U.S. patent application number 10/016249 was filed with the patent office on 2003-05-29 for secreted proteins and polynucleotides encoding them.
This patent application is currently assigned to Genetics Institute, Inc.. Invention is credited to Evans, Cheryl, Jacobs, Kenneth, La Vallie, Edward R., McCoy, John M., Merberg, David, Mi, Sha, Racie, Lisa A., Treacy, Maurice.
Application Number | 20030100053 10/016249 |
Document ID | / |
Family ID | 27373709 |
Filed Date | 2003-05-29 |
United States Patent
Application |
20030100053 |
Kind Code |
A1 |
Jacobs, Kenneth ; et
al. |
May 29, 2003 |
Secreted proteins and polynucleotides encoding them
Abstract
Novel polynucleotides and the proteins encoded thereby are
disclosed.
Inventors: |
Jacobs, Kenneth; (Newton,
MA) ; McCoy, John M.; (Reading, MA) ; La
Vallie, Edward R.; (Harvard, MA) ; Racie, Lisa
A.; (Acton, MA) ; Evans, Cheryl; (Germantown,
MD) ; Merberg, David; (Acton, MA) ; Mi,
Sha; (Belmont, CA) ; Treacy, Maurice;
(Chestnut Hill, MA) |
Correspondence
Address: |
LAHIVE & COCKFIELD
28 STATE STREET
BOSTON
MA
02109
US
|
Assignee: |
Genetics Institute, Inc.
|
Family ID: |
27373709 |
Appl. No.: |
10/016249 |
Filed: |
October 30, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10016249 |
Oct 30, 2001 |
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09175928 |
Oct 20, 1998 |
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6312921 |
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09175928 |
Oct 20, 1998 |
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09080478 |
May 18, 1998 |
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09080478 |
May 18, 1998 |
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08976110 |
Nov 21, 1997 |
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08976110 |
Nov 21, 1997 |
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08686878 |
Jul 26, 1996 |
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5708157 |
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09175928 |
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08721489 |
Sep 27, 1996 |
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5786465 |
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09175928 |
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08721924 |
Sep 27, 1996 |
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5969125 |
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Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 514/19.3; 514/19.8; 514/44R; 530/350;
536/23.5 |
Current CPC
Class: |
C07K 14/47 20130101;
A61K 38/00 20130101 |
Class at
Publication: |
435/69.1 ;
435/325; 435/320.1; 530/350; 536/23.5; 514/12; 514/44 |
International
Class: |
C12P 021/02; C12N
005/06; C07K 014/435; A61K 048/00; A61K 038/17; C07H 021/04 |
Claims
What is claimed is:
1. A composition comprising an isolated polynucleotide selected
from the group consisting of: (a) a polynucleotide comprising the
nucleotide sequence of SEQ ID NO:1; (b) a polynucleotide comprising
the nucleotide sequence of SEQ ID NO:1 from nucleotide 44 to
nucleotide 1204; (c) a polynucleotide comprising the nucleotide
sequence of SEQ ID NO:1 from nucleotide 1 to nucleotide 403; (d) a
polynucleotide comprising the nucleotide sequence of the
full-length protein coding sequence of clone AJ26.sub.--3 deposited
under accession number ATCC 98115; (e) a polynucleotide encoding
the full-length protein encoded by the cDNA insert of clone
AJ26.sub.--3 deposited under accession number ATCC 98115; (f) a
polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone AJ26.sub.--3 deposited under
accession number ATCC 98115; (g) a polynucleotide encoding the
mature protein encoded by the cDNA insert of clone AJ26.sub.--3
deposited under accession number ATCC 98115; (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; (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 capable of hybridizing under stringent conditions to
any one of the polynucleotides specified in (a)-(i).
2. A composition of claim 1 wherein said polynucleotide is operably
linked to at least one expression control sequence.
3. A host cell transformed with a composition 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 a composition 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.
6. A protein produced according to the process of claim 5.
7. The protein of claim 6 comprising a mature protein.
8. 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:2; (b) the amino acid
sequence of SEQ ID NO:2 from amino acid 1 to amino acid 120; (c)
fragments of the amino acid sequence of SEQ ID NO:2; and (d) the
amino acid sequence encoded by the cDNA insert of clone
AJ26.sub.--3 deposited under accession number ATCC 98115; the
protein being substantially free from other mammalian proteins.
9. The composition of claim 8, wherein said protein comprises the
amino acid sequence of SEQ ID NO:2.
10. The composition of claim 8, wherein said protein comprises the
amino acid sequence of SEQ ID NO:2 from amino acid 1 to amino acid
120.
11. The composition of claim 8, further comprising a
pharmaceutically acceptable carrier.
12. A method for preventing, treating or ameliorating a medical
condition which comprises administering to a mammalian subject a
therapeutically effective amount of a composition of claim 11.
13. An isolated gene corresponding to the cDNA sequence of SEQ ID
NO:1.
14. A composition comprising 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 928 to
nucleotide 2541; (c) a polynucleotide comprising the nucleotide
sequence of SEQ ID NO:3 from nucleotide 988 to nucleotide 2541; (d)
a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3
from nucleotide 684 to nucleotide 1128; (e) a polynucleotide
comprising the nucleotide sequence of the full-length protein
coding sequence of clone AJ172.sub.--2 deposited under accession
number ATCC 98115; (f) a polynucleotide encoding the full-length
protein encoded by the cDNA insert of clone AJ172.sub.--2 deposited
under accession number ATCC 98115; (g) a polynucleotide comprising
the nucleotide sequence of the mature protein coding sequence of
clone AJ172.sub.--2 deposited under accession number ATCC 98115;
(h) a polynucleotide encoding the mature protein encoded by the
cDNA insert of clone AJ172.sub.--2 deposited under accession number
ATCC 98115; (i) a polynucleotide encoding a protein comprising the
amino acid sequence of SEQ ID NO:4; a polynucleotide encoding a
protein comprising a fragment of the amino acid sequence of SEQ ID
NO:4 having biological activity; (k) a polynucleotide which is an
allelic variant of a polynucleotide of (a)-(h) above; (l) a
polynucleotide which encodes a species homologue of the protein of
(i) or (j) above; and (m) a polynucleotide capable of hybridizing
under stringent conditions to any one of the polynucleotides
specified in (a)-(j).
15. 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:4; (b) the amino acid
sequence of SEQ ID NO:4 from amino acid 1 to amino acid 67; (c)
fragments of the amino acid sequence of SEQ ID NO:4; and (d) the
amino acid sequence encoded by the cDNA insert of clone
AJ172.sub.--2 deposited under accession number ATCC 98115; the
protein being substantially free from other mammalian proteins.
16. An isolated gene corresponding to the cDNA sequence of SEQ ID
NO:3.
17. An isolated gene corresponding to the cDNA sequence of SEQ ID
NO:6, SEQ ID NO:5 or SEQ ID NO:8.
18. A composition comprising 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 6 to
nucleotide 2408; (c) a polynucleotide comprising the nucleotide
sequence of SEQ ID NO:9 from nucleotide 1295 to nucleotide 1705;
(d) a polynucleotide comprising the nucleotide sequence of the
full-length protein coding sequence of clone BL89.sub.--13
deposited under accession number ATCC 98115; (e) a polynucleotide
encoding the full-length protein encoded by the cDNA insert of
clone BL89.sub.--13 deposited under accession number ATCC 98115;
(f) a polynucleotide comprising the nucleotide sequence of the
mature protein coding sequence of clone BL89.sub.--13 deposited
under accession number ATCC 98115; (g) a polynucleotide encoding
the mature protein encoded by the cDNA insert of clone
BL89.sub.--13 deposited under accession number ATCC 98115; (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; (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 capable of hybridizing
under stringent conditions to any one of the polynucleotides
specified in (a)-(i).
19. A composition comprising a protein, wherein said protein
comprises an amino acid sequence selected from the group consisting
of: (a) the amino acid sequence of SEQ ID NO:10; (b) the amino acid
sequence of SEQ ID NO:10 from amino acid 431 to amino acid 567; (c)
fragments of the amino acid sequence of SEQ ID NO:10; and (d) the
amino acid sequence encoded by the cDNA insert of clone
BL89.sub.--13 deposited under accession number ATCC 98115; the
protein being substantially free from other mammalian proteins.
20. An isolated gene corresponding to the cDNA sequence of SEQ ID
NO:9.
21. A composition comprising 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
2113 to nucleotide 2337; (c) a polynucleotide comprising the
nucleotide sequence of SEQ ID NO:11 from nucleotide 2036 to
nucleotide 2316; (d) a polynucleotide comprising the nucleotide
sequence of the full-length protein coding sequence of clone
BL341.sub.--4 deposited under accession number ATCC 98115; (e) a
polynucleotide encoding the full-length protein encoded by the cDNA
insert of clone BL341.sub.--4 deposited under accession number ATCC
98115; (f) a polynucleotide comprising the nucleotide sequence of
the mature protein coding sequence of clone BL341.sub.--4 deposited
under accession number ATCC 98115; (g) a polynucleotide encoding
the mature protein encoded by the cDNA insert of clone
BL341.sub.--4 deposited under accession number ATCC 98115; (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; (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 capable of hybridizing
under stringent conditions to any one of the polynucleotides
specified in (a)-(i).
22. 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:12; (b) the amino acid
sequence of SEQ ID NO:12 from amino acid 1 to amino acid 68; (c)
fragments of the amino acid sequence of SEQ ID NO:12; and (d) the
amino acid sequence encoded by the cDNA insert of clone
BL341.sub.--4 deposited under accession number ATCC 98115; the
protein being substantially free from other mammalian proteins.
23. An isolated gene corresponding to the cDNA sequence of SEQ ID
NO:11.
24. A composition comprising 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
144 to nucleotide 257; (c) a polynucleotide comprising the
nucleotide sequence of SEQ ID NO:15 from nucleotide 30 to
nucleotide 271; (d) a polynucleotide comprising the nucleotide
sequence of the full-length protein coding sequence of clone
CC25.sub.--17 deposited under accession number ATCC 98115; (e) a
polynucleotide encoding the full-length protein encoded by the cDNA
insert of clone CC25.sub.--17 deposited under accession number ATCC
98115; (f) a polynucleotide comprising the nucleotide sequence of
the mature protein coding sequence of clone CC25.sub.--17 deposited
under accession number ATCC 98115; (g) a polynucleotide encoding
the mature protein encoded by the cDNA insert of clone
CC25.sub.--17 deposited under accession number ATCC 98115; (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; (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 capable of hybridizing
under stringent conditions to any one of the polynucleotides
specified in (a)-(i).
25. 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:16; (b) fragments of
the amino acid sequence of SEQ ID NO:16; and (c) the amino acid
sequence encoded by the cDNA insert of clone CC25.sub.--17
deposited under accession number ATCC 98115; the protein being
substantially free from other mammalian proteins.
26. An isolated gene corresponding to the cDNA sequence of SEQ ID
NO:15.
27. A composition comprising 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
431 to nucleotide 520; (c) a polynucleotide comprising the
nucleotide sequence of SEQ ID NO:17 from nucleotide 266 to
nucleotide 511; (d) a polynucleotide comprising the nucleotide
sequence of the full-length protein coding sequence of clone
CC397.sub.--19 deposited under accession number ATCC 98115; (e) a
polynucleotide encoding the full-length protein encoded by the cDNA
insert of clone CC397.sub.--19 deposited under accession number
ATCC 98115; (f) a polynucleotide comprising the nucleotide sequence
of the mature protein coding sequence of clone CC397.sub.--19
deposited under accession number ATCC 98115; (g) a polynucleotide
encoding the mature protein encoded by the cDNA insert of clone
CC397.sub.--19 deposited under accession number ATCC 98115; (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; 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 capable of hybridizing under
stringent conditions to any one of the polynucleotides specified in
(a)-(i).
28. 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:18; (b) the amino acid
sequence of SEQ ID NO:18 from amino acid 1 to amino acid 27; (c)
fragments of the amino acid sequence of SEQ ID NO:18; and (d) the
amino acid sequence encoded by the cDNA insert of clone
CC397.sub.--19 deposited under accession number ATCC 98115; the
protein being substantially free from other mammalian proteins.
29. An isolated gene corresponding to the cDNA sequence of SEQ ID
NO:17.
30. A composition comprising an isolated polynucleotide selected
from the group consisting of: (a) a polynucleotide comprising the
nucleotide sequence of SEQ ID NO:41; (b) a polynucleotide
comprising the nucleotide sequence of SEQ ID NO:41 from nucleotide
218 to nucleotide 1159; (c) a polynucleotide comprising the
nucleotide sequence of SEQ ID NO:41 from nucleotide 806 to
nucleotide 1159; (d) a polynucleotide comprising the nucleotide
sequence of SEQ ID NO:41 from nucleotide 217 to nucleotide 517; (e)
a polynucleotide comprising the nucleotide sequence of the
full-length protein coding sequence of clone K483.sub.--1 deposited
under accession number ATCC 98115; (f) a polynucleotide encoding
the full-length protein encoded by the cDNA insert of clone
K483.sub.--1 deposited under accession number ATCC 98115; (g) a
polynucleotide comprising the nucleotide sequence of the mature
protein coding sequence of clone K483.sub.--1 deposited under
accession number ATCC 98115; (h) a polynucleotide encoding the
mature protein encoded by the cDNA insert of clone K483.sub.--1
deposited under accession number ATCC 98115; (i) a polynucleotide
encoding a protein comprising the amino acid sequence of SEQ ID
NO:42; (j) a polynucleotide encoding a protein comprising a
fragment of the amino acid sequence of SEQ ID NO:42 having
biological activity; (k) a polynucleotide which is an allelic
variant of a polynucleotide of (a)-(h) above; (l) a polynucleotide
which encodes a species homologue of the protein of (i) or (j)
above; and (m) a polynucleotide capable of hybridizing under
stringent conditions to any one of the polynucleotides specified in
(a)-(j).
31. 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:42; (b) the amino acid
sequence of SEQ ID NO:42 from amino acid 1 to amino acid 100; (c)
fragments of the amino acid sequence of SEQ ID NO:42; and (d) the
amino acid sequence encoded by the cDNA insert of clone
K483.sub.--1 deposited under accession number ATCC 98115; the
protein being substantially free from other mammalian proteins.
32. An isolated gene corresponding to the cDNA sequence of SEQ ID
NO:41.
33. A method of promoting cell-cell fusion, said method comprising
contacting a first cell and a second cell, wherein said first cell
expresses an AJ172.sub.--2 protein.
34. The method of claim 33, wherein said first cell naturally
expresses said AJ172.sub.--2 protein.
35. The method of claim 34, wherein said first cell expresses said
AJ172.sub.--2 protein as a result of transfection of said first
cell.
36. The method of claim 35, wherein said first cell is transfected
with a composition of claim 14.
37. The method of claim 35, wherein said first cell is transfected
with a gene of claim 16.
38. The method of claim 33, wherein said AJ172.sub.--2 protein is a
protein of claim 15.
39. The method of claim 33, wherein said first cell and said second
cell are cells of the same type.
40. The method of claim 33, wherein at least one of said first cell
and said second cell are transfected to express an additional
protein other than said AJ172.sub.--2 protein.
41. A method of inhibiting cell-cell fusion between a first cell
which expresses an AJ172.sub.--2 protein and a second cell, said
method comprising contacting said first cell with an AJ172.sub.--2
protein antagonist.
42. The method of claim 41, wherein said antagonist is selected
from the group consisting of an antibody or antibody fragment
directed to an AJ172.sub.--2 protein, an antisense polynucleotide
directed to a polynucleotide expressing an AJ172.sub.--2 protein, a
nucleotide aptamer directed to an AJ172.sub.--2 protein, a peptide
aptamer directed to an AJ172.sub.--2 protein and a small molecule
which blocks the fusion-inducing activity of an AJ172.sub.--2
protein.
43. The method of claim 41, wherein said first cell is a placental
cell.
44. The method of claim 43, wherein said second cell is a cell from
the maternal uterine lining.
45. The method of claim 41, wherein said first cell is a
cytotrophoblast.
46. A method of inhibiting blastocyst implantation, said method
comprising contacting a cell within said blastocyst which expresses
an AJ172.sub.--2 protein with an AJ172.sub.--2 protein
antagonist.
47. A method of inhibiting trophoblast invasion, said method
comprising contacting a first cell which expresses an AJ172.sub.--2
protein with an AJ172.sub.--2 protein antagonist.
48. The method of claim 42, wherein said antagonist is selected
from the group consisting of an antibody or antibody fragment
directed to an AJ172.sub.--2 protein, an antisense polynucleotide
directed to a polynucleotide expressing an AJ172.sub.--2 protein, a
nucleotide aptamer directed to an AJ172.sub.--2 protein, and a
peptide aptamer directed to an AJ172.sub.--2 protein.
49. The method of claim 48, wherein said antagonist is selected
from the group consisting of an antibody or antibody fragment
directed to an AJ172.sub.--2 protein, and an antisense
polynucleotide directed to a polynucleotide expressing an AJ
172.sub.--2 protein.
50. A method of diagnosing or predicting the existence of a
condition associated with disregulation of AH172.sub.--2 protein in
a mammalian subject, said method comprising (a) determining a first
level of expression of AJ172.sub.--2 protein in said subject, and
(b) comparing said first level of expression to a second level of
expression of AJ172.sub.--2 protein in one or more other mammalian
subjects which do not have said condition.
51. The method of claim 50, wherein said condition is selected from
the group consisting of pre-eclampsia, placental pathology and
cancer.
52. The method of claim 50, wherein said first level of expression
is determined in the serum of said subject.
53. The method of claim 50, wherein said first level of expression
is determined using an antibody or antibody fragment directed to
AJ172.sub.--2 protein.
54. The method of claim 51, wherein said cancer is
choriocarcinoma.
55. A method of treating a neoplastic disease in a mammalian
subject, said method comprising administering to said subject a
therapeutically effective amount of an agent which modulates the
expression or function of AJ172.sub.--2.
56. The method of claim 55, wherein said disease is
choriocarcinoma.
57. A method of inhibiting metastasis in a mammalina subject, said
method comprising administering to said subject a therapeutically
effective amount of an agent which modulates the expression or
function of AJ172.sub.--2.
Description
[0001] This application is a continuation-in-part of application
Ser. No. 09/080,478, filed May 18, 1998, which was a
continuation-in-part of application Ser. No. 08/976,110, filed Nov.
21, 1997, which was a continuation-in-part of the following
applications: Ser. No. 08/686,878, filed Jul. 26, 1996; Ser. No.
08/702,081, filed Aug. 23, 1996, which is a continuation-in-part of
Ser. No. 08/686,878; Ser. No. 08/721,489, filed Sep. 27, 1996,
which is a continuation-in-part of Ser. No. 08/686,878; and
08/721,924, filed Sep. 27, 1996, which is a continuation-in-part of
Ser. No. 08/686,878. All of such applications are incorporated by
reference herein.
[0002] FIELD OF THE INVENTION
[0003] 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
[0004] Technology aimed at the discovery of protein factors
(including e.g., cytokines, such as lymphokines, interferons, CSFs
and interleukins) has matured rapidly over the past decade. The now
routine hybridization cloning and expression cloning techniques
clone novel polynucleotides "directly" in the sense that they rely
on information directly related to the discovered protein (i.e.,
partial DNA/amino acid sequence of the protein in the case of
hybridization cloning; activity of the protein in the case of
expression cloning). More recent "indirect" cloning techniques such
as signal sequence cloning, which isolates DNA sequences based on
the presence of a now well-recognized secretory leader sequence
motif, as well as various PCR-based or low stringency hybridization
cloning techniques, have advanced the state of the art by making
available large numbers of DNA/amino acid sequences for proteins
that are known to have biological activity by virtue of their
secreted nature in the case of leader sequence cloning, or by
virtue of the cell or tissue source in the case of PCR-based
techniques. It is to these proteins and the polynucleotides
encoding them that the present invention is directed.
SUMMARY OF THE INVENTION
[0005] In one embodiment, the present invention provides a
composition comprising an isolated polynucleotide selected from the
group consisting of:
[0006] (a) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:1;
[0007] (b) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:1 from nucleotide 44 to nucleotide 1204;
[0008] (c) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:1 from nucleotide 1 to nucleotide 403;
[0009] (d) a polynucleotide comprising the nucleotide sequence of
the full-length protein coding sequence of clone AJ26.sub.--3
deposited under accession number ATCC 98115;
[0010] (e) a polynucleotide encoding the full-length protein
encoded by the cDNA insert of clone AJ26.sub.--3 deposited under
accession number ATCC 98115;
[0011] (f) a polynucleotide comprising the nucleotide sequence of
the mature protein coding sequence of clone AJ26.sub.--3 deposited
under accession number ATCC 98115;
[0012] (g) a polynucleotide encoding the mature protein encoded by
the cDNA insert of clone AJ26.sub.--3 deposited under accession
number ATCC 98115;
[0013] (h) a polynucleotide encoding a protein comprising the amino
acid sequence of SEQ ID NO:2;
[0014] (i) a polynucleotide encoding a protein comprising a
fragment of the amino acid sequence of SEQ ID NO:2 having
biological activity;
[0015] (j) a polynucleotide which is an allelic variant of a
polynucleotide of (a)-(g) above;
[0016] (k) a polynucleotide which encodes a species homologue of
the protein of (h) or (i) above; and
[0017] (l) a polynucleotide capable of hybridizing under stringent
conditions to any one of the polynucleotides specified in
(a)-(i).
[0018] Preferably, such polynucleotide comprises the nucleotide
sequence of SEQ ID NO:1 from nucleotide 44 to nucleotide 1204; the
nucleotide sequence of SEQ ID NO:1 from nucleotide 1 to nucleotide
403; the nucleotide sequence of the full-length protein coding
sequence of clone AJ26.sub.--3 deposited under accession number
ATCC 98115; or the nucleotide sequence of the mature protein coding
sequence of clone AJ26.sub.--3 deposited under accession number
ATCC 98115. In other preferred embodiments, the polynucleotide
encodes the full-length or mature protein encoded by the cDNA
insert of clone AJ26.sub.--3 deposited under accession number ATCC
98115. In yet other preferred embodiments, the present invention
provides a polynucleotide encoding a protein comprising the amino
acid sequence of SEQ ID NO:2 from amino acid 1 to amino acid
120.
[0019] Other embodiments provide the gene corresponding to the cDNA
sequence of SEQ ID NO:1.
[0020] 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:
[0021] (a) the amino acid sequence of SEQ ID NO:2;
[0022] (b) the amino acid sequence of SEQ ID NO:2 from amino acid 1
to amino acid 120;
[0023] (c) fragments of the amino acid sequence of SEQ ID NO:2;
and
[0024] (d) the amino acid sequence encoded by the cDNA insert of
clone AJ26.sub.--3 deposited under accession number ATCC 98115;
[0025] the protein being substantially free from other mammalian
proteins. Preferably such protein comprises the amino acid sequence
of SEQ ID NO:2 or the amino acid sequence of SEQ ID NO:2 from amino
acid 1 to amino acid 120.
[0026] In one embodiment, the present invention provides a
composition comprising an isolated polynucleotide selected from the
group consisting of:
[0027] (a) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:3
[0028] (b) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:3 from nucleotide 928 to nucleotide 2541;
[0029] (c) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:3 from nucleotide 988 to nucleotide 2541;
[0030] (d) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:3 from nucleotide 684 to nucleotide 1128;
[0031] (e) a polynucleotide comprising the nucleotide sequence of
the full-length protein coding sequence of clone AJ172.sub.--2
deposited under accession number ATCC 98115;
[0032] (f) a polynucleotide encoding the full-length protein
encoded by the cDNA insert of clone AJ172.sub.--2 deposited under
accession number ATCC 98115;
[0033] (g) a polynucleotide comprising the nucleotide sequence of
the mature protein coding sequence of clone AJ172.sub.--2 deposited
under accession number ATCC 98115;
[0034] (h) a polynucleotide encoding the mature protein encoded by
the cDNA insert of clone AJ172.sub.--2 deposited under accession
number ATCC 98115;
[0035] (i) a polynucleotide encoding a protein comprising the amino
acid sequence of SEQ ID NO:4;
[0036] (j) a polynucleotide encoding a protein comprising a
fragment of the amino acid sequence of SEQ ID NO:4 having
biological activity;
[0037] (k) a polynucleotide which is an allelic variant of a
polynucleotide of (a)-(h) above;
[0038] (l) a polynucleotide which encodes a species homologue of
the protein of (i) or (j) above; and
[0039] (m) a polynucleotide capable of hybridizing under stringent
conditions to any one of the polynucleotides specified in
(a)-(j).
[0040] Preferably, such polynucleotide comprises the nucleotide
sequence of SEQ ID NO:3 from nucleotide 928 to nucleotide 2541; the
nucleotide sequence of SEQ ID NO:3 from nucleotide 988 to
nucleotide 2541; the nucleotide sequence of SEQ ID NO:3 from
nucleotide 684 to nucleotide 1128; the nucleotide sequence of the
full-length protein coding sequence of clone AJ172.sub.--2
deposited under accession number ATCC 98115; or the nucleotide
sequence of the mature protein coding sequence of clone
AJ172.sub.--2 deposited under accession number ATCC 98115. In other
preferred embodiments, the polynucleotide encodes the full-length
or mature protein encoded by the cDNA insert of clone AJ172.sub.--2
deposited under accession number ATCC 98115. In yet other preferred
embodiments, the present invention provides a polynucleotide
encoding a protein comprising the amino acid sequence of SEQ ID
NO:4 from amino acid 1 to amino acid 67.
[0041] Other embodiments provide the gene corresponding to the cDNA
sequence of SEQ ID NO:3.
[0042] 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:
[0043] (a) the amino acid sequence of SEQ ID NO:4;
[0044] (b) the amino acid sequence of SEQ ID NO:4 from amino acid 1
to amino acid 67;
[0045] (c) fragments of the amino acid sequence of SEQ ID NO:4;
and
[0046] (d) the amino acid sequence encoded by the cDNA insert of
clone AJ172.sub.--2 deposited under accession number ATCC
98115;
[0047] the protein being substantially free from other mammalian
proteins. Preferably such protein comprises the amino acid sequence
of SEQ ID NO:4 or the amino acid sequence of SEQ ID NO:4 from amino
acid 1 to amino acid 67.
[0048] In one embodiment, the present invention provides a
composition comprising an isolated polynucleotide selected from the
group consisting of:
[0049] (a) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:6;
[0050] (b) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:6 from nucleotide 185 to nucleotide 385;
[0051] (c) a polynucleotide comprising the nucleotide sequence of
the full-length protein coding sequence of clone AP224.sub.--2
deposited under accession number ATCC 98115;
[0052] (d) a polynucleotide encoding the full-length protein
encoded by the cDNA insert of clone AP224.sub.--2 deposited under
accession number ATCC 98115;
[0053] (e) a polynucleotide comprising the nucleotide sequence of
the mature protein coding sequence of clone AP224.sub.--2 deposited
under accession number ATCC 98115;
[0054] (f) a polynucleotide encoding the mature protein encoded by
the cDNA insert of clone AP224.sub.--2 deposited under accession
number ATCC 98115;
[0055] (g) a polynucleotide encoding a protein comprising the amino
acid sequence of SEQ ID NO:7;
[0056] (h) a polynucleotide encoding a protein comprising a
fragment of the amino acid sequence of SEQ ID NO:7 having
biological activity;
[0057] (i) a polynucleotide which is an allelic variant of a
polynucleotide of (a)-(f) above;
[0058] (j) a polynucleotide which encodes a species homologue of
the protein of (g) or (h) above; and
[0059] (k) a polynucleotide capable of hybridizing under stringent
conditions to any one of the polynucleotides specified in
(a)-(h).
[0060] Preferably, such polynucleotide comprises the nucleotide
sequence of SEQ ID NO:6 from nucleotide 185 to nucleotide 385; the
nucleotide sequence of the full-length protein coding sequence of
clone AP224.sub.--2 deposited under accession number ATCC 98115; or
the nucleotide sequence of the mature protein coding sequence of
clone AP224.sub.--2 deposited under accession number ATCC 98115. In
other preferred embodiments, the polynucleotide encodes the
full-length or mature protein encoded by the cDNA insert of clone
AP224.sub.--2 deposited under accession number ATCC 98115. In yet
other preferred embodiments, the present invention provides a
polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID NO:7 from amino acid 1 to amino acid 28.
[0061] Other embodiments provide the gene corresponding to the cDNA
sequence of SEQ ID NO:6, SEQ ID NO:5 or SEQ ID NO:8.
[0062] 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:
[0063] (a) the amino acid sequence of SEQ ID NO:7;
[0064] (b) the amino acid sequence of SEQ ID NO:7 from amino acid 1
to amino acid 28;
[0065] (c) fragments of the amino acid sequence of SEQ ID NO:7;
and
[0066] (d) the amino acid sequence encoded by the cDNA insert of
clone AP224.sub.--2 deposited under accession number ATCC
98115;
[0067] the protein being substantially free from other mammalian
proteins. Preferably such protein comprises the amino acid sequence
of SEQ ID NO:7 or the amino acid sequence of SEQ ID NO:7 from amino
acid 1 to amino acid 28.
[0068] In one embodiment, the present invention provides a
composition comprising an isolated polynucleotide selected from the
group consisting of:
[0069] (a) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:9;
[0070] (b) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:9 from nucleotide 6 to nucleotide 2408;
[0071] (c) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:9 from nucleotide 1295 to nucleotide 1705;
[0072] (d) a polynucleotide comprising the nucleotide sequence of
the full-length protein coding sequence of clone BL89.sub.--13
deposited under accession number ATCC 98115;
[0073] (e) a polynucleotide encoding the full-length protein
encoded by the cDNA insert of clone BL89.sub.--13 deposited under
accession number ATCC 98115;
[0074] (f) a polynucleotide comprising the nucleotide sequence of
the mature protein coding sequence of clone BL89.sub.--13 deposited
under accession number ATCC 98115;
[0075] (g) a polynucleotide encoding the mature protein encoded by
the cDNA insert of clone BL89.sub.--13 deposited under accession
number ATCC 98115;
[0076] (h) a polynucleotide encoding a protein comprising the amino
acid sequence of SEQ ID NO:10;
[0077] (i) a polynucleotide encoding a protein comprising a
fragment of the amino acid sequence of SEQ ID NO:10 having
biological activity;
[0078] (j) a polynucleotide which is an allelic variant of a
polynucleotide of (a)-(g) above;
[0079] (k) a polynucleotide which encodes a species homologue of
the protein of (h) or (i) above; and
[0080] (l) a polynucleotide capable of hybridizing under stringent
conditions to any one of the polynucleotides specified in
(a)-(i).
[0081] Preferably, such polynucleotide comprises the nucleotide
sequence of SEQ ID NO:9 from nucleotide 6 to nucleotide 2408; the
nucleotide sequence of SEQ ID NO:9 from nucleotide 1295 to
nucleotide 1705; the nucleotide sequence of the full-length protein
coding sequence of clone BL89.sub.--13 deposited under accession
number ATCC 98115; or the nucleotide sequence of the mature protein
coding sequence of clone BL89.sub.--13 deposited under accession
number ATCC 98115. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by
the cDNA insert of clone BL89.sub.--13 deposited under accession
number ATCC 98115. In yet other preferred embodiments, the present
invention provides a polynucleotide encoding a protein comprising
the amino acid sequence of SEQ ID NO:10 from amino acid 431 to
amino acid 567.
[0082] Other embodiments provide the gene corresponding to the cDNA
sequence of SEQ ID NO:9.
