U.S. patent application number 12/945589 was filed with the patent office on 2011-03-10 for novel pro258 gene disruptions, and methods relating thereto.
Invention is credited to JOEL A. EDWARDS, Charles Montgomery, Zheng-Zheng Shi, Mary Jean Sparks.
Application Number | 20110061114 12/945589 |
Document ID | / |
Family ID | 35677379 |
Filed Date | 2011-03-10 |
United States Patent
Application |
20110061114 |
Kind Code |
A1 |
EDWARDS; JOEL A. ; et
al. |
March 10, 2011 |
NOVEL PRO258 GENE DISRUPTIONS, AND METHODS RELATING THERETO
Abstract
The present invention relates to transgenic animals, as well as
compositions and methods relating to the characterization of gene
function. Specifically, the present invention provides transgenic
mice comprising disruptions in PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
genes. Such in vivo studies and characterizations may provide
valuable identification and discovery of therapeutics and/or
treatments useful in the prevention, amelioration or correction of
diseases or dysfunctions associated with gene disruptions such as
neurological disorders; cardiovascular, endothelial or angiogenic
disorders; eye abnormalities; immunological disorders; oncological
disorders; bone metabolic abnormalities or disorders; lipid
metabolic disorders; or developmental abnormalities.
Inventors: |
EDWARDS; JOEL A.; (The
Woodlands, TX) ; Montgomery; Charles; (Jay, OK)
; Shi; Zheng-Zheng; (The Woodlands, TX) ; Sparks;
Mary Jean; (Magnolia, TX) |
Family ID: |
35677379 |
Appl. No.: |
12/945589 |
Filed: |
November 12, 2010 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11572702 |
Jan 25, 2007 |
|
|
|
PCT/US05/29782 |
Aug 23, 2005 |
|
|
|
12945589 |
|
|
|
|
60604323 |
Aug 25, 2004 |
|
|
|
Current U.S.
Class: |
800/3 ;
424/172.1; 435/29; 435/325; 435/352; 514/1.7; 514/1.9; 514/13.3;
514/13.5; 514/15.4; 514/15.7; 514/16.4; 514/16.6; 514/16.8;
514/16.9; 514/17.7; 514/19.3; 514/4.3; 514/6.9 |
Current CPC
Class: |
A61P 13/12 20180101;
A61P 11/00 20180101; A61P 27/02 20180101; A61P 35/00 20180101; A61P
25/28 20180101; A61P 37/00 20180101; C12N 15/8509 20130101; A61P
17/06 20180101; A01K 2227/105 20130101; A61P 31/14 20180101; A61P
9/00 20180101; A61P 19/10 20180101; A61P 25/24 20180101; A61P 37/02
20180101; A61P 1/16 20180101; A61P 3/10 20180101; A61P 5/14
20180101; A61P 19/08 20180101; A61P 17/00 20180101; A61P 7/06
20180101; A61P 25/00 20180101; A01K 67/0276 20130101; A61P 9/04
20180101; A61P 9/12 20180101; A61P 17/08 20180101; A01K 2267/03
20130101; A61P 25/18 20180101; A61P 5/16 20180101; A61P 25/02
20180101; A61P 37/06 20180101; A01K 2267/02 20130101; A61P 1/04
20180101; A61P 9/10 20180101; A61P 27/12 20180101; A01K 2217/05
20130101; A61P 25/22 20180101; A61P 17/02 20180101; A61P 19/02
20180101; A61P 25/20 20180101; A61P 37/08 20180101; A61P 11/06
20180101; A61P 29/00 20180101 |
Class at
Publication: |
800/3 ; 435/325;
435/352; 435/29; 424/172.1; 514/17.7; 514/16.4; 514/13.3; 514/6.9;
514/19.3; 514/16.6; 514/15.4; 514/16.9; 514/13.5; 514/1.9; 514/4.3;
514/16.8; 514/1.7; 514/15.7 |
International
Class: |
G01N 33/00 20060101
G01N033/00; C12N 5/10 20060101 C12N005/10; C12Q 1/02 20060101
C12Q001/02; A61K 39/395 20060101 A61K039/395; A61K 38/02 20060101
A61K038/02; A61P 9/00 20060101 A61P009/00; A61P 35/00 20060101
A61P035/00; A61P 3/10 20060101 A61P003/10; A61P 29/00 20060101
A61P029/00; A61P 13/12 20060101 A61P013/12; A61P 19/10 20060101
A61P019/10; A61P 7/06 20060101 A61P007/06; A61P 9/10 20060101
A61P009/10; A61P 31/14 20060101 A61P031/14; A61P 25/00 20060101
A61P025/00; A61P 19/02 20060101 A61P019/02; A61P 25/28 20060101
A61P025/28; A61P 37/02 20060101 A61P037/02; A61P 37/06 20060101
A61P037/06; A61P 37/08 20060101 A61P037/08; A61P 11/06 20060101
A61P011/06; A61P 9/12 20060101 A61P009/12 |
Claims
1. A method of identifying a phenotype associated with a disruption
of a gene which encodes for a PRO258 polypeptide, the method
comprising: (a) providing a non-human transgenic animal whose
genome comprises a disruption of the gene which encodes for a
PRO258 polypeptide; (b) measuring a physiological characteristic of
the non-human transgenic animal; and (c) comparing the measured
physiological characteristic with that of a gender matched
wild-type animal, wherein the physiological characteristic of the
non-human transgenic animal that differs from the physiological
characteristic of the wild-type animal is identified as a phenotype
resulting from the gene disruption in the non-human transgenic
animal.
2. The method of claim 1, wherein the non-human transgenic animal
is heterozygous for the disruption of a gene which encodes for a
PRO258 polypeptide.
3. The method of claim 1, wherein the phenotype exhibited by the
non-human transgenic animal as compared with gender matched
wild-type littermates is at least one of the following: a
neurological disorder; a cardiovascular, endothelial or angiogenic
disorder; an immunological disorder; a bone metabolic abnormality
or disorder; or a lipid metabolic disorder.
4-5. (canceled)
6. The method of claim 3, wherein the neurological disorder is an
abnormal circadian rhythm during home-cage activity testing.
7. The method of claim 3, wherein the neurological disorder is an
enhanced motor coordination during inverted screen testing.
8-17. (canceled)
18. The method of claim 3, wherein the cardiovascular, endothelial
or angiogenic disorders are arterial diseases, such as diabetes
mellitus; papilledema; optic atrophy; atherosclerosis; angina;
myocardial infarctions such as acute myocardial infarctions,
cardiac hypertrophy, and heart failure such as congestive heart
failure; hypertension; inflammatory vasculitides; Reynaud's disease
and Reynaud's phenomenon; aneurysms and arterial restenosis; venous
and lymphatic disorders such as thrombophlebitis, lymphangitis, and
lymphedema; peripheral vascular disease; cancer such as vascular
tumors, e.g., hemangioma (capillary and cavernous), glomus tumors,
telangiectasia, bacillary angiomatosis, hemangioendothelioma,
angiosarcoma, haemangiopericytoma, Kaposi's sarcoma, lymphangioma,
and lymphangiosarcoma; tumor angiogenesis; trauma such as wounds,
burns, and other injured tissue, implant fixation, scarring;
ischemia reperfusion injury; rheumatoid arthritis; cerebrovascular
disease; renal diseases such as acute renal failure, or
osteoporosis.
19. The method of claim 3, wherein the immunological disorders are
systemic lupus erythematosis; rheumatoid arthritis; juvenile
chronic arthritis; spondyloarthropathies; systemic sclerosis
(scleroderma); idiopathic inflammatory myopathies (dermatomyositis,
polymyositis); Sjogren's syndrome; systemic vasculitis;
sarcoidosis; autoimmune hemolytic anemia (immune pancytopenia,
paroxysmal nocturnal hemoglobinuria); autoimmune thrombocytopenia
(idiopathic thrombocytopenic purpura, immune-mediated
thrombocytopenia); thyroiditis (Grave's disease, Hashimoto's
thyroiditis, juvenile lymphocytic thyroiditis, atrophic
thyroiditis); diabetes mellitus; immune-mediated renal disease
(glomerulonephritis, tubulointerstitial nephritis); demyelinating
diseases of the central and peripheral nervous systems such as
multiple sclerosis, idiopathic demyelinating polyneuropathy or
Guillain-Barre syndrome, and chronic inflammatory demyelinating
polyneuropathy; hepatobiliary diseases such as infectious hepatitis
(hepatitis A, B, C, D, E and other non-hepatotropic viruses),
autoimmune chronic active hepatitis, primary biliary cirrhosis,
granulomatous hepatitis, and sclerosing cholangitis; inflammatory
bowel disease (ulcerative colitis: Crohn's disease);
gluten-sensitive enteropathy, and Whipple's disease; autoimmune or
immune-mediated skin diseases including bullous skin diseases,
erythema multiforme and contact dermatitis, psoriasis; allergic
diseases such as asthma, allergic rhinitis, atopic dermatitis, food
hypersensitivity and urticaria; immunologic diseases of the lung
such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and
hypersensitivity pneumonitis; or transplantation associated
diseases including graft rejection and graft-versus-host
disease.
20. The method of claim 3, wherein the bone metabolic abnormality
or disorder is arthritis, osteoporosis or osteopetrosis.
21. The method of claim 1, wherein the non-human transgenic animal
exhibits at least one of the following physiological
characteristics compared with gender matched wild-type littermates:
abnormal circadian rhythm during home-cage activity testing
including decreased ambulatory counts; enhanced motor coordination
during inverted screen testing; increased mean fasting serum
glucose levels; decreased heart rate; increased mean serum
cholesterol levels; increased mean percentage of CD4 cells and
decreased mean percentage of B cells; decreased mean serum IgG2a
response to an ovalbumin challenge; decreased mean total white
blood cell (WBC) counts; decreased absolute lymphocyte counts;
decreased absolute monocyte counts; decreased total femoral
midshaft cross-sectional area; decreased mean vertebral trabecular
bone volume, number and connectivity density.
22. An isolated cell derived from a non-human transgenic animal
whose genome comprises a disruption of the gene which encodes for a
PRO258 polypeptide.
23. The isolated cell of claim 22 which is a murine cell.
24. The isolated cell of claim 23, wherein the murine cell is an
embryonic stem cell.
25. The isolated cell of claim 22, wherein the non-human transgenic
animal exhibits at least one of the following phenotypes compared
with gender matched wild-type littermates: a neurological disorder;
a cardiovascular, endothelial or angiogenic disorder; an
immunological disorder; a bone metabolic abnormality or disorder;
or a lipid metabolic disorder.
26. A method of identifying an agent that modulates a phenotype
associated with a disruption of a gene which encodes for a PRO258
polypeptide, the method comprising: (a) providing a non-human
transgenic animal whose genome comprises a disruption of the gene
which encodes for the PRO258 polypeptide; (b) measuring a
physiological characteristic of the non-human transgenic animal of
(a); (c) comparing the measured physiological characteristic of (b)
with that of a gender matched wild-type animal, wherein the
physiological characteristic of the non-human transgenic animal
that differs from the physiological characteristic of the wild-type
animal is identified as a phenotype resulting from the gene
disruption in the non-human transgenic animal; (d) administering a
test agent to the non-human transgenic animal of (a); and (e)
determining whether the test agent modulates the identified
phenotype associated with gene disruption in the non-human
transgenic animal.
27. The method of claim 26, wherein the phenotype associated with
the gene disruption comprises a neurological disorder; a
cardiovascular, endothelial or angiogenic disorder; an eye
abnormality; an immunological disorder; an oncological disorder; a
bone metabolic abnormality or disorder; a lipid metabolic disorder;
or a developmental abnormality.
28-29. (canceled)
30. The method of claim 27, wherein the neurological disorder is an
abnormal circadian rhythm during home-cage activity testing.
31. The method of claim 27, wherein the neurological disorder is an
enhanced motor coordination during inverted screen testing.
32-41. (canceled)
42. The method of claim 27, wherein the cardiovascular, endothelial
or angiogenic disorders are arterial diseases, such as diabetes
mellitus; papilledema; optic atrophy; atherosclerosis; angina;
myocardial infarctions such as acute myocardial infarctions,
cardiac hypertrophy, and heart failure such as congestive heart
failure; hypertension; inflammatory vasculitides; Reynaud's disease
and Reynaud's phenomenon; aneurysms and arterial restenosis; venous
and lymphatic disorders such as thrombophlebitis, lymphangitis, and
lymphedema; peripheral vascular disease; cancer such as vascular
tumors, e.g., hemangioma (capillary and cavernous), glomus tumors,
telangiectasia, bacillary angiomatosis, hemangioendothelioma,
angiosarcoma, haemangiopericytoma, Kaposi's sarcoma, lymphangioma,
and lymphangiosarcoma; tumor angiogenesis; trauma such as wounds,
burns, and other injured tissue, implant fixation, scarring;
ischemia reperfusion injury; rheumatoid arthritis; cerebrovascular
disease; renal diseases such as acute renal failure, or
osteoporosis.
43. The method of claim 27, wherein the immunological disorders are
systemic lupus erythematosis; rheumatoid arthritis; juvenile
chronic arthritis; spondyloarthropathies; systemic sclerosis
(scleroderma); idiopathic inflammatory myopathies (dermatomyositis,
polymyositis); Sjogren's syndrome; systemic vasculitis;
sarcoidosis; autoimmune hemolytic anemia (immune pancytopenia,
paroxysmal nocturnal hemoglobinuria); autoimmune thrombocytopenia
(idiopathic thrombocytopenic purpura, immune-mediated
thrombocytopenia); thyroiditis (Grave's disease, Hashimoto's
thyroiditis, juvenile lymphocytic thyroiditis, atrophic
thyroiditis); diabetes mellitus; immune-mediated renal disease
(glomerulonephritis, tubulointerstitial nephritis); demyelinating
diseases of the central and peripheral nervous systems such as
multiple sclerosis, idiopathic demyelinating polyneuropathy or
Guillain-Barre syndrome, and chronic inflammatory demyelinating
polyneuropathy; hepatobiliary diseases such as infectious hepatitis
(hepatitis A, B, C, D, E and other non-hepatotropic viruses),
autoimmune chronic active hepatitis, primary biliary cirrhosis,
granulomatous hepatitis, and sclerosing cholangitis; inflammatory
bowel disease (ulcerative colitis: Crohn's disease);
gluten-sensitive enteropathy, and Whipple's disease; autoimmune or
immune-mediated skin diseases including bullous skin diseases,
erythema multiforme and contact dermatitis, psoriasis; allergic
diseases such as asthma, allergic rhinitis, atopic dermatitis, food
hypersensitivity and urticaria; immunologic diseases of the lung
such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and
hypersensitivity pneumonitis; or transplantation-associated
diseases including graft rejection and graft-versus-host
disease.
44. The method of claim 27, wherein said bone metabolic abnormality
or disorder is arthritis, osteoporosis or osteopetrosis.
45. The method of claim 26, wherein the non-human transgenic animal
exhibits at least one of the following physiological
characteristics compared with gender matched wild-type littermates:
abnormal circadian rhythm during home-cage activity testing
including decreased ambulatory counts; enhanced motor coordination
during inverted screen testing; increased mean fasting serum
glucose levels; decreased heart rate; increased mean serum
cholesterol levels; increased mean percentage of CD4 cells and
decreased mean percentage of B cells; decreased mean serum IgG2a
response to an ovalbumin challenge; decreased mean total white
blood cell (WBC) counts; decreased absolute lymphocyte counts;
decreased absolute monocyte counts; decreased total femoral
midshaft cross-sectional area; decreased mean vertebral trabecular
bone volume, number and connectivity density.
46-49. (canceled)
50. A method of identifying an agent that modulates a physiological
characteristic associated with a disruption of the gene which
encodes for a PRO258 polypeptide, the method comprising: (a)
providing a non-human transgenic animal whose genome comprises a
disruption of the gene which encodes for a PRO258 polypeptide; (b)
measuring a physiological characteristic exhibited by the non-human
transgenic animal of (a); (c) comparing the measured physiological
characteristic of (b) with that of a gender matched wild-type
animal, wherein the physiological characteristic exhibited by the
non-human transgenic animal that differs from the physiological
characteristic exhibited by the wild-type animal is identified as a
physiological characteristic associated with gene disruption; (d)
administering a test agent to the non-human transgenic animal of
(a); and (e) determining whether the physiological characteristic
associated with gene disruption is modulated.
51. The method of claim 50, wherein the non-human transgenic animal
exhibits at least one of the following physiological
characteristics compared with gender matched wild-type littermates:
abnormal circadian rhythm during home-cage activity testing
including decreased ambulatory counts; enhanced motor coordination
during inverted screen testing; increased mean fasting serum
glucose levels; decreased heart rate; increased mean serum
cholesterol levels; increased mean percentage of CD4 cells and
decreased mean percentage of B cells; decreased mean serum IgG2a
response to an ovalbumin challenge; decreased mean total white
blood cell (WBC) counts; decreased absolute lymphocyte counts;
decreased absolute monocyte counts; decreased total femoral
midshaft cross-sectional area; decreased mean vertebral trabecular
bone volume, number and connectivity density.
52-55. (canceled)
56. A method of identifying an agent which modulates a behavior
associated with a disruption of the gene which encodes for a PRO258
polypeptide, the method comprising: (a) providing a non-human
transgenic animal whose genome comprises a disruption of the gene
which encodes for a PRO258 polypeptide; (b) observing the behavior
exhibited by the non-human transgenic animal of (a); (c) comparing
the observed behavior of (b) with that of a gender matched
wild-type animal, wherein the observed behavior exhibited by the
non-human transgenic animal that differs from the observed behavior
exhibited by the wild-type animal is identified as a behavior
associated with gene disruption; (d) administering a test agent to
the non-human transgenic animal of (a); and (e) determining whether
the agent modulates the behavior associated with gene
disruption.
57-58. (canceled)
59. The method of claim 56, wherein the behavior is an abnormal
circadian rhythm during home-cage activity testing.
60. The method of claim 56, wherein the behavior is an enhanced
motor coordination during inverted screen testing.
61-66. (canceled)
67. A method of identifying an agent that ameliorates or modulates
a neurological disorder; a cardiovascular, endothelial or
angiogenic disorder; an immunological disorder; a bone metabolic
abnormality or disorder; or a lipid metabolic disorder associated
with a disruption in the gene which encodes for a PRO258
polypeptide, the method comprising: (a) providing a non-human
transgenic animal whose genome comprises a disruption of the gene
which encodes for a PRO258 polypeptide; (b) administering a test
agent to said non-human transgenic animal; and (c) determining
whether said test agent ameliorates or modulates the neurological
disorder; cardiovascular, endothelial or angiogenic disorder;
immunological disorder; bone metabolic abnormality or disorder; or
lipid metabolic disorder; in the non-human transgenic animal.
68-69. (canceled)
70. The method of claim 67, wherein the neurological disorder is an
abnormal circadian rhythm during home-cage activity testing.
71. The method of claim 67, wherein the neurological disorder is an
enhanced motor coordination during inverted screen testing.
72-81. (canceled)
82. The method of claim 67, wherein the cardiovascular, endothelial
or angiogenic disorders are arterial diseases, such as diabetes
mellitus; papilledema; optic atrophy; atherosclerosis; angina;
myocardial infarctions such as acute myocardial infarctions,
cardiac hypertrophy, and heart failure such as congestive heart
failure; hypertension; inflammatory vasculitides; Reynaud's disease
and Reynaud's phenomenon; aneurysms and arterial restenosis; venous
and lymphatic disorders such as thrombophlebitis, lymphangitis, and
lymphedema; peripheral vascular disease; cancer such as vascular
tumors, e.g., hemangioma (capillary and cavernous), glomus tumors,
telangiectasia, bacillary angiomatosis, hemangioendothelioma,
angiosarcoma, haemangiopericytoma, Kaposi's sarcoma, lymphangioma,
and lymphangiosarcoma; tumor angiogenesis; trauma such as wounds,
burns, and other injured tissue, implant fixation, scarring;
ischemia reperfusion injury; rheumatoid arthritis; cerebrovascular
disease; renal diseases such as acute renal failure, or
osteoporosis.
83. The method of claim 67, wherein the immunological disorders are
systemic lupus erythematosis; rheumatoid arthritis; juvenile
chronic arthritis; spondyloarthropathies; systemic sclerosis
(scleroderma); idiopathic inflammatory myopathies (dermatomyositis,
polymyositis); Sjogren's syndrome; systemic vasculitis;
sarcoidosis; autoimmune hemolytic anemia (immune pancytopenia,
paroxysmal nocturnal hemoglobinuria); autoimmune thrombocytopenia
(idiopathic thrombocytopenic purpura, immune-mediated
thrombocytopenia); thyroiditis (Grave's disease, Hashimoto's
thyroiditis, juvenile lymphocytic thyroiditis, atrophic
thyroiditis); diabetes mellitus; immune-mediated renal disease
(glomerulonephritis, tubulointerstitial nephritis); demyelinating
diseases of the central and peripheral nervous systems such as
multiple sclerosis, idiopathic demyelinating polyneuropathy or
Guillain-Barre syndrome, and chronic inflammatory demyelinating
polyneuropathy; hepatobiliary diseases such as infectious hepatitis
(hepatitis A, B, C, D, E and other non-hepatotropic viruses),
autoimmune chronic active hepatitis, primary biliary cirrhosis,
granulomatous hepatitis, and sclerosing cholangitis; inflammatory
bowel disease (ulcerative colitis: Crohn's disease);
gluten-sensitive enteropathy, and Whipple's disease; autoimmune or
immune-mediated skin diseases including bullous skin diseases,
erythema multiforme and contact dermatitis, psoriasis; allergic
diseases such as asthma, allergic rhinitis, atopic dermatitis, food
hypersensitivity and urticaria; immunologic diseases of the lung
such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and
hypersensitivity pneumonitis; or transplantation associated
diseases including graft rejection and graft-versus-host
disease.
84. The method of claim 67, wherein said bone metabolic abnormality
or disorder is arthritis, osteoporosis or osteopetrosis.
85. The method of claim 67, wherein the non-human transgenic animal
exhibits at least one of the following physiological
characteristics compared with gender matched wild-type littermates:
abnormal circadian rhythm during home-cage activity testing
including decreased ambulatory counts; enhanced motor coordination
during inverted screen testing; increased mean fasting serum
glucose levels; decreased heart rate; increased mean serum
cholesterol levels; increased mean percentage of CD4 cells and
decreased mean percentage of B cells; decreased mean serum IgG2a
response to an ovalbumin challenge; decreased mean total white
blood cell (WBC) counts; decreased absolute lymphocyte counts;
decreased absolute monocyte counts; decreased total femoral
midshaft cross-sectional area; decreased mean vertebral trabecular
bone volume, number and connectivity density.
86-90. (canceled)
91. A method of identifying an agent that modulates the expression
of a PRO258 polypeptide, the method comprising: (a) contacting a
test agent with a host cell expressing a PRO258 polypeptide; and
(b) determining whether the test agent modulates the expression of
the PRO258 polypeptide by the host cell.
92-95. (canceled)
96. A method of evaluating a therapeutic agent capable of affecting
a condition associated with a disruption of a gene which encodes
for a PRO258 polypeptide, the method comprising: (a) providing a
non-human transgenic animal whose genome comprises a disruption of
the gene which encodes for the PRO258 polypeptide; (b) measuring a
physiological characteristic of the non-human transgenic animal of
(a); (c) comparing the measured physiological characteristic of (b)
with that of a gender matched wild-type animal, wherein the
physiological characteristic of the non-human transgenic animal
that differs from the physiological characteristic of the wild-type
animal is identified as a condition resulting from the gene
disruption in the non-human transgenic animal; (d) administering a
test agent to the non-human transgenic animal of (a); and (e)
evaluating the effects of the test agent on the identified
condition associated with gene disruption in the non-human
transgenic animal.
97. The method of claim 96, wherein the condition is a neurological
disorder; a cardiovascular, endothelial or angiogenic disorder; an
immunological disorder; a bone metabolic abnormality or disorder;
or a lipid metabolic disorder.
98-102. (canceled)
103. A method of treating or preventing or ameliorating a
neurological disorder; cardiovascular, endothelial or angiogenic
disorder; immunological disorder; bone metabolic abnormality or
disorder; or lipid metabolic disorder associated with the
disruption of a gene which encodes for a PRO258 polypeptide, the
method comprising administering to a subject in need of such
treatment whom may already have the disorder, or may be prone to
have the disorder or may be in whom the disorder is to be
prevented, a therapeutically effective amount of therapeutic agent
identified by the method of claim 91, or agonists or antagonists
thereof, thereby effectively treating or preventing or ameliorating
said disorder.
104-105. (canceled)
106. The method of claim 103, wherein the neurological disorder is
an abnormal circadian rhythm during home-cage activity testing.
107. The method of claim 103, wherein the neurological disorder is
an enhanced motor coordination during inverted screen testing.
108-117. (canceled)
118. The method of claim 103, wherein the cardiovascular,
endothelial or angiogenic disorders are arterial diseases, such as
diabetes mellitus; papilledema; optic atrophy; atherosclerosis;
angina; myocardial infarctions such as acute myocardial
infarctions, cardiac hypertrophy, and heart failure such as
congestive heart failure; hypertension; inflammatory vasculitides;
Reynaud's disease and Reynaud's phenomenon; aneurysms and arterial
restenosis; venous and lymphatic disorders such as
thrombophlebitis, lymphangitis, and lymphedema; peripheral vascular
disease; cancer such as vascular tumors, e.g., hemangioma
(capillary and cavernous), glomus tumors, telangiectasia, bacillary
angiomatosis, hemangioendothelioma, angiosarcoma,
haemangiopericytoma, Kaposi's sarcoma, lymphangioma, and
lymphangiosarcoma; tumor angiogenesis; trauma such as wounds,
burns, and other injured tissue, implant fixation, scarring;
ischemia reperfusion injury; rheumatoid arthritis; cerebrovascular
disease; renal diseases such as acute renal failure, or
osteoporosis.
119. The method of claim 103, wherein the immunological disorders
are systemic lupus erythematosis; rheumatoid arthritis; juvenile
chronic arthritis; spondyloarthropathies; systemic sclerosis
(scleroderma); idiopathic inflammatory myopathies (dermatomyositis,
polymyositis); Sjogren's syndrome; systemic vasculitis;
sarcoidosis; autoimmune hemolytic anemia (immune pancytopenia,
paroxysmal nocturnal hemoglobinuria); autoimmune thrombocytopenia
(idiopathic thrombocytopenic purpura, immune-mediated
thrombocytopenia); thyroiditis (Grave's disease, Hashimoto's
thyroiditis, juvenile lymphocytic thyroiditis, atrophic
thyroiditis); diabetes mellitus; immune-mediated renal disease
(glomerulonephritis, tubulointerstitial nephritis); demyelinating
diseases of the central and peripheral nervous systems such as
multiple sclerosis, idiopathic demyelinating polyneuropathy or
Guillain-Barre syndrome, and chronic inflammatory demyelinating
polyneuropathy; hepatobiliary diseases such as infectious hepatitis
(hepatitis A, B, C, D, E and other non-hepatotropic viruses),
autoimmune chronic active hepatitis, primary biliary cirrhosis,
granulomatous hepatitis, and sclerosing cholangitis; inflammatory
bowel disease (ulcerative colitis: Crohn's disease);
gluten-sensitive enteropathy, and Whipple's disease; autoimmune or
immune-mediated skin diseases including bullous skin diseases,
erythema multiforme and contact dermatitis, psoriasis; allergic
diseases such as asthma, allergic rhinitis, atopic dermatitis, food
hypersensitivity and urticaria; immunologic diseases of the lung
such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and
hypersensitivity pneumonitis; or transplantation associated
diseases including graft rejection and graft-versus-host
disease.
120. The method of claim 103, wherein said bone metabolic
abnormality or disorder is arthritis, osteoporosis or
osteopetrosis.
121. A method of identifying an agent that ameliorates or modulates
a neurological disorder; a cardiovascular, endothelial or
angiogenic disorder; an immunological disorder; a bone metabolic
abnormality or disorder; or a lipid metabolic disorder associated
with a disruption in the gene which encodes for a PRO258
polypeptide, the method comprising: (a) providing a non-human
transgenic animal cell culture, each cell of said culture
comprising a disruption of the gene which encodes for a PRO258
polypeptide; (b) administering a test agent to said cell culture;
and (c) determining whether said test agent ameliorates or
modulates the neurological disorder; cardiovascular, endothelial or
angiogenic disorder; eye abnormality; immunological disorder;
oncological disorder; bone metabolic abnormality or disorder; lipid
metabolic disorder; or developmental abnormality in said cell
culture.
