of clone m1235_2 deposited under accession number ATCC 98636. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert of clone m1235 2 deposited under accession number ATCC 98636. In further preferred 2 0 embodiments, the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:10 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO:20, or a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:10 having 2 5 biological activity, the fragment comprising the amino acid sequence from amino acid 38 to amino acid 47 of SEQ ID NO:10.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:9.
Further embodiments of the invention provide isolated polynucleotides produced 3 0 according to a process selected from the group consisting of:
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(aa) SEQ ID N0:9, but excluding the poly(A) tail at the 3' end of SEQ ID N0:9; and (ab) the nucleotide sequence of the cDNA insert of clone m1235 2 deposited under accession number A TCC 98636;
(ii) hybridizing said probes) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C; and (iii) isolating the DNA polynucleotides detected with the probe(s);
and (b) a process comprising the steps of:
(i) preparing one or more polynucleotfde primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(ba) SEQ ID N0:9, but excluding the poly(A) tail at the 3' end of SEQ ID N0:9; and (bb) the nucleotide sequence of the cDNA insert of clone m1235 2 deposited under accession number ATCC 98636;
(ii) hybridizing said primers) to human genomic DNA in conditions at least as stringent as 9:X SSC at 65 degrees C;
2 0 (iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii).
Preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:9, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID
N0:9 to 2 5 a nucleotide sequence corresponding to the 3' end of SEQ ID N0:9 , but excluding the poly(A) tail at the 3' end of SEQ ID N0:9. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:9 from nucleotide 127 to nucleotide 387, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of 3 0 SEQ ID N0:9 from nucleotide 127 to nucleotide 387, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID N0:9 from nucleotide 127 to nucleotide 387. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
N0:9 from nucleotide 172 to nucleotide 387, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
N0:9 from nucleotide 172 to nucleotide 387, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID N0:9 from nucleotide 172 to nucleotide 387.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:10;
(b) fragments of the amino acid sequence of SEQ ID NO:10, each fragment comprising eight consecutive amino acids of SEQ ID NO:10; and (c} the amino acid sequence encoded by the cDNA insert of clone m1235 2 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID NO:10. In further preferred embodiments, the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID NO:10 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID NO:10, or a protein comprising a fragment of the amino acid sequence of SEQ ID N0:10 having biological activity, the fragment comprising the amino acid sequence from amino acid 38 to amino acid 47 of SEQ ID NO:10.
2 0 In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:11;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
2 5 N0:11 from nucleotide 147 to nucleotide 1163;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:11 from nucleotide 273 to nucleotide 1163;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone mt24 2 deposited under accession 3 0 number ATCC 98636;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone mt24_2 deposited under accession number ATCC 98636;
(f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone mt24_2 deposited under accession number ATCC 98636;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone mt24_2 deposited under accession number ATCC 98636;
(h) a polynudeotide encoding a protein comprising the amino acid sequence of SEQ ID N0:12;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:12 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID N0:12;
(j) a polynucleotide which is an allelic variant of a polynucleotfde of (ar(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and (1) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:11 from nucleotide 147 to nucleotide 1163; the nucleotide sequence of SEQ
ID N0:11 from nucleotide 273 to nucleotide 1163; the nucleotide sequence of the full-length protein 2 0 coding sequence of clone mt24 2 deposited under accession number ATCC
98636; or the nucleotide sequence of a mature protein coding sequence of clone mt24 2 deposited under accession number ATCC 98636. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone mt24 2 deposited under accession number ATCC 98636. In further preferred embodiments, the 2 5 present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:12 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID N0:12, or a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:12 having biological activity, the 3 0 fragment comprising the amino acid sequence from amino acid 164 to amino acid 173 of SEQ ID N0:12.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID NO:11.
Further embodiments of the invention provide isolated polynucleotides produced according to a process selected from the group consisting of:
(a) a process comprising the steps of:
{i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(aa) SEQ ID N0:11, but excluding the poly(A) tail at the 3' end of SEQ ID N0:11; and (ab) the nucleotide sequence of the cDNA insert of clone mt24 2 deposited under accession number ATCC 98636;
(ii} hybridizing said probes) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C; and {iii) isolating the DNA polynucleotides detected with the probe(s);
and (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
2 0 (ba) SEQ ID N0:11, but excluding the poly(A) tail at the 3' end of SEQ ID NO:11; and (bb) the nucleotide sequence of the cDNA insert of clone mt24 2 deposited under accession number ATCC 98636;
(ii) hybridizing said primers) to human genomic DNA in 2 5 conditions at least as stringent as 4X SSC at 65 degrees C;
(iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii).
Preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID NO:11, and 3 0 extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ
ID NO:11 to a nucleotide sequence corresponding to the 3' end of SEQ ID N0:11 , but excluding the poly(A) tail at the 3' end of SEQ ID NO:11. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:11 from nucleotide 147 to nucleotide 1163, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID NO:11 from nucleotide 147 to nucleotide 1163, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:11 from nucleotide 147 to nucleotide 1163. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:11 from nucleotide 273 to nucleotide 1163, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
NO:11 from nucleotide 273 to nucleotide 1163, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID N0:11 from nucleotide 273 to nucleotide 1163.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
{a) the amino acid sequence of SEQ ID N0:12;
(b) fragments of the amino acid sequence of SEQ ID N0:12, each fragment comprising eight consecutive amino acids of SEQ ID N0:12; and (c) the amino acid sequence encoded by the cDNA insert of clone mt24_2 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID N0:12. In further preferred 2 0 embodiments, the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID N0:12 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID N0:12, or a protein comprising a fragment of the amino acid sequence of SEQ ID N0:12 having biological activity, the fragment comprising the amino acid 2 5 sequence from amino acid 164 to amino acid 173 of SEQ ID N0:12.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:13;
3 0 (b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:13 from nucleotide 320 to nucleotide 1681;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:13 from nucleotide 437 to nucleotide 1681;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone pe584_2 deposited under accession number ATCC 98636;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone pe584_2 deposited under accession number ATCC 98636;
(f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone pe584 2 deposited under accession number ATCC 98636;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone pe584 2 deposited under accession number ATCC 98636;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID N0:14;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:14 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID N0:14;
(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 2 0 (1) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a~(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:13 from nucleotide 320 to nucleotide 1681; the nucleotide sequence of SEQ
ID N0:13 from nucleotide 437 to nucleotide 1681; the nucleotide sequence of the full-length protein 2 5 coding sequence of clone pe584_2 deposited under accession number ATCC
98636; or the nucleotide sequence of a mature protein coding sequence of clone pe584_2 deposited under accession number ATCC 98636. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert of clone pe584 2 deposited under accession number ATCC 98636. In further preferred 3 0 embodiments, the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:14 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID N0:14, or a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:14 having biological activity, the fragment comprising the amino acid sequence from amino acid 222 to amino acid 231 of SEQ ID N0:14.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:13.
Further embodiments of the invention provide isolated polynucleotides produced according to a process selected from the group consisting of:
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(aa) SEQ ID N0:13, but excluding the poly(A) tail at the 3' end of SEQ ID N0:13; and (ab) the nucleotide sequence of the cDNA insert of clone pe584 2 deposited under accession number ATCC 98636;
(ii) hybridizing said probes) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C; and (iii) isolating the DNA polynucleotides detected with the probe(s);
and 2 0 (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(ba) SEQ ID N0:13, but excluding the poly(A) tail at the 2 5 3' end of SEQ ID N0:13; and (bb) the nucleotide sequence of the cDNA insert of clone pe584 2 deposited under accession number ATCC 98636;
(ii) hybridizing said primers) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C;
3 0 (iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii).
Preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:13, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ
ID N0:13 to a nucleotide sequence corresponding to the 3' end of SEQ ID N0:13 , but excluding the poly(A) tail at the 3' end of SEQ ID N0:13. Also preferably .the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:13 from nucleotide 320 to nucleotide 1681, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID N0:13 from nucleotide 320 to nucleotide 1681, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID N0:13 from nucleotide 320 to nucleotide 1681. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
N0:13 from nucleotide 437 to nucleotide 1681, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
N0:13 from nucleotide 437 to nucleotide 1681, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID N0:13 from nucleotide 437 to nucleotide 1681.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID N0:14;
(b) fragments of the amino acid sequence of SEQ ID N0:14, each fragment comprising eight consecutive amino acids of SEQ ID N0:14; and 2 0 (c) the amino acid sequence encoded by the cDNA insert of clone pe584 2 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID N0:14. In further preferred embodiments, the present invention provides a protein comprising a fragment of the 2 5 amino acid sequence of SEQ ID N0:14 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID N0:14, or a protein comprising a fragment of the amino acid sequence of SEQ ID N0:14 having biological activity, the fragment comprising the amino acid sequence from amino acid 222 to amino acid 231 of SEQ ID N0:14.
3 0 In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:15;
i~VO 99/37674 PCT/US99/01404 (b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:15 from nucleotide 78 to nucleotide 1502;
- (c} a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:15 from nucleotide 564 to nucleotide 1502;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone pj323_2 deposited under accession number ATCC 98636;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone pj323 2 deposited under accession number ATCC 98636;
(f) a polynucleotide comprising the nucleotide sequence of a mature protein coding sequence of clone pj323 2 deposited under accession number ATCC 98636;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone pj323_2 deposited under accession number ATCC 98636;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID N0:16;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:16 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID N0:16;
2 0 (j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and a polynucleotide that hybridizes under stringent conditions to any 2 5 one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:15 from nucleotide 78 to nucleotide 1502; the nucleotide sequence of SEQ ID
N0:15 from nucleotide 564 to nucleotide 1502; the nucleotide sequence of the full-length protein coding sequence of clone pj323 2 deposited under accession number ATCC 98636;
or the 3 0 nucleotide sequence of a mature protein coding sequence of clone pj323_2 deposited under accession number ATCC 98636. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA
insert of clone pj323 2 deposited under accession number ATCC 98636. In yet other preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID N0:16 from amino acid 54 to amino acid 145. In further preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID
N0:16 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID N0:16, or a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:16 having biological activity, the fragment comprising the amino acid sequence from amino acid 232 to amino acid 241 of SEQ ID N0:16.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:15.
Further embodiments of the invention provide isolated polynucleotides produced according to a process selected from the group consisting of:
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(aa) SEQ ID N0:15, but excluding the poly(A) tail at the 3' end of SEQ ID N0:15; and (ab} the nucleotide sequence of the cDNA insert of clone 2 0 pj323_2 deposited under accession number ATCC 98636;
(ii) hybridizing said probes) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C; and (iii) isolating the DNA polynucleotides detected with the probe(s);
2 5 and (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
3 0 (ba) SEQ ID NO:IS, but excluding the poly(A) tail at the 3' end of SEQ ID N0:15; and (bb) the nucleotide sequence of the cDNA insert of clone pj323_2 deposited under accession number ATCC 98636;
(ii) hybridizing said primers) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C;
(iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii).
Preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:15, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ
ID N0:15 to a nucleotide sequence corresponding to the 3' end of SEQ ID N0:15 , but excluding the poly(A) tail at the 3' end of SEQ ID N0:15. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:15 from nucleotide 78 to nucleotide 1502, and extending contiguously from a nucleotide sequence corresponding to the 5' ~d of said sequence of SEQ ID N0:15 from nucleotide 78 to nucleotide 1502, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ TD N0:15 from nucleotide 78 to nucleotide 1502. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
N0:15 from nucleotide 564 to nucleotide 1502, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
N0:15 from nucleotide 564 to nucleotide 1502, to a nucleotide sequence corresponding to the 3' end 2 0 of said sequence of SEQ ID N0:15 from nucleotide 564 to nucleotide 1502.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID N0:16;
2 5 (b) the amino acid sequence of SEQ ID N0:16 from amino acid 54 to amino acid 145;
(c) fragments of the amino acid sequence of SEQ ID N0:16, each fragment comprising eight consecutive amino acids of SEQ ID N0:16; and (d) the amino acid sequence encoded by the cDNA insert of clone 3 0 pj323 2 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID N0:16 or the amino acid sequence of SEQ ID N0:16 from amino acid 54 to amino acid 145. In further preferred embodiments, the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID N0:16 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID N0:16, or a protein comprising a fragment of the amino acid sequence of SEQ ID N0:16 having biological activity, the fragment comprising the amino acid sequence from amino acid 232 to amino acid 241 of SEQ ID N0:16.
In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:17;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:17 from nucleotide 130 to nucleotide 294;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:17 from nucleotide 241 to nucleotide 294;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone yb24_1 deposited under accession number ATCC 98636;
{e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone yb24_1 deposited under accession number ATCC 98636;
(f) a polynucleotide comprising the nucleotide sequence of a mature 2 0 protein coding sequence of clone yb24_1 deposited under accession number ATCC
98636;
{g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone yb24_1 deposited under accession number ATCC 98636;
(h) a polynucleotide encoding a protein comprising the amino acid 2 5 sequence of SEQ ID N0:18;
(i) a polynucleofide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:18 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID N0:18;
(j) a polynucleotide which is an allelic variant of a polynucleotide of 3 0 (a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and (1) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (ar(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:17 from nucleotide 130 to nucleotide 294; the nucleotide sequence of SEQ ID
N0:17 from nucleotide 241 to nucleotide 294; the nucleotide sequence of the full-length protein coding sequence of clone yb24_1 deposited under accession number ATCC 98636;
or the nucleotide sequence of a mature protein coding sequence of clone yb24_1 deposited under accession number ATCC 98636. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone yb24_1 deposited under accession number ATCC 98636. In further preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:18 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID N0:18, or a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:18 having biological activity, the fragment comprising the amino acid sequence from amino acid 22 to amino acid 31 of SEQ
ID N0:18.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:17.
Further embodiments of the invention provide isolated polynucleotides produced according to a process selected from the group consisting of:
2 0 {a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(aa) SEQ ID N0:17, but excluding the poly(A) tail at the 2 5 3' end of SEQ ID N0:17; and (ab) the nucleotide sequence of the cDNA insert of clone yb24_1 deposited under accession number ATCC 98636;
(ii) hybridizing said probes) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C; and 3 0 (iii) isolating the DNA polynucleotides detected with the probe(s);
and (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected_from the group consisting of:
(ba) SEQ ID N0:17, but excluding the poly(A) tail at the 3' end of SEQ ID N0:17; and (bb) the nucleotide sequence of the cDNA insert of clone yb24_1 deposited under accession number ATCC 98636;
(ii) hybridizing said primers) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C;
(iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii).
Preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:17, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ
ID N0:17 to a nucleotide sequence corresponding to the 3' end of SEQ ID N0:17 , but excluding the poly(A) tail at the 3' end of SEQ ID N0:17. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:17 from nucleotide 130 to nucleotide 294, and extending contiguously from a nucleotide sequence corresponding to the 5' end 2 0 of said sequence of SEQ ID N0:17 from nucleotide 130 to nucleotide 294, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID N0:17 from nucleotide 130 to nucleotide 294. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
N0:17 from nucleotide 241 to nucleotide 294, and extending contiguously from a 2 5 nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
N0:17 from nucleotide 241 to nucleotide 294, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID N0:17 from nucleotide 241 to nucleotide 294.
In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group 3 0 consisting of:
(a) the amino acid sequence of SEQ ID N0:18;
(b) fragments of the amino acid sequence of SEQ ID N0:18, each fragment comprising eight consecutive amino acids of SEQ ID N0:18; and (c) the amino acid sequence encoded by the cDNA insert of clone yb24_1 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID N0:18. In further preferred embodiments, the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID N0:18 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID N0:18, or a protein comprising a fragment of the amino acid sequence of SEQ ID N0:18 having biological activity, the fragment comprising the amino acid sequence from amino acid 22 to amino acid 31 of SEQ ID N0:18.
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
N0:19;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:19 from nucleotide 514 to nucleotide 1707;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
N0:19 from nucleotide 580 to nucleotide 1707;
(d) a polynucleotide comprising the nucleotide sequence of the full-2 0 length protein coding sequence of clone yb44_1 deposited under accession number ATCC 98636;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone yb44_1 deposited under accession number ATCC 98636;
(f) a polynucleotide comprising the nucleotide sequence of a mature 2 5 protein coding sequence of clone yb44_1 deposited under accession number ATCC
98636;
(g) a polynucleotide encoding a mature protein encoded by the cDNA
insert of clone yb44_1 deposited under accession number ATCC 98636;
(h) a polynucleotide encoding a protein comprising the amino acid 3 0 sequence of SEQ ID N0:20;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:20 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID N0:20;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above;
(k) a polynucleotide which encodes a species homologue of the protein of (h) or (i) above ; and (1) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i).
Preferably, such polynucleotide comprises the nucleotide sequence of SEQ ID
N0:19 from nucleotide 514 to nucleotide 1707; the nucleotide sequence of SEQ
ID N0:19 from nucleotide 580 to nucleotide 1707; the nucleotide sequence of the full-length protein coding sequence of clone yb44_1 deposited under accession number ATCC 98636;
or the nucleotide sequence of a mature protein coding sequence of clone yb44_1 deposited under accession number ATCC 98636. In other preferred embodiments, the polynucleotide encodes the full-length or a mature protein encoded by the cDNA insert of clone yb44_1 deposited under accession number ATCC 98636. In further preferred embodiments, the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:20 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID N0:20, or a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID N0:20 having biological activity, the 2 0 fragment comprising the amino acid sequence from amino acid 194 to amino acid 203 of SEQ ID N0:20.
Other embodiments provide the gene corresponding to the cDNA sequence of SEQ
ID N0:19.
Further embodiments of the invention provide isolated polynucleotides produced 2 5 according to a process selected from the group consisting of:
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
3 0 (aa) SEQ ID N0:19, but excluding the poly(A) tail at the 3' end of SEQ ID N0:19; and (ab) the nucleotide sequence of the cDNA insert of clone yb44_1 deposited under accession number ATCC 98636;
(ii) hybridizing said probes) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C; and (iii) isolating the DNA polynucleotides detected with the probe(s);
and (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(ba) SEQ ID N0:19, but excluding the poly(A) tail at the 3' end of SEQ ID N0:19; and (bb) the nucleotide sequence of the cDNA insert of clone yb44_1 deposited under accession number ATCC 98636;
(ii) hybridizing said primers) to human genomic DNA in conditions at least as stringent as 4X SSC at b5 degrees C;
(iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii.).
Preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:19, and 2 0 extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ
ID N0:19 to a nucleotide sequence corresponding to the 3' end of SEQ ID N0:19 , but excluding the poly(A) tail at the 3' end of SEQ ID N0:19. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID N0:19 from nucleotide 514 to nucleotide 2 5 1707, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID N0:19 from nucleotide 514 to nucleotide 1707, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID N0:19 from nucleotide 514 to nucleotide 1707. Also preferably the polynucleotide isolated according to the above process comprises a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
3 0 N0:19 from nucleotide 580 to nucleotide 1707, and extending contiguously from a nucleotide sequence corresponding to the.5' end of said sequence of SEQ ID
N0:19 from nucleotide 580 to nucleotide 1707, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID N0:19 from nucleotide 580 to nucleotide 1707.
'WO 99/37674 PCTNS99/01404 In other embodiments, the present invention provides a composition comprising a protein, wherein said protein comprises an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID N0:20;
(b) fragments of the amino acid sequence of SEQ ID N0:20, each fragment comprising eight consecutive amino acids of SEQ ID N0:20; and (c) the amino acid sequence encoded by the cDNA insert of clone yb44_1 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins. Preferably such protein comprises the amino acid sequence of SEQ ID N0:20. In further preferred embodiments, the present invention provides a protein comprising a fragment of the amino acid sequence of SEQ ID N0:20 having biological activity, the fragment preferably comprising eight (more preferably twenty, most preferably thirty) consecutive amino acids of SEQ ID N0:20, or a protein comprising a fragment of the amino acid sequence of SEQ ID N0:20 having biological activity, the fragment comprising the amino acid sequence from amino acid 194 to amino acid 203 of SEQ ID N0:20.
In certain preferred embodiments, the polynucleotide is operably linked to an expression control sequence. The invention also provides a host cell, including bacterial, yeast, insect and mammalian cells, transformed with such polynucleotide compositions.
2 0 Also provided by the present invention are organisms that have enhanced, reduced, or modified expression of the genes) corresponding to the polynucleotide sequences disclosed herein.
Processes are also provided for producing a protein, which comprise:
(a) growing a culture of the host cell transformed with such 2 5 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.
Protein compositions of the present invention may further comprise a 3 0 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 WO 99/37674 PC"T/US99/01404 effective amount of a composition comprising a protein of the present invention and a pharmaceutically acceptable carrier.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures lA and 1B are schematic representations of the pED6 and pNOTs vectors, respectively, used for deposit of clones disclosed herein.
DETAILED DESCRIPTION
ISOLATED PROTEINS AND POLYNUCLEOTIDES
Nucleotide and amino acid sequences, as presently determined, are reported below for each clone and protein disclosed in the present application. The nucleotide sequence of each clone can readily be determined by sequencing of the deposited clone in accordance with known methods. The predicted amino acid sequence (both full-length and mature forms) can then be determined from such nucleotide sequence. The amino acid sequence of the protein encoded by a particular clone can also be determined by expression of the clone in a suitable host cell, collecting the protein and determining its sequence. For each disclosed protein applicants have identified what they have determined to be the reading frame best identifiable with sequence information available at the time of filing.
2 0 As used herein a "seae~ed" protein is one which, when expressed in a suitable host cell, is transported across or through a membrane, including transport as a result of signal sequences in its amino acid sequence. "Secreted" proteins include without limitation proteins secreted wholly (e.g., soluble proteins) or partially (e.g. , receptors) from the cell in which they are expressed. "Secreted" proteins also include without limitation proteins 2 5 which are transported across the membrane of the endoplasmic reticulum.
Clone "cs756 2"
A polynucleotide of the present invention has been identified as clone "cs756_2".
cs756_2 was isolated from a human fetal 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. cs756_2 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "cs756 2 protein").
VIrO 99/37674 PCTNS99/01404 The nucleotide sequence of cs756_2 as presently determined is reported in SEQ
ID
NOa, and includes a poly(A) tail. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the cs756 2 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID N0:2.
Amino acids 211 to 223 of SEQ ID N0:2 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 224. Due to the hydrophobic nature of the predicted leader/signal sequence, it is likely to act as a transmembrane domain should the predicted leader/signal sequence not be separated from the remainder of the cs756 2 protein. The TopPredII computer program predicts a potential transmembrane domain within the cs756_2 protein sequence of SEQ ID
N0:2, centered around amino acid 15 of SEQ ID N0:2; amino acids 2 to 14 of SEQ ID
N0:2 are also a possible leader/signal sequence, with the predicted mature amino acid sequence in that case beginning at amino acid 15.
Another possible cs756 2 reading frame and predicted amino acid sequence, encoded by base pairs 385 to 825 of SEQ ID NO:1, is reported in SEQ ID N0:30;
the TopPredII computer program predicts a potential transmembrane domain centered around amino acid 100 of SEQ ID N0:30. The open reading frames corresponding to SEQ
ID N0:30 and SEQ ID N0:2 could be joined if a frameshift were introduced into the nucleotide sequence of SEQ ID N0:1.
2 0 The EcoRI/NotI restriction fragment obtainable from the deposit containing clone cs756_2 should be approximately 3000 bp.
The nucleotide sequence disclosed herein for cs756 2 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. cs756_2 demonstrated at least some similarity with sequences 2 5 identified as AA398077 (zt58c03.s1 Soares testis NHT Homo Sapiens cDNA
clone 726532 3'), AA541286 (nf97e03.s1 NCI_CGAP_Co3 Homo Sapiens cDNA clone 1MAGE:927868), W28620 (49c2 Human retina cDNA randomly primed sublibrary Homo Sapiens cDNA), and W47601 (zc35g08.r1 Soares senescent fibroblasts NbHSF Homo sapiens cDNA
clone 324350 5'). The predicted amino acid sequence disclosed herein for SEQ ID
N0:30 was 3 0 searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted SEQ ID N0:30 protein demonstrated at least some similarity to sequences identified as L76938 (Werner syndrome gene, complete cds [Homo sapiensJ). "Werner's syndrome (WS) is an inherited disease with clinical symptoms resembling premature aging ... [the] predicted protein is 1432 amino acids in WO 99/376'14 PCTNS99/01404 length and shows significant similarity to DNA helicases" (Yu et al., 1996, Science 272(5259):258-262, which is incorporated by reference herein). Based upon sequence similarity, cs756 2 proteins and each similar protein or peptide may share at least some activity. The nucleotide sequence of cs756 2 indicates that it may contain one or more of the following repetitive elements: MIR, MER.
Clone "ew150 1"
A polynucleotide of the present invention has been identified as clone "ew150_1".
ew150_1 was isolated from a human adult placenta cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No.
5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. evV150_1 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "ew150_1 protein').
The nucleotide sequence of ew150_1 as presently determined is reported in SEQ
ID N0:3, and includes a poly(A) tail. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the ew150_1 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID N0:4.
Amino acids 26 to 38 of SEQ ID N0:4 are a predicted leader/signal sequence, with the predicted 2 0 mature amino acid sequence beginning at amino acid 39. Due to the hydrophobic nature of the predicted leader/signal sequence, it is likely to act as a transmembrane domain should the predicted leader/signal sequence not be separated from the remainder of the ew150_1 protein.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone 2 5 ew150_1 should be approximately 2000 bp.
The nucleotide sequence disclosed herein for ew150_1 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. ew150_1 demonstrated at least some similarity with sequences identified as AA563938 (nk23b12.s1 NCI_CGAP Col1 Homo sapiens cDNA clone IMAGE
3 0 1014335), D63209 (Human placenta cDNA 5'-end GEN-506F01), M90423 (Bacteriphage US3 lytic-enzyme), W23461 (zb33cOl.r1 Soares parathyroid tumor NbHPA Homo sapiens cDNA clone 305376 5'), and 256916 (H.sapiens CpG DNA, clone 153b7, forward read cpg153b7.ftla). In the region around position 1514 of SEQ ID N0:3, ew150_1 also demonstrated at least some similarity with sequences encoding a mitochondria) energy-transfer proteins signature motif which is found in mitochondrial and other membrane proteins. Based upon sequence similarity, ew150_1 proteins and each similar protein or peptide may share at least some activity. The TopPredII computer program predicts ten potential transmembrane domains within the ew150_1 protein sequence, which are centered around amino acids 70,106,133, 200, 314, 349, 387, 457, 504, and 527 of SEQ ID
N0:4, respectively.
lone "g~894 1~"
A polynucleotide of the present invention has been identified as clone "gg894_13".
gg894_13 was isolated from a human fetal kidney cDNA library using methods which are selective for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637), or was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. gg894_13 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "gg894_13 protein").
The nucleotide sequence of gg894_13 as presently determined is reported in SEQ
ID N0:5, and includes a poly(A) tail. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the gg894_13 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID N0:6.
Amino 2 0 acids 41 to 53 of SEQ ID N0:6 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 54. Due to the hydrophobic nature of the predicted leader/signal sequence, it is likely to act as a transmembrane domain should the predicted leader/signal sequence not be separated from the remainder of the gg894_13 protein. Another possible gg894_13 reading frame and predicted amino acid 2 5 sequence, encoded by base pairs 602 to 1129 of SEQ ID N0:5, is reported in SEQ ID
N0:31. The open reading frames corresponding to SEQ ID N0:31 and SEQ ID N0:6 could be joined if a frameshift were introduced into the nucleotide sequence of SEQ
ID N0:5.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone gg894_13 should be approximately 2400 bp.
3 0 The nucleotide sequence disclosed herein for gg894_13 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. gg894_13 demonstrated at least some similarity with sequences identified as H57424 (yr13a10.s1 Homo sapiens cDNA clone 205146 3'), T23885 (Human gene signature HUMGS05820), and W80358 (zh49a07.s1 Soares fetal liver spleen WO 99!37674 PCT/US99/01404 Sl Homo sapiens cDNA clone 415380 3'). Based upon sequence similarity, gg894_13 proteins and each similar protein or peptide may share at least some activity.
The TopPredII computer program predicts a potential transmembrane domain within the gg894_13 protein sequence centered around amino acid 115 of SEQ ID N0:6. The nucleotide sequence of gg894_13 indicates that it may contain a RBMI
repetitive element.
Clone "it217 2"
A polynucleotide of the present invention has been identified as clone "it217_2".
it217 2 was isolated from a human adult brain (thalamus) 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. it217 2 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "it217_2 protein").
The nucleotide sequence of it217 2 as presently determined is reported in SEQ
ID
N0:7, and includes a poly(A) tail. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the it217_2 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID N0:8.
Another possible it217 2 reading frame and predicted amino acid sequence, encoded by base pairs 2 0 45 to 311 of SEQ ID N0:7, is reported in SEQ ID N0:32. Amino acids 36 to 48 of SEQ ID
N0:32 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 49. Due to the hydrophobic nature of the predicted leader/signal sequence, it is likely to act as a transmembrane domain should the predicted leader/signal sequence not be separated from the remainder of the it217_2 protein. The 2 5 open reading frames corresponding to SEQ ID N0:32 and SEQ ID N0:8 could be joined if at least one frameshift were introduced into the nucleotide sequence of SEQ
ID N0:7.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone it217_2 should be approximately 2250 bp.
The nucleotide sequence disclosed herein for it217 2 was searched against the 3 0 GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. it217_2 demonstrated at least some similarity with sequences identified as AA242969 (zr65h09.r1 Soares NhHMPu S1 Homo sapiens cDNA clone 668321 5' similar to SW SCC2_HUMAN P48594 SQUAMOUS CELL CARCINOMA
ANTIGEN 2 ;contains Alu repetitive element), B44876 (HS-1060-Al-G06-MR.abi CTT
i~NO 99/37674 PGT/US99/01404 Human Genomic Sperm Library C Homo sapien genomic clone Plate CT 782 Col 11 Row M), H82168 (yv78d08.r1 Homo sapiens cDNA clone), 566896 (squamous cell carcinoma antigen), U19556 (Human squamous cell carcinoma antigen 1 (SCCAl) mRNA, complete cds), U19557 (Human squamous cell carcinoma antigen 2 (SCCA2) mRNA, complete cds), S and U35459 (Human bomapin mRNA, complete cds). The predicted amino acid sequence disclosed herein for it217_2 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted it217_2 protein demonstrated at least some similarity to sequences identified as L40377 (cytoplasmic antiproteinase 2 [Homo sapiens]), M34352 (ovalbumin (callus gallus]), M91161 (serpin [Equus caballus]), 825276 (SCC antigen), 848379 (Human megakaryocyte differentiation factor), 566896 (squamous cell carcinoma antigen, SCC antigen serine protease inhibitor [human, Peptide, 390 aa] [Homo sapiens]), U19568 (squamous cell carcinoma antigen [Homo sapiens]), and U19576 (squamous cell carcinoma antigen [Homo sapiens]).
Human bomapin may play a role in the regulation of protease activities during hematopoiesis (Riewald et al.,1995, J. Biol. Chem. 270: 26754, which is incorporated by reference herein). Serpins are SERine Proteinase IlVhibitors and are considered extracellular in localization. Human squamous cell carcinoma antigen (SSCA) is a member of the serpin family of proteinase inhibitors, purified from sera of cancer patients.
Based upon sequence similarity, it217 2 proteins and each similar protein or peptide may 2 0 share at least some activity.
Clong "m1235 2"
A polynucleotide of the present invention has been identified as clone "m1235 2".
m1235_2 was isolated from a human adult brain (caudate nucleus) cDNA library using 2 5 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. m1235 2 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "m1235 2 protein').
3 0 The nucleotide sequence of m1235 2 as presently determined is reported in SEQ
ID N0:9, and includes a poly(A) tail. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the m1235 2 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID
N0:10. Amino acids 3 to 15 of SEQ ID NO:10 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 16. Due to the hydrophobic nature of the predicted leader/signal sequence, it is likely to act as a transmembrane domain should the predicted leader/signal sequence not be separated from the remainder of the m1235 2 protein.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone m1235 2 should be approximately 1400 bp.
The nucleotide sequence disclosed herein for m1235 2 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. m1235_2 demonstrated at least some similarity with sequences identified as AA160887 (zo79b05.s1 Stratagene pancreas (#937208} Homo sapiens cDNA
clone 593073 3'), 814349 (yf79f12.r1 Homo sapiens cDNA clone 28451 5'), and (yg74f07.r1 Homo sapiens cDNA clone 39059 5'). Based upon sequence similarity, m1235 2 proteins and each similar protein or peptide may share at least some activity.
The TopPredII computer program predicts a potential transmembrane domain within the m1235_2 protein sequence centered around amino acid 25 of SEQ ID N0:10.
Clone "mt24 2"
A polynudeotide of the present invention has been identified as clone "mt24 2".
mt24_2 was isolated from a human adult testes cDNA library using methods which are 2 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. mt24 2 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "mt24 2 protein').
2 5 The nucleotide sequence of mt24 2 as presently determined is reported in SEQ ID
N0:11, and includes a poly(A) tail. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the mt24 2 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID
N0:12. Amino acids 30 to 42 of SEQ ID N0:12 are a predicted leader/signal sequence, with the predicted 3 0 mature amino acid sequence beginning at amino acid 43. Due to the hydrophobic nature of the predicted leader/signal sequence, it is likely to act as a transmembrane domain should the predicted leader/signal sequence not be separated from the remainder of the mt24 2 protein.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone mt24 2 should be approximately 1400 bp.
The nucleotide sequence disclosed herein for mt24_2 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. mt24 2 demonstrated at least some similarity with sequences identified as AA062589 (zf68f04.r1 Soares pineal gland N3HPG Homo sapiens cDNA
clone 3821115') and T19332 (b08016t Testis 1 Homo sapiens cDNA clone b08016 5' end).
Based upon sequence similarity, mt24 2 proteins and each similar protein or peptide may share at least some activity. The TopPredII computer program predicts four potential transmembrane domains within the mt24_2 protein sequence centered around amino acids 38,153,167, and 232 of SEQ ID N0:12, respectively.
Clone"pe584 2"
A polynucleotide of the present invention has been identified as clone "pe584_2".
pe584_2 was isolated from a human adult blood (chronic myelogenous leukemia K5) 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. pe584 2 is a full-length clone, including the entire coding sequence of a secreted 2 0 protein (also referred to herein as "pe584 2 protein").
The nucleotide sequence of pe584 2 as presently determined is reported in SEQ
ID N0:13, and includes a poly(A) tail. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the pe584_2 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID
N0:14. Amino 2 5 acids 27 to 39 of SEQ ID N0:14 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 40. Due to the hydrophobic nature of the predicted leader/signal sequence, it is likely to act as a transmembrane domain should the predicted leader/signal sequence not be separated from the remainder of the pe584_2 protein.
3 0 The EcoRI/NotI restriction fragment obtainable from the deposit containing clone pe584 2 should be approximately 3000 bp.
The nucleotide sequence disclosed herein for pe584_2 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. pe584 2 demonstrated at least some similarity with sequences identified as AA303149 (EST13039 Uterus tumor I), AA405004 (zt06e03.s1 NCI_CGAP_GCBl Homo Sapiens cDNA clone IMAGE 712348 3'), AA481230 (aa34gOl.r1 NCI_CGAP GCB1 Homo sapiens cDNA clone 815184 5' similar to SW TCR2_ECOLI
P02981 TETRACYCLINE RESISTANCE PROTEIN), D88315 (Mouse mRNA for tetracycline transporter-like protein, complete cds), and T10077 (seq1295 Homo sapiens cDNA clone b4HB3MA-COT8-HAP-Ft109 5'}. The predicted amino acid sequence disclosed herein for pe584 2 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted pe584_2 protein demonstrated at least some similarity to sequences identified as D88315 (tetracycline transporter-like protein [Mus musculus]). Mouse tetracycline transporter-like protein is a sugar transporter (Matsuo et al.,1997, Biochem. Biophys. Res. Comm. 238: 126-192, which is incorporated by reference herein). Based upon sequence similarity, pe584 2 proteins and each similar protein or peptide may share at least some activity. The TopPredII
computer program predicts eleven potential transmembrane domains within the pe584 2 protein sequence, which are centered around amino acids 32, 55, 78, 114,142, 196, 235, 264, 287, 332, and 375 of SEQ ID N0:14, respectively.