[0083] 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:
[0084] (a) the amino acid sequence of SEQ ID NO:10;
[0085] (b) the amino acid sequence of SEQ ID NO:10 from amino acid
431 to amino acid 567;
[0086] (c) fragments of the amino acid sequence of SEQ ID NO:10;
and
[0087] (d) the amino acid sequence encoded by the cDNA insert of
clone BL89.sub.--13 deposited under accession number ATCC
98115;
[0088] the protein being substantially free from other mammalian
proteins. Preferably such protein comprises the amino acid sequence
of SEQ ID NO:10 or the amino acid sequence of SEQ ID NO:10 from
amino acid 431 to amino acid 567.
[0089] In one embodiment, the present invention provides a
composition comprising an isolated polynucleotide selected from the
group consisting of:
[0090] (a) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:11;
[0091] (b) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:11 from nucleotide 2113 to nucleotide 2337;
[0092] (c) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:11 from nucleotide 2036 to nucleotide 2316;
[0093] (d) a polynucleotide comprising the nucleotide sequence of
the full-length protein coding sequence of clone BL341.sub.--4
deposited under accession number ATCC 98115;
[0094] (e) a polynucleotide encoding the full-length protein
encoded by the cDNA insert of clone BL341.sub.--4 deposited under
accession number ATCC 98115;
[0095] (f) a polynucleotide comprising the nucleotide sequence of
the mature protein coding sequence of clone BL341.sub.--4 deposited
under accession number ATCC 98115;
[0096] (g) a polynucleotide encoding the mature protein encoded by
the cDNA insert of clone BL341.sub.--4 deposited under accession
number ATCC 98115;
[0097] (h) a polynucleotide encoding a protein comprising the amino
acid sequence of SEQ ID NO:12;
[0098] (i) a polynucleotide encoding a protein comprising a
fragment of the amino acid sequence of SEQ ID NO:12 having
biological activity;
[0099] (j) a polynucleotide which is an allelic variant of a
polynucleotide of (a)-(g) above;
[0100] (k) a polynucleotide which encodes a species homologue of
the protein of (h) or (i) above; and
[0101] (l) a polynucleotide capable of hybridizing under stringent
conditions to any one of the polynucleotides specified in
(a)-(i).
[0102] Preferably, such polynucleotide comprises the nucleotide
sequence of SEQ ID NO:11 from nucleotide 2113 to nucleotide 2337;
the nucleotide sequence of SEQ ID NO:11 from nucleotide 2036 to
nucleotide 2316; the nucleotide sequence of the full-length protein
coding sequence of clone BL341.sub.--4 deposited under accession
number ATCC 98115; or the nucleotide sequence of the mature protein
coding sequence of clone BL341.sub.--4 deposited under accession
number ATCC 98115. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by
the cDNA insert of clone BL341.sub.--4 deposited under accession
number ATCC 98115. In yet other preferred embodiments, the present
invention provides a polynucleotide encoding a protein comprising
the amino acid sequence of SEQ ID NO:12 from amino acid 1 to amino
acid 68.
[0103] Other embodiments provide the gene corresponding to the cDNA
sequence of SEQ ID NO:11.
[0104] 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:
[0105] (a) the amino acid sequence of SEQ ID NO:12;
[0106] (b) the amino acid sequence of SEQ ID NO:12 from amino acid
1 to amino acid 68;
[0107] (c) fragments of the amino acid sequence of SEQ ID NO:12;
and
[0108] (d) the amino acid sequence encoded by the cDNA insert of
clone BL341.sub.--4 deposited under accession number ATCC
98115;
[0109] the protein being substantially free from other mammalian
proteins. Preferably such protein comprises the amino acid sequence
of SEQ ID NO:12 or the amino acid sequence of SEQ ID NO:12 from
amino acid 1 to amino acid 68.
[0110] In one embodiment, the present invention provides a
composition comprising an isolated polynucleotide selected from the
group consisting of:
[0111] (a) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:13;
[0112] (b) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:13 from nucleotide 1 to nucleotide 390;
[0113] (c) a polynucleotide comprising the nucleotide sequence of
the full-length protein coding sequence of clone BV239.sub.--3
deposited under accession number ATCC 98115;
[0114] (d) a polynucleotide encoding the full-length protein
encoded by the cDNA insert of clone BV239.sub.--3 deposited under
accession number ATCC 98115;
[0115] (e) a polynucleotide comprising the nucleotide sequence of
the mature protein coding sequence of clone BV239.sub.--3 deposited
under accession number ATCC 98115;
[0116] (f) a polynucleotide encoding the mature protein encoded by
the cDNA insert of clone BV239.sub.--3 deposited under accession
number ATCC 98115;
[0117] (g) a polynucleotide encoding a protein comprising the amino
acid sequence of SEQ ID NO:14;
[0118] (h) a polynucleotide encoding a protein comprising a
fragment of the amino acid sequence of SEQ ID NO:14 having
biological activity;
[0119] (i) a polynucleotide which is an allelic variant of a
polynucleotide of (a)-(f) above;
[0120] (j) a polynucleotide which encodes a species homologue of
the protein of (g) or (h) above; and
[0121] (k) a polynucleotide capable of hybridizing under stringent
conditions to any one of the polynucleotides specified in
(a)-(h).
[0122] Preferably, such polynucleotide comprises the nucleotide
sequence of SEQ ID NO:13 from nucleotide 1 to nucleotide 390; the
nucleotide sequence of the full-length protein coding sequence of
clone BV239.sub.--3 deposited under accession number ATCC 98115; or
the nucleotide sequence of the mature protein coding sequence of
clone BV239.sub.--3 deposited under accession number ATCC 98115. In
other preferred embodiments, the polynucleotide encodes the
full-length or mature protein encoded by the cDNA insert of clone
BV239.sub.--3 deposited under accession number ATCC 98115. In yet
other preferred embodiments, the present invention provides a
polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID NO:14 from amino acid 50 to amino acid 130.
[0123] Other embodiments provide the gene corresponding to the cDNA
sequence of SEQ ID NO:13.
[0124] 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:
[0125] (a) the amino acid sequence of SEQ ID NO:14;
[0126] (b) the amino acid sequence of SEQ ID NO:14 from amino acid
50 to amino acid 130;
[0127] (c) fragments of the amino acid sequence of SEQ ID NO:14;
and
[0128] (d) the amino acid sequence encoded by the cDNA insert of
clone BV239.sub.--3 deposited under accession number ATCC
98115;
[0129] the protein being substantially free from other mammalian
proteins. Preferably such protein comprises the amino acid sequence
of SEQ ID NO:14 or the amino acid sequence of SEQ ID NO:14 from
amino acid 50 to amino acid 130.
[0130] In one embodiment, the present invention provides a
composition comprising an isolated polynucleotide selected from the
group consisting of:
[0131] (a) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:15;
[0132] (b) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:15 from nucleotide 144 to nucleotide 257;
[0133] (c) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:15 from nucleotide 30 to nucleotide 271;
[0134] (d) a polynucleotide comprising the nucleotide sequence of
the full-length protein coding sequence of clone CC25.sub.--17
deposited under accession number ATCC 98115;
[0135] (e) a polynucleotide encoding the full-length protein
encoded by the cDNA insert of clone CC25.sub.--17 deposited under
accession number ATCC 98115;
[0136] (f) a polynucleotide comprising the nucleotide sequence of
the mature protein coding sequence of clone CC25.sub.--17 deposited
under accession number ATCC 98115;
[0137] (g) a polynucleotide encoding the mature protein encoded by
the cDNA insert of clone CC25.sub.--17 deposited under accession
number ATCC 98115;
[0138] (h) a polynucleotide encoding a protein comprising the amino
acid sequence of SEQ ID NO:16;
[0139] (i) a polynucleotide encoding a protein comprising a
fragment of the amino acid sequence of SEQ ID NO:16 having
biological activity;
[0140] (j) a polynucleotide which is an allelic variant of a
polynucleotide of (a)-(g) above;
[0141] (k) a polynucleotide which encodes a species homologue of
the protein of (h) or (i) above; and
[0142] (l) a polynucleotide capable of hybridizing under stringent
conditions to any one of the polynucleotides specified in
(a)-(i).
[0143] Preferably, such polynucleotide comprises the nucleotide
sequence of SEQ ID NO:15 from nucleotide 144 to nucleotide 257; the
nucleotide sequence of SEQ ID NO:15 from nucleotide 30 to
nucleotide 271; the nucleotide sequence of the full-length protein
coding sequence of clone CC25.sub.--17 deposited under accession
number ATCC 98115; or the nucleotide sequence of the mature protein
coding sequence of clone CC25.sub.--17 deposited under accession
number ATCC 98115. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by
the cDNA insert of clone CC25.sub.--17 deposited under accession
number ATCC 98115.
[0144] Other embodiments provide the gene corresponding to the cDNA
sequence of SEQ ID NO:15.
[0145] 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:
[0146] (a) the amino acid sequence of SEQ ID NO:16;
[0147] (b) fragments of the amino acid sequence of SEQ ID NO:16;
and
[0148] (c) the amino acid sequence encoded by the cDNA insert of
clone CC25.sub.--17 deposited under accession number ATCC
98115;
[0149] the protein being substantially free from other mammalian
proteins. Preferably such protein comprises the amino acid sequence
of SEQ ID NO:16.
[0150] In one embodiment, the present invention provides a
composition comprising an isolated polynucleotide selected from the
group consisting of:
[0151] (a) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:17;
[0152] (b) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:17 from nucleotide 431 to nucleotide 520;
[0153] (c) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:17 from nucleotide 266 to nucleotide 511;
[0154] (d) a polynucleotide comprising the nucleotide sequence of
the full-length protein coding sequence of clone CC397.sub.--19
deposited under accession number ATCC 98115;
[0155] (e) a polynucleotide encoding the full-length protein
encoded by the cDNA insert of clone CC397.sub.--19 deposited under
accession number ATCC 98115;
[0156] (f) a polynucleotide comprising the nucleotide sequence of
the mature protein coding sequence of clone CC397.sub.--19
deposited under accession number ATCC 98115;
[0157] (g) a polynucleotide encoding the mature protein encoded by
the cDNA insert of clone CC397.sub.--19 deposited under accession
number ATCC 98115;
[0158] (h) a polynucleotide encoding a protein comprising the amino
acid sequence of SEQ ID NO:18;
[0159] (i) a polynucleotide encoding a protein comprising a
fragment of the amino acid sequence of SEQ ID NO:18 having
biological activity;
[0160] (j) a polynucleotide which is an allelic variant of a
polynucleotide of (a)-(g) above;
[0161] (k) a polynucleotide which encodes a species homologue of
the protein of (h) or (i) above; and
[0162] (l) a polynucleotide capable of hybridizing under stringent
conditions to any one of the polynucleotides specified in
(a)-(i).
[0163] Preferably, such polynucleotide comprises the nucleotide
sequence of SEQ ID NO:17 from nucleotide 431 to nucleotide 520; the
nucleotide sequence of SEQ ID NO:17 from nucleotide 266 to
nucleotide 511; the nucleotide sequence of the full-length protein
coding sequence of clone CC397.sub.--19 deposited under accession
number ATCC 98115; or the nucleotide sequence of the mature protein
coding sequence of clone CC397.sub.--19 deposited under accession
number ATCC 98115. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by
the cDNA insert of clone CC397.sub.--19 deposited under accession
number ATCC 98115. In yet other preferred embodiments, the present
invention provides a polynucleotide encoding a protein comprising
the amino acid sequence of SEQ ID NO:18 from amino acid 1 to amino
acid 27.
[0164] Other embodiments provide the gene corresponding to the cDNA
sequence of SEQ ID NO:17.
[0165] 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:
[0166] (a) the amino acid sequence of SEQ ID NO:18;
[0167] (b) the amino acid sequence of SEQ ID NO:18 from amino acid
1 to amino acid 27;
[0168] (c) fragments of the amino acid sequence of SEQ ID NO:18;
and
[0169] (d) the amino acid sequence encoded by the cDNA insert of
clone CC397.sub.--19 deposited under accession number ATCC
98115;
[0170] the protein being substantially free from other mammalian
proteins. Preferably such protein comprises the amino acid sequence
of SEQ ID NO:18 or the amino acid sequence of SEQ ID NO:18 from
amino acid 1 to amino acid 27.
[0171] In one embodiment, the present invention provides a
composition comprising an isolated polynucleotide selected from the
group consisting of:
[0172] (a) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:20;
[0173] (b) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:20 from nucleotide 253 to nucleotide 519;
[0174] (c) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:20 from nucleotide 298 to nucleotide 519;
[0175] (d) a polynucleotide comprising the nucleotide sequence of
the full-length protein coding sequence of clone D305.sub.--2
deposited under accession number ATCC 98115;
[0176] (e) a polynucleotide encoding the full-length protein
encoded by the cDNA insert of clone D305.sub.--2 deposited under
accession number ATCC 98115;
[0177] (f) a polynucleotide comprising the nucleotide sequence of
the mature protein coding sequence of clone D305.sub.--2 deposited
under accession number ATCC 98115;
[0178] (g) a polynucleotide encoding the mature protein encoded by
the cDNA insert of clone D305.sub.--2 deposited under accession
number ATCC 98115;
[0179] (h) a polynucleotide encoding a protein comprising the amino
acid sequence of SEQ ID NO:21;
[0180] (i) a polynucleotide encoding a protein comprising a
fragment of the amino acid sequence of SEQ ID NO:21 having
biological activity;
[0181] (j) a polynucleotide which is an allelic variant of a
polynucleotide of (a)-(g) above;
[0182] (k) a polynucleotide which encodes a species homologue of
the protein of (h) or (i) above; and
[0183] (l) a polynucleotide capable of hybridizing under stringent
conditions to any one of the polynucleotides specified in
(a)-(i).
[0184] Preferably, such polynucleotide comprises the nucleotide
sequence of SEQ ID NO:20 from nucleotide 253 to nucleotide 519; the
nucleotide sequence of SEQ ID NO:20 from nucleotide 298 to
nucleotide 519; the nucleotide sequence of the full-length protein
coding sequence of clone D305.sub.--2 deposited under accession
number ATCC 98115; or the nucleotide sequence of the mature protein
coding sequence of clone D305.sub.--2 deposited under accession
number ATCC 98115. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by
the cDNA insert of clone D305.sub.--2 deposited under accession
number ATCC 98115.
[0185] Other embodiments provide the gene corresponding to the cDNA
sequence of SEQ ID NO:20, SEQ ID NO:19 or SEQ ID NO:22.
[0186] 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:
[0187] (a) the amino acid sequence of SEQ ID NO:21;
[0188] (b) fragments of the amino acid sequence of SEQ ID NO:21;
and
[0189] (c) the amino acid sequence encoded by the cDNA insert of
clone D305.sub.--2 deposited under accession number ATCC 98115;
[0190] the protein being substantially free from other mammalian
proteins. Preferably such protein comprises the amino acid sequence
of SEQ ID NO:21.
[0191] In one embodiment, the present invention provides a
composition comprising an isolated polynucleotide selected from the
group consisting of:
[0192] (a) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO 23,
[0193] (b) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:23 from nucleotide 194 to nucleotide 622;
[0194] (c) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:23 from nucleotide 524 to nucleotide 622;
[0195] (d) a polynucleotide comprising the nucleotide sequence of
the full-length protein coding sequence of clone G55.sub.--1
deposited under accession number ATCC 98115;
[0196] (e) a polynucleotide encoding the full-length protein
encoded by the cDNA insert of clone G55.sub.--1 deposited under
accession number ATCC 98115;
[0197] (f) a polynucleotide comprising the nucleotide sequence of
the mature protein coding sequence of clone G55.sub.--1 deposited
under accession number ATCC 98115;
[0198] (g) a polynucleotide encoding the mature protein encoded by
the cDNA insert of clone G55.sub.--1 deposited under accession
number ATCC 98115;
[0199] (h) a polynucleotide encoding a protein comprising the amino
acid sequence of SEQ ID NO:24;
[0200] (i) a polynucleotide encoding a protein comprising a
fragment of the amino acid sequence of SEQ ID NO:24 having
biological activity;
[0201] (j) a polynucleotide which is an allelic variant of a
polynucleotide of (a)-(g) above;
[0202] (k) a polynucleotide which encodes a species homologue of
the protein of (h) or (i) above; and
[0203] (l) a polynucleotide capable of hybridizing under stringent
conditions to any one of the polynucleotides specified in
(a)-(i).
[0204] Preferably, such polynucleotide comprises the nucleotide
sequence of SEQ ID NO:23 from nucleotide 194 to nucleotide 622; the
nucleotide sequence of SEQ ID NO:23 from nucleotide 524 to
nucleotide 622; the nucleotide sequence of the full-length protein
coding sequence of clone G55.sub.--1 deposited under accession
number ATCC 98115; or the nucleotide sequence of the mature protein
coding sequence of clone G55.sub.--1 deposited under accession
number ATCC 98115. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by
the cDNA insert of clone G55.sub.--1 deposited under accession
number ATCC 98115. In yet other preferred embodiments, the present
invention provides a polynucleotide encoding a protein comprising
the amino acid sequence of SEQ ID NO:24 from amino acid 1 to amino
acid 32.
[0205] Other embodiments provide the gene corresponding to the cDNA
sequence of SEQ ID NO:23 or SEQ ID NO:25.
[0206] 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:
[0207] (a) the amino acid sequence of SEQ ID NO:24;
[0208] (b) the amino acid sequence of SEQ ID NO:24 from amino acid
1 to amino acid 32;
[0209] (c) fragments of the amino acid sequence of SEQ ID NO:24;
and
[0210] (d) the amino acid sequence encoded by the cDNA insert of
clone G55.sub.--1 deposited under accession number ATCC 98115;
[0211] the protein being substantially free from other mammalian
proteins. Preferably such protein comprises the amino acid sequence
of SEQ ID NO:24 or the amino acid sequence of SEQ ID NO:24 from
amino acid 1 to amino acid 32.
[0212] In one embodiment, the present invention provides a
composition comprising an isolated polynucleotide selected from the
group consisting of:
[0213] (a) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:26;
[0214] (b) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:26 from nucleotide 402 to nucleotide 533;
[0215] (c) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:26 from nucleotide 447 to nucleotide 533;
[0216] (d) a polynucleotide comprising the nucleotide sequence of
the full-length protein coding sequence of clone K39.sub.--7
deposited under accession number ATCC 98115;
[0217] (e) a polynucleotide encoding the full-length protein
encoded by the cDNA insert of clone K39.sub.--7 deposited under
accession number ATCC 98115;
[0218] (f) a polynucleotide comprising the nucleotide sequence of
the mature protein coding sequence of clone K39.sub.--7 deposited
under accession number ATCC 98115;
[0219] (g) a polynucleotide encoding the mature protein encoded by
the cDNA insert of clone K39.sub.--7 deposited under accession
number ATCC 98115;
[0220] (h) a polynucleotide encoding a protein comprising the amino
acid sequence of SEQ ID NO:27;
[0221] (i) a polynucleotide encoding a protein comprising a
fragment of the amino acid sequence of SEQ ID NO:27 having
biological activity;
[0222] (j) a polynucleotide which is an allelic variant of a
polynucleotide of (a)-(g) above;
[0223] (k) a polynucleotide which encodes a species homologue of
the protein of (h) or (i) above; and
[0224] (l) a polynucleotide capable of hybridizing under stringent
conditions to any one of the polynucleotides specified in
(a)-(i).
[0225] Preferably, such polynucleotide comprises the nucleotide
sequence of SEQ ID NO:26 from nucleotide 402 to nucleotide 533; the
nucleotide sequence of SEQ ID NO:26 from nucleotide 447 to
nucleotide 533; the nucleotide sequence of the full-length protein
coding sequence of clone K39.sub.--7 deposited under accession
number ATCC 98115; or the nucleotide sequence of the mature protein
coding sequence of clone K39.sub.--7 deposited under accession
number ATCC 98115. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by
the cDNA insert of clone K39.sub.--7 deposited under accession
number ATCC 98115.
[0226] Other embodiments provide the gene corresponding to the cDNA
sequence of SEQ ID NO:26 or SEQ ID NO:28.
[0227] 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:
[0228] (a) the amino acid sequence of SEQ ID NO:27;
[0229] (b) fragments of the amino acid sequence of SEQ ID NO:27;
and
[0230] (c) the amino acid sequence encoded by the cDNA insert of
clone K39.sub.--7 deposited under accession number ATCC 98115;
[0231] the protein being substantially free from other mammalian
proteins. Preferably such protein comprises the amino acid sequence
of SEQ ID NO:27.
[0232] In one embodiment, the present invention provides a
composition comprising an isolated polynucleotide selected from the
group consisting of:
[0233] (a) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:29;
[0234] (b) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:29 from nucleotide 241 to nucleotide 525;
[0235] (c) a polynucleotide comprising the nucleotide sequence of
the full-length protein coding sequence of clone K330.sub.--3
deposited under accession number ATCC 98115;
[0236] (d) a polynucleotide encoding the full-length protein
encoded by the cDNA insert of clone K330.sub.--3 deposited under
accession number ATCC 98115;
[0237] (e) a polynucleotide comprising the nucleotide sequence of
the mature protein coding sequence of clone K330.sub.--3 deposited
under accession number ATCC 98115;
[0238] (f) a polynucleotide encoding the mature protein encoded by
the cDNA insert of clone K330.sub.--3 deposited under accession
number ATCC 98115;
[0239] (g) a polynucleotide encoding a protein comprising the amino
acid sequence of SEQ ID NO:30;
[0240] (h) a polynucleotide encoding a protein comprising a
fragment of the amino acid sequence of SEQ ID NO:30 having
biological activity;
[0241] (i) a polynucleotide which is an allelic variant of a
polynucleotide of (a)-(f) above;
[0242] (j) a polynucleotide which encodes a species homologue of
the protein of (g) or (h) above; and
[0243] (k) a polynucleotide capable of hybridizing under stringent
conditions to any one of the polynucleotides specified in
(a)-(h).
[0244] Preferably, such polynucleotide comprises the nucleotide
sequence of SEQ ID NO:29 from nucleotide 241 to nucleotide 525; the
nucleotide sequence of the full-length protein coding sequence of
clone K330.sub.--3 deposited under accession number ATCC 98115; or
the nucleotide sequence of the mature protein coding sequence of
clone K330.sub.--3 deposited under accession number ATCC 98115. In
other preferred embodiments, the polynucleotide encodes the
full-length or mature protein encoded by the cDNA insert of clone
K330.sub.--3 deposited under accession number ATCC 98115. In yet
other preferred embodiments, the present invention provides a
polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID NO:30 from amino acid 1 to amino acid 35.
[0245] Other embodiments provide the gene corresponding to the cDNA
sequence of SEQ ID NO:29or SEQ ID NO:31.
[0246] 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:
[0247] (a) the amino acid sequence of SEQ ID NO:30;
[0248] (b) the amino acid sequence of SEQ ID NO:30 from amino acid
1 to amino acid 35;
[0249] (c) fragments of the amino acid sequence of SEQ ID NO:30;
and
[0250] (d) the amino acid sequence encoded by the cDNA insert of
clone K330.sub.--3 deposited under accession number ATCC 98115;
[0251] the protein being substantially free from other mammalian
proteins. Preferably such protein comprises the amino acid sequence
of SEQ ID NO:30 or the amino acid sequence of SEQ ID NO:30 from
amino acid 1 to amino acid 35.
[0252] In one embodiment, the present invention provides a
composition comprising an isolated polynucleotide selected from the
group consisting of:
[0253] (a) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:32;
[0254] (b) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:32 from nucleotide 158 to nucleotide 571;
[0255] (c) a polynucleotide comprising the nucleotide sequence of
the full-length protein coding sequence of clone K363.sub.--3
deposited under accession number ATCC 98115:
[0256] (d) a polynucleotide encoding the full-length protein
encoded by the cDNA insert of clone K363.sub.--3 deposited under
accession number ATCC 98115;
[0257] (e) a polynucleotide comprising the nucleotide sequence of
the mature protein coding sequence of clone K363.sub.--3 deposited
under accession number ATCC 98115;
[0258] (f) a polynucleotide encoding the mature protein encoded by
the cDNA insert of clone K363.sub.--3 deposited under accession
number ATCC 98115;
[0259] (g) a polynucleotide encoding a protein comprising the amino
acid sequence of SEQ ID NO:33;
[0260] (h) a polynucleotide encoding a protein comprising a
fragment of the amino acid sequence of SEQ ID NO:33 having
biological activity;
[0261] (i) a polynucleotide which is an allelic variant of a
polynucleotide of (a)-(f) above;
[0262] (j) a polynucleotide which encodes a species homologue of
the protein of (g) or (h) above; and
[0263] (k) a polynucleotide capable of hybridizing under stringent
conditions to any one of the polynucleotides specified in
(a)-(h).
[0264] Preferably, such polynucleotide comprises the nucleotide
sequence of SEQ ID NO:32 from nucleotide 158 to nucleotide 571; the
nucleotide sequence of the full-length protein coding sequence of
clone K363.sub.--3 deposited under accession number ATCC 98115; or
the nucleotide sequence of the mature protein coding sequence of
clone K363.sub.--3 deposited under accession number ATCC 98115. In
other preferred embodiments, the polynucleotide encodes the
full-length or mature protein encoded by the cDNA insert of clone
K363.sub.--3 deposited under accession number ATCC 98115. In yet
other preferred embodiments, the present invention provides a
polynucleotide encoding a protein comprising the amino acid
sequence of SEQ ID NO:33 from amino acid 24 to amino acid 96.
[0265] Other embodiments provide the gene corresponding to the cDNA
sequence of SEQ ID NO:32 or SEQ ID NO:34.
[0266] 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:
[0267] (a) the amino acid sequence of SEQ ID NO:33;
[0268] (b) the amino acid sequence of SEQ ID NO:33 from amino acid
24 to amino acid 96;
[0269] (c) fragments of the amino acid sequence of SEQ ID NO:33,
and
[0270] (d) the amino acid sequence encoded by the cDNA insert of
clone K363.sub.--3 deposited under accession number ATCC 98115;
[0271] the protein being substantially free from other mammalian
proteins. Preferably such protein comprises the amino acid sequence
of SEQ ID NO:33 or the amino acid sequence of SEQ ID NO:33 from
amino acid 24 to amino acid 96.
[0272] In one embodiment, the present invention provides a
composition comprising an isolated polynucleotide selected from the
group consisting of:
[0273] (a) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:35;
[0274] (b) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:35 from nucleotide 401 to nucleotide 526;
[0275] (c) a polynucleotide comprising the nucleotide sequence of
the full-length protein coding sequence of clone K446.sub.--3
deposited under accession number ATCC 98115;
[0276] (d) a polynucleotide encoding the full-length protein
encoded by the cDNA insert of clone K446.sub.--3 deposited under
accession number ATCC 98115;
[0277] (e) a polynucleotide comprising the nucleotide sequence of
the mature protein coding sequence of clone K446.sub.--3 deposited
under accession number ATCC 98115;
[0278] (f) a polynucleotide encoding the mature protein encoded by
the cDNA insert of clone K446.sub.--3 deposited under accession
number ATCC 98115;
[0279] (g) a polynucleotide encoding a protein comprising the amino
acid sequence of SEQ ID NO:36;
[0280] (h) a polynucleotide encoding a protein comprising a
fragment of the amino acid sequence of SEQ ID NO:36 having
biological activity;
[0281] (i) a polynucleotide which is an allelic variant of a
polynucleotide of (a)-(f) above;
[0282] (j) a polynucleotide which encodes a species homologue of
the protein of (g) or (h) above; and
[0283] (k) a polynucleotide capable of hybridizing under stringent
conditions to any one of the polynucleotides specified in
(a)-(h).
[0284] Preferably, such polynucleotide comprises the nucleotide
sequence of SEQ ID NO:35 from nucleotide 401 to nucleotide 526; the
nucleotide sequence of the full-length protein coding sequence of
clone K446.sub.--3 deposited under accession number ATCC 98115; or
the nucleotide sequence of the mature protein coding sequence of
clone K446.sub.--3 deposited under accession number ATCC 98115. In
other preferred embodiments, the polynucleotide encodes the
full-length or mature protein encoded by the cDNA insert of clone
K446.sub.--3 deposited under accession number ATCC 98115.
[0285] Other embodiments provide the gene corresponding to the cDNA
sequence of SEQ ID NO:35 or SEQ ID NO:37.
[0286] 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:
[0287] (a) the amino acid sequence of SEQ ID NO:36;
[0288] (b) fragments of the amino acid sequence of SEQ ID NO:36;
and
[0289] (c) the amino acid sequence encoded by the cDNA insert of
clone K446.sub.--3 deposited under accession number ATCC 98115;
[0290] the protein being substantially free from other mammalian
proteins. Preferably such protein comprises the amino acid sequence
of SEQ ID NO:36.
[0291] In one embodiment, the present invention provides a
composition comprising an isolated polynucleotide selected from the
group consisting of:
[0292] (a) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:38;
[0293] (b) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:38 from nucleotide 380 to nucleotide 535;
[0294] (c) a polynucleotide comprising the nucleotide sequence of
the full-length protein coding sequence of clone K464.sub.--4
deposited under accession number ATCC 98115;
[0295] (d) a polynucleotide encoding the full-length protein
encoded by the cDNA insert of clone K464.sub.--4 deposited under
accession number ATCC 98115;
[0296] (e) a polynucleotide comprising the nucleotide sequence of
the mature protein coding sequence of clone K464.sub.--4 deposited
under accession number ATCC 98115;
[0297] (f) a polynucleotide encoding the mature protein encoded by
the cDNA insert of clone K464.sub.--4 deposited under accession
number ATCC 98115;
[0298] (g) a polynucleotide encoding a protein comprising the amino
acid sequence of SEQ ID NO:39;
[0299] (h) a polynucleotide encoding a protein comprising a
fragment of the amino acid sequence of SEQ ID NO:39 having
biological activity;
[0300] (i) a polynucleotide which is an allelic variant of a
polynucleotide of (a)-(f) above;
[0301] (j) a polynucleotide which encodes a species homologue of
the protein of (g) or (h) above; and
[0302] (k) a polynucleotide capable of hybridizing under stringent
conditions to any one of the polynucleotides specified in
(a)-(h).
[0303] Preferably, such polynucleotide comprises the nucleotide
sequence of SEQ ID NO:38 from nucleotide 380 to nucleotide 535; the
nucleotide sequence of the full-length protein coding sequence of
clone K464.sub.--4 deposited under accession number ATCC 98115; or
the nucleotide sequence of the mature protein coding sequence of
clone K464.sub.--4 deposited under accession number ATCC 98115. In
other preferred embodiments, the polynucleotide encodes the
full-length or mature protein encoded by the cDNA insert of clone
K464.sub.--4 deposited under accession number ATCC 98115.
[0304] Other embodiments provide the gene corresponding to the cDNA
sequence of SEQ ID NO:38 or SEQ ID NO:40.
[0305] 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:
[0306] (a) the amino acid sequence of SEQ ID NO:39;
[0307] (b) fragments of the amino acid sequence of SEQ ID NO:39;
and
[0308] (c) the amino acid sequence encoded by the cDNA insert of
clone K464.sub.--4 deposited under accession number ATCC 98115;
[0309] the protein being substantially free from other mammalian
proteins. Preferably such protein comprises the amino acid sequence
of SEQ ID NO:39.