122-123. (canceled)
124. The method of claim 121, wherein the neurological disorder is
an abnormal circadian rhythm during home-cage activity testing.
125. The method of claim 121, wherein the neurological disorder is
an enhanced motor coordination during inverted screen testing.
126. (canceled)
127. The method of claim 121, wherein the neurological disorder is
depression, generalized anxiety disorders, attention deficit
disorder, sleep disorder, hyperactivity disorder, obsessive
compulsive disorder, schizophrenia, cognitive disorders,
hyperalgesia or sensory disorders.
128-135. (canceled)
136. The method of claim 121, wherein the cardiovascular,
endothelial or angiogenic disorders are arterial diseases, such as
diabetes mellitus; papilledema; optic atrophy; atherosclerosis;
angina; myocardial infarctions such as acute myocardial
infarctions, cardiac hypertrophy, and heart failure such as
congestive heart failure; hypertension; inflammatory vasculitides;
Reynaud's disease and Reynaud's phenomenon; aneurysms and arterial
restenosis; venous and lymphatic disorders such as
thrombophlebitis, lymphangitis, and lymphedema; peripheral vascular
disease; cancer such as vascular tumors, e.g., hemangioma
(capillary and cavernous), glomus tumors, telangiectasia, bacillary
angiomatosis, hemangioendothelioma, angiosarcoma,
haemangiopericytoma, Kaposi's sarcoma, lymphangioma, and
lymphangiosarcoma; tumor angiogenesis; trauma such as wounds,
burns, and other injured tissue, implant fixation, scarring;
ischemia reperfusion injury; rheumatoid arthritis; cerebrovascular
disease; renal diseases such as acute renal failure, or
osteoporosis.
137. The method of claim 121, wherein the immunological disorders
are systemic lupus erythematosis; rheumatoid arthritis; juvenile
chronic arthritis; spondyloarthropathies; systemic sclerosis
(scleroderma); idiopathic inflammatory myopathies (dermatomyositis,
polymyositis); Sjogren's syndrome; systemic vasculitis;
sarcoidosis; autoimmune hemolytic anemia (immune pancytopenia,
paroxysmal nocturnal hemoglobinuria); autoimmune thrombocytopenia
(idiopathic thrombocytopenic purpura, immune-mediated
thrombocytopenia); thyroiditis (Grave's disease, Hashimoto's
thyroiditis, juvenile lymphocytic thyroiditis, atrophic
thyroiditis); diabetes mellitus; immune-mediated renal disease
(glomerulonephritis, tubulointerstitial nephritis); demyelinating
diseases of the central and peripheral nervous systems such as
multiple sclerosis, idiopathic demyelinating polyneuropathy or
Guillain-Barre syndrome, and chronic inflammatory demyelinating
polyneuropathy; hepatobiliary diseases such as infectious hepatitis
(hepatitis A, B, C, D, E and other non-hepatotropic viruses),
autoimmune chronic active hepatitis, primary biliary cirrhosis,
granulomatous hepatitis, and sclerosing cholangitis; inflammatory
bowel disease (ulcerative colitis: Crohn's disease);
gluten-sensitive enteropathy, and Whipple's disease; autoimmune or
immune-mediated skin diseases including bullous skin diseases,
erythema multiforme and contact dermatitis, psoriasis; allergic
diseases such as asthma, allergic rhinitis, atopic dermatitis, food
hypersensitivity and urticaria; immunologic diseases of the lung
such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and
hypersensitivity pneumonitis; or transplantation associated
diseases including graft rejection and graft-versus-host
disease.
138. The method of claim 121, wherein said bone metabolic
abnormality or disorder is arthritis, osteoporosis or
osteopetrosis.
139-143. (canceled)
144. A method of modulating a phenotype associated with a
disruption of a gene which encodes for a PRO258 polypeptide, the
method comprising administering to a subject whom may already have
the phenotype, or may be prone to have the phenotype or may be in
whom the phenotype is to be prevented, an effective amount of an
agent identified by the method of claim 26, or agonists or
antagonists thereof, thereby effectively modulating the
phenotype.
145. A method of modulating a physiological characteristic
associated with a disruption of a gene which encodes for a PRO258
polypeptide, the method comprising administering to a subject whom
may already exhibit the physiological characteristic, or may be
prone to exhibit the physiological characteristic or may be in whom
the physiological characteristic is to be prevented, an effective
amount of an agent identified by the method of claim 50, or
agonists or antagonists thereof, thereby effectively modulating the
physiological characteristic.
146. A method of modulating a behavior associated with a disruption
of a gene which encodes for a PRO258 polypeptide, the method
comprising administering to a subject whom may already exhibit the
behavior, or may be prone to exhibit the behavior or may be in whom
the exhibited behavior is to be prevented, an effective amount of
an agent identified by the method of claim 56, or agonists or
antagonists thereof, thereby effectively modulating the
behavior.
147. A method of modulating the expression of a PRO258 polypeptide,
the method comprising administering to a host cell expressing said
PRO258 polypeptide, an effective amount of an agent identified by
the method of claim 91, or agonists or antagonists thereof, thereby
effectively modulating the expression of said polypeptide.
148. A method of modulating a condition associated with a
disruption of a gene which encodes for a PRO258 polypeptide, the
method comprising administering to a subject whom may have the
condition, or may be prone to have the condition or may be in whom
the condition is to be prevented, a therapeutically effective
amount of a therapeutic agent identified by the method of claim 96,
or agonists or antagonists thereof, thereby effectively modulating
the condition.
149. A method of treating or preventing or ameliorating a
neurological disorder; cardiovascular, endothelial or angiogenic
disorder; immunological disorder; bone metabolic abnormality or
disorder, or lipid metabloc disorder associated with the disruption
of a gene which encodes for a PRO258 polypeptide, the method
comprising administering to a non-human transgenic animal cell
culture, each cell of said culture comprising a disruption of the
gene which encodes for a PRO258 polypeptide, a therapeutically
effective amount of an agent identified by the method of claim 121,
or agonists or antagonists thereof, thereby effectively treating or
preventing or ameliorating said disorder.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to compositions, including
transgenic and knockout animals and methods of using such
compositions for the diagnosis and treatment of diseases or
disorders.
BACKGROUND OF THE INVENTION
[0002] Extracellular proteins play important roles in, among other
things, the formation, differentiation and maintenance of
multicellular organisms. The fate of many individual cells, e.g.,
proliferation, migration, differentiation, or interaction with
other cells, is typically governed by information received from
other cells and/or the immediate environment. This information is
often transmitted by secreted polypeptides (for instance, mitogenic
factors, survival factors, cytotoxic factors, differentiation
factors, neuropeptides, and hormones) which are, in turn, received
and interpreted by diverse cell receptors or membrane-bound
proteins. These secreted polypeptides or signaling molecules
normally pass through the cellular secretory pathway to reach their
site of action in the extracellular environment.
[0003] Secreted proteins have various industrial applications,
including as pharmaceuticals, diagnostics, biosensors and
bioreactors. Most protein drugs available at present, such as
thrombolytic agents, interferons, interleukins, erythropoietins,
colony stimulating factors, and various other cytokines, are
secretory proteins. Their receptors, which are membrane proteins,
also have potential as therapeutic or diagnostic agents. Efforts
are being undertaken by both industry and academia to identify new,
native secreted proteins. Many efforts are focused on the screening
of mammalian recombinant DNA libraries to identify the coding
sequences for novel secreted proteins. Examples of screening
methods and techniques are described in the literature [see, for
example, Klein et al., Proc. Natl. Acad. Sci. 93:7108-7113 (1996);
U.S. Pat. No. 5,536,637)].
[0004] Membrane-bound proteins and receptors can play important
roles in, among other things, the formation, differentiation and
maintenance of multicellular organisms. The fate of many individual
cells, e.g., proliferation, migration, differentiation, or
interaction with other cells, is typically governed by information
received from other cells and/or the immediate environment. This
information is often transmitted by secreted polypeptides (for
instance, mitogenic factors, survival factors, cytotoxic factors,
differentiation factors, neuropeptides, and hormones) which are, in
turn, received and interpreted by diverse cell receptors or
membrane-bound proteins. Such membrane-bound proteins and cell
receptors include, but are not limited to, cytokine receptors,
receptor kinases, receptor phosphatases, receptors involved in
cell-cell interactions, and cellular adhesion molecules like
selectins and integrins. For instance, transduction of signals that
regulate cell growth and differentiation is regulated in part by
phosphorylation of various cellular proteins. Protein tyrosine
kinases, enzymes that catalyze that process, can also act as growth
factor receptors. Examples include fibroblast growth factor
receptor and nerve growth factor receptor.
[0005] Membrane-bound proteins and receptor molecules have various
industrial applications, including as pharmaceutical and diagnostic
agents. Receptor immuno-adhesions, for instance, can be employed as
therapeutic agents to block receptor-ligand interactions. The
membrane-bound proteins can also be employed for screening of
potential peptide or small molecule inhibitors of the relevant
receptor/ligand interaction.
[0006] Efforts are being undertaken by both industry and academia
to identify new, native receptor or membrane-bound proteins. Many
efforts are focused on the screening of mammalian recombinant DNA
libraries to identify the coding sequences for novel receptor or
membrane-bound proteins.
[0007] Given the importance of secreted and membrane-bound proteins
in biological and disease processes, in vivo studies and
characterizations may provide valuable identification and discovery
of therapeutics and/or treatments useful in the prevention,
amelioration or correction of diseases or dysfunctions. In this
regard, genetically engineered mice have proven to be invaluable
tools for the functional dissection of biological processes
relevant to human disease, including immunology, cancer,
neuro-biology, cardiovascular biology, obesity and many others.
Gene knockouts can be viewed as modeling the biological mechanism
of drug action by presaging the activity of highly specific
antagonists in vivo. Knockout mice have been shown to model drug
activity; phenotypes of mice deficient for specific pharmaceutical
target proteins can resemble the human clinical phenotype caused by
the corresponding antagonist drug. Gene knockouts enable the
discovery of the mechanism of action of the target, the predominant
physiological role of the target, and mechanism-based side-effects
that might result from inhibition of the target in mammals.
Examples of this type include mice deficient in the angiotensin
converting enzyme (ACE) [Esther, C. R. et al., Lab. Invest.,
74:953-965 (1996)] and cyclooxygenase-1 (COX1) genes [Langenbach,
R. et al., Cell, 83:483-492 (1995)]. Conversely, knocking the gene
out in the mouse can have an opposite phenotypic effect to that
observed in humans after administration of an agonist drug to the
corresponding target. Examples include the erythropoietin knockout
[Wu, C. S. et al., Cell, 83:59-67 (1996)], in which a consequence
of the mutation is deficient red blood cell production, and the
GABA(A)-R-.beta.3 knockout [DeLorey, T. M., J. Neurosci.,
18:8505-8514 (1998)], in which the mutant mice show hyperactivity
and hyper-responsiveness. Both these phenotypes are opposite to the
effects of erythropoietin and benzodiazepine administration in
humans. A striking example of a target validated using mouse
genetics is the ACC2 gene. Although the human ACC2 gene had been
identified several years ago, interest in ACC2 as a target for drug
development was stimulated only recently after analysis of ACC2
function using a knockout mouse. ACC2 mutant mice eat more than
their wild-type littermates, yet burn more fat and store less fat
in their adipocytes, making this enzyme a probable target for
chemical antagonism in the treatment of obesity [Abu-Elheiga, L. et
al., Science, 291:2613-2616 (2001)].
[0008] In the instant application, mutated gene disruptions have
resulted in phenotypic observations related to various disease
conditions or dysfunctions including: CNS/neurological disturbances
or disorders such as anxiety; eye abnormalities and associated
diseases; cardiovascular, endothelial or angiogenic disorders
including atherosclerosis; abnormal metabolic disorders including
diabetes and dyslipidemias associated with elevated serum
triglycerides and cholesterol levels; immunological and
inflammatory disorders; oncological disorders; bone metabolic
abnormalities or disorders such as arthritis, osteoporosis and
osteopetrosis; or a developmental disease such as embryonic
lethality.
SUMMARY OF THE INVENTION
A. Embodiments
[0009] The invention provides an isolated nucleic acid molecule
comprising a nucleotide sequence that encodes a PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 polypeptide.
[0010] In one aspect, the isolated nucleic acid molecule comprises
a nucleotide sequence having at least about 80% nucleic acid
sequence identity, alternatively at least about 81% nucleic acid
sequence identity, alternatively at least about 82% nucleic acid
sequence identity, alternatively at least about 83% nucleic acid
sequence identity, alternatively at least about 84% nucleic acid
sequence identity, alternatively at least about 85% nucleic acid
sequence identity, alternatively at least about 86% nucleic acid
sequence identity, alternatively at least about 87% nucleic acid
sequence identity, alternatively at least about 88% nucleic acid
sequence identity, alternatively at least about 89% nucleic acid
sequence identity, alternatively at least about 90% nucleic acid
sequence identity, alternatively at least about 91% nucleic acid
sequence identity, alternatively at least about 92% nucleic acid
sequence identity, alternatively at least about 93% nucleic acid
sequence identity, alternatively at least about 94% nucleic acid
sequence identity, alternatively at least about 95% nucleic acid
sequence identity, alternatively at least about 96% nucleic acid
sequence identity, alternatively at least about 97% nucleic acid
sequence identity, alternatively at least about 98% nucleic acid
sequence identity and alternatively at least about 99% nucleic acid
sequence identity to (a) a DNA molecule encoding a PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 polypeptide having a full-length amino acid sequence as
disclosed herein, an amino acid sequence lacking the signal peptide
as disclosed herein, an extracellular domain of a transmembrane
protein, with or without the signal peptide, as disclosed herein or
any other specifically defined fragment of the full-length amino
acid sequence as disclosed herein, or (b) the complement of the DNA
molecule of (a).
[0011] In other aspects, the isolated nucleic acid molecule
comprises a nucleotide sequence having at least about 80% nucleic
acid sequence identity, alternatively at least about 81% nucleic
acid sequence identity, alternatively at least about 82% nucleic
acid sequence identity, alternatively at least about 83% nucleic
acid sequence identity, alternatively at least about 84% nucleic
acid sequence identity, alternatively at least about 85% nucleic
acid sequence identity, alternatively at least about 86% nucleic
acid sequence identity, alternatively at least about 87% nucleic
acid sequence identity, alternatively at least about 88% nucleic
acid sequence identity, alternatively at least about 89% nucleic
acid sequence identity, alternatively at least about 90% nucleic
acid sequence identity, alternatively at least about 91% nucleic
acid sequence identity, alternatively at least about 92% nucleic
acid sequence identity, alternatively at least about 93% nucleic
acid sequence identity, alternatively at least about 94% nucleic
acid sequence identity, alternatively at least about 95% nucleic
acid sequence identity, alternatively at least about 96% nucleic
acid sequence identity, alternatively at least about 97% nucleic
acid sequence identity, alternatively at least about 98% nucleic
acid sequence identity and alternatively at least about 99% nucleic
acid sequence identity to (a) a DNA molecule comprising the coding
sequence of a full-length PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide cDNA as disclosed herein, the coding sequence of a
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide lacking the signal peptide
as disclosed herein, the coding sequence of an extracellular domain
of a transmembrane PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide, with
or without the signal peptide, as disclosed herein or the coding
sequence of any other specifically defined fragment of the
full-length amino acid sequence as disclosed herein, or (b) the
complement of the DNA molecule of (a).
[0012] In a further aspect, the invention concerns an isolated
nucleic acid molecule comprising a nucleotide sequence having at
least about 80% nucleic acid sequence identity, alternatively at
least about 81% nucleic acid sequence identity, alternatively at
least about 82% nucleic acid sequence identity, alternatively at
least about 83% nucleic acid sequence identity, alternatively at
least about 84% nucleic acid sequence identity, alternatively at
least about 85% nucleic acid sequence identity, alternatively at
least about 86% nucleic acid sequence identity, alternatively at
least about 87% nucleic acid sequence identity, alternatively at
least about 88% nucleic acid sequence identity, alternatively at
least about 89% nucleic acid sequence identity, alternatively at
least about 90% nucleic acid sequence identity, alternatively at
least about 91% nucleic acid sequence identity, alternatively at
least about 92% nucleic acid sequence identity, alternatively at
least about 93% nucleic acid sequence identity, alternatively at
least about 94% nucleic acid sequence identity, alternatively at
least about 95% nucleic acid sequence identity, alternatively at
least about 96% nucleic acid sequence identity, alternatively at
least about 97% nucleic acid sequence identity, alternatively at
least about 98% nucleic acid sequence identity and alternatively at
least about 99% nucleic acid sequence identity to (a) a DNA
molecule that encodes the same mature polypeptide encoded by any of
the human protein cDNAs deposited with the ATCC as disclosed
herein, or (b) the complement of the DNA molecule of (a).
[0013] Another aspect of the invention provides an isolated nucleic
acid molecule comprising a nucleotide sequence encoding a PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide which is either transmembrane
domain-deleted or transmembrane domain-inactivated, or is
complementary to such encoding nucleotide sequence, wherein the
transmembrane domain(s) of such polypeptide are disclosed herein.
Therefore, soluble extracellular domains of the herein described
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptides are contemplated.
[0014] The invention also provides fragments of a PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 polypeptide coding sequence, or the complement thereof, that
may find use as, for example, hybridization probes, for encoding
fragments of a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide that
may optionally encode a polypeptide comprising a binding site for
an anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245,
anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344,
anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732,
anti-PRO1003, anti-PRO1104, anti-PRO1151, anti-PRO1244,
anti-PRO1298, anti-PRO1313, anti-PRO1570, anti-PRO1886,
anti-PRO1891, anti-PRO4409, anti-PRO5725, anti-PRO5994,
anti-PRO6097, anti-PRO7425, anti-PRO10102, anti-PRO10282,
anti-PRO61709 or anti-PRO779 antibody or as antisense
oligonucleotide probes. Such nucleic acid fragments usually are or
are at least about 10 nucleotides in length, alternatively are or
are at least about 15 nucleotides in length, alternatively are or
are at least about 20 nucleotides in length, alternatively are or
are at least about 30 nucleotides in length, alternatively are or
are at least about 40 nucleotides in length, alternatively are or
are at least about 50 nucleotides in length, alternatively are or
are at least about 60 nucleotides in length, alternatively are or
are at least about 70 nucleotides in length, alternatively are or
are at least about 80 nucleotides in length, alternatively are or
are at least about 90 nucleotides in length, alternatively are or
are at least about 100 nucleotides in length, alternatively are or
are at least about 110 nucleotides in length, alternatively are or
are at least about 120 nucleotides in length, alternatively are or
are at least about 130 nucleotides in length, alternatively are or
are at least about 140 nucleotides in length, alternatively are or
are at least about 150 nucleotides in length, alternatively are or
are at least about 160 nucleotides in length, alternatively are or
are at least about 170 nucleotides in length, alternatively are or
are at least about 180 nucleotides in length, alternatively are or
are at least about 190 nucleotides in length, alternatively are or
are at least about 200 nucleotides in length, alternatively are or
are at least about 250 nucleotides in length, alternatively are or
are at least about 300 nucleotides in length, alternatively are or
are at least about 350 nucleotides in length, alternatively are or
are at least about 400 nucleotides in length, alternatively are or
are at least about 450 nucleotides in length, alternatively are or
are at least about 500 nucleotides in length, alternatively are or
are at least about 600 nucleotides in length, alternatively are or
are at least about 700 nucleotides in length, alternatively are or
are at least about 800 nucleotides in length, alternatively are or
are at least about 900 nucleotides in length and alternatively are
or are at least about 1000 nucleotides in length, wherein in this
context the term "about" means the referenced nucleotide sequence
length plus or minus 10% of that referenced length. It is noted
that novel fragments of a PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide-encoding nucleotide sequence may be determined in a
routine manner by aligning the PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide-encoding nucleotide sequence with other known
nucleotide sequences using any of a number of well known sequence
alignment programs and determining which PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide-encoding nucleotide sequence fragment(s) are novel. All
of such PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 polypeptide-encoding
nucleotide sequences are contemplated herein. Also contemplated are
the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide fragments encoded by these
nucleotide molecule fragments, preferably those PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 polypeptide fragments that comprise a binding site for an
anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245,
anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344,
anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732,
anti-PRO1003, anti-PRO1104, anti-PRO1151, anti-PRO1244,
anti-PRO1298, anti-PRO1313, anti-PRO1570, anti-PRO1886,
anti-PRO1891, anti-PRO4409, anti-PRO5725, anti-PRO5994,
anti-PRO6097, anti-PRO7425, anti-PRO10102, anti-PRO10282,
anti-PRO61709 or anti-PRO779 antibody.
[0015] The invention provides isolated PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptides encoded by any of the isolated nucleic acid sequences
hereinabove identified.
[0016] In a certain aspect, the invention concerns an isolated
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide, comprising an amino acid
sequence having at least about 80% amino acid sequence identity,
alternatively at least about 81% amino acid sequence identity,
alternatively at least about 82% amino acid sequence identity,
alternatively at least about 83% amino acid sequence identity,
alternatively at least about 84% amino acid sequence identity,
alternatively at least about 85% amino acid sequence identity,
alternatively at least about 86% amino acid sequence identity,
alternatively at least about 87% amino acid sequence identity,
alternatively at least about 88% amino acid sequence identity,
alternatively at least about 89% amino acid sequence identity,
alternatively at least about 90% amino acid sequence identity,
alternatively at least about 91% amino acid sequence identity,
alternatively at least about 92% amino acid sequence identity,
alternatively at least about 93% amino acid sequence identity,
alternatively at least about 94% amino acid sequence identity,
alternatively at least about 95% amino acid sequence identity,
alternatively at least about 96% amino acid sequence identity,
alternatively at least about 97% amino acid sequence identity,
alternatively at least about 98% amino acid sequence identity and
alternatively at least about 99% amino acid sequence identity to a
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide having a full-length amino
acid sequence as disclosed herein, an amino acid sequence lacking
the signal peptide as disclosed herein, an extracellular domain of
a transmembrane protein, with or without the signal peptide, as
disclosed herein or any other specifically defined fragment of the
full-length amino acid sequence as disclosed herein.
[0017] In a further aspect, the invention concerns an isolated
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide comprising an amino acid
sequence having at least about 80% amino acid sequence identity,
alternatively at least about 81% amino acid sequence identity,
alternatively at least about 82% amino acid sequence identity,
alternatively at least about 83% amino acid sequence identity,
alternatively at least about 84% amino acid sequence identity,
alternatively at least about 85% amino acid sequence identity,
alternatively at least about 86% amino acid sequence identity,
alternatively at least about 87% amino acid sequence identity,
alternatively at least about 88% amino acid sequence identity,
alternatively at least about 89% amino acid sequence identity,
alternatively at least about 90% amino acid sequence identity,
alternatively at least about 91% amino acid sequence identity,
alternatively at least about 92% amino acid sequence identity,
alternatively at least about 93% amino acid sequence identity,
alternatively at least about 94% amino acid sequence identity,
alternatively at least about 95% amino acid sequence identity,
alternatively at least about 96% amino acid sequence identity,
alternatively at least about 97% amino acid sequence identity,
alternatively at least about 98% amino acid sequence identity and
alternatively at least about 99% amino acid sequence identity to an
amino acid sequence encoded by any of the human protein cDNAs
deposited with the ATCC as disclosed herein.
[0018] In one aspect, the invention concerns PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 variant polypeptides which are or are at least about 10
amino acids in length, alternatively are or are at least about 20,
30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170,
180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300,
310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430,
440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560,
570, 580, 590, 600 amino acids in length, or more. Optionally,
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 variant polypeptides will have or have
no more than one conservative amino acid substitution as compared
to the native PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide
sequence, alternatively will have or will have no more than 2, 3,
4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitution as
compared to the native PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide sequence.
[0019] In a specific aspect, the invention provides an isolated
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide without the N-terminal
signal sequence and/or the initiating methionine and is encoded by
a nucleotide sequence that encodes such an amino acid sequence as
hereinbefore described. Processes for producing the same are also
herein described, wherein those processes comprise culturing a host
cell comprising a vector which comprises the appropriate encoding
nucleic acid molecule under conditions suitable for expression of
the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide and recovering the PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide from the cell culture.
[0020] Another aspect the invention provides an isolated PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide which is either transmembrane
domain-deleted or transmembrane domain-inactivated. Processes for
producing the same are also herein described, wherein those
processes comprise culturing a host cell comprising a vector which
comprises the appropriate encoding nucleic acid molecule under
conditions suitable for expression of the PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide and recovering the PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide from the cell culture.
[0021] The invention provides agonists and antagonists of a native
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide as defined herein. In
particular, the agonist or antagonist is an anti-PRO196,
anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246,
anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357,
anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003,
anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298,
anti-PRO1313, anti-PRO1570, anti-PRO1886, anti-PRO1891,
anti-PRO4409, anti-PRO5725, anti-PRO5994, anti-PRO6097,
anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709 or
anti-PRO779 antibody or a small molecule.
[0022] The invention provides a method of identifying agonists or
antagonists to a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide which
comprise contacting the PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide with a candidate molecule and monitoring a biological
activity mediated by said PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide. Preferably, the PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide is a native PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide.
[0023] The invention provides a composition of matter comprising a
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide, or an agonist or
antagonist of a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide as
herein described, or an anti-PRO196, anti-PRO217, anti-PRO231,
anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258, anti-PRO287,
anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526, anti-PRO724,
anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104, anti-PRO1151,
anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570,
anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725,
anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102,
anti-PRO10282, anti-PRO61709 or anti-PRO779 antibody, in
combination with a carrier. Optionally, the carrier is a
pharmaceutically acceptable carrier.
[0024] The invention provides the use of a PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide, or an agonist or antagonist thereof as hereinbefore
described, or an anti-PRO196, anti-PRO217, anti-PRO231,
anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258, anti-PRO287,
anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526, anti-PRO724,
anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104, anti-PRO1151,
anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570,
anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725,
anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102,
anti-PRO10282, anti-PRO61709 or anti-PRO779 antibody, for the
preparation of a medicament useful in the treatment of a condition
which is responsive to the anti-PRO196, anti-PRO217, anti-PRO231,
anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258, anti-PRO287,
anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526, anti-PRO724,
anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104, anti-PRO1151,
anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570,
anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725,
anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102,
anti-PRO10282, anti-PRO61709 or anti-PRO779 antibody.
[0025] The invention provides vectors comprising DNA encoding any
of the herein described polypeptides. Host cell comprising any such
vector are also provided. By way of example, the host cells may be
CHO cells, E. coli, or yeast. A process for producing any of the
herein described polypeptides is further provided and comprises
culturing host cells under conditions suitable for expression of
the desired polypeptide and recovering the desired polypeptide from
the cell culture.
[0026] The invention provides chimeric molecules comprising any of
the herein described polypeptides fused to a heterologous
polypeptide or amino acid sequence. Example of such chimeric
molecules comprise any of the herein described polypeptides fused
to an epitope tag sequence or a Fc region of an immunoglobulin.
[0027] The invention provides an antibody which binds, preferably
specifically, to any of the above or below described polypeptides.
Optionally, the antibody is a monoclonal antibody, humanized
antibody, antibody fragment or single-chain antibody.
[0028] The invention provides oligonucleotide probes which may be
useful for isolating genomic and cDNA nucleotide sequences,
measuring or detecting expression of an associated gene or as
antisense probes, wherein those probes may be derived from any of
the above or below described nucleotide sequences. Preferred probe
lengths are described above.