Clony '~i3,~_' A polynucleotide of the present invention has been identified as clone "pj323 2".
2 0 pj323 2 was isolated from a human fetal carcinoma (NTD2 cells treated with retinoic acid for 23 days) 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. pj323 2 is a full-length clone, including the entire coding sequence 2 5 of a secreted protein (also referred to herein as "pj323 2 protein").
The nucleotide sequence of pj323_2 as presently determined is reported in SEQ
ID
N0:15, and includes a poly(A) tail. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the pj323 2 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID
N0:16. Amino 3 0 acids 150 to 162 of SEQ ID N0:16 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 163. Due to the hydrophobic nature of the predicted leader/signal sequence, it is likely to act as a transmembrane domain should the predicted leader/signal sequence not be separated from the remainder of the pj323_2 protein.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone pj323_2 should be approximately 2500 bp.
The nucleotide sequence disclosed herein for pj323_2 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. pj323 2 demonstrated at least some similarity with sequences identified as AA160454 (zo74g05.r1 Stratagene pancreas (#937208) Homo sapiens cDNA
clone 592664 5'), AA398257 (zt60a08.s1 Scares testis NHT Homo Sapiens cDNA
clone 726710 3'), and T47284 (yb64g11.s1 Homo Sapiens cDNA clone 76004 3'). The predicted amino acid sequence disclosed herein for pj323 2 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted pj323 2 protein demonstrated at least some similarity to human integral nuclear envelope protein, lamin B receptors from several species, and sterol reductases from several species. Lamin B receptors have hydrophobic carboxy terminal portions and hydrophilic amino terminal portions. Antibodies to lamin B receptors have been found in patients with primary biliary cirrhosis. Sterol reductases demonstrate sequence similarity to the hydrophobic portions of lamin B receptors. Based upon sequence similarity, pj323_2 proteins and each similar protein or peptide may share at least some activity. The TopPredII computer program predicts six potential transmembrane domains within the pj323 2 protein sequence, which are centered around amino acids 47,106,164, 2 0 187, 341, and 432 of SEQ ID N0:16, respectively.
pj323 2 protein was expressed in a COS cell expression system, and an expressed protein band of approximately 46 kDa was detected in membrane fractions using SDS
polyacrylamide gel electrophoresis.
2 S Clone "vb24 1"
A polynucleotide of the present invention has been identified as clone "yb24_1".
yb24_1 was isolated from a human fetal brain cDNA library and was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the amino acid sequence of the encoded protein. yb24_1 is a full-length clone, including the 3 0 entire coding sequence of a secreted protein (also referred to herein as "yb24_1 protein").
The nucleotide sequence of yb24_1 as presently determined is reported in SEQ
ID
N0:17, and includes a poly(A) tail. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the yb24_1 protein corresponding to the foregoing nucleotide sequence is reported in SEQ ID
N0:18. Amino acids 25 to 37 of SEQ ID N0:18 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 38. Due to the hydrophobic nature of the predicted leader/signal sequence, it is likely to act as a transmembrane domain should the predicted leader/signal sequence not be separated from the remainder of the yb24_1 protein.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone yb24_1 should be approximately 1700 bp.
The nucleotide sequence disclosed herein for yb24_1 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. yb24_1 demonstrated at least some similarity with sequences identified as AA149807 (z147c09.s1 Soares pregnant uterus NbHPU Homo sapiens cDNA
clone 505072 3') and AB003515 (Rat mRNA for GEF-2, complete cds). Based upon sequence similarity, yb24_1 proteins and each similar protein or peptide may share at least some activity.
A polynucleotide of the present invention has been identified as clone "yb44_1".
yb44_1 was isolated from a human fetal brain cDNA library and was identified as encoding a secreted or transmembrane protein on the basis of computer analysis of the 2 0 amino acid sequence of the encoded protein. yb44_1 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "yb44_1 protein").
The nucleotide sequence of yb44_1 as presently determined is reported in SEQ
ID
N0:19, and includes a poly(A) tail. What applicants presently believe to be the proper reading frame and the predicted amino acid sequence of the yb44_1 protein 2 5 corresponding to the foregoing nucleotide sequence is reported in SEQ ID
N0:20. Amino acids 10 to 22 of SEQ ID N0:20 are a predicted leader/signal sequence, with the predicted mature amino acid sequence beginning at amino acid 23. Due to the hydrophobic nature of the predicted leader/signal sequence, it is likely to act as a transmembrane domain should the predicted leader/signal sequence not be separated from the remainder of the 3 0 yb44_1 protein.
The EcoRI/NotI restriction fragment obtainable from the deposit containing clone yb44_1 should be approximately 2000 bp.
The nucleotide sequence disclosed herein for yb44_1 was searched against the GenBank and GeneSeq nucleotide sequence databases using BLASTN/BLASTX and FASTA search protocols. yb44_1 demonstrated at least some similarity with sequences identified as AC000016 {*** SEQUENCING IN PROGRESS *** EPMl /APECED region of chromosome 21, BAC clone B4P3; HTGS phase 1,10 unordered pieces). The predicted amino acid sequence disclosed herein for yb44_1 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The predicted yb44_1 protein demonstrated at least some similarity to sequences identified as 872377 (Human auxiliary cytochrome P450 species 2D6 variant 2 protein) and (cytochrome P450 [Drosophila melanogaster]). Based upon sequence similarity, yb44_1 proteins and each similar protein or peptide may share at least some activity.
The TopPredII computer program predicts three additional potential transmembrane domains within the yb44_1 protein sequence, which are centered around amino acids 82,128, and 361 of SEQ ID N0:20, respectively. The nucleotide sequence of yb44_1 indicates that it may contain one or more of the following repetitive elements: Alu, AT, TATACA, MER44A, TACA.
Den osit of ~,lones Clones cs756_2, ew150_l, gg894_13, it217_2, m1235_2, mt24_2, pe584 2, pj323 2, yb24_l, and yb44_1 were deposited on January 22,1998 with the American Type Culture Collection (10801 University Boulevard, Manassas, Virginia 20110-2209 U.S.A.) as an 2 0 original deposit under the Budapest Treaty and were given the accession number ATCC
98636, 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), and the term of the deposit will comply with 37 C.F.R. ~
1.806.
2 5 Each clone has been transfected into separate bacterial cells (E. coh~ 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 pEDb or pNOTs vector depicted in Figures lA and 1B, respectively. The pED6dpc2 vector 3 0 ("pED6") was derived from pED6dpc1 by insertion of a new polylinker to facilitate cDNA cloning (Kaufman et al.,1991, Nucleic Acids Res.19: 4485-4490); the pNOTs vector was derived from pMT2 (Kaufman et al.,1989, Mol. Cell. Biol. 9: 946-958) by deletion of the DHFR sequences, insertion of a new polylinker, and insertion of the M13 origin of replication in the CIaI site. In some instances, the deposited clone can become "flipped"
'WO 99/37674 PCT/US99/01404 (i.e., in the reverse orientation) in the deposited isolate. In such instances, the cDNA insert can still be isolated by digestion with EcoRI and NotI. However, NotI will then produce the 5' site and EcoRI will produce the 3' site for placement of the cDNA in proper orientation for expression in a suitable vector. The cDNA may also be expressed from the vectors in which they were deposited.
Bacterial cells containing a particular clone can be obtained from the composite deposit as follows:
An oligonucleotide probe or probes should be designed to the sequence that is known for that particular clone. This sequence can be derived from the sequences provided herein, or from a combination of those sequences. The sequence of an oligonudeotide probe that was used to isolate or to sequence each full-length clone is identified below, and should be most reliable in isolating the clone of interest.
Clone Probe Sequence cs756_2 SEQ ID N0:21 ew150_1 SEQ ID N0:22 gg894_13 SEQ ID N0:23 it217_2 SEQ ID N0:24 m1235 2 SEQ ID N0:25 2 0 mt24_2 SEQ ID N0:26 pe584_2 ~ SEQ ID N0:27 pj323_2 SEQ ID N0:28 yb24_1 SEQ ID N0:29 2 5 In the sequences listed above which include an N at position 2, that position is occupied in preferred probes/primers by a biotinylated phosphoaramidite residue rather than a nucleotide (such as, for example, that produced by use of biotin phosphoramidite (1-dimethoxytrityloxy-2-(N-biotinyl-4-aminobutyl)-propyl-3-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramadite) (Glen Research, cat. no.10-1953)).
3 0 The design of the oligonucleotide probe should preferably follow these parameters:
(a) It should be designed to an area of the sequence which has the fewest ambiguous bases ("N's"), if any;
(b) It should be designed to have a Tm of approx. 80 ° C (assuming 2° for each A or T and 4 degrees for each G or C).
The oligonucleotide should preferably be labeled with y 32P ATP (specific activity 6000 Ci/mmole) and T4 polynucleotide kinase using commonly employed techniques for labeling oligonucleotides. Other labeling techniques can also be used.
Unincorporated label should preferably be removed by gel filtration chromatography or other established methods. The amount of radioactivity incorporated into the probe should be quantitated by measurement in a scintillation counter. Preferably, specific activity of the resulting probe should be approximately 4e+6 dpm/pmole.
The bacterial culture containing the pool of full-length clones should preferably be thawed and 100 Ixl 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 ug/ml and agar at 1.5% in a 150 mm petri dish when grown overnight at 37°C. Other known methods of obtaining distinct, well-separated colonies can also be employed.
Standard colony hybridization procedures should then be used to transfer the 2 0 colonies to nitrocellulose filters and lyre, 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 ~g/ml of yeast RNA, and 10 mM EDTA
(approximately 10 mL per 150 mm filter). Preferably, the probe is then added to the hybridization mix at a concentration greater than or equal to 1e+6 dpm/mL. The filter is then preferably incubated at 65°C with gentle agitation overnight. The filter is then preferably washed in 500 mL of 2X SSC/0.5% SDS at room temperature without agitation, preferably followed by 500 mL of 2X SSC/0.1% SDS at room temperature with gentle shaking for 15 minutes.
A third wash with O.1X SSC/0.5% SDS at 65°C for 30 minutes to 1 hour is optional. The 3 0 filter is then preferably dried and subjected to autoradiography for sufficient time to visualize the positives on the X-ray film. Other known hybridization methods can also be employed.
The positive colonies are picked, grown in culture, and plasmid DNA isolated using standard procedures. The clones can then be verified by restriction analysis, hybridization analysis, or DNA sequencing.
Fragments of the proteins of the present invention which are capable of exhibiting biological activity are also encompassed by the present invention. Fragments of the protein may be in linear form or they may be cyclized using known methods, for example, as described in H.U. Saragovi, et al., Bio/Technology 1~0 773-778 (1992) and in R.S.
McDowell, et al., J. Amer. Chem. Soc. ~~, 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 forms) of such protein may be obtained by expression of the disclosed full-length 2 0 polynucleotide (preferably those deposited with ATCC) in a suitable mammalian cell or other host cell. The sequences) of the mature forms) of the protein may also be determinable from the amino acid sequence of the full-length form.
The present invention also provides genes corresponding to the polynucleotide sequences disclosed herein. "Corresponding genes" are the regions of the genome that 2 5 are transcribed to produce the mRNAs from which cDNA polynucleotide sequences are derived and may include contiguous regions of the genome necessary for the regulated expression of such genes. Corresponding genes may therefore include but are not limited to coding sequences, 5' and 3' untranslated regions, alternatively spliced exons, introns, promoters, enhancers, and silencer or suppressor elements. The corresponding genes can 3 0 be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include the preparation of probes or primers from the disclosed sequence information for identification and/or amplification of genes in appropriate genomic libraries or other sources of genomic materials. An "isolated gene" is a gene that has been separated from the adjacent coding sequences, if any, present in the genome of the organism from which the gene was isolated.
The chromosomal location corresponding to the polynucleotide sequences disclosed herein may also be determined, for example by hybridizing appropriately labeled polynucleotides of the present invention to chromosomes in situ. It may also be possible to determine the corresponding chromosomal location for a disclosed polynucleotide by identifying significantly similar nucleotide sequences in public databases, such as expressed sequence tags (ESTs), that have already been mapped to particular chromosomal locations. For at least some of the polynucleotide sequences disclosed herein, public database sequences having at least some similarity to the polynucleotide of the present invention have been listed by database accession number.
Searches using the GenBank accession numbers of these public database sequences can then be performed at an Internet site provided by the National Center for Biotechnology Information having the address http://www.ncbi.nlm.nih.gov/UniGene/, in order to identify "UniGene clusters" of overlapping sequences. Many of the "UniGene clusters"
so identified will already have been mapped to particular chromosomal sites.
Organisms that have enhanced, reduced, or modified expression of the genes) corresponding to the polynucleotide sequences disclosed herein are provided.
The desired change in gene expression can be achieved through the use of aniisense 2 0 polynucleotides or ribozymes that bind and/or cleave the mRNA transcribed from the gene (Albert and Morris,1994, Trends Phurmacol. Sci.15(~: 250-254; Lavarosky et al.,1997, Biochem. Mol. Med. 62(1):11-22; and Hampel,1998, Prog. Nucleic Acid Res. Mol.
Biol. 58:1-39; all of which are incorporated by reference herein). Transgenic animals that have multiple copies of the genes) corresponding to the polynucleotide sequences disclosed 2 5 herein, preferably produced by transformation of cells with genetic constructs that are stably maintained within the transformed cells and their progeny, are provided.
Transgenic animals that have modified genetic control regions that increase or reduce gene expression levels, or that change temporal or spatial patterns of gene expression, are also provided (see European Patent No. 0 649 464 B1, incorporated by reference herein).
3 0 In addition, organisms are provided in which the genes) corresponding to the polynucleotide sequences disclosed herein have been partially or completely inactivated, through insertion of extraneous sequences into the corresponding genes) or through deletion of all or part of the corresponding gene(s). Partial or complete gene inactivation can be accomplished through insertion, preferably followed by imprecise excision, of transposable elements (Plasterk,1992, Bioessays 14(9): 629-633; Zwaal et al.,1993, Proc. Natl.
Acad. Sci. LISA 90(16): 7431-7435; Clark et al.,1994, Proc. Natl. Acad. Sci.
LISA 91(2): 719-722;
all of which are incorporated by reference herein), or through homologous recombination, preferably detected by positive/negative genetic selection strategies (Mansour et al.,1988, Nature 336: 348-352; U.S. Patent Nos. 5,464,764; 5,487,992; 5,627,059;
5,631,153; 5,614, 396;
5,616,491; and 5,679,523; all of which are incorporated by reference herein).
These organisms with altered gene expression are preferably eukaryotes and more preferably are mammals. Such organisms are useful for the development of non-human models for the study of disorders involving the corresponding gene(s), and for the development of assay systems for the identification of molecules that interact with the protein products) of the corresponding gene(s).
Where the protein of the present inv~tion is membrane-bound (e.g., is a receptor), the present invention also provides for soluble forms of such protein. In such forms, part or all of the intracellular and transmembrane domains of the protein are deleted such that the protein is fully secreted from the cell in which it is expressed. The intracellular and transmembrane domains of proteins of the invention can be identified in accordance with known techniques for determination of such domains from sequence information.
For example, the TopPredII computer program can be used to predict the location of transmembrane domains in an amino acid sequence, domains which are described by the 2 0 location of the center of the transmsmbrane domain, with at least ten transmembrane amino acids on each side of the reported central residue(s).
Proteins and protein fragments of the present invention include proteins with amino acid sequence lengths that are at least 25%(more preferably at least 50%, and most preferably at least 75%) of the length of a disclosed protein and have at least 60% sequence 2 5 identity (more preferably, at least 75% identity; most preferably at least 90% or 95%
identity) with that disclosed protein, where sequence identity is determined by comparing the amino acid sequences of the proteins when aligned so as to maximize overlap and identity while mi~aimizing sequence gaps. Also included in the present invention are proteins and protein fragments that contain a segment preferably comprising 8 or more 3 0 (more preferably 20 or more, most preferably 30 or more) contiguous amino acids that shares at least 75% sequence identity (more preferably, at least 85% identity;
most preferably at least 95% identity) with any such segment of any of the disclosed proteins.
In particular, sequence identity may be determined using WU-BLAST
(Washington University BLAST) version 2.0 software, which builds upon WU-BLAST
version 1.4, which in turn is based on the public domain NCBI-BLAST version 1.4 (Altschul and Gish, 1996, Local alignment statistics, Doolittle ed., Methods in Enzymology 266: 460-480; Altschul et al., 1990, Basic local alignment search tool, Journal of Molecular Biology 2I5: 403-410; Gish and States, 1993, Identification of protein coding regions by database similarity search, Nature Genetics 3: 266-272; Karlin and Altschul, 1993, Applications and statistics for multiple high-scoring segments in molecular sequences, Proc. Natl. Acad. Sci. USA 90: 5873-5877; all of which are incorporated by reference herein). WU-BLAST version 2.0 executable programs for several UNIX
platforms can be downloaded from ftp://blast.wustl.edu/blast/executables. The complete suite of search programs (BLASTP, BLASTN, BLASTX, TBLASTN, and TBLASTX) is provided at that site, in addition to several support programs. WU-BLAST 2.0 is copyrighted and may not be sold or redistributed in any form or manner without the express written consent of the author; but the posted executables may otherwise be freely used for commercial, nonprofit, or academic purposes. In all search programs in the suite -- BLASTP, BLASTN, BLASTX, TBLASTN and TBLASTX -- the gapped alignment routines are integral to the database search itself, and thus yield much better sensitivity and selectivity while producing the more easily interpreted output. Gapping can optionally be fumed off in all of these programs, if desired. The default penalty (Q) for a gap of length one is Q=9 for proteins and BLASTP, and Q=10 for BLASTN, but may be changed to any 2 0 integer value including zero, one through eight, nine, ten, eleven, twelve through twenty, twenty-one through fifty, fifty-one through one hundred, etc. The default per-residue penalty for extending a gap (R) is R=2 for proteins and BLASTP, and R=10 for BLASTN, but may be changed to any integer value including zero, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve through twenty, twenty-one through fifty, fifty-one 2 5 through one hundred, etc. Any combination of values for Q and R can be used in order to align sequences so as to maximize overlap and identity while minimizing sequence gaps.
The default amino acid comparison matrix is BLOSUM62, but other amino acid comparison matrices such as PAM can be utilized.
Species homologues of the disclosed polynucleotides and proteins are also 3 0 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. Preferably, polynucleotide species homologues have at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90%
identity) with the given polynudeotide, and protein species homologues have at least 30%
sequence identity (more preferably, at least 45% identity; most preferably at least 60%
identity) with the given protein, where sequence identity is determined by comparing the nucleotide sequences of the polynucleotides or the amino acid sequences of the proteins when aligned so as to maximize overlap and identity while minimizing sequence gaps.
Species homologues may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from the desired species. Preferably, species homologues are those isolated from mammalian species. Most preferably, species homologues are those isolated from certain mammalian species such as, for example, Pan troglodytes, Gorilla gorilla, Pongo pygmaeus, Hylobates concolor, Macaca mulatta, Papio papio, Papio hamadryas, Cercopithecus aethiops, Cebus capucinus, Aotus trivirgatus, Sanguinus Oedipus, Microcebus murinus, Mus musculus, Rattus norvegicus, CricetuIus griseus, Fells catus, Mustela visor, Canis familiaris, O~yctolagus cuniculus, Bos taurus, Ovis aries, Sus scrota, and Equus caballus, for which genetic maps have been created allowing the identification of syntenic relationships between the genomic organization of genes in one species and the genomic organization of the related genes in another species (O'Brien and Seuanez, 1988, Ann. Rev. Genet. 22: 323-351; OBrien et al., 1993, Nature 2 0 Genetics 3:103-112; Johansson et al.,1995, Genomics 25: 682-690; Lyons et al.,1997, Nature Genetics 15: 47-56; O'Brien et al.,1997, Trends in Genetics 13(10): 393-399;
Carver and Stubbs, 1997, Genome Research 7:1123-1137; all of which are incorporated by reference herein).
The invention also encompasses allelic variants of the disclosed polynucleotides or proteins; that is, naturally-occurring alternative forms of the isolated polynucleotides 2 5 which also encode proteins which are identical or have significantly similar sequences to those encoded by the disclosed polynucleotides. Preferably, allelic variants have at least 60% sequence identity (more preferably, at least 75% identity; most preferably at least 90%
identity} with the given polynudeotide, where sequence identity is determined by comparing the nucleotide sequences of the polynucleotides when aligned so as to maximize 3 0 overlap and identity while minimizing sequence gaps. Allelic variants may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from individuals of the appropriate species.
VlrO 99/37674 PCTNS99/01404 The invention also includes polynucleotides with sequences complementary to those of the polynucleotides disclosed herein.
The present invention also includes polynucleotides that hybridize under reduced stringency conditions, more preferably stringent conditions, and most preferably highly stringent conditions, to polynucleotides described herein. Examples of stringency conditions are shown in the table below: highiy stringent conditions are those that are at least as stringent as, for example, conditions A-F; stringent conditions are at least as stringent as, for example, conditions G-L; and reduced stringency conditions are at least as stringent as, for example, conditions M-R.
StringencyPolynucleotideHybridHybridization TemperatureWash ConditionHybrid Lengthand Temperature (bp)= Buffer' and Buffers _ A DNA:DNA z 50 65C; lxSSC -or- 65C; 0.3xSSC
42C; lxSSC, 50% formamide B DNA:DNA <50 TB*; lxSSC Te*; 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, SO% formamide F RNA:RNA <50 TF*; lxSSC TF*; ixSSC
G DNA:DNA 2 SO 65C; 4xSSC -or- 65C; lxSSC
42C; 4xSSC, 50% formamide 1 H DNA:DNA <50 T"*; 4xSSC TH'; 4xSSC
O
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 2 50 70C; 4xSSC -or- 67C; lxSSC
50C; 4xSSC, 50% formamide L RNA:RNA <50 T~'; 2xSSC T~'; 2xSSC
M DNA:DNA z 50 50C; 4xSSC -or- 50C; 2xSSC
40C; 6xSSC, 50% formamide N DNA:DNA <50 TN*; 6xSSC TN*; 6xSSC
O DNA:RNA a 50 55C; 4xSSC -or- 55C; 2xSSC
42C; 6xSSC, 509 formamide P DNA:RNA <50 TP*; 6xSSC TP'; 6xSSC
Q RNA:RNA a 50 60C; 4xSSC -or- 60C; 2xSSC
45C; 6xSSC, 50~ formamide 2 R RNA:RNA <50 TR'; 4xSSC TR*; 4xSSC
~: The hybrid length is that anticipated for the hybridized regions) of the hybridizing polynudeotides. When hybridizing a polynucleotide to a target polynucleotide of unknown sequence, the hybrid length is assumed to be that of the hybridizing polynucleotide. When polynucleotides of known sequence are hybridized, the 2 5 hybrid length can be determined by aligning the sequences of the polynudeotides and identifying the region or regions of optimai sequence complementarily.
t: SSPE (lxSSPE is 0.15M NaCI, IOmM NaH2P0" and 1.25mM EDTA, pH 7.4) can be substituted for SSC
(lxSSC is O.15M NaCI and lSmM sodium citrate) in the hybridization and wash buffers; washes are performed for 15 minutes after hybridization is complete.
3 0 "Ts - 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 aaording 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'j) + 0.41(~°G+C) (600/N), where N is the number of bases in the hybrid, and [Na'j is the concentration of sodium ions in the 3 5 hybridization buffer ([Na') for lxSSC = 0.165 M).
Additional examples of stringency conditions for polynucleotide hybridization are provided in Sambrook, J., E.F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, chapters 9 and 11, and Current Protocols in Molecular Biology,1995, F.M.
Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, 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 ICaufman et al., Nucleic Acids Res. 12, 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.
ICaufman, Methods in Enzymology ~$~, 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 i~ 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 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 Ae~ricultural Experiment Station Bulletin No. 1555 (198, incorporated herein by reference. As used herein, an insect cell capable of expressing a polynucleotide of the present invention is "transformed."
The protein of the invention may be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant protein. The resulting expressed protein may then be purified from such culture (i.e., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography. The purification of the protein may also include an affinity column 2 0 containing agents which will bind to the protein; one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearl~ or Cibacrom blue 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 BioLabs (Beverly, MA), Pharmacia (Piscataway, NJ) and 3 0 Invitrogen Corporation (Carlsbad, CA), 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 the Eastman Kodak Company (New Haven, CT).
Finally, one or more reverse-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify the protein. Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant protein. The protein thus purified is substantially free of other mammalian proteins and is defined in accordance with the present invention as an "isolated protein."
The protein of the invention may also be expressed as a product of transgeruc 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 struchlral and/or conformational characteristics with proteins may possess biological properties in common therewith, including protein activity. Thus, they may be employed as biologically active or immunological substitutes for natural, purified proteins in screening of therapeutic compounds and in immunological processes for the development of antibodies.
2 0 The proteins provided herein also include proteins characterized by amino acid sequences similar to those of purified proteins but into which modification are naturally provided or deliberately engineered. For example, modifications in the peptide or DNA
sequences can be made by those skilled in the art using known techniques.
Modifications of interest in the protein sequences may include the alteration, substitution, replacement, 2 5 insertion or deletion of a selected amino acid residue in the coding sequence. For example, one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule. Techniques for such alteration, substitution, replacement, insertion or deletion are well known to those skilled in the art (see, e.g., U.S. Patent No. 4,518,584). Preferably, such alteration, substitution, replacement, 3 0 insertion or deletion retains the desired activity of the protein.
Other fragments and derivatives of the sequences of proteins which would be expected to retain profiein activity in whole or in part and may thus be useful for screening or other immunological methodologies may also be easily made by those skilled in the art given the disclosures herein. Such modifications are believed to be encompassed by the present invention.
USES AND BIOLOGICAL ACTIVL~
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 2 0 (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"
known sequences in the process of discovering other novel polynucleotides; for selecting 2 5 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 binds to another protein (such as, for example, in a receptor-ligand interaction), the 3 0 polynucleotide can also be used in interaction trap assays (such as, for example, those described in Gyuris et al.,1993, Cell 75: 791-803 and in Rossi et al.,1997, Proc. Natl. Acad.
Sci. LISA 94: 8405-8410, all of which are incorporated by reference herein) to identify polynucleotides encoding the other protein with which binding occurs or to identify inhibitors of the binding interaction.
The proteins provided by the present invention can similarly be used in assay to determine biological activity, including in a panel of multiple proteins for high-throughput screening; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological fluids; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state); and, of course, to isolate correlative receptors or ligands. Where the protein binds or potentially binds to another protein {such as, for example, in a receptor-ligand interaction), the protein can be used to identify the other protein with which binding occurs or to identify inhibitors of the binding interaction. Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.
Any or alI 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 2 0 Molecular Cloning Techniques", Academic Press, Berger, S.L. and A.R.
ICimmel 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 5 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 3 0 can be added to the medium in or on which the microorganism is cultured.
~,tr,k;np and Cell Proliferation/Differentiation Activity A protein of the present invention may exhibit cytokine, cell proliferation (either inducing or intu'biting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations. Many protein factors discovered to date, including all known cytokines, have exhibited activity in one or more factor-dependent cell proliferation assays, and hence the assays serve as a convenient confirmation of cytokine activity. The activity of a protein of the present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+ (preB M+), 2E8, RBS, DA1,123, T1165, HT2, CTLL2, TF-1, Mo7e and CMK.
The activity of a protein of the invention may, among other means, be measured by the following methods:
Assays for T-cell or thymocyte proliferation include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A.M.
Kruisbeek, D.H.
Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986;
Bertagnolli et al., J. Immunol.145:1706-1712, 1990; Bertagnolli et al., Cellular Immunology 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 2 0 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 Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley and Sons, Toronto.1994.
2 5 Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those described in: Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L.S. and Lipsky, P.E. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto. 1991; deVries et al., J. Exp. Med. 173:1205-1211, 1991; Moreau et al., Nature 3 0 336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A.
80:2931-2938, 1983;
Measurement of mouse and human interleukin 6 - Nordan, R. In Current Protocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.6.1-6.6.5, john Wiley and Sons, Toronto.1991;
Smith et al., Proc. Natl. Acad. Sci. U.S.A. 83:1857-1861, 1986; Measurement of human Interleukin 11- Bennett, F., Giannotti, J., Clark, S.C. and Turner, K. J. In Current Protocols WO 99/3?6?4 PCT/US99/01404 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. Coiigan eds. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto.1991.
Assays for T-cell clone responses to antigens (which will identify, among others, proteins that affect APC-T cell interactions as well as direct T-cell effects by measuring proliferation and cytokine production) include, without limitation, those described in:
Current Protocols in Immunology, Ed by J. E. Coligan, A.M. Kruisbeek, D.H.
Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their cellular receptors; Chapter 7, Immunologic studies in Humans);
Weinberger et al., Proc. Natl. Acad. Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J.
Immun.
11:405-411, 1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol.
140:508-512, 1988.
Immune Stimulating or Su~ressing, 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 2 0 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 infections, or may result from autoimmune disorders. More specifically, infectious 2 5 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. C~f course, in this regard, a protein of the present invention may also be useful where a boost to the immune system generally may be desirable, i.e., in the 3 0 treatment of cancer.
Autoimmune disorders which may be treated using a protein of the present inv~tion include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye disease.
Such a protein of the present invention may also to be useful in the treatment of allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems. Other conditions, in which immune suppression is desired (including, for example, organ transplantation), may also be treatable using a protein of the present invention.
Using the proteins of the invention it may also be possible to regulate 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 2 0 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 transplantation. Typically, in tissue transplants, rejection of the transplant is initiated 2 5 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 monomeric form of a peptide having an activity of another B lymphocyte antigen (e.g., B7-3 0 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 immunosuppressant. Moreover, the lack of costimulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject. Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents. To achieve sufficient immunosuppression or tolerance in a subject, it may also be necessary to block the function of a combination of B lymphocyte antigens.
The efficacy of particular blocking reagents in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans. Examples of appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow et al., Science 257:789-792 (1992) and Turka et al., Proc. Natl. Acad.
Sci USA, 89:11102-11105 {1992). In addition, 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 2 0 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 activation and prevent production of autoantibodies or T cell-derived cytokines which may be involved in the disease process. Additionally, blocking reagents may induce 2 5 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 human autoimmune diseases. Examples include marine experimental autoimmune encephalitis, systemic lupus erythmatosis in MRL/lpr/Ipr mice or NZB hybrid mice, 3 0 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).
Upregulation of an antigen function (preferably a B lymphocyte antigen function), as a means of up regulating immune responses, may also be useful in therapy.
Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response. For example, enhancing an immune response through stimulating B lymphocyte antigen function may be useful in cases of viral infection. In addition, systemic viral diseases such as influenza, the common cold, and encephalitis might be alleviated by the administration of stimulatory forms of B
lymphocyte antigens systemically.
Alternatively, anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen-pulsed APCs either expressing a peptide of the present invention or together with a stimulatory form of a soluble peptide of the present invention and reintroducing the in vitro activated T cells into the patient. Another method of enhancing anti-viral immune responses would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding a protein of the present invention as described herein such that the cells express all or a portion of the protein on their surface, and reintroduce the transfected cells into the patient. The infected cells would now be capable of delivering a 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, 2 0 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 ran be transfected to express a combination of peptides.
For example, tumor cells obtained from a patient can be transfected ex vivo with an expression vector directing the expression of a peptide having B7-2-like activity alone, or in conjunction with a peptide having B7-2-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 to target a tumor cell for transfection in vivo.
The presence of the peptide of the present invention having the activity of a B
3 0 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
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 (iZ
microglobulin protein or an MHC class II a chain protein and an MHC class II
~i chain protein to thereby express MHC class I or MHC class II proteins on the cell surface.
Expression of the appropriate class I or class II MHC in conjunction with a peptide having the activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a T
cell mediated immune response against the transfected tumor cell. Optionally, a gene encoding an antisense construct which blocks expression of an MHC class II associated protein, such as the invariant chain, can also be cotransfected with a DNA encoding a peptide having the activity of a B lymphocyte antigen to promote presentation of tumor associated antigens and induce tumor specific immunity. Thus, the induction of a T cell mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject.
The activity of a protein of the invention may, among other means, be measured by the following methods:
Suitable assays for thymocyte or splenocyte cytotoxicity include, without 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.
2 0 USA 78:248&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, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et ai., 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 immunoglobuiin responses and isotype switching (which will identify, among others, proteins that modulate T-cell dependent antibody responses and that affect Thl /Th2 profiles) include, without limitation, those described 3 0 in: Maliszewski, J. Imrnunol.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.
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.
ICruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol.137:3494-3500,1986; Takai et al., J. Immunol. 140:508-5I2, 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; Macatorua 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, 2 0 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 development include, without limitation, those described in: Antica et al., Blood 84:111-I17, 1994; Fine et al., Cellular Immunology 155:111-122,1994; Galy et al., Blood 2 5 85:2770-2778,1995; Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551,1991.
Hematopoiesis Reg~,, ' a ActivitX
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 3 0 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 progenitox cells alone or in combination with other cytokines, thereby indicating utility, for example, in treating various anemias or for use in conjunction with irradiation/chemotherapy to stimulate the production of erythroid precursors and/or erythroid cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF
activity) useful, for example, in conjunction with chemotherapy to prevent or treat consequent myelo-suppression; in supporting the growth and proliferation of megakaryocytes and consequently of platelets thereby allowing prevention or treatment of various platelet disorders such as thrombocytoperua, and generally for use in place of or complimentary to platelet transfusions; and/or in supporting the growth and proliferation of hematopoietic stem cells which are capable of maturing to any and all of the above-mentioned hematopoietic cells and therefore find therapeutic utility in various stem cell disorders (such as those usually treated with transplantation, including, without limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well as in repopulating the stem cell compartment post irradiation/chemotherapy, either in-vivo or ex-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.
2 0 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 al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al., Blood 81:2903-2915,1993.
2 5 Assays for stem cell survival and differentiation (which will identify, among others, proteins that regulate lympho-hematopoiesis) include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, M.G. In Culture of Hematopoietic Cells. R.L 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 3 0 hematopoietic colony forming cells with high proliferative potential, McNiece, LK. and Briddell, R.A. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds.
Vol pp. 23-39, Wiley-Liss, Inc., New York, NY.1994; Neben et al., Experimental Hematology 22:353-359, 1994; Cobblestone area forming cell assay, Ploemacher, R.E. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. l-21, Wiley-Liss, lnc.., New York, NY.1994; Long term bone marrow cultures in the presence of stromal cells, Spooncer, E., Dexter, M. and Allen, T. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 163-179, Wiley-Liss, Inc., New York, NY.1994; Long term culture initiating cell assay, Sutherland, H.J. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 139-162, Wiley-Liss, Inc., New York, NY. 1994.
Tissue Growth Activity A protein of the present invention also may have utility in compositions used for bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration, as well as for wound healing and tissue repair and replacement, and in the treatment of burns, incisions and ulcers.
A protein of the present invention, which induces cartilage and/or bone growth in circumstances where bone is not normally formed, has application in the healing of bone fractures and cartilage damage or defects in humans and other animals.
Such a preparation employing a protein of the invention may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints. De novo bone formation induced by an osteogeruc agent contributes to the repair of congenital, trauma induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic plastic surgery.
2 0 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 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.
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 3 0 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 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 nova tendon/ligament-like tissue formation induced by a composition of the present invention contributes to the repair of congerutai, 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- ar 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 viva for return in viva 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 2 0 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 invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma and cerebrovascular diseases such as stroke. Peripheral neuropathies resulting 2 5 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 non-healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like.
3 0 It is expected that a protein of the present invention may also exhibit activity for generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac) and vascular (including vascular endothelium) tissue, or for promoting the growth of cells comprising such tissues. Part of the desired effects may be by inhibition or modulation WO 99!37674 PCT/US99/01404 of fibrotic scarring to allow normal tissue to regenerate. A protein of the invention may also exhibit angiogeruc activity.
A protein of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokine damage.
A protein of the present invention may also be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells; or for inhibiting the growth of tissues described above.