[0310] In one embodiment, the present invention provides a
composition comprising an isolated polynucleotide selected from the
group consisting of:
[0311] (a) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:41;
[0312] (b) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:41 from nucleotide 218 to nucleotide 1159;
[0313] (c) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:41 from nucleotide 806 to nucleotide 1159;
[0314] (d) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:41 from nucleotide 217 to nucleotide 517;
[0315] (e) a polynucleotide comprising the nucleotide sequence of
the full-length protein coding sequence of clone K483.sub.--1
deposited under accession number ATCC 98115;
[0316] (f) a polynucleotide encoding the full-length protein
encoded by the cDNA insert of clone K483.sub.--1 deposited under
accession number ATCC 98115;
[0317] (g) a polynucleotide comprising the nucleotide sequence of
the mature protein coding sequence of clone K483.sub.--1 deposited
under accession number ATCC 98115;
[0318] (h) a polynucleotide encoding the mature protein encoded by
the cDNA insert of clone K483.sub.--1 deposited under accession
number ATCC 98115;
[0319] (i) a polynucleotide encoding a protein comprising the amino
acid sequence of SEQ ID NO:42;
[0320] (j) a polynucleotide encoding a protein comprising a
fragment of the amino acid sequence of SEQ ID NO:42 having
biological activity;
[0321] (k) a polynucleotide which is an allelic variant of a
polynucleotide of (a)-(h) above;
[0322] (l) a polynucleotide which encodes a species homologue of
the protein of (i) or (j) above; and
[0323] (m) a polynucleotide capable of hybridizing under stringent
conditions to any one of the polynucleotides specified in
(a)-(j).
[0324] Preferably, such polynucleotide comprises the nucleotide
sequence of SEQ ID NO:41 from nucleotide 218 to nucleotide 1159;
the nucleotide sequence of SEQ ID NO:41 from nucleotide 806 to
nucleotide 1159; the nucleotide sequence of SEQ ID NO:41 from
nucleotide 217 to nucleotide 517; the nucleotide sequence of the
full-length protein coding sequence of clone K483.sub.--1 deposited
under accession number ATCC 98115; or the nucleotide sequence of
the mature protein coding sequence of clone K483.sub.--1 deposited
under accession number ATCC 98115. In other preferred embodiments,
the polynucleotide encodes the full-length or mature protein
encoded by the cDNA insert of clone K483.sub.--1 deposited under
accession number ATCC 98115. In yet other preferred embodiments,
the present invention provides a polynucleotide encoding a protein
comprising the amino acid sequence of SEQ ID NO:42 from amino acid
1 to amino acid 100.
[0325] Other embodiments provide the gene corresponding to the cDNA
sequence of SEQ ID NO:41.
[0326] 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:
[0327] (a) the amino acid sequence of SEQ ID NO:42;
[0328] (b) the amino acid sequence of SEQ ID NO:42 from amino acid
1 to amino acid 100;
[0329] (c) fragments of the amino acid sequence of SEQ ID NO:42;
and
[0330] (d) the amino acid sequence encoded by the cDNA insert of
clone K483.sub.--1 deposited under accession number ATCC 98115;
[0331] the protein being substantially free from other mammalian
proteins. Preferably such protein comprises the amino acid sequence
of SEQ ID NO:42 or the amino acid sequence of SEQ ID NO:42 from
amino acid 1 to amino acid 100.
[0332] In one embodiment, the present invention provides a
composition comprising an isolated polynucleotide selected from the
group consisting of:
[0333] (a) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:43;
[0334] (b) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:43 from nucleotide 446 to nucleotide 835;
[0335] (c) a polynucleotide comprising the nucleotide sequence of
SEQ ID NO:43 from nucleotide 503 to nucleotide 835;
[0336] (d) a polynucleotide comprising the nucleotide sequence of
the full-length protein coding sequence of clone L69.sub.--3
deposited under accession number ATCC 98115;
[0337] (e) a polynucleotide encoding the full-length protein
encoded by the cDNA insert of clone L69.sub.--3 deposited under
accession number ATCC 98115;
[0338] (f) a polynucleotide comprising the nucleotide sequence of
the mature protein coding sequence of clone L69.sub.--3 deposited
under accession number ATCC 98115;
[0339] (g) a polynucleotide encoding the mature protein encoded by
the cDNA insert of clone L69.sub.--3 deposited under accession
number ATCC 98115;
[0340] (h) a polynucleotide encoding a protein comprising the amino
acid sequence of SEQ ID NO:44;
[0341] (i) a polynucleotide encoding a protein comprising a
fragment of the amino acid sequence of SEQ ID NO:44 having
biological activity;
[0342] (j) a polynucleotide which is an allelic variant of a
polynucleotide of (a)-(g) above;
[0343] (k) a polynucleotide which encodes a species homologue of
the protein of (h) or (i) above; and
[0344] (l) a polynucleotide capable of hybridizing under stringent
conditions to any one of the polynucleotides specified in
(a)-(i).
[0345] Preferably, such polynucleotide comprises the nucleotide
sequence of SEQ ID NO:43 from nucleotide 446 to nucleotide 835; the
nucleotide sequence of SEQ ID NO:43 from nucleotide 503 to
nucleotide 835; the nucleotide sequence of the full-length protein
coding sequence of clone L69.sub.--3 deposited under accession
number ATCC 98115; or the nucleotide sequence of the mature protein
coding sequence of clone L69.sub.--3 deposited under accession
number ATCC 98115. In other preferred embodiments, the
polynucleotide encodes the full-length or mature protein encoded by
the cDNA insert of clone L69.sub.--3 deposited under accession
number ATCC 98115. In yet other preferred embodiments, the present
invention provides a polynucleotide encoding a protein comprising
the amino acid sequence of SEQ ID NO:44 from amino acid 1 to amino
acid 93.
[0346] Other embodiments provide the gene corresponding to the cDNA
sequence of SEQ ID NO:43 or SEQ ID NO:45.
[0347] 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:
[0348] (a) the amino acid sequence of SEQ ID NO:44;
[0349] (b) the amino acid sequence of SEQ ID NO:44 from amino acid
1 to amino acid 93;
[0350] (c) fragments of the amino acid sequence of SEQ ID NO:44;
and
[0351] (d) the amino acid sequence encoded by the cDNA insert of
clone L69.sub.--3 deposited under accession number ATCC 98115;
[0352] the protein being substantially free from other mammalian
proteins. Preferably such protein comprises the amino acid sequence
of SEQ ID NO:44 or the amino acid sequence of SEQ ID NO:44 from
amino acid 1 to amino acid 93.
[0353] 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.
[0354] Processes are also provided for producing a protein, which
comprise:
[0355] (a) growing a culture of the host cell transformed with such
polynucleotide compositions in a suitable culture medium; and
[0356] (b) purifying the protein from the culture.
[0357] The protein produced according to such methods is also
provided by the present invention. Preferred embodiments include
those in which the protein produced by such process is a mature
form of the protein.
[0358] Protein compositions of the present invention may further
comprise a pharmaceutically acceptable carrier. Compositions
comprising an antibody which specifically reacts with such protein
are also provided by the present invention.
[0359] Methods are also provided for preventing, treating or
ameliorating a medical condition which comprises administering to a
mammalian subject a therapeutically effective amount of a
composition comprising a protein of the present invention and a
pharmaceutically acceptable carrier.
[0360] Also disclosed are methods of promoting cell-cell fusion.
Such a method comprises contacting a first cell and a second cell,
wherein said first cell expresses an AJ172.sub.--2 protein.
AJ172.sub.--2 expression in the first cell can occur naturally or
be the result of transfection with a polynucleotide encoding an
AJ172.sub.--2 protein. Preferably, the first cell is transfected
with a polynucleotide or gene described above. The first cell and
second cell can be of the same type or of different types. In other
embodiments, at least one of said first cell and said second cell
are transfected to express an additional protein other than the
AJ172.sub.--2 protein.
[0361] In yet other embodiments, a method of inhibiting cell-cell
fusion between a first cell which expresses an AJ172.sub.--2
protein and a second cell is disclosed, wherein the method
comprises contacting said first cell with an AJ172.sub.--2 protein
antagonist. Preferably, the antagonist is selected from the group
consisting of an antibody or antibody fragment directed to an
AJ172.sub.--2 protein, an antisense polynucleotide directed to a
polynucleotide expressing an AJ172.sub.--2 protein, a nucleotide
aptamer directed to an AJ172.sub.--2 protein, a peptide aptamer
directed to an AJ172.sub.--2 protein and a small molecule which
blocks the fusion-inducing activity of an AJ172.sub.--2 protein. In
other preferred embodiments, the first cell is a placental cell
(such as a cytotrophoblast) and the second cell is a cell from the
maternal uterine lining.
[0362] Other embodiments provide for a method of inhibiting
blastocyst implantation, wherein the method comprises contacting a
cell within said blastocyst which expresses an AJ172.sub.--2
protein with an AJ172.sub.--2 protein antagonist.
[0363] Yet other embodiments provide for a method of inhibiting
trophoblast invasion, wherein the method comprises contacting a
first cell which expresses an AJ172.sub.--2 protein with an
AJ172.sub.--2 protein antagonist.
[0364] Further embodiments provide for a method of diagnosing or
predicting the existence of a condition associated with
disregulation of AH172.sub.--2 protein in a mammalian subject, such
method comprising (a) determining a first level of expression of
AJ172.sub.--2 protein in the subject, and (b) comparing such first
level of expression to a second level of expression of
AJ172.sub.--2 protein in one or more other mammalian subjects which
do not have said condition. Preferably, the condition is selected
from the group consisting of pre-eclampsia, placental pathology and
cancer (including choriocarcinoma). In preferred embodiments, such
first level of expression is determined in the serum of the
subject, using an antibody or antibody fragment directed to
AJ172.sub.--2 protein.
[0365] Other embodiments provide for a method of treating a
neoplastic disease (including choriocarcinoma) in a mammalian
subject, such method comprising administering to said subject a
therapeutically effective amount of an agent which promotes the
expression or function of AJ172.sub.--2.
[0366] Yet other embodiments provide for a method of inhibiting
metastasis in a mammalian subject, such method comprising
administering to the subject a therapeutically effective amount of
an agent which inhibits the expression or function of
AJ172.sub.--2.
BRIEF DESCRIPTION OF THE DRAWINGS
[0367] FIGS. 1A and 1B are schematic representations of the pED6
and pNOTs vectors, respectively, used for deposit of clones
disclosed herein.
[0368] FIG. 2 presents the results of Northern analyses of multiple
human tissues which indicate that AJ172.sub.--2 is expressed highly
in the placenta and weakly in the testes.
[0369] FIG. 3 presents the results of in situ antisense-RNA
hybridizations which localize AJ172.sub.--2 expression to placental
syncytiotrophoblasts.
[0370] FIG. 4 demonstrates that AJ172.sub.--2 expression in
transfected COS cells can cause the formation of giant
multinucleated syncytia by a fusigenic mechanism. The cells
expressing AJ172.sub.--2 can be seen to have formed multinucleate
syncytia, while the non-transfected cells remained
mononucleate.
[0371] FIG. 5 demonstrates that AJ172.sub.--2 mediates actual cell
fusion and does not operate through a mechanism of arrested cell
division. A first cell line was transfected with AJ172.sub.--2,
luciferase and ERK. A second cell line was transfected with
AJ172.sub.--2 and MEK. When the cells were mixed, fusion occurred
resulting in production of luciferase activity.
[0372] FIG. 6 demonstrates that AJ172.sub.--2 can mediate fusion
between cells of differing types and between a cell expressing
AJ172.sub.--2 and a cell not expressing AJ172.sub.--2. HELA cells
were transfected with a cDNA encoding a P-selectin glycoprotein
ligand-1/Fc fusion protein (PSGL-Fc). COS cells were transfected
with AJ172.sub.--2. Another batch of COS cells was transfected with
AJ172.sub.--2 in reverse orientation. The transfected HELA cells
were mixed with each type of COS cells. As shown in FIG. 6, mixture
with the AJ172.sub.--2 transfected COS cells caused fusion with the
HELA cells, resulting in multinucleate fusions. Mixture with the
COS cells transfected with AJ172.sub.--2 in reverse orientation
resulted in no fusion (mononucleate cells remained).
[0373] FIG. 7 demonstrates that the mechanism of AJ172.sub.--2
induced cell fusion does not require homophilic or heterophilic
protein-protein interactions. COS cells transfected with
AJ172.sub.--2 were mixed with liposomes containing a green
fluorescent protein (GFP) expression plasmid. As shown in FIG. 7,
the COS cells fused with the liposomes, took up the expression
plasmid, and began expressing GFP.
[0374] FIGS. 8-10 present data which demonstrate that AJ172.sub.--2
is expressed in the formation of cytotrophoblasts associated with
choriocarcinoma (see Example 2).
[0375] FIGS. 11 and 12 present data which demonstrate that
AJ172.sub.--2 is disregulated in pre-eclampsia (see Example 3).
[0376] FIGS. 13 and 14 present data which demonstrate activity of
AJ172.sub.--2 in remodeling of extracellular matrices (see Example
4).
DETAILED DESCRIPTION
[0377] Isolated Proteins and Polynucleotides
[0378] 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) 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.
[0379] As used herein a "secreted" protein is one which, when
expressed in a suitable host cell, is transported across or through
a membrane, including transport as a result of signal sequences in
its amino acid sequence. "Secreted" proteins include without
limitation proteins secreted wholly (e.g., soluble proteins) or
partially (e.g., receptors) from the cell in which they are
expressed. "Secreted" proteins also include without limitation
proteins which are transported across the membrane of the
endoplasmic reticulum.
[0380] Clone "AJ26.sub.--3"
[0381] A polynucleotide of the present invention has been
identified as clone "AJ26.sub.--3". AJ26.sub.--3 was isolated from
a human adult testes cDNA library using methods which are selective
for cDNAs encoding secreted proteins (see U.S. Pat. No. 5,536,637),
or was identified as encoding a secreted or transmembrane protein
on the basis of computer analysis of the amino acid sequence of the
encoded protein. AJ26.sub.--3 is a full-length clone, including the
entire coding sequence of a secreted protein (also referred to
herein as "AJ26.sub.--3 protein").
[0382] The nucleotide sequence of AJ26.sub.--3 as presently
determined is reported in SEQ ID NO:1. What applicants presently
believe to be the proper reading frame and the predicted amino acid
sequence of the AJ26.sub.--3 protein corresponding to the foregoing
nucleotide sequence is reported in SEQ ID NO:2.
[0383] The EcoRI/NotI restriction fragment obtainable from the
deposit containing clone AJ26.sub.--3 should be approximately 2100
bp.
[0384] The nucleotide sequence disclosed herein for AJ26.sub.--3
was searched against the GenBank and GeneSeq nucleotide sequence
databases using BLASTN/BLASTX and FASTA search protocols.
AJ26.sub.--3 demonstrated at least some similarity with sequences
identified as U46493 (Cloning vector pFlp recombinase gene,
complete cds). The predicted amino acid sequence disclosed herein
for AJ26.sub.--3 was searched against the GenPept and GeneSeq amino
acid sequence databases using the BLASTX search protocol. The
predicted AJ26.sub.--3 protein demonstrated at least some
similarity to sequences identified as J01917 (DNA polymerase [Human
adenovirus type 2]), J01969 (DNA polymerase [Human adenovirus type
5]), L24893 (HUMAAC02.sub.--1 myelin protein zero [Homo sapiens]),
U43330 (CTX [Xenopus laevis]), and U43394 (CTX [Xenopus laevis]).
Based upon sequence similarity, AJ26.sub.--3 proteins and each
similar protein or peptide may share at least some activity. The
TopPredII computer program predicts four potential transmembrane
domains within the AJ26.sub.--3 protein sequence, centered around
amino acids 11, 41, 163, and 246 of SEQ ID NO:2. The AJ26.sub.--3
protein also has a possible signal sequence that could be cleaved
to produce a mature protein starting at amino acid 17 of SEQ ID
NO:2.
[0385] Clone "AJ172.sub.--2"
[0386] A polynucleotide of the present invention has been
identified as clone "AJ172.sub.--2". AJ172.sub.--2 was isolated
from a human adult testes cDNA library using methods which are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No.
5,536,637), or was identified as encoding a secreted or
transmembrane protein on the basis of computer analysis of the
amino acid sequence of the encoded protein. AJ172.sub.--2 is a
full-length clone, including the entire coding sequence of a
secreted protein (also referred to herein as "AJ172.sub.--2
protein").
[0387] The nucleotide sequence of AJ172.sub.--2 as presently
determined is reported in SEQ ID NO:3. What applicants presently
believe to be the proper reading frame and the predicted amino acid
sequence of the AJ172.sub.--2 protein corresponding to the
foregoing nucleotide sequence is reported in SEQ ID NO:4. Amino
acids 8 to 20 are a predicted leader/signal sequence, with the
predicted mature amino acid sequence beginning at amino acid 21, or
are a transmembrane domain.
[0388] The EcoRI/NotI restriction fragment obtainable from the
deposit containing clone AJ172.sub.--2 should be approximately 3000
bp.
[0389] The nucleotide sequence disclosed herein for AJ172.sub.--2
was searched against the GenBank and GeneSeq nucleotide sequence
databases using BLASTN/BLASTX and FASTA search protocols.
AJ172.sub.--2 demonstrated at least some similarity with sequences
identified as AA077794 (7H01C09 Chromosome 7 HeLa cDNA Library Homo
sapiens cDNA clone 7H01C09), AC000064 (Human BAC clone RG083M05
from 7q21-7q22, complete sequence), D78692 (Human placenta cDNA
5'-end GEN-503H08), H12439 (yj11h10.r1 Homo sapiens cDNA clone
148483 5'), R27389 (yh46a09.s1 Homo sapiens cDNA clone 132760 3'),
and T09280 (Novel AMP/MCF virus clone 24 genome). The predicted
amino acid sequence disclosed herein for AJ172.sub.--2 was searched
against the GenPept and GeneSeq amino acid sequence databases using
the BLASTX search protocol. The predicted AJ172.sub.--2 protein
demonstrated at least some similarity to sequences identified as
M26927 (pol polyprotein [Gibbon leukemia virus]), M93134 (pol
protein [Friend murine leukemia virus]), and R75189 (Osteoinductive
retrovirus RFB-14 pol gene product). AJ172.sub.--2 protein is
similar to a number of viral env proteins, including those of
baboon endogenous virus and many leukemia viruses, which associate
with the membrane portion of the viral envelope. Based upon
sequence similarity, AJ172.sub.--2 proteins and each similar
protein or peptide may share at least some activity. The TopPredII
computer program predicts five potential transmembrane domains
within the AJ172.sub.--2 protein sequence, centered around amino
acids 104, 267, 292, 328, and 457 of SEQ ID NO:4.
[0390] Clone "AP224.sub.--2"
[0391] A polynucleotide of the present invention has been
identified as clone "AP224.sub.--2". AP224.sub.--2 was isolated
from a human adult placenta cDNA library using methods which are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No.
5,536,637), or was identified as encoding a secreted or
transmembrane protein on the basis of computer analysis of the
amino acid sequence of the encoded protein. AP224.sub.--2 is a
full-length clone, including the entire coding sequence of a
secreted protein (also referred to herein as "AP224.sub.--2
protein").
[0392] The nucleotide sequence of the 5' portion of AP224.sub.--2
as presently determined is reported in SEQ ID NO:5. An additional
internal nucleotide sequence from AP224.sub.--2 as presently
determined is reported in SEQ ID NO:6. What applicants believe is
the proper reading frame and the predicted amino acid sequence
encoded by such internal sequence is reported in SEQ ID NO:7.
[0393] Additional nucleotide sequence from the 3' portion of
AP224.sub.--2, including the polyA tail, is reported in SEQ ID
NO:8.
[0394] The EcoRI/NotI restriction fragment obtainable from the
deposit containing clone AP224.sub.--2 should be approximately 2100
bp.
[0395] The nucleotide sequence disclosed herein for AP224.sub.--2
was searched against the GenBank and GeneSeq nucleotide sequence
databases using BLASTN/BLASTX and FASTA search protocols.
AP224.sub.--2 demonstrated at least some similarity with sequences
identified as R37675 (yf61f08.s1 Homo sapiens cDNA clone 26687 3').
Based upon sequence similarity. AP224.sub.--2 proteins and each
similar protein or peptide may share at least some activity.
[0396] Clone "BL89.sub.--13"
[0397] A polynucleotide of the present invention has been
identified as clone "BL89.sub.--13". BL89.sub.--13 was isolated
from a human adult testes cDNA library using methods which are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No.
5,536,637), or was identified as encoding a secreted or
transmembrane protein on the basis of computer analysis of the
amino acid sequence of the encoded protein. BL89.sub.--13 is a
full-length clone, including the entire coding sequence of a
secreted protein (also referred to herein as "BL89.sub.--13
protein").
[0398] The nucleotide sequence of BL89.sub.--13 as presently
determined is reported in SEQ ID NO:9. What applicants presently
believe to be the proper reading frame and the predicted amino acid
sequence of the BL89.sub.--13 protein corresponding to the
foregoing nucleotide sequence is reported in SEQ ID NO:10.
[0399] The EcoRI/NotI restriction fragment obtainable from the
deposit containing clone BL89.sub.--13 should be approximately 3200
bp.
[0400] The nucleotide sequence disclosed herein for BL89.sub.--13
was searched against the GenBank and GeneSeq nucleotide sequence
databases using BLASTN/BLASTX and FASTA search protocols. No hits
were found in the database. The TopPredII computer program predicts
a potential transmembrane domain within the BL89.sub.--13 protein
sequence centered around amino acid 625 of SEQ ID NO:10.
[0401] Clone "BL341.sub.--4"
[0402] A polynucleotide of the present invention has been
identified as clone "BL341.sub.--4". BL341.sub.--4 was isolated
from a human adult testes cDNA library using methods which are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No.
5,536,637), or was identified as encoding a secreted or
transmembrane protein on the basis of computer analysis of the
amino acid sequence of the encoded protein. BL341.sub.--4 is a
full-length clone, including the entire coding sequence of a
secreted protein (also referred to herein as "BL341.sub.--4
protein").
[0403] The nucleotide sequence of BL341.sub.--4 as presently
determined is reported in SEQ ID NO:11. What applicants presently
believe to be the proper reading frame and the predicted amino acid
sequence of the BL341.sub.--4 protein corresponding to the
foregoing nucleotide sequence is reported in SEQ ID NO:12.
[0404] The EcoRI/NotI restriction fragment obtainable from the
deposit containing clone BL341.sub.--4 should be approximately 2600
bp.
[0405] The nucleotide sequence disclosed herein for BL341.sub.--4
was searched against the GenBank and GeneSeq nucleotide sequence
databases using BLASTN/BLASTX and FASTA search protocols.
BL341.sub.--4 demonstrated at least some similarity with sequences
identified as AA460103 (zx50a12.r1 Soares testis NHT Homo sapiens
cDNA clone) and Z63359 (H.sapiens CpG island DNA genomic Mse1
fragment, clone 81e7, reverse read cpg81e7.rt1a). Based upon
sequence similarity, BL341.sub.--4 proteins and each similar
protein or peptide may share at least some activity.
[0406] Clone "BV239.sub.--3"
[0407] A polynucleotide of the present invention has been
identified as clone "BV239.sub.--3". BV239.sub.--3 was isolated
from a human adult brain cDNA library using methods which are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No.
5,536,637), or was identified as encoding a secreted or
transmembrane protein on the basis of computer analysis of the
amino acid sequence of the encoded protein. BV239.sub.--3 is a
full-length clone, including the entire coding sequence of a
secreted protein (also referred to herein as "BV239.sub.--3
protein").
[0408] The nucleotide sequence of BV239.sub.--3 as presently
determined is reported in SEQ ID NO:13. What applicants presently
believe to be the proper reading frame and the predicted amino acid
sequence of the BV239.sub.--3 protein corresponding to the
foregoing nucleotide sequence is reported in SEQ ID NO:14.
[0409] The EcoRI/NotI restriction fragment obtainable from the
deposit containing clone BV239.sub.--3 should be approximately 310
bp.
[0410] The nucleotide sequence disclosed herein for BV239.sub.--3
was searched against the GenBank and GeneSeq nucleotide sequence
databases using BLASTN/BLASTX and FASTA search protocols.
BV239.sub.--3 demonstrated at least some similarity with sequences
identified as U46493 (Cloning vector pFlp recombinase gene,
complete cds). Based upon sequence similarity, BV239.sub.--3
proteins and each similar protein or peptide may share at least
some activity.
[0411] Clone "CC25.sub.--17"
[0412] A polynucleotide of the present invention has been
identified as clone "CC25.sub.--17". CC25.sub.--17 was isolated
from a human adult brain cDNA library using methods which are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No.
5,536,637), or was identified as encoding a secreted or
transmembrane protein on the basis of computer analysis of the
amino acid sequence of the encoded protein. CC25.sub.--17 is a
full-length clone, including the entire coding sequence of a
secreted protein (also referred to herein as "CC25.sub.--17
protein").
[0413] The nucleotide sequence of CC25.sub.--17 as presently
determined is reported in SEQ ID NO:15. What applicants presently
believe to be the proper reading frame and the predicted amino acid
sequence of the CC25.sub.--17 protein corresponding to the
foregoing nucleotide sequence is reported in SEQ ID NO:16.
[0414] The EcoRI/NotI restriction fragment obtainable from the
deposit containing clone CC25.sub.--17 should be approximately 300
bp.
[0415] The nucleotide sequence disclosed herein for CC25.sub.--17
was searched against the GenBank and GeneSeq nucleotide sequence
databases using BLASTN/BLASTX and FASTA search protocols.
CC25.sub.--17 demonstrated at least some similarity with sequences
identified as U46493 (Cloning vector pFlp recombinase gene,
complete cds). Based upon sequence similarity, CC25.sub.--17
proteins and each similar protein or peptide may share at least
some activity.
[0416] Clone "CC397.sub.--19"
[0417] A polynucleotide of the present invention has been
identified as clone "CC397.sub.--19". CC397.sub.--19 was isolated
from a human adult brain cDNA library using methods which are
selective for cDNAs encoding secreted proteins (see U.S. Pat. No.
5,536,637), or was identified as encoding a secreted or
transmembrane protein on the basis of computer analysis of the
amino acid sequence of the encoded protein. CC397.sub.--19 is a
full-length clone, including the entire coding sequence of a
secreted protein (also referred to herein as "CC397.sub.--19
protein").
[0418] The nucleotide sequence of CC397.sub.--19 as presently
determined is reported in SEQ ID NO:17. What applicants presently
believe to be the proper reading frame and the predicted amino acid
sequence of the CC397.sub.--19 protein corresponding to the
foregoing nucleotide sequence is reported in SEQ ID NO:18.
[0419] The EcoRI/NotI restriction fragment obtainable from the
deposit containing clone CC397.sub.--19 should be approximately
1700 bp.
[0420] The nucleotide sequence disclosed herein for CC397.sub.--19
was searched against the GenBank and GeneSeq nucleotide sequence
databases using BLASTN/BLASTX and FASTA search protocols.
CC397.sub.--19 demonstrated at least some similarity with sequences
identified as AC002129 (Human DNA from chromsome 19 cosmid R33743,
genomic sequence, complete sequence), D82019 (Mouse gene for
basigin precursor, basigin signal precursor), G08688 (human STS
CHLC.GATA29D08.P14592 clone GATA29DO8), M68516 (Human protein C
inhibitor gene, complete cds), and Z68756 (Human DNA sequence from
cosmid L191F1, Huntington's Disease Region, chromosome 4p16.3
contains Huntington Disease (HD) gene, CpG island ESTs and U7 small
nuclear RNA). The predicted amino acid sequence disclosed herein
for CC397.sub.--19 was searched against the GenPept and GeneSeq
amino acid sequence databases using the BLASTX search protocol. The
predicted CC397.sub.--19 protein demonstrated at least some
similarity to sequences identified as X52164 (Q300 protein (AA
1-77) [Mus musculus]). Based upon sequence similarity,
CC397.sub.--19 proteins and each similar protein or peptide may
share at least some activity. The nucleotide sequence of
CC397.sub.--19 indicates that it may contain an Alu repetitive
element.
[0421] Clone "D3052"
[0422] A polynucleotide of the present invention has been
identified as clone "D305.sub.--2". D305.sub.--2 was isolated from
a human adult blood (peripheral blood mononuclear cells treated
with concanavalin A and phorbol myristate acetate) 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. D305.sub.--2 is a full-length clone, including the entire
coding sequence of a secreted protein (also referred to herein as
"D305.sub.--2 protein").
[0423] The nucleotide sequence of the 5' portion of D305.sub.--2 as
presently determined is reported in SEQ ID NO:19. An additional
internal nucleotide sequence from D305.sub.--2 as presently
determined is reported in SEQ ID NO:20. What applicants believe is
the proper reading frame and the predicted amino acid sequence
encoded by such internal sequence is reported in SEQ ID NO:21.
Amino acids 3 to 15 of SEQ ID NO:21 are a predicted leader/signal
sequence, with the predicted mature amino acid sequence beginning
at amino acid 16, or are a transmembrane domain. Additional
nucleotide sequence from the 3' portion of D305.sub.--2, including
the polyA tail, is reported in SEQ ID NO:22.
[0424] The EcoRI/NotI restriction fragment obtainable from the
deposit containing clone D305.sub.--2 should be approximately 2400
bp.
[0425] The nucleotide sequence disclosed herein for D305.sub.--2
was searched against the GenBank and GeneSeq nucleotide sequence
databases using BLASTN/BLASTX and FASTA search protocols.
D305.sub.--2 demonstrated at least some similarity with sequences
identified as AA055703 (zl75d04.r1 Stratagene colon (#937204) Homo
sapiens cDNA clone 510439 5'), N49593 (yy58d05.s1 Homo sapiens cDNA
clone 277737 3'), R66646 (yi35b08.r1 Homo sapiens cDNA clone 141207
5' similar to SP P24A_YEAST P32802 P24A PROTEIN), U81006 (Human p76
mRNA, complete cds), and Z48758 (S.cerevisiae chromosome IV cosmid
9727). The predicted amino acid sequence disclosed herein for
D305.sub.--2 was searched against the GenPept and GeneSeq amino
acid sequence databases using the BLASTX search protocol. The
predicted D305.sub.--2 protein demonstrated at least some
similarity to sequences identified as U53880 (P24A protein (unknown
function) (Swiss Prot. accession number P32802) [Saccharomyces
cerevisiae]), U81006 (p76 [Homo sapiens]), X67316 (SCEMP70.sub.--1
p24a 70 kDa precursor [Saccharomyces cerevisiae]), and Z48758
(unknown [Saccharomyces cerevisiae]). Based upon sequence
similarity, D305.sub.--2 proteins and each similar protein or
peptide may share at least some activity.
[0426] Clone "G55.sub.--1"
[0427] A polynucleotide of the present invention has been
identified as clone "G55.sub.--1". G55.sub.--1 was isolated from a
human adult blood (peripheral blood mononuclear cells treated with
concanavalin A and phorbol myristate acetate) 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. G55.sub.--1 is a
full-length clone, including the entire coding sequence of a
secreted protein (also referred to herein as "G55.sub.--1
protein").
[0428] The nucleotide sequence of the 5' portion of G55.sub.--1 as
presently determined is reported in SEQ ID NO:23. What applicants
presently believe is the proper reading frame for the coding region
is indicated in SEQ ID NO:24. The predicted amino acid sequence of
the G55.sub.--1 protein corresponding to the foregoing nucleotide
sequence is reported in SEQ ID NO:24. Amino acids 98 to 110 are a
predicted leader/signal sequence, with the predicted mature amino
acid sequence beginning at amino acid 111, or are a transmembrane
domain. Additional nucleotide sequence from the 3' portion of
G55.sub.--1, including the polyA tail, is reported in SEQ ID
NO:25.
[0429] The EcoRI/NotI restriction fragment obtainable from the
deposit containing clone G55.sub.--1 should be approximately 2000
bp.
[0430] The nucleotide sequence disclosed herein for G55.sub.--1 was
searched against the GenBank and GeneSeq nucleotide sequence
databases using BLASTN/BLASTX and FASTA search protocols.