[0029] The invention also provides a method of identifying a
phenotype associated with a disruption of a gene which encodes for
a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide, the method
comprising:
[0030] (a) providing a non-human transgenic animal whose genome
comprises a disruption of the gene which encodes for a PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide;
[0031] (b) measuring a physiological characteristic of the
non-human transgenic animal; and
[0032] (c) comparing the measured physiological characteristic with
that of a gender matched wild-type animal, wherein the
physiological characteristic of the non-human transgenic animal
that differs from the physiological characteristic of the wild-type
animal is identified as a phenotype resulting from the gene
disruption in the non-human transgenic animal. In one aspect, the
non-human transgenic animal is a mammal. In another aspect, the
mammal is a rodent. In still another aspect, the mammal is a rat or
a mouse. In one aspect, the non-human transgenic animal is
heterozygous for the disruption of a gene which encodes for a
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide. In another aspect, the
phenotype exhibited by the non-human transgenic animal as compared
with gender matched wild-type littermates is at least one of the
following: a neurological disorder; a cardiovascular, endothelial
or angiogenic disorder; an eye abnormality; an immunological
disorder; an oncological disorder; a bone metabolic abnormality or
disorder; a lipid metabolic disorder; or a developmental
abnormality.
[0033] In yet another aspect, the neurological disorder is an
increased anxiety-like response during open field activity testing.
In yet another aspect, the neurological disorder is a decreased
anxiety-like response during open field activity testing. In yet
another aspect, the neurological disorder is an abnormal circadian
rhythm during home-cage activity testing. In yet another aspect,
the neurological disorder is an enhanced motor coordination during
inverted screen testing. In yet another aspect, the neurological
disorder is impaired motor coordination during inverted screen
testing. In yet another aspect, the neurological disorder includes
depression, generalized anxiety disorders, attention deficit
disorder, sleep disorder, hyperactivity disorder, obsessive
compulsive disorder, schizophrenia, cognitive disorders,
hyperalgesia and sensory disorders. Such neurological disorders
include the category defined as "anxiety disorders" which include
but are not limited to: mild to moderate anxiety, anxiety disorder
due to a general medical condition, anxiety disorder not otherwise
specified, generalized anxiety disorder, panic attack, panic
disorder with agoraphobia, panic disorder without agoraphobia,
posttraumatic stress disorder, social phobia, social anxiety,
autism, specific phobia, substance-induced anxiety disorder, acute
alcohol withdrawal, obsessive compulsive disorder, agoraphobia,
monopolar disorders, bipolar disorder I or II, bipolar disorder not
otherwise specified, cyclothymic disorder, depressive disorder,
major depressive disorder, mood disorder, substance-induced mood
disorder, enhancement of cognitive function, loss of cognitive
function associated with but not limited to Alzheimer's disease,
stroke, or traumatic injury to the brain, seizures resulting from
disease or injury including but not limited to epilepsy, learning
disorders/disabilities, cerebral palsy. In addition, anxiety
disorders may apply to personality disorders including but not
limited to the following types: paranoid, antisocial, avoidant
behavior, borderline personality disorders, dependent, histronic,
narcissistic, obsessive-compulsive, schizoid, and schizotypal.
[0034] In another aspect, the eye abnormality is a retinal
abnormality. In still another aspect, the eye abnormality is
consistent with vision problems or blindness. In yet another
aspect, the retinal abnormality is consistent with retinitis
pigmentosa or is characterized by retinal degeneration or retinal
dysplasia.
[0035] In still another aspect, the retinal abnormalities are
consistent with retinal dysplasia, various retinopathies, including
retinopathy of prematurity, retrolental fibroplasia, neovascular
glaucoma, age-related macular degeneration, diabetic macular edema,
corneal neovascularization, corneal graft neovascularization,
corneal graft rejection, retinal/choroidal neovascularization,
neovascularization of the angle (rubeosis), ocular neovascular
disease, vascular restenosis, arteriovenous malformations (AVM),
meningioma, hemangioma, angiofibroma, thyroid hyperplasias
(including Grave's disease), corneal and other tissue
transplantation, retinal artery obstruction or occlusion; retinal
degeneration causing secondary atrophy of the retinal vasculature,
retinitis pigmentosa, macular dystrophies, Stargardt's disease,
congenital stationary night blindness, choroideremia, gyrate
atrophy, Leber's congenital amaurosis, retinoschisis disorders,
Wagner's syndrome, Usher syndromes, Zellweger syndrome,
Saldino-Mainzer syndrome, Senior-Loken syndrome, Bardet-Biedl
syndrome, Alport's syndrome, Alstrom's syndrome, Cockayne's
syndrome, dysplaisa spondyloepiphysaria congentia, Flynn-Aird
syndrome, Friedreich ataxia, Hallgren syndrome, Marshall syndrome,
Albers-Schnoberg disease, Refsum's disease, Kearns-Sayre syndrome,
Waardenburg's syndrome, Alagile syndrome, myotonic dystrophy,
olivopontocerebellar atrophy, Pierre-Marie dunsdrome, Stickler
syndrome, carotinemeia, cystinosis, Wolfram syndrome,
Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentia
pigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria,
or mannosidosis.
[0036] In still another aspect, the eye abnormality is a cataract.
In still yet another aspect, the cataract is a systemic disease
such as human Down's syndrome, Hallerman-Streiff syndrome, Lowe
syndrome, galactosemia, Marfan syndrome, Trismoy 13-15, Alport
syndrome, myotonic dystrophy, Fabry disease, hypoparathroidism or
Conradi syndrome.
[0037] In still another aspect, the developmental abnormality
comprises embryonic lethality or reduced viability.
[0038] In still yet another aspect, the cardiovascular, endothelial
or angiogenic disorders are arterial diseases, such as diabetes
mellitus; papilledema; optic atrophy; atherosclerosis; angina;
myocardial infarctions such as acute myocardial infarctions,
cardiac hypertrophy, and heart failure such as congestive heart
failure; hypertension; inflammatory vasculitides; Reynaud's disease
and Reynaud's phenomenon; aneurysms and arterial restenosis; venous
and lymphatic disorders such as thrombophlebitis, lymphangitis, and
lymphedema; peripheral vascular disease; cancer such as vascular
tumors, e.g., hemangioma (capillary and cavernous), glomus tumors,
telangiectasia, bacillary angiomatosis, hemangioendothelioma,
angiosarcoma, haemangiopericytoma, Kaposi's sarcoma, lymphangioma,
and lymphangiosarcoma; tumor angiogenesis; trauma such as wounds,
burns, and other injured tissue, implant fixation, scarring;
ischemia reperfusion injury; rheumatoid arthritis; cerebrovascular
disease; renal diseases such as acute renal failure, or
osteoporosis.
[0039] In still another aspect, the immunological disorders are
consistent with systemic lupus erythematosis; rheumatoid arthritis;
juvenile chronic arthritis; spondyloarthropathies; systemic
sclerosis (scleroderma); idiopathic inflammatory myopathies
(dermatomyositis, polymyositis); Sjogren's syndrome; systemic
vasculitis; sarcoidosis; autoimmune hemolytic anemia (immune
pancytopenia, paroxysmal nocturnal hemoglobinuria); autoimmune
thrombocytopenia (idiopathic thrombocytopenic purpura,
immune-mediated thrombocytopenia); thyroiditis (Grave's disease,
Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic
thyroiditis); diabetes mellitus; immune-mediated renal disease
(glomerulonephritis, tubulointerstitial nephritis); demyelinating
diseases of the central and peripheral nervous systems such as
multiple sclerosis, idiopathic demyelinating polyneuropathy or
Guillain-Barre syndrome, and chronic inflammatory demyelinating
polyneuropathy; hepatobiliary diseases such as infectious hepatitis
(hepatitis A, B, C, D, E and other non-hepatotropic viruses),
autoimmune chronic active hepatitis, primary biliary cirrhosis,
granulomatous hepatitis, and sclerosing cholangitis; inflammatory
bowel disease (ulcerative colitis: Crohn's disease);
gluten-sensitive enteropathy, and Whipple's disease; autoimmune or
immune-mediated skin diseases including bullous skin diseases,
erythema multiforme and contact dermatitis, psoriasis; allergic
diseases such as asthma, allergic rhinitis, atopic dermatitis, food
hypersensitivity and urticaria; immunologic diseases of the lung
such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and
hypersensitivity pneumonitis; or transplantation associated
diseases including graft rejection and graft-versus-host
disease.
[0040] In still another aspect, the bone metabolic abnormality or
disorder is arthritis, osteoporosis, osteopenia or
osteopetrosis.
[0041] In another aspect, the non-human transgenic animal exhibits
at least one of the following physiological characteristics
compared with gender matched wild-type littermates: increased
anxiety-like response during open field testing; decreased
anxiety-like response during open field activity testing; abnormal
circadian rhythm during home-cage activity testing including
decreased ambulatory counts; increased exploratory activity during
open-field testing; increased stress induced hyperthermia; enhanced
motor coordination during inverted screen testing; impaired motor
coordination during inverted screen testing; increase in retinal
artery tortuosity; retinal degeneration marked by attenuated
retinal vessels; opthamological abnormalities; increased mean
systolic blood pressure; increased mean fasting serum glucose
levels; decreased mean serum glucose levels; increased mean serum
cholesterol levels; increased mean serum triglyceride levels;
decreased mean serum cholesterol levels; decreased mean serum
triglyceride levels; enhanced glucose tolerance; impaired glucose
tolerance; increased mean serum insulin levels; decreased mean
serum insulin levels; increased uric acid levels; decreased serum
phosphate levels; increased alkaline phosphatase levels and
increased alanine amino transferase levels; liver disease;
increased mean percentage of CD25+ in both spleen and lymph nodes;
decreased mean percentage of natural killer cells; decreased mean
percentage of CD21HiCD23Med cells in spleen and lymph nodes;
increased mean percentage of CD4 cells and decreased mean
percentage of B cells; increased mean percentage of CD8+ cells;
decreased mean percentage of eosinophils; decreased mean serum IgG1
response to an ovalbumin challenge; decreased mean serum IgG2a
response to an ovalbumin challenge; increased mean serum IgG1
response to an ovalbumin challenge; increased mean serum IgG2a
response to an ovalbumin challenge; increased mean serum MCP-1
response to a LPS challenge; increased mean serum TNF-alpha
response to a LPS challenge; decreased mean serum MCP-1 response to
a LPS challenge; decreased mean serum IL-6 response to a LPS
challenge; decreased TNF-alpha response to a LPS challenge;
increased mean serum IL6 response to a LPS challenge; increased
mean platelet counts; decreased mean total white blood cell (WBC)
counts; decreased absolute lymphocyte counts; decreased absolute
monocyte counts; decreased skin fibroblast proliferation; increased
skin fibroblast proliferation; increased mean percent of total body
fat and total fat mass; increased mean body weight; increased mean
body length; increased organ weights; increased total tissue mass
(TTM); increased lean body mass (LBM); increased bone mineral
density (BMD) in total body, femur and vertebrae; increased bone
mineral content (BMC) in total body, femur and vertebrae; increased
volumetric bone mineral density (vBMD) in total body, femur and
vertebrae; increased mean femoral midshaft cortical thickness and
cross-sectional area; increased mean vertebral trabecular bone
volume, number and connectivity density; decreased mean percent of
total body fat and total fat mass; decreased mean body weight;
decreased mean body length; decreased total tissue mass (TTM);
decreased lean body mass (LBM); decreased bone mineral density
(BMD) in total body, femur and vertebrae; decreased bone mineral
content (BMC) in total body, femur and vertebrae; decreased
volumetric bone mineral density (vBMD) in total body, femur and
vertebrae; decreased mean femoral midshaft cortical thickness and
cross-sectional area; decreased mean vertebral trabecular bone
volume, number and connectivity density; severe depletion of
abdominal and subcutaneous body fat deposits; decreased organ
weights; growth retardation; hydrocephalus; sebaceous gland
hyperplasia and growth retardation; apoptosis of olfactory
neuroepithelial cells; lymphoid hyperplasia and tissue
inflammation; development abnormalities; male infertility; growth
retardation with reduced viability; and embryonic lethality.
[0042] The invention also provides an isolated cell derived from a
non-human transgenic animal whose genome comprises a disruption of
the gene which encodes for a PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide. In one aspect, the isolated cell is a murine cell. In
yet another aspect, the murine cell is an embryonic stem cell. In
still another aspect, the isolated cell is derived from a non-human
transgenic animal which exhibits at least one of the following
phenotypes compared with gender matched wild-type littermates: a
neurological disorder; a cardiovascular, endothelial or angiogenic
disorder; an eye abnormality; an immunological disorder; an
oncological disorder; a bone metabolic abnormality or disorder; a
lipid metabolic disorder; or a developmental abnormality. The
invention also provides a method of identifying an agent that
modulates a phenotype associated with a disruption of a gene which
encodes for a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide, the
method comprising:
[0043] (a) providing a non-human transgenic animal whose genome
comprises a disruption of the gene which encodes for the PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide;
[0044] (b) measuring a physiological characteristic of the
non-human transgenic animal of (a);
[0045] (c) comparing the measured physiological characteristic of
(b) with that of a gender matched wild-type animal, wherein the
physiological characteristic of the non-human transgenic animal
that differs from the physiological characteristic of the wild-type
animal is identified as a phenotype resulting from the gene
disruption in the non-human transgenic animal;
[0046] (d) administering a test agent to the non-human transgenic
animal of (a); and
[0047] (e) determining whether the test agent modulates the
identified phenotype associated with gene disruption in the
non-human transgenic animal.
[0048] In one aspect, the phenotype associated with the gene
disruption comprises a neurological disorder; a cardiovascular,
endothelial or angiogenic disorder; an eye abnormality; an
immunological disorder; an oncological disorder; a bone metabolic
abnormality or disorder; a lipid metabolic disorder; or a
developmental abnormality.
[0049] In yet another aspect, the neurological disorder is an
increased anxiety-like response during open field activity testing.
In yet another aspect, the neurological disorder is a decreased
anxiety-like response during open field activity testing. In yet
another aspect, the neurological disorder is an abnormal circadian
rhythm during home-cage activity testing. In yet another aspect,
the neurological disorder is an enhanced motor coordination during
inverted screen testing. In yet another aspect, the neurological
disorder is impaired motor coordination during inverted screen
testing. In yet another aspect, the neurological disorder includes
depression, generalized anxiety disorders, attention deficit
disorder, sleep disorder, hyperactivity disorder, obsessive
compulsive disorder, schizophrenia, cognitive disorders,
hyperalgesia and sensory disorders. Such neurological disorders
include the category defined as "anxiety disorders" which include
but are not limited to: mild to moderate anxiety, anxiety disorder
due to a general medical condition, anxiety disorder not otherwise
specified, generalized anxiety disorder, panic attack, panic
disorder with agoraphobia, panic disorder without agoraphobia,
posttraumatic stress disorder, social phobia, social anxiety,
autism, specific phobia, substance-induced anxiety disorder, acute
alcohol withdrawal, obsessive compulsive disorder, agoraphobia,
monopolar disorders, bipolar disorder I or II, bipolar disorder not
otherwise specified, cyclothymic disorder, depressive disorder,
major depressive disorder, mood disorder, substance-induced mood
disorder, enhancement of cognitive function, loss of cognitive
function associated with but not limited to Alzheimer's disease,
stroke, or traumatic injury to the brain, seizures resulting from
disease or injury including but not limited to epilepsy, learning
disorders/disabilities, cerebral palsy. In addition, anxiety
disorders may apply to personality disorders including but not
limited to the following types: paranoid, antisocial, avoidant
behavior, borderline personality disorders, dependent, histronic,
narcissistic, obsessive-compulsive, schizoid, and schizotypal.
[0050] In yet another aspect, the eye abnormality is a retinal
abnormality. In still another aspect, the eye abnormality is
consistent with vision problems or blindness. In yet another
aspect, the retinal abnormality is consistent with retinitis
pigmentosa or is characterized by retinal degeneration or retinal
dysplasia.
[0051] In still another aspect, the retinal abnormalities are
consistent with retinal dysplasia, various retinopathies, including
retinopathy of prematurity, retrolental fibroplasia, neovascular
glaucoma, age-related macular degeneration, diabetic macular edema,
corneal neovascularization, corneal graft neovascularization,
corneal graft rejection, retinal/choroidal neovascularization,
neovascularization of the angle (rubeosis), ocular neovascular
disease, vascular restenosis, arteriovenous malformations (AVM),
meningioma, hemangioma, angiofibroma, thyroid hyperplasias
(including Grave's disease), corneal and other tissue
transplantation, retinal artery obstruction or occlusion; retinal
degeneration causing secondary atrophy of the retinal vasculature,
retinitis pigmentosa, macular dystrophies, Stargardt's disease,
congenital stationary night blindness, choroideremia, gyrate
atrophy, Leber's congenital amaurosis, retinoschisis disorders,
Wagner's syndrome, Usher syndromes, Zellweger syndrome,
Saldino-Mainzer syndrome, Senior-Loken syndrome, Bardet-Biedl
syndrome, Alport's syndrome, Alstrom's syndrome, Cockayne's
syndrome, dysplaisa spondyloepiphysaria congentia, Flynn-Aird
syndrome, Friedreich ataxia, Hallgren syndrome, Marshall syndrome,
Albers-Schnoberg disease, Refsum's disease, Kearns-Sayre syndrome,
Waardenburg's syndrome, Alagile syndrome, myotonic dystrophy,
olivopontocerebellar atrophy, Pierre-Marie dunsdrome, Stickler
syndrome, carotinemeia, cystinosis, Wolfram syndrome,
Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentia
pigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria,
or mannosidosis.
[0052] In still another aspect, the eye abnormality is a cataract.
In still yet another aspect, the cataract is a systemic disease
such as human Down's syndrome, Hallerman-Streiff syndrome, Lowe
syndrome, galactosemia, Marfan syndrome, Trismoy 13-15, Alport
syndrome, myotonic dystrophy, Fabry disease, hypoparathroidism, or
Conradi syndrome.
[0053] In still another aspect, the developmental abnormality
comprises embryonic lethality or reduced viability.
[0054] In still another aspect, the cardiovascular, endothelial or
angiogenic disorders are arterial diseases, such as diabetes
mellitus; papilledema; optic atrophy; atherosclerosis; angina;
myocardial infarctions such as acute myocardial infarctions,
cardiac hypertrophy, and heart failure such as congestive heart
failure; hypertension; inflammatory vasculitides; Reynaud's disease
and Reynaud's phenomenon; aneurysms and arterial restenosis; venous
and lymphatic disorders such as thrombophlebitis, lymphangitis, and
lymphedema; peripheral vascular disease; cancer such as vascular
tumors, e.g., hemangioma (capillary and cavernous), glomus tumors,
telangiectasia, bacillary angiomatosis, hemangioendothelioma,
angiosarcoma, haemangiopericytoma, Kaposi's sarcoma, lymphangioma,
and lymphangiosarcoma; tumor angiogenesis; trauma such as wounds,
burns, and other injured tissue, implant fixation, scarring;
ischemia reperfusion injury; rheumatoid arthritis; cerebrovascular
disease; renal diseases such as acute renal failure, or
osteoporosis.
[0055] In still another aspect, the immunological disorders are
consistent with systemic lupus erythematosis; rheumatoid arthritis;
juvenile chronic arthritis; spondyloarthropathies; systemic
sclerosis (scleroderma); idiopathic inflammatory myopathies
(dermatomyositis, polymyositis); Sjogren's syndrome; systemic
vasculitis; sarcoidosis; autoimmune hemolytic anemia (immune
pancytopenia, paroxysmal nocturnal hemoglobinuria); autoimmune
thrombocytopenia (idiopathic thrombocytopenic purpura,
immune-mediated thrombocytopenia); thyroiditis (Grave's disease,
Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic
thyroiditis); diabetes mellitus; immune-mediated renal disease
(glomerulonephritis, tubulointerstitial nephritis); demyelinating
diseases of the central and peripheral nervous systems such as
multiple sclerosis, idiopathic demyelinating polyneuropathy or
Guillain-Barre syndrome, and chronic inflammatory demyelinating
polyneuropathy; hepatobiliary diseases such as infectious hepatitis
(hepatitis A, B, C, D, E and other non-hepatotropic viruses),
autoimmune chronic active hepatitis, primary biliary cirrhosis,
granulomatous hepatitis, and sclerosing cholangitis; inflammatory
bowel disease (ulcerative colitis: Crohn's disease);
gluten-sensitive enteropathy, and Whipple's disease; autoimmune or
immune-mediated skin diseases including bullous skin diseases,
erythema multiforme and contact dermatitis, psoriasis; allergic
diseases such as asthma, allergic rhinitis, atopic dermatitis, food
hypersensitivity and urticaria; immunologic diseases of the lung
such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and
hypersensitivity pneumonitis; or transplantation associated
diseases including graft rejection and graft-versus-host
disease.
[0056] In yet another aspect, the bone metabolic abnormality or
disorder is arthritis, osteoporosis, osteopenia or
osteopetrosis.
[0057] In another aspect, the non-human transgenic animal exhibits
at least one of the following physiological characteristics
compared with gender matched wild-type littermates: increased
anxiety-like response during open field testing; decreased
anxiety-like response during open field activity testing; abnormal
circadian rhythm during home-cage activity testing including
decreased ambulatory counts; increased exploratory activity during
open-field testing; increased stress induced hyperthermia; enhanced
motor coordination during inverted screen testing; impaired motor
coordination during inverted screen testing; increase in retinal
artery tortuosity; retinal degeneration marked by attenuated
retinal vessels; opthamological abnormalities; increased mean
systolic blood pressure; increased mean fasting serum glucose
levels; decreased mean serum glucose levels; increased mean serum
cholesterol levels; increased mean serum triglyceride levels;
decreased mean serum cholesterol levels; decreased mean serum
triglyceride levels; enhanced glucose tolerance; impaired glucose
tolerance; increased mean serum insulin levels; decreased mean
serum insulin levels; increased uric acid levels; decreased serum
phosphate levels; increased alkaline phosphatase levels and
increased alanine amino transferase levels; liver disease;
increased mean percentage of CD25+ in both spleen and lymph nodes;
decreased mean percentage of natural killer cells; decreased mean
percentage of CD21HiCD23Med cells in spleen and lymph nodes;
increased mean percentage of CD4 cells and decreased mean
percentage of B cells; increased mean percentage of CD8+ cells;
decreased mean percentage of eosinophils; decreased mean serum IgG1
response to an ovalbumin challenge; decreased mean serum IgG2a
response to an ovalbumin challenge; increased mean serum IgG1
response to an ovalbumin challenge; increased mean serum IgG2a
response to an ovalbumin challenge; increased mean serum MCP-1
response to a LPS challenge; increased mean serum TNF-alpha
response to a LPS challenge; decreased mean serum MCP-1 response to
a LPS challenge; decreased mean serum IL-6 response to a LPS
challenge; decreased TNF-alpha response to a LPS challenge;
increased mean serum IL6 response to a LPS challenge; increased
mean platelet counts; decreased mean total white blood cell (WBC)
counts; decreased absolute lymphocyte counts; decreased absolute
monocyte counts; decreased skin fibroblast proliferation; increased
skin fibroblast proliferation; increased mean percent of total body
fat and total fat mass; increased mean body weight; increased mean
body length; increased organ weights; increased total tissue mass
(TTM); increased lean body mass (LBM); increased bone mineral
density (BMD) in total body, femur and vertebrae; increased bone
mineral content (BMC) in total body, femur and vertebrae; increased
volumetric bone mineral density (vBMD) in total body, femur and
vertebrae; increased mean femoral midshaft cortical thickness and
cross-sectional area; increased mean vertebral trabecular bone
volume, number and connectivity density; decreased mean percent of
total body fat and total fat mass; decreased mean body weight;
decreased mean body length; decreased total tissue mass (TTM);
decreased lean body mass (LBM); decreased bone mineral density
(BMD) in total body, femur and vertebrae; decreased bone mineral
content (BMC) in total body, femur and vertebrae; decreased
volumetric bone mineral density (vBMD) in total body, femur and
vertebrae; decreased mean femoral midshaft cortical thickness and
cross-sectional area; decreased mean vertebral trabecular bone
volume, number and connectivity density; severe depletion of
abdominal and subcutaneous body fat deposits; decreased organ
weights; growth retardation; hydrocephalus; sebaceous gland
hyperplasia and growth retardation; apoptosis of olfactory
neuroepithelial cells; lymphoid hyperplasia and tissue
inflammation; development abnormalities; male infertility; growth
retardation with reduced viability; and embryonic lethality. The
invention also provides an agent which modulates the phenotype
associated with gene disruption. In one aspect, the agent is an
agonist or antagonist of a PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide. In yet another aspect, the agonist agent is an
anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245,
anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344,
anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732,
anti-PRO1003, anti-PRO1104, anti-PRO1151, anti-PRO1244,
anti-PRO1298, anti-PRO1313, anti-PRO1570, anti-PRO1886,
anti-PRO1891, anti-PRO4409, anti-PRO5725, anti-PRO5994,
anti-PRO6097, anti-PRO7425, anti-PRO10102, anti-PRO10282,
anti-PRO61709 or anti-PRO779 antibody. In still another aspect, the
antagonist agent is an anti-PRO196, anti-PRO217, anti-PRO231,
anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258, anti-PRO287,
anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526, anti-PRO724,
anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104, anti-PRO1151,
anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570,
anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725,
anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102,
anti-PRO10282, anti-PRO61709 or anti-PRO779 antibody.
[0058] The invention also provides a method of identifying an agent
that modulates a physiological characteristic associated with a
disruption of the gene which encodes for a PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide, the method comprising:
[0059] (a) providing a non-human transgenic animal whose genome
comprises a disruption of the gene which encodes for a PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide;
[0060] (b) measuring a physiological characteristic exhibited by
the non-human transgenic animal of (a);
[0061] (c) comparing the measured physiological characteristic of
(b) with that of a gender matched wild-type animal, wherein the
physiological characteristic exhibited by the non-human transgenic
animal that differs from the physiological characteristic exhibited
by the wild-type animal is identified as a physiological
characteristic associated with gene disruption;
[0062] (d) administering a test agent to the non-human transgenic
animal of (a); and
[0063] (e) determining whether the physiological characteristic
associated with gene disruption is modulated.
[0064] In one aspect, the non-human transgenic animal exhibits at
least one of the following physiological characteristics compared
with gender matched wild-type littermates:
[0065] In another aspect, the non-human transgenic animal exhibits
at least one of the following physiological characteristics
compared with gender matched wild-type littermates: increased
anxiety-like response during open field testing; decreased
anxiety-like response during open field activity testing; abnormal
circadian rhythm during home-cage activity testing including
decreased ambulatory counts; increased exploratory activity during
open-field testing; increased stress induced hyperthermia; enhanced
motor coordination during inverted screen testing; impaired motor
coordination during inverted screen testing; increase in retinal
artery tortuosity; retinal degeneration marked by attenuated
retinal vessels; opthamological abnormalities; increased mean
systolic blood pressure; increased mean fasting serum glucose
levels; decreased mean serum glucose levels; increased mean serum
cholesterol levels; increased mean serum triglyceride levels;
decreased mean serum cholesterol levels; decreased mean serum
triglyceride levels; enhanced glucose tolerance; impaired glucose
tolerance; increased mean serum insulin levels; decreased mean
serum insulin levels; increased uric acid levels; decreased serum
phosphate levels; increased alkaline phosphatase levels and
increased alanine amino transferase levels; liver disease;
increased mean percentage of CD25+ in both spleen and lymph nodes;
decreased mean percentage of natural killer cells; decreased mean
percentage of CD21HiCD23Med cells in spleen and lymph nodes;
increased mean percentage of CD4 cells and decreased mean
percentage of B cells; increased mean percentage of CD8+ cells;
decreased mean percentage of eosinophils; decreased mean serum IgG1
response to an ovalbumin challenge; decreased mean serum IgG2a
response to an ovalbumin challenge; increased mean serum IgG1
response to an ovalbumin challenge; increased mean serum IgG2a
response to an ovalbumin challenge; increased mean serum MCP-1
response to a LPS challenge; increased mean serum TNF-alpha
response to a LPS challenge; decreased mean serum MCP-1 response to
a LPS challenge; decreased mean serum IL-6 response to a LPS
challenge; decreased TNF-alpha response to a LPS challenge;
increased mean serum IL6 response to a LPS challenge; increased
mean platelet counts; decreased mean total white blood cell (WBC)
counts; decreased absolute lymphocyte counts; decreased absolute
monocyte counts; decreased skin fibroblast proliferation; increased
skin fibroblast proliferation; increased mean percent of total body
fat and total fat mass; increased mean body weight; increased mean
body length; increased organ weights; increased total tissue mass
(TTM); increased lean body mass (LBM); increased bone mineral
density (BMD) in total body, femur and vertebrae; increased bone
mineral content (BMC) in total body, femur and vertebrae; increased
volumetric bone mineral density (vBMD) in total body, femur and
vertebrae; increased mean femoral midshaft cortical thickness and
cross-sectional area; increased mean vertebral trabecular bone
volume, number and connectivity density; decreased mean percent of
total body fat and total fat mass; decreased mean body weight;
decreased mean body length; decreased total tissue mass (TTM);
decreased lean body mass (LBM); decreased bone mineral density
(BMD) in total body, femur and vertebrae; decreased bone mineral
content (BMC) in total body, femur and vertebrae; decreased
volumetric bone mineral density (vBMD) in total body, femur and
vertebrae; decreased mean femoral midshaft cortical thickness and
cross-sectional area; decreased mean vertebral trabecular bone
volume, number and connectivity density; severe depletion of
abdominal and subcutaneous body fat deposits; decreased organ
weights; growth retardation; hydrocephalus; sebaceous gland
hyperplasia and growth retardation; apoptosis of olfactory
neuroepithelial cells; lymphoid hyperplasia and tissue
inflammation; development abnormalities; male infertility; growth
retardation with reduced viability; and embryonic lethality.