The activity of a protein of the invention may, among other means, be measured by the following methods:
Assays for tissue generation activity include, without limitation, those described in: International Patent Publication No. W095/16035 (bone, cartilage, tendon);
International Patent Publication No. W095/05846 (nerve, neuronal);
International Patent Publication No. WCr91/07491 (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).
2 0 Activin /Inhibin ActivitX
A protein of the present invention may also exhibit activin- or inhibin-related activities. Inhibins are characterized by their ability to inhibit the release of follicle stimulating hormone (FSH), while activins and are characterized by their ability to stimulate the release of follicle stimulating hormone (FSH). Thus, a protein of the present 2 5 invention, alone or in heterodimers with a member of the inhibin a family, may be useful as a contraceptive based on the ability of inhibins to decrease fertility in female mammals and decrease spermatogenesis in male mammals. Administration of sufficient amounts of other inhibins can induce infertility in these mammals. Alternatively, the protein of the invention, as a homodimer or as a heterodimer with other protein subunits of the inhibin-3 0 ~ group, may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells of the anterior pituitary.
See, for example, United States Patent 4,798,885. A protein of the invention may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as cows, sheep and pigs.
The activity of a protein of the invention may, among other means, be measured by the following methods:
Assays for activin/inhibin activity include, without limitation, those described in:
Vale et al., Endocrinology 91:562-572,1972; Ling et al., Nature 321:779-782,1986; Vale et al., Nature 321:776-779,1986; Mason et al., Nature 318:659-663,1985; Forage et al., Proc.
Natl. Acad. Sci. USA 83:3091-3095,1986.
Chemotactic/Chemokinetic ActivitX
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 2 0 population. Preferably, the protein or peptide has the ability to directly stimulate directed movement of cells. Whether a particular protein has chemotactic activity for a population of cells can be readily determined by employing such protein or peptide in any known assay for cell chemotaxis.
The activity of a protein of the invention may, among other means, be measured 2 5 by the following methods:
Assays for chemotactic activity (which will identify proteins that induce or prevent chemotaxis) consist of assays that measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population. Suitable assays for movement and adhesion 3 0 include, without limitation, those described in: Current Protocols in Immunology, Ed by J.E. Coligan, A.M. ICruisbeek, D.H. Margulies, E.M. Shevach, W.Strober, Pub.
Greene Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:1370-1376,1995; Lind et al.
APMIS 103:140-146, 1995; Muller et al Eur. J. Immunol. 25: 1744-1748; Gruber et al. J. of Immunol. 152:5860-5867,1994; Johnston et al. J. of Immunol. 153: 1762-1768,1994.
HemoStatic and Thrombolvtic Activity A protein of the invention may also exhibit hemostatic or thrombolytic activity.
As a result, such a protein is expected to be useful in treatment of various coagulation disorders (including hereditary disorders, such as hemophilias) or to enhance coagulation and other hemostatic events in treating wounds resulting from trauma, surgery or other causes. A protein of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system vessels (e.g., stroke).
The activity of a protein of the invention may, among other means, be measured by the following methods:
Assay for hemostatic and thrombolytic activity include, without limitation, those described in: Linet et 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.
2 0 Receptor/Ligand,~, Acrivitv A protein of the present invention may also demonstrate activity as receptors, receptor ligands or inhibitors or agonists of receptor/ligand interactions.
Examples of such receptors and ligands include, without limitation, cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, 2 5 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 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 3 0 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.
The activity of a protein of the invention may, among other means, be measured by the following methods:
VlrO 99/37674 PCTNS99/01404 Suitable assays for receptor-ligand activity include without limitation those described in:Current Protocols in Immunology, Ed by J.E. Coligan, A.M.
Kruisbeek, D.H.
Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7.28.1-7.28.22), Takai et al., Proc. Natl. Acad. Sci: USA 84:6864-6868, 1987;
Bierer et al., J. Exp. Med.168:1145-1156, 1988; Rosenstein et al., J. Exp.
Med.169:149-160 1989; Stoltenborg et al., J. Immunol. Methods 175:59-68,1994; Stitt et al., Cell 80:661-670, 1995.
Anti-Inflammatory Activity Proteins of the present invention may also exhibit anti-inflammatory activity.
The anti-inflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or promoting cell-cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or 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 2 0 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 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 Invasio~,Su~~ressor ActivitX
Cadherins are calcium-dependent adhesion molecules that appear to play major roles during development, particularly in defining specific cell types. Loss or alteration of normal cadherin expression can lead to changes in cell adhesion properties linked to 3 0 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.
The cadherin superfamily includes well over forty members, each with a distinct pattern of expression. All members of the superfamily have in common conserved extracellular repeats (cadherin domains), but structural differences are found in other parts of the molecule. The cadherin domains bind calcium to form their tertiary structure and thus calcium is required to mediate their adhesion. Only a few amino acids in the first cadherin domain provide the basis for homophilic adhesion; modification of this recognition site can change the specificity of a cadherin so that instead of recognizing only itself, the mutant molecule can now also bind to a different cadherin. In addition, some cadherins engage in heterophilic adhesion with other cadherins.
E-cadherin, one member of the cadherin superfamily, is expressed in epithelial cell types. Pathologically, if E-cadherin expression is lost in a tumor, the malignant cells become invasive and the cancer metastasizes. Transfection of cancer cell lines with polynucleotides expressing E-cadherin has reversed cancer-associated changes by returning altered cell shapes to normal, restoring cells' adhesiveness to each other and to their substrate, decreasing the cell growth rate, and drastically reducing anchorage-independent cell growth. Thus, reintroducing E-cadherin expression reverts carcinomas to a less advanced stage. It is likely that other cadherins have the same invasion suppressor role in carcinomas derived from other tissue types. Therefore, proteins of the present invention with cadherin activity, and polynucleotides of the present invention encoding such proteins, can be used to treat cancer. Introducing such proteins or polynudeotides into cancer cells can reduce or eliminate the cancerous changes observed 2 0 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 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 2 5 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 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 3 0 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 cancer, the less cadherin expression there will be, and this decrease in cadherin expression can be detected by the use of a cadherin-binding antibody.
Fragments of proteins of the present invention with cadherin activity, preferably a polypeptide comprising a decapeptide of the cadherin recognition site, and poly-nucleotides of the present invention encoding such protein fragments, can also be used to block cadherin function by binding to cadherins and preventing them from binding in ways that produce undesirable effects. Additionally, fragments of proteins of the present invention with cadherin activity, preferably truncated soluble cadherin fragments which have been found to be stable in the circulation of cancer patients, and polynucleotides encoding such protein fragments, can be used to disturb proper cell-cell adhesion.
Assays for cadherin adhesive and invasive suppressor activity include, without limitation, those described in: Hortsch et al. J Biol Chem 270 (32): 18809-18817, 1995;
Miyaki et al. Oncogene 11: 2547-2552,1995; Ozawa et al. Cell 63: 1033-1038,1990.
Tumor Inhibition Activity In addition to the activities described above for immunological treatment or prevention of tumors, a protein of the invention may exhibit other anti-tumor activities.
A protein may inhibit tumor growth directly or indirectly (such as, for example, via antibody-dependent cell-mediated cytotoxicity (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 2 0 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.
9~her Activities 2 5 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, weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ 3 0 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, carbohydrate, vitamins, minerals, cofactors or other nutritional factors or component(s);
effecting behavioral characteristics, including, without limitation, appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders) and violent behaviors; providing analgesic effects or other pain reducing effects;
promoting differentiation and growth of embryonic stem cells in lineages other than hematopoietic lineages; hormonal or endocrine activity; in the case of enzymes, correcting deficiencies of the enzyme and treating deficiency-related diseases; treatment of hyperproliferative disorders (such as, for example, psoriasis); immunoglobulin-like activity (such as, for example, the ability to bind antigens or complement); and the ability to act as an antigen in a vaccine composition to raise an immune response against such protein or another material or entity which is cross-reactive with such protein.
ADMINISTRATION ANA DOSI_NG
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 2 0 effectiveness of the biological activity of the active ingredient(s). The characteristics of the carrier will depend on the route of administration. The pharmaceutical composition of the invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL.-2, IL.-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IFN, TNFO, TNF1, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem 2 5 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 3 0 in formulations of the particular cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent.
A protein of the present invention may be active in multimers (e.g., heterodimers 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 muitimeric or complexed form.
The pharmaceutical composition of the invention may be in the form of a complex of the proteins) of present invention along with protein or peptide antigens.
The protein and/or peptide antigen will deliver a stimulatory signal to both B and T
lymphocytes. B
lymphocytes will respond to antigen through their surface immunoglobulin receptor. T
lymphocytes will respond to antigen through the T cell receptor (TCR) following presentation of the antigen by MHC proteins. MHC and structurally related proteins including those encoded by class I and class II NiHC 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 2 0 pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the 2 5 art, as disclosed, for example, in U.S. Patent No. 4,235,871; U.S. Patent No. 4,501,728; U.S.
Patent No. 4,837,028; and U.S. Patent No. 4,737,323, all of which are incorporated herein by reference.
As used herein, the term "therapeutically effective amount" means the total amount of each active component of the pharmaceutical composition or method that is 3 0 sufficient to show a meaningful patient benefit, i.e., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
In practicing the method of treatment or use of the present invention, a therapeutically effective amount of protein of the present invention is administered to a mammal having a condition to be treated. Protein of the present invention may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing cytolcines, lympholcines or other hematopoietic factors. When co-administered with one or more cytolcines, lymphokines or other hematopoietic factors, protein of the present invention may be administered either simultaneously with the cytolcine(s), lympholcine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or sequentially. If administered sequentially, the attending physician will decide on the appropriate sequence of administering protein of the present invention in combination with cytolcine(s), lympholcine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors.
Administration of protein of the present invention used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection.
2 0 Intravenous administration to the patient is preferred.
When a therapeutically effective amount of protein of the present invention is administered orally, protein of the present invention will be in the form of a tablet, capsule, powder, solution or elixir. When administered in tablet form, the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or 2 5 an adjuvant. The tablet, capsule, and powder contain from about 5 to 95%
protein of the present invention, and preferably from about 25 to 90% protein of the present invention.
When administered in liquid form, a liquid Garner such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. The liquid form of the pharmaceutical composition may further contain 3 0 physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90% by weight of protein of the present invention, and preferably from about 1 to 50% protein of the present invention.
When a therapeutically effective amount of protein of the present invention is administered by intravenous, cutaneous or subcutaneous injection, protein of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution.
The preparation of such parenterally acceptable protein solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. A
preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to protein of the present invention, an isotonic vehicle such as Sodium Chloride Injeckion, Ringei 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 2 0 increased further. It is contemplated that the various pharmaceutical compositions used to practice the method of the present invention should contain about 0.01 lzg to about 100 mg (preferably about O.lng to about 10 mg, more preferably about 0.1 Ilg to about 1 mg) of protein of the present invention per kg body weight.
The duration of intravenous therapy using the pharmaceutical composition of the 2 5 present invention will vary, depending on the severity of the disease being treated and the condition and potential idiosyncratic response of each individual patient.
It is contemplated that the duration of each application of the protein of the present invention will be in the range of 12 to 24 hours of continuous intravenous administration.
Ultimately the attending physician will decide on the appropriate duration of intravenous 3 0 therapy using the pharmaceutical composition of the present invention.
Protein of the invention may also be used to immunize animals to obtain polyclonal and monoclonal antibodies which specifically react with the protein. Such antibodies may be obtained using either the entire protein or fragments thereof as an immunogen. The peptide immunogens additionally may contain a cysteine residue at the WO 99/37674 PG"T/US99/01404 carboxyl terminus, and are conjugated to a hapten such as keyhole limpet hemocyanin (KLH). Methods for synthesizing such peptides are known in the art, for example, as in R.P. Merrifield, J. Amer.Chem.Soc. $,~, 2149-2154 (1963); J.L. Krstenansky, et al., FEBS Lett.
21~, 10 (1987). Monoclonal antibodies binding to the protein of the invention may be useful diagnostic agents for the immunodetection of the protein. Neutralizing monoclonal antibodies binding to the protein may also be useful therapeutics for both conditions associated with the protein and also in the treatment of some forms of cancer where abnormal expression of the protein is involved. In the case of cancerous cells or leukemic cells, neutralizing monoclonal antibodies against the protein may be useful in detecting and preventing the metastatic spread of the cancerous cells, which may be mediated by the protein.
For compositions of the present invention which are useful for bone, cartilage, tendon or ligament regeneration, the therapeutic method includes administering the composition topically, systematically, or locally as an implant or device.
When administered, the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form. Further, the composition may desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage or 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 2 0 optionally be included in the composition as described above, may alternatively or additionally, be administered simultaneously or sequentially with the composition in the methods of the invention. Preferably for bone and/or cartilage formation, the composition would include a matrix capable of delivering the protein-containing composition to the site of bone and/or cartilage damage, providing a structure for the 2 5 developing bone and cartilage and optimally capable of being resorbed into the body.
Such matrices may be formed of materials presently in use for other implanted medical applications.
The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties. The particular 3 0 application of the compositions will define the appropriate formulation.
Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides. Other potential materials are biodegradable and biologically well-defined, such as bone or dermal collagen. Further matrices are comprised of pure proteins or extracellular matrix components. Other potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxapatite, bioglass, aluminates, or other _ ceramics. Matrices may be comprised of combinations of any of the above mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalciumphosphate. The bioceramics may be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradability.
Presently preferred is a 50:50 (mole weight) copolymer of lactic acid and glycolic acid in the form of porous particles having diameters ranging from 150 to 800 microns.
In some applications, it will be useful to utilize a sequestering agent, such as carboxymethyl cellulose or autologous blood clot, to prevent the protein compositions from disassociating from the matrix.
A preferred family of sequestering agents is cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl methylcellulose, and carboxymethylcellulose, the most preferred being cationic salts of carboxymethylcellulose {CMC). Other preferred sequestering agents include hyaluronic acid, sodium alginate, polyethylene glycol), polyoxyethyiene oxide, carboxyvinyl polymer and polyvinyl alcohol). The amount of sequestering agent useful herein is 0.5-20 2 0 wt%, preferably 1-10 wt% based on total formulation weight, which represents the amount necessary to prevent desorbtion of the protein from the polymer matrix and to provide appropriate handling of the composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix, thereby providing the protein the opportunity to assist the osteogenic activity of the progenitor cells.
2 5 In further compositions, proteins of the invention may be combined with other agents beneficial to the treatment of the bone and/or cartilage defect, wound, or tissue in question. These agents include various growth factors such as epidermal growth factor (EGF), platelet derived growth factor {PDGF), transforming growth factors (TGF-a and TGF (i), and insulin-like growth factor (IGF).
3 0 The therapeutic compositions are also presently valuable for veterinary applications. Particularly domestic animals and thoroughbred horses, in addition to humans, are desired patients for such treatment with proteins of the present invention.
The dosage regimen of a protein-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the proteins, e.g., amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors. The dosage may vary with the type of matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical composition. For example, the addition of other known growth factors, such as IGF I (insulin like growth factor I), to the final composition, may also effect the dosage. Progress can be monitored by periodic assessment of tissue/bone growth and/or repair, for example, X-rays, histomorphometric determinations and tetracycline labeling.
Polynucleotides of the present invention can also be used for gene therapy.
Such polynucleotides can be introduced either in vivo or ex vivo into cells for expression in a mammalian subject. Polynucleotides of the invention may also be administered by other known methods for introduction of nucleic acid into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA).
Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells.
Treated cells can then be introduced in vivo for therapeutic purposes.
2 0 Patent and literature references cited herein are incorporated by reference as if fully set forth.
SEQUENCE LISTING
<110> Jacobs, Kenneth MCCoy, John M.
LaVallie, Edward R.
Collins-Racie, Lisa A.
Merberg, David Treacy, Maurice Agostino, Michael J.
Steininger II, Robert J.
Wong, Gordon G.
Clark, Hilary Fechtel, Kim Genetics Institute, Inc.
<120> SECRETED PROTEINS AND POLYNUCLEOTIDES ENCODING THEM
<130> GI 6062A
<140>
<141>
<160> 32 <170> PatentIn Ver. 2.0 <210> 1 <211> 3149 <212> DNA
<213> Homo sapiens <400> 1 ggtctttaac gtgagcccgc tgcaggtgtg cggcccagtc cgagacagca gatgaggaga 60 ctgtccttcc tgtttcgcag atgaggaaac tgaggcttag agaagtttgg caaattggct 120 aagttcctac agctaccaca gcagaaagtg ctgggcagta gagagctgcc ccctccagaa 180 gatgatcagc tgcactccag tgcccccaga tcctcgtgga aggaacggat ccttaaagca 240 aaggtggtga cggtgtctca ggaggcagar tgggatcaaa tcgagccctt gcttagaagt 300 gaattagaag attttccagt acttggaatt gactgtgagt gggtaaattt ggaaggcaaa 360 gcctgccctc tgtcacttct acaaatggcc tccccaagtg gcctgtgtgt cttggttcgc 420 ctgcccaagc taatctgtgg aggaaaaaca ctaccaagaa cgttattgga tattttggca 480 gatggcacca ttttgaaagt tggagtggga tgctcagaag atgccagcaa gcttctgcag 540 gattatggcc tcgttgttag ggggtgcctg gacctccgat acctagccat gcggcagaga 600 aacaatttgc tctgtaatgg gcttagcctg aagtccctcg ctgagactgt tttgaacttt 660 ccccttgaca agtcccttct acttcgttgc agcaactggg atgctgagac tctcacagag 720 gaccaggtaa tttatgctgc cagggatgcc cagatttcag tggctctctt tcttcatctt 780 cttggatacc ctttctctag gaattcacct ggagaaaaaa aacgatgacc acagtagctg 840 gagaaaagtc ttggasaaat gccagggtgt ggtcgacatc ccatttcgaa gcaaaggaat 900 gagcagattg ggagaagagg ttaatgggga agcaacagaa tctcagcaga agccaagaaa 960 taagaagtct aagatggatg ggatggtgcc aggcaaccac caagggagag accccagaaa 1020 acataaaaga aagcctctgg gggtgggcta ttctgccaga aaatcacctc tttatgataa 1080 ctgctttctc catgctcctg atggacagcc cctctgcact tgtgatagaa gaaaagctca 1140 gtggtacctg gacaaaggca ttggtgagct ggtgagtgaa gagccctttg tggtgaagct 1200 gcggtttgaa cctgcaggaa ggcccgaatc tcctggagac tattacttga tggttaaaga 1260 gaacctgtgt gtagtgtgtg gcaagagaga ctcctacatt cggaagaacg tgattccaca 1320 tgagtaccgg aagcacttcc ccatcgagat gaaggaccac aactcccacg atgtgctgct 1380 gctctgcacc tcctgccatg ccatttccaa ctactatgac aaccatctga agcagcagct 1440 ggccaaggag ttccaggccc ccatcggctc tgaggagggc ttgcgcctgc tggaagatcc 1500 tgagcgccgg caggtgcgtt ctggggccag ggccctgctc aacgcggaga gcctgcctac 1560 tcatcgaaag gaggagctgc tgcaagcact cagagagttt tataacacag acgtggtcac 1620 agaggagatg cttcaagagg ctgccagcct ggagaccaga atctccaatg aaaactatgt 1680 tcctcacggg ctgaaggtgg tgcagtgtca cagccagggt ggcctgcgct ccctcatgca 1740 gctggagagc cgctggcgtc agcacttcct ggactccatg cagcccaagc acctgcccca 1800 gcagtggtca gtggaccaca accatcagaa gctgctccgg aaattcgggg aagatcttcc 1860 catccagctg tcttgatagc tgctttcctc ccagttagga caagtgggaa gctggagcca 1920 aggttgaaga gtcacctctt cccattttag tacatcatta attgtcaaag cctgtgtgac 1980 acaactcaga atactaacct agactaatcc caggatgctt ctgctggagc aaagatattg 2040 tttgaaggag agtttatggt tttggatttt aaacgggcag ggtctttttt cctctcattt 2100 ttgtggacaa gagaggcctt cgcctttatt tttactctcc ctcttctgct gtccctgtgc 2160 agaggaaaaa tgaagaattc tcccagaagt gacttgtcaa gacttaaaaa aaatgttttt 2220 aatgcatttc ttccttgtct agtgcctcgg tttatctcta acaggggctg tccagtatat 2280 cggtcctgtt aggaggggag aaaaagttct tccaaaggct ggagaagtga acaaggagtc 2340 aaatttattt tcccaattca acttcataat tatcatttct ttggcttcat gctctcccgt 2400 aactcatgtg gttgggatcc atcccatctg ggtcacttca gtctacttca cgtacttgaa 2460 aaggctttcc tttacacttc caggaccaaa cagcaacttc ctgccacaca cttccaccct 2520 atcactggga gaaatccttt tctggacatg agcctttgac ctgggtgggg cagaaagaac 2580 cacaaactcc atctcccaat agaactttga aattcactca gcttttcctt tcatgctgtt 2640 tgttgcctgc ttgttgcact cctcctgccc cagaactgca agatttttag cttcacccct 2700 ttctgagagt aatgttatct tttatcagaa tcagtatcag ttcccctgta ttctgtgctt 2760 catcgaattt gcaagactga cctcttttaa gcatttaatt cactcccaga gtcatctggt 2820 caggttgcaa tatgaggact tctctgtctc ctctgaagcc tgggacactg agcttactta 2880 atacattaga tgttcaaaag aggagcgttg tttcatcttt caaaatgtta ggccattact 2940 ttgaatataa aatcgactta ttaatgatta gtaatttttc taaagtattg ggaaaacttt 3000 cttattttat aagatcttaa caagcttaaa aaagaatttt atgaccagaa tccaacaaga 3060 gctctatttt ggaattgtgc ccaagttggt gatgtttact ctaaaattaa taataaaact 3120 acttgtaagc aaaaaaaaaa aaaaaaaaa 3149 <210> 2 <211> 380 <212> PRT
<213> Homo sapiens <400> 2 Met Leu Pro Gly Met Pro Arg Phe Gln Trp Leu Ser Phe Phe Ile Phe Leu Asp Thr Leu Ser Leu Gly Ile His Leu Glu Lys Lys Asn Asp Asp His Ser Ser Trp Arg Lys Val Leu Glu Lys Cys Gln Gly Val Val Asp Ile Pro Phe Arg Ser Lys Gly Met Ser Arg Leu Gly Glu Glu Val Asn Gly Glu Ala Thr Glu Ser Gln Gln Lys Pro Arg Asn Lys Lys Ser Lys Met Asp Gly Met Val Pro Gly Asn His Gln Gly Arg Asp Pro Arg Lys His Lys Arg Lys Pro Leu Gly Val Gly Tyr Ser Ala Arg Lys Ser Pro Leu Tyr Asp Asn Cys Phe Leu His Ala Pro Asp Gly Gln Pro Leu Cys Thr Cys Asp Arg Arg Lys Ala Gln Trp Tyr Leu Asp Lys Gly Ile Gly Glu Leu Val Ser Glu Glu Pro Phe Val Val Lys Leu Arg Phe Glu Pro Ala G1y Arg Pro Glu Ser Pro Gly Asp Tyr Tyr Leu Met Val Lys Glu Asn Leu Cys Val Val Cys Gly Lys Arg Asp Ser Tyr Ile Arg Lys Asn leo le5 190 Val Ile Pro His Glu Tyr Arg Lys His Phe Pro Ile Glu Met Lys Asp His Asn Ser His Asp Val Leu Leu Leu Cys Thr Ser Cys His Ala Ile Ser Asn Tyr Tyr Asp Asn His Leu Lys Gln Gln Leu Ala Lys Glu Phe Gln Ala Pro Ile Gly Ser Glu Glu Gly Leu Arg Leu Leu Glu Asp Pro Glu Arg Arg Gln Val Arg Ser Gly Ala Arg Ala Leu Leu Asn Ala Glu Ser Leu Pro Thr His Arg Lys Glu Glu Leu Leu Gln Ala Leu Arg Glu Phe Tyr Asn Thr Asp Val Val Thr Glu Glu Met Leu Gln Glu Ala Ala Ser Leu Glu Thr Arg Ile Ser Asn Glu Asn Tyr Val Pro His Gly Leu Lys Val Val Gln Cys His Ser Gln Gly Gly Leu Arg Ser Leu Met Gln Leu Glu Ser Arg Trp Arg Gln His Phe Leu Asp Ser Met Gln Pro Lys His Leu Pro Gln Gln Trp Ser Val Asp His Asn His Gln Lys Leu Leu Arg Lys Phe Gly Glu Asp Leu Pro Ile Gln Leu Ser <210> 3 <211> 1861 <212> DNA
<213> Homo sapiens <400> 3 agagccaggg gggtcgcgta gtgtcatgac cagggcggga gatcacaacc gccagagagg 60 atgctgtgga tccttggcgg actacctgac ctctgcaaaa ttccttctct accttggtca 120 ttctctctct acttggggag atcggatgtg gcactttgcg gtgtctgtgt ttctggtaga 180 gctctatgga aacagcctcc ttttgacagc agtctacggg ctggtggtgg cagggtctgt 240 tctggtcctg ggagccatca tcggtgactg ggtggacaag aatgctagac ttaaagtggc 300 ccagacctcg ctggtggtac agaatgtttc agtcatcctg tgtggaatca tcctgatgat 360 ggttttctta cataaacatg agcttctgac catgtaccat ggatgggttc tcacttcctg 420 ctatatcctg atcatcacta ttgcaaatat tgcaaatttg gccagtactg ctactgcaat 480 cacaatccaa agggattgga ttgttgttgt tgcaggagaa gacagaagca aactagcaaa 540 tatgaatgcc acaatacgaa ggattgacca gttaaccaac atcttagccc ccatggctgt 600 tggccagatt atgacatttg gctccccart catcggctgt ggctttattt cgggatggaa 660 cttggtatcc atgtgcgtgg agtacgttct gctctggaag gtttaccaga aaaccccagc 720 tctagctgtg aaagctggtc ttaaagaaga ggaaactgaa ttgaaacagc tgaatttaca 780 caaagatact gagccaaaac ccctggaggg aactcatcta atgggtgtga aagactctaa 840 catccatgag cttgaacatg agcaagagcc tacttgtgcc tcccagatgg ctgagccctt 900 ccgtaccttc cgagatggat gggtctccta ctacaaccag cctgtgtttc tggctggcat 960 gggtcttgct ttcctttata tgactgtcct gggctttgac tgcatcacca cagggtacgc 1020 ctacactcag ggactgagtg gttccatcct cagtattttg atgggagcat cagctataac 1080 tggaataatg ggaactgtag cttttacttg gctacgtcga aaatgtggtt tggttcggac 1140 aggtctgatc tcaggattgg cacagctttc ctgtttgatc ttgtgtgtga tctctgtatt 1200 catgcctgga agccccctgg acttgtccgt ttctcctttt gaagatatcc gatcaaggtt 1260 cattcaagga gagtcaatta cacctaccaa gatacctgaa attacaactg aaatatacat 1320 gtctaatggg tctaattctg ctaatattgt cccggagaca agtcctgaat ctgtgcccat 1380 aatctctgtc agtctgctgt ttgcaggcgt cattgctgct agaatcggtc tttggtcctt 1440 tgatttaact gtgacacagt tgctgcaaga aaatgtaatt gaatctgaaa gaggcattat 1500 aaatggtgta cagaactcca tgaactatct tcttratctt ctgcatttca tcatggtcat 1560 cctggctcca aatcctgaag cttttggctt gctcgtattg atttcagtct cctttgtggc 1620 aatgggccac attatgtatt tccgatttgc ccaaaatact ctgggaaaca agctctttgc 1680 ttgcggtcct gatgcaaaag aagttaggaa ggaaaatcaa gcaaatacat ctgttgtttg 1740 agacagttta actgttgcta tcctgttact agattatata gagcacatgt gcttattttg 1800 tactgcagaa ttccaataaa tggctgggtg tr_ttgctctg tttttaaaaa aaaaaaaaaa 1860 a <210> 4 <211> 571 <212> PRT
<213> Homo sapiens <220>
<221> UNSURE
<222> (202) <220>
<221> UNSURE
<222> (504) <400> 4 Met Thr Arg Ala Gly Asp His Asn Arg Gln Arg Gly Cys Cys Gly Ser Leu Ala Asp Tyr Leu Thr Ser Ala Lys Phe Leu Leu Tyr Leu Gly His Ser Leu Ser Thr Trp Gly Asp Arg Met Trp His Phe Ala Val Ser Val Phe Leu Val Glu Leu Tyr Gly Asn Ser Leu Leu Leu Thr Ala Val Tyr Gly Leu Val Val Ala Gly Ser Val Leu Val Leu Gly Ala Ile Ile Gly Asp Trp Val Asp Lys Asn Ala Arg Leu Lys Val Ala Gln Thr Ser Leu Val Val Gln Asn Val Ser Val Ile Leu Cys Gly Ile Ile Leu Met Met Val Phe Leu His Lys His Glu Leu Leu Thr Met Tyr His Gly Trp Val WO 99/376'74 PCTNS99/01404 Leu Thr Ser Cys Tyr Ile Leu Ile Ile Thr Ile Ala Asn Ile Ala Asn Leu Ala Ser Thr Ala Thr Ala Ile Thr Ile Gln Arg Asp Trp Ile Val Val Val Ala Gly Glu Asp Arg Ser Lys Leu Ala Asn Met Asn Ala Thr Ile Arg Arg Ile Asp Gln Leu Thr Asn Ile Leu Als Pro Met Ala Val Gly Gln Ile Met Thr Phe Gly Ser Pro Xaa Ile Gly Cys Gly Phe Ile Ser Gly Trp Asn Leu Val Ser Met Cys Val Glu Tyr Val Leu Leu Trp Lys Val Tyr Gln Lys Thr Pro Ala Leu Ala Val Lys Ala Gly Leu Lys Glu Glu Glu Thr Glu Leu Lys Gln Leu Asn Leu His Lys Asp Thr Glu Pro Lys Pro Leu Glu Gly Thr His Leu Met Gly Val Lys Asp Ser Asn Ile His Glu Leu Glu His Glu Gln Glu Pro Thr Cys Ala Ser Gln Met Ala Glu Pro Phe Arg Thr Phe Arg Asp Gly Trp Val Ser Tyr Tyr Asn Gln Pro Val Phe Leu Ala Gly Met Gly Leu Ala Phe Leu Tyr Met Thr Val Leu Gly Phe Asp Cys Ile Thr Thr Gly Tyr Ala Tyr Thr Gln Gly Leu Ser Gly Ser Ile Leu Ser Ile Leu Met Gly Ala Ser Ala Ile Thr Gly Ile Met Gly Thr Val Ala Phe Thr Trp Leu Arg Arg Lys Cys Gly Leu Val Arg Thr Gly Leu Ile Ser Gly Leu Ala Gln Leu Ser Cys Leu Ile Leu Cys Val Ile Ser Val Phe Met Pro Gly Ser Pro Leu Asp Leu Ser Val Ser Pro Phe Glu Asp Ile Arg Ser Arg Phe Ile Gln Gly Glu Ser Ile Thr Pro Thr Lys Ile Pro Glu Ile Thr Thr Glu Ile Tyr Met Ser Asn Gly Ser Asn Ser Ala Asn Ile Val Pro Glu Thr Ser Pro Glu Ser Val Pro Ile hle Ser Val Ser Leu Leu Phe Ala Gly Val Ile Ala Ala Arg Ile Gly Leu Trp Ser Phe Asp Leu Thr Val Thr Gln Leu Leu Gln Glu Asn Val Ile Glu Ser Glu Arg Gly Ile Ile Asn Gly Val Gln Asn Ser Met Asn Tyr Leu Leu Xaa Leu Leu His Phe Ile Met Val Ile Leu Ala Pro Asn Pro Glu Ala Phe Gly Leu Leu Val Leu Ile Ser Val Ser Phe Val Ala Met Gly His Ile Met Tyr Phe Arg Phe Ala Gln Asn Thr Leu Gly Asn Lys Leu Phe Ala Cys Gly Pro Asp Ala Lys Glu Val Arg Lys Glu Asn Gln Ala Asn Thr Ser Val Val <210> 5 <211> 2157 <212> DNA