G55.sub.--1 demonstrated at least some similarity with sequences
identified as R83586 (yp16a07.r1 Homo sapiens cDNA clone 187572
5'). Based upon sequence similarity, G55.sub.--1 proteins and each
similar protein or peptide may share at least some activity.
[0431] Clone "K39.sub.--7"
[0432] A polynucleotide of the present invention has been
identified as clone "K39.sub.--7". K39.sub.--7 was referred to as
K39.sub.--2 in previous applications. K39.sub.--7 was isolated from
a murine adult bone marrow (stromal cell line FCM4) 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. K39.sub.--7 is a full-length clone, including the entire
coding sequence of a secreted protein (also referred to herein as
"K39.sub.--7 protein").
[0433] The nucleotide sequence of the 5' portion of K39.sub.--7 as
presently determined is reported in SEQ ID NO:26. What applicants
presently believe is the proper reading frame for the coding region
is indicated in SEQ ID NO:27. The predicted amino acid sequence of
the K39.sub.--7 protein corresponding to the foregoing nucleotide
sequence is reported in SEQ ID NO:27. Amino acids 3 to 15 are a
predicted leader/signal sequence, with the predicted mature amino
acid sequence beginning at amino acid 16, or are a transmembrane
domain. Additional nucleotide sequence from the 3' portion of
K39.sub.--7, including the polyA tail, is reported in SEQ ID
NO:28.
[0434] The EcoRI/NotI restriction fragment obtainable from the
deposit containing clone K39.sub.--7 should be approximately 1675
bp.
[0435] The nucleotide sequence disclosed herein for K39.sub.--7 was
searched against the GenBank and GeneSeq nucleotide sequence
databases using BLASTN/BLASTX and FASTA search protocols.
K39.sub.--7 demonstrated at least some similarity with sequences
identified as AA254326 (va15d06.r1 Soares mouse lymph node NbMILN
Mus musculus cDNA clone 722987 5' similar to WP:C09G4.1 CE03978),
D18935 (Mouse 3'-directed cDNA, MUSGS01125, clone mc0564), H14129
(ym65b04.r1 Homo sapiens cDNA clone 163759 5'), and R20230 (hUOG-1,
DNA segment encoding a mammalian GDF-1 protein). The predicted
amino acid sequence disclosed herein for K39.sub.--7 was searched
against the GenPept and GeneSeq amino acid sequence databases using
the BLASTX search protocol. The predicted K39.sub.--7 protein
demonstrated at least some similarity to sequences identified as
R86811 (Saccharomyces cerevisiae mutant LAG1 protein) and U42438
(similar to S. cerevisiae longevity-assurance protein 1 (SP P38703)
[Caenorhabditis elegans]). Based upon sequence similarity,
K39.sub.--7 proteins and each similar protein or peptide may share
at least some activity.
[0436] Clone "K330.sub.--3"
[0437] A polynucleotide of the present invention has been
identified as clone "K330.sub.--3". K330.sub.--3 was referred to as
K330.sub.--2 in previous applications. K330.sub.--3 was isolated
from a murine adult bone marrow (stromal cell line FCM-4) 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. K330.sub.--3 is a full-length clone, including the entire
coding sequence of a secreted protein (also referred to herein as
"K330.sub.--3 protein").
[0438] The nucleotide sequence of the 5' portion of K330.sub.--3 as
presently determined is reported in SEQ ID NO:29. What applicants
presently believe is the proper reading frame for the coding region
is indicated in SEQ ID NO:30. The predicted amino acid sequence of
the K330.sub.--3 protein corresponding to the foregoing nucleotide
sequence is reported in SEQ ID NO:30. Additional nucleotide
sequence from the 3' portion of K330.sub.--3, including the polyA
tail, is reported in SEQ ID NO:31.
[0439] The EcoRI/NotI restriction fragment obtainable from the
deposit containing clone K330.sub.--3 should be approximately 1300
bp.
[0440] The nucleotide sequence disclosed herein for K330.sub.--3
was searched against the GenBank and GeneSeq nucleotide sequence
databases using BLASTN/BLASTX and FASTA search protocols.
K330.sub.--3 demonstrated at least some similarity with sequences
identified as A03900 (H.sapiens HuV(NP) gene), AA038010 (mi80a11.r1
Soares mouse p3NMF19.5 Mus musculus cDNA clone 472892 5'), M30775
(Mouse thymidylate synthase pseudogene, 3' flank), R40824
(yf82c07.s1 Homo sapiens cDNA clone 28939 3'), T23245 (Human gene
signature HUMGS05046), and U23512 (Caenorhabditis elegans cosmid
M01G4). Based upon sequence similarity, K330.sub.--3 proteins and
each similar protein or peptide may share at least some
activity.
[0441] Clone "K363.sub.--3"
[0442] A polynucleotide of the present invention has been
identified as clone "K363.sub.--3". K363.sub.--3 was referred to as
K363.sub.--2 in previous applications. K363.sub.--3 was isolated
from a murine adult bone marrow (stromal cell line FCM-4) 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. K363.sub.--3 is a full-length clone, including the entire
coding sequence of a secreted protein (also referred to herein as
"K363.sub.--3 protein").
[0443] The nucleotide sequence of the 5' portion of K363.sub.--3 as
presently determined is reported in SEQ ID NO:32. What applicants
presently believe is the proper reading frame for the coding region
is indicated in SEQ ID NO:33. The predicted amino acid sequence of
the K363.sub.--3 protein corresponding to the foregoing nucleotide
sequence is reported in SEQ ID NO:33. Additional nucleotide
sequence from the 3' portion of K363.sub.--3, including the polyA
tail, is reported in SEQ ID NO:34.
[0444] The EcoRI/NotI restriction fragment obtainable from the
deposit containing clone K363.sub.--3 should be approximately 2690
bp.
[0445] The nucleotide sequence disclosed herein for K363.sub.--3
was searched against the GenBank and GeneSeq nucleotide sequence
databases using BLASTN/BLASTX and FASTA search protocols.
K363.sub.--3 demonstrated at least some similarity with sequences
identified as AA437876 (vd20h06.s1 Knowles Solter mouse 2 cell Mus
musculus cDNA clone 793115 5'), D21554 (Mouse embryonal carcinoma
F9 cell cDNA, 67F09), and Y08460 (Mus musculus mRNA for Mdes
transmembrane protein). The predicted amino acid sequence disclosed
herein for K363.sub.--3 was searched against the GenPept and
GeneSeq amino acid sequence databases using the BLASTX search
protocol. The predicted K363.sub.--3 protein demonstrated at least
some similarity to sequences identified as Y08460 (Mdes protein
[Mus musculus]). Based upon sequence similarity, K363.sub.--3
proteins and each similar protein or peptide may share at least
some activity.
[0446] Clone "K446.sub.--3"
[0447] A polynucleotide of the present invention has been
identified as clone "K446.sub.--3". K446.sub.--3 was referred to as
K446.sub.--2 in previous applications. K446.sub.--3 was isolated
from a murine adult bone marrow (stromal cell line FCM-4) 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. K446.sub.--3 is a full-length clone, including the entire
coding sequence of a secreted protein (also referred to herein as
"K446.sub.--3 protein").
[0448] The nucleotide sequence of the 5' portion of K446.sub.--3 as
presently determined is reported in SEQ ID NO:35. What applicants
presently believe is the proper reading frame for the coding region
is indicated in SEQ ID NO:36. The predicted amino acid sequence of
the K446.sub.--3 protein corresponding to the foregoing nucleotide
sequence is reported in SEQ ID NO:36. Additional nucleotide
sequence from the 3' portion of K446.sub.--3, including the polyA
tail, is reported in SEQ ID NO:37.
[0449] The EcoRI/NotI restriction fragment obtainable from the
deposit containing clone K446.sub.--3 should be approximately 2150
bp.
[0450] The nucleotide sequence disclosed herein for K446.sub.--3
was searched against the GenBank and GeneSeq nucleotide sequence
databases using BLASTN/BLASTX and FASTA search protocols. No hits
were found in the database.
[0451] Clone "K464.sub.--4"
[0452] A polynucleotide of the present invention has been
identified as clone "K464.sub.--4". K464.sub.--4 was referred to as
K464.sub.--3 in previous applications. K464.sub.--4 was isolated
from a murine adult bone marrow (stromal cell line FCM-4) 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. K464.sub.--4 is a full-length clone, including the entire
coding sequence of a secreted protein (also referred to herein as
"K464.sub.--4 protein").
[0453] The nucleotide sequence of the 5' portion of K464.sub.--4 as
presently determined is reported in SEQ ID NO:38. What applicants
presently believe is the proper reading frame for the coding region
is indicated in SEQ ID NO:39. The predicted amino acid sequence of
the K464.sub.--4 protein corresponding to the foregoing nucleotide
sequence is reported in SEQ ID NO:39. Additional nucleotide
sequence from the 3' portion of K464.sub.--4, including the polyA
tail, is reported in SEQ ID NO:40.
[0454] The EcoRI/NotI restriction fragment obtainable from the
deposit containing clone K464.sub.--4 should be approximately 1250
bp.
[0455] The nucleotide sequence disclosed herein for K464.sub.--4
was searched against the GenBank and GeneSeq nucleotide sequence
databases using BLASTN/BLASTX and FASTA search protocols.
K464.sub.--4 demonstrated at least some similarity with sequences
identified as AA260484 (va95a09.r1 Soares mouse NML Mus musculus
cDNA clone 747160 5'), AA419864 (vf49b08.r1 Soares mouse NbM),
L25338 (Mus musculus folate-binding protein gene, 5' end), M22527
(Mouse cytotoxic T lymphocyte-specific serine protease), T01176
(P815A antigen precursor gene P1A), T21224 (Human gene signature
HUMGS02538), T41900 (Vector pAPEX-3p), U46493 (Cloning vector pFlp
recombinase gene, complete cds), U89673 (Cloning vector pIRES1neo,
complete plasmid sequence), W32699 (zc06b11.s1 Soares parathyroid
tumor NbHPA Homo sapiens cDNA clone 321501 3'), and W36926
(mb82b10.r1 Soares mouse p3NMF19.5 Mus musculus cDNA clone 335899
5'). The predicted amino acid sequence disclosed herein for
K464.sub.--4 was searched against the GenPept and GeneSeq amino
acid sequence databases using the BLASTX search protocol. The
predicted K464.sub.--4 protein demonstrated at least some
similarity to sequences identified as L33768 (JAK3 [Mus musculus])
and X16213 (MHC T7 class I antigen (64 AA) (119 is 2nd base in
codon) [Mus musculus]). Based upon sequence similarity,
K464.sub.--4 proteins and each similar protein or peptide may share
at least some activity.
[0456] Clone "K483.sub.--1"
[0457] A polynucleotide of the present invention has been
identified as clone "K483.sub.--1". K483.sub.--1 was isolated from
a murine adult bone marrow (stromal cell line FCM-4) 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. K483.sub.--1 is a full-length clone, including the entire
coding sequence of a secreted protein (also referred to herein as
"K483.sub.--1 protein").
[0458] The nucleotide sequence of K483.sub.--1 as presently
determined is reported in SEQ ID NO:41. What applicants presently
believe to be the proper reading frame and the predicted amino acid
sequence of the K483.sub.--1 protein corresponding to the foregoing
nucleotide sequence is reported in SEQ ID NO:42. Amino acids 184 to
196 are a predicted leader/signal sequence, with the predicted
mature amino acid sequence beginning at amino acid 197, or are a
transmembrane domain.
[0459] The EcoRI/NotI restriction fragment obtainable from the
deposit containing clone K483.sub.--1 should be approximately 1500
bp.
[0460] The nucleotide sequence disclosed herein for K483.sub.--1
was searched against the GenBank and GeneSeq nucleotide sequence
databases using BLASTN/BLASTX and FASTA search protocols.
K483.sub.--1 demonstrated at least some similarity with sequences
identified as AA110914 (mm02c03.r1 Stratagene mouse kidney
(#937315) Mus musculus cDNA clone 520324 5'), AA318160 (EST20431
Retina II Homo sapiens cDNA 5' end), AA500150 (vi97c09.r1 Barstead
mouse pooled organs MPLRB4 Mus musculus cDNA clone 920176 5'), and
N41895 (yw86b03.r1 Homo sapiens cDNA clone 259085 5'). Based upon
sequence similarity, K483.sub.--1 proteins and each similar protein
or peptide may share at least some activity. The TopPredII computer
program predicts three potential transmembrane domains within the
K483.sub.--1 protein sequence, centered around amino acids 18, 179,
and 270 of SEQ ID NO:42. The K483.sub.--1 protein also has a
possible signal sequence that could be cleaved to produce a mature
protein starting at amino acid 34 of SEQ ID NO:42.
[0461] Clone "L69.sub.--3"
[0462] A polynucleotide of the present invention has been
identified as clone "L69.sub.--3". L69.sub.--3 was referred to as
L69.sub.--2 in previous applications. L69.sub.--3 was isolated from
a murine adult thymus 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. L69.sub.--3 is a
full-length clone, including the entire coding sequence of a
secreted protein (also referred to herein as "L69.sub.--3
protein").
[0463] The nucleotide sequence of the 5' portion of L69.sub.--3 as
presently determined is reported in SEQ ID NO:43. What applicants
presently believe is the proper reading frame for the coding region
is indicated in SEQ ID NO:44. The predicted amino acid sequence of
the L69.sub.--3 protein corresponding to the foregoing nucleotide
sequence is reported in SEQ ID NO:44. Amino acids 7 to 19 are a
predicted leader/signal sequence, with the predicted mature amino
acid sequence beginning at amino acid 20, or are a transmembrane
domain. Additional nucleotide sequence from the 3' portion of
L69.sub.--3, including the polyA tail, is reported in SEQ ID
NO:45.
[0464] The EcoRI/NotI restriction fragment obtainable from the
deposit containing clone L69.sub.--3 should be approximately 1200
bp.
[0465] The nucleotide sequence disclosed herein for L69.sub.--3 was
searched against the GenBank and GeneSeq nucleotide sequence
databases using BLASTN/BLASTX and FASTA search protocols.
L69.sub.--3 demonstrated at least some similarity with sequences
identified as H35162 (EST108034 Rattus sp. cDNA similar to
H.sapiens hypothetical protein (PIR:S25641)), U02442 (Cloning
vector pADbeta, complete sequence), W74864 (md91b10.r1 Soares mouse
embryo NbME13.5 14.5 Mus musculus cDNA), and X67698 (H.sapiens
tissue specific mRNA). The predicted amino acid sequence disclosed
herein for L69.sub.--3 was searched against the GenPept and GeneSeq
amino acid sequence databases using the BLASTX search protocol. The
predicted L69.sub.--3 protein demonstrated at least some similarity
to sequences identified as A18921 (tissue-specific secretory
protein [unidentified]). Based upon sequence similarity,
L69.sub.--3 proteins and each similar protein or peptide may share
at least some activity.
[0466] Deposit of Clones
[0467] Clones AJ26.sub.--3, AJ172.sub.--2, AP224.sub.--2,
BL89.sub.--10, BL341.sub.--4, BV239.sub.--2, CC25.sub.--16,
CC397.sub.--11, D305.sub.--2, G55.sub.--1, K39.sub.--7,
K330.sub.--3, K363.sub.--3, K446.sub.--3, K464.sub.--4,
K483.sub.--1, and L69.sub.--3 were deposited on Jul. 25, 1996 with
the American Type Culture Collection as an original deposit under
the Budapest Treaty and were given the accession number ATCC 98115,
from which each clone comprising a particular polynucleotide is
obtainable. Clones K39.sub.--7, K330.sub.--3, K363.sub.--3,
K446.sub.--3, K464.sub.--4, and L69.sub.--3 were referred to as
K39.sub.--2, K330.sub.--2, K363.sub.--2, K446.sub.--2,
K464.sub.--3, and L69.sub.--2, respectively, when the Jul. 25, 1996
deposit was made. An additional isolate of each of clones
BL89.sub.--10, BV239.sub.--2, CC25.sub.--16, and CC397.sub.--11
(namely isolates BL89.sub.--13, BV239.sub.--3, CC25.sub.--17, and
CC397.sub.--19, respectively) were deposited with the American Type
Culture Collection on Aug. 23, 1996 under accession number 98153,
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. .sctn.1.808(b).
[0468] Each clone has been transfected into separate bacterial
cells (E. coli) in this composite deposit. Each clone can be
removed from the vector in which it was deposited by performing an
EcoRI/NotI digestion (5' site, EcoRI; 3' site, NotI) to produce the
appropriate fragment for such clone. Each clone was deposited in
either the pED6 or pNOTs vector depicted in FIG. 1. The pED6dpc2
vector ("pED6") was derived from pED6dpc1 by insertion of a new
polylinker to facilitate cDNA cloning (Kaufman et al., 1991,
Nucleic Acids Res. 19: 4485-4490); the pNOTs vector was derived
from pMT2 (Kaufman et al., 1989, Mol. Cell. Biol. 9: 946-958) by
deletion of the DHFR sequences, insertion of a new polylinker, and
insertion of the M13 origin of replication in the ClaI site. In
some instances, the deposited clone can become "flipped" (i.e., in
the reverse orientation) in the deposited isolate. In such
instances, the cDNA insert can still be isolated by digestion with
EcoRI and NotI. However, NotI will then produce the 5' site and
EcoRI will produce the 3' site for placement of the cDNA in proper
orientation for expression in a suitable vector. The cDNA may also
be expressed from the vectors in which they were deposited.
[0469] Bacterial cells containing a particular clone can be
obtained from the composite deposit as follows:
[0470] An oligonucleotide probe or probes should be designed to the
sequence that is known for that particular clone. This sequence can
be derived from the sequences provided herein, or from a
combination of those sequences. The sequence of the oligonucleotide
probe that was used to isolate each full-length clone is identified
below, and should be most reliable in isolating the clone of
interest.
1 Clone Probe Sequence AJ26_3 SEQ ID NO:46 AJ172_2 SEQ ID NO:47
AP224_2 SEQ ID NO:48 BL89_13 SEQ ID NO:49 BL341_4 SEQ ID NO:50
BV239_3 SEQ ID NO:51 CC25_17 SEQ ID NO:52 CC397_19 SEQ ID NO:53
D305_2 SEQ ID NO:54 G55_1 SEQ ID NO:55 K39_7 SEQ ID NO:56 K330_3
SEQ ID NO:57 K363_3 SEQ ID NO:58 K446_3 SEQ ID NO:59 K464_4 SEQ ID
NO:60 K483_1 SEQ ID NO:61 L69_3 SEQ ID NO:62
[0471] 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)-phosph-
oramadite) (Glen Research, cat. no. 10-1953)).
[0472] The design of the oligonucleotide probe should preferably
follow these parameters:
[0473] (a) It should be designed to an area of the sequence which
has the fewest ambiguous bases ("N's"), if any;
[0474] (b) It should be designed to have a T.sub.m of approx.
80.degree. C. (assuming 2.degree. for each A or T and 4 degrees for
each G or C).
[0475] The oligonucleotide should preferably be labeled with
g-.sup.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.
[0476] The bacterial culture containing the pool of full-length
clones should preferably be thawed and 100 .mu.l of the stock used
to inoculate a sterile culture flask containing 25 ml of sterile
L-broth containing ampicillin at 100 .mu.g/mil. The culture should
preferably be grown to saturation at 37.degree. 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 .mu.g/ml and
agar at 1.5% in a 150 mm petri dish when grown overnight at
37.degree. C. Other known methods of obtaining distinct,
well-separated colonies can also be employed.
[0477] Standard colony hybridization procedures should then be used
to transfer the colonies to nitrocellulose filters and lyse,
denature and bake them.
[0478] The filter is then preferably incubated at 65.degree. C. for
1 hour with gentle agitation in 6.times. SSC (20.times. stock is
175.3 g NaCl/liter, 88.2 g Na citrate/liter, adjusted to pH 7.0
with NaOH) containing 0.5% SDS, 100 .mu.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.degree. C. with gentle agitation overnight. The
filter is then preferably washed in 500 mL of 2.times. SSC/0.5% SDS
at room temperature without agitation, preferably followed by 500
mL of 2.times. SSC/0.1% SDS at room temperature with gentle shaking
for 15 minutes. A third wash with 0.1.times. SSC/0.5% SDS at
65.degree. C. for 30 minutes to 1 hour is optional. The filter is
then preferably dried and subjected to autoradiography for
sufficient time to visualize the positives on the X-ray film. Other
known hybridization methods can also be employed.
[0479] 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.
[0480] Fragments of the proteins of the present invention which are
capable of exhibiting biological activity are also encompassed by
the present invention. Fragments of the protein may be in linear
form or they may be cyclized using known methods, for example, as
described in H. U. Saragovi, et al., Bio/Technology 10, 773-778
(1992) and in R. S. McDowell, et al., J. Amer. Chem. Soc. 114,
9245-9253 (1992), both of which are incorporated herein by
reference. Such fragments may be fused to carrier molecules such as
immunoglobulins for many purposes, including increasing the valency
of protein binding sites. For example, fragments of the protein may
be fused through "linker" sequences to the Fc portion of an
immunoglobulin. For a bivalent form of the protein, such a fusion
could be to the Fc portion of an IgG molecule. Other immunoglobulin
isotypes may also be used to generate such fusions. For example, a
protein -IgM fusion would generate a decavalent form of the protein
of the invention.
[0481] The present invention also provides both full-length and
mature forms of the disclosed proteins. The full-length form of the
such proteins is identified in the sequence listing by translation
of the nucleotide sequence of each disclosed clone. The mature form
of such protein may be obtained by expression of the disclosed
full-length polynucleotide (preferably those deposited with ATCC)
in a suitable mammalian cell or other host cell. The sequence of
the mature form of the protein may also be determinable from the
amino acid sequence of the full-length form.
[0482] The present invention also provides genes corresponding to
the cDNA sequences disclosed herein. "Corresponding genes" are the
regions of the genome that are transcribed to produce the mRNAs
from which the cDNA sequences are derived and any contiguous
regions of the genome necessary for the regulated expression of
such genes, including but not limited to coding sequences, 5' and
3' untranslated regions, alternatively spliced exons, introns,
promoters, enhancers, and silencer or suppressor elements. The
corresponding genes can be isolated in accordance with known
methods using the sequence information disclosed herein. Such
methods include the preparation of probes or primers from the
disclosed sequence information for identification and/or
amplification of genes in appropriate genomic libraries or other
sources of genomic materials.
[0483] Where the protein of the present invention is membrane-bound
(e.g., is a receptor), the present invention also provides for
soluble forms of such protein. In such forms part or all of the
intracellular and transmembrane domains of the protein are deleted
such that the protein is fully secreted from the cell in which it
is expressed. The intracellular and transmembrane domains of
proteins of the invention can be identified in accordance with
known techniques for determination of such domains from sequence
information.
[0484] Proteins and protein fragments of the present invention
include proteins with amino acid sequence lengths that are at least
25%c(more preferably at least 50%, and most preferably at least
75%) of the length of a disclosed protein and have at least 60%
sequence identity (more preferably, at least 75% identity; most
preferably at least 90% or 95% identity) with that disclosed
protein, where sequence identity is determined by comparing the
amino acid sequences of the proteins when aligned so as to maximize
overlap and identity while minimizing sequence gaps. Also included
in the present invention are proteins and protein fragments that
contain a segment preferably comprising 8 or more (more preferably
20 or more, most preferably 30 or more) contiguous amino acids that
shares at least 75% sequence identity (more preferably, at least
85% identity; most preferably at least 95% identity) with any such
segment of any of the disclosed proteins.
[0485] Species homologs of the disclosed polynucleotides and
proteins are also provided by the present invention. As used
herein, a "species homologue" is a protein or polynucleotide with a
different species of origin from that of a given protein or
polynucleotide, but with significant sequence similarity to the
given protein or polynucleotide, as determined by those of skill in
the art. Species homologs may be isolated and identified by making
suitable probes or primers from the sequences provided herein and
screening a suitable nucleic acid source from the desired
species.
[0486] The invention also encompasses allelic variants of the
disclosed polynucleotides or proteins;
[0487] that is, naturally-occurring alternative forms of the
isolated polynucleotide which also encode proteins which are
identical, homologous, or related to that encoded by the
polynucleotides.
[0488] The invention also includes polynucleotides with sequences
complementary to those of the polynucleotides disclosed herein.
[0489] The present invention also includes polynucleotides capable
of hybridizing under reduced stringency conditions, more preferably
stringent conditions, and most preferably highly stringent
conditions, to polynucleotides described herein. Examples of
stringency conditions are shown in the table below: highly
stringent conditions are those that are at least as stringent as,
for example, conditions A-F; stringent conditions are at least as
stringent as, for example, conditions G-L; and reduced stringency
conditions are at least as stringent as, for example, conditions
M-R.
2 Hybrid Hybridization Wash Stringency Polynucleotide Length
Temperature and Temperature Condition Hybrid (bp).sup..xi.
Buffer.sup..differential. and Buffer.sup..differential. A DNA:DNA
.gtoreq.50 65.degree. C.; 1 .times. SSC 65.degree. C.; -or-
42.degree. C.; 1 .times. 0.3 .times. SSC SSC, 50% formamide B
DNA:DNA <50 T.sub.B*; 1 .times. SSC T.sub.B*; 1 .times. SSC C
DNA:RNA .gtoreq.50 67.degree. C., 1 .times. SSC 67.degree. C., -or-
45.degree. C.; 0.3 .times. SSC 1 .times. SSC, 50% formamide D
DNA:RNA <50 T.sub.D*; 1 .times. SSC T.sub.D*, 1 .times. SSC E
RNA:RNA .gtoreq.50 70.degree. C.; 1 .times. SSC 70.degree. C.; -or-
50.degree. C.; 0.3 .times. SSC 1 .times. SSC, 50% formamide F
RNA:RNA <50 T.sub.F*; 1 .times. SSC T.sub.F*; 1 .times. SSC G
DNA:DNA .gtoreq.50 65.degree. C.; 4 .times. SSC 65.degree. C.; -or-
42.degree. C.; 1 .times. SSC 4 .times. SSC, 50% formamide H DNA:DNA
<50 T.sub.H*; 4 .times. SSC T.sub.H*; 4 .times. SSC I DNA:RNA
.gtoreq.50 67.degree. C.; 4 .times. SSC 67.degree. C.; -or-
45.degree. C.; 1 .times. SSC 4 .times. SSC, 50% formamide J DNA:RNA
<50 T.sub.J*; 4 .times. SSC T.sub.J*; 4 .times. SSC K RNA:RNA
.gtoreq.50 70.degree. C.; 4 .times. SSC 67.degree. C.; -or-
50.degree. C.; 1 .times. SSC 4 .times. SSC, 50% formamide L RNA:RNA
<50 T.sub.L*; 2 .times. SSC T.sub.L*; 2 .times. SSC M DNA:DNA
.gtoreq.50 50.degree. C.; 4 .times. SSC 50.degree. C.; -or-
40.degree. C.; 2 .times. SSC 6 .times. SSC, 50% formamide N DNA:DNA
<50 T.sub.N*; 6 .times. SSC T.sub.N*; 6 .times. SSC O DNA:RNA
.gtoreq.50 55.degree. C.; 4 .times. SSC 55.degree. C.; -or-
42.degree. C.; 2 .times. SSC 6 .times. SSC, 50% formamide P DNA:RNA
<50 T.sub.P*; 6 .times. SSC T.sub.P*; 6 .times. SSC Q RNA:RNA
.gtoreq.50 60.degree. C.; 4 .times. SSC 60.degree. C.; -or-
45.degree. C.; 2 .times. SSC 6 .times. SSC, 50% formamide R RNA:RNA
<50 T.sub.R*; 4 .times. SSC T.sub.R*; 4 .times. SSC .sup..xi.The
hybrid length is that anticipated for the hybridized region(s) of
the hybridizing polynucleotides. When hybridizing a polynucleotide
to a target polynucleotide of unknown sequence, the hybrid length
is assumed to be that of the hybridizing polynucleotide. When
polynucleotides of known sequence are hybridized, the hybrid length
can be determined by aligning the sequences of the polynucleotides
and identifying the region or regions of optimal sequence
complementarity. .sup..differential.SSPE (1 .times. SSPE is 0.15M
NaCl, 10mM NaH.sub.2PO.sub.4, and 1.25mM EDTA, pH 7.4) can be
substituted for SSC (1 .times. SSC is 0.15M NaCl and 15mM sodium
citrate) in the hybridization and wash buffers; washes are
performed for 15 minutes after hybridization is complete. *T.sub.B-
T.sub.R: The hybridization temperature for hybrids anticipated to
be less than 50 base pairs in length should be 5-10.degree. C. less
than the melting temperature (T.sub.m) of the hybrid, where T.sub.m
is determined according to the following equations. For hybrids
less than 18 base pairs in length, T.sub.m(.degree. C.) = 2(# of A
+ T bases) + 4(# of G + C bases). For hybrids between 18 and 49
base pairs in length, # T.sub.m(.degree. C.) = 81.5 +
16.6(log.sub.10[Na.sup.+]) + 0.41 (%G + C) - (600/N), where N is
the number of bases in the hybrid, and [Na.sup.+] is the
concentration of sodium ions in the hybridization buffer
([Na.sup.+] for 1 .times. SSC = 0.165 M).
[0490] 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,
N.Y., 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.
[0491] 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.
[0492] The isolated polynucleotide of the invention may be operably
linked to an expression control sequence such as the pMT2 or pED
expression vectors disclosed in Kaufman et al., Nucleic Acids Res.
19, 4485-4490 (1991), in order to produce the protein
recombinantly. Many suitable expression control sequences are known
in the art. General methods of expressing recombinant proteins are
also known and are exemplified in R. Kaufman, Methods in Enzymology
185, 537-566 (1990). As defined herein "operably 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.
[0493] A number of types of cells may act as suitable host cells
for expression of the protein. Mammalian host cells include, for
example, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human
kidney 293 cells, human epidermal A431 cells, human Colo205 cells,
3T3 cells, CV-1 cells, other transformed primate cell lines, normal
diploid cells, cell strains derived from in vitro culture of
primary tissue, primary explants, HeLa cells, mouse L cells, BHK,
HL-60, U937, HaK or Jurkat cells.
[0494] 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.
[0495] 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, Calif.,
U.S.A. (the MaxBac.RTM. kit), and such methods are well known in
the art, as described in Summers and Smith, Texas Agricultural
Experiment Station Bulletin No. 1555 (1987), incorporated herein by
reference. As used herein, an insect cell capable of expressing a
polynucleotide of the present invention is "transformed."
[0496] 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 containing agents which
will bind to the protein;
[0497] one or more column steps over such affinity resins as
concanavalin A-agarose, heparin-toyopearl.RTM. or Cibacrom blue 3GA
Sepharose.RTM.; one or more steps involving hydrophobic interaction
chromatography using such resins as phenyl ether, butyl ether, or
propyl ether; or immunoaffinity chromatography.
[0498] 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 (MB P), glutathione-S-transferase (GST) or
thioredoxin (TRX). Kits for expression and purification of such
fusion proteins are commercially available from New England BioLab
(Beverly, Mass.), Pharmacia (Piscataway, N.J.) and InVitrogen,
respectively. The protein can also be tagged with an epitope and
subsequently purified by using a specific antibody directed to such
epitope. One such epitope ("Flag") is commercially available from
Kodak (New Haven, Conn.).
[0499] 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."
[0500] The protein of the invention may also be expressed as a
product of transgenic animals, e.g., as a component of the milk of
transgenic cows, goats, pigs, or sheep which are characterized by
somatic or germ cells containing a nucleotide sequence encoding the
protein.