[0066] The invention also provides an agent that modulates a
physiological characteristic which is associated with gene
disruption. In one aspect, the agent is an agonist or antagonist of
the phenotype associated with a disruption of a gene which encodes
for a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 polypeptide. In yet another
aspect, the agent is an agonist or antagonist of a PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 polypeptide. In yet another aspect, the agonist agent is an
anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245,
anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344,
anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732,
anti-PRO1003, anti-PRO1104, anti-PRO1151, anti-PRO1244,
anti-PRO1298, anti-PRO1313, anti-PRO1570, anti-PRO1886,
anti-PRO1891, anti-PRO4409, anti-PRO5725, anti-PRO5994,
anti-PRO6097, anti-PRO7425, anti-PRO10102, anti-PRO10282,
anti-PRO61709 or anti-PRO779 antibody. In still another aspect, the
antagonist agent is an anti-PRO196, anti-PRO217, anti-PRO231,
anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258, anti-PRO287,
anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526, anti-PRO724,
anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104, anti-PRO1151,
anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570,
anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725,
anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102,
anti-PRO10282, anti-PRO61709 or anti-PRO779 antibody.
[0067] The invention also provides a method of identifying an agent
which modulates a behavior associated with a disruption of the gene
which encodes for a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide, the
method comprising:
[0068] (a) providing a non-human transgenic animal whose genome
comprises a disruption of the gene which encodes for a PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide;
[0069] (b) observing the behavior exhibited by the non-human
transgenic animal of (a);
[0070] (c) comparing the observed behavior of (b) with that of a
gender matched wild-type animal, wherein the observed behavior
exhibited by the non-human transgenic animal that differs from the
observed behavior exhibited by the wild-type animal is identified
as a behavior associated with gene disruption;
[0071] (d) administering a test agent to the non-human transgenic
animal of (a); and
[0072] (e) determining whether the agent modulates the behavior
associated with gene disruption.
[0073] In one aspect, the observed behavior is an increased
anxiety-like response during open field activity testing. In yet
another aspect, the observed behavior is a decreased anxiety-like
response during open field activity testing. In yet another aspect,
the observed behavior is an abnormal circadian rhythm during
home-cage activity testing. In yet another aspect, the observed
behavior is an enhanced motor coordination during inverted screen
testing. In yet another aspect, the observed behavior is impaired
motor coordination during inverted screen testing. In yet another
aspect, the observed behavior includes depression, generalized
anxiety disorders, attention deficit disorder, sleep disorder,
hyperactivity disorder, obsessive compulsive disorder,
schizophrenia, cognitive disorders, hyperalgesia and sensory
disorders. Such disorders include the category defined as "anxiety
disorders" which include but are not limited to: mild to moderate
anxiety, anxiety disorder due to a general medical condition,
anxiety disorder not otherwise specified, generalized anxiety
disorder, panic attack, panic disorder with agoraphobia, panic
disorder without agoraphobia, posttraumatic stress disorder, social
phobia, social anxiety, autism, specific phobia, substance-induced
anxiety disorder, acute alcohol withdrawal, obsessive compulsive
disorder, agoraphobia, monopolar disorders, bipolar disorder I or
II, bipolar disorder not otherwise specified, cyclothymic disorder,
depressive disorder, major depressive disorder, mood disorder,
substance-induced mood disorder, enhancement of cognitive function,
loss of cognitive function associated with but not limited to
Alzheimer's disease, stroke, or traumatic injury to the brain,
seizures resulting from disease or injury including but not limited
to epilepsy, learning disorders/disabilities, cerebral palsy. In
addition, anxiety disorders may apply to personality disorders
including but not limited to the following types: paranoid,
antisocial, avoidant behavior, borderline personality disorders,
dependent, histronic, narcissistic, obsessive-compulsive, schizoid,
and schizotypal.
[0074] The invention also provides an agent that modulates a
behavior which is associated with gene disruption. In one aspect,
the agent is an agonist or antagonist of the phenotype associated
with a disruption of a gene which encodes for a PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 polypeptide. In yet another aspect, the agent is an agonist
or antagonist of a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide. In yet
another aspect, the agonist agent is an anti-PRO196, anti-PRO217,
anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258,
anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526,
anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104,
anti-PRO1151, anti-PRO1244, anti-PRO1298, anti-PRO1313,
anti-PRO1570, anti-PRO1886, anti-PRO1891, anti-PRO4409,
anti-PRO5725, anti-PRO5994, anti-PRO6097, anti-PRO7425,
anti-PRO10102, anti-PRO10282, anti-PRO61709 or anti-PRO779
antibody. In still another aspect, the antagonist agent is an
anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245,
anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344,
anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732,
anti-PRO1003, anti-PRO1104, anti-PRO1151, anti-PRO1244,
anti-PRO1298, anti-PRO1313, anti-PRO1570, anti-PRO1886,
anti-PRO1891, anti-PRO4409, anti-PRO5725, anti-PRO5994,
anti-PRO6097, anti-PRO7425, anti-PRO10102, anti-PRO10282,
anti-PRO61709 or anti-PRO779 antibody.
[0075] The invention also provides a method of identifying an agent
that ameliorates or modulates a neurological disorder; a
cardiovascular, endothelial or angiogenic disorder; an eye
abnormality; an immunological disorder; an oncological disorder; a
bone metabolic abnormality or disorder; a lipid metabolic disorder;
or a developmental abnormality associated with a disruption in the
gene which encodes for a PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide, the method comprising:
[0076] (a) providing a non-human transgenic animal whose genome
comprises a disruption of the gene which encodes for a PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide;
[0077] (b) administering a test agent to said non-human transgenic
animal; and
[0078] (c) determining whether the test agent ameliorates or
modulates the neurological disorder; cardiovascular, endothelial or
angiogenic disorder; eye abnormality; immunological disorder;
oncological disorder; bone metabolic abnormality or disorder; lipid
metabolic disorder; or developmental abnormality associated with
the gene disruption in the non-human transgenic animal.
[0079] In yet another aspect, the neurological disorder is an
increased anxiety-like response during open field activity testing.
In yet another aspect, the neurological disorder is a decreased
anxiety-like response during open field activity testing. In yet
another aspect, the neurological disorder is an abnormal circadian
rhythm during home-cage activity testing. In yet another aspect,
the neurological disorder is an enhanced motor coordination during
inverted screen testing. In yet another aspect, the neurological
disorder is impaired motor coordination during inverted screen
testing. In yet another aspect, the neurological disorder includes
depression, generalized anxiety disorders, attention deficit
disorder, sleep disorder, hyperactivity disorder, obsessive
compulsive disorder, schizophrenia, cognitive disorders,
hyperalgesia and sensory disorders. Such neurological disorders
include the category defined as "anxiety disorders" which include
but are not limited to: mild to moderate anxiety, anxiety disorder
due to a general medical condition, anxiety disorder not otherwise
specified, generalized anxiety disorder, panic attack, panic
disorder with agoraphobia, panic disorder without agoraphobia,
posttraumatic stress disorder, social phobia, social anxiety,
autism, specific phobia, substance-induced anxiety disorder, acute
alcohol withdrawal, obsessive compulsive disorder, agoraphobia,
monopolar disorders, bipolar disorder I or II, bipolar disorder not
otherwise specified, cyclothymic disorder, depressive disorder,
major depressive disorder, mood disorder, substance-induced mood
disorder, enhancement of cognitive function, loss of cognitive
function associated with but not limited to Alzheimer's disease,
stroke, or traumatic injury to the brain, seizures resulting from
disease or injury including but not limited to epilepsy, learning
disorders/disabilities, cerebral palsy. In addition, anxiety
disorders may apply to personality disorders including but not
limited to the following types: paranoid, antisocial, avoidant
behavior, borderline personality disorders, dependent, histronic,
narcissistic, obsessive-compulsive, schizoid, and schizotypal.
[0080] In another aspect, the eye abnormality is a retinal
abnormality. In still another aspect, the eye abnormality is
consistent with vision problems or blindness. In yet another
aspect, the retinal abnormality is consistent with retinitis
pigmentosa or is characterized by retinal degeneration or retinal
dysplasia.
[0081] In still another aspect, the retinal abnormalities the
retinal abnormalities are consistent with retinal dysplasia,
various retinopathies, including retinopathy of prematurity,
retrolental fibroplasia, neovascular glaucoma, age-related macular
degeneration, diabetic macular edema, corneal neovascularization,
corneal graft neovascularization, corneal graft rejection,
retinal/choroidal neovascularization, neovascularization of the
angle (rubeosis), ocular neovascular disease, vascular restenosis,
arteriovenous malformations (AVM), meningioma, hemangioma,
angiofibroma, thyroid hyperplasias (including Grave's disease),
corneal and other tissue transplantation, retinal artery
obstruction or occlusion; retinal degeneration causing secondary
atrophy of the retinal vasculature, retinitis pigmentosa, macular
dystrophies, Stargardt's disease, congenital stationary night
blindness, choroideremia, gyrate atrophy, Leber's congenital
amaurosis, retinoschisis disorders, Wagner's syndrome, Usher
syndromes, Zellweger syndrome, Saldino-Mainzer syndrome,
Senior-Loken syndrome, Bardet-Biedl syndrome, Alport's syndrome,
Alstrom's syndrome, Cockayne's syndrome, dysplaisa
spondyloepiphysaria congentia, Flynn-Aird syndrome, Friedreich
ataxia, Hallgren syndrome, Marshall syndrome, Albers-Schnoberg
disease, Refsum's disease, Kearns-Sayre syndrome, Waardenburg's
syndrome, Alagile syndrome, myotonic dystrophy,
olivopontocerebellar atrophy, Pierre-Marie dunsdrome, Stickler
syndrome, carotinemeia, cystinosis, Wolfram syndrome,
Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentia
pigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria,
or mannosidosis.
[0082] In still another aspect, the eye abnormality is a cataract.
In still yet another aspect, the cataract is a systemic disease
such as human Down's syndrome, Hallerman-Streiff syndrome, Lowe
syndrome, galactosemia, Marfan syndrome, Trismoy 13-15, Alport
syndrome, myotonic dystrophy, Fabry disease, hypoparathroidism, or
Conradi syndrome.
[0083] In still another aspect, the developmental abnormality
comprises embryonic lethality or reduced viability.
[0084] In yet another aspect, the cardiovascular, endothelial or
angiogenic disorders are arterial diseases, such as diabetes
mellitus; papilledema; optic atrophy; atherosclerosis; angina;
myocardial infarctions such as acute myocardial infarctions,
cardiac hypertrophy, and heart failure such as congestive heart
failure; hypertension; inflammatory vasculitides; Reynaud's disease
and Reynaud's phenomenon; aneurysms and arterial restenosis; venous
and lymphatic disorders such as thrombophlebitis, lymphangitis, and
lymphedema; peripheral vascular disease; cancer such as vascular
tumors, e.g., hemangioma (capillary and cavernous), glomus tumors,
telangiectasia, bacillary angiomatosis, hemangioendothelioma,
angiosarcoma, haemangiopericytoma, Kaposi's sarcoma, lymphangioma,
and lymphangiosarcoma; tumor angiogenesis; trauma such as wounds,
burns, and other injured tissue, implant fixation, scarring;
ischemia reperfusion injury; rheumatoid arthritis; cerebrovascular
disease; renal diseases such as acute renal failure, or
osteoporosis.
[0085] In still yet another aspect, the immunological disorders are
consistent with systemic lupus erythematosis; rheumatoid arthritis;
juvenile chronic arthritis; spondyloarthropathies; systemic
sclerosis (scleroderma); idiopathic inflammatory myopathies
(dermatomyositis, polymyositis); Sjogren's syndrome; systemic
vasculitis; sarcoidosis; autoimmune hemolytic anemia (immune
pancytopenia, paroxysmal nocturnal hemoglobinuria); autoimmune
thrombocytopenia (idiopathic thrombocytopenic purpura,
immune-mediated thrombocytopenia); thyroiditis (Grave's disease,
Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic
thyroiditis); diabetes mellitus; immune-mediated renal disease
(glomerulonephritis, tubulointerstitial nephritis); demyelinating
diseases of the central and peripheral nervous systems such as
multiple sclerosis, idiopathic demyelinating polyneuropathy or
Guillain-Barre syndrome, and chronic inflammatory demyelinating
polyneuropathy; hepatobiliary diseases such as infectious hepatitis
(hepatitis A, B, C, D, E and other non-hepatotropic viruses),
autoimmune chronic active hepatitis, primary biliary cirrhosis,
granulomatous hepatitis, and sclerosing cholangitis; inflammatory
bowel disease (ulcerative colitis: Crohn's disease);
gluten-sensitive enteropathy, and Whipple's disease; autoimmune or
immune-mediated skin diseases including bullous skin diseases,
erythema multiforme and contact dermatitis, psoriasis; allergic
diseases such as asthma, allergic rhinitis, atopic dermatitis, food
hypersensitivity and urticaria; immunologic diseases of the lung
such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and
hypersensitivity pneumonitis; or transplantation associated
diseases including graft rejection and graft-versus-host
disease.
[0086] In yet another aspect, the bone metabolic abnormality or
disorder is arthritis, osteoporosis, osteopenia or
osteopetrosis.
[0087] In another aspect, the non-human transgenic animal exhibits
at least one of the following physiological characteristics
compared with gender matched wild-type littermates: increased
anxiety-like response during open field testing; decreased
anxiety-like response during open field activity testing; abnormal
circadian rhythm during home-cage activity testing including
decreased ambulatory counts; increased exploratory activity during
open-field testing; increased stress induced hyperthermia; enhanced
motor coordination during inverted screen testing; impaired motor
coordination during inverted screen testing; increase in retinal
artery tortuosity; retinal degeneration marked by attenuated
retinal vessels; opthamological abnormalities; increased mean
systolic blood pressure; increased mean fasting serum glucose
levels; decreased mean serum glucose levels; increased mean serum
cholesterol levels; increased mean serum triglyceride levels;
decreased mean serum cholesterol levels; decreased mean serum
triglyceride levels; enhanced glucose tolerance; impaired glucose
tolerance; increased mean serum insulin levels; decreased mean
serum insulin levels; increased uric acid levels; decreased serum
phosphate levels; increased alkaline phosphatase levels and
increased alanine amino transferase levels; liver disease;
increased mean percentage of CD25+ in both spleen and lymph nodes;
decreased mean percentage of natural killer cells; decreased mean
percentage of CD21HiCD23Med cells in spleen and lymph nodes;
increased mean percentage of CD4 cells and decreased mean
percentage of B cells; increased mean percentage of CD8+ cells;
decreased mean percentage of eosinophils; decreased mean serum IgG1
response to an ovalbumin challenge; decreased mean serum IgG2a
response to an ovalbumin challenge; increased mean serum IgG1
response to an ovalbumin challenge; increased mean serum IgG2a
response to an ovalbumin challenge; increased mean serum MCP-1
response to a LPS challenge; increased mean serum TNF-alpha
response to a LPS challenge; decreased mean serum MCP-1 response to
a LPS challenge; decreased mean serum IL-6 response to a LPS
challenge; decreased TNF-alpha response to a LPS challenge;
increased mean serum IL6 response to a LPS challenge; increased
mean platelet counts; decreased mean total white blood cell (WBC)
counts; decreased absolute lymphocyte counts; decreased absolute
monocyte counts; decreased skin fibroblast proliferation; increased
skin fibroblast proliferation; increased mean percent of total body
fat and total fat mass; increased mean body weight; increased mean
body length; increased organ weights; increased total tissue mass
(TTM); increased lean body mass (LBM); increased bone mineral
density (BMD) in total body, femur and vertebrae; increased bone
mineral content (BMC) in total body, femur and vertebrae; increased
volumetric bone mineral density (vBMD) in total body, femur and
vertebrae; increased mean femoral midshaft cortical thickness and
cross-sectional area; increased mean vertebral trabecular bone
volume, number and connectivity density; decreased mean percent of
total body fat and total fat mass; decreased mean body weight;
decreased mean body length; decreased total tissue mass (TTM);
decreased lean body mass (LBM); decreased bone mineral density
(BMD) in total body, femur and vertebrae; decreased bone mineral
content (BMC) in total body, femur and vertebrae; decreased
volumetric bone mineral density (vBMD) in total body, femur and
vertebrae; decreased mean femoral midshaft cortical thickness and
cross-sectional area; decreased mean vertebral trabecular bone
volume, number and connectivity density; severe depletion of
abdominal and subcutaneous body fat deposits; decreased organ
weights; growth retardation; hydrocephalus; sebaceous gland
hyperplasia and growth retardation; apoptosis of olfactory
neuroepithelial cells; lymphoid hyperplasia and tissue
inflammation; development abnormalities; male infertility; growth
retardation with reduced viability; and embryonic lethality. The
invention also provides an agent that ameliorates or modulates a
neurological disorder; a cardiovascular, endothelial or angiogenic
disorder; an eye abnormality; an immunological disorder; an
oncological disorder; a bone metabolic abnormality or disorder; a
lipid metabolic disorder; or a developmental abnormality which is
associated with gene disruption. In one aspect, the agent is an
agonist or antagonist of the phenotype associated with a disruption
of a gene which encodes for a PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide. In yet another aspect, the agent is an agonist or
antagonist of a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide. In yet
another aspect, the agonist agent is an anti-PRO196, anti-PRO217,
anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258,
anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526,
anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104,
anti-PRO1151, anti-PRO1244, anti-PRO1298, anti-PRO1313,
anti-PRO1570, anti-PRO1886, anti-PRO1891, anti-PRO4409,
anti-PRO5725, anti-PRO5994, anti-PRO6097, anti-PRO7425,
anti-PRO10102, anti-PRO10282, anti-PRO61709 or anti-PRO779
antibody. In still another aspect, the antagonist agent is an
anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245,
anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344,
anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732,
anti-PRO1003, anti-PRO1104, anti-PRO1151, anti-PRO1244,
anti-PRO1298, anti-PRO1313, anti-PRO1570, anti-PRO1886,
anti-PRO1891, anti-PRO4409, anti-PRO5725, anti-PRO5994,
anti-PRO6097, anti-PRO7425, anti-PRO10102, anti-PRO10282,
anti-PRO61709 or anti-PRO779 antibody.
[0088] The invention also provides a therapeutic agent for the
treatment of a neurological disorder; a cardiovascular, endothelial
or angiogenic disorder; an eye abnormality; an immunological
disorder; an oncological disorder; a bone metabolic abnormality or
disorder; a lipid metabolic disorder; or a developmental
abnormality.
[0089] The invention also provides a method of identifying an agent
that modulates the expression of a PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide, the method comprising:
[0090] (a) contacting a test agent with a host cell expressing a
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide; and
[0091] (b) determining whether the test agent modulates the
expression of the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide by the
host cell.
[0092] The invention also provides an agent that modulates the
expression of a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide. In one
aspect, the agent is an agonist or antagonist of the phenotype
associated with a disruption of a gene which encodes for a PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide. In yet another aspect, the agent is
an agonist or antagonist of a PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide. In yet another aspect, the agonist agent is an
anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245,
anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344,
anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732,
anti-PRO1003, anti-PRO1104, anti-PRO1151, anti-PRO1244,
anti-PRO1298, anti-PRO1313, anti-PRO1570, anti-PRO1886,
anti-PRO1891, anti-PRO4409, anti-PRO5725, anti-PRO5994,
anti-PRO6097, anti-PRO7425, anti-PRO10102, anti-PRO10282,
anti-PRO61709 or anti-PRO779 antibody. In still another aspect, the
antagonist agent is an anti-PRO196, anti-PRO217, anti-PRO231,
anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258, anti-PRO287,
anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526, anti-PRO724,
anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104, anti-PRO1151,
anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570,
anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725,
anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102,
anti-PRO10282, anti-PRO61709 or anti-PRO779 antibody.
[0093] The invention also provides a method of evaluating a
therapeutic agent capable of affecting a condition associated with
a disruption of a gene which encodes for a PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide, the method comprising:
[0094] (a) providing a non-human transgenic animal whose genome
comprises a disruption of the gene which encodes for the PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide;
[0095] (b) measuring a physiological characteristic of the
non-human transgenic animal of (a);
[0096] (c) comparing the measured physiological characteristic of
(b) with that of a gender matched wild-type animal, wherein the
physiological characteristic of the non-human transgenic animal
that differs from the physiological characteristic of the wild-type
animal is identified as a condition resulting from the gene
disruption in the non-human transgenic animal;
[0097] (d) administering a test agent to the non-human transgenic
animal of (a); and
[0098] (e) evaluating the effects of the test agent on the
identified condition associated with gene disruption in the
non-human transgenic animal.
[0099] In one aspect, the condition is a neurological disorder; a
cardiovascular, endothelial or angiogenic disorder; an eye
abnormality; an immunological disorder; an oncological disorder; a
bone metabolic abnormality or disorder; a lipid metabolic disorder;
or a developmental abnormality.
[0100] The invention also provides a therapeutic agent which is
capable of affecting a condition associated with gene disruption.
In one aspect, the agent is an agonist or antagonist of the
phenotype associated with a disruption of a gene which encodes for
a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide. In yet another aspect,
the agent is an agonist or antagonist of a PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide. In yet another aspect, the agonist agent is an
anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245,
anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344,
anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732,
anti-PRO1003, anti-PRO1104, anti-PRO1151, anti-PRO1244,
anti-PRO1298, anti-PRO1313, anti-PRO1570, anti-PRO1886,
anti-PRO1891, anti-PRO4409, anti-PRO5725, anti-PRO5994,
anti-PRO6097, anti-PRO7425, anti-PRO10102, anti-PRO10282,
anti-PRO61709 or anti-PRO779 antibody. In still another aspect, the
antagonist agent is an anti-PRO196, anti-PRO217, anti-PRO231,
anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258, anti-PRO287,
anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526, anti-PRO724,
anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104, anti-PRO1151,
anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570,
anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725,
anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102,
anti-PRO10282, anti-PRO61709 or anti-PRO779 antibody.
[0101] The invention also provides a pharmaceutical composition
comprising a therapeutic agent capable of affecting the condition
associated with gene disruption.
[0102] The invention also provides a method of treating or
preventing or ameliorating a neurological disorder; cardiovascular,
endothelial or angiogenic disorder; immunological disorder;
oncological disorder; bone metabolic abnormality or disorder, or
embryonic lethality associated with the disruption of a gene which
encodes for a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide, the
method comprising administering to a subject in need of such
treatment whom may already have the disorder, or may be prone to
have the disorder or may be in whom the disorder is to be
prevented, a therapeutically effective amount of a therapeutic
agent, or agonists or antagonists thereof, thereby effectively
treating or preventing or ameliorating said disorder or
disease.
[0103] In yet another aspect, the neurological disorder is an
increased anxiety-like response during open field activity testing.
In yet another aspect, the neurological disorder is a decreased
anxiety-like response during open field activity testing. In yet
another aspect, the neurological disorder is an abnormal circadian
rhythm during home-cage activity testing. In yet another aspect,
the neurological disorder is an enhanced motor coordination during
inverted screen testing. In yet another aspect, the neurological
disorder is impaired motor coordination during inverted screen
testing. In yet another aspect, the neurological disorder includes
depression, generalized anxiety disorders, attention deficit
disorder, sleep disorder, hyperactivity disorder, obsessive
compulsive disorder, schizophrenia, cognitive disorders,
hyperalgesia and sensory disorders. Such neurological disorders
include the category defined as "anxiety disorders" which include
but are not limited to: mild to moderate anxiety, anxiety disorder
due to a general medical condition, anxiety disorder not otherwise
specified, generalized anxiety disorder, panic attack, panic
disorder with agoraphobia, panic disorder without agoraphobia,
posttraumatic stress disorder, social phobia, social anxiety,
autism, specific phobia, substance-induced anxiety disorder, acute
alcohol withdrawal, obsessive compulsive disorder, agoraphobia,
monopolar disorders, bipolar disorder I or II, bipolar disorder not
otherwise specified, cyclothymic disorder, depressive disorder,
major depressive disorder, mood disorder, substance-induced mood
disorder, enhancement of cognitive function, loss of cognitive
function associated with but not limited to Alzheimer's disease,
stroke, or traumatic injury to the brain, seizures resulting from
disease or injury including but not limited to epilepsy, learning
disorders/disabilities, cerebral palsy. In addition, anxiety
disorders may apply to personality disorders including but not
limited to the following types: paranoid, antisocial, avoidant
behavior, borderline personality disorders, dependent, histronic,
narcissistic, obsessive-compulsive, schizoid, and schizotypal.
[0104] In another aspect, the eye abnormality is a retinal
abnormality. In still another aspect, the eye abnormality is
consistent with vision problems or blindness. In yet another
aspect, the retinal abnormality is consistent with retinitis
pigmentosa or is characterized by retinal degeneration or retinal
dysplasia.
[0105] In still another aspect, the retinal abnormalities are
consistent with retinal dysplasia, various retinopathies, including
retinopathy of prematurity, retrolental fibroplasia, neovascular
glaucoma, age-related macular degeneration, diabetic macular edema,
corneal neovascularization, corneal graft neovascularization,
corneal graft rejection, retinal/choroidal neovascularization,
neovascularization of the angle (rubeosis), ocular neovascular
disease, vascular restenosis, arteriovenous malformations (AVM),
meningioma, hemangioma, angiofibroma, thyroid hyperplasias
(including Grave's disease), corneal and other tissue
transplantation, retinal artery obstruction or occlusion; retinal
degeneration causing secondary atrophy of the retinal vasculature,
retinitis pigmentosa, macular dystrophies, Stargardt's disease,
congenital stationary night blindness, choroideremia, gyrate
atrophy, Leber's congenital amaurosis, retinoschisis disorders,
Wagner's syndrome, Usher syndromes, Zellweger syndrome,
Saldino-Mainzer syndrome, Senior-Loken syndrome, Bardet-Biedl
syndrome, Alport's syndrome, Alstrom's syndrome, Cockayne's
syndrome, dysplaisa spondyloepiphysaria congentia, Flynn-Aird
syndrome, Friedreich ataxia, Hallgren syndrome, Marshall syndrome,
Albers-Schnoberg disease, Refsum's disease, Kearns-Sayre syndrome,
Waardenburg's syndrome, Alagile syndrome, myotonic dystrophy,
olivopontocerebellar atrophy, Pierre-Marie dunsdrome, Stickler
syndrome, carotinemeia, cystinosis, Wolfram syndrome,
Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentia
pigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria,
or mannosidosis.
[0106] In still another aspect, the eye abnormality is a cataract.
In still yet another aspect, the cataract is a systemic disease
such as human Down's syndrome, Hallerman-Streiff syndrome, Lowe
syndrome, galactosemia, Marfan syndrome, Trismoy 13-15, Alport
syndrome, myotonic dystrophy, Fabry disease, hypoparathroidism or
Conradi syndrome.
[0107] In still another aspect, the developmental abnormality
comprises embryonic lethality or reduced viability.