<213> Homo Sapiens <400> 5 ctctctttaa tatcttcacc tctaccatgt gtctttcttt taatatagtt ataattttcc 60 aaccacgtag atcaatattt actcatcatg accataaaat gcagtttagc catatagaaa 120 actatgatta cttttcttta taatttccct tcagttaata cttattttat tttctgtttt 180 tatcatctag tcaactcgca aacttccagc atttgtctaa atctactcaa tatattccag 240 tacatcagat aatatatcag tttcatcctc ctgaaaaact cttttccagt gtatcctgac 300 ctgctctaat tttgacttga tgctttctgt atctggtgca cagctgttac cttggaatct 360 tcccttcatc attattcaga gtgtttctgt agtttttctc ttgcattgga ttttgtgctt 420 cctgaatccc tctctCtctt tttttttttt tttttacttg gcttactcct tgctttgatg 480 gatctcaggc tccagtagct tccttggaaa gagtgtttgg aagttgcttc tgcaggaagc 540 ctttttggtg gcatggtcct caagaagttc ctaaaaggtt gatgaaaagc ccagaacctt 600 gatgacagat tgtctggtta taaagcattt tttacgtaaa atcatcatgg tgcaccctaa 660 ggtcagattt catttcagtg taaaggtaaa tggaatcctc tccacagaga tctttggggt 720 ggagaatgaa cccactttga accttgggaa tggaattgct cttttggtcg actcccagca 780 ttatgtgagt agaccaaatt ttggtacaat tgaatcacac tgcagcagaa ttcaccctgt 840 gctaggacat ccagtaatgc ttttcatccc tgaagacgtg gctggcatgg acttgttggg 900 agaactgata ctgactccag cagctgcact gtgccccagc ccaaaggttt cttccaacca 960 gcttaacagg atttcttcag tttccatatt tctatatgga cctttgggtc tgcctctgat 1020 attgtcaact tgggagcagc cgatgactac tttcttcaaa gatacctctt ctttagttga 1080 ctggaaaata ccatttgtgt atgataccca atttggatct caatttggat agagatttgg 1140 tgcttccaga tgtgagttat caggtggaat ccagtgagga ggatcagtct cagactatgg 1200 atcctcaagg acasactctg ctgctttttc tctttgtgga tttccacagt gcatttccag 1260 tccagcaaat ggaaatctgg ggagtctata ctttgctcac aactcatctc aatgccatcc 1320 ttgtggagag ccacagtgta gtgcaaggtt ccatccaatt cactgtggac aaggtcttgg 1380 agcaacatca ccaggctgcc aaggctcagc agaaactaca ggcctcactc tcagtggctg 1440 tgaactccat catgagtatt ctgactggaa gcactaggag cagcttccga aagatgtgtc 1500 tccagaccct tcaagcagct gacacacaag agttcaggac caaactgcac aaagtatttc 1560 gtgagatcac ccaacaccaa tttcttcacc actgctcatg tgaggtgaag cagctaaccc 1620 tagaaaaaaaggactcagcc cagggcactg tgataacagcagcctggagc1680 aggacgcacc tcctagcagtgcttaaacag ccttcccagc aggggtacagcagctctcac1740 ccacagcagc attcagtcactagcagagat gccagatacc cagaaaacaagaggctcaag1800 agcgggcaag aggggcagcccccgcataga ggagatgcga ctgccagggccccgagcccg1860 gctctgcgct tcagaggccgccccgcgccg cccggaagcc ccctcactcytagaggaagg1920 accgcggccc gagcaccgcgaggctcacgg cagggccctg gggcgagcctcggaagccgc1980 gcgccgggca ctggaggacgtcctgtggct gcaggaggtc cagagtggctgagtcccagc2040 tccaacctgt cctgggccctgagccgggtc cccttccgca gatccggaggctgcgggcag2100 agcgcccacc ccgttatcccgtggtttaat aaagctgccg aaaaaaaaaaaaaaaaa 2157 cgcgctcacc <210>
<211>
<212>
PRT
<213> Sapiens Homo <400>
Met Ile Asn Leu Asp Leu Asn Leu Asp Leu Leu Pro Pro Asp Arg Val Asp Val Tyr Gln Val Glu Ser Ser Asp Gln Gln Thr Ser Glu Glu Ser Met Asp Gln Gly Gln Thr Leu Leu Leu Phe Asp Phe Pro Leu Phe Val His Ser Phe Pro Val Gln Gln Met Trp Gly Tyr Thr Ala Glu Ile Val Leu Leu Thr His Leu Asn Ala Ile Glu Ser Ser Val Thr Leu Val His Val Gln Ser Ile Gln Phe Thr Val Val Leu Gln His Gly Asp Lys Glu His Gln Ala Lys Ala Gln Gln Lys Ala Ser Ser Val Ala Leu Gln Leu Ala Val Ser Ile Met Ser Ile Leu Ser Thr Ser Ser Asn Thr Gly Arg Phe Arg Met Cys Leu Gln Thr Leu Ala Asp Gln Glu Lys Gln Ala Thr Phe Arg Lys Leu His Lys Val Phe Ile Thr His Gln Thr Arg Glu Gln Phe Leu His Cys Ser Cys Glu Val Leu Thr Glu Lys His Lys Gln Leu Lys Asp Ala Gln Gly Thr Glu Asp Asp Asn Ser Leu Ser Ala Pro Ser Glu Leu Ala Val Leu Lys Gln Pro Pro Thr Ala Gly Leu Ser Gln Ala Val Gln Leu Ser His Ser Val Thr Asp Ala Tyr Gln Gln Ser Arg Arg Arg Ala Arg Lys Gln Glu Ala Gln Gln Pro His Arg Ser Glu Gly Pro Gly Asp Ala Ser Ser Ala Leu Cys Gln Gly Pro GIu Pro Val Arg Gly Arg Pro Ala Pro Pro Gly Ser His Arg Gly Pro Pro His Ser <210> 7 <211> 1607 <212> DNA
<213> Homo Sapiens <400> 7 gtgaacttca ctactggaaa gcaacaaagg cagtcggcat aaaaatgggt tctctcagca 60 cagctaacgt tgaattttgc cttgatgtgt tcaaagagct gaacagtaac aacataggag 120 ataacatctt cttttcttcg ctgagtctgc tttatgctct aagcatggtc ctccttggtg 180 ccaggggaga gactgcagag caattggaga aggtgcttca ttttagtcat actgtagact 240 cattaaaacc agggttcaag gactcaccta agtgcagcca agctggaaga attcattccg 300 agtttggtgt ctaattctct caaatcaacc agccagactc taactgtacc ctcagcattg 360 ccaacaggct ctacgggaca aagacgatgg catttcatca ggaaaagtcg caaatctctt 420 tggaaagagc acaattgacc cttcatctgt aatggtcctg gtgaatacca tatatttcaa 480 aggacaatgg caaaataaat ttcaagtaag agagacagtt aaaagtcctt ttcagctaag 540 tgagggtaaa aatgtaactg tggaaatgat gtatcaaatt ggaacattta aactggcctt 600 tgtaaaggag ccgcagatgc aagttcttga gctgccctac gttaacaaca aattaagcat 660 gattattctg cttccagtag gcatagctaa tctgaaacag atagaaaagc agctgaattc 720 ggggacgttt catgagtgga caagctcttc taacatgatg gaaagagaag ttgaagtaca 780 cctccccaga ttcaaacttg aaattaagta tgagctaaat tccctgttaa aacctctagg 840 ggtgacagat ctcttcaacc aggtcaaagc tgatctttct ggaatgtcac caaccaaggg 900 cctatattta tcaaaagcca tccacaagtc atacctggat gtcagcgaag agggcacgga 960 ggcagcagca gccactgggg acagcatcgc tgtaaaaagc ctaccaatga gagctcagtt 1020 caaggcgaac caccccttcc tgttctttat aaggcacact cataccaaca cgatcctatt 1080 ctgtggcaag cttgcctctc cctaatcaga tggggttgag taaggctcag agttgcagat 1140 gaggtgcaga gacaatcctg tgactttccc acggccaaaa agctgttcac acctcacaca 1200 cctctgtgcc tcagtttgct catctgcaaa ataggtctag gatttcttcc aaccatttca 1260 tgagttgtga agctaaggct ttgttaatca tggaaaaagg tagacttatg cagaaagcct 1320 ttctggcttt cttatctgtg gtgtctcatt tgagtgctgt ccagtgacat gatcasgtca 1380 atgagtaaaa ttttaaggga ttagattttc ttgacttgta kgtatctgtg agatcttgaa 1440 taagtgacct gacatctctg cttaaagaaa accagctgaa gggcttcaac tttgcttgga 1500 tttttaaata ttttccttgc atatgtaaat agaatgtggt gagttttagt tcaasattct 1560 ctgttgagaa taataaatgc atgaaatacc ttaaaaaaaa aaaaaaa 1607 <210> 8 <211> 217 <212> PRT
<213> Homo Sapiens <400> 8 Met Val Leu Val Asn Thr Ile Tyr Phe Lys Gly Gln Trp Gln Asn Lys Phe Gln Val Arg Glu Thr Val Lys Ser Pro Phe Gln Leu Ser GIu Gly Lys Asn Val Thr Val Glu Met Met Tyr Gln Ile Gly Thr Phe Lys Leu Ala Phe Val Lys Glu Pro Gln Met Gln Val Leu Glu Leu Pro Tyr Val Asn Asn Lys Leu Ser Met Ile Ile Leu Leu Pro Val Gly Ile Ala Asn Leu Lys Gln Ile Glu Lys Gln Leu Asn Ser Gly Thr Phe His Glu Trp Thr Ser Ser Ser Asn Met Met Glu Arg Glu Val Glu Val His Leu Pro Arg Phe Lys Leu Glu Ile Lys Tyr Glu Leu Asn Ser Leu Leu Lys Pro Leu Gly Val Thr Asp Leu Phe Asn Gln Val Lys Ala Asp Leu Ser Gly Met Ser Pro Thr Lys Gly Leu Tyr Leu Ser Lys Ala Ile His Lys Ser Tyr Leu Asp Val Ser Glu Glu Gly Thr Glu Ala Ala Ala Ala Thr Gly Asp Ser Ile Ala Val Lys Ser Leu Pro Met Arg Ala Gln Phe Lys Ala Asn His Pro Phe Leu Phe Phe Ile Arg His Thr His Thr Asn Thr Ile Leu Phe Cys Gly Lys Leu Ala Ser Pro <210> 9 <211> 1537 <212> DNA
<213> Homo sapiens <400> 9 gtaggatttg gggatgtgga tatttaagac aatttctttt ttcttttggt ttaatagggg 60 cgggtatagg gaccaactgg gaccgagtgc ccagggggcc gagcacggtc atgctggccg 120 gcctgcatgc atgcgtgtgc cgggctgggc tgggcggccg gcggtcgtgg ggcagggttg 190 ggggtctgtg ctcagctgat aactgccatg cactgtactg cacacgtccc tagagcctac 240 cgggacccga cgcttttcag ggcatttctc cctccagcca gggcccaact cccacctgcc 300 tgggcgaatc tcctccaagg aagtcccagg aggatgggga ccaggaaggc tgtggacccc 360 catctccagg gggccttccc agcctgatcc ctgtcctcca agttctggag gaggccgctg 420 tagggtctgg ctgagcttcc cacccacttt ccctggtccc aatcctttct tgtcctatac 480 ccagctgggg ttgctgccct gaacgaactg cgtgtggggc cggcacatcc tagcaggcag 540 cccctggcgc ctgctgcctc agggatgctc caaccaccct cgttctcctc gcagtggccc 600 tggctcccac ctcccgcccc agcctgccgt ggggcccgtc agcctggtcc cacccccatg 660 gagaacccaa agtcttactg tatataactc caggtgacgt ttctatattt atagcagtgt 720 tgaaaaccca cgtgttttac acagaaccac cctctccaac ccctcccttc ccgaccccaa 780 caaaacgttt tcaaacccct tacagttcct ggggcaggcg gaaacaggct cacagattgt 840 gtgtcggctg cagcagtgat tccaacaagc agctattggg ggggaaacac agcatttaaa 900 aagatcatca ttaaaaaaca agatttatac aacaattact taggatgttt gtgatctgcc 960 gaccttgcta tagatgccat gttaccaatg atttcctgtg gtgggggctt gccattgttt 1020 actctcttat ttaccaactt ctggcctagg catgacagtg ggcaccttcc cccagccctg 1080 gctgggccca gcgcctgtgt tytgtgttag aaaggtttta tatatatata aaattacata 1140 tatakgtaga aatatatgta attttggggg ccctgttcct tgcacatttt acagttacct 1200 catttttccc atgtatgtat ttgagaaaat gctaatatat agagaaaaaa atggttctta 1260 aaacttaaat gtgtggtttt ttccattcca tgggattcac attggtttgt agcatttaac 1320 ataactagta tgttgtatta tatatatgtg tatactgatt gaaattttta acagatttgt 1380 WO 99/37674 PC"TNS99/01404 acttttttta aaatgaaagt tgctagttct gcttgaccaa gtagtgcaat cattattttt 1440 tttaatattg ttgctgattt cagagggata ttcactaata aatgtatgat gtatacccac 1500 graaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa 1537 <210> 10 <211> 86 <212> PRT
<213> Homo sapiens <400> 10 Met His Ala Cys Ala Gly Leu Gly Trp Ala Ala Gly Gly Arg Gly Ala Gly Leu Gly Val Cys Ala Gln Leu Ile Thr Ala Met His Cys Thr Ala His Val Pro Arg Ala Tyr Arg Asp Pro Thr Leu Phe Arg Ala Phe Leu Pro Pro Ala Arg Ala Gln Leu Pro Pro Ala Trp Ala Asn Leu Leu Gln Gly Ser Pro Arg Arg Met Gly Thr Arg Lys Ala Val Asp Pro His Leu Gln Gly Ala Phe Pro Ala <210> 11 <211> 1302 <212> DNA
<213> Homo sapiens <400> 11 cttcatggcc tacacacacc accttacccc tctgctggca agaggggacc tgattcatcc 60 tcacgctaaa cactcattct acccaactga ttgagacaga acagaagata aactgaaact 120 tctctgcctt cccgctgcaa gagtgaatga gcgatccctc tcaactgact caaaatgttt 180 gcctcaccca ggagatggag ctctcgaagg ccttctctgg ccagcggaca ctcctatctg 240 ccatcctcag catgctatca ctcagcttct ccacaacatc cctgctcagc aactactggt 300 ttgtgggcac acagaaggtg cccaagcccc tgtgcgagaa aggtctggca gccaagtgct 360 ttgacatgcc agtgtccctg gatggagata ccaacacatc cacccaggag gtggtacaat 420 acaactggga gactggggat gaccggttct ccttccggag cttccggagt ggcatgtggc 480 tatcctgtga ggaaactgtg gaagaaccag gggagaggtg ccgaagtttc attgaactta 540 caccaccagc caagagagaa atcctatggt tatccctggg aacgcagatc acctacatcg 600 gacttcaatt catcagcttc ctcctgctac taacagactt gctactcact gggaaccctg 660 cctgtgggct caaactgagc gcctttgctg ctgtttcctc tgtcctgtca ggtctcctgg 720 ggatggtggc ccacatgatg tattcacaag tcttccaagc gactgtcaac ttgggtccag 780 aagactggag accacatgtt tggaattatg gctgggcctt ctacatggcc tggctctcct 840 tcacctgctg catggcgtcg gctgtcacca ccttcaacac gtacaccagg atggtgctgg 900 agttcaagtg caagcatagt aagagcttca aggaaaaccc gaactgccta ccacatcacc 960 atcagtgttt ccctcggcgg ctgtcaagtg cagcccccac cgtgggtcct ttgaccagct 1020 accaccagta tcataatcag cccatccact ctgtctctga gggagtcgac ttctactccg 1080 agctgcggaa caagggattt caaagagggg ccagccagga gctgaaagaa gcagttaggt 1140 catctgtaga ggaagagcag tgttaggagt taagcgggtt tggggagtag gcttgagccc 1200 taccttacac gtctgctgat tatcaacatg tgcttaagcc aaaaaaaaaa aaaaaaaaaa 1260 aaseaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa as 1302 <210> 12 <211> 339 lU
<212> PRT
<213> Homo sapiens <400> 12 Met Ser Asp Pro Ser Gln Leu Thr Gln Asn Val Cys Leu Thr Gln Glu Met Glu Leu Ser Lys Ala Phe Ser Gly Gln Arg Thr Leu Leu Ser Ala Ile Leu Ser Met Leu Ser Leu Ser Phe Ser Thr Thr Ser Leu Leu Ser Asn Tyr Trp Phe Val Gly Thr Gln Lys Val Pro Lys Pro Leu Cys Glu Lys Gly Leu Ala Ala Lys Cys Phe Asp Met Pro Val Ser Leu Asp Gly Asp Thr Asn Thr Ser Thr Gln Glu Val Val Gln Tyr Asn Trp Glu Thr Gly Asp Asp Arg Phe Ser Phe Arg Ser Phe Arg Ser Gly Met Trp Leu Ser Cys Glu Glu Thr Val Glu Glu Pro Gly Glu Arg Cys Arg Ser Phe Ile Glu Leu Thr Pro Pro Ala Lys Arg Glu Ile Leu Trp Leu Ser Leu Gly Thr Gln Ile Thr Tyr Ile Gly Leu Gln Phe I1e Ser Phe Leu Leu Leu Leu Thr Asp Leu Leu Leu Thr Gly Asn Pro Ala Cys Gly Leu Lys Leu Ser Ala Phe Ala Ala Val Ser Ser Val Leu Ser Gly Leu Leu Gly Met Val Ala His Met Met Tyr Ser Gln Val Phe Gln Ala Thr Val Asn Leu Gly Pro Glu Asp Trp Arg Pro His Val Trp Asn Tyr Gly Trp Ala Phe Tyr Met Ala Trp Leu Ser Phe Thr Cys Cys Met Ala Ser Ala Val Thr Thr Phe Asn Thr Tyr Thr Arg Met Val Leu Glu Phe Lys Cys Lys His Ser Lys Ser Phe Lys Glu Asn Pro Asri Cys Leu Pro His His His Gln Cys Phe Pro Arg Arg Leu Ser Ser Ala Ala Pro Thr Val Gly Pro Leu Thr Ser Tyr His Gln Tyr His Asn Gln Pro Ile His Ser Val Ser VlrO 99/37674 PCT/US99/01404 Glu Gly Val Asp Phe Tyr Ser Glu Leu Arg Asn Lys Gly Phe Gln Arg Gly Ala Ser Gln Glu Leu Lys Glu Ala Val Arg Ser Ser Val Glu Glu Glu Gln Cys <210> 13 <211> 3184 <212> DNA
<213> Homo sapiens <220>
<221> unsure <222> (1644) <400> 13 gtgcatgctt gtaatcgcag ctacttcgga gcctgagaga ctccttcagg gtgagcaaag 60 gcctggaaaa acctgtatgc agataaagaa aaggaaagaa agagataatc agtgcatgca 120 gttgtcagct ggctgggacc tgaggagagt cacttgtgga ggcaactggt ctttatcccc 180 attgtccggt acaaggcagg cattaatcct gtgatcctta tctgaagctc agctacaagg 240 ctttggccga ccaagtgtgt accatgctgc tattgtcatc ttccttgaat tctttgcgtg 300 gggcctgttg acaactccaa tgttgactgt tctacatgaa acattttctc aacacacatt 360 cctcatgaat ggtctcattc aaggtgtaaa gggcctgctc tcttttttga gtgccccact 420 cattggtgcc ctgtctgatg tgtgggggag gaagcccttt ctcctcggca ctgtattctt 480 tacctgcttc ccaatcccac tgatgaggat cagcccatgg tggtattttg cgatgatttc 540 tgtgtctgga gtcttctcgg tcacgttttc tgttatattt gcctatgtag ctgatgtcac 600 tcaggagcac gagcgaagta cagcttatgg atgggtctca gccacctttg cggctagtct 660 tgtcagcagc ccggccattg gagcatatct ttctgccagt tacggagaca gcctcgttgt 720 gctggtggcc acagtggtgg ctcttctgga catctgcttc atcttagtgg ctgttccaga 780 atctctgcct gagaaaatga gaccggtttc ctggggagct cagatttctt ggaaacaagc 840 agaccctttt gcgtcgttga agaaagttgg aaaagattct actgtcttac taatctgcat 900 caccgtgttt ctttcatacc ttcctgaagc tggacagtat tcaagttttt ttctctatct 960 caggcaggtc ataggttttg gatctgttaa aattgcagca ttcatagcta tggtaggaat 1020 tctgtctatt gtggctcaga cggcctttct tagcatcttg atgagatcat taggaaataa 1080 gaatactgtc ctccttggct tgggcttcca gatgctccag ttagcctggt acggttttgg 1140 atcacaggcc tggatgatgt gggcagcagg gaccgtggct gccatgtcca gcatcacgtt 1200 tccggcaatc agtgccctcg tctctcggaa tgcagagtca gatcagcaag gagttgccca 1260 ggggatcata actggaataa gaggactatg caatggcctg gggccagcac tgtatggctt 1320 catattctac atgttccatg tggaactgac tgagttgggc ccgaaattga attctaacaa 1380 cgttcccctg cagggagctg tcatcccagg cccgccgttt ttatttgggg catgtatagt 1440 ccttatgtct tttctggttg ccttattcat tcctgaatac agtaaagcca gtggagttca 1500 aaaacacagt aacagcagca gcggcagcct gaccaacacc ccagaacggg gcagtgatga 1560 ggacattgag ccactactgc aagacagcag catctgggag ctctcttcat ttgaggagcc 1620 tgggaatcag tgcactgagc tgtnaactcg gcagaaagtg ggattctgca tacgccatct 1680 ctgagagcca tggagggagc cacacccctg gtgacttcat ggtgctggat gggagacgct 1740 agcggcatcc ttcagggcca agtttgataa ataccaccgc catcattctg ctcatcctcc 1800 tcctgttttt tttttttctc ttacattctt tttttttttc ctgtttatac attagaacaa 1860 gataagattt gaaatacttc cttgcaaata atgtgcaact cccaaggtga aactcaaata 1920 gaaaaagtca tctctctggt agaaaggatg gctttcctgt aatgactata gagtaagagt 1980 ggcagcaatc tttccatgcc cttttcagca gaaggcacag aacagtagcg ggactgccat 2040 ctctggcaag atttcaggta aagaatctct tcttaatttc taccttcctg tttctctgaa 2100 tcagcccata ggtgttgatg agtggccact cttaaagagt cactcagtat cagggatcta 2160 ctgtctttgt tcaaaggtca aataaaaacc tagtctcctt ttattctact ttctattctt 2220 agctagaatg aaactcagca tatatacact tctggacata ataatattga atagtaatta 2280 cctttactag atgaaagaaa ttttcattac aaacttasat catgtaaaac tcaacaactc 2340 agattcctgg acctggtgtc ctggttgggt ccaaggtgat tttacagaag aaaaaaacaa 2900 ctcaagcatt ctggtggcaa catagagatt gtaggctgct tctaagaaag ttattaacaa 2460 tttggaaatt cctaagtagg atgagagtta gtaactggat acgagtgaag tttatatcca 2520 agttcagact caaaggcatt attatgattt gcttcttccc atgtcttcca tgtcctgctt 2580 ctcaaagttt ttctcatcca tcacactcct gccttaactg ctctgagtat gcatttgttt 2640 tcaattcatc tttatttcaa tctgtttaac ttttgaatcg catgggaata cgcacattaa 2700 gttcctttct aaaataaggt tttatgaagc tgagtttcac gataagtgtc ttgctatttt 2760 ttgagatgtt ttatggacaa agaaaacttt acagatttat atgtattttg ctgcaccagt 2820 aaatggacca ttaactaggg cccaccttta acagagcacc cctttgaaag ttttataggt 2880 atgaaatata tgtagatatt tgtaaagggt tttaattttt tttttttgat ggggtgctgt 2940 gtaaatcttg tatttataaa tgtaatgaag gtattgacag aaaaaaatat atacaacttt 3000 tataaaggat tgtgtactga ctgaatacat ttaaaagaaa atatattttg aaacctgttc 3060 tgctatgaac agagataaca tatcttttta ctatgctatt ggtttttagg ttaagcttcc 3120 taatgcataa taaatttaca gtggttaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3180 aaaa 3184 <210> 14 <211> 454 <212> PRT
<213> Homo Sapiens <220>
<221> UNSURE
<222> (442) <400> 14 Met Leu Thr Val Leu His Glu Thr Phe Ser Gln His Thr Phe Leu Met Asn Gly Leu Ile Gln Gly Val Lys Gly Leu Leu Ser Phe Leu Ser Ala Pro Leu Ile Gly Ala Leu Ser Asp Val Trp Gly Arg Lys Pro Phe Leu Leu Gly Thr Val Phe Phe Thr Cys Phe Pro Ile Pro Leu Met Arg Ile Ser Pro Trp Trp Tyr Phe Ala Met Ile Ser Val Ser Gly Val Phe Ser Val Thr Phe Ser Val Ile Phe Ala Tyr Val Ala Asp Val Thr Gln Glu His Glu Arg Ser Thr Ala Tyr Gly Trp Val Ser Ala Thr Phe Ala Ala Ser Leu Val Ser Ser Pro Ala Ile Gly Ala Tyr Leu Ser Ala Ser Tyr Gly Asp Ser Leu Val Val Leu Val Ala Thr Val Val Ala Leu Leu Asp Ile Cys Phe Ile Leu Val Ala Val Pro Glu Ser Leu Pro Glu Lys Met Arg Pro Val Ser Trp Gly Ala Gln Ile Ser Trp Lys Gln Ala Asp Pro Phe Ala Ser Leu Lys Lys Val Gly Lys Asp Ser Thr Val Leu Leu Ile lao las 190 Cys Ile Thr Val Phe Leu Ser Tyr Leu Pro Glu Ala Gly Gln Tyr Ser Ser Phe Phe Leu Tyr Leu Arg Gln Val Ile Gly Phe Gly Ser Val Lys Ile Ala Ala Phe Ile Ala Met Val Gly Ile Leu Ser Ile Val Ala Gln Thr Ala Phe Leu Ser Ile Leu Met Arg Ser Leu Gly Asn Lys Asn Thr Val Leu Leu Gly Leu Gly Phe Gln Met Leu Gln Leu Ala Trp Tyr Gly Phe Gly Ser Gln Ala Trp Met Met Trp Ala Ala Gly Thr Val Ala Ala Met Ser Ser Ile Thr Phe Pro Ala Ile Ser Ala Leu Val Ser Arg Asn Ala Glu Ser Asp Gln Gln Gly Val Ala Gln Gly Ile Ile Thr Gly Ile Arg Gly Leu Cys Asn Gly Leu Gly Pro Ala Leu Tyr Gly Phe Ile Phe Tyr Met Phe His Val Glu Leu Thr Glu Leu Gly Pro Lys Leu Asn Ser Asn Asn Val Pro Leu Gln Gly Ala Val Ile Pro Gly Pro Pro Phe Leu Phe Gly Ala Cys Ile Val Leu Met Ser Phe Leu Val Ala Leu Phe Ile Pro Glu Tyr Ser Lys Ala Ser Gly Val Gln Lys His Ser Asn Ser Ser Ser Gly Ser Leu Thr Asn Thr Pro Glu Arg Gly Ser Asp Glu Asp Ile Glu Pro Leu Leu Gln Asp Ser Ser Ile Trp Glu Leu Ser Ser Phe Glu Glu Pro Gly Asn Gln Cys Thr Glu Leu Xaa Thr Arg Gln Lys Val Gly Phe Cys Ile Arg His Leu <210> 15 <211> 2481 <212> DNA
<213> Homo sapiens <400> 15 WO 99/37674 PC1'/US99/01404 aggtctagaa ttcaatcggg aagaaggaaa agttcccttc tgctgtgaaa ctatttggca 60 agaggctgga gggcccaatg gctgcaaaat cgcaacccaa cattcccaaa gccaagagtc 120 tagatggcgt caccaatgac agaaccgcat ctcaagggca gtggggccgt gcctgggagg 180 tggactggtt ttcactggcg agcgtcatct tcctactgct gttcgccccc ttcatcgtct 240 actacttcat catggcttgt gaccaataca gctgcgccct gaccggccct gtggtggaca 300 tcgtcaccgg acatgctcgg ctctcggaca tctgggccaa gactccacct ataacgagga 360 aagccgccca gctctatacc ttgtgggtca ccttccaggt gcttctgtac acgtctctcc 420 ctgacttctg ccataagttt ctacccggct acgtaggagg catccaggag ggggccgtga 480 ctcctgcagg ggttgtgaac aagtatcaga tcaacggcct gcaagcctgg ctcctcacgc 540 acctgctctg gtttgcaaac gctcatctcc tgtcctggtt ctcgcccacc atcatcttcg 600 acaactggat cccactgctg tggtgcgcca acatccttgg ctatgccgtc tccaccttcg 660 ccatggtcaa gggctacttc ttccccacca gcgccagaga ctgcaaattc acaggcaatt 720 tcttttacaa ctacatgatg ggcatcgagt ttaaccctcg gatcgggaag tggtttgact 780 tcaagctgtt cttcaatggg cgccccggga tcgtcgcctg gaccctcatc aacctgtcct 840 tcgcagcgaa gcagcgggag ctccacagcc atgtgaccaa tgccatggtc ctggtcaacg 900 tcctgcaggc catctacgtg attgacttct tctggaacga aacctggtac ctgaagacca 960 ttgacatctg ccatgaccac ttcgggtggt acctgggctg gggcgactgt gtctggctgc 1020 cttatcttta cacgctgcag ggtctgtact tggtgtacca ccccgtgcag ctgtccaccc 1080 cgcacgccgt gggcgtcctg ctgctgggcc tggtgggcta ctacatcttc cgggtggcca 1140 accaccagaa ggacctgttc cgccgcacgg atgggcgctg cctcatctgg ggcaggaagc 1200 ccaaggtcat cgagtgctcc tacacatccg ccgacgggca gaggcaccac agcaagctgc 1260 tggtgtcggg cttctggggc gtggcccgcc acttcaacta cgtcggcgac ctgatgggca 1320 gcctggccta ctgcctggcc tgtggcggtg gccacctgct gccctacttc tacatcatct 1380 acatggccat cctgctgacc caccgctgcc tccgggacga gcaccgctgc gccagcaagt 1440 acggccggga ctgggagcgc tacaccgccg cagtgcctta ccgcctgctg cctggaatct 1500 tctaagggca cgccctaggg agaagccctg tggggctgtc aagagcgtgt tctgccaggt 1560 ccatgggggc tggcatccca gctccaactc gaggagcctc agtttcctca tctgtaaact 1620 ggagagagcc cagcacttgg caggtgtcca gtacctaatc acgctctgtt ccttgctttt 1680 gccttcaagg gaattccgag tgtccagcac tgccgtattg ccagcacaga cggattttct 1740 ctaatcagtg tccctggggc aggaggatga cccagtcacc tttactagtc ctttggagac 1800 aatttacctg tattaggagc ccaggccacg ctacactctg cccacactgg tgagcaggag 1860 gtcttcccac gccctgtcat taggctgcat ttactcttgc taaataaaag tgggagtggg 1920 gcgtgcgcgt tatccatgta ttgcctttca gctctagatc cccctcccct gcctgctctg 1980 cagtcgtggg tggggcccgt gcgccgtttc tccttggtag cgtgcacggt gttgaactgg 2040 gacactgggg agaaaggggc tttcatgtcg tttccttcct gctcctgctg macagctgcc 2100 aggagtgctc tgcctggagt ctgcagacct cagagaggtc ccagcactgg ctgtggcctt 2160 tcaggtgtag gcaggtgggc tctgcttccc gattccctgt gagcgcccac cctctcgaaa 2220 gaattttctg cttgccctgt gactgtgcag actctggctc gagcaacccg gggaacttca 2280 ccctcagggg cctcccacac cttctccagc gaggaggtyt cagtcccagc ctcgggaggg 2340 cacctccttt tctgtgcttt cttccctgag gcattcttcc tcatccctag ggtgttgtgt 2400 agaactcttt ttaaactcta tgctccgagt agagttcatc tttatattaa acttcccctg 2460 ttcaaataaa aaaaaaaaaa a 2481 <210> 16 <211> 475 <212> PRT
<213> Homo sapiens <400> 16 Met Ala Ala Lys Ser Gln Pro Asn Ile Pro Lys Ala Lys Ser Leu Asp Gly Val Thr Asn Asp Arg Thr Ala Ser Gln Gly Gln Trp Gly Arg Ala Trp Glu Val Asp Trp Phe Ser Leu Ala Ser Val Ile Phe Leu Leu Leu Phe Ala Pro Phe Ile Val Tyr Tyr Phe Ile Met Ala Cys Asp Gln Tyr Ser Cys Ala Leu Thr Gly Pro Val Val Asp Ile Val Thr Gly His Ala Arg Leu Ser Asp Ile Trp Ala Lys Thr Pro Pro Ile Thr Arg Lys Ala Ala Gln Leu Tyr Thr Leu Trp Val Thr Phe Gln Val Leu Leu Tyr Thr Ser Leu Pro Asp Phe Cys His Lys Phe Leu Pro Gly Tyr Val Gly Gly Ile Gln Glu Gly Ala Val Thr Pro Ala Gly Val Val Asn Lys..Tyr Gln Ile Asn Gly Leu Gln Ala Trp Leu Leu Thr His Leu Leu Trp Phe Ala Asn Ala His Leu Leu Ser Trp Phe Ser Pro Thr Ile Ile Phe Asp Asn Trp Ile Pro Leu Leu Trp Cys Ala Asn Ile Leu Gly Tyr Ala Val Ser Thr Phe Ala Met Val Lys Gly Tyr Phe Phe Pro Thr Ser Ala Arg Asp Cys Lys Phe Thr Gly Asn Phe Phe Tyr Asn Tyr Met Met Gly Ile Glu Phe Asn Pro Arg Ile Gly Lys Trp Phe Asp Phe Lys Leu Phe Phe Asn Gly Arg Pro Gly Ile Val Ala Trp Thr Leu Ile Asn Leu Ser Phe Ala Ala Lys Gln Arg Glu Leu His Ser His Val Thr Asn Ala Met Val Leu Val Asn Val Leu Gln Ala Ile Tyr Val Ile Asp Phe Phe Trp Asn Glu Thr Trp Tyr Leu Lys Thr Ile Asp Ile Cys His Asp His Phe Gly Trp Tyr Leu Gly Trp Gly Asp Cys Val Trp Leu Pro Tyr Leu Tyr Thr Leu Gln Gly Leu Tyr Leu Val Tyr His Pro Val Gln Leu Ser Thr Pro His Ala Val Gly Val Leu Leu Leu Gly Leu Val Gly Tyr Tyr Ile Phe Arg Val Ala Asn His Gln Lys Asp Leu Phe Arg Arg Thr Asp Gly Arg Cys Leu Ile Trp Gly Arg Lys Pro Lys Val Ile Glu Cys Ser Tyr Thr Ser Ala Asp Gly Gln Arg His His Ser Lys Leu Leu Val Ser Gly Phe Trp Gly Val Ala Arg His Phe Asn Tyr Val Gly Asp Leu Met Gly Ser Leu Ala Tyr Cys Leu Ala Cys Gly Gly Gly His Leu Leu Pro Tyr Phe Tyr Ile Ile Tyr Met Ala Ile Leu Leu Thr His Arg Cys Leu Arg Asp Glu His Arg Cys Ala Ser Lys Tyr Gly Arg Asp Trp Glu Arg Tyr Thr Ala Ala Val Pro Tyr Arg Leu Leu Pro Gly Ile Phe <210> 17 <211> 1518 <212> DNA
<213> Homo sapiens <400> 17 cttccccact ggctcttggt ttatgagttc cccttttaag gatctgttgt gacttaccta 60 tctgggctag tgacctcaga tgtctcagac tgagcatctt accactgttt ctggttgatc 120 ccttcactca tggtcttaac acatttgcac ttcctctcat ctcagagagt acagtcacgg 180 ggcagagctt gcatagggat ccaggtgtta ctagtcttac tctggagctg gtccaactca 240 gtttcatggc acagaactag attaggtctc cactgcgcag tctgttttac tgcttaggga 300 aagccagctt ttctacccac acacgtttag tttgaagagt atctattttt ggagggttct 360 ttgggaggtt gggcaggctt ctttggatcc cagatacatt tagagctttt tgcattaagt 420 gtgaggaaaa taacttctct ttgatgatgt tgatacacca tgtkggcacc ytggggcaca 480 gcggtttagc tggggagatt ccatgagaat gaacecaaac tactcttctt tgctagggtc 540 ctttacccac acagaggtga gcctttcagg ttcttcattt tgcttagttt cttcccttgt 600 ccttggcatt taagaggcat ccatgtgtta gccagccaaa gccccctgaa ggagctggct 660 gctttaaagg atttacttgg gaggatgtca satggctttg ccttctgcag acttcattta 720 ttttaatctt tttatggctc ctttctcttg ctttaaaaca ggattataag cacacagcag 780 gtactgacac ctgaagtctt actaaattcc tgtcctcagg ccatcctttt tctcctgaaa 840 cctggactcc aattttcaat gacgtttttg tttttctctt tcaagcctaa ctatgggaca 900 gctttacgag aaggaaaaag atgaagatgg attcttatat gtggcctaca gcggagagaa 960 cacttttggc ttctgagggc cattgctggg ctaggtgcac cgtaactgct tgtgtatctt 1020 gtaaatagcc asccattttc agttattawa ccagaacctc ttmacataga cctattagtg 1080 catttgtaac tggatttatt tcttaatata tkggaaggtt ttgtttcctt agactagtaa 1140 attatcatac agagttttat tttgagtttt tctttttgtg cattgtcctc atgcctgtat 1200 tctccaggaa acttgtcctt ctggaaatca tatkgaatga tatttctata tcgaagtgag 1260 gtaggtgcgg tattaaagtg aaagggaagg tgatgcattt attctgggtt atgcttgaag 1320 tgttagatgg ctaagtatta aaattatcca aattaaatcc ttagcagtca gaacacttgc 1380 ttcactagaa tatgccaact gccaatcatg ttggactgag ctaatttgtt cctctttctg 1440 aaactattaa ggtaaataat taacaataaa aattctctta taaaggcaaa aaaaaaaaaa 1500 aaaaaaaaaa aaaaaasa 1518 <210> 18 <211> 55 <212> PRT
<213> Homo sapiens <400> 18 Met Val Leu Thr His Leu His Phe Leu Ser Ser Gln Arg Val Gln Ser Arg Gly Arg Ala Cys Ile Gly Ile Gln Val Leu Leu Val Leu Leu Trp Ser Trp Ser Asn Ser Val Ser Trp His Arg Thr Arg Leu Gly Leu His Cys Ala Val Cys Phe Thr Ala <210> 19 <211> 2097 <212> DNA
<213> Homo sapiens <400> 19 ctcttgagta cctggggctt gcagatgcat gccaccacac ccggctaatt tttttttttt 60 ttaaatagag atggggtctt gttctgttgc ccargctggt ctggaactcc tggcttcaat 120 cagtcctccc acctcagctt cccaaagctc tgggsttata ggcatgagcc actgtacctg 180 tccacctgag aaattttcta agcctggatt cagtcttatg aaatataata ctttgaaatg 240 cacaataact ttgaaaatga aactcattgc ttttcatttc accaggagtt actaactata 300 ataagcttta gagcaaattc tccttagata tgatttttgt tattattaga aacacatact 360 atcttgataa ctaaattttg ccaatcattc ttcttgacta gtggtcttta tatatacata 420 catatatata tatatataca tatatatata tatgaggaat tttccataag tgacttgaaa 480 aatacagaat gcactccatg gtaggtctgt tcagtgttat caggaatact gtttctcatc 540 ttcctttctt ggtgtccctt tgcaggggtt gtgtttgcac attatggtcc cgtctggaga 600 caacaaagga agttctctca ttcaactctt cgtcattttg ggttgggaaa acttagcttg 660 gagcccaaga ttattgagga gttcaaatat gtgaaagcag aaatgcaaaa gcacggagaa 720 gaccccttct gccctttctc catcatcagc aatgccgtct ctaacatcat ttgctccttg 780 tgctttggcc agcgctttga ttacactaat a9tgagttca agaaaatgct tggttttatg 840 tcacgaggcc tagaaatctg tctgaacagt caagtcctcc tggtcaacat atgcccttgg 900 ctttattacc ttccctttgg accatttaag gaattaagac aaattgaaaa ggatataacc 960 agtttcctta aaaaaatcat caaagaccat caagagtctc tggatagaga gaaccctcag 1020 gacttcatag acatgtacct tctccacatg gaagaggaga ggaaaaataa tagtaacagc 1080 agttttgatg aagagtactt attttatatc attggggatc tctttattgc tgggactgat 1140 accacaacta actctttgct ctggtgcctg ctgtatatgt cgctgaaccc cgatgtacaa 1200 gaaaaggttc atgaagaaat tgaaagagtc attggcgcca accgagctcc ttccctcaca 1260 gacaaggccc agatgcccta cacagaagcc accatcatgg aagtgcagag gctaactgtg 1320 gtggtgccgc ttgccattcc tcatatgacc tcagagaaca cagtgctcca agggtatacc 1380 attcctaaag gcacattgat cttacccaac ctgtggtcag tacatagaga cccagccatt 1440 tgggagaaac cggaggattt ctaccctaat cgatttctgg atgaccaagg acaactaatt 1500 aaaaaagaaa cctttattcc ttttgggata gggaagcggg tgtgtatggg agaacaactg 1560 gcaaagatgg aattattcct aatgtttgtg agcctaatgc agagtttcgc atttgcttta 1620 cctgaggatt ctaagaagcc cctcctgast ggaagatttg gtctaacttt agccccacat 1680 ccattta8ta taactatttc aaggagatga agagcatctc caagaagaga tggtaaaaag 1740 atatatsaat acatatcctt ctaagcagat tcttcctact gcaaaggaca gtgaatccag 1800 caactcagtg gatccaagct gggctcagag gtcggaagga gggtagagca cactgggagg 1860 tttcatcttg gaggattcct cagcaggata cttcagccat tttagtaatg caggtctgtg 1920 atttggggga tagaaaacaa agtacctatg aaacgggata tctggatttt acttgcagtg 1980 gcttccaccg atgggccaat cttctcattt cttagtgcct cagacatccc atatgtasaa 2040 tgagagtaat aaaacttggc ttctctctac ctctcagcac taaaaaaaaa aaaaaaa 2097 <210> 20 <211> 398 <212> PRT
<213> Homo sapiens <220>
1$
<221> UNSURE
<222> (379) <400> 20 -Val Leu Ser Gly Ile Leu Phe Leu Ile Phe Leu Ser Trp Cys Pro Phe Ala Gly Val Val Phe Ala His Tyr Gly Pro Val Trp Arg Gln Gln Arg Lys Phe Ser His Ser Thr Leu Arg His Phe Gly Leu Gly Lys Leu Ser Leu Glu Pro Lys Ile Ile Glu Glu Phe Lys Tyr Val Lys Ala Glu Met Gln Lys His Gly Glu Asp Pro Phe Cys Pro Phe Ser Ile Ile Ser Asn Ala Val Ser Asn Ile Ile Cys Ser Leu Cys Phe Gly Gln Arg Phe Asp Tyr Thr Asn Ser Glu Phe Lys Lys Met Leu Gly Phe Met Ser Arg Gly Leu Glu Ile Cys Leu Asn Ser Gln Val Leu Leu Val Asn Ile Cys Pro Trp Leu Tyr Tyr Leu Pro Phe Gly Pro Phe Lys Glu Leu Arg Gln Ile Glu Lys Asp Ile Thr Ser Phe Leu Lys Lys Ile Ile Lys Asp His Gln Glu Ser Leu Asp Arg Glu Asn Pro Gln Asp Phe Ile Asp Met Tyr Leu Leu His Met Glu Glu Glu Arg Lys Asn Asn Ser Asn Ser Ser Phe Asp Glu Glu Tyr Leu Phe Tyr Ile Ile Gly Asp Leu Phe Ile Ala Gly Thr Asp Thr Thr Thr Asn Ser Leu Leu Trp Cys Leu Leu Tyr Met Ser Leu Asn Pro Asp Val Gln Glu Lys Val His Glu Glu Ile Glu Arg Val Ile Gly Ala Asn Arg Ala Pro Ser Leu Thr Asp Lys Ala Gln Met Pro Tyr Thr Glu Ala Thr Ile Met Glu Val Gln Arg Leu Thr Val Val Val Pro Leu Ala Ile Pro His Met Thr Sex Glu Asn Thr Val Leu Gln Gly Tyr Thr Ile Pro Lys Gly Thr Leu Ile Leu Pro Asn Leu Trp Ser Val His Arg Asp Pro Ala Ile Trp Glu Lys Pro Glu Asp Phe Tyr Pro Asn Arg Phe Leu Asp Asp Gln Gly Gln Leu Ile Lys Lys Glu Thr Phe Ile Pro Phe Gly Ile Gly Lys Arg Val Cys Met Gly Glu Gln Leu Ala Lys Met Glu Leu Phe Leu Met Phe Vai Ser Leu Met Gln Ser Phe Ala Phe Ala Leu Pro Glu Asp Ser Lys Lys Pro Leu Leu Xaa Gly Arg Phe Gly Leu Thr Leu Ala Pro His Pro Phe Asn Ile Thr Ile Ser Arg Arg <210> 21 <211> 29 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotide <220>
<221> misc_feature <222> (2) <223> biotinylated phosphoaramidite residue <400> 21 cnaagttcta ttgggagatg gagtttgtg 29 <210> 22 <211> 29 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotide <220>
<221> misc_feature <222> (2) <223> biotinylated phosphoaramidite residue <400> 22 cnatccatgg tacatggtca gaagctcat 29 <210> 23 <211> 29 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotide <220>
<221> misc_feature <222> (2) <223> biotinylated phosphoaramiditeresidue <400> 23 tngagcaggt caggatacac tggaaaaga 29 <210> 24 <211> 29 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotide <220>
<221> misc_feature <222> (2) <223> biotinylated phosphoaramiditeresidue <400> 24 cnactgcctt tgttgctttc cagtagtga 29 <210> 25 <211> 29 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotide <220>
<221> misc_feature <222> (2) <223> biotinylated phosphoaramiditeresidue <400> 25 tnaatatcca catccccaaa tcctacacg 29 <210> 26 <211> 29 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotide <220>
<221> misc_feature <222> (2) <223> biotinylated phosphoaramiditeresidue <400> 26 cncttgcagc gggaaggcag agaagtttc 29 <210> 27 <211> 29 <212> DNA
<213> Artificial Sequence VSO 99/376'74 PCT/US99/01404 <220>
<223> oligonucleotide <220>
<221> misc_feature <222> (2) <223> biotinylated phosphoaramidite residue <400> 27 cntgagccac aatagacaga attcctacc 29 <210> 28 <211> 29 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotide <220>
<221> misc_feature <222> (2) <223> biotinylated phosphoaramidite residue <400> 28 cngtcagggc gcagctgtat tggtcacaa 29 <210> 29 <211> 19 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotide <400> 29 acccacacag aagtgagcc 19 <210> 30 <211> 147 <212> PRT
<213> Homo sapiens <400> 30 Met Ala Ser Pro Ser Gly Leu Cys Val Leu Val Arg Leu Pro Lys Leu Ile Cys Gly Gly Lys Thr Leu Pro Arg Thr Leu Leu Asp Ile Leu Ala Asp Gly Thr Ile Leu Lys Val Gly Val Gly Cys Ser Glu Asp Ala Ser Lys Leu Leu Gln Asp Tyr Gly Leu Val Val Arg Gly Cys Leu Asp Leu Arg Tyr Leu Ala Met Arg Gln Arg Asn Asn Leu Leu Cys Asn Gly Leu Ser Leu Lys Ser Leu Ala Glu Thr Val Leu Asn Phe Pro Leu Asp Lys Ser Leu Leu Leu Arg Cys Ser Asn Trp Asp Ala Glu Thr Leu Thr Glu ' Asp Gln Val Ile Tyr Ala Ala Arg Asp Ala Gln Ile Ser Val Ala Leu Phe Leu His Leu Leu Gly Tyr Pro Phe Ser Arg Asn Ser Pro Gly Glu Lys Lys Arg <210> 31 <211> 176 <212> PRT
<213> Homo sapiens <400> 31 Met Thr Asp Cys Leu Val Ile Lys His Phe Leu Arg Lys Ile Ile Met Val His Pro Lys Val Arg Phe His Phe Ser Val Lys Val Asn Gly Ile Leu Ser Thr Glu Ile Phe Gly Val Glu Asn Glu Pro Thr Leu Asn Leu Gly Asn Gly Ile Ala Leu Leu Val Asp Ser Gln His Tyr Val Ser Arg Pro Asn Phe Gly Thr Ile Glu Ser His Cys Ser Arg Ile His Pro Val Leu Gly His Pro Val Met Leu Phe Ile Pro Glu Asp Val Ala Gly Met Asp Leu Leu Gly Glu Leu Ile Leu Thr Pro Ala Ala Ala Leu Cys Pro Ser Pro Lys Val Ser Ser Asn Gln Leu Asn Arg Ile Ser Ser Val Ser Ile Phe Leu Tyr Gly Pro Leu Gly Leu Pro Leu Ile Leu Ser Thr Trp Glu Gln Pro Met Thr Thr Phe Phe Lys Asp Thr Ser Ser Leu Val Asp Trp Lys Ile Pro Phe Val Tyr Asp Thr Gln Phe Gly Ser Gln Phe Gly <210> 32 <211> 89 <212> PRT
<213> Homo Sapiens VIrO 99/37674 PCT/US99/OI404 <400> 32 Met Gly Ser Leu Ser Thr Ala Asn Val Glu Phe Cys Leu Asp Val Phe Lys Glu Leu Asn Ser Asn Asn Ile Gly Asp Asn Ile Phe Phe Ser Ser Leu Ser Leu Leu Tyr Ala Leu Ser Met Val Leu Leu Gly Ala Arg Gly Glu Thr Ala Glu Gln Leu Glu Lys Val Leu His Phe Ser His Thr Val Asp Ser Leu Lys Pro Gly Phe Lys Asp Ser Pro Lys Cys Ser Gln Ala Gly Arg Ile His Ser Glu Phe Gly Val
140:508-512, 1988.