[0501] The protein may also be produced by known conventional
chemical synthesis. Methods for constructing the proteins of the
present invention by synthetic means are known to those skilled in
the art. The synthetically-constructed protein sequences, by virtue
of sharing primary, secondary or tertiary structural and/or
conformational characteristics with proteins may possess biological
properties in common therewith, including protein activity. Thus,
they may be employed as biologically active or immunological
substitutes for natural, purified proteins in screening of
therapeutic compounds and in immunological processes for the
development of antibodies.
[0502] 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,
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. Pat. No. 4,518,584).
Preferably, such alteration, substitution, replacement, insertion
or deletion retains the desired activity of the protein.
[0503] Other fragments and derivatives of the sequences of proteins
which would be expected to retain protein activity in whole or in
part and may thus be useful for screening or other immunological
methodologies may also be easily made by those skilled in the art
given the disclosures herein. Such modifications are believed to be
encompassed by the present invention.
[0504] Uses and Biological Activity
[0505] 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).
[0506] Research uses and Utilities
[0507] The polynucleotides provided by the present invention can be
used by the research community for various purposes. The
polynucleotides can be used to express recombinant protein for
analysis, characterization or therapeutic use; as markers for
tissues in which the corresponding protein is preferentially
expressed (either constitutively or at a particular stage of tissue
differentiation or development or in disease states); as molecular
weight markers on Southern gels; as chromosome markers or tags
(when labeled) to identify chromosomes or to map related gene
positions; to compare with endogenous DNA sequences in patients to
identify potential genetic disorders; as probes to hybridize and
thus discover novel, related DNA sequences; as a source of
information to derive PCR primers for genetic fingerprinting; as a
probe to "subtract-out" known sequences in the process of
discovering other novel polynucleotides; for selecting and making
oligomers for attachment to a "gene chip" or other support,
including for examination of expression patterns; to raise
anti-protein antibodies using DNA immunization techniques; and as
an antigen to raise anti-DNA antibodies or elicit another immune
response. Where the polynucleotide encodes a protein which binds or
potentially binds to another protein (such as, for example, in a
receptor-ligand interaction), the polynucleotide can also be used
in interaction trap assays (such as, for example, that described in
Gyuris et al., Cell 75:791-803 (1993)) to identify polynucleotides
encoding the other protein with which binding occurs or to identify
inhibitors of the binding interaction.
[0508] 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.
[0509] Any or all of these research utilities are capable of being
developed into reagent grade or kit format for commercialization as
research products.
[0510] Methods for performing the uses listed above are well known
to those skilled in the art.
[0511] References disclosing such methods include without
limitation "Molecular Cloning: A Laboratory Manual", 2d ed., Cold
Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T.
Maniatis eds., 1989, and "Methods in Enzymology: Guide to Molecular
Cloning Techniques", Academic Press, Berger, S. L. and A. R. Kimmel
eds., 1987.
[0512] Nutritional uses
[0513] Polynucleotides and proteins of the present invention can
also be used as nutritional sources or supplements. Such uses
include without limitation use as a protein or amino acid
supplement, use as a carbon source, use as a nitrogen source and
use as a source of carbohydrate. In such cases the protein or
polynucleotide of the invention can be added to the feed of a
particular organism or can be administered as a separate solid or
liquid preparation, such as in the form of powder, pills,
solutions, suspensions or capsules. In the case of microorganisms,
the protein or polynucleotide of the invention can be added to the
medium in or on which the microorganism is cultured.
[0514] Cytokine and Cell Proliferation/Differentiation Activity
[0515] A protein of the present invention may exhibit cytokine,
cell proliferation (either inducing or inhibiting) or cell
differentiation (either inducing or inhibiting) activity or may
induce production of other cytokines in certain cell populations.
Many protein factors discovered to date, including all known
cytokines, have exhibited activity in one or more factor dependent
cell proliferation assays, and hence the assays serve as a
convenient confirmation of cytokine activity. The activity of a
protein of the present invention is evidenced by any one of a
number of routine factor dependent cell proliferation assays for
cell lines including, without limitation, 32D, DA2, DA1G, T10, B9,
B9/11, BaF3, MC9/G, M+(preB M+), 2E8, RB5, DA1, 123, T1165, HT2,
CTLL2, TF-1, Mo7e and CMK.
[0516] The activity of a protein of the invention may, among other
means, be measured by the following methods:
[0517] 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.
[0518] Assays for cytokine production and/or proliferation of
spleen cells, lymph node cells or thymocytes include, without
limitation, those described in: Polyclonal T cell stimulation,
Kruisbeek, A. M. and Shevach, E. M. In Current Protocols in
Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John
Wiley and Sons, Toronto. 1994; and Measurement of mouse and human
Interferon .gamma., 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.
[0519] 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
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 in Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 6.15.1
John Wiley and Sons, Toronto. 1991; Measurement of mouse and human
Interleukin 9--Ciarletta, A., Giannotti, J., Clark, S. C. and
Turner, K. J. In Current Protocols in Immunology. J. E. e.a.
Coligan eds. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto.
1991.
[0520] 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.
[0521] Immune Stimulating or Suppressing Activity
[0522] A protein of the present invention may also exhibit immune
stimulating or immune suppressing activity, including without
limitation the activities for which assays are described herein. A
protein may be useful in the treatment of various immune
deficiencies and disorders (including severe combined
immunodeficiency (SCID)), e.g., in regulating (up or down) growth
and proliferation of T and/or B lymphocytes, as well as effecting
the cytolytic activity of NK cells and other cell populations.
These immune deficiencies may be genetic or be caused by viral
(e.g., HIV) as well as bacterial or fungal infections, or may
result from autoimmune disorders. More specifically, infectious
diseases causes by viral, bacterial, fungal or other infection may
be treatable using a protein of the present invention, including
infections by HIV, hepatitis viruses, herpesviruses, mycobacteria,
Leishmania spp., malaria spp. and various fungal infections such as
candidiasis. Of course, in this regard, a protein of the present
invention may also be useful where a boost to the immune system
generally may be desirable, i.e., in the treatment of cancer.
[0523] Autoimmune disorders which may be treated using a protein of
the present invention include, for example, connective tissue
disease, multiple sclerosis, systemic lupus erythematosus,
rheumatoid arthritis, autoimmune pulmonary inflammation,
Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent
diabetes mellitis, myasthenia gravis, graft-versus-host disease and
autoimmune inflammatory eye disease. Such a protein of the present
invention may also to be useful in the treatment of allergic
reactions and conditions, such as asthma (particularly allergic
asthma) or other respiratory problems. Other conditions, in which
immune suppression is desired (including, for example, organ
transplantation), may also be treatable using a protein of the
present invention.
[0524] Using the proteins of the invention it may also be possible
to immune responses, in a number of ways. Down regulation may be in
the form of inhibiting or blocking an immune response already in
progress or may involve preventing the induction of an immune
response. The functions of activated T cells may be inhibited by
suppressing T cell responses or by inducing specific tolerance in T
cells, or both. Immunosuppression of T cell responses is generally
an active, non-antigen-specific, process which requires continuous
exposure of the T cells to the suppressive agent. Tolerance, which
involves inducing non-responsiveness or anergy in T cells, is
distinguishable from immunosuppression in that it is generally
antigen-specific and persists after exposure to the tolerizing
agent has ceased. Operationally, tolerance can be demonstrated by
the lack of a T cell response upon reexposure to specific antigen
in the absence of the tolerizing agent.
[0525] Down regulating or preventing one or more antigen functions
(including without limitation B lymphocyte antigen functions (such
as, for example, B7)), e.g., preventing high level lymphokine
synthesis by activated T cells, will be useful in situations of
tissue, skin and organ transplantation and in graft-versus-host
disease (GVHD). For example, blockage of T cell function should
result in reduced tissue destruction in tissue transplantation.
Typically, in tissue transplants, rejection of the transplant is
initiated through its recognition as foreign by T cells, followed
by an immune reaction that destroys the transplant. The
administration of a molecule which inhibits or blocks interaction
of a B7 lymphocyte antigen with its natural ligand(s) on immune
cells (such as a soluble, monomeric form of a peptide having B7-2
activity alone or in conjunction with a monomeric form of a peptide
having an activity of another B lymphocyte antigen (e.g., B7-1,
B7-3) or blocking antibody), prior to transplantation can lead to
the binding of the molecule to the natural ligand(s) on the immune
cells without transmitting the corresponding costimulatory signal.
Blocking B lymphocyte antigen function in this matter prevents
cytokine synthesis by immune cells, such as T cells, and thus acts
as an 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.
[0526] The efficacy of particular blocking reagents in preventing
organ transplant rejection or GVHD can be assessed using animal
models that are predictive of efficacy in humans. Examples of
appropriate systems which can be used include allogeneic cardiac
grafts in rats and xenogeneic pancreatic islet cell grafts in mice,
both of which have been used to examine the immunosuppressive
effects of CTLA4Ig fusion proteins in vivo as described in Lenschow
et al., Science 257:789-792 (1992) and Turka et al., Proc. Natl.
Acad. Sci USA, 89:11102-11105 (1992). In addition, murine models of
GVHD (see Paul ed., Fundamental Immunology, Raven Press, New York,
1989, pp. 846-847) can be used to determine the effect of blocking
B lymphocyte antigen function in vivo on the development of that
disease.
[0527] Blocking antigen function may also be therapeutically useful
for treating autoimmune diseases. Many autoimmune disorders are the
result of inappropriate activation of T cells that are reactive
against self tissue and which promote the production of cytokines
and autoantibodies involved in the pathology of the diseases.
Preventing the activation of autoreactive T cells may reduce or
eliminate disease symptoms. Administration of reagents which block
costimulation of T cells by disrupting receptor:ligand interactions
of B lymphocyte antigens can be used to inhibit T cell activation
and prevent production of autoantibodies or T cell-derived
cytokines which may be involved in the disease process.
Additionally, blocking reagents may induce antigen-specific
tolerance of autoreactive T cells which could lead to long-term
relief from the disease. The efficacy of blocking reagents in
preventing or alleviating autoimmune disorders can be determined
using a number of well-characterized animal models of human
autoimmune diseases. Examples include murine experimental
autoimmune encephalitis, systemic lupus erythmatosis in MRL/lpr/lpr
mice or NZB hybrid mice, murine autoimmune collagen arthritis,
diabetes mellitus in NOD mice and BB rats, and murine experimental
myasthenia gravis (see Paul ed., Fundamental Immunology, Raven
Press, New York, 1989, pp. 840-856).
[0528] 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.
[0529] 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.
[0530] In another application, up regulation or enhancement of
antigen function (preferably B lymphocyte antigen function) may be
useful in the induction of tumor immunity. Tumor cells (e.g.,
sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma)
transfected with a nucleic acid encoding at least one peptide of
the present invention can be administered to a subject to overcome
tumor-specific tolerance in the subject. If desired, the tumor cell
can be transfected to express a combination of peptides. For
example, tumor cells obtained from a patient can be transfected ex
vivo with an expression vector directing the expression of a
peptide having B7-2-like activity alone, or in conjunction with a
peptide having B7-1-like activity and/or B7-3-like activity. The
transfected tumor cells are returned to the patient to result in
expression of the peptides on the surface of the transfected cell.
Alternatively, gene therapy techniques can be used to target a
tumor cell for transfection in vivo.
[0531] The presence of the peptide of the present invention having
the activity of a B lymphocyte antigen(s) on the surface of the
tumor cell provides the necessary 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 .alpha.
chain protein and .beta..sub.2 microglobulin protein or an MHC
class II .alpha. chain protein and an MHC class II .beta. 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.
[0532] The activity of a protein of the invention may, among other
means, be measured by the following methods:
[0533] Suitable assays for thymocyte or splenocyte cytotoxicity
include, without limitation, those described in: Current Protocols
in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H.
Margulies, E. M. Shevach, W Strober, Pub. Greene Publishing
Associates and Wiley-Interscience (Chapter 3, In Vitro assays for
Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies
in Humans); Herrmann et al., Proc. Natl. Acad. Sci. USA
78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974,
1982; Handaet 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 al., J. Immunol. 135:1564-1572, 1985; Takai et al.,
J. Immunol. 137:3494-3500, 1986; Bowmanet al., J. Virology
61:1992-1998; Takai et al., J. Immunol. 140:508-512, 1988;
Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Brown et
al., J. Immunol. 153:3079-3092, 1994.
[0534] Assays for T-cell-dependent immunoglobulin responses and
isotype switching (which will identify, among others, proteins that
modulate T-cell dependent antibody responses and that affect
Th1/Th2 profiles) include, without limitation, those described in:
Maliszewski, J. Immunol. 144:3028-3033, 1990; and Assays for B cell
function: In vitro antibody production, Mond, J. J. and Brunswick,
M. In Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol
1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. 1994.
[0535] Mixed lymphocyte reaction (MLR) assays (which will identify,
among others, proteins that generate predominantly Th1 and CTL
responses) include, without limitation, those described in:
[0536] 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; Takai et al., J. Immunol. 140:508-512, 1988;
Bertagnolli et al., J. Immunol. 149:3778-3783, 1992.
[0537] Dendritic cell-dependent assays (which will identify, among
others, proteins expressed by dendritic cells that activate naive
T-cells) include, without limitation, those described in: Guery et
al., J. Immunol. 134:536-544, 1995; Inaba et al., Journal of
Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal
of Immunology 154:5071-5079, 1995; Porgador et al., Journal of
Experimental Medicine 182:255-260, 1995; Nair et al., Journal of
Virology 67:4062-4069, 1993; Huang et al., Science 264:961-965,
1994; Macatonia et al., Journal of Experimental Medicine
169:1255-1264, 1989; Bhardwaj et al., Journal of Clinical
Investigation 94:797-807, 1994; and Inaba et al., Journal of
Experimental Medicine 172:631-640, 1990.
[0538] Assays for lymphocyte survival/apoptosis (which will
identify, among others, proteins that prevent apoptosis after
superantigen induction and proteins that regulate lymphocyte
homeostasis) include, without limitation, those described in:
Darzynkiewicz et al., Cytometry 13:795-808, 1992; Gorczyca et al.,
Leukemia 7:659-670, 1993; Gorczyca et al., Cancer Research
53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991; Zacharchuk,
Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry
14:891-897, 1993; Gorczyca et al., International Journal of
Oncology 1:639-648, 1992.
[0539] Assays for proteins that influence early steps of T-cell
commitment and development include, without limitation, those
described in: Antica et al., Blood 84:111-117, 1994; Fine et al.,
Cellular Immunology 155:111-122, 1994; Galy et al., Blood
85:2770-2778, 1995; Toki et al., Proc. Nat. Acad Sci. USA
88:7548-7551, 1991.
[0540] Hematopoiesis Regulating Activity
[0541] A protein of the present invention may be useful in
regulation of hematopoiesis and, consequently, in the treatment of
myeloid or lymphoid cell deficiencies. Even marginal biological
activity in support of colony forming cells or of factor-dependent
cell lines indicates involvement in regulating hematopoiesis, e.g.
in supporting the growth and proliferation of erythroid progenitor
cells alone or in combination with other cytokines, thereby
indicating utility, for example, in treating various anemias or for
use in conjunction with irradiation/chemotherapy to stimulate the
production of erythroid precursors and/or erythroid cells; in
supporting the growth and proliferation of myeloid cells such as
granulocytes and monocytes/macrophages (i.e., traditional CSF
activity) useful, for example, in conjunction with chemotherapy to
prevent or treat consequent myelo-suppression; in supporting the
growth and proliferation of megakaryocytes and consequently of
platelets thereby allowing prevention or treatment of various
platelet disorders such as thrombocytopenia, and generally for use
in place of or complimentary to platelet transfusions; and/or in
supporting the growth and proliferation of hematopoietic stem cells
which are capable of maturing to any and all of the above-mentioned
hematopoietic cells and therefore find therapeutic utility in
various stem cell disorders (such as those usually treated with
transplantation, including, without limitation, aplastic anemia and
paroxysmal nocturnal hemoglobinuria), as well as in repopulating
the stem cell compartment post irradiation/chemotherapy, either
in-vivo or ex-vivo (i.e., in conjunction with bone marrow
transplantation or with peripheral progenitor cell transplantation
(homologous or heterologous)) as normal cells or genetically
manipulated for gene therapy.
[0542] The activity of a protein of the invention may, among other
means, be measured by the following methods:
[0543] Suitable assays for proliferation and differentiation of
various hematopoietic lines are cited above.
[0544] 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.
[0545] 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. I. Freshney, et al. eds. Vol pp.
265-268, Wiley-Liss, Inc., New York, N.Y. 1994; Hirayama et al.,
Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992; Primitive
hematopoietic colony forming cells with high proliferative
potential, McNiece, I. K. and Briddell, R. A. In Culture of
Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 23-39,
Wiley-Liss, Inc., New York, N.Y. 1994; Neben et al., Experimental
Hematology 22:353-359, 1994; Cobblestone area forming cell assay,
Ploemacher, R. E. In Culture of Hematopoietic Cells. R. I.
Freshney, et al. eds. Vol pp. 1-21, Wiley-Liss, Inc., New York,
N.Y. 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, N.Y. 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, N.Y. 1994.
[0546] Tissue Growth Activity
[0547] 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.
[0548] A protein of the present invention, which induces cartilage
and/or bone growth in circumstances where bone is not normally
formed, has application in the healing of bone fractures and
cartilage damage or defects in humans and other animals. Such a
preparation employing a protein of the invention may have
prophylactic use in closed as well as open fracture reduction and
also in the improved fixation of artificial joints. De novo bone
formation induced by an osteogenic agent contributes to the repair
of congenital, trauma induced, or oncologic resection induced
craniofacial defects, and also is useful in cosmetic plastic
surgery.
[0549] 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.
[0550] Another category of tissue regeneration activity that may be
attributable to the protein of the present invention is
tendon/ligament formation. A protein of the present invention,
which induces tendon/ligament-like tissue or other tissue formation
in circumstances where such tissue is not normally formed, has
application in the healing of tendon or ligament tears, deformities
and other tendon or ligament defects in humans and other animals.
Such a preparation employing a tendon/ligament-like tissue inducing
protein may have prophylactic use in preventing damage to tendon or
ligament tissue, as well as use in the improved fixation of tendon
or ligament to bone or other tissues, and in repairing defects to
tendon or ligament tissue. De novo tendon/ligament-like tissue
formation induced by a composition of the present invention
contributes to the repair of congenital, trauma induced, or other
tendon or ligament defects of other origin, and is also useful in
cosmetic plastic surgery for attachment or repair of tendons or
ligaments. The compositions of the present invention may provide an
environment to attract tendon- or ligament-forming cells, stimulate
growth of tendon- or ligament-forming cells, induce differentiation
of progenitors of tendon- or ligament-forming cells, or induce
growth of tendon/ligament cells or progenitors ex vivo for return
in vivo to effect tissue repair. The compositions of the invention
may also be useful in the treatment of tendinitis, carpal tunnel
syndrome and other tendon or ligament defects. The compositions may
also include an appropriate matrix and/or sequestering agent as a
carrier as is well known in the art.
[0551] The protein of the present invention may also be useful for
proliferation of neural cells and for regeneration of nerve and
brain tissue, i.e. for the treatment of central and peripheral
nervous system diseases and neuropathies, as well as mechanical and
traumatic disorders, which involve degeneration, death or trauma to
neural cells or nerve tissue. More specifically, a protein may be
used in the treatment of diseases of the peripheral nervous system,
such as peripheral nerve injuries, peripheral neuropathy and
localized neuropathies, and central nervous system diseases, such
as Alzheimer's, Parkinson's disease, Huntington's disease,
amyotrophic lateral sclerosis, and Shy-Drager syndrome. Further
conditions which may be treated in accordance with the present
invention include mechanical and traumatic disorders, such as
spinal cord disorders, head trauma and cerebrovascular diseases
such as stroke. Peripheral neuropathies resulting from chemotherapy
or other medical therapies may also be treatable using a protein of
the invention.
[0552] 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.
[0553] It is expected that a protein of the present invention may
also exhibit activity for generation or regeneration of other
tissues, such as organs (including, for example, pancreas, liver,
intestine, kidney, skin, endothelium), muscle (smooth, skeletal or
cardiac) and vascular (including vascular endothelium) tissue, or
for promoting the growth of cells comprising such tissues. Part of
the desired effects may be by inhibition or modulation of fibrotic
scarring to allow normal tissue to regenerate. A protein of the
invention may also exhibit angiogenic activity.
[0554] 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.
[0555] 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.
[0556] The activity of a protein of the invention may, among other
means, be measured by the following methods:
[0557] Assays for tissue generation activity include, without
limitation, those described in:
[0558] International Patent Publication No. WO95/16035 (bone,
cartilage, tendon); International Patent Publication No. WO95/05846
(nerve, neuronal); International Patent Publication No. WO91/07491
(skin, endothelium).
[0559] Assays for wound healing activity include, without
limitation, those described in: Winter, Epidermal Wound Healing,
pps. 71-112 (Maibach, H I and Rovee, D T, eds.), Year Book Medical
Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J.
Invest. Dermatol 71:382-84 (1978).
[0560] Activin/Inhibin Activity
[0561] 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 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-.beta. 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, U.S. Pat. No. 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.
[0562] The activity of a protein of the invention may, among other
means, be measured by the following methods:
[0563] 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.
[0564] Chemotactic/Chemokinetic Activity
[0565] 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.
[0566] A protein or peptide has chemotactic activity for a
particular cell population if it can stimulate, directly or
indirectly, the directed orientation or movement of such cell
population. Preferably, the protein or peptide has the ability to
directly stimulate directed movement of cells. Whether a particular
protein has chemotactic activity for a population of cells can be
readily determined by employing such protein or peptide in any
known assay for cell chemotaxis.
[0567] The activity of a protein of the invention may, among other
means, be measured by the following methods:
[0568] 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 include, without
limitation, those described in: Current Protocols in Immunology, Ed
by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach,
W. Strober, Pub. Greene Publishing Associates and
Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta
Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest.
95:1370-1376, 1995; Lind et al. APMIS 103:140-146, 1995; Muller et
al Eur. J. Immunol. 25: 1744-1748; Gruber et al. J. of Immunol.
152:5860-5867, 1994; Johnston et al. J. of Immunol. 153: 1762-1768,
1994.
[0569] Hemostatic and Thrombolytic Activity
[0570] 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).
[0571] The activity of a protein of the invention may, among other
means, be measured by the following methods:
[0572] 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.
45:413419, 1987; Humphrey et al., Fibrinolysis 5:71-79 (1991);
Schaub, Prostaglandins 35:467-474, 1988.
[0573] Receptor/Ligand Activity
[0574] A protein of the present invention may also demonstrate
activity as receptors, receptor ligands or inhibitors or agonists
of receptor/ligand interactions. Examples of such receptors and
ligands include, without limitation, cytokine receptors and their
ligands, receptor kinases and their ligands, receptor phosphatases
and their ligands, receptors involved in cell-cell interactions and
their ligands (including without limitation, cellular adhesion
molecules (such as selecting, integrins and their ligands) and
receptor/ligand pairs involved in antigen presentation, antigen
recognition and development of cellular and humoral immune
responses). Receptors and ligands are also useful for screening of
potential peptide or small molecule inhibitors of the relevant
receptor/ligand interaction. A protein of the present invention
(including, without limitation, fragments of receptors and ligands)
may themselves be useful as inhibitors of receptor/ligand
interactions.
[0575] The activity of a protein of the invention may, among other
means, be measured by the following methods:
[0576] 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.
[0577] Anti-Inflammatory Activity
[0578] Proteins of the present invention may also exhibit
anti-inflammatory activity. The anti-inflammatory activity may be
achieved by providing a stimulus to cells involved in the
inflammatory response, by inhibiting or promoting cell-cell
interactions (such as, for example, cell adhesion), by inhibiting
or promoting chemotaxis of cells involved in the inflammatory
process, inhibiting or promoting cell extravasation, or by
stimulating or suppressing production of other factors which more
directly inhibit or promote an inflammatory response. Proteins
exhibiting such activities can be used to treat inflammatory
conditions including chronic or acute conditions), including
without limitation inflammation associated with infection (such as
septic shock, sepsis or systemic inflammatory response syndrome
(SIRS)), ischemia-reperfusion injury, endotoxin lethality,
arthritis, complement-mediated hyperacute rejection, nephritis,
cytokine or chemokine-induced lung injury, inflammatory bowel
disease, Crohn's disease or resulting 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.
[0579] Cadherin/Tumor Invasion Suppressor Activity
[0580] Cadherins are calcium-dependent adhesion molecules that
appear to play major roles during development, particularly in
defining specific cell types. Loss or alteration of normal cadherin
expression can lead to changes in cell adhesion properties linked
to tumor growth and metastasis. Cadherin malfunction is also
implicated in other human diseases, such as pemphigus vulgaris and
pemphigus foliaceus (auto-immune blistering skin diseases), Crohn's
disease, and some developmental abnormalities.
[0581] 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.
[0582] E-cadherin, one member of the cadherin superfamily, is
expressed in epithelial cell types. Pathologically, if E-cadherin
expression is lost in a tumor, the malignant cells become invasive
and the cancer metastasizes. Transfection of cancer cell lines with
polynucleotides expressing E-cadherin has reversed
cancer-associated changes by returning altered cell shapes to
normal, restoring cells' adhesiveness to each other and to their
substrate, decreasing the cell growth rate, and drastically
reducing anchorage-independent cell growth. Thus, reintroducing
E-cadherin expression reverts carcinomas to a less advanced stage.
It is likely that other cadherins have the same invasion suppressor
role in carcinomas derived from other tissue types. Therefore,
proteins of the present invention with cadherin activity, and
polynucleotides of the present invention encoding such proteins,
can be used to treat cancer. Introducing such proteins or
polynucleotides into cancer cells can reduce or eliminate the
cancerous changes observed in these cells by providing normal
cadherin expression.
[0583] Cancer cells have also been shown to express cadherins of a
different tissue type than their origin, thus allowing these cells
to invade and metastasize in a different tissue in the body.
Proteins of the present invention with cadherin activity, and
polynucleotides of the present invention encoding such proteins,
can be substituted in these cells for the inappropriately expressed
cadherins, restoring normal cell adhesive properties and reducing
or eliminating the tendency of the cells to metastasize.
[0584] Additionally, proteins of the present invention with
cadherin activity, and polynucleotides of the present invention
encoding such proteins, can used to generate antibodies recognizing
and binding to cadherins. Such antibodies can be used to block the
adhesion of inappropriately expressed tumor-cell cadherins,
preventing the cells from 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.
[0585] 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.
[0586] 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.
[0587] Tumor Inhibition Activity
[0588] In addition to the activities described above for
immunological treatment or prevention of tumors, a protein of the
invention may exhibit other anti-tumor activities. A protein may
inhibit tumor growth directly or indirectly (such as, for example,
via ADCC). A protein may exhibit its tumor inhibitory activity by
acting on tumor tissue or tumor precursor tissue, by inhibiting
formation of tissues necessary to support tumor growth (such as,
for example, by inhibiting angiogenesis), by causing production of
other factors, agents or cell types which inhibit tumor growth, or
by suppressing, eliminating or inhibiting factors, agents or cell
types which promote tumor growth.
[0589] Other Activities
[0590] 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 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.
[0591] Administration and Dosing
[0592] A protein of the present invention (from whatever source
derived, including without limitation from recombinant and
non-recombinant sources) may be used in a pharmaceutical
composition when combined with a pharmaceutically acceptable
carrier. Such a composition may also contain (in addition to
protein and a carrier) diluents, fillers, salts, buffers,
stabilizers, solubilizers, and other materials well known in the
art. The term "pharmaceutically acceptable" means a non-toxic
material that does not interfere with the effectiveness of the
biological activity of the active ingredient(s). The
characteristics of the carrier will depend on the route of
administration. The pharmaceutical composition of the invention may
also contain cytokines, lymphokines, or other hematopoietic factors
such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL4, IL-5, IL-6,
IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IFN,
TNF0, TNF1, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor,
and erythropoietin. The pharmaceutical composition may further
contain other agents which either enhance the activity of the
protein or compliment its activity or use in treatment. Such
additional factors and/or agents may be included in the
pharmaceutical composition to produce a synergistic effect with
protein of the invention, or to minimize side effects. Conversely,
protein of the present invention may be included in formulations of
the particular cytokine, lymphokine, other hematopoietic factor,
thrombolytic or anti-thrombotic factor, or anti-inflammatory agent
to minimize side effects of the cytokine, lymphokine, other
hematopoietic factor, thrombolytic or anti-thrombotic factor, or
anti-inflammatory agent.
[0593] A protein of the present invention may be active in
multimers (e.g., heterodimers or homodimers) or complexes with
itself or other proteins. As a result, pharmaceutical compositions
of the invention may comprise a protein of the invention in such
multimeric or complexed form.
[0594] The pharmaceutical composition of the invention may be in
the form of a complex of the protein(s) of present invention along
with protein or peptide antigens. The protein and/or peptide
antigen will deliver a stimulatory signal to both B and T
lymphocytes. B lymphocytes will respond to antigen through their
surface immunoglobulin receptor. T lymphocytes will respond to
antigen through the T cell receptor (TCR) following presentation of
the antigen by MHC proteins. MHC and structurally related proteins
including those encoded by class I and class II MHC genes on host
cells will serve to present the peptide antigen(s) 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.
[0595] The pharmaceutical composition of the invention may be in
the form of a liposome in which protein of the present invention is
combined, in addition to other pharmaceutically acceptable
carriers, with amphipathic agents such as lipids which exist in
aggregated form as micelles, insoluble monolayers, liquid crystals,
or lamellar layers in aqueous solution. Suitable lipids for
liposomal formulation include, without limitation, monoglycerides,
diglycerides, sulfatides, lysolecithin, phospholipids, saponin,
bile acids, and the like. Preparation of such liposomal
formulations is within the level of skill in the art, as disclosed,
for example, in U.S. Pat. No. 4,235,871; U.S. Pat. No. 4,501,728;
U.S. Pat. No. 4,837,028; and U.S. Pat. No. 4,737,323, all of which
are incorporated herein by reference.
[0596] As used herein, the term "therapeutically effective amount"
means the total amount of each active component of the
pharmaceutical composition or method that is sufficient to show a
meaningful patient benefit, i.e., treatment, healing, prevention or
amelioration of the relevant medical condition, or an increase in
rate of treatment, healing, prevention or amelioration of such
conditions. When applied to an individual active 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.
[0597] In practicing the method of treatment or use of the present
invention, a therapeutically effective amount of protein of the
present invention is administered to a mammal having a condition to
be treated. Protein of the present invention may be administered in
accordance with the method of the invention either alone or in
combination with other therapies such as treatments employing
cytokines, lymphokines or other hematopoietic factors. When
co-administered with one or more cytokines, lymphokines or other
hematopoietic factors, protein of the present invention may be
administered either simultaneously with the cytokine(s),
lymphokine(s), other hematopoietic factor(s), thrombolytic or
anti-thrombotic factors, or sequentially. If administered
sequentially, the attending physician will decide on the
appropriate sequence of administering protein of the present
invention in combination with cytokine(s), lymphokine(s), other
hematopoietic factor(s), thrombolytic or anti-thrombotic
factors.
[0598] Administration of protein of the present invention used in
the pharmaceutical composition or to practice the method of the
present invention can be carried out in a variety of conventional
ways, such as oral ingestion, inhalation, topical application or
cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous
injection. Intravenous administration to the patient is
preferred.