[0108] In yet another aspect, the cardiovascular, endothelial or
angiogenic disorders are arterial diseases, such as diabetes
mellitus; papilledema; optic atrophy; atherosclerosis; angina;
myocardial infarctions such as acute myocardial infarctions,
cardiac hypertrophy, and heart failure such as congestive heart
failure; hypertension; inflammatory vasculitides; Reynaud's disease
and Reynaud's phenomenon; aneurysms and arterial restenosis; venous
and lymphatic disorders such as thrombophlebitis, lymphangitis, and
lymphedema; peripheral vascular disease; cancer such as vascular
tumors, e.g., hemangioma (capillary and cavernous), glomus tumors,
telangiectasia, bacillary angiomatosis, hemangioendothelioma,
angiosarcoma, haemangiopericytoma, Kaposi's sarcoma, lymphangioma,
and lymphangiosarcoma; tumor angiogenesis; trauma such as wounds,
burns, and other injured tissue, implant fixation, scarring;
ischemia reperfusion injury; rheumatoid arthritis; cerebrovascular
disease; renal diseases such as acute renal failure, or
osteoporosis.
[0109] In still yet another aspect, the immunological disorders are
consistent with systemic lupus erythematosis; rheumatoid arthritis;
juvenile chronic arthritis; spondyloarthropathies; systemic
sclerosis (scleroderma); idiopathic inflammatory myopathies
(dermatomyositis, polymyositis); Sjogren's syndrome; systemic
vasculitis; sarcoidosis; autoimmune hemolytic anemia (immune
pancytopenia, paroxysmal nocturnal hemoglobinuria); autoimmune
thrombocytopenia (idiopathic thrombocytopenic purpura,
immune-mediated thrombocytopenia); thyroiditis (Grave's disease,
Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic
thyroiditis); diabetes mellitus; immune-mediated renal disease
(glomerulonephritis, tubulointerstitial nephritis); demyelinating
diseases of the central and peripheral nervous systems such as
multiple sclerosis, idiopathic demyelinating polyneuropathy or
Guillain-Barre syndrome, and chronic inflammatory demyelinating
polyneuropathy; hepatobiliary diseases such as infectious hepatitis
(hepatitis A, B, C, D, E and other non-hepatotropic viruses),
autoimmune chronic active hepatitis, primary biliary cirrhosis,
granulomatous hepatitis, and sclerosing cholangitis; inflammatory
bowel disease (ulcerative colitis: Crohn's disease);
gluten-sensitive enteropathy, and Whipple's disease; autoimmune or
immune-mediated skin diseases including bullous skin diseases,
erythema multiforme and contact dermatitis, psoriasis; allergic
diseases such as asthma, allergic rhinitis, atopic dermatitis, food
hypersensitivity and urticaria; immunologic diseases of the lung
such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and
hypersensitivity pneumonitis; or transplantation associated
diseases including graft rejection and graft-versus-host
disease.
[0110] In yet another aspect, the bone metabolic abnormality or
disorder is arthritis, osteoporosis, osteopenia or
osteopetrosis.
[0111] In another aspect the therapeutic agent is an agonist or
antagonist of the phenotype associated with a disruption of a gene
which encodes for a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide. In yet
another aspect, the agent is an agonist or antagonist of a PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide. In yet another aspect, the agonist
agent is an anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236,
anti-PRO245, anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328,
anti-PRO344, anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731,
anti-PRO732, anti-PRO1003, anti-PRO1104, anti-PRO1151,
anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570,
anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725,
anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102,
anti-PRO10282, anti-PRO61709 or anti-PRO779 antibody. In still
another aspect, the antagonist agent is an anti-PRO196,
anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246,
anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357,
anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003,
anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298,
anti-PRO1313, anti-PRO1570, anti-PRO1886, anti-PRO1891,
anti-PRO4409, anti-PRO5725, anti-PRO5994, anti-PRO6097,
anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709 or
anti-PRO779 antibody.
[0112] The invention also provides a method of identifying an agent
that ameliorates or modulates a neurological disorder; a
cardiovascular, endothelial or angiogenic disorder; an eye
abnormality; an immunological disorder; an oncological disorder; a
bone metabolic abnormality or disorder; a lipid metabolic disorder;
or a developmental abnormality associated with a disruption in the
gene which encodes for a PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide, the method comprising:
[0113] (a) providing a non-human transgenic animal cell culture,
each cell of said culture comprising a disruption of the gene which
encodes for a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide;
[0114] (b) administering a test agent to said cell culture; and
[0115] (c) determining whether the test agent ameliorates or
modulates the neurological disorder; cardiovascular, endothelial or
angiogenic disorder; eye abnormality; immunological disorder;
oncological disorder; bone metabolic abnormality or disorder; lipid
metabolic disorder; or developmental abnormality in said culture.
In yet another aspect, the neurological disorder is an increased
anxiety-like response during open field activity testing. In yet
another aspect, the neurological disorder is a decreased
anxiety-like response during open field activity testing. In yet
another aspect, the neurological disorder is an abnormal circadian
rhythm during home-cage activity testing. In yet another aspect,
the neurological disorder is an enhanced motor coordination during
inverted screen testing. In yet another aspect, the neurological
disorder is impaired motor coordination during inverted screen
testing. In yet another aspect, the neurological disorder includes
depression, generalized anxiety disorders, attention deficit
disorder, sleep disorder, hyperactivity disorder, obsessive
compulsive disorder, schizophrenia, cognitive disorders,
hyperalgesia and sensory disorders. Such neurological disorders
include the category defined as "anxiety disorders" which include
but are not limited to: mild to moderate anxiety, anxiety disorder
due to a general medical condition, anxiety disorder not otherwise
specified, generalized anxiety disorder, panic attack, panic
disorder with agoraphobia, panic disorder without agoraphobia,
posttraumatic stress disorder, social phobia, social anxiety,
autism, specific phobia, substance-induced anxiety disorder, acute
alcohol withdrawal, obsessive compulsive disorder, agoraphobia,
monopolar disorders, bipolar disorder I or II, bipolar disorder not
otherwise specified, cyclothymic disorder, depressive disorder,
major depressive disorder, mood disorder, substance-induced mood
disorder, enhancement of cognitive function, loss of cognitive
function associated with but not limited to Alzheimer's disease,
stroke, or traumatic injury to the brain, seizures resulting from
disease or injury including but not limited to epilepsy, learning
disorders/disabilities, cerebral palsy. In addition, anxiety
disorders may apply to personality disorders including but not
limited to the following types: paranoid, antisocial, avoidant
behavior, borderline personality disorders, dependent, histronic,
narcissistic, obsessive-compulsive, schizoid, and schizotypal.
[0116] In another aspect, the eye abnormality is a retinal
abnormality. In still another aspect, the eye abnormality is
consistent with vision problems or blindness. In yet another
aspect, the retinal abnormality is consistent with retinitis
pigmentosa or is characterized by retinal degeneration or retinal
dysplasia.
[0117] In still another aspect, the retinal abnormalities are
consistent with retinal dysplasia, various retinopathies, including
retinopathy of prematurity, retrolental fibroplasia, neovascular
glaucoma, age-related macular degeneration, diabetic macular edema,
corneal neovascularization, corneal graft neovascularization,
corneal graft rejection, retinal/choroidal neovascularization,
neovascularization of the angle (rubeosis), ocular neovascular
disease, vascular restenosis, arteriovenous malformations (AVM),
meningioma, hemangioma, angiofibroma, thyroid hyperplasias
(including Grave's disease), corneal and other tissue
transplantation, retinal artery obstruction or occlusion; retinal
degeneration causing secondary atrophy of the retinal vasculature,
retinitis pigmentosa, macular dystrophies, Stargardt's disease,
congenital stationary night blindness, choroideremia, gyrate
atrophy, Leber's congenital amaurosis, retinoschisis disorders,
Wagner's syndrome, Usher syndromes, Zellweger syndrome,
Saldino-Mainzer syndrome, Senior-Loken syndrome, Bardet-Biedl
syndrome, Alport's syndrome, Alstrom's syndrome, Cockayne's
syndrome, dysplaisa spondyloepiphysaria congentia, Flynn-Aird
syndrome, Friedreich ataxia, Hallgren syndrome, Marshall syndrome,
Albers-Schnoberg disease, Refsum's disease, Kearns-Sayre syndrome,
Waardenburg's syndrome, Alagile syndrome, myotonic dystrophy,
olivopontocerebellar atrophy, Pierre-Marie dunsdrome, Stickler
syndrome, carotinemeia, cystinosis, Wolfram syndrome,
Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentia
pigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria,
or mannosidosis.
[0118] In still another aspect, the eye abnormality is a cataract.
In still yet another aspect, the cataract is a systemic disease
such as human Down's syndrome, Hallerman-Streiff syndrome, Lowe
syndrome, galactosemia, Marfan syndrome, Trismoy 13-15, Alport
syndrome, myotonic dystrophy, Fabry disease, hypoparathroidism or
Conradi syndrome.
[0119] In still another aspect, the developmental abnormality
comprises embryonic lethality or reduced viability.
[0120] In yet another aspect, the cardiovascular, endothelial or
angiogenic disorders are arterial diseases, such as diabetes
mellitus; papilledema; optic atrophy; atherosclerosis; angina;
myocardial infarctions such as acute myocardial infarctions,
cardiac hypertrophy, and heart failure such as congestive heart
failure; hypertension; inflammatory vasculitides; Reynaud's disease
and Reynaud's phenomenon; aneurysms and arterial restenosis; venous
and lymphatic disorders such as thrombophlebitis, lymphangitis, and
lymphedema; peripheral vascular disease; cancer such as vascular
tumors, e.g., hemangioma (capillary and cavernous), glomus tumors,
telangiectasia, bacillary angiomatosis, hemangioendothelioma,
angiosarcoma, haemangiopericytoma, Kaposi's sarcoma, lymphangioma,
and lymphangiosarcoma; tumor angiogenesis; trauma such as wounds,
burns, and other injured tissue, implant fixation, scarring;
ischemia reperfusion injury; rheumatoid arthritis; cerebrovascular
disease; renal diseases such as acute renal failure, or
osteoporosis.
[0121] In still yet another aspect, the immunological disorders are
consistent with systemic lupus erythematosis; rheumatoid arthritis;
juvenile chronic arthritis; spondyloarthropathies; systemic
sclerosis (scleroderma); idiopathic inflammatory myopathies
(dermatomyositis, polymyositis); Sjogren's syndrome; systemic
vasculitis; sarcoidosis; autoimmune hemolytic anemia (immune
pancytopenia, paroxysmal nocturnal hemoglobinuria); autoimmune
thrombocytopenia (idiopathic thrombocytopenic purpura,
immune-mediated thrombocytopenia); thyroiditis (Grave's disease,
Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic
thyroiditis); diabetes mellitus; immune-mediated renal disease
(glomerulonephritis, tubulointerstitial nephritis); demyelinating
diseases of the central and peripheral nervous systems such as
multiple sclerosis, idiopathic demyelinating polyneuropathy or
Guillain-Barre syndrome, and chronic inflammatory demyelinating
polyneuropathy; hepatobiliary diseases such as infectious hepatitis
(hepatitis A, B, C, D, E and other non-hepatotropic viruses),
autoimmune chronic active hepatitis, primary biliary cirrhosis,
granulomatous hepatitis, and sclerosing cholangitis; inflammatory
bowel disease (ulcerative colitis: Crohn's disease);
gluten-sensitive enteropathy, and Whipple's disease; autoimmune or
immune-mediated skin diseases including bullous skin diseases,
erythema multiforme and contact dermatitis, psoriasis; allergic
diseases such as asthma, allergic rhinitis, atopic dermatitis, food
hypersensitivity and urticaria; immunologic diseases of the lung
such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and
hypersensitivity pneumonitis; or transplantation associated
diseases including graft rejection and graft-versus-host
disease.
[0122] In yet another aspect, the bone metabolic abnormality or
disorder is arthritis, osteoporosis, osteopenia or
osteopetrosis.
[0123] The invention also provides an agent that ameliorates or
modulates a neurological disorder; a cardiovascular, endothelial or
angiogenic disorder; an eye abnormality; an immunological disorder;
an oncological disorder; a bone metabolic abnormality or disorder;
a lipid metabolic disorder; or a developmental abnormality which is
associated with gene disruption in said culture. In one aspect, the
agent is an agonist or antagonist of the phenotype associated with
a disruption of a gene which encodes for a PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide. In yet another aspect, the agent is an agonist or
antagonist of a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide. In yet
another aspect, the agonist agent is an anti-PRO196, anti-PRO217,
anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258,
anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526,
anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104,
anti-PRO1151, anti-PRO1244, anti-PRO1298, anti-PRO1313,
anti-PRO1570, anti-PRO1886, anti-PRO1891, anti-PRO4409,
anti-PRO5725, anti-PRO5994, anti-PRO6097, anti-PRO7425,
anti-PRO10102, anti-PRO10282, anti-PRO61709 or anti-PRO779
antibody. In still another aspect, the antagonist agent is an
anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245,
anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344,
anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732,
anti-PRO1003, anti-PRO1104, anti-PRO1151, anti-PRO1244,
anti-PRO1298, anti-PRO1313, anti-PRO1570, anti-PRO1886,
anti-PRO1891, anti-PRO4409, anti-PRO5725, anti-PRO5994,
anti-PRO6097, anti-PRO7425, anti-PRO10102, anti-PRO10282,
anti-PRO61709 or anti-PRO779 antibody.
[0124] The invention also provides a method of modulating a
phenotype associated with a disruption of a gene which encodes for
a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide, the method comprising
administering to a subject whom may already have the phenotype, or
may be prone to have the phenotype or may be in whom the phenotype
is to be prevented, an effective amount of an agent identified as
modulating said phenotype, or agonists or antagonists thereof,
thereby effectively modulating the phenotype.
[0125] The invention also provides a method of modulating a
physiological characteristic associated with a disruption of a gene
which encodes for a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide, the
method comprising administering to a subject whom may already
exhibit the physiological characteristic, or may be prone to
exhibit the physiological characteristic or may be in whom the
physiological characteristic is to be prevented, an effective
amount of an agent identified as modulating said physiological
characteristic, or agonists or antagonists thereof, thereby
effectively modulating the physiological characteristic.
[0126] The invention also provides a method of modulating a
behavior associated with a disruption of a gene which encodes for a
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide, the method comprising
administering to a subject whom may already exhibit the behavior,
or may be prone to exhibit the behavior or may be in whom the
exhibited behavior is to be prevented, an effective amount of an
agent identified as modulating said behavior, or agonists or
antagonists thereof, thereby effectively modulating the
behavior.
[0127] The invention also provides a method of modulating the
expression of a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide, the
method comprising administering to a host cell expressing said
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide, an effective amount of an
agent identified as modulating said expression, or agonists or
antagonists thereof, thereby effectively modulating the expression
of said polypeptide.
[0128] The invention also provides a method of modulating a
condition associated with a disruption of a gene which encodes for
a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide, the method comprising
administering to a subject whom may have the condition, or may be
prone to have the condition or may be in whom the condition is to
be prevented, a therapeutically effective amount of a therapeutic
agent identified as modulating said condition, or agonists or
antagonists thereof, thereby effectively modulating the
condition.
[0129] The invention also provides a method of treating or
preventing or ameliorating a neurological disorder; cardiovascular,
endothelial or angiogenic disorder; immunological disorder;
oncological disorder; bone metabolic abnormality or disorder, or
embryonic lethality associated with the disruption of a gene which
encodes for a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide, the
method comprising administering to a non-human transgenic animal
cell culture, each cell of said culture comprising a disruption of
the gene which encodes for a PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide, an effective amount of an agent identified as treating
or preventing or ameliorating said disorder, or agonists or
antagonists thereof, thereby effectively treating or preventing or
ameliorating said disorder.
B. Further Embodiments
[0130] In yet further embodiments, the invention is directed to the
following set of potential claims for this application: [0131] A
method of identifying a phenotype associated with a disruption of a
gene which encodes for a PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide, the method comprising:
[0132] (a) providing a non-human transgenic animal whose genome
comprises a disruption of the gene which encodes for a PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide;
[0133] (b) measuring a physiological characteristic of the
non-human transgenic animal; and
[0134] (c) comparing the measured physiological characteristic with
that of a gender matched wild-type animal, wherein the
physiological characteristic of the non-human transgenic animal
that differs from the physiological characteristic of the wild-type
animal is identified as a phenotype resulting from the gene
disruption in the non-human transgenic animal. [0135] The method of
Claim 1, wherein the non-human transgenic animal is heterozygous
for the disruption of a gene which encodes for a PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 polypeptide. [0136] The method of Claim 1, wherein the
phenotype exhibited by the non-human transgenic animal as compared
with gender matched wild-type littermates is at least one of the
following: a neurological disorder; a cardiovascular, endothelial
or angiogenic disorder; an eye abnormality; an immunological
disorder; an oncological disorder; a bone metabolic abnormality or
disorder; a lipid metabolic disorder; or a developmental
abnormality. [0137] 4. The method of Claim 3, wherein the
neurological disorder is an increased anxiety-like response during
open field activity testing. [0138] 5. The method of Claim 3,
wherein the neurological disorder is a decreased anxiety-like
response during open field activity testing. [0139] 6. The method
of Claim 3, wherein the neurological disorder is an abnormal
circadian rhythm during home-cage activity testing. [0140] 7. The
method of Claim 3, wherein the neurological disorder is an enhanced
motor coordination during inverted screen testing. [0141] 8. The
method of Claim 3, wherein the neurological disorder is an impaired
motor coordination during inverted screen testing. [0142] 9. The
method of Claim 3, wherein the neurological disorder is depression,
generalized anxiety disorders, attention deficit disorder, sleep
disorder, hyperactivity disorder, obsessive compulsive disorder,
schizophrenia, cognitive disorders, hyperalgesia or sensory
disorders. [0143] 10. The method of Claim 3, wherein the eye
abnormality is a retinal abnormality. [0144] 11. The method of
Claim 3, wherein the eye abnormality is consistent with vision
problems or blindness. [0145] 12. The method of Claim 10, wherein
the retinal abnormality is consistent with retinitis pigmentosa.
[0146] 13. The method of Claim 10, wherein the retinal abnormality
is characterized by retinal degeneration or retinal dysplasia.
[0147] 14. The method of Claim 10, wherein the retinal abnormality
is consistent with retinal dysplasia, various retinopathies,
including retinopathy of prematurity, retrolental fibroplasia,
neovascular glaucoma, age-related macular degeneration, diabetic
macular edema, corneal neovascularization, corneal graft
neovascularization, corneal graft rejection, retinal/choroidal
neovascularization, neovascularization of the angle (rubeosis),
ocular neovascular disease, vascular restenosis, arteriovenous
malformations (AVM), meningioma, hemangioma, angiofibroma, thyroid
hyperplasias (including Grave's disease), corneal and other tissue
transplantation, retinal artery obstruction or occlusion; retinal
degeneration causing secondary atrophy of the retinal vasculature,
retinitis pigmentosa, macular dystrophies, Stargardt's disease,
congenital stationary night blindness, choroideremia, gyrate
atrophy, Leber's congenital amaurosis, retinoschisis disorders,
Wagner's syndrome, Usher syndromes, Zellweger syndrome,
Saldino-Mainzer syndrome, Senior-Loken syndrome, Bardet-Biedl
syndrome, Alport's syndrome, Alstrom's syndrome, Cockayne's
syndrome, dysplaisa spondyloepiphysaria congentia, Flynn-Aird
syndrome, Friedreich ataxia, Hallgren syndrome, Marshall syndrome,
Albers-Schnoberg disease, Refsum's disease, Kearns-Sayre syndrome,
Waardenburg's syndrome, Alagile syndrome, myotonic dystrophy,
olivopontocerebellar atrophy, Pierre-Marie dunsdrome, Stickler
syndrome, carotinemeia, cystinosis, Wolfram syndrome,
Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentia
pigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria,
or mannosidosis. [0148] 15. The method of Claim 3, wherein the eye
abnormality is a cataract. [0149] 16. The method of Claim 15,
wherein the cataract is consistent with systemic diseases such as
human Down's syndrome, Hallerman-Streiff syndrome, Lowe syndrome,
galactosemia, Marfan syndrome, Trismoy 13-15, Alport syndrome,
myotonic dystrophy, Fabry disease, hypoparathroidism or Conradi
syndrome. [0150] 17. The method of Claim 3, wherein the
developmental abnormality comprises embryonic lethality or reduced
viability. [0151] 18. The method of Claim 3, wherein the
cardiovascular, endothelial or angiogenic disorders are arterial
diseases, such as diabetes mellitus; papilledema; optic atrophy;
atherosclerosis; angina; myocardial infarctions such as acute
myocardial infarctions, cardiac hypertrophy, and heart failure such
as congestive heart failure; hypertension; inflammatory
vasculitides; Reynaud's disease and Reynaud's phenomenon; aneurysms
and arterial restenosis; venous and lymphatic disorders such as
thrombophlebitis, lymphangitis, and lymphedema; peripheral vascular
disease; cancer such as vascular tumors, e.g., hemangioma
(capillary and cavernous), glomus tumors, telangiectasia, bacillary
angiomatosis, hemangioendothelioma, angiosarcoma,
haemangiopericytoma, Kaposi's sarcoma, lymphangioma, and
lymphangiosarcoma; tumor angiogenesis; trauma such as wounds,
burns, and other injured tissue, implant fixation, scarring;
ischemia reperfusion injury; rheumatoid arthritis; cerebrovascular
disease; renal diseases such as acute renal failure, or
osteoporosis. [0152] 19. The method of Claim 3, wherein the
immunological disorders are systemic lupus erythematosis;
rheumatoid arthritis; juvenile chronic arthritis;
spondyloarthropathies; systemic sclerosis (scleroderma); idiopathic
inflammatory myopathies (dermatomyositis, polymyositis); Sjogren's
syndrome; systemic vasculitis; sarcoidosis; autoimmune hemolytic
anemia (immune pancytopenia, paroxysmal nocturnal hemoglobinuria);
autoimmune thrombocytopenia (idiopathic thrombocytopenic purpura,
immune-mediated thrombocytopenia); thyroiditis (Grave's disease,
Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic
thyroiditis); diabetes mellitus; immune-mediated renal disease
(glomerulonephritis, tubulointerstitial nephritis); demyelinating
diseases of the central and peripheral nervous systems such as
multiple sclerosis, idiopathic demyelinating polyneuropathy or
Guillain-Barre syndrome, and chronic inflammatory demyelinating
polyneuropathy; hepatobiliary diseases such as infectious hepatitis
(hepatitis A, B, C, D, E and other non-hepatotropic viruses),
autoimmune chronic active hepatitis, primary biliary cirrhosis,
granulomatous hepatitis, and sclerosing cholangitis; inflammatory
bowel disease (ulcerative colitis: Crohn's disease);
gluten-sensitive enteropathy, and Whipple's disease; autoimmune or
immune-mediated skin diseases including bullous skin diseases,
erythema multiforme and contact dermatitis, psoriasis; allergic
diseases such as asthma, allergic rhinitis, atopic dermatitis, food
hypersensitivity and urticaria; immunologic diseases of the lung
such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and
hypersensitivity pneumonitis; or transplantation associated
diseases including graft rejection and graft-versus-host disease.
[0153] 20. The method of Claim 3, wherein the bone metabolic
abnormality or disorder is arthritis, osteoporosis or
osteopetrosis. [0154] 21. The method of Claim 1, wherein the
non-human transgenic animal exhibits at least one of the following
physiological characteristics compared with gender matched
wild-type littermates: increased anxiety-like response during open
field testing; decreased anxiety-like response during open field
activity testing; abnormal circadian rhythm during home-cage
activity testing including decreased ambulatory counts; increased
exploratory activity during open-field testing; increased stress
induced hyperthermia; enhanced motor coordination during inverted
screen testing; impaired motor coordination during inverted screen
testing; increase in retinal artery tortuosity; retinal
degeneration marked by attenuated retinal vessels; opthamological
abnormalities; increased mean systolic blood pressure; increased
mean fasting serum glucose levels; decreased mean serum glucose
levels; increased mean serum cholesterol levels; increased mean
serum triglyceride levels; decreased mean serum cholesterol levels;
decreased mean serum triglyceride levels; enhanced glucose
tolerance; impaired glucose tolerance; increased mean serum insulin
levels; decreased mean serum insulin levels; increased uric acid
levels; decreased serum phosphate levels; increased alkaline
phosphatase levels and increased alanine amino transferase levels;
liver disease; increased mean percentage of CD25+ in both spleen
and lymph nodes; decreased mean percentage of natural killer cells;
decreased mean percentage of CD21HiCD23Med cells in spleen and
lymph nodes; increased mean percentage of CD4 cells and decreased
mean percentage of B cells; increased mean percentage of CD8+
cells; decreased mean percentage of eosinophils; decreased mean
serum IgG1 response to an ovalbumin challenge; decreased mean serum
IgG2a response to an ovalbumin challenge; increased mean serum IgG1
response to an ovalbumin challenge; increased mean serum IgG2a
response to an ovalbumin challenge; increased mean serum MCP-1
response to a LPS challenge; increased mean serum TNF-alpha
response to a LPS challenge; decreased mean serum MCP-1 response to
a LPS challenge; decreased mean serum IL-6 response to a LPS
challenge; decreased TNF-alpha response to a LPS challenge;
increased mean serum IL6 response to a LPS challenge; increased
mean platelet counts; decreased mean total white blood cell (WBC)
counts; decreased absolute lymphocyte counts; decreased absolute
monocyte counts; decreased skin fibroblast proliferation; increased
skin fibroblast proliferation; increased mean percent of total body
fat and total fat mass; increased mean body weight; increased mean
body length; increased organ weights; increased total tissue mass
(TTM); increased lean body mass (LBM); increased bone mineral
density (BMD) in total body, femur and vertebrae; increased bone
mineral content (BMC) in total body, femur and vertebrae; increased
volumetric bone mineral density (vBMD) in total body, femur and
vertebrae; increased mean femoral midshaft cortical thickness and
cross-sectional area; increased mean vertebral trabecular bone
volume, number and connectivity density; decreased mean percent of
total body fat and total fat mass; decreased mean body weight;
decreased mean body length; decreased total tissue mass (TTM);
decreased lean body mass (LBM); decreased bone mineral density
(BMD) in total body, femur and vertebrae; decreased bone mineral
content (BMC) in total body, femur and vertebrae; decreased
volumetric bone mineral density (vBMD) in total body, femur and
vertebrae; decreased mean femoral midshaft cortical thickness and
cross-sectional area; decreased mean vertebral trabecular bone
volume, number and connectivity density; severe depletion of
abdominal and subcutaneous body fat deposits; decreased organ
weights; growth retardation; hydrocephalus; sebaceous gland
hyperplasia and growth retardation; apoptosis of olfactory
neuroepithelial cells; lymphoid hyperplasia and tissue
inflammation; development abnormalities; male infertility; growth
retardation with reduced viability; and embryonic lethality. [0155]
22. An isolated cell derived from a non-human transgenic animal
whose genome comprises a disruption of the gene which encodes for a
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide. [0156] 23. The isolated
cell of Claim 22 which is a murine cell. [0157] 24. The isolated
cell of Claim 23, wherein the murine cell is an embryonic stem
cell. [0158] 25. The isolated cell of Claim 22, wherein the
non-human transgenic animal exhibits at least one of the following
phenotypes compared with gender matched wild-type littermates: a
neurological disorder; a cardiovascular, endothelial or angiogenic
disorder; an eye abnormality; an immunological disorder; an
oncological disorder; a bone metabolic abnormality or disorder; a
lipid metabolic disorder; or a developmental abnormality. [0159]
26. A method of identifying an agent that modulates a phenotype
associated with a disruption of a gene which encodes for a PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide, the method comprising:
[0160] (a) providing a non-human transgenic animal whose genome
comprises a disruption of the gene which encodes for the PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide;
[0161] (b) measuring a physiological characteristic of the
non-human transgenic animal of (a);
[0162] (c) comparing the measured physiological characteristic of
(b) with that of a gender matched wild-type animal, wherein the
physiological characteristic of the non-human transgenic animal
that differs from the physiological characteristic of the wild-type
animal is identified as a phenotype resulting from the gene
disruption in the non-human transgenic animal;
[0163] (d) administering a test agent to the non-human transgenic
animal of (a); and
[0164] (e) determining whether the test agent modulates the
identified phenotype associated with gene disruption in the
non-human transgenic animal. [0165] 27. The method of Claim 26,
wherein the phenotype associated with the gene disruption comprises
a neurological disorder; a cardiovascular, endothelial or
angiogenic disorder; an eye abnormality; an immunological disorder;
an oncological disorder; a bone metabolic abnormality or disorder;
a lipid metabolic disorder; or a developmental abnormality. [0166]
28. The method of Claim 27, wherein the neurological disorder is an
increased anxiety-like response during open field activity testing.