Immune Stimulating or Su~ressing, 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 2 0 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 infections, or may result from autoimmune disorders. More specifically, infectious 2 5 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. C~f course, in this regard, a protein of the present invention may also be useful where a boost to the immune system generally may be desirable, i.e., in the 3 0 treatment of cancer.
Autoimmune disorders which may be treated using a protein of the present inv~tion include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye disease.
Such a protein of the present invention may also to be useful in the treatment of allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems. Other conditions, in which immune suppression is desired (including, for example, organ transplantation), may also be treatable using a protein of the present invention.
Using the proteins of the invention it may also be possible to regulate 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 2 0 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 transplantation. Typically, in tissue transplants, rejection of the transplant is initiated 2 5 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 monomeric form of a peptide having an activity of another B lymphocyte antigen (e.g., B7-3 0 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 immunosuppressant. Moreover, the lack of costimulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject. Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents. To achieve sufficient immunosuppression or tolerance in a subject, it may also be necessary to block the function of a combination of B lymphocyte antigens.
The efficacy of particular blocking reagents in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans. Examples of appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow et al., Science 257:789-792 (1992) and Turka et al., Proc. Natl. Acad.
Sci USA, 89:11102-11105 {1992). In addition, 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 2 0 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 activation and prevent production of autoantibodies or T cell-derived cytokines which may be involved in the disease process. Additionally, blocking reagents may induce 2 5 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 human autoimmune diseases. Examples include marine experimental autoimmune encephalitis, systemic lupus erythmatosis in MRL/lpr/Ipr mice or NZB hybrid mice, 3 0 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).
Upregulation of an antigen function (preferably a B lymphocyte antigen function), as a means of up regulating immune responses, may also be useful in therapy.
Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response. For example, enhancing an immune response through stimulating B lymphocyte antigen function may be useful in cases of viral infection. In addition, systemic viral diseases such as influenza, the common cold, and encephalitis might be alleviated by the administration of stimulatory forms of B
lymphocyte antigens systemically.
Alternatively, anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen-pulsed APCs either expressing a peptide of the present invention or together with a stimulatory form of a soluble peptide of the present invention and reintroducing the in vitro activated T cells into the patient. Another method of enhancing anti-viral immune responses would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding a protein of the present invention as described herein such that the cells express all or a portion of the protein on their surface, and reintroduce the transfected cells into the patient. The infected cells would now be capable of delivering a 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, 2 0 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 ran be transfected to express a combination of peptides.
For example, tumor cells obtained from a patient can be transfected ex vivo with an expression vector directing the expression of a peptide having B7-2-like activity alone, or in conjunction with a peptide having B7-2-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 to target a tumor cell for transfection in vivo.
The presence of the peptide of the present invention having the activity of a B
3 0 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
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 (iZ
microglobulin protein or an MHC class II a chain protein and an MHC class II
~i chain protein to thereby express MHC class I or MHC class II proteins on the cell surface.
Expression of the appropriate class I or class II MHC in conjunction with a peptide having the activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a T
cell mediated immune response against the transfected tumor cell. Optionally, a gene encoding an antisense construct which blocks expression of an MHC class II associated protein, such as the invariant chain, can also be cotransfected with a DNA encoding a peptide having the activity of a B lymphocyte antigen to promote presentation of tumor associated antigens and induce tumor specific immunity. Thus, the induction of a T cell mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject.
The activity of a protein of the invention may, among other means, be measured by the following methods:
Suitable assays for thymocyte or splenocyte cytotoxicity include, without 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.
2 0 USA 78:248&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, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et ai., 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 immunoglobuiin responses and isotype switching (which will identify, among others, proteins that modulate T-cell dependent antibody responses and that affect Thl /Th2 profiles) include, without limitation, those described 3 0 in: Maliszewski, J. Imrnunol.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.
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.
ICruisbeek, D.H. Margulies, E.M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol.137:3494-3500,1986; Takai et al., J. Immunol. 140:508-5I2, 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; Macatorua 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, 2 0 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 development include, without limitation, those described in: Antica et al., Blood 84:111-I17, 1994; Fine et al., Cellular Immunology 155:111-122,1994; Galy et al., Blood 2 5 85:2770-2778,1995; Toki et al., Proc. Nat. Acad Sci. USA 88:7548-7551,1991.
Hematopoiesis Reg~,, ' a ActivitX
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 3 0 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 progenitox cells alone or in combination with other cytokines, thereby indicating utility, for example, in treating various anemias or for use in conjunction with irradiation/chemotherapy to stimulate the production of erythroid precursors and/or erythroid cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF
activity) useful, for example, in conjunction with chemotherapy to prevent or treat consequent myelo-suppression; in supporting the growth and proliferation of megakaryocytes and consequently of platelets thereby allowing prevention or treatment of various platelet disorders such as thrombocytoperua, and generally for use in place of or complimentary to platelet transfusions; and/or in supporting the growth and proliferation of hematopoietic stem cells which are capable of maturing to any and all of the above-mentioned hematopoietic cells and therefore find therapeutic utility in various stem cell disorders (such as those usually treated with transplantation, including, without limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well as in repopulating the stem cell compartment post irradiation/chemotherapy, either in-vivo or ex-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.
2 0 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 al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al., Blood 81:2903-2915,1993.
2 5 Assays for stem cell survival and differentiation (which will identify, among others, proteins that regulate lympho-hematopoiesis) include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, M.G. In Culture of Hematopoietic Cells. R.L 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 3 0 hematopoietic colony forming cells with high proliferative potential, McNiece, LK. and Briddell, R.A. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds.
Vol pp. 23-39, Wiley-Liss, Inc., New York, NY.1994; Neben et al., Experimental Hematology 22:353-359, 1994; Cobblestone area forming cell assay, Ploemacher, R.E. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. l-21, Wiley-Liss, lnc.., New York, NY.1994; Long term bone marrow cultures in the presence of stromal cells, Spooncer, E., Dexter, M. and Allen, T. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 163-179, Wiley-Liss, Inc., New York, NY.1994; Long term culture initiating cell assay, Sutherland, H.J. In Culture of Hematopoietic Cells. R.I. Freshney, et al. eds. Vol pp. 139-162, Wiley-Liss, Inc., New York, NY. 1994.
Tissue Growth Activity A protein of the present invention also may have utility in compositions used for bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration, as well as for wound healing and tissue repair and replacement, and in the treatment of burns, incisions and ulcers.
A protein of the present invention, which induces cartilage and/or bone growth in circumstances where bone is not normally formed, has application in the healing of bone fractures and cartilage damage or defects in humans and other animals.
Such a preparation employing a protein of the invention may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints. De novo bone formation induced by an osteogeruc agent contributes to the repair of congenital, trauma induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic plastic surgery.
2 0 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 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.
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 3 0 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 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 nova tendon/ligament-like tissue formation induced by a composition of the present invention contributes to the repair of congerutai, 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- ar 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 viva for return in viva 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 2 0 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 invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma and cerebrovascular diseases such as stroke. Peripheral neuropathies resulting 2 5 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 non-healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like.
3 0 It is expected that a protein of the present invention may also exhibit activity for generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac) and vascular (including vascular endothelium) tissue, or for promoting the growth of cells comprising such tissues. Part of the desired effects may be by inhibition or modulation WO 99!37674 PCT/US99/01404 of fibrotic scarring to allow normal tissue to regenerate. A protein of the invention may also exhibit angiogeruc activity.
A protein of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokine damage.
A protein of the present invention may also be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells; or for inhibiting the growth of tissues described above.
The activity of a protein of the invention may, among other means, be measured by the following methods:
Assays for tissue generation activity include, without limitation, those described in: International Patent Publication No. W095/16035 (bone, cartilage, tendon);
International Patent Publication No. W095/05846 (nerve, neuronal);
International Patent Publication No. WCr91/07491 (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).
2 0 Activin /Inhibin ActivitX
A protein of the present invention may also exhibit activin- or inhibin-related activities. Inhibins are characterized by their ability to inhibit the release of follicle stimulating hormone (FSH), while activins and are characterized by their ability to stimulate the release of follicle stimulating hormone (FSH). Thus, a protein of the present 2 5 invention, alone or in heterodimers with a member of the inhibin a family, may be useful as a contraceptive based on the ability of inhibins to decrease fertility in female mammals and decrease spermatogenesis in male mammals. Administration of sufficient amounts of other inhibins can induce infertility in these mammals. Alternatively, the protein of the invention, as a homodimer or as a heterodimer with other protein subunits of the inhibin-3 0 ~ group, may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells of the anterior pituitary.
See, for example, United States Patent 4,798,885. A protein of the invention may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as cows, sheep and pigs.
The activity of a protein of the invention may, among other means, be measured by the following methods:
Assays for activin/inhibin activity include, without limitation, those described in:
Vale et al., Endocrinology 91:562-572,1972; Ling et al., Nature 321:779-782,1986; Vale et al., Nature 321:776-779,1986; Mason et al., Nature 318:659-663,1985; Forage et al., Proc.
Natl. Acad. Sci. USA 83:3091-3095,1986.
Chemotactic/Chemokinetic ActivitX
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 2 0 population. Preferably, the protein or peptide has the ability to directly stimulate directed movement of cells. Whether a particular protein has chemotactic activity for a population of cells can be readily determined by employing such protein or peptide in any known assay for cell chemotaxis.
The activity of a protein of the invention may, among other means, be measured 2 5 by the following methods:
Assays for chemotactic activity (which will identify proteins that induce or prevent chemotaxis) consist of assays that measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population. Suitable assays for movement and adhesion 3 0 include, without limitation, those described in: Current Protocols in Immunology, Ed by J.E. Coligan, A.M. ICruisbeek, D.H. Margulies, E.M. Shevach, W.Strober, Pub.
Greene Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:1370-1376,1995; Lind et al.
APMIS 103:140-146, 1995; Muller et al Eur. J. Immunol. 25: 1744-1748; Gruber et al. J. of Immunol. 152:5860-5867,1994; Johnston et al. J. of Immunol. 153: 1762-1768,1994.
HemoStatic and Thrombolvtic Activity A protein of the invention may also exhibit hemostatic or thrombolytic activity.
As a result, such a protein is expected to be useful in treatment of various coagulation disorders (including hereditary disorders, such as hemophilias) or to enhance coagulation and other hemostatic events in treating wounds resulting from trauma, surgery or other causes. A protein of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system vessels (e.g., stroke).
The activity of a protein of the invention may, among other means, be measured by the following methods:
Assay for hemostatic and thrombolytic activity include, without limitation, those described in: Linet et 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.
2 0 Receptor/Ligand,~, Acrivitv A protein of the present invention may also demonstrate activity as receptors, receptor ligands or inhibitors or agonists of receptor/ligand interactions.
Examples of such receptors and ligands include, without limitation, cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, 2 5 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 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 3 0 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.
The activity of a protein of the invention may, among other means, be measured by the following methods:
VlrO 99/37674 PCTNS99/01404 Suitable assays for receptor-ligand activity include without limitation those described in:Current Protocols in Immunology, Ed by J.E. Coligan, A.M.
Kruisbeek, D.H.
Margulies, E.M. Shevach, W.Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7.28.1-7.28.22), Takai et al., Proc. Natl. Acad. Sci: USA 84:6864-6868, 1987;
Bierer et al., J. Exp. Med.168:1145-1156, 1988; Rosenstein et al., J. Exp.
Med.169:149-160 1989; Stoltenborg et al., J. Immunol. Methods 175:59-68,1994; Stitt et al., Cell 80:661-670, 1995.
Anti-Inflammatory Activity Proteins of the present invention may also exhibit anti-inflammatory activity.
The anti-inflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or promoting cell-cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or 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 2 0 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 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 Invasio~,Su~~ressor ActivitX
Cadherins are calcium-dependent adhesion molecules that appear to play major roles during development, particularly in defining specific cell types. Loss or alteration of normal cadherin expression can lead to changes in cell adhesion properties linked to 3 0 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.
The cadherin superfamily includes well over forty members, each with a distinct pattern of expression. All members of the superfamily have in common conserved extracellular repeats (cadherin domains), but structural differences are found in other parts of the molecule. The cadherin domains bind calcium to form their tertiary structure and thus calcium is required to mediate their adhesion. Only a few amino acids in the first cadherin domain provide the basis for homophilic adhesion; modification of this recognition site can change the specificity of a cadherin so that instead of recognizing only itself, the mutant molecule can now also bind to a different cadherin. In addition, some cadherins engage in heterophilic adhesion with other cadherins.
E-cadherin, one member of the cadherin superfamily, is expressed in epithelial cell types. Pathologically, if E-cadherin expression is lost in a tumor, the malignant cells become invasive and the cancer metastasizes. Transfection of cancer cell lines with polynucleotides expressing E-cadherin has reversed cancer-associated changes by returning altered cell shapes to normal, restoring cells' adhesiveness to each other and to their substrate, decreasing the cell growth rate, and drastically reducing anchorage-independent cell growth. Thus, reintroducing E-cadherin expression reverts carcinomas to a less advanced stage. It is likely that other cadherins have the same invasion suppressor role in carcinomas derived from other tissue types. Therefore, proteins of the present invention with cadherin activity, and polynucleotides of the present invention encoding such proteins, can be used to treat cancer. Introducing such proteins or polynudeotides into cancer cells can reduce or eliminate the cancerous changes observed 2 0 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 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 2 5 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 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 3 0 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 cancer, the less cadherin expression there will be, and this decrease in cadherin expression can be detected by the use of a cadherin-binding antibody.
Fragments of proteins of the present invention with cadherin activity, preferably a polypeptide comprising a decapeptide of the cadherin recognition site, and poly-nucleotides of the present invention encoding such protein fragments, can also be used to block cadherin function by binding to cadherins and preventing them from binding in ways that produce undesirable effects. Additionally, fragments of proteins of the present invention with cadherin activity, preferably truncated soluble cadherin fragments which have been found to be stable in the circulation of cancer patients, and polynucleotides encoding such protein fragments, can be used to disturb proper cell-cell adhesion.
Assays for cadherin adhesive and invasive suppressor activity include, without limitation, those described in: Hortsch et al. J Biol Chem 270 (32): 18809-18817, 1995;
Miyaki et al. Oncogene 11: 2547-2552,1995; Ozawa et al. Cell 63: 1033-1038,1990.
Tumor Inhibition Activity In addition to the activities described above for immunological treatment or prevention of tumors, a protein of the invention may exhibit other anti-tumor activities.
A protein may inhibit tumor growth directly or indirectly (such as, for example, via antibody-dependent cell-mediated cytotoxicity (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 2 0 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.
9~her Activities 2 5 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, weight, hair color, eye color, skin, fat to lean ratio or other tissue pigmentation, or organ 3 0 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, carbohydrate, vitamins, minerals, cofactors or other nutritional factors or component(s);
effecting behavioral characteristics, including, without limitation, appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders) and violent behaviors; providing analgesic effects or other pain reducing effects;
promoting differentiation and growth of embryonic stem cells in lineages other than hematopoietic lineages; hormonal or endocrine activity; in the case of enzymes, correcting deficiencies of the enzyme and treating deficiency-related diseases; treatment of hyperproliferative disorders (such as, for example, psoriasis); immunoglobulin-like activity (such as, for example, the ability to bind antigens or complement); and the ability to act as an antigen in a vaccine composition to raise an immune response against such protein or another material or entity which is cross-reactive with such protein.
ADMINISTRATION ANA DOSI_NG
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 2 0 effectiveness of the biological activity of the active ingredient(s). The characteristics of the carrier will depend on the route of administration. The pharmaceutical composition of the invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL.-2, IL.-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IFN, TNFO, TNF1, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem 2 5 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 3 0 in formulations of the particular cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent.
A protein of the present invention may be active in multimers (e.g., heterodimers 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 muitimeric or complexed form.
The pharmaceutical composition of the invention may be in the form of a complex of the proteins) of present invention along with protein or peptide antigens.
The protein and/or peptide antigen will deliver a stimulatory signal to both B and T
lymphocytes. B
lymphocytes will respond to antigen through their surface immunoglobulin receptor. T
lymphocytes will respond to antigen through the T cell receptor (TCR) following presentation of the antigen by MHC proteins. MHC and structurally related proteins including those encoded by class I and class II NiHC 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 2 0 pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the 2 5 art, as disclosed, for example, in U.S. Patent No. 4,235,871; U.S. Patent No. 4,501,728; U.S.
Patent No. 4,837,028; and U.S. Patent No. 4,737,323, all of which are incorporated herein by reference.
As used herein, the term "therapeutically effective amount" means the total amount of each active component of the pharmaceutical composition or method that is 3 0 sufficient to show a meaningful patient benefit, i.e., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.
In practicing the method of treatment or use of the present invention, a therapeutically effective amount of protein of the present invention is administered to a mammal having a condition to be treated. Protein of the present invention may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing cytolcines, lympholcines or other hematopoietic factors. When co-administered with one or more cytolcines, lymphokines or other hematopoietic factors, protein of the present invention may be administered either simultaneously with the cytolcine(s), lympholcine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or sequentially. If administered sequentially, the attending physician will decide on the appropriate sequence of administering protein of the present invention in combination with cytolcine(s), lympholcine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors.
Administration of protein of the present invention used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection.
2 0 Intravenous administration to the patient is preferred.
When a therapeutically effective amount of protein of the present invention is administered orally, protein of the present invention will be in the form of a tablet, capsule, powder, solution or elixir. When administered in tablet form, the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or 2 5 an adjuvant. The tablet, capsule, and powder contain from about 5 to 95%
protein of the present invention, and preferably from about 25 to 90% protein of the present invention.
When administered in liquid form, a liquid Garner such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. The liquid form of the pharmaceutical composition may further contain 3 0 physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90% by weight of protein of the present invention, and preferably from about 1 to 50% protein of the present invention.
When a therapeutically effective amount of protein of the present invention is administered by intravenous, cutaneous or subcutaneous injection, protein of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution.
The preparation of such parenterally acceptable protein solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. A
preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to protein of the present invention, an isotonic vehicle such as Sodium Chloride Injeckion, Ringei 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 2 0 increased further. It is contemplated that the various pharmaceutical compositions used to practice the method of the present invention should contain about 0.01 lzg to about 100 mg (preferably about O.lng to about 10 mg, more preferably about 0.1 Ilg to about 1 mg) of protein of the present invention per kg body weight.
The duration of intravenous therapy using the pharmaceutical composition of the 2 5 present invention will vary, depending on the severity of the disease being treated and the condition and potential idiosyncratic response of each individual patient.
It is contemplated that the duration of each application of the protein of the present invention will be in the range of 12 to 24 hours of continuous intravenous administration.
Ultimately the attending physician will decide on the appropriate duration of intravenous 3 0 therapy using the pharmaceutical composition of the present invention.
Protein of the invention may also be used to immunize animals to obtain polyclonal and monoclonal antibodies which specifically react with the protein. Such antibodies may be obtained using either the entire protein or fragments thereof as an immunogen. The peptide immunogens additionally may contain a cysteine residue at the WO 99/37674 PG"T/US99/01404 carboxyl terminus, and are conjugated to a hapten such as keyhole limpet hemocyanin (KLH). Methods for synthesizing such peptides are known in the art, for example, as in R.P. Merrifield, J. Amer.Chem.Soc. $,~, 2149-2154 (1963); J.L. Krstenansky, et al., FEBS Lett.
21~, 10 (1987). Monoclonal antibodies binding to the protein of the invention may be useful diagnostic agents for the immunodetection of the protein. Neutralizing monoclonal antibodies binding to the protein may also be useful therapeutics for both conditions associated with the protein and also in the treatment of some forms of cancer where abnormal expression of the protein is involved. In the case of cancerous cells or leukemic cells, neutralizing monoclonal antibodies against the protein may be useful in detecting and preventing the metastatic spread of the cancerous cells, which may be mediated by the protein.
For compositions of the present invention which are useful for bone, cartilage, tendon or ligament regeneration, the therapeutic method includes administering the composition topically, systematically, or locally as an implant or device.
When administered, the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form. Further, the composition may desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage or 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 2 0 optionally be included in the composition as described above, may alternatively or additionally, be administered simultaneously or sequentially with the composition in the methods of the invention. Preferably for bone and/or cartilage formation, the composition would include a matrix capable of delivering the protein-containing composition to the site of bone and/or cartilage damage, providing a structure for the 2 5 developing bone and cartilage and optimally capable of being resorbed into the body.
Such matrices may be formed of materials presently in use for other implanted medical applications.
The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties. The particular 3 0 application of the compositions will define the appropriate formulation.
Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, tricalciumphosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides. Other potential materials are biodegradable and biologically well-defined, such as bone or dermal collagen. Further matrices are comprised of pure proteins or extracellular matrix components. Other potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxapatite, bioglass, aluminates, or other _ ceramics. Matrices may be comprised of combinations of any of the above mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalciumphosphate. The bioceramics may be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradability.
Presently preferred is a 50:50 (mole weight) copolymer of lactic acid and glycolic acid in the form of porous particles having diameters ranging from 150 to 800 microns.
In some applications, it will be useful to utilize a sequestering agent, such as carboxymethyl cellulose or autologous blood clot, to prevent the protein compositions from disassociating from the matrix.
A preferred family of sequestering agents is cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl methylcellulose, and carboxymethylcellulose, the most preferred being cationic salts of carboxymethylcellulose {CMC). Other preferred sequestering agents include hyaluronic acid, sodium alginate, polyethylene glycol), polyoxyethyiene oxide, carboxyvinyl polymer and polyvinyl alcohol). The amount of sequestering agent useful herein is 0.5-20 2 0 wt%, preferably 1-10 wt% based on total formulation weight, which represents the amount necessary to prevent desorbtion of the protein from the polymer matrix and to provide appropriate handling of the composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix, thereby providing the protein the opportunity to assist the osteogenic activity of the progenitor cells.
2 5 In further compositions, proteins of the invention may be combined with other agents beneficial to the treatment of the bone and/or cartilage defect, wound, or tissue in question. These agents include various growth factors such as epidermal growth factor (EGF), platelet derived growth factor {PDGF), transforming growth factors (TGF-a and TGF (i), and insulin-like growth factor (IGF).
3 0 The therapeutic compositions are also presently valuable for veterinary applications. Particularly domestic animals and thoroughbred horses, in addition to humans, are desired patients for such treatment with proteins of the present invention.
The dosage regimen of a protein-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the proteins, e.g., amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors. The dosage may vary with the type of matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical composition. For example, the addition of other known growth factors, such as IGF I (insulin like growth factor I), to the final composition, may also effect the dosage. Progress can be monitored by periodic assessment of tissue/bone growth and/or repair, for example, X-rays, histomorphometric determinations and tetracycline labeling.
Polynucleotides of the present invention can also be used for gene therapy.
Such polynucleotides can be introduced either in vivo or ex vivo into cells for expression in a mammalian subject. Polynucleotides of the invention may also be administered by other known methods for introduction of nucleic acid into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA).
Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells.
Treated cells can then be introduced in vivo for therapeutic purposes.
2 0 Patent and literature references cited herein are incorporated by reference as if fully set forth.
SEQUENCE LISTING
<110> Jacobs, Kenneth MCCoy, John M.
LaVallie, Edward R.
Collins-Racie, Lisa A.
Merberg, David Treacy, Maurice Agostino, Michael J.
Steininger II, Robert J.
Wong, Gordon G.
Clark, Hilary Fechtel, Kim Genetics Institute, Inc.