[0599] When a therapeutically effective amount of protein of the
present invention is administered orally, protein of the present
invention will be in the form of a tablet, capsule, powder,
solution or elixir. When administered in tablet form, the
pharmaceutical composition of the invention may additionally
contain a solid carrier such as a gelatin or an adjuvant. The
tablet, capsule, and powder contain from about 5 to 95% protein of
the present invention, and preferably from about 25 to 90% protein
of the present invention. When administered in liquid form, a
liquid carrier such as water, petroleum, oils of animal or plant
origin such as peanut oil, mineral oil, soybean oil, or sesame oil,
or synthetic oils may be added. The liquid form of the
pharmaceutical composition may further contain 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.
[0600] When a therapeutically effective amount of protein of the
present invention is administered by intravenous, cutaneous or
subcutaneous injection, protein of the present invention will be in
the form of a pyrogen-free, parenterally acceptable aqueous
solution. The preparation of such parenterally acceptable protein
solutions, having due regard to pH, isotonicity, stability, and the
like, is within the skill in the art. A preferred pharmaceutical
composition for intravenous, cutaneous, or subcutaneous injection
should contain, in addition to protein of the present invention, an
isotonic vehicle such as Sodium Chloride Injection, Ringer's
Injection, Dextrose Injection, Dextrose and Sodium Chloride
Injection, Lactated Ringer's Injection, or other vehicle as known
in the art. The pharmaceutical composition of the present invention
may also contain stabilizers, preservatives, buffers, antioxidants,
or other additives known to those of skill in the art.
[0601] The amount of protein of the present invention in the
pharmaceutical composition of the present invention will depend
upon the nature and severity of the condition being treated, and on
the nature of prior treatments which the patient has undergone.
Ultimately, the attending physician will decide the amount of
protein of the present invention with which to treat each
individual patient. Initially, the attending physician will
administer low doses of protein of the present invention and
observe the patient's response. Larger doses of protein of the
present invention may be administered until the optimal therapeutic
effect is obtained for the patient, and at that point the dosage is
not increased further. It is contemplated that the various
pharmaceutical compositions used to practice the method of the
present invention should contain about 0.01 .mu.g to about 100 mg
(preferably about 0.1 ng to about 10 mg, more preferably about 0.1
.mu.g to about 1 mg) of protein of the present invention per kg
body weight.
[0602] The duration of intravenous therapy using the pharmaceutical
composition of the present invention will vary, depending on the
severity of the disease being treated and the condition and
potential idiosyncratic response of each individual patient. It is
contemplated that the duration of each application of the protein
of the present invention will be in the range of 12 to 24 hours of
continuous intravenous administration. Ultimately the attending
physician will decide on the appropriate duration of intravenous
therapy using the pharmaceutical composition of the present
invention.
[0603] 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 carboxyl terminus, and are conjugated to a
hapten such as keyhole limpet hemocyanin (KLH). Methods for
synthesizing such peptides are known in the art, for example, as in
R. P. Merrifield, J. Amer.Chem.Soc. 85, 2149-2154 (1963); J. L.
Krstenansky, et al., FEBS Lett. 211, 10 (1987). Monoclonal
antibodies binding to the protein of the invention may be useful
diagnostic agents for the immunodetection of the protein.
Neutralizing monoclonal antibodies binding to the protein may also
be useful therapeutics for both conditions associated with the
protein and also in the treatment of some forms of cancer where
abnormal expression of the protein is involved. In the case of
cancerous cells or leukemic cells, neutralizing monoclonal
antibodies against the protein may be useful in detecting and
preventing the metastatic spread of the cancerous cells, which may
be mediated by the protein.
[0604] For compositions of the present invention which are useful
for bone, cartilage, tendon or ligament regeneration, the
therapeutic method includes administering the composition
topically, systematically, or locally as an implant or device. When
administered, the therapeutic composition for use in this invention
is, of course, in a pyrogen-free, physiologically acceptable form.
Further, the composition may desirably be encapsulated or injected
in a viscous form for delivery to the site of bone, cartilage or
tissue damage. Topical administration may be suitable for wound
healing and tissue repair. Therapeutically useful agents other than
a protein of the invention which may also optionally be included in
the composition as described above, may alternatively or
additionally, be administered simultaneously or sequentially with
the composition in the methods of the invention. Preferably for
bone and/or cartilage formation, the composition would include a
matrix capable of delivering the protein-containing composition to
the site of bone and/or cartilage damage, providing a structure for
the developing bone and cartilage and optimally capable of being
resorbed into the body. Such matrices may be formed of materials
presently in use for other implanted medical applications.
[0605] The choice of matrix material is based on biocompatibility,
biodegradability, mechanical properties, cosmetic appearance and
interface properties. The particular application of the
compositions will define the appropriate formulation. Potential
matrices for the compositions may be biodegradable and chemically
defined calcium sulfate, tricalciumphosphate, hydroxyapatite,
polylactic acid, polyglycolic acid and 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, alurninates, 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.
[0606] 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.
[0607] 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, poly(ethylene glycol), polyoxyethylene oxide,
carboxyvinyl polymer and poly(vinyl alcohol). The amount of
sequestering agent useful herein is 0.5-20 wt %, preferably 1-10 wt
% based on total formulation weight, which represents the amount
necessary to prevent desorbtion of the protein from the polymer
matrix and to provide appropriate handling of the composition, yet
not so much that the progenitor cells are prevented from
infiltrating the matrix, thereby providing the protein the
opportunity to assist the osteogenic activity of the progenitor
cells.
[0608] 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-.alpha. and TGF-.beta. ), and insulin-like growth
factor (IGF).
[0609] 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.
[0610] 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.
[0611] 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).
[0612] 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.
EXAMPLE 1
Characterization of AJ172.sub.--2 DNA and Protein
[0613] AJ172.sub.--2, a novel human cDNA isolated using a yeast
signal sequence trap, encodes a protein that exhibits significant
homology to a baboon endogenous retrovirus envelope protein.
Genomic DNA sequences flanking the AJ172.sub.--2 gene reveal that
it is part of a previously uncharacterized defective provirus,
indicating that the sequence may be an example of a retroviral gene
that has been "captured" by the human host. Southern blot analyses
show that AJ172.sub.--2-hybridizing sequences are restricted to
humans and monkeys; being absent from cows, dogs, rats, mice,
rabbits, chickens and yeast. Northern blot analyses demonstrate
that although the AJ172.sub.--2 gene is very highly transcribed in
the human placenta and weakly transcribed in the testes, it is not
expressed at all in 21 additional human tissues. In situ antisense
RNA hybridizations performed on full-term human placental sections
revealed that AJ172.sub.--2 transcripts are specifically localized
to villous syncytiotrophoblasts, a fused, multinucleated cell type
derived from fetal trophoblast tissue. We find that AJ172.sub.--2
expression in COS cells reproducibly causes the formation of giant
multinucleated COS-cell syncytia which closely resemble these fused
placental syncytiotrophoblasts, suggesting that AJ172.sub.--2 plays
a role in mediating cell fusion events in human placenta and fusion
of other types of cells expressing AJ172.sub.--2. A number of
independent binary reporter gene approaches indicate that
AJ172.sub.--2-induced COS syncytia are truly derived from cell-cell
fusions and do not result from incomplete cell divisions.
AJ172.sub.--2 can mediate fusion of many different cell types,
including human, monkey, rodent and insect lines. Moreover even
simple liposomes can fuse readily to AJ172.sub.--2-transfected COS
cells, suggesting that AJ172.sub.--2 requires neither homophilic
nor heterophilic protein-protein interactions to promote membrane
fusion events.
[0614] We have also found that co-transfection of AJ172.sub.--2
with IL-11 or IL-12 into COS cells reproducibly leads to 2-5 fold
increases in secreted cytokine yields. This phenomenon can be used
in applications for AJ172.sub.--2 in enhancing mammalian cell
recombinant gene expression.
[0615] The full-length AJ172.sub.--2 cDNA encodes a typical signal
sequence, an extracellular domain, a transmembrane domain and a
short cytoplasmic tail. The recent appearance in public databases
of many very closely related EST sequences derived from many
independent cDNA libraries provides additional supporting evidence
for the expression of AJ172.sub.--2 in vivo. We also find that the
DNA sequence of a segment of human chromosome 7 (genbank accession
#AC000064) carries the entire AJ172.sub.--2 sequence. A closer
examination of this chromosomal segment reveals the presence of a
complete, previously uncharacterized, defective retrovirus. This
provirus exhibits a typical morphology; 5' and 3'-long terminal
repeat sequences, putative gag and pol genes, and a third open
reading frame encoding a putative viral envelope protein. The gag
and pol genes are punctuated by multiple chain termination codons
and are defective, whereas the third, envelope ORF is intact. This
third ORF is identical to AJ172.sub.--2, which we conclude to be
the envelope gene of an ancient retrovirus.
[0616] We have demonstrated that AJ172.sub.--2 is expressed highly
and very specifically in human placental syncytiotrophoblasts. By
means of in vitro transfection experiments we go on to show that
AJ172.sub.--2, like many previously described viral envelope
proteins, can mediate cell to cell fusion events leading to the
formation of giant syncytia. In a series of further experiments we
demonstrate that neither homophilic nor heterophilic
protein-protein interactions are required for AJ172.sub.--2
function, indeed the molecule can mediate efficient cell fusion to
simple liposomes. We suspect that AJ172.sub.--2 may play a critical
role in the normal placental biology of humans and primates,
mediating cell fusion events which may be important in processes
such as blastocyst implantation, the control of uterine wall
infiltration by fetal trophoblasts, and in optimizing the
efficiency of placental transporter and secretory function.
AJ172.sub.--2 may thus be the first described example of a captured
viral gene performing an important biological role in a mammalian
host organism.
[0617] FIG. 2 presents the results of Northern analyses of multiple
human tissues which indicate that AJ172.sub.--2 is expressed highly
in the placenta and weakly in the testes.
[0618] As shown in FIG. 3, in situ antisense-RNA hybridizations
were used to specifically localize AJ172.sub.--2 expression to
placental syncytiotrophoblasts.
[0619] FIG. 4 demonstrates that AJ172.sub.--2 expression in
transfected COS cells can cause the formation of giant
multinucleated syncytia by a fusigenic mechanism. The cells
expressing AJ172.sub.--2 can be seen to have formed multinucleate
syncytia, while the non-transfected cells remained
mononucleate.
[0620] FIG. 5 demonstrates that AJ172.sub.--2 mediates actual cell
fusion and does not operate through a mechanism of arrested cell
division. A first cell line was transfected with AJ172.sub.--2,
luciferase and ERK. A second cell line was transfected with
AJ172.sub.--2 and MEK. When the cells were mixed, fusion occurred
resulting in production of luciferase activity.
[0621] FIG. 6 demonstrates that AJ172.sub.--2 can mediate fusion
between cells of differing types and between a cell expressing
AJ172.sub.--2 and a cell not expressing AJ172.sub.--2. HELA cells
were transfected with a cDNA encoding a P-selectin glycoprotein
ligand-1/Fc fusion protein (PSGL-Fc). COS cells were transfected
with AJ172.sub.--2. Another batch of COS cells was transfected with
AJ172.sub.--2 in reverse orientation. The transfected HELA cells
were mixed with each type of COS cells. As shown in FIG. 6, mixture
with the AJ172_transfected COS cells caused fusion with the HELA
cells, resulting in multinucleate fusions. Mixture with the COS
cells transfected with AJ172.sub.--2 in reverse orientation
resulted in no fusion (mononucleate cells remained).
[0622] FIG. 7 demonstrates that the mechanism of AJ172.sub.--2
induced cell fusion does not require homophilic or heterophilic
protein-protein interactions. COS cells transfected with
AJ172.sub.--2 were mixed with liposomes containing a green
fluorescent protein (GFP) expression plasmid. As shown in FIG. 7,
the COS cells fused with the liposomes, took up the expression
plasmid, and began expressing GFP.
[0623] Cell fusion in the human placenta has been implicated in a
number of critical processes. Early in human placental development,
embryonic trophoblastic cells are thought to fuse with epithelial
cells during blastocyst implantation into the uterine wall.
Subsequently, the carefully orchestrated invasion of fetal
cytotrophoblast through the maternal decidua into the endometrium
is perhaps controlled by cell fusion events, since invasive
cytotrophoblasts become non-invasive when they fuse to form
multinucleated syncytiotrophoblasts. Choriocarcinomas arise when
cytotrophoblast fusion fails to occur and the invasive process
continues unabated. Finally syncytiotrophoblasts found in the
placental villi form a continuous boundary layer between maternal
and fetal tissue. This vitally important structure is responsible
for the efficient transfer to the fetus of nutrients, growth
factors and antibodies, and for the removal of waste products. It
is possible that cytotrophoblast cell fusion is an adaptation to
increase the efficiency of these transport and secretory
processes.
[0624] In 1991 Harris proposed that the invasiveness of the fetal
trophoblast was a trait acquired by a mammalian antecedent
following an ancient retroviral infection. He went on to suggest
that this event may even have been seminal in the evolution of all
modern placental mammals. Harris based his hypothesis on
circumstantial evidence, namely the frequent observation of
retrovirus-like particles in placental preparations, and the
presence in placenta of fused cells with a morphology reminiscent
of retrovirally-induced syncytia. The ability of AJ172.sub.--2 to
mediate cell fusion events, its highly specific pattern of
expression in placental syncytiotrophoblasts, and its presence in
the genome as a part of a cryptic provirus all tend to support
Harris's view. AJ172.sub.--2 may be the first example of a viral
gene co-opted to serve a vital role in a mammalian host.
[0625] Although other mammalian placentas possess fused cell types,
to date we have found AJ172.sub.--2-related sequences only in human
and primate genomes. The simplest explanation for this would be
that the protypical AJ172.sub.--2-like sequence has diverged
extensively during the course of mammalian evolution. An
alternative explanation would be that sequestration of a retrovirus
to perform a role in placental development was a relatively common
evolutionary event, and that the antecedents of different mammalian
lineages simply used different retroviruses to achieve the same
goal The fact that AJ172.sub.--2 needs no other receptor protein to
function, the common ability of many known viruses to mediate
cell-cell fusions, and the great diversity observed in placental
morphology even among closely related mammalian species would all
be consistent with this explanation. Notwithstanding this, it is
also possible that early primates acquired the AJ172.sub.--2
provirus for some unknown, primate-specific reason.
[0626] Trophoblast syncytia may make a number of important
contributions to overall placental efficiency. One of these may
simply be to provide an extended surface area for maternal/fetal
exchange. In this regard the theoretical geometry of syncytia
formation predicts that the ratio of overall cell surface area to
cytoplasmic volume will decrease progressively as cells fuse
together. If overall cytoplasmic volume and cell surface area
remain constant during cell fusion, then the growing syncytium will
progressively accumulate "excess" cell membrane over and above that
which is required simply to enclose the cytoplasmic contents. This
extra membrane should enable the syncytium to "spread" and cover a
larger area of the substratum than would the equivalent number of
unfused cells. Thus in the placenta the formation of syncytia would
tend to increase placental efficiency by enlarging the area of the
exchange surface. In support of this theoretical prediction we have
observed in vitro that COS cells fused as a result of AJ172.sub.--2
expression adopt a "fried egg" appearance, with the nuclei gathered
tightly together in one location and the cytoplasm covering an
extended surface area.
[0627] Antagonists to AJ172.sub.--2 (either antibodies, antibody
fragments, nucleotide aptamers, peptide aptamers, antisense
nucleotides or small molecules) may be useful as birth control
agents, either by preventing initial implantation of embryos into
the uterine wall or by interrupting normal placental development
and leading to abortion of the conceptus.
[0628] Since the regulation of AJ172.sub.--2 expression is very
tight, small molecules designed to positively or negatively
modulate the control of AJ172.sub.--2 gene expression may be useful
as birth control agents. They may also be useful in treatment of
placental pathologies such as pre-eclampsia or choriocarcinoma,
where aberrant cytotrophoblast fusion events have been
observed.
[0629] AJ172.sub.--2 as a cell fusion agent may have applications
as a means of increasing DNA transfection efficiencies in vitro
(research applications, moving genes into cultured cell lines or
primary cell lines with greater efficiency) or in vivo (gene
therapy applications, moving genes into cells in the intact
organism with greater efficiency).
[0630] AJ172.sub.--2 may also be implicated in osteoclast fusion.
Therefore AJ172.sub.--2 antagonists or small molecules directed to
control of AJ172.sub.--2 gene expression may be useful in the
treatment of bone disorders such as osteoporosis or
osteopetrosis.
EXAMPLE 2
Additional Evidence for AJ172 Expression in Choriocarcinoma
Lines
[0631] Although the above experiments demonstrate that
AJ172.sub.--2 can induce cell fusion, to establish that
AJ172.sub.--2 expression could be correlated with cytotrophoblast
fusion, we examined in vitro the fusion of BeWo cells, and
monitored levels of AJ172.sub.--2 transcription in response to
forskolin treatment. BeWo is a human trophoblastic choriocarcinoma
line which can be induced by forskolin to form syncytiotrophblasts.
(BeWo-derived syncytiotrophblasts are morpologically very similar
to AJ172.sub.--2-fused COS cells). FIG. 8 shows that AJ172.sub.--2
transcription in BeWo cells increases at least five fold in
response to forskolin treatment, correlating well with cell fusion
(FIG. 9). In contrast a control choriocarcinoma line which fails to
fuse in response to forskolin, JEG3, showed no expression of
AJ172.sub.--2. Taken together these results establish that
AJ172.sub.--2 can indeed mediate cell fusion in a trophoblastic
cell type. Moreover DNA synthesis is arrested in BeWo cells which
have been treated with forskolin (FIG. 10), perhaps as a result of
AJ172 expression and induction of cell fusion. Thus induction of
AJ172 synthesis in vivo may be a therapy useful for controlling the
growth of choricarcinomas.
EXAMPLE 3
Additional Evidence for AJ172 Disregulation in Pre-Eclampsia
[0632] We performed in situ hybridizations on tissue sections
prepared from the villous region of human pre-eclamptic placenta to
examine if there were differences in the distribution of AJ172
expression compared to normal placenta. In the normal situation
hybridization of a digoxygenin-labelled antisense AJ172.sub.--2 RNA
probe is observed only to syncytiotrophoblasts comprising the layer
of fused cells on the edges of the villi, bordering on the maternal
blood space (FIG. 11). In contrast for pre-eclamptic samples
hybridization was observed in patches of fused cells throughout the
placental villi (FIG. 12). Thus it appears that AJ172 expression is
disregulated in pre-eclampsia. This would indicate that
pre-eclampsia and other diseases of placental morphogenesis may be
caused by temporal or spatial disregulation of AJ172 expression,
quantitative disregulation in AJ172 expression levels, or by
mutations in the AJ172 gene.
[0633] In addition to uses and therapies discussed above,
antibodies to AJ172 are useful in detecting serum levels of AJ172,
and thus are useful as a dignostic tools to monitor for
pre-eclampsia, for other placental pathologies or for cancerous
conditions. Agents modulating AJ172 expression or function have
therapeutic potential in treatment of neoplastic diseases in
addition to choriocarcinoma.
EXAMPLE 4
Additional Data Supporting a Role for AJ172 in Tissue Remodelling
and Extracellular Matrix Degradation
[0634] We have observed that in BeWo choriocarcinoma cells,
following treatments with forskolin and resulting in induction of
both AJ172 and cell fusion, an increase in collagenase A expression
at the RNA (FIG. 13) and protein levels (FIG. 14). These changes in
expression may reflect the normal course of events in placenta,
where it is known that a large amount of extracellular matrix
remodelling occurs during placental morphogenesis. We see similar
changes in COS cells transfected with AJ172, leading to the
conclusion that changes in collagenase A expression levels may be a
general cellular response to the cell fusion process. It is further
possible that disregulated AJ172 expression, leading to increased
local extracellular matrix breakdown, could be a contributing
factor to metastatic processes in certain tumors. Thus, correction
of this disregulation using AJ172 DNA, protein and/or antibodies
would inhibit such breakdown and the resulting metastases.
[0635] Patent and literature references cited herein are
incorporated by reference as if fully set forth.
Sequence CWU 1
1
62 1 2166 DNA Homo sapiens 1 ggcaagctac tggcacctgc tgctctcaac
taacctccac acaatggtgt tcgcattttg 60 gaaggtcttt ctgatcctaa