[0167] 29. The method of Claim 27, wherein the neurological
disorder is a decreased anxiety-like response during open field
activity testing. [0168] 30. The method of Claim 27, wherein the
neurological disorder is an abnormal circadian rhythm during
home-cage activity testing. [0169] 31. The method of Claim 27,
wherein the neurological disorder is an enhanced motor coordination
during inverted screen testing. [0170] 32. The method of Claim 27,
wherein the neurological disorder is an impaired motor coordination
during inverted screen testing. [0171] 33. The method of Claim 27,
wherein the neurological disorder is depression, generalized
anxiety disorders, attention deficit disorder, sleep disorder,
hyperactivity disorder, obsessive compulsive disorder,
schizophrenia, cognitive disorders, hyperalgesia or sensory
disorders. [0172] 34. The method of Claim 27, wherein the eye
abnormality is a retinal abnormality. [0173] 35. The method of
Claim 27, wherein the eye abnormality is consistent with vision
problems or blindness. [0174] 36. The method of Claim 34, wherein
the retinal abnormality is consistent with retinitis pigmentosa.
[0175] 37. The method of Claim 34, wherein the retinal abnormality
is characterized by retinal degeneration or retinal dysplasia.
[0176] 38. The method of Claim 34, wherein the retinal abnormality
is consistent with retinal dysplasia, various retinopathies,
including retinopathy of prematurity, retrolental fibroplasia,
neovascular glaucoma, age-related macular degeneration, diabetic
macular edema, corneal neovascularization, corneal graft
neovascularization, corneal graft rejection, retinal/choroidal
neovascularization, neovascularization of the angle (rubeosis),
ocular neovascular disease, vascular restenosis, arteriovenous
malformations (AVM), meningioma, hemangioma, angiofibroma, thyroid
hyperplasias (including Grave's disease), corneal and other tissue
transplantation, retinal artery obstruction or occlusion; retinal
degeneration causing secondary atrophy of the retinal vasculature,
retinitis pigmentosa, macular dystrophies, Stargardt's disease,
congenital stationary night blindness, choroideremia, gyrate
atrophy, Leber's congenital amaurosis, retinoschisis disorders,
Wagner's syndrome, Usher syndromes, Zellweger syndrome,
Saldino-Mainzer syndrome, Senior-Loken syndrome, Bardet-Biedl
syndrome, Alport's syndrome, Alstrom's syndrome, Cockayne's
syndrome, dysplaisa spondyloepiphysaria congentia, Flynn-Aird
syndrome, Friedreich ataxia, Hallgren syndrome, Marshall syndrome,
Albers-Schnoberg disease, Refsum's disease, Kearns-Sayre syndrome,
Waardenburg's syndrome, Alagile syndrome, myotonic dystrophy,
olivopontocerebellar atrophy, Pierre-Marie dunsdrome, Stickler
syndrome, carotinemeia, cystinosis, Wolfram syndrome,
Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentia
pigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria,
or mannosidosis. [0177] 39. The method of Claim 27, wherein the eye
abnormality is a cataract. [0178] 40. The method of Claim 39,
wherein the cataract is consistent with systemic diseases such as
human Down's syndrome, Hallerman-Streiff syndrome, Lowe syndrome,
galactosemia, Marfan syndrome, Trismoy 13-15, Alport syndrome,
myotonic dystrophy, Fabry disease, hypoparathroidism or Conradi
syndrome. [0179] 41. The method of Claim 27, wherein the
developmental abnormality comprises embryonic lethality or reduced
viability. [0180] 42. The method of Claim 27, wherein the
cardiovascular, endothelial or angiogenic disorders are arterial
diseases, such as diabetes mellitus; papilledema; optic atrophy;
atherosclerosis; angina; myocardial infarctions such as acute
myocardial infarctions, cardiac hypertrophy, and heart failure such
as congestive heart failure; hypertension; inflammatory
vasculitides; Reynaud's disease and Reynaud's phenomenon; aneurysms
and arterial restenosis; venous and lymphatic disorders such as
thrombophlebitis, lymphangitis, and lymphedema; peripheral vascular
disease; cancer such as vascular tumors, e.g., hemangioma
(capillary and cavernous), glomus tumors, telangiectasia, bacillary
angiomatosis, hemangioendothelioma, angiosarcoma,
haemangiopericytoma, Kaposi's sarcoma, lymphangioma, and
lymphangiosarcoma; tumor angiogenesis; trauma such as wounds,
burns, and other injured tissue, implant fixation, scarring;
ischemia reperfusion injury; rheumatoid arthritis; cerebrovascular
disease; renal diseases such as acute renal failure, or
osteoporosis. [0181] 43. The method of Claim 27, wherein the
immunological disorders are systemic lupus erythematosis;
rheumatoid arthritis; juvenile chronic arthritis;
spondyloarthropathies; systemic sclerosis (scleroderma); idiopathic
inflammatory myopathies (dermatomyositis, polymyositis); Sjogren's
syndrome; systemic vasculitis; sarcoidosis; autoimmune hemolytic
anemia (immune pancytopenia, paroxysmal nocturnal hemoglobinuria);
autoimmune thrombocytopenia (idiopathic thrombocytopenic purpura,
immune-mediated thrombocytopenia); thyroiditis (Grave's disease,
Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic
thyroiditis); diabetes mellitus; immune-mediated renal disease
(glomerulonephritis, tubulointerstitial nephritis); demyelinating
diseases of the central and peripheral nervous systems such as
multiple sclerosis, idiopathic demyelinating polyneuropathy or
Guillain-Barre syndrome, and chronic inflammatory demyelinating
polyneuropathy; hepatobiliary diseases such as infectious hepatitis
(hepatitis A, B, C, D, E and other non-hepatotropic viruses),
autoimmune chronic active hepatitis, primary biliary cirrhosis,
granulomatous hepatitis, and sclerosing cholangitis; inflammatory
bowel disease (ulcerative colitis: Crohn's disease);
gluten-sensitive enteropathy, and Whipple's disease; autoimmune or
immune-mediated skin diseases including bullous skin diseases,
erythema multiforme and contact dermatitis, psoriasis; allergic
diseases such as asthma, allergic rhinitis, atopic dermatitis, food
hypersensitivity and urticaria; immunologic diseases of the lung
such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and
hypersensitivity pneumonitis; or transplantation-associated
diseases including graft rejection and graft-versus-host disease.
[0182] 44. The method of Claim 27, wherein said bone metabolic
abnormality or disorder is arthritis, osteoporosis or
osteopetrosis. [0183] 45. The method of Claim 26, wherein the
non-human transgenic animal exhibits at least one of the following
physiological characteristics compared with gender matched
wild-type littermates: increased anxiety-like response during open
field testing; decreased anxiety-like response during open field
activity testing; abnormal circadian rhythm during home-cage
activity testing including decreased ambulatory counts; increased
exploratory activity during open-field testing; increased stress
induced hyperthermia; enhanced motor coordination during inverted
screen testing; impaired motor coordination during inverted screen
testing; increase in retinal artery tortuosity; retinal
degeneration marked by attenuated retinal vessels; opthamological
abnormalities; increased mean systolic blood pressure; increased
mean fasting serum glucose levels; decreased mean serum glucose
levels; increased mean serum cholesterol levels; increased mean
serum triglyceride levels; decreased mean serum cholesterol levels;
decreased mean serum triglyceride levels; enhanced glucose
tolerance; impaired glucose tolerance; increased mean serum insulin
levels; decreased mean serum insulin levels; increased uric acid
levels; decreased serum phosphate levels; increased alkaline
phosphatase levels and increased alanine amino transferase levels;
liver disease; increased mean percentage of CD25+ in both spleen
and lymph nodes; decreased mean percentage of natural killer cells;
decreased mean percentage of CD21HiCD23Med cells in spleen and
lymph nodes; increased mean percentage of CD4 cells and decreased
mean percentage of B cells; increased mean percentage of CD8+
cells; decreased mean percentage of eosinophils; decreased mean
serum IgG1 response to an ovalbumin challenge; decreased mean serum
IgG2a response to an ovalbumin challenge; increased mean serum IgG1
response to an ovalbumin challenge; increased mean serum IgG2a
response to an ovalbumin challenge; increased mean serum MCP-1
response to a LPS challenge; increased mean serum TNF-alpha
response to a LPS challenge; decreased mean serum MCP-1 response to
a LPS challenge; decreased mean serum IL-6 response to a LPS
challenge; decreased TNF-alpha response to a LPS challenge;
increased mean serum IL6 response to a LPS challenge; increased
mean platelet counts; decreased mean total white blood cell (WBC)
counts; decreased absolute lymphocyte counts; decreased absolute
monocyte counts; decreased skin fibroblast proliferation; increased
skin fibroblast proliferation; increased mean percent of total body
fat and total fat mass; increased mean body weight; increased mean
body length; increased organ weights; increased total tissue mass
(TTM); increased lean body mass (LBM); increased bone mineral
density (BMD) in total body, femur and vertebrae; increased bone
mineral content (BMC) in total body, femur and vertebrae; increased
volumetric bone mineral density (vBMD) in total body, femur and
vertebrae; increased mean femoral midshaft cortical thickness and
cross-sectional area; increased mean vertebral trabecular bone
volume, number and connectivity density; decreased mean percent of
total body fat and total fat mass; decreased mean body weight;
decreased mean body length; decreased total tissue mass (TTM);
decreased lean body mass (LBM); decreased bone mineral density
(BMD) in total body, femur and vertebrae; decreased bone mineral
content (BMC) in total body, femur and vertebrae; decreased
volumetric bone mineral density (vBMD) in total body, femur and
vertebrae; decreased mean femoral midshaft cortical thickness and
cross-sectional area; decreased mean vertebral trabecular bone
volume, number and connectivity density; severe depletion of
abdominal and subcutaneous body fat deposits; decreased organ
weights; growth retardation; hydrocephalus; sebaceous gland
hyperplasia and growth retardation; apoptosis of olfactory
neuroepithelial cells; lymphoid hyperplasia and tissue
inflammation; development abnormalities; male infertility; growth
retardation with reduced viability; and embryonic lethality. [0184]
46. An agent identified by the method of Claim 26. [0185] 47. The
agent of Claim 46 which is an agonist or antagonist of a PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide. [0186] 48. The agent of Claim 47,
wherein the agonist is an anti-PRO196, anti-PRO217, anti-PRO231,
anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258, anti-PRO287,
anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526, anti-PRO724,
anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104, anti-PRO1151,
anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570,
anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725,
anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102,
anti-PRO10282, anti-PRO61709 or anti-PRO779 antibody. [0187] 49.
The agent of Claim 47, wherein the antagonist is an anti-PRO196,
anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246,
anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357,
anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003,
anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298,
anti-PRO1313, anti-PRO1570, anti-PRO1886, anti-PRO1891,
anti-PRO4409, anti-PRO5725, anti-PRO5994, anti-PRO6097,
anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709 or
anti-PRO779 antibody. [0188] 50. A method of identifying an agent
that modulates a physiological characteristic associated with a
disruption of the gene which encodes for a PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide, the method comprising:
[0189] (a) providing a non-human transgenic animal whose genome
comprises a disruption of the gene which encodes for a PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide;
[0190] (b) measuring a physiological characteristic exhibited by
the non-human transgenic animal of (a);
[0191] (c) comparing the measured physiological characteristic of
(b) with that of a gender matched wild-type animal, wherein the
physiological characteristic exhibited by the non-human transgenic
animal that differs from the physiological characteristic exhibited
by the wild-type animal is identified as a physiological
characteristic associated with gene disruption;
[0192] (d) administering a test agent to the non-human transgenic
animal of (a); and
[0193] (e) determining whether the physiological characteristic
associated with gene disruption is modulated. [0194] 51. The method
of Claim 50, wherein the non-human transgenic animal exhibits at
least one of the following physiological characteristics compared
with gender matched wild-type littermates: increased anxiety-like
response during open field testing; decreased anxiety-like response
during open field activity testing; abnormal circadian rhythm
during home-cage activity testing including decreased ambulatory
counts; increased exploratory activity during open-field testing;
increased stress induced hyperthermia; enhanced motor coordination
during inverted screen testing; impaired motor coordination during
inverted screen testing; increase in retinal artery tortuosity;
retinal degeneration marked by attenuated retinal vessels;
opthamological abnormalities; increased mean systolic blood
pressure; increased mean fasting serum glucose levels; decreased
mean serum glucose levels; increased mean serum cholesterol levels;
increased mean serum triglyceride levels; decreased mean serum
cholesterol levels; decreased mean serum triglyceride levels;
enhanced glucose tolerance; impaired glucose tolerance; increased
mean serum insulin levels; decreased mean serum insulin levels;
increased uric acid levels; decreased serum phosphate levels;
increased alkaline phosphatase levels and increased alanine amino
transferase levels; liver disease; increased mean percentage of
CD25+ in both spleen and lymph nodes; decreased mean percentage of
natural killer cells; decreased mean percentage of CD21HiCD23Med
cells in spleen and lymph nodes; increased mean percentage of CD4
cells and decreased mean percentage of B cells; increased mean
percentage of CD8+ cells; decreased mean percentage of eosinophils;
decreased mean serum IgG1 response to an ovalbumin challenge;
decreased mean serum IgG2a response to an ovalbumin challenge;
increased mean serum IgG1 response to an ovalbumin challenge;
increased mean serum IgG2a response to an ovalbumin challenge;
increased mean serum MCP-1 response to a LPS challenge; increased
mean serum TNF-alpha response to a LPS challenge; decreased mean
serum MCP-1 response to a LPS challenge; decreased mean serum IL-6
response to a LPS challenge; decreased TNF-alpha response to a LPS
challenge; increased mean serum IL6 response to a LPS challenge;
increased mean platelet counts; decreased mean total white blood
cell (WBC) counts; decreased absolute lymphocyte counts; decreased
absolute monocyte counts; decreased skin fibroblast proliferation;
increased skin fibroblast proliferation; increased mean percent of
total body fat and total fat mass; increased mean body weight;
increased mean body length; increased organ weights; increased
total tissue mass (TTM); increased lean body mass (LBM); increased
bone mineral density (BMD) in total body, femur and vertebrae;
increased bone mineral content (BMC) in total body, femur and
vertebrae; increased volumetric bone mineral density (vBMD) in
total body, femur and vertebrae; increased mean femoral midshaft
cortical thickness and cross-sectional area; increased mean
vertebral trabecular bone volume, number and connectivity density;
decreased mean percent of total body fat and total fat mass;
decreased mean body weight; decreased mean body length; decreased
total tissue mass (TTM); decreased lean body mass (LBM); decreased
bone mineral density (BMD) in total body, femur and vertebrae;
decreased bone mineral content (BMC) in total body, femur and
vertebrae; decreased volumetric bone mineral density (vBMD) in
total body, femur and vertebrae; decreased mean femoral midshaft
cortical thickness and cross-sectional area; decreased mean
vertebral trabecular bone volume, number and connectivity density;
severe depletion of abdominal and subcutaneous body fat deposits;
decreased organ weights; growth retardation; hydrocephalus;
sebaceous gland hyperplasia and growth retardation; apoptosis of
olfactory neuroepithelial cells; lymphoid hyperplasia and tissue
inflammation; development abnormalities; male infertility; growth
retardation with reduced viability; and embryonic lethality. [0195]
52. An agent identified by the method of Claim 50. [0196] 53. The
agent of Claim 52 which is an agonist or antagonist of a PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide. [0197] 54. The agent of Claim 53,
wherein the agonist is an anti-PRO196, anti-PRO217, anti-PRO231,
anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258, anti-PRO287,
anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526, anti-PRO724,
anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104, anti-PRO1151,
anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570,
anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725,
anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102,
anti-PRO10282, anti-PRO61709 or anti-PRO779 antibody. [0198] 55.
The agent of Claim 53, wherein the antagonist is an anti-PRO196,
anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246,
anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357,
anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003,
anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298,
anti-PRO1313, anti-PRO1570, anti-PRO1886, anti-PRO1891,
anti-PRO4409, anti-PRO5725, anti-PRO5994, anti-PRO6097,
anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709 or
anti-PRO779 antibody. [0199] 56. A method of identifying an agent
which modulates a behavior associated with a disruption of the gene
which encodes for a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide, the
method comprising:
[0200] (a) providing a non-human transgenic animal whose genome
comprises a disruption of the gene which encodes for a PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide;
[0201] (b) observing the behavior exhibited by the non-human
transgenic animal of (a);
[0202] (c) comparing the observed behavior of (b) with that of a
gender matched wild-type animal, wherein the observed behavior
exhibited by the non-human transgenic animal that differs from the
observed behavior exhibited by the wild-type animal is identified
as a behavior associated with gene disruption;
[0203] (d) administering a test agent to the non-human transgenic
animal of (a); and
[0204] (e) determining whether the agent modulates the behavior
associated with gene disruption. [0205] 57. The method of Claim 56,
wherein the behavior is an increased anxiety-like response during
open field activity testing. [0206] 58. The method of Claim 56,
wherein the behavior is a decreased anxiety-like response during
open field activity testing. [0207] 59. The method of Claim 56,
wherein the behavior is an abnormal circadian rhythm during
home-cage activity testing. [0208] 60. The method of Claim 56,
wherein the behavior is an enhanced motor coordination during
inverted screen testing. [0209] 61. The method of Claim 56, wherein
the behavior is an impaired motor coordination during inverted
screen testing. [0210] 62. The method of Claim 56, wherein the
behavior is depression, generalized anxiety disorders, attention
deficit disorder, sleep disorder, hyperactivity disorder, obsessive
compulsive disorder, schizophrenia, cognitive disorders,
hyperalgesia or sensory disorders. [0211] 63. An agent identified
by the method of Claim 56. [0212] 64. The agent of Claim 63 which
is an agonist or antagonist of a PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide. [0213] 65. The agent of Claim 64, wherein the agonist
is an anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236,
anti-PRO245, anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328,
anti-PRO344, anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731,
anti-PRO732, anti-PRO1003, anti-PRO1104, anti-PRO1151,
anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570,
anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725,
anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102,
anti-PRO10282, anti-PRO61709 or anti-PRO779 antibody. [0214] 66.
The agent of Claim 64, wherein the antagonist is an anti-PRO196,
anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246,
anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357,
anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003,
anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298,
anti-PRO1313, anti-PRO1570, anti-PRO1886, anti-PRO1891,
anti-PRO4409, anti-PRO5725, anti-PRO5994, anti-PRO6097,
anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709 or
anti-PRO779 antibody. [0215] 67. A method of identifying an agent
that ameliorates or modulates a neurological disorder; a
cardiovascular, endothelial or angiogenic disorder; an eye
abnormality; an immunological disorder; an oncological disorder; a
bone metabolic abnormality or disorder; a lipid metabolic disorder;
or a developmental abnormality associated with a disruption in the
gene which encodes for a PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide, the method comprising:
[0216] (a) providing a non-human transgenic animal whose genome
comprises a disruption of the gene which encodes for a PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide;
[0217] (b) administering a test agent to said non-human transgenic
animal; and
[0218] (c) determining whether said test agent ameliorates or
modulates the neurological disorder; cardiovascular, endothelial or
angiogenic disorder; eye abnormality; immunological disorder;
oncological disorder; bone metabolic abnormality or disorder; lipid
metabolic disorder; or developmental abnormality in the non-human
transgenic animal. [0219] 68. The method of Claim 67, wherein the
neurological disorder is an increased anxiety-like response during
open field activity testing. [0220] 69. The method of Claim 67,
wherein the neurological disorder is a decreased anxiety-like
response during open field activity testing. [0221] 70. The method
of Claim 67, wherein the neurological disorder is an abnormal
circadian rhythm during home-cage activity testing. [0222] 71. The
method of Claim 67, wherein the neurological disorder is an
enhanced motor coordination during inverted screen testing. [0223]
72. The method of Claim 67, wherein the neurological disorder is an
impaired motor coordination during inverted screen testing. [0224]
73. The method of Claim 73, wherein the neurological disorder is
depression, generalized anxiety disorders, attention deficit
disorder, sleep disorder, hyperactivity disorder, obsessive
compulsive disorder, schizophrenia, cognitive disorders,
hyperalgesia or sensory disorders. [0225] 74. The method of Claim
67, wherein the eye abnormality is a retinal abnormality. [0226]
75. The method of Claim 67, wherein the eye abnormality is
consistent with vision problems or blindness. [0227] 76. The method
of Claim 74, wherein the retinal abnormality is consistent with
retinitis pigmentosa. [0228] 77. The method of Claim 74, wherein
the retinal abnormality is characterized by retinal degeneration or
retinal dysplasia. [0229] 78. The method of Claim 74, wherein the
retinal abnormality is consistent with retinal dysplasia, various
retinopathies, including retinopathy of prematurity, retrolental
fibroplasia, neovascular glaucoma, age-related macular
degeneration, diabetic macular edema, corneal neovascularization,
corneal graft neovascularization, corneal graft rejection,
retinal/choroidal neovascularization, neovascularization of the
angle (rubeosis), ocular neovascular disease, vascular restenosis,
arteriovenous malformations (AVM), meningioma, hemangioma,
angiofibroma, thyroid hyperplasias (including Grave's disease),
corneal and other tissue transplantation, retinal artery
obstruction or occlusion; retinal degeneration causing secondary
atrophy of the retinal vasculature, retinitis pigmentosa, macular
dystrophies, Stargardt's disease, congenital stationary night
blindness, choroideremia, gyrate atrophy, Leber's congenital
amaurosis, retinoschisis disorders, Wagner's syndrome, Usher
syndromes, Zellweger syndrome, Saldino-Mainzer syndrome,
Senior-Loken syndrome, Bardet-Biedl syndrome, Alport's syndrome,
Alstrom's syndrome, Cockayne's syndrome, dysplaisa
spondyloepiphysaria congentia, Flynn-Aird syndrome, Friedreich
ataxia, Hallgren syndrome, Marshall syndrome, Albers-Schnoberg
disease, Refsum's disease, Kearns-Sayre syndrome, Waardenburg's
syndrome, Alagile syndrome, myotonic dystrophy,
olivopontocerebellar atrophy, Pierre-Marie dunsdrome, Stickler
syndrome, carotinemeia, cystinosis, Wolfram syndrome,
Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentia
pigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria,
or mannosidosis. [0230] 79. The method of Claim 67, wherein the eye
abnormality is a cataract. [0231] 80. The method of Claim 79,
wherein the cataract is a systemic disease such as human Down's
syndrome, Hallerman-Streiff syndrome, Lowe syndrome, galactosemia,
Marfan syndrome, Trismoy 13-15, Alport syndrome, myotonic
dystrophy, Fabry disease, hypoparathroidism or Conradi syndrome.
[0232] 81. The method of Claim 67, wherein the developmental
abnormality comprises embryonic lethality or reduced viability.
[0233] 82. The method of Claim 67, wherein the cardiovascular,
endothelial or angiogenic disorders are arterial diseases, such as
diabetes mellitus; papilledema; optic atrophy; atherosclerosis;
angina; myocardial infarctions such as acute myocardial
infarctions, cardiac hypertrophy, and heart failure such as
congestive heart failure; hypertension; inflammatory vasculitides;
Reynaud's disease and Reynaud's phenomenon; aneurysms and arterial
restenosis; venous and lymphatic disorders such as
thrombophlebitis, lymphangitis, and lymphedema; peripheral vascular
disease; cancer such as vascular tumors, e.g., hemangioma
(capillary and cavernous), glomus tumors, telangiectasia, bacillary
angiomatosis, hemangioendothelioma, angiosarcoma,
haemangiopericytoma, Kaposi's sarcoma, lymphangioma, and
lymphangiosarcoma; tumor angiogenesis; trauma such as wounds,
burns, and other injured tissue, implant fixation, scarring;
ischemia reperfusion injury; rheumatoid arthritis; cerebrovascular
disease; renal diseases such as acute renal failure, or
osteoporosis. [0234] 83. The method of Claim 67, wherein the
immunological disorders are systemic lupus erythematosis;
rheumatoid arthritis; juvenile chronic arthritis;
spondyloarthropathies; systemic sclerosis (scleroderma); idiopathic
inflammatory myopathies (dermatomyositis, polymyositis); Sjogren's
syndrome; systemic vasculitis; sarcoidosis; autoimmune hemolytic
anemia (immune pancytopenia, paroxysmal nocturnal hemoglobinuria);
autoimmune thrombocytopenia (idiopathic thrombocytopenic purpura,
immune-mediated thrombocytopenia); thyroiditis (Grave's disease,
Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic
thyroiditis); diabetes mellitus; immune-mediated renal disease
(glomerulonephritis, tubulointerstitial nephritis); demyelinating
diseases of the central and peripheral nervous systems such as
multiple sclerosis, idiopathic demyelinating polyneuropathy or
Guillain-Barre syndrome, and chronic inflammatory demyelinating
polyneuropathy; hepatobiliary diseases such as infectious hepatitis
(hepatitis A, B, C, D, E and other non-hepatotropic viruses),
autoimmune chronic active hepatitis, primary biliary cirrhosis,
granulomatous hepatitis, and sclerosing cholangitis; inflammatory
bowel disease (ulcerative colitis: Crohn's disease);
gluten-sensitive enteropathy, and Whipple's disease; autoimmune or
immune-mediated skin diseases including bullous skin diseases,
erythema multiforme and contact dermatitis, psoriasis; allergic
diseases such as asthma, allergic rhinitis, atopic dermatitis, food
hypersensitivity and urticaria; immunologic diseases of the lung
such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and
hypersensitivity pneumonitis; or transplantation associated
diseases including graft rejection and graft-versus-host disease.
[0235] 84. The method of Claim 67, wherein said bone metabolic
abnormality or disorder is arthritis, osteoporosis or
osteopetrosis. [0236] 85. The method of Claim 67, wherein the
non-human transgenic animal exhibits at least one of the following
physiological characteristics compared with gender matched
wild-type littermates: increased anxiety-like response during open
field testing; decreased anxiety-like response during open field
activity testing; abnormal circadian rhythm during home-cage
activity testing including decreased ambulatory counts; increased
exploratory activity during open-field testing; increased stress
induced hyperthermia; enhanced motor coordination during inverted
screen testing; impaired motor coordination during inverted screen
testing; increase in retinal artery tortuosity; retinal
degeneration marked by attenuated retinal vessels; opthamological
abnormalities; increased mean systolic blood pressure; increased
mean fasting serum glucose levels; decreased mean serum glucose
levels; increased mean serum cholesterol levels; increased mean
serum triglyceride levels; decreased mean serum cholesterol levels;
decreased mean serum triglyceride levels; enhanced glucose
tolerance; impaired glucose tolerance; increased mean serum insulin
levels; decreased mean serum insulin levels; increased uric acid
levels; decreased serum phosphate levels; increased alkaline
phosphatase levels and increased alanine amino transferase levels;
liver disease; increased mean percentage of CD25+ in both spleen
and lymph nodes; decreased mean percentage of natural killer cells;
decreased mean percentage of CD21HiCD23Med cells in spleen and
lymph nodes; increased mean percentage of CD4 cells and decreased
mean percentage of B cells; increased mean percentage of CD8+
cells; decreased mean percentage of eosinophils; decreased mean
serum IgG1 response to an ovalbumin challenge; decreased mean serum
IgG2a response to an ovalbumin challenge; increased mean serum IgG1
response to an ovalbumin challenge; increased mean serum IgG2a
response to an ovalbumin challenge; increased mean serum MCP-1
response to a LPS challenge; increased mean serum TNF-alpha
response to a LPS challenge; decreased mean serum MCP-1 response to
a LPS challenge; decreased mean serum IL-6 response to a LPS
challenge; decreased TNF-alpha response to a LPS challenge;
increased mean serum IL6 response to a LPS challenge; increased
mean platelet counts; decreased mean total white blood cell (WBC)
counts; decreased absolute lymphocyte counts; decreased absolute
monocyte counts; decreased skin fibroblast proliferation; increased
skin fibroblast proliferation; increased mean percent of total body
fat and total fat mass; increased mean body weight; increased mean
body length; increased organ weights; increased total tissue mass
(TTM); increased lean body mass (LBM); increased bone mineral
density (BMD) in total body, femur and vertebrae; increased bone
mineral content (BMC) in total body, femur and vertebrae; increased
volumetric bone mineral density (vBMD) in total body, femur and
vertebrae; increased mean femoral midshaft cortical thickness and
cross-sectional area; increased mean vertebral trabecular bone
volume, number and connectivity density; decreased mean percent of
total body fat and total fat mass; decreased mean body weight;
decreased mean body length; decreased total tissue mass (TTM);
decreased lean body mass (LBM); decreased bone mineral density
(BMD) in total body, femur and vertebrae; decreased bone mineral
content (BMC) in total body, femur and vertebrae; decreased
volumetric bone mineral density (vBMD) in total body, femur and
vertebrae; decreased mean femoral midshaft cortical thickness and
cross-sectional area; decreased mean vertebral trabecular bone
volume, number and connectivity density; severe depletion of
abdominal and subcutaneous body fat deposits; decreased organ
weights; growth retardation; hydrocephalus; sebaceous gland
hyperplasia and growth retardation; apoptosis of olfactory
neuroepithelial cells; lymphoid hyperplasia and tissue
inflammation; development abnormalities; male infertility; growth
retardation with reduced viability; and embryonic lethality. [0237]
86. An agent identified by the method of Claim 67. [0238] 87. The
agent of Claim 86 which is an agonist or antagonist of a PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide. [0239] 88. The agent of Claim 87,
wherein the agonist is an anti-PRO196, anti-PRO217, anti-PRO231,
anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258, anti-PRO287,
anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526, anti-PRO724,
anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104, anti-PRO1151,
anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570,
anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725,
anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102,
anti-PRO10282, anti-PRO61709 or anti-PRO779 antibody. [0240] 89.