<120> SECRETED PROTEINS AND POLYNUCLEOTIDES ENCODING THEM
<130> GI 6062A
<140>
<141>
<160> 32 <170> PatentIn Ver. 2.0 <210> 1 <211> 3149 <212> DNA
<213> Homo sapiens <400> 1 ggtctttaac gtgagcccgc tgcaggtgtg cggcccagtc cgagacagca gatgaggaga 60 ctgtccttcc tgtttcgcag atgaggaaac tgaggcttag agaagtttgg caaattggct 120 aagttcctac agctaccaca gcagaaagtg ctgggcagta gagagctgcc ccctccagaa 180 gatgatcagc tgcactccag tgcccccaga tcctcgtgga aggaacggat ccttaaagca 240 aaggtggtga cggtgtctca ggaggcagar tgggatcaaa tcgagccctt gcttagaagt 300 gaattagaag attttccagt acttggaatt gactgtgagt gggtaaattt ggaaggcaaa 360 gcctgccctc tgtcacttct acaaatggcc tccccaagtg gcctgtgtgt cttggttcgc 420 ctgcccaagc taatctgtgg aggaaaaaca ctaccaagaa cgttattgga tattttggca 480 gatggcacca ttttgaaagt tggagtggga tgctcagaag atgccagcaa gcttctgcag 540 gattatggcc tcgttgttag ggggtgcctg gacctccgat acctagccat gcggcagaga 600 aacaatttgc tctgtaatgg gcttagcctg aagtccctcg ctgagactgt tttgaacttt 660 ccccttgaca agtcccttct acttcgttgc agcaactggg atgctgagac tctcacagag 720 gaccaggtaa tttatgctgc cagggatgcc cagatttcag tggctctctt tcttcatctt 780 cttggatacc ctttctctag gaattcacct ggagaaaaaa aacgatgacc acagtagctg 840 gagaaaagtc ttggasaaat gccagggtgt ggtcgacatc ccatttcgaa gcaaaggaat 900 gagcagattg ggagaagagg ttaatgggga agcaacagaa tctcagcaga agccaagaaa 960 taagaagtct aagatggatg ggatggtgcc aggcaaccac caagggagag accccagaaa 1020 acataaaaga aagcctctgg gggtgggcta ttctgccaga aaatcacctc tttatgataa 1080 ctgctttctc catgctcctg atggacagcc cctctgcact tgtgatagaa gaaaagctca 1140 gtggtacctg gacaaaggca ttggtgagct ggtgagtgaa gagccctttg tggtgaagct 1200 gcggtttgaa cctgcaggaa ggcccgaatc tcctggagac tattacttga tggttaaaga 1260 gaacctgtgt gtagtgtgtg gcaagagaga ctcctacatt cggaagaacg tgattccaca 1320 tgagtaccgg aagcacttcc ccatcgagat gaaggaccac aactcccacg atgtgctgct 1380 gctctgcacc tcctgccatg ccatttccaa ctactatgac aaccatctga agcagcagct 1440 ggccaaggag ttccaggccc ccatcggctc tgaggagggc ttgcgcctgc tggaagatcc 1500 tgagcgccgg caggtgcgtt ctggggccag ggccctgctc aacgcggaga gcctgcctac 1560 tcatcgaaag gaggagctgc tgcaagcact cagagagttt tataacacag acgtggtcac 1620 agaggagatg cttcaagagg ctgccagcct ggagaccaga atctccaatg aaaactatgt 1680 tcctcacggg ctgaaggtgg tgcagtgtca cagccagggt ggcctgcgct ccctcatgca 1740 gctggagagc cgctggcgtc agcacttcct ggactccatg cagcccaagc acctgcccca 1800 gcagtggtca gtggaccaca accatcagaa gctgctccgg aaattcgggg aagatcttcc 1860 catccagctg tcttgatagc tgctttcctc ccagttagga caagtgggaa gctggagcca 1920 aggttgaaga gtcacctctt cccattttag tacatcatta attgtcaaag cctgtgtgac 1980 acaactcaga atactaacct agactaatcc caggatgctt ctgctggagc aaagatattg 2040 tttgaaggag agtttatggt tttggatttt aaacgggcag ggtctttttt cctctcattt 2100 ttgtggacaa gagaggcctt cgcctttatt tttactctcc ctcttctgct gtccctgtgc 2160 agaggaaaaa tgaagaattc tcccagaagt gacttgtcaa gacttaaaaa aaatgttttt 2220 aatgcatttc ttccttgtct agtgcctcgg tttatctcta acaggggctg tccagtatat 2280 cggtcctgtt aggaggggag aaaaagttct tccaaaggct ggagaagtga acaaggagtc 2340 aaatttattt tcccaattca acttcataat tatcatttct ttggcttcat gctctcccgt 2400 aactcatgtg gttgggatcc atcccatctg ggtcacttca gtctacttca cgtacttgaa 2460 aaggctttcc tttacacttc caggaccaaa cagcaacttc ctgccacaca cttccaccct 2520 atcactggga gaaatccttt tctggacatg agcctttgac ctgggtgggg cagaaagaac 2580 cacaaactcc atctcccaat agaactttga aattcactca gcttttcctt tcatgctgtt 2640 tgttgcctgc ttgttgcact cctcctgccc cagaactgca agatttttag cttcacccct 2700 ttctgagagt aatgttatct tttatcagaa tcagtatcag ttcccctgta ttctgtgctt 2760 catcgaattt gcaagactga cctcttttaa gcatttaatt cactcccaga gtcatctggt 2820 caggttgcaa tatgaggact tctctgtctc ctctgaagcc tgggacactg agcttactta 2880 atacattaga tgttcaaaag aggagcgttg tttcatcttt caaaatgtta ggccattact 2940 ttgaatataa aatcgactta ttaatgatta gtaatttttc taaagtattg ggaaaacttt 3000 cttattttat aagatcttaa caagcttaaa aaagaatttt atgaccagaa tccaacaaga 3060 gctctatttt ggaattgtgc ccaagttggt gatgtttact ctaaaattaa taataaaact 3120 acttgtaagc aaaaaaaaaa aaaaaaaaa 3149 <210> 2 <211> 380 <212> PRT
<213> Homo sapiens <400> 2 Met Leu Pro Gly Met Pro Arg Phe Gln Trp Leu Ser Phe Phe Ile Phe Leu Asp Thr Leu Ser Leu Gly Ile His Leu Glu Lys Lys Asn Asp Asp His Ser Ser Trp Arg Lys Val Leu Glu Lys Cys Gln Gly Val Val Asp Ile Pro Phe Arg Ser Lys Gly Met Ser Arg Leu Gly Glu Glu Val Asn Gly Glu Ala Thr Glu Ser Gln Gln Lys Pro Arg Asn Lys Lys Ser Lys Met Asp Gly Met Val Pro Gly Asn His Gln Gly Arg Asp Pro Arg Lys His Lys Arg Lys Pro Leu Gly Val Gly Tyr Ser Ala Arg Lys Ser Pro Leu Tyr Asp Asn Cys Phe Leu His Ala Pro Asp Gly Gln Pro Leu Cys Thr Cys Asp Arg Arg Lys Ala Gln Trp Tyr Leu Asp Lys Gly Ile Gly Glu Leu Val Ser Glu Glu Pro Phe Val Val Lys Leu Arg Phe Glu Pro Ala G1y Arg Pro Glu Ser Pro Gly Asp Tyr Tyr Leu Met Val Lys Glu Asn Leu Cys Val Val Cys Gly Lys Arg Asp Ser Tyr Ile Arg Lys Asn leo le5 190 Val Ile Pro His Glu Tyr Arg Lys His Phe Pro Ile Glu Met Lys Asp His Asn Ser His Asp Val Leu Leu Leu Cys Thr Ser Cys His Ala Ile Ser Asn Tyr Tyr Asp Asn His Leu Lys Gln Gln Leu Ala Lys Glu Phe Gln Ala Pro Ile Gly Ser Glu Glu Gly Leu Arg Leu Leu Glu Asp Pro Glu Arg Arg Gln Val Arg Ser Gly Ala Arg Ala Leu Leu Asn Ala Glu Ser Leu Pro Thr His Arg Lys Glu Glu Leu Leu Gln Ala Leu Arg Glu Phe Tyr Asn Thr Asp Val Val Thr Glu Glu Met Leu Gln Glu Ala Ala Ser Leu Glu Thr Arg Ile Ser Asn Glu Asn Tyr Val Pro His Gly Leu Lys Val Val Gln Cys His Ser Gln Gly Gly Leu Arg Ser Leu Met Gln Leu Glu Ser Arg Trp Arg Gln His Phe Leu Asp Ser Met Gln Pro Lys His Leu Pro Gln Gln Trp Ser Val Asp His Asn His Gln Lys Leu Leu Arg Lys Phe Gly Glu Asp Leu Pro Ile Gln Leu Ser <210> 3 <211> 1861 <212> DNA
<213> Homo sapiens <400> 3 agagccaggg gggtcgcgta gtgtcatgac cagggcggga gatcacaacc gccagagagg 60 atgctgtgga tccttggcgg actacctgac ctctgcaaaa ttccttctct accttggtca 120 ttctctctct acttggggag atcggatgtg gcactttgcg gtgtctgtgt ttctggtaga 180 gctctatgga aacagcctcc ttttgacagc agtctacggg ctggtggtgg cagggtctgt 240 tctggtcctg ggagccatca tcggtgactg ggtggacaag aatgctagac ttaaagtggc 300 ccagacctcg ctggtggtac agaatgtttc agtcatcctg tgtggaatca tcctgatgat 360 ggttttctta cataaacatg agcttctgac catgtaccat ggatgggttc tcacttcctg 420 ctatatcctg atcatcacta ttgcaaatat tgcaaatttg gccagtactg ctactgcaat 480 cacaatccaa agggattgga ttgttgttgt tgcaggagaa gacagaagca aactagcaaa 540 tatgaatgcc acaatacgaa ggattgacca gttaaccaac atcttagccc ccatggctgt 600 tggccagatt atgacatttg gctccccart catcggctgt ggctttattt cgggatggaa 660 cttggtatcc atgtgcgtgg agtacgttct gctctggaag gtttaccaga aaaccccagc 720 tctagctgtg aaagctggtc ttaaagaaga ggaaactgaa ttgaaacagc tgaatttaca 780 caaagatact gagccaaaac ccctggaggg aactcatcta atgggtgtga aagactctaa 840 catccatgag cttgaacatg agcaagagcc tacttgtgcc tcccagatgg ctgagccctt 900 ccgtaccttc cgagatggat gggtctccta ctacaaccag cctgtgtttc tggctggcat 960 gggtcttgct ttcctttata tgactgtcct gggctttgac tgcatcacca cagggtacgc 1020 ctacactcag ggactgagtg gttccatcct cagtattttg atgggagcat cagctataac 1080 tggaataatg ggaactgtag cttttacttg gctacgtcga aaatgtggtt tggttcggac 1140 aggtctgatc tcaggattgg cacagctttc ctgtttgatc ttgtgtgtga tctctgtatt 1200 catgcctgga agccccctgg acttgtccgt ttctcctttt gaagatatcc gatcaaggtt 1260 cattcaagga gagtcaatta cacctaccaa gatacctgaa attacaactg aaatatacat 1320 gtctaatggg tctaattctg ctaatattgt cccggagaca agtcctgaat ctgtgcccat 1380 aatctctgtc agtctgctgt ttgcaggcgt cattgctgct agaatcggtc tttggtcctt 1440 tgatttaact gtgacacagt tgctgcaaga aaatgtaatt gaatctgaaa gaggcattat 1500 aaatggtgta cagaactcca tgaactatct tcttratctt ctgcatttca tcatggtcat 1560 cctggctcca aatcctgaag cttttggctt gctcgtattg atttcagtct cctttgtggc 1620 aatgggccac attatgtatt tccgatttgc ccaaaatact ctgggaaaca agctctttgc 1680 ttgcggtcct gatgcaaaag aagttaggaa ggaaaatcaa gcaaatacat ctgttgtttg 1740 agacagttta actgttgcta tcctgttact agattatata gagcacatgt gcttattttg 1800 tactgcagaa ttccaataaa tggctgggtg tr_ttgctctg tttttaaaaa aaaaaaaaaa 1860 a <210> 4 <211> 571 <212> PRT
<213> Homo sapiens <220>
<221> UNSURE
<222> (202) <220>
<221> UNSURE
<222> (504) <400> 4 Met Thr Arg Ala Gly Asp His Asn Arg Gln Arg Gly Cys Cys Gly Ser Leu Ala Asp Tyr Leu Thr Ser Ala Lys Phe Leu Leu Tyr Leu Gly His Ser Leu Ser Thr Trp Gly Asp Arg Met Trp His Phe Ala Val Ser Val Phe Leu Val Glu Leu Tyr Gly Asn Ser Leu Leu Leu Thr Ala Val Tyr Gly Leu Val Val Ala Gly Ser Val Leu Val Leu Gly Ala Ile Ile Gly Asp Trp Val Asp Lys Asn Ala Arg Leu Lys Val Ala Gln Thr Ser Leu Val Val Gln Asn Val Ser Val Ile Leu Cys Gly Ile Ile Leu Met Met Val Phe Leu His Lys His Glu Leu Leu Thr Met Tyr His Gly Trp Val WO 99/376'74 PCTNS99/01404 Leu Thr Ser Cys Tyr Ile Leu Ile Ile Thr Ile Ala Asn Ile Ala Asn Leu Ala Ser Thr Ala Thr Ala Ile Thr Ile Gln Arg Asp Trp Ile Val Val Val Ala Gly Glu Asp Arg Ser Lys Leu Ala Asn Met Asn Ala Thr Ile Arg Arg Ile Asp Gln Leu Thr Asn Ile Leu Als Pro Met Ala Val Gly Gln Ile Met Thr Phe Gly Ser Pro Xaa Ile Gly Cys Gly Phe Ile Ser Gly Trp Asn Leu Val Ser Met Cys Val Glu Tyr Val Leu Leu Trp Lys Val Tyr Gln Lys Thr Pro Ala Leu Ala Val Lys Ala Gly Leu Lys Glu Glu Glu Thr Glu Leu Lys Gln Leu Asn Leu His Lys Asp Thr Glu Pro Lys Pro Leu Glu Gly Thr His Leu Met Gly Val Lys Asp Ser Asn Ile His Glu Leu Glu His Glu Gln Glu Pro Thr Cys Ala Ser Gln Met Ala Glu Pro Phe Arg Thr Phe Arg Asp Gly Trp Val Ser Tyr Tyr Asn Gln Pro Val Phe Leu Ala Gly Met Gly Leu Ala Phe Leu Tyr Met Thr Val Leu Gly Phe Asp Cys Ile Thr Thr Gly Tyr Ala Tyr Thr Gln Gly Leu Ser Gly Ser Ile Leu Ser Ile Leu Met Gly Ala Ser Ala Ile Thr Gly Ile Met Gly Thr Val Ala Phe Thr Trp Leu Arg Arg Lys Cys Gly Leu Val Arg Thr Gly Leu Ile Ser Gly Leu Ala Gln Leu Ser Cys Leu Ile Leu Cys Val Ile Ser Val Phe Met Pro Gly Ser Pro Leu Asp Leu Ser Val Ser Pro Phe Glu Asp Ile Arg Ser Arg Phe Ile Gln Gly Glu Ser Ile Thr Pro Thr Lys Ile Pro Glu Ile Thr Thr Glu Ile Tyr Met Ser Asn Gly Ser Asn Ser Ala Asn Ile Val Pro Glu Thr Ser Pro Glu Ser Val Pro Ile hle Ser Val Ser Leu Leu Phe Ala Gly Val Ile Ala Ala Arg Ile Gly Leu Trp Ser Phe Asp Leu Thr Val Thr Gln Leu Leu Gln Glu Asn Val Ile Glu Ser Glu Arg Gly Ile Ile Asn Gly Val Gln Asn Ser Met Asn Tyr Leu Leu Xaa Leu Leu His Phe Ile Met Val Ile Leu Ala Pro Asn Pro Glu Ala Phe Gly Leu Leu Val Leu Ile Ser Val Ser Phe Val Ala Met Gly His Ile Met Tyr Phe Arg Phe Ala Gln Asn Thr Leu Gly Asn Lys Leu Phe Ala Cys Gly Pro Asp Ala Lys Glu Val Arg Lys Glu Asn Gln Ala Asn Thr Ser Val Val <210> 5 <211> 2157 <212> DNA
<213> Homo Sapiens <400> 5 ctctctttaa tatcttcacc tctaccatgt gtctttcttt taatatagtt ataattttcc 60 aaccacgtag atcaatattt actcatcatg accataaaat gcagtttagc catatagaaa 120 actatgatta cttttcttta taatttccct tcagttaata cttattttat tttctgtttt 180 tatcatctag tcaactcgca aacttccagc atttgtctaa atctactcaa tatattccag 240 tacatcagat aatatatcag tttcatcctc ctgaaaaact cttttccagt gtatcctgac 300 ctgctctaat tttgacttga tgctttctgt atctggtgca cagctgttac cttggaatct 360 tcccttcatc attattcaga gtgtttctgt agtttttctc ttgcattgga ttttgtgctt 420 cctgaatccc tctctCtctt tttttttttt tttttacttg gcttactcct tgctttgatg 480 gatctcaggc tccagtagct tccttggaaa gagtgtttgg aagttgcttc tgcaggaagc 540 ctttttggtg gcatggtcct caagaagttc ctaaaaggtt gatgaaaagc ccagaacctt 600 gatgacagat tgtctggtta taaagcattt tttacgtaaa atcatcatgg tgcaccctaa 660 ggtcagattt catttcagtg taaaggtaaa tggaatcctc tccacagaga tctttggggt 720 ggagaatgaa cccactttga accttgggaa tggaattgct cttttggtcg actcccagca 780 ttatgtgagt agaccaaatt ttggtacaat tgaatcacac tgcagcagaa ttcaccctgt 840 gctaggacat ccagtaatgc ttttcatccc tgaagacgtg gctggcatgg acttgttggg 900 agaactgata ctgactccag cagctgcact gtgccccagc ccaaaggttt cttccaacca 960 gcttaacagg atttcttcag tttccatatt tctatatgga cctttgggtc tgcctctgat 1020 attgtcaact tgggagcagc cgatgactac tttcttcaaa gatacctctt ctttagttga 1080 ctggaaaata ccatttgtgt atgataccca atttggatct caatttggat agagatttgg 1140 tgcttccaga tgtgagttat caggtggaat ccagtgagga ggatcagtct cagactatgg 1200 atcctcaagg acasactctg ctgctttttc tctttgtgga tttccacagt gcatttccag 1260 tccagcaaat ggaaatctgg ggagtctata ctttgctcac aactcatctc aatgccatcc 1320 ttgtggagag ccacagtgta gtgcaaggtt ccatccaatt cactgtggac aaggtcttgg 1380 agcaacatca ccaggctgcc aaggctcagc agaaactaca ggcctcactc tcagtggctg 1440 tgaactccat catgagtatt ctgactggaa gcactaggag cagcttccga aagatgtgtc 1500 tccagaccct tcaagcagct gacacacaag agttcaggac caaactgcac aaagtatttc 1560 gtgagatcac ccaacaccaa tttcttcacc actgctcatg tgaggtgaag cagctaaccc 1620 tagaaaaaaaggactcagcc cagggcactg tgataacagcagcctggagc1680 aggacgcacc tcctagcagtgcttaaacag ccttcccagc aggggtacagcagctctcac1740 ccacagcagc attcagtcactagcagagat gccagatacc cagaaaacaagaggctcaag1800 agcgggcaag aggggcagcccccgcataga ggagatgcga ctgccagggccccgagcccg1860 gctctgcgct tcagaggccgccccgcgccg cccggaagcc ccctcactcytagaggaagg1920 accgcggccc gagcaccgcgaggctcacgg cagggccctg gggcgagcctcggaagccgc1980 gcgccgggca ctggaggacgtcctgtggct gcaggaggtc cagagtggctgagtcccagc2040 tccaacctgt cctgggccctgagccgggtc cccttccgca gatccggaggctgcgggcag2100 agcgcccacc ccgttatcccgtggtttaat aaagctgccg aaaaaaaaaaaaaaaaa 2157 cgcgctcacc <210>
<211>
<212>
PRT
<213> Sapiens Homo <400>
Met Ile Asn Leu Asp Leu Asn Leu Asp Leu Leu Pro Pro Asp Arg Val Asp Val Tyr Gln Val Glu Ser Ser Asp Gln Gln Thr Ser Glu Glu Ser Met Asp Gln Gly Gln Thr Leu Leu Leu Phe Asp Phe Pro Leu Phe Val His Ser Phe Pro Val Gln Gln Met Trp Gly Tyr Thr Ala Glu Ile Val Leu Leu Thr His Leu Asn Ala Ile Glu Ser Ser Val Thr Leu Val His Val Gln Ser Ile Gln Phe Thr Val Val Leu Gln His Gly Asp Lys Glu His Gln Ala Lys Ala Gln Gln Lys Ala Ser Ser Val Ala Leu Gln Leu Ala Val Ser Ile Met Ser Ile Leu Ser Thr Ser Ser Asn Thr Gly Arg Phe Arg Met Cys Leu Gln Thr Leu Ala Asp Gln Glu Lys Gln Ala Thr Phe Arg Lys Leu His Lys Val Phe Ile Thr His Gln Thr Arg Glu Gln Phe Leu His Cys Ser Cys Glu Val Leu Thr Glu Lys His Lys Gln Leu Lys Asp Ala Gln Gly Thr Glu Asp Asp Asn Ser Leu Ser Ala Pro Ser Glu Leu Ala Val Leu Lys Gln Pro Pro Thr Ala Gly Leu Ser Gln Ala Val Gln Leu Ser His Ser Val Thr Asp Ala Tyr Gln Gln Ser Arg Arg Arg Ala Arg Lys Gln Glu Ala Gln Gln Pro His Arg Ser Glu Gly Pro Gly Asp Ala Ser Ser Ala Leu Cys Gln Gly Pro GIu Pro Val Arg Gly Arg Pro Ala Pro Pro Gly Ser His Arg Gly Pro Pro His Ser <210> 7 <211> 1607 <212> DNA
<213> Homo Sapiens <400> 7 gtgaacttca ctactggaaa gcaacaaagg cagtcggcat aaaaatgggt tctctcagca 60 cagctaacgt tgaattttgc cttgatgtgt tcaaagagct gaacagtaac aacataggag 120 ataacatctt cttttcttcg ctgagtctgc tttatgctct aagcatggtc ctccttggtg 180 ccaggggaga gactgcagag caattggaga aggtgcttca ttttagtcat actgtagact 240 cattaaaacc agggttcaag gactcaccta agtgcagcca agctggaaga attcattccg 300 agtttggtgt ctaattctct caaatcaacc agccagactc taactgtacc ctcagcattg 360 ccaacaggct ctacgggaca aagacgatgg catttcatca ggaaaagtcg caaatctctt 420 tggaaagagc acaattgacc cttcatctgt aatggtcctg gtgaatacca tatatttcaa 480 aggacaatgg caaaataaat ttcaagtaag agagacagtt aaaagtcctt ttcagctaag 540 tgagggtaaa aatgtaactg tggaaatgat gtatcaaatt ggaacattta aactggcctt 600 tgtaaaggag ccgcagatgc aagttcttga gctgccctac gttaacaaca aattaagcat 660 gattattctg cttccagtag gcatagctaa tctgaaacag atagaaaagc agctgaattc 720 ggggacgttt catgagtgga caagctcttc taacatgatg gaaagagaag ttgaagtaca 780 cctccccaga ttcaaacttg aaattaagta tgagctaaat tccctgttaa aacctctagg 840 ggtgacagat ctcttcaacc aggtcaaagc tgatctttct ggaatgtcac caaccaaggg 900 cctatattta tcaaaagcca tccacaagtc atacctggat gtcagcgaag agggcacgga 960 ggcagcagca gccactgggg acagcatcgc tgtaaaaagc ctaccaatga gagctcagtt 1020 caaggcgaac caccccttcc tgttctttat aaggcacact cataccaaca cgatcctatt 1080 ctgtggcaag cttgcctctc cctaatcaga tggggttgag taaggctcag agttgcagat 1140 gaggtgcaga gacaatcctg tgactttccc acggccaaaa agctgttcac acctcacaca 1200 cctctgtgcc tcagtttgct catctgcaaa ataggtctag gatttcttcc aaccatttca 1260 tgagttgtga agctaaggct ttgttaatca tggaaaaagg tagacttatg cagaaagcct 1320 ttctggcttt cttatctgtg gtgtctcatt tgagtgctgt ccagtgacat gatcasgtca 1380 atgagtaaaa ttttaaggga ttagattttc ttgacttgta kgtatctgtg agatcttgaa 1440 taagtgacct gacatctctg cttaaagaaa accagctgaa gggcttcaac tttgcttgga 1500 tttttaaata ttttccttgc atatgtaaat agaatgtggt gagttttagt tcaasattct 1560 ctgttgagaa taataaatgc atgaaatacc ttaaaaaaaa aaaaaaa 1607 <210> 8 <211> 217 <212> PRT
<213> Homo Sapiens <400> 8 Met Val Leu Val Asn Thr Ile Tyr Phe Lys Gly Gln Trp Gln Asn Lys Phe Gln Val Arg Glu Thr Val Lys Ser Pro Phe Gln Leu Ser GIu Gly Lys Asn Val Thr Val Glu Met Met Tyr Gln Ile Gly Thr Phe Lys Leu Ala Phe Val Lys Glu Pro Gln Met Gln Val Leu Glu Leu Pro Tyr Val Asn Asn Lys Leu Ser Met Ile Ile Leu Leu Pro Val Gly Ile Ala Asn Leu Lys Gln Ile Glu Lys Gln Leu Asn Ser Gly Thr Phe His Glu Trp Thr Ser Ser Ser Asn Met Met Glu Arg Glu Val Glu Val His Leu Pro Arg Phe Lys Leu Glu Ile Lys Tyr Glu Leu Asn Ser Leu Leu Lys Pro Leu Gly Val Thr Asp Leu Phe Asn Gln Val Lys Ala Asp Leu Ser Gly Met Ser Pro Thr Lys Gly Leu Tyr Leu Ser Lys Ala Ile His Lys Ser Tyr Leu Asp Val Ser Glu Glu Gly Thr Glu Ala Ala Ala Ala Thr Gly Asp Ser Ile Ala Val Lys Ser Leu Pro Met Arg Ala Gln Phe Lys Ala Asn His Pro Phe Leu Phe Phe Ile Arg His Thr His Thr Asn Thr Ile Leu Phe Cys Gly Lys Leu Ala Ser Pro <210> 9 <211> 1537 <212> DNA
<213> Homo sapiens <400> 9 gtaggatttg gggatgtgga tatttaagac aatttctttt ttcttttggt ttaatagggg 60 cgggtatagg gaccaactgg gaccgagtgc ccagggggcc gagcacggtc atgctggccg 120 gcctgcatgc atgcgtgtgc cgggctgggc tgggcggccg gcggtcgtgg ggcagggttg 190 ggggtctgtg ctcagctgat aactgccatg cactgtactg cacacgtccc tagagcctac 240 cgggacccga cgcttttcag ggcatttctc cctccagcca gggcccaact cccacctgcc 300 tgggcgaatc tcctccaagg aagtcccagg aggatgggga ccaggaaggc tgtggacccc 360 catctccagg gggccttccc agcctgatcc ctgtcctcca agttctggag gaggccgctg 420 tagggtctgg ctgagcttcc cacccacttt ccctggtccc aatcctttct tgtcctatac 480 ccagctgggg ttgctgccct gaacgaactg cgtgtggggc cggcacatcc tagcaggcag 540 cccctggcgc ctgctgcctc agggatgctc caaccaccct cgttctcctc gcagtggccc 600 tggctcccac ctcccgcccc agcctgccgt ggggcccgtc agcctggtcc cacccccatg 660 gagaacccaa agtcttactg tatataactc caggtgacgt ttctatattt atagcagtgt 720 tgaaaaccca cgtgttttac acagaaccac cctctccaac ccctcccttc ccgaccccaa 780 caaaacgttt tcaaacccct tacagttcct ggggcaggcg gaaacaggct cacagattgt 840 gtgtcggctg cagcagtgat tccaacaagc agctattggg ggggaaacac agcatttaaa 900 aagatcatca ttaaaaaaca agatttatac aacaattact taggatgttt gtgatctgcc 960 gaccttgcta tagatgccat gttaccaatg atttcctgtg gtgggggctt gccattgttt 1020 actctcttat ttaccaactt ctggcctagg catgacagtg ggcaccttcc cccagccctg 1080 gctgggccca gcgcctgtgt tytgtgttag aaaggtttta tatatatata aaattacata 1140 tatakgtaga aatatatgta attttggggg ccctgttcct tgcacatttt acagttacct 1200 catttttccc atgtatgtat ttgagaaaat gctaatatat agagaaaaaa atggttctta 1260 aaacttaaat gtgtggtttt ttccattcca tgggattcac attggtttgt agcatttaac 1320 ataactagta tgttgtatta tatatatgtg tatactgatt gaaattttta acagatttgt 1380 WO 99/37674 PC"TNS99/01404 acttttttta aaatgaaagt tgctagttct gcttgaccaa gtagtgcaat cattattttt 1440 tttaatattg ttgctgattt cagagggata ttcactaata aatgtatgat gtatacccac 1500 graaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa 1537 <210> 10 <211> 86 <212> PRT
<213> Homo sapiens <400> 10 Met His Ala Cys Ala Gly Leu Gly Trp Ala Ala Gly Gly Arg Gly Ala Gly Leu Gly Val Cys Ala Gln Leu Ile Thr Ala Met His Cys Thr Ala His Val Pro Arg Ala Tyr Arg Asp Pro Thr Leu Phe Arg Ala Phe Leu Pro Pro Ala Arg Ala Gln Leu Pro Pro Ala Trp Ala Asn Leu Leu Gln Gly Ser Pro Arg Arg Met Gly Thr Arg Lys Ala Val Asp Pro His Leu Gln Gly Ala Phe Pro Ala <210> 11 <211> 1302 <212> DNA
<213> Homo sapiens <400> 11 cttcatggcc tacacacacc accttacccc tctgctggca agaggggacc tgattcatcc 60 tcacgctaaa cactcattct acccaactga ttgagacaga acagaagata aactgaaact 120 tctctgcctt cccgctgcaa gagtgaatga gcgatccctc tcaactgact caaaatgttt 180 gcctcaccca ggagatggag ctctcgaagg ccttctctgg ccagcggaca ctcctatctg 240 ccatcctcag catgctatca ctcagcttct ccacaacatc cctgctcagc aactactggt 300 ttgtgggcac acagaaggtg cccaagcccc tgtgcgagaa aggtctggca gccaagtgct 360 ttgacatgcc agtgtccctg gatggagata ccaacacatc cacccaggag gtggtacaat 420 acaactggga gactggggat gaccggttct ccttccggag cttccggagt ggcatgtggc 480 tatcctgtga ggaaactgtg gaagaaccag gggagaggtg ccgaagtttc attgaactta 540 caccaccagc caagagagaa atcctatggt tatccctggg aacgcagatc acctacatcg 600 gacttcaatt catcagcttc ctcctgctac taacagactt gctactcact gggaaccctg 660 cctgtgggct caaactgagc gcctttgctg ctgtttcctc tgtcctgtca ggtctcctgg 720 ggatggtggc ccacatgatg tattcacaag tcttccaagc gactgtcaac ttgggtccag 780 aagactggag accacatgtt tggaattatg gctgggcctt ctacatggcc tggctctcct 840 tcacctgctg catggcgtcg gctgtcacca ccttcaacac gtacaccagg atggtgctgg 900 agttcaagtg caagcatagt aagagcttca aggaaaaccc gaactgccta ccacatcacc 960 atcagtgttt ccctcggcgg ctgtcaagtg cagcccccac cgtgggtcct ttgaccagct 1020 accaccagta tcataatcag cccatccact ctgtctctga gggagtcgac ttctactccg 1080 agctgcggaa caagggattt caaagagggg ccagccagga gctgaaagaa gcagttaggt 1140 catctgtaga ggaagagcag tgttaggagt taagcgggtt tggggagtag gcttgagccc 1200 taccttacac gtctgctgat tatcaacatg tgcttaagcc aaaaaaaaaa aaaaaaaaaa 1260 aaseaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa as 1302 <210> 12 <211> 339 lU
<212> PRT
<213> Homo sapiens <400> 12 Met Ser Asp Pro Ser Gln Leu Thr Gln Asn Val Cys Leu Thr Gln Glu Met Glu Leu Ser Lys Ala Phe Ser Gly Gln Arg Thr Leu Leu Ser Ala Ile Leu Ser Met Leu Ser Leu Ser Phe Ser Thr Thr Ser Leu Leu Ser Asn Tyr Trp Phe Val Gly Thr Gln Lys Val Pro Lys Pro Leu Cys Glu Lys Gly Leu Ala Ala Lys Cys Phe Asp Met Pro Val Ser Leu Asp Gly Asp Thr Asn Thr Ser Thr Gln Glu Val Val Gln Tyr Asn Trp Glu Thr Gly Asp Asp Arg Phe Ser Phe Arg Ser Phe Arg Ser Gly Met Trp Leu Ser Cys Glu Glu Thr Val Glu Glu Pro Gly Glu Arg Cys Arg Ser Phe Ile Glu Leu Thr Pro Pro Ala Lys Arg Glu Ile Leu Trp Leu Ser Leu Gly Thr Gln Ile Thr Tyr Ile Gly Leu Gln Phe I1e Ser Phe Leu Leu Leu Leu Thr Asp Leu Leu Leu Thr Gly Asn Pro Ala Cys Gly Leu Lys Leu Ser Ala Phe Ala Ala Val Ser Ser Val Leu Ser Gly Leu Leu Gly Met Val Ala His Met Met Tyr Ser Gln Val Phe Gln Ala Thr Val Asn Leu Gly Pro Glu Asp Trp Arg Pro His Val Trp Asn Tyr Gly Trp Ala Phe Tyr Met Ala Trp Leu Ser Phe Thr Cys Cys Met Ala Ser Ala Val Thr Thr Phe Asn Thr Tyr Thr Arg Met Val Leu Glu Phe Lys Cys Lys His Ser Lys Ser Phe Lys Glu Asn Pro Asri Cys Leu Pro His His His Gln Cys Phe Pro Arg Arg Leu Ser Ser Ala Ala Pro Thr Val Gly Pro Leu Thr Ser Tyr His Gln Tyr His Asn Gln Pro Ile His Ser Val Ser VlrO 99/37674 PCT/US99/01404 Glu Gly Val Asp Phe Tyr Ser Glu Leu Arg Asn Lys Gly Phe Gln Arg Gly Ala Ser Gln Glu Leu Lys Glu Ala Val Arg Ser Ser Val Glu Glu Glu Gln Cys <210> 13 <211> 3184 <212> DNA
<213> Homo sapiens <220>
<221> unsure <222> (1644) <400> 13 gtgcatgctt gtaatcgcag ctacttcgga gcctgagaga ctccttcagg gtgagcaaag 60 gcctggaaaa acctgtatgc agataaagaa aaggaaagaa agagataatc agtgcatgca 120 gttgtcagct ggctgggacc tgaggagagt cacttgtgga ggcaactggt ctttatcccc 180 attgtccggt acaaggcagg cattaatcct gtgatcctta tctgaagctc agctacaagg 240 ctttggccga ccaagtgtgt accatgctgc tattgtcatc ttccttgaat tctttgcgtg 300 gggcctgttg acaactccaa tgttgactgt tctacatgaa acattttctc aacacacatt 360 cctcatgaat ggtctcattc aaggtgtaaa gggcctgctc tcttttttga gtgccccact 420 cattggtgcc ctgtctgatg tgtgggggag gaagcccttt ctcctcggca ctgtattctt 480 tacctgcttc ccaatcccac tgatgaggat cagcccatgg tggtattttg cgatgatttc 540 tgtgtctgga gtcttctcgg tcacgttttc tgttatattt gcctatgtag ctgatgtcac 600 tcaggagcac gagcgaagta cagcttatgg atgggtctca gccacctttg cggctagtct 660 tgtcagcagc ccggccattg gagcatatct ttctgccagt tacggagaca gcctcgttgt 720 gctggtggcc acagtggtgg ctcttctgga catctgcttc atcttagtgg ctgttccaga 780 atctctgcct gagaaaatga gaccggtttc ctggggagct cagatttctt ggaaacaagc 840 agaccctttt gcgtcgttga agaaagttgg aaaagattct actgtcttac taatctgcat 900 caccgtgttt ctttcatacc ttcctgaagc tggacagtat tcaagttttt ttctctatct 960 caggcaggtc ataggttttg gatctgttaa aattgcagca ttcatagcta tggtaggaat 1020 tctgtctatt gtggctcaga cggcctttct tagcatcttg atgagatcat taggaaataa 1080 gaatactgtc ctccttggct tgggcttcca gatgctccag ttagcctggt acggttttgg 1140 atcacaggcc tggatgatgt gggcagcagg gaccgtggct gccatgtcca gcatcacgtt 1200 tccggcaatc agtgccctcg tctctcggaa tgcagagtca gatcagcaag gagttgccca 1260 ggggatcata actggaataa gaggactatg caatggcctg gggccagcac tgtatggctt 1320 catattctac atgttccatg tggaactgac tgagttgggc ccgaaattga attctaacaa 1380 cgttcccctg cagggagctg tcatcccagg cccgccgttt ttatttgggg catgtatagt 1440 ccttatgtct tttctggttg ccttattcat tcctgaatac agtaaagcca gtggagttca 1500 aaaacacagt aacagcagca gcggcagcct gaccaacacc ccagaacggg gcagtgatga 1560 ggacattgag ccactactgc aagacagcag catctgggag ctctcttcat ttgaggagcc 1620 tgggaatcag tgcactgagc tgtnaactcg gcagaaagtg ggattctgca tacgccatct 1680 ctgagagcca tggagggagc cacacccctg gtgacttcat ggtgctggat gggagacgct 1740 agcggcatcc ttcagggcca agtttgataa ataccaccgc catcattctg ctcatcctcc 1800 tcctgttttt tttttttctc ttacattctt tttttttttc ctgtttatac attagaacaa 1860 gataagattt gaaatacttc cttgcaaata atgtgcaact cccaaggtga aactcaaata 1920 gaaaaagtca tctctctggt agaaaggatg gctttcctgt aatgactata gagtaagagt 1980 ggcagcaatc tttccatgcc cttttcagca gaaggcacag aacagtagcg ggactgccat 2040 ctctggcaag atttcaggta aagaatctct tcttaatttc taccttcctg tttctctgaa 2100 tcagcccata ggtgttgatg agtggccact cttaaagagt cactcagtat cagggatcta 2160 ctgtctttgt tcaaaggtca aataaaaacc tagtctcctt ttattctact ttctattctt 2220 agctagaatg aaactcagca tatatacact tctggacata ataatattga atagtaatta 2280 cctttactag atgaaagaaa ttttcattac aaacttasat catgtaaaac tcaacaactc 2340 agattcctgg acctggtgtc ctggttgggt ccaaggtgat tttacagaag aaaaaaacaa 2900 ctcaagcatt ctggtggcaa catagagatt gtaggctgct tctaagaaag ttattaacaa 2460 tttggaaatt cctaagtagg atgagagtta gtaactggat acgagtgaag tttatatcca 2520 agttcagact caaaggcatt attatgattt gcttcttccc atgtcttcca tgtcctgctt 2580 ctcaaagttt ttctcatcca tcacactcct gccttaactg ctctgagtat gcatttgttt 2640 tcaattcatc tttatttcaa tctgtttaac ttttgaatcg catgggaata cgcacattaa 2700 gttcctttct aaaataaggt tttatgaagc tgagtttcac gataagtgtc ttgctatttt 2760 ttgagatgtt ttatggacaa agaaaacttt acagatttat atgtattttg ctgcaccagt 2820 aaatggacca ttaactaggg cccaccttta acagagcacc cctttgaaag ttttataggt 2880 atgaaatata tgtagatatt tgtaaagggt tttaattttt tttttttgat ggggtgctgt 2940 gtaaatcttg tatttataaa tgtaatgaag gtattgacag aaaaaaatat atacaacttt 3000 tataaaggat tgtgtactga ctgaatacat ttaaaagaaa atatattttg aaacctgttc 3060 tgctatgaac agagataaca tatcttttta ctatgctatt ggtttttagg ttaagcttcc 3120 taatgcataa taaatttaca gtggttaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3180 aaaa 3184 <210> 14 <211> 454 <212> PRT
<213> Homo Sapiens <220>
<221> UNSURE
<222> (442) <400> 14 Met Leu Thr Val Leu His Glu Thr Phe Ser Gln His Thr Phe Leu Met Asn Gly Leu Ile Gln Gly Val Lys Gly Leu Leu Ser Phe Leu Ser Ala Pro Leu Ile Gly Ala Leu Ser Asp Val Trp Gly Arg Lys Pro Phe Leu Leu Gly Thr Val Phe Phe Thr Cys Phe Pro Ile Pro Leu Met Arg Ile Ser Pro Trp Trp Tyr Phe Ala Met Ile Ser Val Ser Gly Val Phe Ser Val Thr Phe Ser Val Ile Phe Ala Tyr Val Ala Asp Val Thr Gln Glu His Glu Arg Ser Thr Ala Tyr Gly Trp Val Ser Ala Thr Phe Ala Ala Ser Leu Val Ser Ser Pro Ala Ile Gly Ala Tyr Leu Ser Ala Ser Tyr Gly Asp Ser Leu Val Val Leu Val Ala Thr Val Val Ala Leu Leu Asp Ile Cys Phe Ile Leu Val Ala Val Pro Glu Ser Leu Pro Glu Lys Met Arg Pro Val Ser Trp Gly Ala Gln Ile Ser Trp Lys Gln Ala Asp Pro Phe Ala Ser Leu Lys Lys Val Gly Lys Asp Ser Thr Val Leu Leu Ile lao las 190 Cys Ile Thr Val Phe Leu Ser Tyr Leu Pro Glu Ala Gly Gln Tyr Ser Ser Phe Phe Leu Tyr Leu Arg Gln Val Ile Gly Phe Gly Ser Val Lys Ile Ala Ala Phe Ile Ala Met Val Gly Ile Leu Ser Ile Val Ala Gln Thr Ala Phe Leu Ser Ile Leu Met Arg Ser Leu Gly Asn Lys Asn Thr Val Leu Leu Gly Leu Gly Phe Gln Met Leu Gln Leu Ala Trp Tyr Gly Phe Gly Ser Gln Ala Trp Met Met Trp Ala Ala Gly Thr Val Ala Ala Met Ser Ser Ile Thr Phe Pro Ala Ile Ser Ala Leu Val Ser Arg Asn Ala Glu Ser Asp Gln Gln Gly Val Ala Gln Gly Ile Ile Thr Gly Ile Arg Gly Leu Cys Asn Gly Leu Gly Pro Ala Leu Tyr Gly Phe Ile Phe Tyr Met Phe His Val Glu Leu Thr Glu Leu Gly Pro Lys Leu Asn Ser Asn Asn Val Pro Leu Gln Gly Ala Val Ile Pro Gly Pro Pro Phe Leu Phe Gly Ala Cys Ile Val Leu Met Ser Phe Leu Val Ala Leu Phe Ile Pro Glu Tyr Ser Lys Ala Ser Gly Val Gln Lys His Ser Asn Ser Ser Ser Gly Ser Leu Thr Asn Thr Pro Glu Arg Gly Ser Asp Glu Asp Ile Glu Pro Leu Leu Gln Asp Ser Ser Ile Trp Glu Leu Ser Ser Phe Glu Glu Pro Gly Asn Gln Cys Thr Glu Leu Xaa Thr Arg Gln Lys Val Gly Phe Cys Ile Arg His Leu <210> 15 <211> 2481 <212> DNA