gctgccttgc aggtcaggtt agtgtggtgc aagtgaccat 120 cccagacggt
ttcgtgaacg tgactgttgg atctaatgtc actctcatct gcatctacac 180
caccactgtg gcctcccgag aacagctttc catccagtgg tctttcttcc ataagaagga
240 gatggagcca atttctattt acttttctca aggtggacaa gctgtagcca
tcgggcaatt 300 taaagatcga attacagggt ccaacgatcc aggtaatgca
tctatcacta tctcgcatat 360 gcagccagca gacagtggaa tttacatctg
cgatgttaac aaccccccag actttctcgg 420 ccaaaaccaa ggcatcctca
acgtcagtgt gttagtgaaa ccttctaagc ccctttgtag 480 cgttcaagga
agaccagaaa ctggccacac tatttccctt tcctgtctct ctgcgcttgg 540
aacaccttcc cctgtgtact actggcataa acttgaggga agagacatcg tgccagtgaa
600 agaaaacttc aacccaacca ccgggatttt ggtcattgga aatctgacaa
attttgaaca 660 aggttattac cagtgtactg ccatcaacag acttggcaat
agttcctgcg aaatcgatct 720 cacttcttca catccagaag ttggaatcat
tgttggggcc ttgattggta gcctggtagg 780 tgccgccatc atcatctctg
ttgtgtgctt cgcaaggaat aaggcaaaag caaaggcaaa 840 agaaagaaat
tctaagacca tcgcggaact tgagccaatg acaaagataa acccaagggg 900
agaaagcgaa gcaatgccaa gagaagacgc tacccaacta gaagtaactc taccatcttc
960 cattcatgag actggccctg ataccatcca agaaccagac tatgagccaa
agcctactca 1020 ggagcctgcc ccagagcctg ccccaggatc agagcctatg
gcagtgcctg accttgacat 1080 cgagctggag ctggagccag aaacgcagtc
ggaattggag ccagagccag agccagagcc 1140 agagtcagag cctggggttg
tagttgagcc cttaagtgaa gatgaaaagg gagtggttaa 1200 ggcataggct
ggtggcctaa gtacagcatt aatcattaag gaacccatta ctgccatttg 1260
gaattcaaat aacctaacca acctccacct cctccttcca ttttgaccaa ccttcttcta
1320 acaaggtgct cattcctact atgaatccag aataaacacg ccaagataac
agctaaatca 1380 gcaagggttc ctgtattacc aatatagaat actaacaatt
ttactaacac gtaagcataa 1440 caaatgacag ggcaagtgat ttctaactta
gttgagtttt gcaacagtac ctgtgttgtt 1500 atttcagaaa atattatttc
tctcttttta actactcttt ttttttattt tagacagagt 1560 cgcttgagcc
caggaggtgg aggttgcagt gggccgagat tgtgccactg cactccaacc 1620
tgggtgacag agtgagattc catctgaaaa acaaaaacaa aaacagaaaa caaacaaaca
1680 aaaaacaaaa aatccccaca actttgtcaa ataatgtaca ggcaaacact
ttcaaatata 1740 atttccttca gtgaatacaa aatgttgata tcataggtga
tgtacaattt agttttgaat 1800 gagttattat gttatcactg tgtctgatgt
tatctacttt gaaaggcagt ccagaaaagt 1860 gttctaagtg aactcttaag
atctatttta gataatttca actaattaaa taacctgttt 1920 tactgcctgt
acattccaca ttaataaagc gataccaatc ttatatgaat gctaatatta 1980
ctaaaatgca ctgatatcac ttcttcttcc cctgttgaaa agctttctca tgatcatatt
2040 tcacccacat ctcaccttga agaaacttac aggtagactt accttttcac
ttgtggaatt 2100 aatcatattt aaatcttact ttaaggctca ataaataata
ctcataaaaa aaaaaaaaaa 2160 aaaaaa 2166 2 387 PRT Homo sapiens 2 Met
Val Phe Ala Phe Trp Lys Val Phe Leu Ile Leu Ser Cys Leu Ala 1 5 10
15 Gly Gln Val Ser Val Val Gln Val Thr Ile Pro Asp Gly Phe Val Asn
20 25 30 Val Thr Val Gly Ser Asn Val Thr Leu Ile Cys Ile Tyr Thr
Thr Thr 35 40 45 Val Ala Ser Arg Glu Gln Leu Ser Ile Gln Trp Ser
Phe Phe His Lys 50 55 60 Lys Glu Met Glu Pro Ile Ser Ile Tyr Phe
Ser Gln Gly Gly Gln Ala 65 70 75 80 Val Ala Ile Gly Gln Phe Lys Asp
Arg Ile Thr Gly Ser Asn Asp Pro 85 90 95 Gly Asn Ala Ser Ile Thr
Ile Ser His Met Gln Pro Ala Asp Ser Gly 100 105 110 Ile Tyr Ile Cys
Asp Val Asn Asn Pro Pro Asp Phe Leu Gly Gln Asn 115 120 125 Gln Gly
Ile Leu Asn Val Ser Val Leu Val Lys Pro Ser Lys Pro Leu 130 135 140
Cys Ser Val Gln Gly Arg Pro Glu Thr Gly His Thr Ile Ser Leu Ser 145
150 155 160 Cys Leu Ser Ala Leu Gly Thr Pro Ser Pro Val Tyr Tyr Trp
His Lys 165 170 175 Leu Glu Gly Arg Asp Ile Val Pro Val Lys Glu Asn
Phe Asn Pro Thr 180 185 190 Thr Gly Ile Leu Val Ile Gly Asn Leu Thr
Asn Phe Glu Gln Gly Tyr 195 200 205 Tyr Gln Cys Thr Ala Ile Asn Arg
Leu Gly Asn Ser Ser Cys Glu Ile 210 215 220 Asp Leu Thr Ser Ser His
Pro Glu Val Gly Ile Ile Val Gly Ala Leu 225 230 235 240 Ile Gly Ser
Leu Val Gly Ala Ala Ile Ile Ile Ser Val Val Cys Phe 245 250 255 Ala
Arg Asn Lys Ala Lys Ala Lys Ala Lys Glu Arg Asn Ser Lys Thr 260 265
270 Ile Ala Glu Leu Glu Pro Met Thr Lys Ile Asn Pro Arg Gly Glu Ser
275 280 285 Glu Ala Met Pro Arg Glu Asp Ala Thr Gln Leu Glu Val Thr
Leu Pro 290 295 300 Ser Ser Ile His Glu Thr Gly Pro Asp Thr Ile Gln
Glu Pro Asp Tyr 305 310 315 320 Glu Pro Lys Pro Thr Gln Glu Pro Ala
Pro Glu Pro Ala Pro Gly Ser 325 330 335 Glu Pro Met Ala Val Pro Asp
Leu Asp Ile Glu Leu Glu Leu Glu Pro 340 345 350 Glu Thr Gln Ser Glu
Leu Glu Pro Glu Pro Glu Pro Glu Pro Glu Ser 355 360 365 Glu Pro Gly
Val Val Val Glu Pro Leu Ser Glu Asp Glu Lys Gly Val 370 375 380 Val
Lys Ala 385 3 2946 DNA Homo sapiens 3 tcgggctgcc ttatcgccaa
gctccttcag gagaacaaag aacaggccat taccctggag 60 aagactggca
actgatttta cccacaagcc caaacctcag ggatttcagt atctactagt 120
ctgggtagat actttcacgg gttgggcaga ggccttcccc tgtaggacag aaaaggccca
180 agaggtaata aaggcactag ttcatgaaat aattcccaga ttcggacttc
cccgaggctt 240 acagagtgac aatagccctg ctttccaggc cacagtaacc
cagggagtat cccaggcgtt 300 aggtatacga tatcacttac actgcgcctg
aaggccacag tcctcaggga aggtcgagaa 360 aatgaatgaa acactcaaag
gacatctaaa aaagcaaacc caggaaaccc acctcacatg 420 gcctgctctg
ttgcctatag ccttaaaaag aatctgcaac tttccccaaa aagcaggact 480
tagcccatac gaaatgctgt atggaagccc cttcataacc aatgaccttg tgcttgaccc
540 aagacagcca acttagttgc agacatcacc tccttagcca aatatcaaca
agttcttaaa 600 acattacaag gaacctatcc ctgagaagag ggaaaagaac
tattccaccc ttgtgacatg 660 gtattagtca agtcccttcc ctctaattcc
ccatccctag atacatcctg ggaaggaccc 720 tacccagtca ttttatctac
cccaactgcg gttaaagtgg ctggagtgga gtcttggata 780 catcacactt
gagtcaaatc ctggatactg ccaaaggaac ctgaaaatcc aggagacaac 840
gctagctatt cctgtgaacc tctagaggat ttgcgcctgc tcttcaaaca acaaccagga
900 ggaaagtaac taaaatcata aatccccatg gccctccctt atcatatttt
tctctttact 960 gttcttttac cctctttcac tctcactgca ccccctccat
gccgctgtat gaccagtagc 1020 tccccttacc aagagtttct atggagaatg
cagcgtcccg gaaatattga tgccccatcg 1080 tataggagtc tttctaaggg
aacccccacc ttcactgccc acacccatat gccccgcaac 1140 tgctatcact
ctgccactct ttgcatgcat gcaaatactc attattggac aggaaaaatg 1200
attaatccta gttgtcctgg aggacttgga gtcactgtct gttggactta cttcacccaa
1260 actggtatgt ctgatggggg tggagttcaa gatcaggcaa gagaaaaaca
tgtaaaagaa 1320 gtaatctccc aactcacccg ggtacatggc acctctagcc
cctacaaagg actagatctc 1380 tcaaaactac atgaaaccct ccgtacccat
actcgcctgg taagcctatt taataccacc 1440 ctcactgggc tccatgaggt
ctcggcccaa aaccctacta actgttggat atgcctcccc 1500 ctgaacttca
ggccatatgt ttcaatccct gtacctgaac aatggaacaa cttcagcaca 1560
gaaataaaca ccacttccgt tttagtagga cctcttgttt ccaatctgga aataacccat
1620 acctcaaacc tcacctgtgt aaaatttagc aatactacat acacaaccaa
ctcccaatgc 1680 atcaggtggg taactcctcc cacacaaata gtctgcctac
cctcaggaat attttttgtc 1740 tgtggtacct cagcctatcg ttgtttgaat
ggctcttcag aatctatgtg cttcctctca 1800 ttcttagtgc cccctatgac
catctacact gaacaagatt tatacaatta tgtcatatct 1860 aagccccgca
acaaaagagt acccattctt ccttttgtta taggagcagg agtgctaggt 1920
gcactaggta ctggcattgg cggtatcaca acctctactc agttctacta caaactatct
1980 caagaactaa atggggacat ggaacgggtc gccgactccc tggtcacctt
gcaagatcaa 2040 cttaactccc tagcagcagt agtccttcaa aatcgaagag
ctttagactt gctaaccgct 2100 gaaagagggg gaacctgttt atttttaggg
gaagaatgct gttattatgt taatcaatcc 2160 ggaatcgtca ctgagaaagt
taaagaaatt cgagatcgaa tacaacgtag agcagaggag 2220 cttcgaaaca
ctggaccctg gggcctcctc agccaatgga tgccctggat tctccccttc 2280
ttaggacctc tagcagctat aatattgcta ctcctctttg gaccctgtat ctttaacctc
2340 cttgttaact ttgtctcttc cagaatcgaa gctgtaaaac tacaaatgga
gcccaagatg 2400 cagtccaaga ctaagatcta ccgcagaccc ctggaccggc
ctgctagccc acgatctgat 2460 gttaatgaca tcaaaggcac ccctcctgag
gaaatctcag ctgcacaacc tctactacgc 2520 cccaattcag caggaagcag
ttagagcggt cgtcggccaa cctccccaac agcacttagg 2580 ttttcctgtt
gagatggggg actgagagac aggactagct ggatttccta ggctgactaa 2640
gaatccctaa gcctagctgg gaaggtgacc acatccacct ttaaacacgg ggcttgcaac
2700 ttagctcaca cctgaccaat cagagagctc actaaaatgc taattaggca
aaaacaggag 2760 gtaaagaaat agccaatcat ctattgcctg agagcacagc
aggagggaca atgatcggga 2820 tataaaccca agtcttcgag ccggcaacgg
caaccccctt tgggtcccct ccctttgtat 2880 gggagctctg ttttcatgct
atttcactct attaaatctt gcaactgcaa aaaaaaaaaa 2940 aaaaaa 2946 4 538
PRT Homo sapiens 4 Met Ala Leu Pro Tyr His Ile Phe Leu Phe Thr Val
Leu Leu Pro Ser 1 5 10 15 Phe Thr Leu Thr Ala Pro Pro Pro Cys Arg
Cys Met Thr Ser Ser Ser 20 25 30 Pro Tyr Gln Glu Phe Leu Trp Arg
Met Gln Arg Pro Gly Asn Ile Asp 35 40 45 Ala Pro Ser Tyr Arg Ser
Leu Ser Lys Gly Thr Pro Thr Phe Thr Ala 50 55 60 His Thr His Met
Pro Arg Asn Cys Tyr His Ser Ala Thr Leu Cys Met 65 70 75 80 His Ala
Asn Thr His Tyr Trp Thr Gly Lys Met Ile Asn Pro Ser Cys 85 90 95
Pro Gly Gly Leu Gly Val Thr Val Cys Trp Thr Tyr Phe Thr Gln Thr 100
105 110 Gly Met Ser Asp Gly Gly Gly Val Gln Asp Gln Ala Arg Glu Lys
His 115 120 125 Val Lys Glu Val Ile Ser Gln Leu Thr Arg Val His Gly
Thr Ser Ser 130 135 140 Pro Tyr Lys Gly Leu Asp Leu Ser Lys Leu His
Glu Thr Leu Arg Thr 145 150 155 160 His Thr Arg Leu Val Ser Leu Phe
Asn Thr Thr Leu Thr Gly Leu His 165 170 175 Glu Val Ser Ala Gln Asn
Pro Thr Asn Cys Trp Ile Cys Leu Pro Leu 180 185 190 Asn Phe Arg Pro
Tyr Val Ser Ile Pro Val Pro Glu Gln Trp Asn Asn 195 200 205 Phe Ser
Thr Glu Ile Asn Thr Thr Ser Val Leu Val Gly Pro Leu Val 210 215 220
Ser Asn Leu Glu Ile Thr His Thr Ser Asn Leu Thr Cys Val Lys Phe 225
230 235 240 Ser Asn Thr Thr Tyr Thr Thr Asn Ser Gln Cys Ile Arg Trp
Val Thr 245 250 255 Pro Pro Thr Gln Ile Val Cys Leu Pro Ser Gly Ile
Phe Phe Val Cys 260 265 270 Gly Thr Ser Ala Tyr Arg Cys Leu Asn Gly
Ser Ser Glu Ser Met Cys 275 280 285 Phe Leu Ser Phe Leu Val Pro Pro
Met Thr Ile Tyr Thr Glu Gln Asp 290 295 300 Leu Tyr Asn Tyr Val Ile
Ser Lys Pro Arg Asn Lys Arg Val Pro Ile 305 310 315 320 Leu Pro Phe
Val Ile Gly Ala Gly Val Leu Gly Ala Leu Gly Thr Gly 325 330 335 Ile
Gly Gly Ile Thr Thr Ser Thr Gln Phe Tyr Tyr Lys Leu Ser Gln 340 345
350 Glu Leu Asn Gly Asp Met Glu Arg Val Ala Asp Ser Leu Val Thr Leu
355 360 365 Gln Asp Gln Leu Asn Ser Leu Ala Ala Val Val Leu Gln Asn
Arg Arg 370 375 380 Ala Leu Asp Leu Leu Thr Ala Glu Arg Gly Gly Thr
Cys Leu Phe Leu 385 390 395 400 Gly Glu Glu Cys Cys Tyr Tyr Val Asn
Gln Ser Gly Ile Val Thr Glu 405 410 415 Lys Val Lys Glu Ile Arg Asp
Arg Ile Gln Arg Arg Ala Glu Glu Leu 420 425 430 Arg Asn Thr Gly Pro
Trp Gly Leu Leu Ser Gln Trp Met Pro Trp Ile 435 440 445 Leu Pro Phe
Leu Gly Pro Leu Ala Ala Ile Ile Leu Leu Leu Leu Phe 450 455 460 Gly
Pro Cys Ile Phe Asn Leu Leu Val Asn Phe Val Ser Ser Arg Ile 465 470
475 480 Glu Ala Val Lys Leu Gln Met Glu Pro Lys Met Gln Ser Lys Thr
Lys 485 490 495 Ile Tyr Arg Arg Pro Leu Asp Arg Pro Ala Ser Pro Arg
Ser Asp Val 500 505 510 Asn Asp Ile Lys Gly Thr Pro Pro Glu Glu Ile
Ser Ala Ala Gln Pro 515 520 525 Leu Leu Arg Pro Asn Ser Ala Gly Ser
Ser 530 535 5 338 DNA Homo sapiens unsure (259) unsure (261) unsure
(282) unsure (285) unsure (312) unsure (334) 5 agcggccgcg
ccatccccat caagcagggg atcctgctaa agcggagcgg caagtccctg 60
aacaaggagt ggaagaagaa gtatgtgacg ctctgtgaca acgggctgct cacctatcac
120 cccagcctgc atcttggtgc gctgtctgtg ccctctgcca acagtggagg
cagcgaggat 180 gaagaggagt ggcaaggggt gtcttggatg tggaaaaaaa
tgtgggttgt ggggttgggc 240 tgggttttgg tttcagtana ngaaacacag
ccagctggag ancanaactc acgggggttg 300 gtggcttttc anaatcaccc
ggctggtggc tganctaa 338 6 387 DNA Homo sapiens unsure (30) unsure
(163) 6 aagtaggcaa gggataataa ccaaagaagn aaatttcatg aagactagac
atcataaagc 60 ataattttaa tagtcactca accaagtatt ttttattttt
tatggatact ctgaatggca 120 attaaatgtg aaacccagtt tcttgggcaa
gtcaaattst ggnatcacat ccacctaaat 180 taaaatgact agctcgtatt
ttccccatct tcaagtttca catcctggtc atcaaaagac 240 tcgacagcaa
gacttagaat gmaaaagggt acttgtttat attaatattt tttacttgaa 300
cacgtgtagc ttgcagcagg ttcttgatga atgtgctttg tgtccaaaat gcctccccat
360 tgtacacagg tgtacaccat gcatgca 387 7 67 PRT Homo sapiens UNSURE
(26) 7 Met Thr Ser Ser Tyr Phe Pro His Leu Gln Val Ser His Pro Gly
His 1 5 10 15 Gln Lys Thr Arg Gln Gln Asp Leu Glu Xaa Lys Arg Val
Leu Val Tyr 20 25 30 Ile Asn Ile Phe Tyr Leu Asn Thr Cys Ser Leu
Gln Gln Val Leu Asp 35 40 45 Glu Cys Ala Leu Cys Pro Lys Cys Leu
Pro Ile Val His Arg Cys Thr 50 55 60 Pro Cys Met 65 8 348 DNA Homo
sapiens unsure (59) unsure (70) unsure (72) unsure (87) unsure (89)
unsure (92) unsure (94) unsure (134) unsure (138) unsure (202)
unsure (216) unsure (223) unsure (303) 8 caaaccctaa accctggcag
gaagcatgtc gaggaaggag ttccggcaac tccagaggnt 60 ccgacagaan
tntgggctga gcctggntnt cntntccagc aagggtttcg cctgagcccc 120
aagggcatcg ggantggnga ctcacctatg gatgggggcc ggggagacag gacacacaga
180 agatgagttt gtgggccagc cntgagcccc gcgccngatt ttngccggcc
caagagagcc 240 cgccgcagct tcccccattt tgcagccagc ggagccattc
acacaatcac cttctgttaa 300 ttntatctgc aacatcaatt aaattgtttg
tagaaactaa aaaaaaaa 348 9 3153 DNA Homo sapiens 9 taatcatgcc
tcttggaagt aagttaacgg gcgtgattgt ggaaaatgar aatattacca 60
aagaaggtgg cttaktggac atggccaaga aagaaaatga cttaaatgca gagcccaatt
120 taaagcagac aattaaagca acagtagara atggcaagaa ggatggcatt
gctgttgatc 180 atgttgtagg cctgaataca gaaaaatatg ctgaaactgt
cmaacttaag cataaaagaa 240 scccaggtaa agtaaaagac atwtcmattg
atgttgaaag aaggaatgaa aacagtgagg 300 tagacaccag tgctggaagt
ggctctgcac cctctgtttt acaccaaagg aacggacaaa 360 ctgaggatgt
ggcaactggg cctaggagag cagaaaagac ttctgttgcc actagtactg 420
aagggaagga caaagatgtc accttaagtc cagtgaaggc tgggcctgcc acaaccactt
480 cttcagaaac aagacaaagt gaggtggctt tgccttgcac cagcattgag
gcagatgaag 540 gcctcataat aggaacacat tccagaaata atcctcttca
tgttggtgca gaagccagtg 600 aatgcactgt ttttgctgca gctgaaaaag
gtggggctgt tgtcacagag ggatttgctg 660 aaagtgaaac cttcctcaca
agcactaagg aaggggaaag tggggagtgt gctgtggctg 720 aatctgagga
cagagcagca gacctactgg ctgtgcatgc agttaaaatc gaagccaatg 780
taaatagcgt tgtgacagag gaaaaggatg atgctgtaac cagtgcaggc tctgaagaaa
840 aatgtgatgg ttctttaagt agagactcag aaatagttga aggaactatt
acttttatta 900 gtgaagttga aagtgatgga gcagttacaa gtgctggaac
agagataaga gcaggatcta 960 taagcagtga agaggtggat ggctcccagg
gaaatatgat gagaatgggt cccaaaaaag 1020 aaacagaggg cactgtgaca
tgtacaggag cagaaggcag aagtgataac tttgtgatct 1080 gctcagtaac
tggagcaggg ccccgggagg aacgcatggt tacaggtgca ggtgttgtcc 1140
tgggagataa tgatgcacca ccaggaacaa gtgccagcca agaaggagat ggttctgtga
1200 atgatggtac agaaggtgag agtgcagtca ccagcacggg gataacagaa
gatggagagg 1260 ggccagcaag ttgcacaggt tcagaagatw gcakcgaagg
ctttgctata agttctgaat 1320 cggaagaaaa tggagagagt gcaatggaca
gcacagtggc caaagaaggc actaatgtac 1380 cattagttgc tgctggtcct
tgtgatgatg aaggcattgt gactagcaca ggcgcaaaag 1440 aggaagacga
ggaaggggag gatgttgtga ctagtactgg aagaggaaat gaaattgggc 1500
atgcttcaac ttgtacaggg ttaggagaag aaagtgaagg ggtcttgatt tgtgaaagtg
1560 cagaagggga cagtcagatt ggtactgtgg tagagcatgt ggaagctgag
gctggagctg 1620 ccatcatgaa tgcaaatgaa aataatgttg acagcatgag
tggcacagag aaaggaagta 1680 aagacacaga tatctgctcc agtgcmaaag
ggattgtaga aagcagtgtg accagtgcag 1740 tctcaggaaa ggatgaagtg
acaccagttc caggaggttg tgagggtcct atgactagtg 1800 ctgcatctga
tcaaagtgac agtcagctcg aaaaagttga agataccact atttccactg 1860
gcctggtcgg gggtagttac gatgttcttg tatctggtga agtcccagaa tgtgaagttg
1920 ctcacacatc accaagtgaa aaagaagatg aggacatcat cacctctgta
gaaaatgaag 1980 agtgtgatgg tttcatggca actacagcca gtggtgatat
taccaaccag aatagcttag 2040 cagggggtaa aaatcaaggc aaagttttga
ttatttccac cagtaccaca aatgattaca 2100 cccctcaggt aagcgcaatt
acagatgtgg aaggaggtct ttcagatgct ctgagaactg 2160 aagaaaatat
ggaaggtacc agagtaacca cagaagaatt
tgaggccccc atgcccagtg 2220 cagtctcagg agatgacagc caactcactg
ccagcagaag tgaagagaaa gatgagtgtg 2280 ccatgatttc cacaagcata
ggggaagaat tcgaattgcc tatctccagt gcaacaacca 2340 tcaagtgtgc
tgaaagtttc agccggttgc tgcagcagtg gaagaaaggg ctacaggtcc 2400
agtcttgata agcaccgccg actttgaggg gcctatgccc agtgcgcccc cagaagctga
2460 aagtcctctt gcctcaacca gcaaggagga gaaggatgaa tgtgctctca
tttccactag 2520 catagcagaa gaatgtgagg cttctgtttc cggtgtagtt
gttgaaagtg aaaatgagcg 2580 agctggcaca gtcatggaag aaaaagacgg
gagtggcatc atctttacga gctcggtgga 2640 agactgtgag ggcccagtgt
ccagtgctgt ccctcaagag gaaggcgacc cctcagtcac 2700 accagcggaa
gagatgggtg acaccgccat gatttccaca agcacctctg aagggtgtga 2760
agcagtcatg attggtgctg tcctccagga tgaagatcgg ctcaccatca caagagtaga
2820 agacttgagc gatgctgcca tcatctccac cagcacagca gaatgtatgc
caatttccgc 2880 cagcattgac agacatgaag agaatcagct gactgcagac
aacccagaag ggaacggtga 2940 cytgtcagcc acagaagtga gcaagcacaa
gktccccatg cccagcytaa ttgctgagaa 3000 taactgtcgg tgtcctgggc
cagtcagggg aggcaaagaa ctgggtcccg tgttggcagt 3060 gagcaccgag
gaggggcaca acgggccatc agtccacaag ccctctgcag ggcaaggcca 3120
tcaagtgctg tttgtgcgga aaaaaaaaaa aaa 3153 10 800 PRT Homo sapiens
UNSURE (24) UNSURE (73) UNSURE (79) UNSURE (429)..(430) 10 Met Pro
Leu Gly Ser Lys Leu Thr Gly Val Ile Val Glu Asn Glu Asn 1 5 10 15
Ile Thr Lys Glu Gly Gly Leu Xaa Asp Met Ala Lys Lys Glu Asn Asp 20
25 30 Leu Asn Ala Glu Pro Asn Leu Lys Gln Thr Ile Lys Ala Thr Val
Glu 35 40 45 Asn Gly Lys Lys Asp Gly Ile Ala Val Asp His Val Val
Gly Leu Asn 50 55 60 Thr Glu Lys Tyr Ala Glu Thr Val Xaa Leu Lys
His Lys Arg Xaa Pro 65 70 75 80 Gly Lys Val Lys Asp Ile Ser Ile Asp
Val Glu Arg Arg Asn Glu Asn 85 90 95 Ser Glu Val Asp Thr Ser Ala
Gly Ser Gly Ser Ala Pro Ser Val Leu 100 105 110 His Gln Arg Asn Gly
Gln Thr Glu Asp Val Ala Thr Gly Pro Arg Arg 115 120 125 Ala Glu Lys
Thr Ser Val Ala Thr Ser Thr Glu Gly Lys Asp Lys Asp 130 135 140 Val
Thr Leu Ser Pro Val Lys Ala Gly Pro Ala Thr Thr Thr Ser Ser 145 150
155 160 Glu Thr Arg Gln Ser Glu Val Ala Leu Pro Cys Thr Ser Ile Glu
Ala 165 170 175 Asp Glu Gly Leu Ile Ile Gly Thr His Ser Arg Asn Asn
Pro Leu His 180 185 190 Val Gly Ala Glu Ala Ser Glu Cys Thr Val Phe
Ala Ala Ala Glu Lys 195 200 205 Gly Gly Ala Val Val Thr Glu Gly Phe
Ala Glu Ser Glu Thr Phe Leu 210 215 220 Thr Ser Thr Lys Glu Gly Glu
Ser Gly Glu Cys Ala Val Ala Glu Ser 225 230 235 240 Glu Asp Arg Ala
Ala Asp Leu Leu Ala Val His Ala Val Lys Ile Glu 245 250 255 Ala Asn
Val Asn Ser Val Val Thr Glu Glu Lys Asp Asp Ala Val Thr 260 265 270
Ser Ala Gly Ser Glu Glu Lys Cys Asp Gly Ser Leu Ser Arg Asp Ser 275
280 285 Glu Ile Val Glu Gly Thr Ile Thr Phe Ile Ser Glu Val Glu Ser
Asp 290 295 300 Gly Ala Val Thr Ser Ala Gly Thr Glu Ile Arg Ala Gly
Ser Ile Ser 305 310 315 320 Ser Glu Glu Val Asp Gly Ser Gln Gly Asn
Met Met Arg Met Gly Pro 325 330 335 Lys Lys Glu Thr Glu Gly Thr Val
Thr Cys Thr Gly Ala Glu Gly Arg 340 345 350 Ser Asp Asn Phe Val Ile
Cys Ser Val Thr Gly Ala Gly Pro Arg Glu 355 360 365 Glu Arg Met Val
Thr Gly Ala Gly Val Val Leu Gly Asp Asn Asp Ala 370 375 380 Pro Pro
Gly Thr Ser Ala Ser Gln Glu Gly Asp Gly Ser Val Asn Asp 385 390 395
400 Gly Thr Glu Gly Glu Ser Ala Val Thr Ser Thr Gly Ile Thr Glu Asp
405 410 415 Gly Glu Gly Pro Ala Ser Cys Thr Gly Ser Glu Asp Xaa Xaa
Glu Gly 420 425 430 Phe Ala Ile Ser Ser Glu Ser Glu Glu Asn Gly Glu
Ser Ala Met Asp 435 440 445 Ser Thr Val Ala Lys Glu Gly Thr Asn Val
Pro Leu Val Ala Ala Gly 450 455 460 Pro Cys Asp Asp Glu Gly Ile Val
Thr Ser Thr Gly Ala Lys Glu Glu 465 470 475 480 Asp Glu Glu Gly Glu
Asp Val Val Thr Ser Thr Gly Arg Gly Asn Glu 485 490 495 Ile Gly His
Ala Ser Thr Cys Thr Gly Leu Gly Glu Glu Ser Glu Gly 500 505 510 Val
Leu Ile Cys Glu Ser Ala Glu Gly Asp Ser Gln Ile Gly Thr Val 515 520
525 Val Glu His Val Glu Ala Glu Ala Gly Ala Ala Ile Met Asn Ala Asn
530 535 540 Glu Asn Asn Val Asp Ser Met Ser Gly Thr Glu Lys Gly Ser
Lys Asp 545 550 555 560 Thr Asp Ile Cys Ser Ser Ala Lys Gly Ile Val
Glu Ser Ser Val Thr 565 570 575 Ser Ala Val Ser Gly Lys Asp Glu Val
Thr Pro Val Pro Gly Gly Cys 580 585 590 Glu Gly Pro Met Thr Ser Ala
Ala Ser Asp Gln Ser Asp Ser Gln Leu 595 600 605 Glu Lys Val Glu Asp
Thr Thr Ile Ser Thr Gly Leu Val Gly Gly Ser 610 615 620 Tyr Asp Val
Leu Val Ser Gly Glu Val Pro Glu Cys Glu Val Ala His 625 630 635 640
Thr Ser Pro Ser Glu Lys Glu Asp Glu Asp Ile Ile Thr Ser Val Glu 645
650 655 Asn Glu Glu Cys Asp Gly Phe Met Ala Thr Thr Ala Ser Gly Asp
Ile 660 665 670 Thr Asn Gln Asn Ser Leu Ala Gly Gly Lys Asn Gln Gly
Lys Val Leu 675 680 685 Ile Ile Ser Thr Ser Thr Thr Asn Asp Tyr Thr
Pro Gln Val Ser Ala 690 695 700 Ile Thr Asp Val Glu Gly Gly Leu Ser
Asp Ala Leu Arg Thr Glu Glu 705 710 715 720 Asn Met Glu Gly Thr Arg
Val Thr Thr Glu Glu Phe Glu Ala Pro Met 725 730 735 Pro Ser Ala Val
Ser Gly Asp Asp Ser Gln Leu Thr Ala Ser Arg Ser 740 745 750 Glu Glu
Lys Asp Glu Cys Ala Met Ile Ser Thr Ser Ile Gly Glu Glu 755 760 765
Phe Glu Leu Pro Ile Ser Ser Ala Thr Thr Ile Lys Cys Ala Glu Ser 770
775 780 Phe Ser Arg Leu Leu Gln Gln Trp Lys Lys Gly Leu Gln Val Gln
Ser 785 790 795 800 11 2426 DNA Homo sapiens 11 tctgttcccg
agctggagct gcgttgggac ccgtcggatc gtaaatccca tgtaaggtat 60
ctgccgtcgg aagatttgaa ctttctaatt ggacacctaa cacccacagt cctccaggtg
120 ggtcctaagg atcttaggag caacgatggg gggtcctaag ccaggggggg
atgagggtct 180 ggctctcagt ccccgcctcg cggggagtgc ctcccccctc
tgcgatgggg gtcctaagag 240 ccagtggggg aaccaggggc tggctctcag
tccctgcctc gcggggggtg cctccccccc 300 tgtgatgggg gtactaacag
ccaggggcgg aagaggggat agctctcagt ccccaccttc 360 gcggggggtg
cctccccctc gtgcgatggg ggtcctaaga tccagggggg gaagagggac 420
tggctctcag tccctgcctc gcggggggtg cctccccccc tgcgatgggg gtactcacag
480 ccaggggtgg aagaggggat agctctcagt ccccactctc gtggggggtg
cctccccctc 540 ctgcgatggg ggtcctcaga gccggggggg aagagggtct
ggctctcagt aatcccacgt 600 aaggtacctg ctgtcggaag atttgaactt
tctacttgga caactaacac ccacagtcct 660 ccaggtgggt cctaaggatc
ttaggatcaa tgatgggggg tcctaagccg gtgggggaag 720 agggtctggc
tctcagtccc cgcctcgcgg ggggtgcctc ccccctctgc gatgggggtc 780
ctaagagcca gtgggggaac caggggctgg ctctcaatcc ctgcctcgcg ggggttactc
840 ccccctcctg cgatgggggt accaacagcc aggggcggaa gaggggatag
ctctcagtcc 900 ccaccctcgt ggggttgcct ccccctcctg cgatgggggt
cctaagatcc tggggaggaa 960 gagggactgg ctctcagtaa tcccacctaa
ggtacctgcc gtcggaagat ttgaactttc 1020 tacttggaca actaacaccc
acagtcctcc aggtgggtcc taaggatctt aggatcaatg 1080 atggggggtc
ctaagccagg ggggaagagg gtctggcact cagtccctgc ctcgcggggg 1140
gtgcctccgc ccccagcgat gggggtccta agagcaaagg ggggaagagg ggctccctct
1200 cagtccccgc gtcgcgaggg gtgcctcccc ccctgcgatg gcggtgcaaa
gagccagggg 1260 aggaaagagg gaggttcgca gtccccgcct cgcgggaatt
gcctcccccc ctgctatggt 1320 ggtcccaaga gccagggggg gaagaggggt
tggctctgag tccccgcctc gcggggggtg 1380 cctccccccc tgcgatggga
gtcccaagag ccagggggta agaggggatg gatctcagcc 1440 atcacaaaat
ggggggcctt tatgttcagg ttttacccaa gaatcagctt atttgcttct 1500
tgtactagca gggcagttgc tgccaaggcc ctcaaatagg ggggccatcc tttagcaacc
1560 ctgtctagtt gtttagagac gtaggctacg ggcctcagcc agggccccac
agtttgggtt 1620 aaaagtccag ctgccatctt ttctctctct gacgcataca
atggaaaagg ctttgtcagg 1680 tcgggtgggc tgccagaaga ttttcttgta
actcatgaaa aacttgctgt tgttgggatc 1740 cccatttcaa aagttccggg
tccccgcccc atttgtgacc tcatacaaag gcttggctaa 1800 tactgcagtt
tgggatccac agcctacaaa accccacagc tcctaagaat tctctcacct 1860
gccttctgcc cttaagctcc ggtagattgc aaataacctg ctttctttct gttcccgagc
1920 tgtgttcgga cccgtcggat cgtaaatccc acgtaaggtc ggaagatttg
aactttctac 1980 ttggacacct aacacccaca gtcctccagg tacctgccgt
cggaagattt gaacgttcta 2040 cttggacaac taacacccac agtcctccag
acagaaagac aacaggtaca aagccctaag 2100 gattataaag gtatgctgct
taccatcatc ttagtgacca aggcagcgaa gctgtttctg 2160 taccttggaa
cagtcttccc tgacaagcca gagaacagtg ataaagccac cagccttggg 2220
atcaggactg aaaaggcaag agtgatggag atttctcctg cgctaagcca agagaaggtt
2280 tcagcacttc agacagctcc caccgaagta gccgcgctcc cagctgcttg
cagatgttga 2340 aaaggaaagc ctcggtttgt cttgaggttg tcagcaggtg
caagacacgt aataaaatgc 2400 aatgtgttcc taaaaaaaaa aaaaaa 2426 12 75
PRT Homo sapiens 12 Met Leu Leu Thr Ile Ile Leu Val Thr Lys Ala Ala
Lys Leu Phe Leu 1 5 10 15 Tyr Leu Gly Thr Val Phe Pro Asp Lys Pro
Glu Asn Ser Asp Lys Ala 20 25 30 Thr Ser Leu Gly Ile Arg Thr Glu
Lys Ala Arg Val Met Glu Ile Ser 35 40 45 Pro Ala Leu Ser Gln Glu
Lys Val Ser Ala Leu Gln Thr Ala Pro Thr 50 55 60 Glu Val Ala Ala
Leu Pro Ala Ala Cys Arg Cys 65 70 75 13 429 DNA Homo sapiens unsure
(10) unsure (18)..(19) unsure (24) unsure (97) unsure (117) unsure
(142)..(143) 13 cttggtccan ttggtttnnt tcgnttcccc ctttttcttc
cccttggttt tctttttttt 60 cgggcaacaa tattttccaa ggctaatacc
aaggcanacc aattcaactc ccaaggntcg 120 ggaattttta accttttaat