The agent of Claim 87, wherein the antagonist is an anti-PRO196,
anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246,
anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357,
anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003,
anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298,
anti-PRO1313, anti-PRO1570, anti-PRO1886, anti-PRO1891,
anti-PRO4409, anti-PRO5725, anti-PRO5994, anti-PRO6097,
anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709 or
anti-PRO779 antibody. [0241] 90. A therapeutic agent identified by
the method of Claim 67. [0242] 91. A method of identifying an agent
that modulates the expression of a PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide, the method comprising:
[0243] (a) contacting a test agent with a host cell expressing a
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide; and
[0244] (b) determining whether the test agent modulates the
expression of the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide by the
host cell. [0245] 92. An agent identified by the method of Claim
91. [0246] 93. The agent of Claim 92 which is an agonist or
antagonist of a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide. [0247]
94. The agent of Claim 93, wherein the agonist is an anti-PRO196,
anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246,
anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357,
anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003,
anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298,
anti-PRO1313, anti-PRO1570, anti-PRO1886, anti-PRO1891,
anti-PRO4409, anti-PRO5725, anti-PRO5994, anti-PRO6097,
anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709 or
anti-PRO779 antibody. [0248] 95. The agent of Claim 93, wherein the
antagonist is an anti-PRO196, anti-PRO217, anti-PRO231,
anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258, anti-PRO287,
anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526, anti-PRO724,
anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104, anti-PRO1151,
anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570,
anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725,
anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102,
anti-PRO10282, anti-PRO61709 or anti-PRO779 antibody. [0249] 96. A
method of evaluating a therapeutic agent capable of affecting a
condition associated with a disruption of a gene which encodes for
a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide, the method
comprising:
[0250] (a) providing a non-human transgenic animal whose genome
comprises a disruption of the gene which encodes for the PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide;
[0251] (b) measuring a physiological characteristic of the
non-human transgenic animal of (a);
[0252] (c) comparing the measured physiological characteristic of
(b) with that of a gender matched wild-type animal, wherein the
physiological characteristic of the non-human transgenic animal
that differs from the physiological characteristic of the wild-type
animal is identified as a condition resulting from the gene
disruption in the non-human transgenic animal;
[0253] (d) administering a test agent to the non-human transgenic
animal of (a); and
[0254] (e) evaluating the effects of the test agent on the
identified condition associated with gene disruption in the
non-human transgenic animal. [0255] 97. The method of Claim 96,
wherein the condition is a neurological disorder; a cardiovascular,
endothelial or angiogenic disorder; an eye abnormality; an
immunological disorder; an oncological disorder; a bone metabolic
abnormality or disorder; a lipid metabolic disorder; or a
developmental abnormality. [0256] 98. A therapeutic agent
identified by the method of Claim 96. [0257] 99. The therapeutic
agent of Claim 98 which is an agonist or antagonist of a PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide. [0258] 100. The therapeutic agent
of Claim 99, wherein the agonist is an anti-PRO196, anti-PRO217,
anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258,
anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526,
anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104,
anti-PRO1151, anti-PRO1244, anti-PRO1298, anti-PRO1313,
anti-PRO1570, anti-PRO1886, anti-PRO1891, anti-PRO4409,
anti-PRO5725, anti-PRO5994, anti-PRO6097, anti-PRO7425,
anti-PRO10102, anti-PRO10282, anti-PRO61709 or anti-PRO779
antibody. [0259] 101. The therapeutic agent of Claim 99, wherein
the antagonist is an anti-PRO196, anti-PRO217, anti-PRO231,
anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258, anti-PRO287,
anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526, anti-PRO724,
anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104, anti-PRO1151,
anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570,
anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725,
anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102,
anti-PRO10282, anti-PRO61709 or anti-PRO779 antibody. [0260] 102. A
pharmaceutical composition comprising the therapeutic agent of
Claim 98. [0261] 103. A method of treating or preventing or
ameliorating a neurological disorder; cardiovascular, endothelial
or angiogenic disorder; immunological disorder; oncological
disorder; bone metabolic abnormality or disorder, or embryonic
lethality associated with the disruption of a gene which encodes
for a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 polypeptide, the method
comprising administering to a subject in need of such treatment
whom may already have the disorder, or may be prone to have the
disorder or may be in whom the disorder is to be prevented, a
therapeutically effective amount of the therapeutic agent of Claim
94, or agonists or antagonists thereof, thereby effectively
treating or preventing or ameliorating said disorder. [0262] 104.
The method of Claim 103, wherein the neurological disorder is an
increased anxiety-like response during open field activity testing.
[0263] 105. The method of Claim 103, wherein the neurological
disorder is a decreased anxiety-like response during open field
activity testing. [0264] 106. The method of Claim 103, wherein the
neurological disorder is an abnormal circadian rhythm during
home-cage activity testing. [0265] 107. The method of Claim 103,
wherein the neurological disorder is an enhanced motor coordination
during inverted screen testing. [0266] 108. The method of Claim
103, wherein the neurological disorder is an impaired motor
coordination during inverted screen testing. [0267] 109. The method
of Claim 103, wherein the neurological disorder is depression,
generalized anxiety disorders, attention deficit disorder, sleep
disorder, hyperactivity disorder, obsessive compulsive disorder,
schizophrenia, cognitive disorders, hyperalgesia or sensory
disorders. [0268] 110. The method of Claim 103, wherein the eye
abnormality is a retinal abnormality. [0269] 111. The method of
Claim 103, wherein the eye abnormality is consistent with vision
problems or blindness. [0270] 112. The method of Claim 110, wherein
the retinal abnormality is consistent with retinitis pigmentosa.
[0271] 113. The method of Claim 110, wherein the retinal
abnormality is characterized by retinal degeneration or retinal
dysplasia. [0272] 114. The method of Claim 110, wherein the retinal
abnormality is consistent with retinal dysplasia, various
retinopathies, including retinopathy of prematurity, retrolental
fibroplasia, neovascular glaucoma, age-related macular
degeneration, diabetic macular edema, corneal neovascularization,
corneal graft neovascularization, corneal graft rejection,
retinal/choroidal neovascularization, neovascularization of the
angle (rubeosis), ocular neovascular disease, vascular restenosis,
arteriovenous malformations (AVM), meningioma, hemangioma,
angiofibroma, thyroid hyperplasias (including Grave's disease),
corneal and other tissue transplantation, retinal artery
obstruction or occlusion; retinal degeneration causing secondary
atrophy of the retinal vasculature, retinitis pigmentosa, macular
dystrophies, Stargardt's disease, congenital stationary night
blindness, choroideremia, gyrate atrophy, Leber's congenital
amaurosis, retinoschisis disorders, Wagner's syndrome, Usher
syndromes, Zellweger syndrome, Saldino-Mainzer syndrome,
Senior-Loken syndrome, Bardet-Biedl syndrome, Alport's syndrome,
Alstrom's syndrome, Cockayne's syndrome, dysplaisa
spondyloepiphysaria congentia, Flynn-Aird syndrome, Friedreich
ataxia, Hallgren syndrome, Marshall syndrome, Albers-Schnoberg
disease, Refsum's disease, Kearns-Sayre syndrome, Waardenburg's
syndrome, Alagile syndrome, myotonic dystrophy,
olivopontocerebellar atrophy, Pierre-Marie dunsdrome, Stickler
syndrome, carotinemeia, cystinosis, Wolfram syndrome,
Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentia
pigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria,
or mannosidosis. [0273] 115. The method of Claim 103, wherein the
eye abnormality is a cataract. [0274] 116. The method of Claim 115,
wherein the cataract is a systemic disease such as human Down's
syndrome, Hallerman-Streiff syndrome, Lowe syndrome, galactosemia,
Marfan syndrome, Trismoy 13-15, Alport syndrome, myotonic
dystrophy, Fabry disease, hypoparathroidism or Conradi syndrome.
[0275] 117. The method of Claim 103, wherein the developmental
abnormality comprises embryonic lethality or reduced viability.
[0276] 118. The method of Claim 103, wherein the cardiovascular,
endothelial or angiogenic disorders are arterial diseases, such as
diabetes mellitus; papilledema; optic atrophy; atherosclerosis;
angina; myocardial infarctions such as acute myocardial
infarctions, cardiac hypertrophy, and heart failure such as
congestive heart failure; hypertension; inflammatory vasculitides;
Reynaud's disease and Reynaud's phenomenon; aneurysms and arterial
restenosis; venous and lymphatic disorders such as
thrombophlebitis, lymphangitis, and lymphedema; peripheral vascular
disease; cancer such as vascular tumors, e.g., hemangioma
(capillary and cavernous), glomus tumors, telangiectasia, bacillary
angiomatosis, hemangioendothelioma, angiosarcoma,
haemangiopericytoma, Kaposi's sarcoma, lymphangioma, and
lymphangiosarcoma; tumor angiogenesis; trauma such as wounds,
burns, and other injured tissue, implant fixation, scarring;
ischemia reperfusion injury; rheumatoid arthritis; cerebrovascular
disease; renal diseases such as acute renal failure, or
osteoporosis. [0277] 119. The method of Claim 103, wherein the
immunological disorders are systemic lupus erythematosis;
rheumatoid arthritis; juvenile chronic arthritis;
spondyloarthropathies; systemic sclerosis (scleroderma); idiopathic
inflammatory myopathies (dermatomyositis, polymyositis); Sjogren's
syndrome; systemic vasculitis; sarcoidosis; autoimmune hemolytic
anemia (immune pancytopenia, paroxysmal nocturnal hemoglobinuria);
autoimmune thrombocytopenia (idiopathic thrombocytopenic purpura,
immune-mediated thrombocytopenia); thyroiditis (Grave's disease,
Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic
thyroiditis); diabetes mellitus; immune-mediated renal disease
(glomerulonephritis, tubulointerstitial nephritis); demyelinating
diseases of the central and peripheral nervous systems such as
multiple sclerosis, idiopathic demyelinating polyneuropathy or
Guillain-Barre syndrome, and chronic inflammatory demyelinating
polyneuropathy; hepatobiliary diseases such as infectious hepatitis
(hepatitis A, B, C, D, E and other non-hepatotropic viruses),
autoimmune chronic active hepatitis, primary biliary cirrhosis,
granulomatous hepatitis, and sclerosing cholangitis; inflammatory
bowel disease (ulcerative colitis: Crohn's disease);
gluten-sensitive enteropathy, and Whipple's disease; autoimmune or
immune-mediated skin diseases including bullous skin diseases,
erythema multiforme and contact dermatitis, psoriasis; allergic
diseases such as asthma, allergic rhinitis, atopic dermatitis, food
hypersensitivity and urticaria; immunologic diseases of the lung
such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and
hypersensitivity pneumonitis; or transplantation associated
diseases including graft rejection and graft-versus-host disease.
[0278] 120. The method of Claim 103, wherein said bone metabolic
abnormality or disorder is arthritis, osteoporosis or
osteopetrosis. [0279] 121. A method of identifying an agent that
ameliorates or modulates a neurological disorder; a cardiovascular,
endothelial or angiogenic disorder; an eye abnormality; an
immunological disorder; an oncological disorder; a bone metabolic
abnormality or disorder; a lipid metabolic disorder; or a
developmental abnormality associated with a disruption in the gene
which encodes for a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide, the
method comprising:
[0280] (a) providing a non-human transgenic animal cell culture,
each cell of said culture comprising a disruption of the gene which
encodes for a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide;
[0281] (b) administering a test agent to said cell culture; and
[0282] (c) determining whether said test agent ameliorates or
modulates the neurological disorder; cardiovascular, endothelial or
angiogenic disorder; eye abnormality; immunological disorder;
oncological disorder; bone metabolic abnormality or disorder; lipid
metabolic disorder; or developmental abnormality in said cell
culture. [0283] 122. The method of Claim 121, wherein the
neurological disorder is an increased anxiety-like response during
open field activity testing. [0284] 123. The method of Claim 121,
wherein the neurological disorder is a decreased anxiety-like
response during open field activity testing. [0285] 124. The method
of Claim 121, wherein the neurological disorder is an abnormal
circadian rhythm during home-cage activity testing. [0286] 125. The
method of Claim 121, wherein the neurological disorder is an
enhanced motor coordination during inverted screen testing. [0287]
126. The method of Claim 121, wherein the neurological disorder is
an impaired motor coordination during inverted screen testing.
[0288] 127. The method of Claim 121, wherein the neurological
disorder is depression, generalized anxiety disorders, attention
deficit disorder, sleep disorder, hyperactivity disorder, obsessive
compulsive disorder, schizophrenia, cognitive disorders,
hyperalgesia or sensory disorders. [0289] 128. The method of Claim
121, wherein the eye abnormality is a retinal abnormality. [0290]
129. The method of Claim 121, wherein the eye abnormality is
consistent with vision problems or blindness. [0291] 130. The
method of Claim 128, wherein the retinal abnormality is consistent
with retinitis pigmentosa. [0292] 131. The method of Claim 128,
wherein the retinal abnormality is characterized by retinal
degeneration or retinal dysplasia. [0293] 132. The method of Claim
128, wherein the retinal abnormality is consistent with retinal
dysplasia, various retinopathies, including retinopathy of
prematurity, retrolental fibroplasia, neovascular glaucoma,
age-related macular degeneration, diabetic macular edema, corneal
neovascularization, corneal graft neovascularization, corneal graft
rejection, retinal/choroidal neovascularization, neovascularization
of the angle (rubeosis), ocular neovascular disease, vascular
restenosis, arteriovenous malformations (AVM), meningioma,
hemangioma, angiofibroma, thyroid hyperplasias (including Grave's
disease), corneal and other tissue transplantation, retinal artery
obstruction or occlusion; retinal degeneration causing secondary
atrophy of the retinal vasculature, retinitis pigmentosa, macular
dystrophies, Stargardt's disease, congenital stationary night
blindness, choroideremia, gyrate atrophy, Leber's congenital
amaurosis, retinoschisis disorders, Wagner's syndrome, Usher
syndromes, Zellweger syndrome, Saldino-Mainzer syndrome,
Senior-Loken syndrome, Bardet-Biedl syndrome, Alport's syndrome,
Alstrom's syndrome, Cockayne's syndrome, dysplaisa
spondyloepiphysaria congentia, Flynn-Aird syndrome, Friedreich
ataxia, Hallgren syndrome, Marshall syndrome, Albers-Schnoberg
disease, Refsum's disease, Kearns-Sayre syndrome, Waardenburg's
syndrome, Alagile syndrome, myotonic dystrophy,
olivopontocerebellar atrophy, Pierre-Marie dunsdrome, Stickler
syndrome, carotinemeia, cystinosis, Wolfram syndrome,
Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentia
pigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria,
or mannosidosis. [0294] 133. The method of Claim 121, wherein the
eye abnormality is a cataract. [0295] 134. The method of Claim 133,
wherein the cataract is a systemic disease such as human Down's
syndrome, Hallerman-Streiff syndrome, Lowe syndrome, galactosemia,
Marfan syndrome, Trismoy 13-15, Alport syndrome, myotonic
dystrophy, Fabry disease, hypoparathroidism or Conradi syndrome.
[0296] 135. The method of Claim 121, wherein the developmental
abnormality comprises embryonic lethality or reduced viability.
[0297] 136. The method of Claim 121, wherein the cardiovascular,
endothelial or angiogenic disorders are arterial diseases, such as
diabetes mellitus; papilledema; optic atrophy; atherosclerosis;
angina; myocardial infarctions such as acute myocardial
infarctions, cardiac hypertrophy, and heart failure such as
congestive heart failure; hypertension; inflammatory vasculitides;
Reynaud's disease and Reynaud's phenomenon; aneurysms and arterial
restenosis; venous and lymphatic disorders such as
thrombophlebitis, lymphangitis, and lymphedema; peripheral vascular
disease; cancer such as vascular tumors, e.g., hemangioma
(capillary and cavernous), glomus tumors, telangiectasia, bacillary
angiomatosis, hemangioendothelioma, angiosarcoma,
haemangiopericytoma, Kaposi's sarcoma, lymphangioma, and
lymphangiosarcoma; tumor angiogenesis; trauma such as wounds,
burns, and other injured tissue, implant fixation, scarring;
ischemia reperfusion injury; rheumatoid arthritis; cerebrovascular
disease; renal diseases such as acute renal failure, or
osteoporosis. [0298] 137. The method of Claim 121, wherein the
immunological disorders are systemic lupus erythematosis;
rheumatoid arthritis; juvenile chronic arthritis;
spondyloarthropathies; systemic sclerosis (scleroderma); idiopathic
inflammatory myopathies (dermatomyositis, polymyositis); Sjogren's
syndrome; systemic vasculitis; sarcoidosis; autoimmune hemolytic
anemia (immune pancytopenia, paroxysmal nocturnal hemoglobinuria);
autoimmune thrombocytopenia (idiopathic thrombocytopenic purpura,
immune-mediated thrombocytopenia); thyroiditis (Grave's disease,
Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic
thyroiditis); diabetes mellitus; immune-mediated renal disease
(glomerulonephritis, tubulointerstitial nephritis); demyelinating
diseases of the central and peripheral nervous systems such as
multiple sclerosis, idiopathic demyelinating polyneuropathy or
Guillain-Barre syndrome, and chronic inflammatory demyelinating
polyneuropathy; hepatobiliary diseases such as infectious hepatitis
(hepatitis A, B, C, D, E and other non-hepatotropic viruses),
autoimmune chronic active hepatitis, primary biliary cirrhosis,
granulomatous hepatitis, and sclerosing cholangitis; inflammatory
bowel disease (ulcerative colitis: Crohn's disease);
gluten-sensitive enteropathy, and Whipple's disease; autoimmune or
immune-mediated skin diseases including bullous skin diseases,
erythema multiforme and contact dermatitis, psoriasis; allergic
diseases such as asthma, allergic rhinitis, atopic dermatitis, food
hypersensitivity and urticaria; immunologic diseases of the lung
such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and
hypersensitivity pneumonitis; or transplantation associated
diseases including graft rejection and graft-versus-host disease.
[0299] 138. The method of Claim 121, wherein said bone metabolic
abnormality or disorder is arthritis, osteoporosis or
osteopetrosis. [0300] 139. An agent identified by the method of
Claim 121. [0301] 140. The agent of Claim 139 which is an agonist
or antagonist of a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide. [0302]
141. The agent of Claim 140, wherein the agonist is an anti-PRO196,
anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246,
anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357,
anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003,
anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298,
anti-PRO1313, anti-PRO1570, anti-PRO1886, anti-PRO1891,
anti-PRO4409, anti-PRO5725, anti-PRO5994, anti-PRO6097,
anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709 or
anti-PRO779 antibody. [0303] 142. The agent of Claim 140, wherein
the antagonist is an anti-PRO196, anti-PRO217, anti-PRO231,
anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258, anti-PRO287,
anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526, anti-PRO724,
anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104, anti-PRO1151,
anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570,
anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725,
anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102,
anti-PRO10282, anti-PRO61709 or anti-PRO779 antibody. [0304] 143. A
therapeutic agent identified by the method of Claim 121. [0305]
144. A method of modulating a phenotype associated with a
disruption of a gene which encodes for a PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide, the method comprising administering to a subject whom
may already have the phenotype, or may be prone to have the
phenotype or may be in whom the phenotype is to be prevented, an
effective amount of the agent of Claim 46, or agonists or
antagonists thereof, thereby effectively modulating the phenotype.
[0306] 145. A method of modulating a physiological characteristic
associated with a disruption of a gene which encodes for a PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide, the method comprising administering
to a subject whom may already exhibit the physiological
characteristic, or may be prone to exhibit the physiological
characteristic or may be in whom the physiological characteristic
is to be prevented, an effective amount of the agent of Claim 52,
or agonists or antagonists thereof, thereby effectively modulating
the physiological characteristic. [0307] 146. A method of
modulating a behavior associated with a disruption of a gene which
encodes for a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide, the
method comprising administering to a subject whom may already
exhibit the behavior, or may be prone to exhibit the behavior or
may be in whom the exhibited behavior is to be prevented, an
effective amount of the agent of Claim 63, or agonists or
antagonists thereof, thereby effectively modulating the behavior.
[0308] 147. A method of modulating the expression of a PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide, the method comprising administering
to a host cell expressing said PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide, an effective amount of the agent of claim 92, or
agonists or antagonists thereof, thereby effectively modulating the
expression of said polypeptide. [0309] 148. A method of modulating
a condition associated with a disruption of a gene which encodes
for a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 polypeptide, the method
comprising administering to a subject whom may have the condition,
or may be prone to have the condition or may be in whom the
condition is to be prevented, a therapeutically effective amount of
the therapeutic agent of claim 98, or agonists or antagonists
thereof, thereby effectively modulating the condition. [0310] 149.
A method of treating or preventing or ameliorating a neurological
disorder; cardiovascular, endothelial or angiogenic disorder;
immunological disorder; oncological disorder; bone metabolic
abnormality or disorder, or embryonic lethality associated with the
disruption of a gene which encodes for a PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide, the method comprising administering to a non-human
transgenic animal cell culture, each cell of said culture
comprising a disruption of the gene which encodes for a PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide, a therapeutically effective amount
of the agent of Claim 139, or agonists or antagonists thereof,
thereby effectively treating or preventing or ameliorating said
disorder.
BRIEF DESCRIPTION OF THE DRAWINGS
[0311] FIG. 1 shows a nucleotide sequence (SEQ ID NO:1) of a native
sequence PRO196 cDNA, wherein SEQ ID NO:1 is a clone designated
herein as "DNA22779-1130" (UNQ170).
[0312] FIG. 2 shows the amino acid sequence (SEQ ID NO:2) derived
from the coding sequence of SEQ ID NO:1 shown in FIG. 1.
[0313] FIG. 3 shows a nucleotide sequence (SEQ ID NO:3) of a native
sequence PRO217 cDNA, wherein SEQ ID NO:3 is a clone designated
herein as "DNA33094-1131" (UNQ191).
[0314] FIG. 4 shows the amino acid sequence (SEQ ID NO:4) derived
from the coding sequence of SEQ ID NO:3 shown in FIG. 3.
[0315] FIG. 5 shows a nucleotide sequence (SEQ ID NO:5) of a native
sequence PRO231 cDNA, wherein SEQ ID NO:5 is a clone designated
herein as "DNA34434-1139" (UNQ205).
[0316] FIG. 6 shows the amino acid sequence (SEQ ID NO:6) derived
from the coding sequence of SEQ ID NO:5 shown in FIG. 5.
[0317] FIG. 7 shows a nucleotide sequence (SEQ ID NO:7) of a native
sequence PRO236 cDNA, wherein SEQ ID NO:7 is a clone designated
herein as "DNA35599-1168" (UNQ210).
[0318] FIG. 8 shows the amino acid sequence (SEQ ID NO:8) derived
from the coding sequence of SEQ ID NO:7 shown in FIG. 7.
[0319] FIG. 9 shows a nucleotide sequence (SEQ ID NO:9) of a native
sequence PRO245 cDNA, wherein SEQ ID NO:9 is a clone designated
herein as "DNA35638-1141" (UNQ219).
[0320] FIG. 10 shows the amino acid sequence (SEQ ID NO:10) derived
from the coding sequence of SEQ ID NO:9 shown in FIG. 9.
[0321] FIG. 11 shows a nucleotide sequence (SEQ ID NO:11) of a
native sequence PRO246 cDNA, wherein SEQ ID NO:11 is a clone
designated herein as "DNA35639-1172" (UNQ220).
[0322] FIG. 12 shows the amino acid sequence (SEQ ID NO:12) derived
from the coding sequence of SEQ ID NO:11 shown in FIG. 11.
[0323] FIG. 13 shows a nucleotide sequence (SEQ ID NO:13) of a
native sequence PRO258 cDNA, wherein SEQ ID NO:13 is a clone
designated herein as "DNA35918-1174" (UNQ225).
[0324] FIG. 14 shows the amino acid sequence (SEQ ID NO:14) derived
from the coding sequence of SEQ ID NO:13 shown in FIG. 13.
[0325] FIG. 15 shows a nucleotide sequence (SEQ ID NO:15) of a
native sequence PRO287 cDNA, wherein SEQ ID NO:15 is a clone
designated herein as "DNA39969-1185" (UNQ250).
[0326] FIG. 16 shows the amino acid sequence (SEQ ID NO:16) derived
from the coding sequence of SEQ ID NO:15 shown in FIG. 15.
[0327] FIG. 17 shows a nucleotide sequence (SEQ ID NO:17) of a
native sequence PRO328 cDNA, wherein SEQ ID NO:17 is a clone
designated herein as "DNA40587-1231" (UNQ289).
[0328] FIG. 18 shows the amino acid sequence (SEQ ID NO:18) derived
from the coding sequence of SEQ ID NO:17 shown in FIG. 17.
[0329] FIG. 19 shows a nucleotide sequence (SEQ ID NO:19) of a
native sequence PRO344 cDNA, wherein SEQ ID NO:19 is a clone
designated herein as "DNA40592-1242" (UNQ303).
[0330] FIG. 20 shows the amino acid sequence (SEQ ID NO:20) derived
from the coding sequence of SEQ ID NO:19 shown in FIG. 19.
[0331] FIG. 21 shows a nucleotide sequence (SEQ ID NO:21) of a
native sequence PRO357 cDNA, wherein SEQ ID NO:21 is a clone
designated herein as "DNA44804-1248" (UNQ314).
[0332] FIG. 22 shows the amino acid sequence (SEQ ID NO:22) derived
from the coding sequence of SEQ ID NO:21 shown in FIG. 21.
[0333] FIG. 23 shows a nucleotide sequence (SEQ ID NO:23) of a
native sequence PRO526 cDNA, wherein SEQ ID NO:23 is a clone
designated herein as "DNA44184-1319" (UNQ330).
[0334] FIG. 24 shows the amino acid sequence (SEQ ID NO:24) derived
from the coding sequence of SEQ ID NO:23 shown in FIG. 23.
[0335] FIG. 25 shows a nucleotide sequence (SEQ ID NO:25) of a
native sequence PRO724 cDNA, wherein SEQ ID NO:25 is a clone
designated herein as "DNA49631-1328" (UNQ389).
[0336] FIG. 26 shows the amino acid sequence (SEQ ID NO:26) derived
from the coding sequence of SEQ ID NO:25 shown in FIG. 25.