<213> Homo sapiens <400> 15 WO 99/37674 PC1'/US99/01404 aggtctagaa ttcaatcggg aagaaggaaa agttcccttc tgctgtgaaa ctatttggca 60 agaggctgga gggcccaatg gctgcaaaat cgcaacccaa cattcccaaa gccaagagtc 120 tagatggcgt caccaatgac agaaccgcat ctcaagggca gtggggccgt gcctgggagg 180 tggactggtt ttcactggcg agcgtcatct tcctactgct gttcgccccc ttcatcgtct 240 actacttcat catggcttgt gaccaataca gctgcgccct gaccggccct gtggtggaca 300 tcgtcaccgg acatgctcgg ctctcggaca tctgggccaa gactccacct ataacgagga 360 aagccgccca gctctatacc ttgtgggtca ccttccaggt gcttctgtac acgtctctcc 420 ctgacttctg ccataagttt ctacccggct acgtaggagg catccaggag ggggccgtga 480 ctcctgcagg ggttgtgaac aagtatcaga tcaacggcct gcaagcctgg ctcctcacgc 540 acctgctctg gtttgcaaac gctcatctcc tgtcctggtt ctcgcccacc atcatcttcg 600 acaactggat cccactgctg tggtgcgcca acatccttgg ctatgccgtc tccaccttcg 660 ccatggtcaa gggctacttc ttccccacca gcgccagaga ctgcaaattc acaggcaatt 720 tcttttacaa ctacatgatg ggcatcgagt ttaaccctcg gatcgggaag tggtttgact 780 tcaagctgtt cttcaatggg cgccccggga tcgtcgcctg gaccctcatc aacctgtcct 840 tcgcagcgaa gcagcgggag ctccacagcc atgtgaccaa tgccatggtc ctggtcaacg 900 tcctgcaggc catctacgtg attgacttct tctggaacga aacctggtac ctgaagacca 960 ttgacatctg ccatgaccac ttcgggtggt acctgggctg gggcgactgt gtctggctgc 1020 cttatcttta cacgctgcag ggtctgtact tggtgtacca ccccgtgcag ctgtccaccc 1080 cgcacgccgt gggcgtcctg ctgctgggcc tggtgggcta ctacatcttc cgggtggcca 1140 accaccagaa ggacctgttc cgccgcacgg atgggcgctg cctcatctgg ggcaggaagc 1200 ccaaggtcat cgagtgctcc tacacatccg ccgacgggca gaggcaccac agcaagctgc 1260 tggtgtcggg cttctggggc gtggcccgcc acttcaacta cgtcggcgac ctgatgggca 1320 gcctggccta ctgcctggcc tgtggcggtg gccacctgct gccctacttc tacatcatct 1380 acatggccat cctgctgacc caccgctgcc tccgggacga gcaccgctgc gccagcaagt 1440 acggccggga ctgggagcgc tacaccgccg cagtgcctta ccgcctgctg cctggaatct 1500 tctaagggca cgccctaggg agaagccctg tggggctgtc aagagcgtgt tctgccaggt 1560 ccatgggggc tggcatccca gctccaactc gaggagcctc agtttcctca tctgtaaact 1620 ggagagagcc cagcacttgg caggtgtcca gtacctaatc acgctctgtt ccttgctttt 1680 gccttcaagg gaattccgag tgtccagcac tgccgtattg ccagcacaga cggattttct 1740 ctaatcagtg tccctggggc aggaggatga cccagtcacc tttactagtc ctttggagac 1800 aatttacctg tattaggagc ccaggccacg ctacactctg cccacactgg tgagcaggag 1860 gtcttcccac gccctgtcat taggctgcat ttactcttgc taaataaaag tgggagtggg 1920 gcgtgcgcgt tatccatgta ttgcctttca gctctagatc cccctcccct gcctgctctg 1980 cagtcgtggg tggggcccgt gcgccgtttc tccttggtag cgtgcacggt gttgaactgg 2040 gacactgggg agaaaggggc tttcatgtcg tttccttcct gctcctgctg macagctgcc 2100 aggagtgctc tgcctggagt ctgcagacct cagagaggtc ccagcactgg ctgtggcctt 2160 tcaggtgtag gcaggtgggc tctgcttccc gattccctgt gagcgcccac cctctcgaaa 2220 gaattttctg cttgccctgt gactgtgcag actctggctc gagcaacccg gggaacttca 2280 ccctcagggg cctcccacac cttctccagc gaggaggtyt cagtcccagc ctcgggaggg 2340 cacctccttt tctgtgcttt cttccctgag gcattcttcc tcatccctag ggtgttgtgt 2400 agaactcttt ttaaactcta tgctccgagt agagttcatc tttatattaa acttcccctg 2460 ttcaaataaa aaaaaaaaaa a 2481 <210> 16 <211> 475 <212> PRT
<213> Homo sapiens <400> 16 Met Ala Ala Lys Ser Gln Pro Asn Ile Pro Lys Ala Lys Ser Leu Asp Gly Val Thr Asn Asp Arg Thr Ala Ser Gln Gly Gln Trp Gly Arg Ala Trp Glu Val Asp Trp Phe Ser Leu Ala Ser Val Ile Phe Leu Leu Leu Phe Ala Pro Phe Ile Val Tyr Tyr Phe Ile Met Ala Cys Asp Gln Tyr Ser Cys Ala Leu Thr Gly Pro Val Val Asp Ile Val Thr Gly His Ala Arg Leu Ser Asp Ile Trp Ala Lys Thr Pro Pro Ile Thr Arg Lys Ala Ala Gln Leu Tyr Thr Leu Trp Val Thr Phe Gln Val Leu Leu Tyr Thr Ser Leu Pro Asp Phe Cys His Lys Phe Leu Pro Gly Tyr Val Gly Gly Ile Gln Glu Gly Ala Val Thr Pro Ala Gly Val Val Asn Lys..Tyr Gln Ile Asn Gly Leu Gln Ala Trp Leu Leu Thr His Leu Leu Trp Phe Ala Asn Ala His Leu Leu Ser Trp Phe Ser Pro Thr Ile Ile Phe Asp Asn Trp Ile Pro Leu Leu Trp Cys Ala Asn Ile Leu Gly Tyr Ala Val Ser Thr Phe Ala Met Val Lys Gly Tyr Phe Phe Pro Thr Ser Ala Arg Asp Cys Lys Phe Thr Gly Asn Phe Phe Tyr Asn Tyr Met Met Gly Ile Glu Phe Asn Pro Arg Ile Gly Lys Trp Phe Asp Phe Lys Leu Phe Phe Asn Gly Arg Pro Gly Ile Val Ala Trp Thr Leu Ile Asn Leu Ser Phe Ala Ala Lys Gln Arg Glu Leu His Ser His Val Thr Asn Ala Met Val Leu Val Asn Val Leu Gln Ala Ile Tyr Val Ile Asp Phe Phe Trp Asn Glu Thr Trp Tyr Leu Lys Thr Ile Asp Ile Cys His Asp His Phe Gly Trp Tyr Leu Gly Trp Gly Asp Cys Val Trp Leu Pro Tyr Leu Tyr Thr Leu Gln Gly Leu Tyr Leu Val Tyr His Pro Val Gln Leu Ser Thr Pro His Ala Val Gly Val Leu Leu Leu Gly Leu Val Gly Tyr Tyr Ile Phe Arg Val Ala Asn His Gln Lys Asp Leu Phe Arg Arg Thr Asp Gly Arg Cys Leu Ile Trp Gly Arg Lys Pro Lys Val Ile Glu Cys Ser Tyr Thr Ser Ala Asp Gly Gln Arg His His Ser Lys Leu Leu Val Ser Gly Phe Trp Gly Val Ala Arg His Phe Asn Tyr Val Gly Asp Leu Met Gly Ser Leu Ala Tyr Cys Leu Ala Cys Gly Gly Gly His Leu Leu Pro Tyr Phe Tyr Ile Ile Tyr Met Ala Ile Leu Leu Thr His Arg Cys Leu Arg Asp Glu His Arg Cys Ala Ser Lys Tyr Gly Arg Asp Trp Glu Arg Tyr Thr Ala Ala Val Pro Tyr Arg Leu Leu Pro Gly Ile Phe <210> 17 <211> 1518 <212> DNA
<213> Homo sapiens <400> 17 cttccccact ggctcttggt ttatgagttc cccttttaag gatctgttgt gacttaccta 60 tctgggctag tgacctcaga tgtctcagac tgagcatctt accactgttt ctggttgatc 120 ccttcactca tggtcttaac acatttgcac ttcctctcat ctcagagagt acagtcacgg 180 ggcagagctt gcatagggat ccaggtgtta ctagtcttac tctggagctg gtccaactca 240 gtttcatggc acagaactag attaggtctc cactgcgcag tctgttttac tgcttaggga 300 aagccagctt ttctacccac acacgtttag tttgaagagt atctattttt ggagggttct 360 ttgggaggtt gggcaggctt ctttggatcc cagatacatt tagagctttt tgcattaagt 420 gtgaggaaaa taacttctct ttgatgatgt tgatacacca tgtkggcacc ytggggcaca 480 gcggtttagc tggggagatt ccatgagaat gaacecaaac tactcttctt tgctagggtc 540 ctttacccac acagaggtga gcctttcagg ttcttcattt tgcttagttt cttcccttgt 600 ccttggcatt taagaggcat ccatgtgtta gccagccaaa gccccctgaa ggagctggct 660 gctttaaagg atttacttgg gaggatgtca satggctttg ccttctgcag acttcattta 720 ttttaatctt tttatggctc ctttctcttg ctttaaaaca ggattataag cacacagcag 780 gtactgacac ctgaagtctt actaaattcc tgtcctcagg ccatcctttt tctcctgaaa 840 cctggactcc aattttcaat gacgtttttg tttttctctt tcaagcctaa ctatgggaca 900 gctttacgag aaggaaaaag atgaagatgg attcttatat gtggcctaca gcggagagaa 960 cacttttggc ttctgagggc cattgctggg ctaggtgcac cgtaactgct tgtgtatctt 1020 gtaaatagcc asccattttc agttattawa ccagaacctc ttmacataga cctattagtg 1080 catttgtaac tggatttatt tcttaatata tkggaaggtt ttgtttcctt agactagtaa 1140 attatcatac agagttttat tttgagtttt tctttttgtg cattgtcctc atgcctgtat 1200 tctccaggaa acttgtcctt ctggaaatca tatkgaatga tatttctata tcgaagtgag 1260 gtaggtgcgg tattaaagtg aaagggaagg tgatgcattt attctgggtt atgcttgaag 1320 tgttagatgg ctaagtatta aaattatcca aattaaatcc ttagcagtca gaacacttgc 1380 ttcactagaa tatgccaact gccaatcatg ttggactgag ctaatttgtt cctctttctg 1440 aaactattaa ggtaaataat taacaataaa aattctctta taaaggcaaa aaaaaaaaaa 1500 aaaaaaaaaa aaaaaasa 1518 <210> 18 <211> 55 <212> PRT
<213> Homo sapiens <400> 18 Met Val Leu Thr His Leu His Phe Leu Ser Ser Gln Arg Val Gln Ser Arg Gly Arg Ala Cys Ile Gly Ile Gln Val Leu Leu Val Leu Leu Trp Ser Trp Ser Asn Ser Val Ser Trp His Arg Thr Arg Leu Gly Leu His Cys Ala Val Cys Phe Thr Ala <210> 19 <211> 2097 <212> DNA
<213> Homo sapiens <400> 19 ctcttgagta cctggggctt gcagatgcat gccaccacac ccggctaatt tttttttttt 60 ttaaatagag atggggtctt gttctgttgc ccargctggt ctggaactcc tggcttcaat 120 cagtcctccc acctcagctt cccaaagctc tgggsttata ggcatgagcc actgtacctg 180 tccacctgag aaattttcta agcctggatt cagtcttatg aaatataata ctttgaaatg 240 cacaataact ttgaaaatga aactcattgc ttttcatttc accaggagtt actaactata 300 ataagcttta gagcaaattc tccttagata tgatttttgt tattattaga aacacatact 360 atcttgataa ctaaattttg ccaatcattc ttcttgacta gtggtcttta tatatacata 420 catatatata tatatataca tatatatata tatgaggaat tttccataag tgacttgaaa 480 aatacagaat gcactccatg gtaggtctgt tcagtgttat caggaatact gtttctcatc 540 ttcctttctt ggtgtccctt tgcaggggtt gtgtttgcac attatggtcc cgtctggaga 600 caacaaagga agttctctca ttcaactctt cgtcattttg ggttgggaaa acttagcttg 660 gagcccaaga ttattgagga gttcaaatat gtgaaagcag aaatgcaaaa gcacggagaa 720 gaccccttct gccctttctc catcatcagc aatgccgtct ctaacatcat ttgctccttg 780 tgctttggcc agcgctttga ttacactaat a9tgagttca agaaaatgct tggttttatg 840 tcacgaggcc tagaaatctg tctgaacagt caagtcctcc tggtcaacat atgcccttgg 900 ctttattacc ttccctttgg accatttaag gaattaagac aaattgaaaa ggatataacc 960 agtttcctta aaaaaatcat caaagaccat caagagtctc tggatagaga gaaccctcag 1020 gacttcatag acatgtacct tctccacatg gaagaggaga ggaaaaataa tagtaacagc 1080 agttttgatg aagagtactt attttatatc attggggatc tctttattgc tgggactgat 1140 accacaacta actctttgct ctggtgcctg ctgtatatgt cgctgaaccc cgatgtacaa 1200 gaaaaggttc atgaagaaat tgaaagagtc attggcgcca accgagctcc ttccctcaca 1260 gacaaggccc agatgcccta cacagaagcc accatcatgg aagtgcagag gctaactgtg 1320 gtggtgccgc ttgccattcc tcatatgacc tcagagaaca cagtgctcca agggtatacc 1380 attcctaaag gcacattgat cttacccaac ctgtggtcag tacatagaga cccagccatt 1440 tgggagaaac cggaggattt ctaccctaat cgatttctgg atgaccaagg acaactaatt 1500 aaaaaagaaa cctttattcc ttttgggata gggaagcggg tgtgtatggg agaacaactg 1560 gcaaagatgg aattattcct aatgtttgtg agcctaatgc agagtttcgc atttgcttta 1620 cctgaggatt ctaagaagcc cctcctgast ggaagatttg gtctaacttt agccccacat 1680 ccattta8ta taactatttc aaggagatga agagcatctc caagaagaga tggtaaaaag 1740 atatatsaat acatatcctt ctaagcagat tcttcctact gcaaaggaca gtgaatccag 1800 caactcagtg gatccaagct gggctcagag gtcggaagga gggtagagca cactgggagg 1860 tttcatcttg gaggattcct cagcaggata cttcagccat tttagtaatg caggtctgtg 1920 atttggggga tagaaaacaa agtacctatg aaacgggata tctggatttt acttgcagtg 1980 gcttccaccg atgggccaat cttctcattt cttagtgcct cagacatccc atatgtasaa 2040 tgagagtaat aaaacttggc ttctctctac ctctcagcac taaaaaaaaa aaaaaaa 2097 <210> 20 <211> 398 <212> PRT
<213> Homo sapiens <220>
1$
<221> UNSURE
<222> (379) <400> 20 -Val Leu Ser Gly Ile Leu Phe Leu Ile Phe Leu Ser Trp Cys Pro Phe Ala Gly Val Val Phe Ala His Tyr Gly Pro Val Trp Arg Gln Gln Arg Lys Phe Ser His Ser Thr Leu Arg His Phe Gly Leu Gly Lys Leu Ser Leu Glu Pro Lys Ile Ile Glu Glu Phe Lys Tyr Val Lys Ala Glu Met Gln Lys His Gly Glu Asp Pro Phe Cys Pro Phe Ser Ile Ile Ser Asn Ala Val Ser Asn Ile Ile Cys Ser Leu Cys Phe Gly Gln Arg Phe Asp Tyr Thr Asn Ser Glu Phe Lys Lys Met Leu Gly Phe Met Ser Arg Gly Leu Glu Ile Cys Leu Asn Ser Gln Val Leu Leu Val Asn Ile Cys Pro Trp Leu Tyr Tyr Leu Pro Phe Gly Pro Phe Lys Glu Leu Arg Gln Ile Glu Lys Asp Ile Thr Ser Phe Leu Lys Lys Ile Ile Lys Asp His Gln Glu Ser Leu Asp Arg Glu Asn Pro Gln Asp Phe Ile Asp Met Tyr Leu Leu His Met Glu Glu Glu Arg Lys Asn Asn Ser Asn Ser Ser Phe Asp Glu Glu Tyr Leu Phe Tyr Ile Ile Gly Asp Leu Phe Ile Ala Gly Thr Asp Thr Thr Thr Asn Ser Leu Leu Trp Cys Leu Leu Tyr Met Ser Leu Asn Pro Asp Val Gln Glu Lys Val His Glu Glu Ile Glu Arg Val Ile Gly Ala Asn Arg Ala Pro Ser Leu Thr Asp Lys Ala Gln Met Pro Tyr Thr Glu Ala Thr Ile Met Glu Val Gln Arg Leu Thr Val Val Val Pro Leu Ala Ile Pro His Met Thr Sex Glu Asn Thr Val Leu Gln Gly Tyr Thr Ile Pro Lys Gly Thr Leu Ile Leu Pro Asn Leu Trp Ser Val His Arg Asp Pro Ala Ile Trp Glu Lys Pro Glu Asp Phe Tyr Pro Asn Arg Phe Leu Asp Asp Gln Gly Gln Leu Ile Lys Lys Glu Thr Phe Ile Pro Phe Gly Ile Gly Lys Arg Val Cys Met Gly Glu Gln Leu Ala Lys Met Glu Leu Phe Leu Met Phe Vai Ser Leu Met Gln Ser Phe Ala Phe Ala Leu Pro Glu Asp Ser Lys Lys Pro Leu Leu Xaa Gly Arg Phe Gly Leu Thr Leu Ala Pro His Pro Phe Asn Ile Thr Ile Ser Arg Arg <210> 21 <211> 29 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotide <220>
<221> misc_feature <222> (2) <223> biotinylated phosphoaramidite residue <400> 21 cnaagttcta ttgggagatg gagtttgtg 29 <210> 22 <211> 29 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotide <220>
<221> misc_feature <222> (2) <223> biotinylated phosphoaramidite residue <400> 22 cnatccatgg tacatggtca gaagctcat 29 <210> 23 <211> 29 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotide <220>
<221> misc_feature <222> (2) <223> biotinylated phosphoaramiditeresidue <400> 23 tngagcaggt caggatacac tggaaaaga 29 <210> 24 <211> 29 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotide <220>
<221> misc_feature <222> (2) <223> biotinylated phosphoaramiditeresidue <400> 24 cnactgcctt tgttgctttc cagtagtga 29 <210> 25 <211> 29 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotide <220>
<221> misc_feature <222> (2) <223> biotinylated phosphoaramiditeresidue <400> 25 tnaatatcca catccccaaa tcctacacg 29 <210> 26 <211> 29 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotide <220>
<221> misc_feature <222> (2) <223> biotinylated phosphoaramiditeresidue <400> 26 cncttgcagc gggaaggcag agaagtttc 29 <210> 27 <211> 29 <212> DNA
<213> Artificial Sequence VSO 99/376'74 PCT/US99/01404 <220>
<223> oligonucleotide <220>
<221> misc_feature <222> (2) <223> biotinylated phosphoaramidite residue <400> 27 cntgagccac aatagacaga attcctacc 29 <210> 28 <211> 29 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotide <220>
<221> misc_feature <222> (2) <223> biotinylated phosphoaramidite residue <400> 28 cngtcagggc gcagctgtat tggtcacaa 29 <210> 29 <211> 19 <212> DNA
<213> Artificial Sequence <220>
<223> oligonucleotide <400> 29 acccacacag aagtgagcc 19 <210> 30 <211> 147 <212> PRT
<213> Homo sapiens <400> 30 Met Ala Ser Pro Ser Gly Leu Cys Val Leu Val Arg Leu Pro Lys Leu Ile Cys Gly Gly Lys Thr Leu Pro Arg Thr Leu Leu Asp Ile Leu Ala Asp Gly Thr Ile Leu Lys Val Gly Val Gly Cys Ser Glu Asp Ala Ser Lys Leu Leu Gln Asp Tyr Gly Leu Val Val Arg Gly Cys Leu Asp Leu Arg Tyr Leu Ala Met Arg Gln Arg Asn Asn Leu Leu Cys Asn Gly Leu Ser Leu Lys Ser Leu Ala Glu Thr Val Leu Asn Phe Pro Leu Asp Lys Ser Leu Leu Leu Arg Cys Ser Asn Trp Asp Ala Glu Thr Leu Thr Glu ' Asp Gln Val Ile Tyr Ala Ala Arg Asp Ala Gln Ile Ser Val Ala Leu Phe Leu His Leu Leu Gly Tyr Pro Phe Ser Arg Asn Ser Pro Gly Glu Lys Lys Arg <210> 31 <211> 176 <212> PRT
<213> Homo sapiens <400> 31 Met Thr Asp Cys Leu Val Ile Lys His Phe Leu Arg Lys Ile Ile Met Val His Pro Lys Val Arg Phe His Phe Ser Val Lys Val Asn Gly Ile Leu Ser Thr Glu Ile Phe Gly Val Glu Asn Glu Pro Thr Leu Asn Leu Gly Asn Gly Ile Ala Leu Leu Val Asp Ser Gln His Tyr Val Ser Arg Pro Asn Phe Gly Thr Ile Glu Ser His Cys Ser Arg Ile His Pro Val Leu Gly His Pro Val Met Leu Phe Ile Pro Glu Asp Val Ala Gly Met Asp Leu Leu Gly Glu Leu Ile Leu Thr Pro Ala Ala Ala Leu Cys Pro Ser Pro Lys Val Ser Ser Asn Gln Leu Asn Arg Ile Ser Ser Val Ser Ile Phe Leu Tyr Gly Pro Leu Gly Leu Pro Leu Ile Leu Ser Thr Trp Glu Gln Pro Met Thr Thr Phe Phe Lys Asp Thr Ser Ser Leu Val Asp Trp Lys Ile Pro Phe Val Tyr Asp Thr Gln Phe Gly Ser Gln Phe Gly <210> 32 <211> 89 <212> PRT
<213> Homo Sapiens VIrO 99/37674 PCT/US99/OI404 <400> 32 Met Gly Ser Leu Ser Thr Ala Asn Val Glu Phe Cys Leu Asp Val Phe Lys Glu Leu Asn Ser Asn Asn Ile Gly Asp Asn Ile Phe Phe Ser Ser Leu Ser Leu Leu Tyr Ala Leu Ser Met Val Leu Leu Gly Ala Arg Gly Glu Thr Ala Glu Gln Leu Glu Lys Val Leu His Phe Ser His Thr Val Asp Ser Leu Lys Pro Gly Phe Lys Asp Ser Pro Lys Cys Ser Gln Ala Gly Arg Ile His Ser Glu Phe Gly Val
Claims (41)
1. An isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:1;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:1 from nucleotide 734 to nucleotide 1873;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:1 from nucleotide 1403 to nucleotide 1873;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone cs756_2 deposited under accession number ATCC 98636;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone cs756_2 deposited under accession number ATCC 98636;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone cs756_2 deposited under accession number ATCC 98636;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone cs756_2 deposited under accession number ATCC 98636;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:2;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:2 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO:2;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above; and (k) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i).
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:1;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:1 from nucleotide 734 to nucleotide 1873;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:1 from nucleotide 1403 to nucleotide 1873;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone cs756_2 deposited under accession number ATCC 98636;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone cs756_2 deposited under accession number ATCC 98636;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone cs756_2 deposited under accession number ATCC 98636;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone cs756_2 deposited under accession number ATCC 98636;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:2;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:2 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO:2;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above; and (k) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i).
2. The polynucleotide of claim 1 wherein said polynucleotide is operably linked to at least one expression control sequence.
3. A host cell transformed with the polynucleotide of claim 2.
4. The host cell of claim 3, wherein said cell is a mammalian cell.
5. A process for producing a protein encoded by the polynucleotide of claim 2, which process comprises:
(a) growing a culture of the host cell of claim 3 in a suitable culture medium; and (b) purifying said protein from the culture.
(a) growing a culture of the host cell of claim 3 in a suitable culture medium; and (b) purifying said protein from the culture.
6. A protein produced according to the process of claim 5.
7. An isolated polynucleotide encoding the protein of claim 6.
8. The polynucleotide of claim 7, wherein the polynucleotide comprises the cDNA insert of clone cs756_2 deposited under accession number ATCC 98636.
9. A protein comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID N0:2;
(b) fragments of the amino acid sequence of SEQ ID N0:2, each fragment comprising eight consecutive amino acids of SEQ ID N0:2; and (c) the amino acid sequence encoded by the cDNA insert of clone cs756_2 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins.
(a) the amino acid sequence of SEQ ID N0:2;
(b) fragments of the amino acid sequence of SEQ ID N0:2, each fragment comprising eight consecutive amino acids of SEQ ID N0:2; and (c) the amino acid sequence encoded by the cDNA insert of clone cs756_2 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins.
10. The protein of claim 9, wherein said protein comprises the amino acid sequence of SEQ ID N0:2.
11. A composition comprising the protein of claim 9 and a pharmaceutically acceptable carrier.
12. A process for producing an isolated polynucleotide, wherein the process is selected from the group consisting of:
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(aa) SEQ ID NO:1, but excluding the poly(A) tail at the 3' end of SEQ ID NO:1; and (ab) the nucleotide sequence of the cDNA insert of clone cs756_2 deposited under accession number ATCC 98636;
(ii) hybridizing said probe(s) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C; and (iii) isolating the DNA polynucleotides detected with the probe(s);
and (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(ba) SEQ ID NO:1, but excluding the poly(A) tail at the 3' end of SEQ ID NO:1; and (bb) the nucleotide sequence of the cDNA insert of clone cs756_2 deposited under accession number ATCC 98636;
(ii) hybridizing said primer(s) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C;
(iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii);
wherein at least one isolated polynucleotide comprises a nucleotide sequence selected from the group consisting of:
(v) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:1, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID NO:1 to a nucleotide sequence corresponding to the 3' end of SEQ ID NO:1, but excluding the poly(A) tail at the 3' end of SEQ ID NO:1;
(w) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:1 from nucleotide 734 to nucleotide 1873, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
NO:1 from nucleotide 734 to nucleotide 1873, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:1 from nucleotide 734 to nucleotide 1873; and (x) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:1 from nucleotide 1403 to nucleotide 1873, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
NO:1 from nucleotide 1403 to nucleotide 1873, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:1 from nucleotide to nucleotide 1873.
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(aa) SEQ ID NO:1, but excluding the poly(A) tail at the 3' end of SEQ ID NO:1; and (ab) the nucleotide sequence of the cDNA insert of clone cs756_2 deposited under accession number ATCC 98636;
(ii) hybridizing said probe(s) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C; and (iii) isolating the DNA polynucleotides detected with the probe(s);
and (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(ba) SEQ ID NO:1, but excluding the poly(A) tail at the 3' end of SEQ ID NO:1; and (bb) the nucleotide sequence of the cDNA insert of clone cs756_2 deposited under accession number ATCC 98636;
(ii) hybridizing said primer(s) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C;
(iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii);
wherein at least one isolated polynucleotide comprises a nucleotide sequence selected from the group consisting of:
(v) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:1, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID NO:1 to a nucleotide sequence corresponding to the 3' end of SEQ ID NO:1, but excluding the poly(A) tail at the 3' end of SEQ ID NO:1;
(w) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:1 from nucleotide 734 to nucleotide 1873, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
NO:1 from nucleotide 734 to nucleotide 1873, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:1 from nucleotide 734 to nucleotide 1873; and (x) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:1 from nucleotide 1403 to nucleotide 1873, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
NO:1 from nucleotide 1403 to nucleotide 1873, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:1 from nucleotide to nucleotide 1873.
13. An isolated polynucleotide produced according to the process of claim 12.
14. An isolated polynucleotide comprising the polynucleotide of claim 13.
15. An isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:3;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:3 from nucleotide 26 to nucleotide 1738;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:3 from nucleotide 140 to nucleotide 1738;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone ew150_1 deposited under accession number ATCC 98636;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone ew150_1 deposited under accession number ATCC 98636;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone ew150_1 deposited under accession number ATCC 98636;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone ew150_1 deposited under accession number ATCC 98636;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:4;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:4 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO:4;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above; and (k) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i).
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:3;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:3 from nucleotide 26 to nucleotide 1738;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:3 from nucleotide 140 to nucleotide 1738;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone ew150_1 deposited under accession number ATCC 98636;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone ew150_1 deposited under accession number ATCC 98636;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone ew150_1 deposited under accession number ATCC 98636;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone ew150_1 deposited under accession number ATCC 98636;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:4;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:4 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO:4;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above; and (k) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i).
16. A protein comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:4;
(b) the amino acid sequence of SEQ ID NO:4 from amino acid 108 to amino acid 166;
(c) fragments of the amino acid sequence of SEQ ID NO:4, each fragment comprising eight consecutive amino acids of SEQ ID NO:4; and (d) the amino acid sequence encoded by the cDNA insert of clone ew150_1 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins.
(a) the amino acid sequence of SEQ ID NO:4;
(b) the amino acid sequence of SEQ ID NO:4 from amino acid 108 to amino acid 166;
(c) fragments of the amino acid sequence of SEQ ID NO:4, each fragment comprising eight consecutive amino acids of SEQ ID NO:4; and (d) the amino acid sequence encoded by the cDNA insert of clone ew150_1 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins.
17. A process for producing an isolated polynucleotide, wherein the process is selected from the group consisting of:
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(aa) SEQ ID NO:3, but excluding the poly(A) tail at the 3' end of SEQ ID NO:3; and (ab) the nucleotide sequence of the cDNA insert of clone ew150_1 deposited under accession number ATCC 98636;
(ii) hybridizing said probe(s) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C; and (iii) isolating the DNA polynucleotides detected with the probe(s);
and (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(ba) SEQ ID NO:3, but excluding the poly(A) tail at the 3' end of SEQ ID NO:3; and (bb) the nucleotide sequence of the cDNA insert of clone ew150_1 deposited under accession number ATCC 98636;
(ii) hybridizing said primer(s) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C;
(iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii);
wherein at least one isolated polynucleotide comprises a nucleotide sequence selected from the group consisting of:
(v) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:3, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID NO:3 to a nucleotide sequence corresponding to the 3' end of SEQ ID NO:3 , but excluding the poly(A) tail at the 3' end of SEQ ID NO:3;
(w) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:3 from nucleotide 26 to nucleotide 1738, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
NO:3 from nucleotide 26 to nucleotide 1738, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:3 from nucleotide 26 to nucleotide 1738;
and (x) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:3 from nucleotide 140 to nucleotide 1738, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
NO:3 from nucleotide 140 to nucleotide 1738, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:3 from nucleotide 140 to nucleotide 1738.