tnnatggccc ctcccactcc ttttctacgg cgatttgtct 180 gtgtctggcc
cccacccact gcccatcccc cattgttgtc tggatgtggt tctatttttt 240
atcggtctcc tttcccctcc tccccgttct cgcccccgcc ccaccccctg ctcccactac
300 cctttgtctc ttgctctttc ttgggcttct gtacaactca acttgtatac
actgtgtaca 360 cacaaccagc caaacgaaaa cccaacggcr aamaaaaaaa
aaaaaaaaaa aaaaaaaaaa 420 aaaaaaaaa 429 14 130 PRT Homo sapiens
UNSURE (4) UNSURE (6)..(7) UNSURE (33) UNSURE (48) 14 Leu Gly Pro
Xaa Gly Xaa Xaa Arg Phe Pro Leu Phe Leu Pro Leu Gly 1 5 10 15 Phe
Leu Phe Phe Arg Ala Thr Ile Phe Ser Lys Ala Asn Thr Lys Ala 20 25
30 Xaa Gln Phe Asn Ser Gln Gly Ser Gly Ile Phe Asn Leu Leu Ile Xaa
35 40 45 Trp Pro Leu Pro Leu Leu Phe Tyr Gly Asp Leu Ser Val Ser
Gly Pro 50 55 60 His Pro Leu Pro Ile Pro His Cys Cys Leu Asp Val
Val Leu Phe Phe 65 70 75 80 Ile Gly Leu Leu Ser Pro Pro Pro Arg Ser
Arg Pro Arg Pro Thr Pro 85 90 95 Cys Ser His Tyr Pro Leu Ser Leu
Ala Leu Ser Trp Ala Ser Val Gln 100 105 110 Leu Asn Leu Tyr Thr Leu
Cys Thr His Asn Gln Pro Asn Glu Asn Pro 115 120 125 Thr Ala 130 15
271 DNA Homo sapiens 15 gccccttcca cctcttctcc tatgacttkg aggactcctc
cctgtccacc aaggagaagg 60 aagcagagtc ccagaaggaa aacagataca
gcaattttgg caataactct tatcactcct 120 caagaccctc atctggatcc
agtgtgccca ccacccccac atcatccgtc tcacccccac 180 aggaggccag
gttggaaagg tcatcaccga gtggtcttct cacatcatcc ttcaggcagc 240
accaagagtc actggcaaaa aaaaaaaaaa a 271 16 38 PRT Homo sapiens 16
Val Pro Thr Thr Pro Thr Ser Ser Val Ser Pro Pro Gln Glu Ala Arg 1 5
10 15 Leu Glu Arg Ser Ser Pro Ser Gly Leu Leu Thr Ser Ser Phe Arg
Gln 20 25 30 His Gln Glu Ser Leu Ala 35 17 1630 DNA Homo sapiens
unsure (1622) 17 cctgacctca ggtgatctgc ccgcctcggc ctctgaaagt
gctgggatta taggcatgag 60 ccaacatgcc tgacctgtta tttattttaa
attatatcag gaatacacac acacacacac 120 acacacacac acacacacac
acaacttata aagataatgg tctccttggc actcccaccc 180 acccacccat
ccaaatttac acaagtaaat ctgtaatcaa tttggttaga agggatttat 240
tttaatattt ttggggattg cttatgatgc agtataattt ttagttatat tagtagtaat
300 tggaaatgtg tatttttgtg actgaagtca ccttctaaat aatttctaga
ataaaatttt 360 tatattgaag aagttggtct taaccatttt tttttcagga
gcatgcattt tgaaatcatt 420 ctgtgggaag atgaaaacaa atttagttct
atgtctcccc tttttagaga tgttgacact 480 ttccttaaat gtaccatgca
tgatttgtct accacccttt tagcttgtta tacttaaatc 540 ccagatctct
gtcttcccat ttcagtttct ctagaatttc tggctgcttc caatgggtca 600
aatttatgag tgaaccatta agaatcactt agtgtagaaa taaaccatgg gttaggagtt
660 tgaacactgc ctaggttctg tttctgattt gattatgact cagctgtgtg
gccttgggaa 720 accaccttac tggtatccct atccttgcag aagcaagaga
gttaatgatg gttgacttaa 780 tctcttgtgg ttattatgaa gatcagataa
gatacattaa cacattttgc caactgaatt 840 aggttattta tttacatgtg
tgtccatgga cctggggatc aggtgctatg tctcagcctt 900 atctttgttt
ttaatcctgt gtctctaatt gtgtttgtca gtaaaggagt gagtcattta 960
atggttgcta gatgtttgag taaaacaaac aagcaaacaa atggtaaatt agtactattt
1020 ctttttaaaa aatttttttt tacattttaa aaattataga taaatacaga
gatgaggtct 1080 caccatgttg cccagtctgg tttcaaactc ctaaactcaa
gtgatcctct ctcctcagcc 1140 tcccaaagtg ctaggattac aggcgtgagc
caccatgcct ggccagtagt actatttctt 1200 tgggaaaata tttagtagta
gtcaacaaag ttgagcatac tgtgacctgg cagttttgat 1260 gctaagtawa
tacccaacag aaatgcaaac atatacttac caaaactcat gtccaagaat 1320
attcgtagaa gcacaattct tatgatagca aaaaggtaga aaacaacyta aatgtttyta
1380 agcagtagca taagagtaat accgtgtggt ttgtttatac agtgagatcc
tgtacagcca 1440 tgtaaaagac caaaatattc cctgtaacaa tgagaatgaa
tctcctgtgc ttgcttcggc 1500 agcacataca ctaaaattgg aacgatacag
agattagcat ggcccctgtg caaggagaat 1560 gaatyttcgt aatgttcagc
aaaagaagcc agatataaat gaatattcca ttttataaaa 1620 anaaaaaaaa 1630 18
30 PRT Homo sapiens 18 Met Lys Thr Asn Leu Val Leu Cys Leu Pro Phe
Leu Glu Met Leu Thr 1 5 10 15 Leu Ser Leu Asn Val Pro Cys Met Ile
Cys Leu Pro Pro Phe 20 25 30 19 456 DNA Homo sapiens 19 aagaaggaga
ctgtaagctt gtttgtacaa aaacatacca tacagagaaa gctgaagaca 60
aacaaaagtt agaattcttg aaaaaaagca tgttattgaa ttatcaacat cactggattg
120 tggataatat gcctgtaacg tggtgttacg atgttgaaga tggtcaggtt
ctgtaatcct 180 ggatttccta ttggctgtta cattacagat aaaggccatg
caaaagatgc ctgtgttatt 240 agttcagatt tccatgaaag agatacattt
tacatcttca accatgttga catcaaaata 300 tactatcatg ttgttgaaac
tgggtccatg ggagcaagat tagtggctgc taaacttgaa 360 ccgaaaagct
tcaaacatac ccatatagat aaaccagact gctcagggcc ccccatggac 420
ataagtaaca aggcttctgg ggagataaaa attgca 456 20 519 DNA Homo sapiens
unsure (4) unsure (12) unsure (28) unsure (35)..(36) unsure (51)
unsure (63)..(65) unsure (90) unsure (111)..(112) unsure (123)
unsure (136) unsure (148) unsure (157) unsure (161) unsure (204)
unsure (239) unsure (305) 20 caantaataa ancttttgtt tccctcgnca
ttgtnntcgt tcccctgtcc ngccttgttt 60 ccnnngtcct gcaccaatat
ttccaaaccn aatacccaag catacaatcc nnactccaag 120 ctnggaattc
gcccanagag accgtcgngg gaagaanttg nctggaaact tgttcatggt 180
gatatatacc gtcctccaag aaangggatg ctgctatcag tctttctagg agccgggana
240 cagatattaa ttatgacctt tgtgactcta tttttcgctt gcctgggagt
tttgtcacct 300 cccanccgag gagcgctgat gacgtgtgct gtggtcctgt
gggtgctgct gggcacccct 360 gcaggctatg tttctgccag attctataag
tcctttggag gtgagaagtg gaaaacaaat 420 gttttattaa catcatttct
ttgtcctggg attgtatttg ctgacttctt tataatgaat 480 ctgatcctct
ggtcaacggc ctctttggcc ctcgagaca 519 21 89 PRT Homo sapiens UNSURE
(18) 21 Met Thr Phe Val Thr Leu Phe Phe Ala Cys Leu Gly Val Leu Ser
Pro 1 5 10 15 Pro Xaa Arg Gly Ala Leu Met Thr Cys Ala Val Val Leu
Trp Val Leu 20 25 30 Leu Gly Thr Pro Ala Gly Tyr Val Ser Ala Arg
Phe Tyr Lys Ser Phe 35 40 45 Gly Gly Glu Lys Trp Lys Thr Asn Val
Leu Leu Thr Ser Phe Leu Cys 50 55 60 Pro Gly Ile Val Phe Ala Asp
Phe Phe Ile Met Asn Leu Ile Leu Trp 65 70 75 80 Ser Thr Ala Ser Leu
Ala Leu Glu Thr 85 22 507 DNA Homo sapiens 22 ttcttcccat acacctttcc
cccataagat gtgtcttcaa cactataaag catttgtatt 60 gtgatttgat
taagtatata tttggttgtt ctcaatgaag agcaaattta aatattatgt 120
gcatttgtaa atacagtagc tataaaattt tccatacttc taatggcaga atagaggagg
180 ccatattaaa taatactgat gaaaggcagg acactgcatt gtaaatagga
ttttctaggc 240 tcggtaggca gaaagaatta tttttctttg aaggaaataa
ctttttatca tggtaatttt 300 gaaggatgat
tcctatgatg tgttcaccag gggaatgtgg cttttaaaga aaatcttcta 360
ttggttgtaa ctgttcatat cttcttactt ttctgtgttg acttcattat tcccatggta
420 ttggcctttt aaactatgtg cctctgagtc tttcaattta taaatttgta
tcttaataaa 480 tattataaaa atgaaaaaaa aaaaaaa 507 23 622 DNA Homo
sapiens unsure (32) unsure (57) unsure (66) unsure (72) unsure
(105) 23 ggttcttcgg gacacccgtg gatggacacg gnaaggaaac accaggccaa
ccacagntgg 60 ggatanaata gnacaaccac accctgccgt ccagagcctc
ccagnctgtg ccccgtccta 120 gtaccaccag caaccatcaa tcccgtctcc
tcctgcctcc tctcctgcaa tccaccccgc 180 cacgactatc gccatggcag
ccctgatcgc agagaacttc cgcttcctgt cacttttctt 240 caagagcaag
gatgtgatga ttttcaacgg cctggtggca ctgggcacgg tgggcagcca 300
ggagctgttc tctgtggtgg ccttccactg cccctgctcg ccggcccgga actacctgta
360 cgggctggcg gccatcggcg tgcccgccct ggtgctcttc atcattggca
tcatcctcaa 420 caaccacacc tggaacctcg tggccgagtg ccagcaccgg
aggaccaaga actgctccgc 480 cgcccccacc ttcctccttc taagctccat
cctgggacgt gcggctgtgg cccctgtcac 540 ctggtctgtc atctccctgc
tgcgtggtga ggcttatgtc tgtgctctca gtgagttcgt 600 ggacccttcc
tcactcacgg cc 622 24 143 PRT Homo sapiens 24 Met Ala Ala Leu Ile
Ala Glu Asn Phe Arg Phe Leu Ser Leu Phe Phe 1 5 10 15 Lys Ser Lys
Asp Val Met Ile Phe Asn Gly Leu Val Ala Leu Gly Thr 20 25 30 Val
Gly Ser Gln Glu Leu Phe Ser Val Val Ala Phe His Cys Pro Cys 35 40
45 Ser Pro Ala Arg Asn Tyr Leu Tyr Gly Leu Ala Ala Ile Gly Val Pro
50 55 60 Ala Leu Val Leu Phe Ile Ile Gly Ile Ile Leu Asn Asn His
Thr Trp 65 70 75 80 Asn Leu Val Ala Glu Cys Gln His Arg Arg Thr Lys
Asn Cys Ser Ala 85 90 95 Ala Pro Thr Phe Leu Leu Leu Ser Ser Ile
Leu Gly Arg Ala Ala Val 100 105 110 Ala Pro Val Thr Trp Ser Val Ile
Ser Leu Leu Arg Gly Glu Ala Tyr 115 120 125 Val Cys Ala Leu Ser Glu
Phe Val Asp Pro Ser Ser Leu Thr Ala 130 135 140 25 314 DNA Homo
sapiens unsure (38) unsure (50) unsure (58) unsure (63) unsure (65)
unsure (70) unsure (77) unsure (82) unsure (84) unsure (94) unsure
(113) unsure (132) unsure (144) unsure (155) unsure (165) unsure
(171) unsure (183) unsure (198) unsure (216) unsure (234) unsure
(249) unsure (254) unsure (256) 25 ttttaaaaaa cttttatctt cttggccagg
ggaaaggncc cccaggcaan ctggggtntg 60 gananaccca naaaacnatg
gnanccccaa ccancagggc caggttacag tgnaactccc 120 cagtgggccc
cnttatggga ctcnattcag ttaanattta tctancttca nagggacacc 180
cancccaaca gttccccnct ggggagtggc ccccanttca acctctggcc ttantttaaa
240 aaattaaant tttnanaaag tttttcttac taaaagggaa aaaaaaaaaa
aaaaaaaaaa 300 aaaaaaaaaa aaaa 314 26 533 DNA Mus musculus unsure
(32) unsure (38) 26 gggatatccc atacaggtat gaaaaaaccc cntatgtnat
agtgttctat agcacacaat 60 accttatgaa ggaagggttt satgaataca
tggcagaaga caatcatgaa agamttatyt 120 tgaggggyta gaartaatga
gtttggaggt gtgcccctta ggtcctgart gtcctgggat 180 ccctmacccc
taatttctct cccaragcat yatcccttct cagtattggt actacatgat 240
tgaactttcc ttctastggt ccctgytctt cagcattgcc tctgatgtcw agcgaaagga
300 ttttaaggaa cagatcatcc accatgtggc cactatcatt ctcctctgct
tctcctggtt 360 tgccaattac gtccgggcag ggaccctcat catggctctg
catgacgctt ctgactacct 420 gctggagtct gccaagatgt ttaactacgc
gggatggaag aacacctgca acaacctctt 480 cattgtgttc gccatcgttt
tcatcatcac tcggctggtt atcatgcctt tct 533 27 44 PRT Mus musculus 27
Met Thr Leu Leu Thr Thr Cys Trp Ser Leu Pro Arg Cys Leu Thr Thr 1 5
10 15 Arg Asp Gly Arg Thr Pro Ala Thr Thr Ser Ser Leu Cys Ser Pro
Ser 20 25 30 Phe Ser Ser Ser Leu Gly Trp Leu Ser Cys Leu Ser 35 40
28 313 DNA Mus musculus unsure (4) unsure (33) unsure (35) unsure
(94) unsure (226) 28 aaanacaagt caatgaagtg aaggagggta tgnanacatg
cccctcacca taccccaggg 60 accatggttc ctaggatctc actgcctccc
tttntggcct tcctgtcccc tcccttcagc 120 tatgacagct ggtgtggagt
agaagggcaa ctagttctgt tatttattga acatttgggg 180 tttcagttgt
aaagccacaa ctacaggtag gacctgatat ttcggngagg gaccatttca 240
gaccaaaatg tactgttaat tttttttaat taaagtatat taaaggttaa ataaaaaaaa
300 aaaaaaaaaa aaa 313 29 525 DNA Mus musculus unsure (22) unsure
(52) unsure (55) unsure (59) 29 aaagacatcc actttgcctt tntctccaca
ggtgtccact cccaggtcca antgnaggng 60 agcctgaatt cggccaaaga
ggcctaatta caatcatttc aaattttgaa tttttaagtt 120 gatgggctct
taagtggtcc gttctgaata raaaccaatt tgctagtttc ggttttgttt 180
tgttttgttt tgttttgttt tgttttgttt ttttaaggaa tcagatagcc agaaaaaaaa
240 atgctattgc ttgttttcat gaacttcagt tgtctctttt tagtaaaccc
agtactttcc 300 acaaagtctt ctctgacctt ccccatcact ggacggttca
cccatcttct tctccaagtg 360 tttatccccc agcccaagcc tttcctgctg
caagccaagc ctgctacatt tgttacagac 420 caagcttata cacagctcga
caactgcact cccactgtag gctccggtgt gtactcttgt 480 cttgtgttgg
gaaggggaag tgaagtgata agccagaatt ttttt 525 30 95 PRT Mus musculus
30 Met Leu Leu Leu Val Phe Met Asn Phe Ser Cys Leu Phe Leu Val Asn
1 5 10 15 Pro Val Leu Ser Thr Lys Ser Ser Leu Thr Phe Pro Ile Thr
Gly Arg 20 25 30 Phe Thr His Leu Leu Leu Gln Val Phe Ile Pro Gln
Pro Lys Pro Phe 35 40 45 Leu Leu Gln Ala Lys Pro Ala Thr Phe Val
Thr Asp Gln Ala Tyr Thr 50 55 60 Gln Leu Asp Asn Cys Thr Pro Thr
Val Gly Ser Gly Val Tyr Ser Cys 65 70 75 80 Leu Val Leu Gly Arg Gly
Ser Glu Val Ile Ser Gln Asn Phe Phe 85 90 95 31 270 DNA Mus
musculus unsure (47) unsure (71) unsure (91) unsure (94) unsure
(105) unsure (170) unsure (189) unsure (192) unsure (210) unsure
(246)..(247) 31 aggtttcttg ggaacagctc agcagatttt tgagaccaat
caaatgncct cattaagaac 60 tttatctgtt nggaaacatg gcttccttcc
nggntctgct aaacngaaag ctcatttgtt 120 gttgctgttg ttgttgtttg
tttgtccatt tctctttaat tctaatgttn acatcatgtc 180 gtgctgtang
antctagaaa gccttaattn acttccacca agaaataaag caatatgttg 240
gtaatnngaa aaaaaaaaaa aaaaaaaaaa 270 32 574 DNA Mus musculus unsure
(9) unsure (29) unsure (37) unsure (53) unsure (56) unsure (68)
unsure (72) unsure (85) unsure (118) unsure (131) unsure
(169)..(170) unsure (172) unsure (180) unsure (253) unsure (448)
unsure (452) unsure (455) unsure (457) unsure (459) unsure (475)
unsure (550) unsure (572) 32 tttggtcana aaagacaatt tttttgttnt
caagctngag gtgtggcagg ctnganattt 60 ggccaaanaa tngagggaca
aaganatcca ctttgccttt ttttccacag gtgtccantc 120 ccaggtccaa
ntgcaggcgg gtccacaggc cgcagccatg ggtagccgnn tntcccgagn 180
ggarttcgaa tgggtytaca cggaccarcc ccacgccgcc cggcgcaagg agatcttagc
240 aaagtatcca ganatcaagt ccttgatgaa acctgaccac aatctgatct
ggattgtagc 300 catgatgctt ctcgtccagc tggcttcatt ttacttagtc
aaagatttgg actggaaatg 360 ggtcatattt tggtcctatg tctttggcag
ctgccttaac cactccatga ctctggctat 420 ccatgagatt tcccacaatt
tccccttngg cnccncnang gcctgtggaa ccgcnggttt 480 ggaatgtttg
ctaacctctc tctccgaatg gcctactcca tttcctttaa aaaaaacaca 540
tggatcaccn ccggtactcc gaacggataa antr 574 33 138 PRT Mus musculus
UNSURE (5) UNSURE (8) UNSURE (32) UNSURE (97) UNSURE (99)..(101) 33
Met Gly Ser Arg Xaa Ser Arg Xaa Glu Phe Glu Trp Val Tyr Thr Asp 1 5
10 15 Gln Pro His Ala Ala Arg Arg Lys Glu Ile Leu Ala Lys Tyr Pro
Xaa 20 25 30 Ile Lys Ser Leu Met Lys Pro Asp His Asn Leu Ile Trp
Ile Val Ala 35 40 45 Met Met Leu Leu Val Gln Leu Ala Ser Phe Tyr
Leu Val Lys Asp Leu 50 55 60 Asp Trp Lys Trp Val Ile Phe Trp Ser
Tyr Val Phe Gly Ser Cys Leu 65 70 75 80 Asn His Ser Met Thr Leu Ala
Ile His Glu Ile Ser His Asn Phe Pro 85 90 95 Xaa Gly Xaa Xaa Xaa
Ala Cys Gly Thr Ala Gly Leu Glu Cys Leu Leu 100 105 110 Thr Ser Leu
Ser Glu Trp Pro Thr Pro Phe Pro Leu Lys Lys Thr His 115 120 125 Gly
Ser Pro Pro Val Leu Arg Thr Asp Lys 130 135 34 216 DNA Mus musculus
unsure (69) unsure (86) unsure (114) unsure (116) unsure (119)
unsure (142) 34 atgaagtgct ttttggagga gcttttgttt agtccaacag
gagtccaagg atgcagatta 60 gagttttgng agtttgctgc ccttgntggg
ctaggcattt cattgttgta actncntcng 120 agtaactgat gatcctataa
gnaaccccaa taaatttttt ggtttactaa aaaaaaaaaa 180 aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaa 216 35 526 DNA Mus musculus unsure (6)
unsure (25) unsure (53) 35 acaggngtcc aatcccaggt ccaantgcag
gggagcctga attcggccaa agnggcctag 60 cctcccaagt gstgggatta
aaggsgtgtg ccaccatgcc ccacttcata tgttatattt 120 ttaatgaata
aagagtggaa aaattatgta tcacatgtgt taatttgggg agaagcgctt 180
tataacagag ggcttactyt caattaaaga gaacaaaggr aaatgtgtty tacaggcagt
240 gtataccttt gacctctgaa aaaacctata tagtttctcc tacagacacc
ttgccagtaa 300 ccttacaggt cttataggag agcagatcca agttgccagg
ctgatctgca agcacaaaca 360 tttgtcaagg gaaagcacag gtcgttactt
tcagtacaaa atggttcttt gctatggatg 420 gattctcttc ttcttgcccc
atgtcctgtt cccaaggacc gacttcctgc agcactgtgg 480 tggactcttc
tatgaggaga caacatctgg gccttattca atagcc 526 36 42 PRT Mus musculus
36 Met Val Leu Cys Tyr Gly Trp Ile Leu Phe Phe Leu Pro His Val Leu
1 5 10 15 Phe Pro Arg Thr Asp Phe Leu Gln His Cys Gly Gly Leu Phe
Tyr Glu 20 25 30 Glu Thr Thr Ser Gly Pro Tyr Ser Ile Ala 35 40 37
208 DNA Mus musculus unsure (8) unsure (29) unsure (31) unsure (42)
unsure (55) unsure (65) unsure (75) unsure (86) unsure (91) unsure
(98) unsure (100)..(101) unsure (113) unsure (119) unsure (121)
unsure (128) unsure (130)..(131) unsure (133) unsure (137) unsure
(161) 37 tttgaaangg caacagaaat attttttgna ntagaaaaag gnatggaacg
tggtnccaat 60 tgttnatttt ccttnattta ttcccngtaa ntttgtcngn
ngataaattg aanataacng 120 ngattaangn ntnatgntaa aaaaaaaaaa
aaaaaaaaaa naaaaaaaaa aaaaaaaaaa 180 aaaaaaaaaa aaaaaaaaaa aaaaaaaa
208 38 535 DNA Mus musculus unsure (6) unsure (30) unsure (60)
unsure (67) unsure (83) unsure (99) unsure (115) unsure (145)
unsure (160) unsure (165) 38 atttgntcag aaaagacaat ttttttgttn
tcaagcttga ggtgtggcag gcttgagatn 60 tgcccanaca cttgagggac
aangacatcc aataacccnt tctctccaca ggtgnccact 120 cccaggtcca
actgcaggcg agccngaatt cggccaaagn ggccnaagat cagttagctc 180
cctgggtcgg aacaaggtga aaagcagctt tcttgctttt gaaatcatyt ttgtgacaag
240 gacacatggg gtcagggtag ggtgtccart taaaatagtg tcactgctta
gaaaggggwa 300 cttggattcc tttagttagc ttagctctgt ctcttgtttc
ataaaacaca ctgggttaga 360 ataraggctc ctgcattaca tggtttgtgt
cactgttttt tgttgggttt tctttttggt 420 ttttcgagac agggtttctc
tgtatagccc tggctgtcct araactcact ctgtagacca 480 ggctggcctc
gaactcagaa atctgcccgc ttctgcctcc caagtgctgg gatta 535 39 52 PRT Mus
musculus UNSURE (28) 39 Met Val Cys Val Thr Val Phe Cys Trp Val Phe
Phe Leu Val Phe Arg 1 5 10 15 Asp Arg Val Ser Leu Tyr Ser Pro Gly
Cys Pro Xaa Thr His Ser Val 20 25 30 Asp Gln Ala Gly Leu Glu Leu
Arg Asn Leu Pro Ala Ser Ala Ser Gln 35 40 45 Val Leu Gly Leu 50 40
308 DNA Mus musculus unsure (43) unsure (115) unsure (134) 40
ggattaaagg catgtgtcac gttttaaatt gatagttata acntcgatgc cacgaatcct
60 gcagtttctc ctgtgctcct ttctttgtgt cagatgggtt aagggttatc
agttngggga 120 agaattgtcc ttgnaccccc tggaattatt tttctcaaaa
atccaagact ccaaagaaca 180 tgggaaaaat tgttctgtcc acttttgacg
ttgaagattt tggttatcct tttcgtactt 240 tctatgtatt ttctatgtaa
aattttacac aattaaaaat gtttttttgt ctagtaaaaa 300 aaaaaaaa 308 41
1351 DNA Mus musculus unsure (134) 41 cagcgcgcgg agccggcgtc
ccgttggcgc gctctggcct ggcttcgggt cgtcgcttcg 60 gccccgagga
gccgctcgct gtctccggag cggcggagag gatggtgcgg ggcagcccgg 120
ggcccgccgc gcgccgccgc gagtgaacag ggccaggccg cgggcgtccg cgggctcgar
180 ccgccagtct gcggggcggt tgccgctggt gggaagcatg ttcagtatca
accccctgga 240 gaacctgaag ctgtacatca gcagccggcc gcccttggtg
gtttttatga tcagtgtcag 300 cgccatggcc atcgccttcc tcaccctggg
ctacttcttc aagatcaagg agattaagtc 360 cccagaaatg gctgaggatt
ggaatacttt tctgctccgg tttaatgatt tggacttgtg 420 tgtatcagaa
aacgagacac tgaagcatct ctccaacgat accaccacac cagagagcac 480
catgaccgtc gggcaggcca gatcgtctac ccagccgccc cagtccctgg aggagtcagg
540 ccccatcaat atttcagtgg ccattacctt gaccttggac cctctcaagc
cctttggagg 600 gtactctcga aatgttacac acctgtactc caccatcctc
gggcatcaga ttggattgtc 660 aggcagggaa gcccacgagg agatcaacat
caccttcacc ctgcctgctg cctggaacgc 720 cgatgactgt gccctccatg
gccactgtga gcaggcggtg ttcacagcat gcatgaccct 780 cacagctgcc
cccggagtct tccccgtcac tgttcagcca cctcactgta tccccgacac 840
atacagcaac gccacgctct ggtacaagat cttcacaact gccagagatg ccaacacgaa
900 atatgctcaa gactacaatc ctttctggtg ttataagggt gccattggga
aagtctacca 960 tgctttaaat cccaaactca ctgttgttgt tccagatgac
gaccgctcat taataaacct 1020 gcatctcatg cacaccagtt acttcctttt
cgtgatggtg ataacgatgt tctgctatgc 1080 agtcatcaaa ggcagaccca
gcaaactgcg gcagagcaat cctgaatttt gccmtgagaa 1140 ggtggytctg
gctgacgcct aatcctacag ctccccattt tytgagagac caagaaccat 1200
gatcattgcc tgctgaatcg gccagggcct ggccactctg tgaatacatg atcttgcaat
1260 gttgggttat tccagccaaa gacatttcaa gtgcctgtaa ctgatttgtc
catatttata 1320 aacactgatc tggnaaaaaa aaaaaaaaaa a 1351 42 314 PRT
Mus musculus UNSURE (306) UNSURE (310) 42 Met Phe Ser Ile Asn Pro
Leu Glu Asn Leu Lys Leu Tyr Ile Ser Ser 1 5 10 15 Arg Pro Pro Leu
Val Val Phe Met Ile Ser Val Ser Ala Met Ala Ile 20 25 30 Ala Phe
Leu Thr Leu Gly Tyr Phe Phe Lys Ile Lys Glu Ile Lys Ser 35 40 45
Pro Glu Met Ala Glu Asp Trp Asn Thr Phe Leu Leu Arg Phe Asn Asp 50
55 60 Leu Asp Leu Cys Val Ser Glu Asn Glu Thr Leu Lys His Leu Ser
Asn 65 70 75 80 Asp Thr Thr Thr Pro Glu Ser Thr Met Thr Val Gly Gln
Ala Arg Ser 85 90 95 Ser Thr Gln Pro Pro Gln Ser Leu Glu Glu Ser
Gly Pro Ile Asn Ile 100 105 110 Ser Val Ala Ile Thr Leu Thr Leu Asp
Pro Leu Lys Pro Phe Gly Gly 115 120 125 Tyr Ser Arg Asn Val Thr His
Leu Tyr Ser Thr Ile Leu Gly His Gln 130 135 140 Ile Gly Leu Ser Gly
Arg Glu Ala His Glu Glu Ile Asn Ile Thr Phe 145 150 155 160 Thr Leu
Pro Ala Ala Trp Asn Ala Asp Asp Cys Ala Leu His Gly His 165 170 175
Cys Glu Gln Ala Val Phe Thr Ala Cys Met Thr Leu Thr Ala Ala Pro 180
185 190 Gly Val Phe Pro Val Thr Val Gln Pro Pro His Cys Ile Pro Asp
Thr 195 200 205 Tyr Ser Asn Ala Thr Leu Trp Tyr Lys Ile Phe Thr Thr
Ala Arg Asp 210 215 220 Ala Asn Thr Lys Tyr Ala Gln Asp Tyr Asn Pro
Phe Trp Cys Tyr Lys 225 230 235 240 Gly Ala Ile Gly Lys Val Tyr His
Ala Leu Asn Pro Lys Leu Thr Val 245 250 255 Val Val Pro Asp Asp Asp
Arg Ser Leu Ile Asn Leu His Leu Met His 260 265 270 Thr Ser Tyr Phe
Leu Phe Val Met Val Ile Thr Met Phe Cys Tyr Ala 275 280 285 Val Ile
Lys Gly Arg Pro Ser Lys Leu Arg Gln Ser Asn Pro Glu Phe 290 295 300
Cys Xaa Glu Lys Val Xaa Leu Ala Asp Ala 305 310 43 848 DNA Mus
musculus unsure (11) unsure (30) unsure (137) unsure (183) unsure
(370) unsure (649) unsure (712) unsure (725) unsure (727) unsure
(729) unsure (746) unsure (760) unsure (840) 43 agctgttggg
ntcgcggttg aggacaaatn ttcgcggtct ttccagtatt cttggatcgg 60
aaacccgtcg gcttccgaac ggtactccgc caccgaggga cctgagcgag tccgcatcga
120 ccggatcgga aaacctntcg actgttgggg tgagtactcc ctctcaaaag
cgggcatgac 180 ttntgcgcta agattgtcag tttccaaaaa cgaggaggat
ttgatattca cctggcccgc 240 ggtgatgcct ttgagggtgg ccgcgtccat
ctggtcagaa aagacaatct ttttgttgtc 300 aagcttgagg tgtggcaggc
ttgagatctg gccatacact tgagtgacaa tgacatccac 360 tttgcctttn
tctccacagg tgtccactcc caggtccaac tgcagacttc gaattcggcc 420
aaagaggcct actttcatat ccacgatgcg ttttctggcc gccacgatcc tgctgctggc
480 gctggtcgct gccagccagg cggagcccct gcacttcaag gactgcggct
ctaaggtggg 540 agttataaag gaggtgaatg tgagcccatg tcccaccgat
ccctgtcagc tgcacaaagg 600 ccagtcctac agtgtcaaca tcacctttac
cagcggcact cagtcccana acagcacggc 660 cttggtccac ggcatcctgg
aagggatccg ggtccccttc cctattcctg ancctgacgg 720 ttgtnanant
ggaatcaact gccccntcca gaaagacaan gtctacagct acctgaataa 780
gcttccggtg aaaaatgaat acccctctat aaaactggtg gtggaatgga aactttgaan
840 atgacaaa 848 44 130 PRT Mus musculus UNSURE (68) UNSURE (89)
UNSURE (94)..(95) UNSURE (101) UNSURE (105) 44 Met Arg Phe Leu Ala
Ala Thr Ile Leu Leu Leu Ala Leu Val Ala Ala 1 5 10 15 Ser Gln Ala
Glu Pro Leu His Phe Lys Asp Cys Gly Ser Lys Val Gly 20
25 30 Val Ile Lys Glu Val Asn Val Ser Pro Cys Pro Thr Asp Pro Cys
Gln 35 40 45 Leu His Lys Gly Gln Ser Tyr Ser Val Asn Ile Thr Phe
Thr Ser Gly 50 55 60 Thr Gln Ser Xaa Asn Ser Thr Ala Leu Val His
Gly Ile Leu Glu Gly 65 70 75 80 Ile Arg Val Pro Phe Pro Ile Pro Xaa
Pro Asp Gly Cys Xaa Xaa Gly 85 90 95 Ile Asn Cys Pro Xaa Gln Lys
Asp Xaa Val Tyr Ser Tyr Leu Asn Lys 100 105 110 Leu Pro Val Lys Asn
Glu Tyr Pro Ser Ile Lys Leu Val Val Glu Trp 115 120 125 Lys Leu 130
45 265 DNA Mus musculus unsure (2) unsure (67) unsure (75) unsure
(79) unsure (101) unsure (104) unsure (111) unsure (121) unsure
(133) unsure (136) unsure (157) unsure (162) unsure (164) unsure
(172) unsure (175) unsure (183) unsure (187) unsure (192) unsure
(199)..(200) unsure (208) unsure (211) unsure (223) unsure (225)
unsure (227) unsure (243)..(244) unsure (246) 45 gngttacctc
ccctgtttct aagtgcctcc tgagtcccca gcccctggct tatcagtcag 60
atgagtntcc ttggnagcnt ctgccccatc gcttcagcag nagngactag ntttcctcgg
120 natccagact ggntgngggg cagtctgccg cagaaanttg tntntgagtg
gntgngtctt 180 tgnggtnagc tntcgttcnn tggtagtntt nattaaagcc
aanantnggt tgcaaaaaaa 240 aanngnaaaa aaaaaaaaaa aaaaa 265 46 29 DNA
Artificial Sequence oligonucleotide 46 tnagatccaa cagtcacgtt
cacgaaacc 29 47 29 DNA Artificial Sequence oligonucleotide 47
cntcctggtt gttgtttgaa gagcaggcg 29 48 29 DNA Artificial Sequence
oligonucleotide 48 tngcccaaga aactgggttt cacatttaa 29 49 29 DNA
Artificial Sequence oligonucleotide 49 gntgaagcat gcccaatttc
atttcctct 29 50 29 DNA Artificial Sequence oligonucleotide 50
antgttctct ggcttgtcag ggaagactg 29 51 29 DNA Artificial Sequence
oligonucleotide 51 tncaagttga gttgtacaga agcccaaga 29 52 29 DNA
Artificial Sequence oligonucleotide 52 gntgtgagaa gaccactcgg
tgatgacct 29 53 29 DNA Artificial Sequence oligonucleotide 53
tngagtctgg gtggtagaca aatcatgca 29 54 29 DNA Artificial Sequence
oligonucleotide 54 anggacggta tatatcacca tgaacaagt 29 55 29 DNA
Artificial Sequence oligonucleotide 55 anaggcagga ggagacggga
ttgatggtt 29 56 29 DNA Artificial Sequence oligonucleotide 56
anaagcgtca tgcagagcca tgatgaggg 29 57 29 DNA Artificial Sequence
oligonucleotide 57 anaaatgtag caggcttggc ttgcagcag 29 58 29 DNA
Artificial Sequence oligonucleotide 58 angacccatt tccagtccaa
atctttgac 29 59 28 DNA Artificial Sequence oligonucleotide 59
gncaaggtgt ctgtaggaga aactatat 28 60 29 DNA Artificial Sequence
oligonucleotide 60 anccagggct atacagagaa accctgtct 29 61 29 DNA
Artificial Sequence oligonucleotide 61 gntcttgaag aagtagccca
gggtgagga 29 62 29 DNA Artificial Sequence oligonucleotide 62
cnggtaaagg tgatgttgac actgtagga 29
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