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(aa) SEQ ID NO:3, but excluding the poly(A) tail at the 3' end of SEQ ID NO:3; and (ab) the nucleotide sequence of the cDNA insert of clone ew150_1 deposited under accession number ATCC 98636;
(ii) hybridizing said probe(s) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C; and (iii) isolating the DNA polynucleotides detected with the probe(s);
and (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(ba) SEQ ID NO:3, but excluding the poly(A) tail at the 3' end of SEQ ID NO:3; and (bb) the nucleotide sequence of the cDNA insert of clone ew150_1 deposited under accession number ATCC 98636;
(ii) hybridizing said primer(s) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C;
(iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii);
wherein at least one isolated polynucleotide comprises a nucleotide sequence selected from the group consisting of:
(v) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:3, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID NO:3 to a nucleotide sequence corresponding to the 3' end of SEQ ID NO:3 , but excluding the poly(A) tail at the 3' end of SEQ ID NO:3;
(w) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:3 from nucleotide 26 to nucleotide 1738, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
NO:3 from nucleotide 26 to nucleotide 1738, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:3 from nucleotide 26 to nucleotide 1738;
and (x) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:3 from nucleotide 140 to nucleotide 1738, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
NO:3 from nucleotide 140 to nucleotide 1738, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:3 from nucleotide 140 to nucleotide 1738.
18. An isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:5;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:5 from nucleotide 1101 to nucleotide 1910;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:5 from nucleotide 1260 to nucleotide 1910;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone gg894_13 deposited under accession number ATCC 98636;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone gg894_13 deposited under accession number ATCC 98636;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone gg894_13 deposited under accession number ATCC 98636;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone gg894_13 deposited under accession number ATCC 98636;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:6;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:6 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO:6;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above; and (k) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i).
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:5;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:5 from nucleotide 1101 to nucleotide 1910;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:5 from nucleotide 1260 to nucleotide 1910;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone gg894_13 deposited under accession number ATCC 98636;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone gg894_13 deposited under accession number ATCC 98636;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone gg894_13 deposited under accession number ATCC 98636;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone gg894_13 deposited under accession number ATCC 98636;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:6;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:6 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO:6;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above; and (k) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i).
19. A protein comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:6;
(b) fragments of the amino acid sequence of SEQ ID NO:6, each fragment comprising eight consecutive amino acids of SEQ ID NO:6; and (c) the amino acid sequence encoded by the cDNA insert of clone gg894_13 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins.
(a) the amino acid sequence of SEQ ID NO:6;
(b) fragments of the amino acid sequence of SEQ ID NO:6, each fragment comprising eight consecutive amino acids of SEQ ID NO:6; and (c) the amino acid sequence encoded by the cDNA insert of clone gg894_13 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins.
20. A process for producing an isolated polynucleotide, wherein the process is selected from the group consisting of:
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(aa) SEQ ID NO:5, but excluding the poly(A) tail at the 3' end of SEQ ID NO:5; and (ab) the nucleotide sequence of the cDNA insert of clone gg894_13 deposited under accession number ATCC 98636;
(ii) hybridizing said probe(s) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C; and (iii) isolating the DNA polynucleotides detected with the probe(s);
and (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(ba) SEQ ID NO:5, but excluding the poly(A) tail at the 3' end of SEQ ID NO:5; and (bb) the nucleotide sequence of the cDNA insert of clone gg894_13 deposited under accession number ATCC 98636;
(ii) hybridizing said primer(s) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C;
(iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii);
wherein at least one isolated polynucleotide comprises a nucleotide sequence selected from the group consisting of:
(v) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:5, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID NO:5 to a nucleotide sequence corresponding to the 3' end of SEQ ID NO:5, but excluding the poly(A) tail at the 3' end of SEQ ID N0:5;
(w) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:5 from nucleotide 1101 to nucleotide 1910, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
NO:5 from nucleotide 1101 to nucleotide 1910, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:5 from nucleotide to nucleotide 1910; and (x) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:5 from nucleotide 1260 to nucleotide 1910, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
NO:5 from nucleotide 1260 to nucleotide 1910, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:5 from nucleotide to nucleotide 1910.
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(aa) SEQ ID NO:5, but excluding the poly(A) tail at the 3' end of SEQ ID NO:5; and (ab) the nucleotide sequence of the cDNA insert of clone gg894_13 deposited under accession number ATCC 98636;
(ii) hybridizing said probe(s) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C; and (iii) isolating the DNA polynucleotides detected with the probe(s);
and (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(ba) SEQ ID NO:5, but excluding the poly(A) tail at the 3' end of SEQ ID NO:5; and (bb) the nucleotide sequence of the cDNA insert of clone gg894_13 deposited under accession number ATCC 98636;
(ii) hybridizing said primer(s) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C;
(iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii);
wherein at least one isolated polynucleotide comprises a nucleotide sequence selected from the group consisting of:
(v) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:5, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID NO:5 to a nucleotide sequence corresponding to the 3' end of SEQ ID NO:5, but excluding the poly(A) tail at the 3' end of SEQ ID N0:5;
(w) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:5 from nucleotide 1101 to nucleotide 1910, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
NO:5 from nucleotide 1101 to nucleotide 1910, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:5 from nucleotide to nucleotide 1910; and (x) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:5 from nucleotide 1260 to nucleotide 1910, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
NO:5 from nucleotide 1260 to nucleotide 1910, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:5 from nucleotide to nucleotide 1910.
21. An isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:7;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:7 from nucleotide 452 to nucleotide 1102;
(c) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone it217_2 deposited under accession number ATCC 98636;
(d) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone it217_2 deposited under accession number ATCC 98636;
(e) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:8;
(f) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:8 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO:8;
(g) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(f) above; and (h) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(f).
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:7;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:7 from nucleotide 452 to nucleotide 1102;
(c) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone it217_2 deposited under accession number ATCC 98636;
(d) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone it217_2 deposited under accession number ATCC 98636;
(e) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:8;
(f) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:8 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO:8;
(g) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(f) above; and (h) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(f).
22. A protein comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:8;
(b) fragments of the amino acid sequence of SEQ ID NO:8, each fragment comprising eight consecutive amino acids of SEQ ID NO:8; and (c) the amino acid sequence encoded by the cDNA insert of clone it217_2 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins.
(a) the amino acid sequence of SEQ ID NO:8;
(b) fragments of the amino acid sequence of SEQ ID NO:8, each fragment comprising eight consecutive amino acids of SEQ ID NO:8; and (c) the amino acid sequence encoded by the cDNA insert of clone it217_2 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins.
23. A process for producing an isolated polynucleotide, wherein the process is selected from the group consisting of:
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(aa) SEQ ID NO:7, but excluding the poly(A) tail at the 3' end of SEQ ID NO:7; and (ab) the nucleotide sequence of the cDNA insert of clone it217_2 deposited under accession number ATCC 98636;
(ii) hybridizing said probe(s) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C; and (iii) isolating the DNA polynucleotides detected with the probe(s);
and (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(ba) SEQ ID NO:7, but excluding the poly(A) tail at the 3' end of SEQ ID NO:7; and (bb) the nucleotide sequence of the cDNA insert of clone it217_2 deposited under accession number ATCC 98636;
(ii) hybridizing said primer(s) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C;
(iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii);
wherein at least one isolated polynucleotide comprises a nucleotide sequence selected from the group consisting of:
(v) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:7, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID NO:7 to a nucleotide sequence corresponding to the 3' end of SEQ ID NO:7, but excluding the poly(A) tail at the 3' end of SEQ ID NO:7; and (w) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:7 from nucleotide 452 to nucleotide 1102, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
NO:7 from nucleotide 452 to nucleotide 1102, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:7 from nucleotide 452 to nucleotide 1102.
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(aa) SEQ ID NO:7, but excluding the poly(A) tail at the 3' end of SEQ ID NO:7; and (ab) the nucleotide sequence of the cDNA insert of clone it217_2 deposited under accession number ATCC 98636;
(ii) hybridizing said probe(s) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C; and (iii) isolating the DNA polynucleotides detected with the probe(s);
and (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(ba) SEQ ID NO:7, but excluding the poly(A) tail at the 3' end of SEQ ID NO:7; and (bb) the nucleotide sequence of the cDNA insert of clone it217_2 deposited under accession number ATCC 98636;
(ii) hybridizing said primer(s) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C;
(iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii);
wherein at least one isolated polynucleotide comprises a nucleotide sequence selected from the group consisting of:
(v) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:7, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID NO:7 to a nucleotide sequence corresponding to the 3' end of SEQ ID NO:7, but excluding the poly(A) tail at the 3' end of SEQ ID NO:7; and (w) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:7 from nucleotide 452 to nucleotide 1102, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
NO:7 from nucleotide 452 to nucleotide 1102, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:7 from nucleotide 452 to nucleotide 1102.
24. An isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:9;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:9 from nucleotide 127 to nucleotide 387;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:9 from nucleotide 172 to nucleotide 387;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone m1235 2 deposited under accession number ATCC 98636;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone m1235 2 deposited under accession number ATCC 98636;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone m1235 2 deposited under accession number ATCC 98636;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone m1235_2 deposited under accession number ATCC 98636;
(h} a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:10;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:10 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO:10;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a~(g) above; and (k) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i).
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:9;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:9 from nucleotide 127 to nucleotide 387;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:9 from nucleotide 172 to nucleotide 387;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone m1235 2 deposited under accession number ATCC 98636;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone m1235 2 deposited under accession number ATCC 98636;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone m1235 2 deposited under accession number ATCC 98636;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone m1235_2 deposited under accession number ATCC 98636;
(h} a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:10;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:10 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO:10;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a~(g) above; and (k) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i).
25. A protein comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:10;
(b) fragments of the amino acid sequence of SEQ ID NO:10, each fragment comprising eight consecutive amino acids of SEQ ID NO:10; and (c) the amino acid sequence encoded by the cDNA insert of clone m1235_2 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins.
(a) the amino acid sequence of SEQ ID NO:10;
(b) fragments of the amino acid sequence of SEQ ID NO:10, each fragment comprising eight consecutive amino acids of SEQ ID NO:10; and (c) the amino acid sequence encoded by the cDNA insert of clone m1235_2 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins.
26. A process for producing an isolated polynucleotide, wherein the process is selected from the group consisting of:
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(aa) SEQ ID NO:9, but excluding the poly(A) tail at the 3' end of SEQ ID NO:9; and (ab) the nucleotide sequence of the cDNA insert of clone m1235_2 deposited under accession number ATCC 98636;
(ii) hybridizing said probe(s) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C; and (iii) isolating the DNA polynucleotides detected with the probe(s);
and (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(ba) SEQ ID NO:9, but excluding the poly(A) tail at the 3' end of SEQ ID NO:9; and (bb) the nucleotide sequence of the cDNA insert of clone m1235_2 deposited under accession number ATCC 98636;
(ii) hybridizing said primer(s) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C;
(iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii);
wherein at least one isolated polynucleotide comprises a nucleotide sequence selected from the group consisting of:
(v) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:9, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID NO:9 to a nucleotide sequence corresponding to the 3' end of SEQ ID NO:9, but excluding the poly(A) tail at the 3' end of SEQ ID NO:9;
(w) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:9 from nucleotide 127 to nucleotide 387, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
NO:9 from nucleotide 127 to nucleotide 387, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:9 from nucleotide 127 to nucleotide 387;
and (x) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:9 from nucleotide 172 to nucleotide 387, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
NO:9 from nucleotide 172 to nucleotide 387, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:9 from nucleotide 172 to nucleotide 387.
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(aa) SEQ ID NO:9, but excluding the poly(A) tail at the 3' end of SEQ ID NO:9; and (ab) the nucleotide sequence of the cDNA insert of clone m1235_2 deposited under accession number ATCC 98636;
(ii) hybridizing said probe(s) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C; and (iii) isolating the DNA polynucleotides detected with the probe(s);
and (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(ba) SEQ ID NO:9, but excluding the poly(A) tail at the 3' end of SEQ ID NO:9; and (bb) the nucleotide sequence of the cDNA insert of clone m1235_2 deposited under accession number ATCC 98636;
(ii) hybridizing said primer(s) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C;
(iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii);
wherein at least one isolated polynucleotide comprises a nucleotide sequence selected from the group consisting of:
(v) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:9, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID NO:9 to a nucleotide sequence corresponding to the 3' end of SEQ ID NO:9, but excluding the poly(A) tail at the 3' end of SEQ ID NO:9;
(w) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:9 from nucleotide 127 to nucleotide 387, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
NO:9 from nucleotide 127 to nucleotide 387, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:9 from nucleotide 127 to nucleotide 387;
and (x) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:9 from nucleotide 172 to nucleotide 387, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
NO:9 from nucleotide 172 to nucleotide 387, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:9 from nucleotide 172 to nucleotide 387.
27. An isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:11;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:11 from nucleotide 147 to nucleotide 1163;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:11 from nucleotide 273 to nucleotide 1163;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone mt24_2 deposited under accession number ATCC 98636;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone mt24_2 deposited under accession number ATCC 98636;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone mt24_2 deposited under accession number ATCC 98636;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone mt24_2 deposited under accession number ATCC 98636;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:12;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:12 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO:12;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above; and (k) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i).
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:11;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:11 from nucleotide 147 to nucleotide 1163;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:11 from nucleotide 273 to nucleotide 1163;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone mt24_2 deposited under accession number ATCC 98636;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone mt24_2 deposited under accession number ATCC 98636;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone mt24_2 deposited under accession number ATCC 98636;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone mt24_2 deposited under accession number ATCC 98636;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:12;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:12 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO:12;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above; and (k) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i).
28. A protein comprising an amino acid sequence selected from the group consisting of:
{a) the amino acid sequence of SEQ ID NO:12;
(b) fragments of the amino acid sequence of SEQ ID NO:12; each fragment comprising eight consecutive amino acids of SEQ ID NO:12; and (c) the amino acid sequence encoded by the cDNA insert of clone mt24_2 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins.
{a) the amino acid sequence of SEQ ID NO:12;
(b) fragments of the amino acid sequence of SEQ ID NO:12; each fragment comprising eight consecutive amino acids of SEQ ID NO:12; and (c) the amino acid sequence encoded by the cDNA insert of clone mt24_2 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins.
29. A process for producing an isolated polynucleotide, wherein the process is selected from the group consisting of:
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(aa) SEQ ID NO:11, but excluding the poly(A) tail at the 3' end of SEQ ID NO:11; and (ab) the nucleotide sequence of the cDNA insert of clone mt24_2 deposited under accession number ATCC 98636;
(ii) hybridizing said probe(s) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C; and (iii) isolating the DNA polynucleotides detected with the probe(s);
and (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(ba) SEQ ID NO:11, but excluding the poly(A) tail at the 3' end of SEQ ID NO:11; and (bb) the nucleotide sequence of the cDNA insert of clone mt24_2 deposited under accession number ATCC 98636;
(ii) hybridizing said primer(s) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C;
(iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii);
wherein at least one isolated polynucleotide comprises a nucleotide sequence selected from the group consisting of:
(v) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:11, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID NO:11 to a nucleotide sequence corresponding to the 3' end of SEQ ID NO:11, but excluding the poly(A) tail at the 3' end of SEQ ID NO:11;
(w) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:11 from nucleotide 147 to nucleotide 1163, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
NO:11 from nucleotide 147 to nucleotide 1163, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:11 from nucleotide to nucleotide 1163; and (x) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:11 from nucleotide 273 to nucleotide 1163, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
NO:11 from nucleotide 273 to nucleotide 1163, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:11 from nucleotide to nucleotide 1163.
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(aa) SEQ ID NO:11, but excluding the poly(A) tail at the 3' end of SEQ ID NO:11; and (ab) the nucleotide sequence of the cDNA insert of clone mt24_2 deposited under accession number ATCC 98636;
(ii) hybridizing said probe(s) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C; and (iii) isolating the DNA polynucleotides detected with the probe(s);
and (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(ba) SEQ ID NO:11, but excluding the poly(A) tail at the 3' end of SEQ ID NO:11; and (bb) the nucleotide sequence of the cDNA insert of clone mt24_2 deposited under accession number ATCC 98636;
(ii) hybridizing said primer(s) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C;
(iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii);
wherein at least one isolated polynucleotide comprises a nucleotide sequence selected from the group consisting of:
(v) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:11, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID NO:11 to a nucleotide sequence corresponding to the 3' end of SEQ ID NO:11, but excluding the poly(A) tail at the 3' end of SEQ ID NO:11;
(w) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:11 from nucleotide 147 to nucleotide 1163, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
NO:11 from nucleotide 147 to nucleotide 1163, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:11 from nucleotide to nucleotide 1163; and (x) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:11 from nucleotide 273 to nucleotide 1163, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
NO:11 from nucleotide 273 to nucleotide 1163, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:11 from nucleotide to nucleotide 1163.
30. An isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:13;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:13 from nucleotide 320 to nucleotide 1681;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:13 from nucleotide 437 to nucleotide 1681;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone pe584 2 deposited under accession number ATCC 98636;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone pe584_2 deposited under accession number ATCC 98636;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone pe584_2 deposited under accession number ATCC 98636;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone pe584_2 deposited under accession number ATCC 98636;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:14;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:14 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO:14;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above; and (k) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i).
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:13;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:13 from nucleotide 320 to nucleotide 1681;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:13 from nucleotide 437 to nucleotide 1681;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone pe584 2 deposited under accession number ATCC 98636;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone pe584_2 deposited under accession number ATCC 98636;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone pe584_2 deposited under accession number ATCC 98636;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone pe584_2 deposited under accession number ATCC 98636;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:14;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:14 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO:14;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above; and (k) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i).
31. A protein comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:14;
(b) fragments of the amino acid sequence of SEQ ID NO:14, each fragment comprising eight consecutive amino acids of SEQ ID NO:14; and (c) the amino acid sequence encoded by the cDNA insert of clone pe584_2 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins.
(a) the amino acid sequence of SEQ ID NO:14;
(b) fragments of the amino acid sequence of SEQ ID NO:14, each fragment comprising eight consecutive amino acids of SEQ ID NO:14; and (c) the amino acid sequence encoded by the cDNA insert of clone pe584_2 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins.
32. A process for producing an isolated polynucleotide, wherein the process is selected from the group consisting of:
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(aa) SEQ ID NO:13, but excluding the poly(A) tail at the 3' end of SEQ ID NO:13; and (ab) the nucleotide sequence of the cDNA insert of clone pe584_2 deposited under accession number ATCC 98636;
(ii) hybridizing said probe(s) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C; and (iii) isolating the DNA polynucleotides detected with the probe(s);
and (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(ba) SEQ ID NO:13, but excluding the poly(A) tail at the 3' end of SEQ ID NO:13; and (bb) the nucleotide sequence of the cDNA insert of clone pe584_2 deposited under accession number ATCC 98636;
(ii) hybridizing said primer(s) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C;
(iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii);
wherein at least one isolated polynucleotide comprises a nucleotide sequence selected from the group consisting of:
(v) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:13, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID NO:13 to a nucleotide sequence corresponding to the 3' end of SEQ ID NO:13, but excluding the poly(A) tail at the 3' end of SEQ ID NO:13;
(w) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:13 from nucleotide 320 to nucleotide 1681, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
NO:13 from nucleotide 320 to nucleotide 1681, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:13 from nucleotide to nucleotide 1681; and (x) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:13 from nucleotide 437 to nucleotide 1681, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
NO:13 from nucleotide 437 to nucleotide 1681, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:13 from nucleotide to nucleotide 1681.
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(aa) SEQ ID NO:13, but excluding the poly(A) tail at the 3' end of SEQ ID NO:13; and (ab) the nucleotide sequence of the cDNA insert of clone pe584_2 deposited under accession number ATCC 98636;
(ii) hybridizing said probe(s) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C; and (iii) isolating the DNA polynucleotides detected with the probe(s);
and (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(ba) SEQ ID NO:13, but excluding the poly(A) tail at the 3' end of SEQ ID NO:13; and (bb) the nucleotide sequence of the cDNA insert of clone pe584_2 deposited under accession number ATCC 98636;
(ii) hybridizing said primer(s) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C;
(iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii);
wherein at least one isolated polynucleotide comprises a nucleotide sequence selected from the group consisting of:
(v) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:13, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID NO:13 to a nucleotide sequence corresponding to the 3' end of SEQ ID NO:13, but excluding the poly(A) tail at the 3' end of SEQ ID NO:13;
(w) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:13 from nucleotide 320 to nucleotide 1681, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
NO:13 from nucleotide 320 to nucleotide 1681, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:13 from nucleotide to nucleotide 1681; and (x) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:13 from nucleotide 437 to nucleotide 1681, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
NO:13 from nucleotide 437 to nucleotide 1681, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:13 from nucleotide to nucleotide 1681.
33. An isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:15;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:15 from nucleotide 78 to nucleotide 1502;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:15 from nucleotide 564 to nucleotide 1502;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone pj323_2 deposited under accession number ATCC 98636;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone pj323_2 deposited under accession number ATCC 98636;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone pj323_2 deposited under accession number ATCC 98636;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone pj323_2 deposited under accession number ATCC 98636;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:16;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:16 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO:16;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above; and (k) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i).
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:15;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:15 from nucleotide 78 to nucleotide 1502;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:15 from nucleotide 564 to nucleotide 1502;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone pj323_2 deposited under accession number ATCC 98636;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone pj323_2 deposited under accession number ATCC 98636;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone pj323_2 deposited under accession number ATCC 98636;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone pj323_2 deposited under accession number ATCC 98636;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:16;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:16 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO:16;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above; and (k) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i).
34. A protein comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:16;
(b) the amino acid sequence of SEQ ID NO:16 from amino acid 54 to amino acid 145;
(c) fragments of the amino acid sequence of SEQ ID NO:16, each fragment comprising eight consecutive amino acids of SEQ ID NO:16; and (d) the amino acid sequence encoded by the cDNA insert of clone pj323_2 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins.
(a) the amino acid sequence of SEQ ID NO:16;
(b) the amino acid sequence of SEQ ID NO:16 from amino acid 54 to amino acid 145;
(c) fragments of the amino acid sequence of SEQ ID NO:16, each fragment comprising eight consecutive amino acids of SEQ ID NO:16; and (d) the amino acid sequence encoded by the cDNA insert of clone pj323_2 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins.
35. A process for producing an isolated polynucleotide, wherein the process is selected from the group consisting of:
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(aa) SEQ ID NO:15, but excluding the poly(A) tail at the 3' end of SEQ ID NO:15; and (ab) the nucleotide sequence of the cDNA insert of clone pj323_2 deposited under accession number ATCC 98636;
(ii) hybridizing said probe(s) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C; and (iii) isolating the DNA polynucleotides detected with the probe(s);
and (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(ba) SEQ ID NO:15, but excluding the poly(A) tail at the 3' end of SEQ ID NO:15; and (bb) the nucleotide sequence of the cDNA insert of clone pj323_2 deposited under accession number ATCC 98636;
(ii) hybridizing said primer(s) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C;
(iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii);
wherein at least one isolated polynucleotide comprises a nucleotide sequence selected from the group consisting of:
(v) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:15, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID NO:15 to a nucleotide sequence corresponding to the 3' end of SEQ ID NO:15, but excluding the poly(A) tail at the 3' end of SEQ ID NO:15;
(w) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:15 from nucleotide 78 to nucleotide 1502, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
NO:15 from nucleotide 78 to nucleotide 1502, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:15 from nucleotide 78 to nucleotide 1502; and (x) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:15 from nucleotide 564 to nucleotide 1502, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
NO:15 from nucleotide 564 to nucleotide 1502, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:15 from nucleotide to nucleotide 1502.
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(aa) SEQ ID NO:15, but excluding the poly(A) tail at the 3' end of SEQ ID NO:15; and (ab) the nucleotide sequence of the cDNA insert of clone pj323_2 deposited under accession number ATCC 98636;
(ii) hybridizing said probe(s) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C; and (iii) isolating the DNA polynucleotides detected with the probe(s);
and (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(ba) SEQ ID NO:15, but excluding the poly(A) tail at the 3' end of SEQ ID NO:15; and (bb) the nucleotide sequence of the cDNA insert of clone pj323_2 deposited under accession number ATCC 98636;
(ii) hybridizing said primer(s) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C;
(iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii);
wherein at least one isolated polynucleotide comprises a nucleotide sequence selected from the group consisting of:
(v) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:15, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID NO:15 to a nucleotide sequence corresponding to the 3' end of SEQ ID NO:15, but excluding the poly(A) tail at the 3' end of SEQ ID NO:15;
(w) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:15 from nucleotide 78 to nucleotide 1502, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
NO:15 from nucleotide 78 to nucleotide 1502, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:15 from nucleotide 78 to nucleotide 1502; and (x) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:15 from nucleotide 564 to nucleotide 1502, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
NO:15 from nucleotide 564 to nucleotide 1502, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:15 from nucleotide to nucleotide 1502.
36. An isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:17;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:17 from nucleotide 130 to nucleotide 294;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:17 from nucleotide 241 to nucleotide 294;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone yb24_1 deposited under accession number ATCC 98636;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone yb24_1 deposited under accession number ATCC 98636;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone yb24_1 deposited under accession number ATCC
98636;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone yb24_1 deposited under accession number ATCC 98636;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:18;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:18 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO:18;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above; and (k) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i).
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:17;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:17 from nucleotide 130 to nucleotide 294;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:17 from nucleotide 241 to nucleotide 294;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone yb24_1 deposited under accession number ATCC 98636;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone yb24_1 deposited under accession number ATCC 98636;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone yb24_1 deposited under accession number ATCC
98636;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone yb24_1 deposited under accession number ATCC 98636;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:18;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:18 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO:18;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above; and (k) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i).
37. A protein comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:18;
(b) fragments of the amino acid sequence of SEQ ID NO:18, each fragment comprising eight consecutive amino acids of SEQ ID NO:18; and (c) the amino acid sequence encoded by the cDNA insert of clone yb24_1 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins.
(a) the amino acid sequence of SEQ ID NO:18;
(b) fragments of the amino acid sequence of SEQ ID NO:18, each fragment comprising eight consecutive amino acids of SEQ ID NO:18; and (c) the amino acid sequence encoded by the cDNA insert of clone yb24_1 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins.
38. A process for producing an isolated polynucleotide, wherein the process is selected from the group consisting of:
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(aa) SEQ ID NO:17, but excluding the poly(A} tail at the 3' end of SEQ ID NO:17; and (ab) the nucleotide sequence of the cDNA insert of clone yb24_1 deposited under accession number ATCC 98636;
(ii) hybridizing said probe(s) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C; and (iii) isolating the DNA polynucleotides detected with the probe(s);
and (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(ba) SEQ ID NO:17, but excluding the poly(A) tail at the 3' end of SEQ ID NO:17; and (bb) the nucleotide sequence of the cDNA insert of clone yb24_1 deposited under accession number ATCC 98636;
(ii) hybridizing said primers) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C;
(iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii);
wherein at least one isolated polynucleotide comprises a nucleotide sequence selected from the group consisting of:
(v) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:17, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID NO:17 to a nucleotide sequence corresponding to the 3' end of SEQ ID NO:17, but excluding the poly(A) tail at the 3' end of SEQ ID NO:17;
(w) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:17 from nucleotide 130 to nucleotide 294, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
NO:17 from nucleotide 130 to nucleotide 294, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:17 from nucleotide 130 to nucleotide 294; and (x) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:17 from nucleotide 241 to nucleotide 294, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
NO:17 from nucleotide 241 to nucleotide 294, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:17 from nucleotide 241 to nucleotide 294.
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(aa) SEQ ID NO:17, but excluding the poly(A} tail at the 3' end of SEQ ID NO:17; and (ab) the nucleotide sequence of the cDNA insert of clone yb24_1 deposited under accession number ATCC 98636;
(ii) hybridizing said probe(s) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C; and (iii) isolating the DNA polynucleotides detected with the probe(s);
and (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(ba) SEQ ID NO:17, but excluding the poly(A) tail at the 3' end of SEQ ID NO:17; and (bb) the nucleotide sequence of the cDNA insert of clone yb24_1 deposited under accession number ATCC 98636;
(ii) hybridizing said primers) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C;
(iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii);
wherein at least one isolated polynucleotide comprises a nucleotide sequence selected from the group consisting of:
(v) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:17, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID NO:17 to a nucleotide sequence corresponding to the 3' end of SEQ ID NO:17, but excluding the poly(A) tail at the 3' end of SEQ ID NO:17;
(w) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:17 from nucleotide 130 to nucleotide 294, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
NO:17 from nucleotide 130 to nucleotide 294, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:17 from nucleotide 130 to nucleotide 294; and (x) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:17 from nucleotide 241 to nucleotide 294, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
NO:17 from nucleotide 241 to nucleotide 294, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:17 from nucleotide 241 to nucleotide 294.
39. An isolated polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:19;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:19 from nucleotide 514 to nucleotide 1707;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:19 from nucleotide 580 to nucleotide 1707;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone yb44_1 deposited under accession number ATCC 98636;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone yb44_1 deposited under accession number ATCC 98636;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone yb44_1 deposited under accession number ATCC
98636;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone yb44_1 deposited under accession number ATCC 98636;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:20;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:20 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO:20;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above; and (k) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i).
(a) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:19;
(b) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:19 from nucleotide 514 to nucleotide 1707;
(c) a polynucleotide comprising the nucleotide sequence of SEQ ID
NO:19 from nucleotide 580 to nucleotide 1707;
(d) a polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone yb44_1 deposited under accession number ATCC 98636;
(e) a polynucleotide encoding the full-length protein encoded by the cDNA insert of clone yb44_1 deposited under accession number ATCC 98636;
(f) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone yb44_1 deposited under accession number ATCC
98636;
(g) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone yb44_1 deposited under accession number ATCC 98636;
(h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO:20;
(i) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO:20 having biological activity, the fragment comprising eight consecutive amino acids of SEQ ID NO:20;
(j) a polynucleotide which is an allelic variant of a polynucleotide of (a)-(g) above; and (k) a polynucleotide that hybridizes under stringent conditions to any one of the polynucleotides specified in (a)-(i).
40. A protein comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence of SEQ ID NO:20;
(b) fragments of the amino acid sequence of SEQ ID NO:20, each fragment comprising eight consecutive amino acids of SEQ ID NO:20; and (c) the amino acid sequence encoded by the cDNA insert of clone yb44_1 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins.
(a) the amino acid sequence of SEQ ID NO:20;
(b) fragments of the amino acid sequence of SEQ ID NO:20, each fragment comprising eight consecutive amino acids of SEQ ID NO:20; and (c) the amino acid sequence encoded by the cDNA insert of clone yb44_1 deposited under accession number ATCC 98636;
the protein being substantially free from other mammalian proteins.
41. A process for producing an isolated polynucleotide, wherein the process is selected from the group consisting of:
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(aa) SEQ ID NO:19, but excluding the poly(A) tail at the 3' end of SEQ ID NO:19; and (ab) the nucleotide sequence of the cDNA insert of clone yb44_1 deposited under accession number ATCC 98636;
(ii) hybridizing said probes) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C; and (iii) isolating the DNA polynucleotides detected with the probe(s);
and (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(ba) SEQ ID NO:19, but excluding the poly(A) tail at the 3' end of SEQ ID NO:19; and (bb) the nucleotide sequence of the cDNA insert of clone yb44_1 deposited under accession number ATCC 98636;
(ii) hybridizing said primer(s) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C;
(iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii);
wherein at least one isolated polynucleotide comprises a nucleotide sequence selected from the group consisting of:
(v) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:19, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID NO:19 to a nucleotide sequence corresponding to the 3' end of SEQ ID NO:19, but excluding the poly(A) tail at the 3' end of SEQ ID NO:19;
(w) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:19 from nucleotide 514 to nucleotide 1707, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
NO:19 from nucleotide 514 to nucleotide 1707, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:19 from nucleotide to nucleotide 1707; and (x) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:19 from nucleotide 580 to nucleotide 1707, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
NO:19 from nucleotide 580 to nucleotide 1707, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:19 from nucleotide to nucleotide 1707.
(a) a process comprising the steps of:
(i) preparing one or more polynucleotide probes that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(aa) SEQ ID NO:19, but excluding the poly(A) tail at the 3' end of SEQ ID NO:19; and (ab) the nucleotide sequence of the cDNA insert of clone yb44_1 deposited under accession number ATCC 98636;
(ii) hybridizing said probes) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C; and (iii) isolating the DNA polynucleotides detected with the probe(s);
and (b) a process comprising the steps of:
(i) preparing one or more polynucleotide primers that hybridize in 6X SSC at 65 degrees C to a nucleotide sequence selected from the group consisting of:
(ba) SEQ ID NO:19, but excluding the poly(A) tail at the 3' end of SEQ ID NO:19; and (bb) the nucleotide sequence of the cDNA insert of clone yb44_1 deposited under accession number ATCC 98636;
(ii) hybridizing said primer(s) to human genomic DNA in conditions at least as stringent as 4X SSC at 65 degrees C;
(iii) amplifying human DNA sequences; and (iv) isolating the polynucleotide products of step (b)(iii);
wherein at least one isolated polynucleotide comprises a nucleotide sequence selected from the group consisting of:
(v) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:19, and extending contiguously from a nucleotide sequence corresponding to the 5' end of SEQ ID NO:19 to a nucleotide sequence corresponding to the 3' end of SEQ ID NO:19, but excluding the poly(A) tail at the 3' end of SEQ ID NO:19;
(w) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:19 from nucleotide 514 to nucleotide 1707, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
NO:19 from nucleotide 514 to nucleotide 1707, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:19 from nucleotide to nucleotide 1707; and (x) a nucleotide sequence corresponding to the cDNA sequence of SEQ ID
NO:19 from nucleotide 580 to nucleotide 1707, and extending contiguously from a nucleotide sequence corresponding to the 5' end of said sequence of SEQ ID
NO:19 from nucleotide 580 to nucleotide 1707, to a nucleotide sequence corresponding to the 3' end of said sequence of SEQ ID NO:19 from nucleotide to nucleotide 1707.
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US7213498P | 1998-01-22 | 1998-01-22 | |
US60/072,134 | 1998-01-22 | ||
US23560999A | 1999-01-20 | 1999-01-20 | |
US09/235,609 | 1999-01-20 | ||
PCT/US1999/001404 WO1999037674A1 (en) | 1998-01-22 | 1999-01-21 | Secreted proteins and polynucleotides encoding them |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2318303A1 true CA2318303A1 (en) | 1999-07-29 |
Family
ID=26753034
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002318303A Abandoned CA2318303A1 (en) | 1998-01-22 | 1999-01-21 | Secreted proteins and polynucleotides encoding them |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP1049714A4 (en) |
JP (1) | JP2002508936A (en) |
AU (1) | AU2466499A (en) |
CA (1) | CA2318303A1 (en) |
WO (1) | WO1999037674A1 (en) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU2001250216A1 (en) * | 2000-04-20 | 2001-11-07 | Cytochroma Inc. | A thymus expressed human cytochrome P450 (P450TEC) |
US20020072488A1 (en) * | 2000-12-12 | 2002-06-13 | Merkulov Gennady V. | Isolated human transporter proteins, nucleic acid molecules encoding human transporter proteins, and uses thereof |
-
1999
- 1999-01-21 WO PCT/US1999/001404 patent/WO1999037674A1/en not_active Application Discontinuation
- 1999-01-21 EP EP99904216A patent/EP1049714A4/en not_active Withdrawn
- 1999-01-21 AU AU24664/99A patent/AU2466499A/en not_active Abandoned
- 1999-01-21 JP JP2000528595A patent/JP2002508936A/en not_active Withdrawn
- 1999-01-21 CA CA002318303A patent/CA2318303A1/en not_active Abandoned
Also Published As
Publication number | Publication date |
---|---|
JP2002508936A (en) | 2002-03-26 |
AU2466499A (en) | 1999-08-09 |
WO1999037674A1 (en) | 1999-07-29 |
EP1049714A1 (en) | 2000-11-08 |
EP1049714A4 (en) | 2003-01-22 |
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