[0337] FIG. 27 shows a nucleotide sequence (SEQ ID NO:27) of a
native sequence PRO731 cDNA, wherein SEQ ID NO:27 is a clone
designated herein as "DNA48331-1329" (UNQ395).
[0338] FIG. 28 shows the amino acid sequence (SEQ ID NO:28) derived
from the coding sequence of SEQ ID NO:27 shown in FIG. 27.
[0339] FIG. 29 shows a nucleotide sequence (SEQ ID NO:29) of a
native sequence PRO732 cDNA, wherein SEQ ID NO:29 is a clone
designated herein as "DNA48334-1435" (UNQ396).
[0340] FIG. 30 shows the amino acid sequence (SEQ ID NO:30) derived
from the coding sequence of SEQ ID NO:29 shown in FIG. 29.
[0341] FIG. 31 shows a nucleotide sequence (SEQ ID NO:31) of a
native sequence PRO1003 cDNA, wherein SEQ ID NO:31 is a clone
designated herein as "DNA58846-1409" (UNQ487).
[0342] FIG. 32 shows the amino acid sequence (SEQ ID NO:32) derived
from the coding sequence of SEQ ID NO:31 shown in FIG. 31.
[0343] FIG. 33 shows a nucleotide sequence (SEQ ID NO:33) of a
native sequence PRO1104 cDNA, wherein SEQ ID NO:33 is a clone
designated herein as "DNA59616-1465" (UNQ547).
[0344] FIG. 34 shows the amino acid sequence (SEQ ID NO:34) derived
from the coding sequence of SEQ ID NO:33 shown in FIG. 33.
[0345] FIG. 35 shows a nucleotide sequence (SEQ ID NO:35) of a
native sequence PRO1151 cDNA, wherein SEQ ID NO:35 is a clone
designated herein as "DNA44694-1500" (UNQ581).
[0346] FIG. 36 shows the amino acid sequence (SEQ ID NO:36) derived
from the coding sequence of SEQ ID NO:35 shown in FIG. 35.
[0347] FIG. 37 shows a nucleotide sequence (SEQ ID NO:37) of a
native sequence PRO1244 cDNA, wherein SEQ ID NO:37 is a clone
designated herein as "DNA64883-1526" (UNQ628).
[0348] FIG. 38 shows the amino acid sequence (SEQ ID NO:38) derived
from the coding sequence of SEQ ID NO:37 shown in FIG. 37.
[0349] FIG. 39 shows a nucleotide sequence (SEQ ID NO:39) of a
native sequence PRO1298 cDNA, wherein SEQ ID NO:39 is a clone
designated herein as "DNA66511-1563" (UNQ666).
[0350] FIG. 40 shows the amino acid sequence (SEQ ID NO:40) derived
from the coding sequence of SEQ ID NO:39 shown in FIG. 39.
[0351] FIG. 41 shows a nucleotide sequence (SEQ ID NO:41) of a
native sequence PRO1313 cDNA, wherein SEQ ID NO:41 is a clone
designated herein as "DNA64966-1575" (UNQ679).
[0352] FIG. 42 shows the amino acid sequence (SEQ ID NO:42) derived
from the coding sequence of SEQ ID NO:41 shown in FIG. 41.
[0353] FIG. 43 shows a nucleotide sequence (SEQ ID NO:43) of a
native sequence PRO1570 cDNA, wherein SEQ ID NO:43 is a clone
designated herein as "DNA68885-1678" (UNQ776).
[0354] FIG. 44 shows the amino acid sequence (SEQ ID NO:44) derived
from the coding sequence of SEQ ID NO:43 shown in FIG. 43.
[0355] FIG. 45 shows a nucleotide sequence (SEQ ID NO:45) of a
native sequence PRO1886 cDNA, wherein SEQ ID NO:45 is a clone
designated herein as "DNA80796-2523" (UNQ870).
[0356] FIG. 46 shows the amino acid sequence (SEQ ID NO:46) derived
from the coding sequence of SEQ ID NO:45 shown in FIG. 45.
[0357] FIG. 47 shows a nucleotide sequence (SEQ ID NO:47) of a
native sequence PRO1891 cDNA, wherein SEQ ID NO:47 is a clone
designated herein as "DNA76788-2526" (UNQ873).
[0358] FIG. 48 shows the amino acid sequence (SEQ ID NO:48) derived
from the coding sequence of SEQ ID NO:47 shown in FIG. 47.
[0359] FIG. 49 shows a nucleotide sequence (SEQ ID NO:49) of a
native sequence PRO4409 cDNA, wherein SEQ ID NO:49 is a clone
designated herein as "DNA88004-2575" (UNQ1934).
[0360] FIG. 50 shows the amino acid sequence (SEQ ID NO:50) derived
from the coding sequence of SEQ ID NO:49 shown in FIG. 49.
[0361] FIG. 51 shows a nucleotide sequence (SEQ ID NO:51) of a
native sequence PRO5725 cDNA, wherein SEQ ID NO:51 is a clone
designated herein as "DNA92265-2669" (UNQ2446).
[0362] FIG. 52 shows the amino acid sequence (SEQ ID NO:52) derived
from the coding sequence of SEQ ID NO:51 shown in FIG. 51.
[0363] FIG. 53 shows a nucleotide sequence (SEQ ID NO:53) of a
native sequence PRO5994 cDNA, wherein SEQ ID NO:53 is a clone
designated herein as "DNA98591" (UNQ2506).
[0364] FIG. 54 shows the amino acid sequence (SEQ ID NO:54) derived
from the coding sequence of SEQ ID NO:53 shown in FIG. 53.
[0365] FIG. 55 shows a nucleotide sequence (SEQ ID NO:55) of a
native sequence PRO6097 cDNA, wherein SEQ ID NO:55 is a clone
designated herein as "DNA107701-2711" (UNQ2545).
[0366] FIG. 56 shows the amino acid sequence (SEQ ID NO:56) derived
from the coding sequence of SEQ ID NO:55 shown in FIG. 55.
[0367] FIG. 57 shows a nucleotide sequence (SEQ ID NO:57) of a
native sequence PRO7425 cDNA, wherein SEQ ID NO:57 is a clone
designated herein as "DNA108792-2753" (UNQ2966).
[0368] FIG. 58 shows the amino acid sequence (SEQ ID NO:58) derived
from the coding sequence of SEQ ID NO:57 shown in FIG. 57.
[0369] FIG. 59 shows a nucleotide sequence (SEQ ID NO:59) of a
native sequence PRO10102 cDNA, wherein SEQ ID NO:59 is a clone
designated herein as "DNA129542-2808" (UNQ3103).
[0370] FIG. 60 shows the amino acid sequence (SEQ ID NO:60) derived
from the coding sequence of SEQ ID NO:59 shown in FIG. 59.
[0371] FIG. 61 shows a nucleotide sequence (SEQ ID NO:61) of a
native sequence PRO10282 cDNA, wherein SEQ ID NO:61 is a clone
designated herein as "DNA148380-2827" (UNQ3126).
[0372] FIG. 62 shows the amino acid sequence (SEQ ID NO:62) derived
from the coding sequence of SEQ ID NO:61 shown in FIG. 61.
[0373] FIG. 63 shows a nucleotide sequence (SEQ ID NO:63) of a
native sequence PRO61709 cDNA, wherein SEQ ID NO:63 is a clone
designated herein as "DNA347767" (UNQ14964).
[0374] FIG. 64 shows the amino acid sequence (SEQ ID NO:64) derived
from the coding sequence of SEQ ID NO:63 shown in FIG. 63.
[0375] FIG. 65 shows a nucleotide sequence (SEQ ID NO:65) of a
native sequence PRO779 cDNA, wherein SEQ ID NO:65 is a clone
designated herein as "DNA58801-1052" (UNQ455).
[0376] FIG. 66 shows the amino acid sequence (SEQ ID NO:66) derived
from the coding sequence of SEQ ID NO:65 shown in FIG. 65.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
I. Definitions
[0377] The terms "PRO polypeptide" and "PRO" as used herein and
when immediately followed by a numerical designation refer to
various polypeptides, wherein the complete designation (i.e.,
PRO/number) refers to specific polypeptide sequences as described
herein. The terms "PRO/number polypeptide" and "PRO/number" wherein
the term "number" is provided as an actual numerical designation as
used herein encompass native sequence polypeptides and polypeptide
variants (which are further defined herein). The PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 polypeptides described herein may be isolated from a variety
of sources, such as from human tissue types or from another source,
or prepared by recombinant or synthetic methods. The term "PRO
polypeptide" refers to each individual PRO/number polypeptide
disclosed herein. All disclosures in this specification which refer
to the "PRO polypeptide" refer to each of the polypeptides
individually as well as jointly. For example, descriptions of the
preparation of, purification of, derivation of, formation of
antibodies to or against, administration of, compositions
containing, treatment of a disease with, etc., pertain to each
polypeptide of the invention individually. The term "PRO
polypeptide" also includes variants of the PRO/number polypeptides
disclosed herein.
[0378] A "native sequence PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide" comprises a polypeptide having the same amino acid
sequence as the corresponding PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide derived from nature. Such native sequence PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptides can be isolated from nature or can
be produced by recombinant or synthetic means. The term "native
sequence PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 polypeptide" specifically
encompasses naturally-occurring truncated or secreted forms of the
specific PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 polypeptide (e.g., an
extracellular domain sequence), naturally-occurring variant forms
(e.g., alternatively spliced forms) and naturally-occurring allelic
variants of the polypeptide. The invention provides native sequence
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptides disclosed herein which
are mature or full-length native sequence polypeptides comprising
the full-length amino acids sequences shown in the accompanying
figures. Start and stop codons are shown in bold font and
underlined in the figures. However, while the PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 polypeptide disclosed in the accompanying figures are shown
to begin with methionine residues designated herein as amino acid
position 1 in the figures, it is conceivable and possible that
other methionine residues located either upstream or downstream
from the amino acid position 1 in the figures may be employed as
the starting amino acid residue for the PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptides.
[0379] The PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 polypeptide "extracellular
domain" or "ECD" refers to a form of the PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide which is essentially free of the transmembrane and
cytoplasmic domains. Ordinarily, a PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide ECD will have less than 1% of such transmembrane and/or
cytoplasmic domains and preferably, will have less than 0.5% of
such domains. It will be understood that any transmembrane domains
identified for the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptides of the
present invention are identified pursuant to criteria routinely
employed in the art for identifying that type of hydrophobic
domain. The exact boundaries of a transmembrane domain may vary but
most likely by no more than about 5 amino acids at either end of
the domain as initially identified herein. Optionally, therefore,
an extracellular domain of a PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide may contain from about 5 or fewer amino acids on either
side of the transmembrane domain/extracellular domain boundary as
identified in the Examples or specification and such polypeptides,
with or without the associated signal peptide, and nucleic acid
encoding them, are contemplated by the present invention.
[0380] The approximate location of the "signal peptides" of the
various PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 polypeptides disclosed
herein are shown in the present specification and/or the
accompanying figures. It is noted, however, that the C-terminal
boundary of a signal peptide may vary, but most likely by no more
than about 5 amino acids on either side of the signal peptide
C-terminal boundary as initially identified herein, wherein the
C-terminal boundary of the signal peptide may be identified
pursuant to criteria routinely employed in the art for identifying
that type of amino acid sequence element (e.g., Nielsen et al.,
Prot. Eng. 10:1-6 (1997) and von Heinje et al., Nucl. Acids. Res.
14:4683-4690 (1986)). Moreover, it is also recognized that, in some
cases, cleavage of a signal sequence from a secreted polypeptide is
not entirely uniform, resulting in more than one secreted species.
These mature polypeptides, where the signal peptide is cleaved
within no more than about 5 amino acids on either side of the
C-terminal boundary of the signal peptide as identified herein, and
the polynucleotides encoding them, are contemplated by the present
invention.
[0381] "PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 polypeptide variant" means a
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide, preferably an active
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide, as defined herein having
at least about 80% amino acid sequence identity with a full-length
native sequence PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide
sequence as disclosed herein, a PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide sequence lacking the signal peptide as disclosed
herein, an extracellular domain of a PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide, with or without the signal peptide, as disclosed
herein or any other fragment of a full-length PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 polypeptide sequence as disclosed herein (such as those
encoded by a nucleic acid that represents only a portion of the
complete coding sequence for a full-length PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide). Such PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide
variants include, for instance, PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptides wherein one or more amino acid residues are added, or
deleted, at the N- or C-terminus of the full-length native amino
acid sequence. Ordinarily, a PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide variant will have or will have at least about 80% amino
acid sequence identity, alternatively will have or will have at
least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence
identity, to a full-length native sequence PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide sequence as disclosed herein, a PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide sequence lacking the signal peptide as disclosed
herein, an extracellular domain of a PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide, with or without the signal peptide, as disclosed
herein or any other specifically defined fragment of a full-length
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide sequence as disclosed
herein. Ordinarily, PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 variant
polypeptides are or are at least about 10 amino acids in length,
alternatively are or are at least about 20, 30, 40, 50, 60, 70, 80,
90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210,
220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340,
350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470,
480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600
amino acids in length, or more. Optionally, PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
variant polypeptides will have no more than one conservative amino
acid substitution as compared to the native PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide sequence, alternatively will have or will have no more
than 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid
substitution as compared to the native PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide sequence.
[0382] "Percent (%) amino acid sequence identity" with respect to
the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide sequences identified
herein is defined as the percentage of amino acid residues in a
candidate sequence that are identical with the amino acid residues
in the specific PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide
sequence, after aligning the sequences and introducing gaps, if
necessary, to achieve the maximum percent sequence identity, and
not considering any conservative substitutions as part of the
sequence identity. Alignment for purposes of determining percent
amino acid sequence identity can be achieved in various ways that
are within the skill in the art, for instance, using publicly
available computer software such as BLAST, BLAST-2, ALIGN or
Megalign (DNASTAR) software. Those skilled in the art can determine
appropriate parameters for measuring alignment, including any
algorithms needed to achieve maximal alignment over the full length
of the sequences being compared. For purposes herein, however, %
amino acid sequence identity values are generated using the
sequence comparison computer program ALIGN-2, wherein the complete
source code for the ALIGN-2 program is provided in Table 1 below.
The ALIGN-2 sequence comparison computer program was authored by
Genentech, Inc. and the source code shown in Table 1 below has been
filed with user documentation in the U.S. Copyright Office,
Washington D.C., 20559, where it is registered under U.S. Copyright
Registration No. TXU510087. The ALIGN-2 program is publicly
available through Genentech, Inc., South San Francisco, Calif. or
may be compiled from the source code provided in Table 1 below. The
ALIGN-2 program should be compiled for use on a UNIX operating
system, preferably digital UNIX V4.0D. All sequence comparison
parameters are set by the ALIGN-2 program and do not vary.
[0383] In situations where ALIGN-2 is employed for amino acid
sequence comparisons, the % amino acid sequence identity of a given
amino acid sequence A to, with, or against a given amino acid
sequence B (which can alternatively be phrased as a given amino
acid sequence A that has or comprises a certain % amino acid
sequence identity to, with, or against a given amino acid sequence
B) is calculated as follows:
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical
matches by the sequence alignment program ALIGN-2 in that program's
alignment of A and B, and where Y is the total number of amino acid
residues in B. It will be appreciated that where the length of
amino acid sequence A is not equal to the length of amino acid
sequence B, the % amino acid sequence identity of A to B will not
equal the % amino acid sequence identity of B to A. As examples of
% amino acid sequence identity calculations using this method,
Tables 2 and 3 demonstrate how to calculate the % amino acid
sequence identity of the amino acid sequence designated "Comparison
Protein" to the amino acid sequence designated "PRO", wherein "PRO"
represents the amino acid sequence of a hypothetical PRO
polypeptide of interest, "Comparison Protein" represents the amino
acid sequence of a polypeptide against which the "PRO" polypeptide
of interest is being compared, and "X, "Y" and "Z" each represent
different hypothetical amino acid residues. Unless specifically
stated otherwise, all % amino acid sequence identity values used
herein are obtained as described in the immediately preceding
paragraph using the ALIGN-2 computer program.
[0384] "PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 variant polynucleotide" or
"PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 variant nucleic acid sequence" means a
nucleic acid molecule which encodes a PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide, preferably an active PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide, as defined herein and which has at least about 80%
nucleic acid sequence identity with a nucleotide acid sequence
encoding a full-length native sequence PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide sequence as disclosed herein, a full-length native
sequence PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 polypeptide sequence lacking
the signal peptide as disclosed herein, an extracellular domain of
a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide, with or without the
signal peptide, as disclosed herein or any other fragment of a
full-length PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 polypeptide sequence as
disclosed herein (such as those encoded by a nucleic acid that
represents only a portion of the complete coding sequence for a
full-length PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 polypeptide). Ordinarily, a
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 variant polynucleotide will have or
will have at least about 80% nucleic acid sequence identity,
alternatively will have or will have at least about 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% nucleic acid sequence identity with a nucleic acid
sequence encoding a full-length native sequence PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 polypeptide sequence as disclosed herein, a full-length
native sequence PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide
sequence lacking the signal peptide as disclosed herein, an
extracellular domain of a PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide, with or without the signal sequence, as disclosed
herein or any other fragment of a full-length PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 polypeptide sequence as disclosed herein. Variants do not
encompass the native nucleotide sequence.
[0385] Ordinarily, PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 variant
polynucleotides are or are at least about 5 nucleotides in length,
alternatively are or are at least about 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110,
115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175,
180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280,
290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410,
420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540,
550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670,
680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800,
810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930,
940, 950, 960, 970, 980, 990, or 1000 nucleotides in length,
wherein in this context the term "about" means the referenced
nucleotide sequence length plus or minus 10% of that referenced
length.
[0386] "Percent (%) nucleic acid sequence identity" with respect to
PRO196-, PRO217-, PRO231-, PRO236-, PRO245-, PRO246-, PRO258-,
PRO287-, PRO328-, PRO344-, PRO357-, PRO526-, PRO724-, PRO731-,
PRO732-, PRO1003-, PRO1104-, PRO1151-, PRO1244-, PRO1298-,
PRO1313-, PRO1570-, PRO1886-, PRO1891-, PRO4409-, PRO5725-,
PRO5994-, PRO6097-, PRO7425-, PRO10102-, PRO10282-, PRO61709- or
PRO779-encoding nucleic acid sequences identified herein is defined
as the percentage of nucleotides in a candidate sequence that are
identical with the nucleotides in the PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
nucleic acid sequence of interest, after aligning the sequences and
introducing gaps, if necessary, to achieve the maximum percent
sequence identity. Alignment for purposes of determining percent
nucleic acid sequence identity can be achieved in various ways that
are within the skill in the art, for instance, using publicly
available computer software such as BLAST, BLAST-2, ALIGN or
Megalign (DNASTAR) software. For purposes herein, however, %
nucleic acid sequence identity values are generated using the
sequence comparison computer program ALIGN-2, wherein the complete
source code for the ALIGN-2 program is provided in Table 1 below.
The ALIGN-2 sequence comparison computer program was authored by
Genentech, Inc. and the source code shown in Table 1 below has been
filed with user documentation in the U.S. Copyright Office,
Washington D.C., 20559, where it is registered under U.S. Copyright
Registration No. TXU510087. The ALIGN-2 program is publicly
available through Genentech, Inc., South San Francisco, Calif. or
may be compiled from the source code provided in Table 1 below. The
ALIGN-2 program should be compiled for use on a UNIX operating
system, preferably digital UNIX V4.0D. All sequence comparison
parameters are set by the ALIGN-2 program and do not vary.
[0387] In situations where ALIGN-2 is employed for nucleic acid
sequence comparisons, the % nucleic acid sequence identity of a
given nucleic acid sequence C to, with, or against a given nucleic
acid sequence D (which can alternatively be phrased as a given
nucleic acid sequence C that has or comprises a certain % nucleic
acid sequence identity to, with, or against a given nucleic acid
sequence D) is calculated as follows:
100 times the fraction W/Z
where W is the number of nucleotides scored as identical matches by
the sequence alignment program ALIGN-2 in that program's alignment
of C and D, and where Z is the total number of nucleotides in D. It
will be appreciated that where the length of nucleic acid sequence
C is not equal to the length of nucleic acid sequence D, the %
nucleic acid sequence identity of C to D will not equal the %
nucleic acid sequence identity of D to C. As examples of % nucleic
acid sequence identity calculations, Tables 4 and 5, demonstrate
how to calculate the % nucleic acid sequence identity of the
nucleic acid sequence designated "Comparison DNA" to the nucleic
acid sequence designated "PRO-DNA", wherein "PRO-DNA" represents a
hypothetical PRO-encoding nucleic acid sequence of interest,
"Comparison DNA" represents the nucleotide sequence of a nucleic
acid molecule against which the "PRO-DNA" nucleic acid molecule of
interest is being compared, and "N", "L" and "V" each represent
different hypothetical nucleotides. Unless specifically stated
otherwise, all % nucleic acid sequence identity values used herein
are obtained as described in the immediately preceding paragraph
using the ALIGN-2 computer program.
[0388] The invention also provides PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
variant polynucleotides which are nucleic acid molecules that
encode a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 polypeptide and which are
capable of hybridizing, preferably under stringent hybridization
and wash conditions, to nucleotide sequences encoding a full-length
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide as disclosed herein.
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 variant polypeptides may be those that
are encoded by a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 variant
polynucleotide.
[0389] The term "full-length coding region" when used in reference
to a nucleic acid encoding a PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide refers to the sequence of nucleotides which encode the
full-length PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 polypeptide of the invention
(which is often shown between start and stop codons, inclusive
thereof, in the accompanying figures). The term "full-length coding
region" when used in reference to an ATCC deposited nucleic acid
refers to the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide-encoding portion of the cDNA that is inserted into the
vector deposited with the ATCC (which is often shown between start
and stop codons, inclusive thereof, in the accompanying
figures).
[0390] "Isolated," when used to describe the various polypeptides
disclosed herein, means polypeptide that has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials that would typically interfere with diagnostic or
therapeutic uses for the polypeptide, and may include enzymes,
hormones, and other proteinaceous or non-proteinaceous solutes. The
invention provides that the polypeptide will be purified (1) to a
degree sufficient to obtain at least 15 residues of N-terminal or
internal amino acid sequence by use of a spinning cup sequenator,
or (2) to homogeneity by SDS-PAGE under non-reducing or reducing
conditions using Coomassie blue or, preferably, silver stain.
Isolated polypeptide includes polypeptide in situ within
recombinant cells, since at least one component of the PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide natural environment will not be
present. Ordinarily, however, isolated polypeptide will be prepared
by at least one purification step.
[0391] An "isolated" PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide-encoding nucleic acid or other polypeptide-encoding
nucleic acid is a nucleic acid molecule that is identified and
separated from at least one contaminant nucleic acid molecule with
which it is ordinarily associated in the natural source of the
polypeptide-encoding nucleic acid. An isolated polypeptide-encoding
nucleic acid molecule is other than in the form or setting in which
it is found in nature. Isolated polypeptide-encoding nucleic acid
molecules therefore are distinguished from the specific
polypeptide-encoding nucleic acid molecule as it exists in natural
cells. However, an isolated polypeptide-encoding nucleic acid
molecule includes polypeptide-encoding nucleic acid molecules
contained in cells that ordinarily express the polypeptide where,
for example, the nucleic acid molecule is in a chromosomal location
different from that of natural cells.
[0392] The term "control sequences" refers to DNA sequences
necessary for the expression of an operably linked coding sequence
in a particular host organism. The control sequences that are
suitable for prokaryotes, for example, include a promoter,
optionally an operator sequence, and a ribosome binding site.
Eukaryotic cells are known to utilize promoters, polyadenylation
signals, and enhancers.
[0393] Nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous, and, in the case of a
secretory leader, contiguous and in reading phase. However,
enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not
exist, the synthetic oligonucleotide adaptors or linkers are used
in accordance with conventional practice.
[0394] "Stringency" of hybridization reactions is readily
determinable by one of ordinary skill in the art, and generally is
an empirical calculation dependent upon probe length, washing
temperature, and salt concentration. In general, longer probes
require higher temperatures for proper annealing, while shorter
probes need lower temperatures. Hybridization generally depends on
the ability of denatured DNA to reanneal when complementary strands
are present in an environment below their melting temperature. The
higher the degree of desired homology between the probe and
hybridizable sequence, the higher the relative temperature which
can be used. As a result, it follows that higher relative
temperatures would tend to make the reaction conditions more
stringent, while lower temperatures less so. For additional details
and explanation of stringency of hybridization reactions, see
Ausubel et al., Current Protocols in Molecular Biology, Wiley
Interscience Publishers, (1995).
[0395] "Stringent conditions" or "high stringency conditions", as
defined herein, may be identified by those that: (1) employ low
ionic strength and high temperature for washing, for example 0.015
M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl
sulfate at 50.degree. C.; (2) employ during hybridization a
denaturing agent, such as formamide, for example, 50% (v/v)
formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%
polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with
750 mM sodium chloride, 75 mM sodium citrate at 42.degree. C.; or
(3) employ 50% formamide, 5.times.SSC (0.75 M NaCl, 0.075 M sodium
citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium
pyrophosphate, 5.times.Denhardt's solution, sonicated salmon sperm
DNA (50 .mu.g/ml), 0.1% SDS, and 10% dextran sulfate at 42.degree.
C., with washes at 42.degree. C. in 0.2.times.SSC (sodium
chloride/sodium citrate) and 50% formamide at 55.degree. C.,
followed by a high-stringency wash consisting of 0.1.times.SSC
containing EDTA at 55.degree. C.
[0396] "Moderately stringent conditions" may be identified as
described by Sambrook et al., Molecular Cloning: A Laboratory
Manual, New York: Cold Spring Harbor Press, 1989, and include the
use of washing solution and hybridization conditions (e.g.,
temperature, ionic strength and % SDS) less stringent that those
described above. An example of moderately stringent conditions is
overnight incubation at 37.degree. C. in a solution comprising: 20%
formamide, 5.times.SSC (150 mM NaCl, 15 mM trisodium citrate), 50
mM sodium phosphate (pH 7.6), 5.times.Denhardt's solution, 10%
dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA,
followed by washing the filters in 1.times.SSC at about
37-50.degree. C. The skilled artisan will recognize how to adjust
the temperature, ionic strength, etc. as necessary to accommodate
factors such as probe length and the like.
[0397] The term "epitope tagged" when used herein refers to a
chimeric polypeptide comprising a PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide fused to a "tag polypeptide". The tag polypeptide has
enough residues to provide an epitope against which an antibody can
be made, yet is short enough such that it does not interfere with
activity of the polypeptide to which it is fused. The tag
polypeptide preferably also is fairly unique so that the antibody
does not substantially cross-react with other epitopes. Suitable
tag polypeptides generally have at least six amino acid residues
and usually between about 8 and 50 amino acid residues (preferably,
between about 10 and 20 amino acid residues).
[0398] "Active" or "activity" for the purposes herein refers to
form(s) of a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide which
retain a biological and/or an immunological activity of native or
naturally-occurring PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide,
wherein "biological" activity refers to a biological function
(either inhibitory or stimulatory) caused by a native or
naturally-occurring PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide other
than the ability to induce the production of an antibody against an
antigenic epitope possessed by a native or naturally-occurring
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide and an "immunological"
activity refers to the ability to induce the production of an
antibody against an antigenic epitope possessed by a native or
naturally-occurring PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide.
[0399] The term "antagonist" is used in the broadest sense [unless
otherwise qualified], and includes any molecule that partially or
fully blocks, inhibits, or neutralizes a biological activity of a
native PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 polypeptide disclosed
herein. In a similar manner, the term "agonist" is used in the
broadest sense [unless otherwise qualified] and includes any
molecule that mimics a biological activity of a native PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide disclosed herein. Suitable agonist
or antagonist molecules specifically include agonist or antagonist
antibodies or antibody fragments, fragments or amino acid sequence
variants of native PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptides,
peptides, antisense oligonucleotides, small organic molecules, etc.
Methods for identifying agonists or antagonists of a PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide may comprise contacting a PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide with a candidate agonist or
antagonist molecule and measuring a detectable change in one or
more biological activities normally associated with the PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide.
[0400] "Treating" or "treatment" or "alleviation" refers to both
therapeutic treatment and prophylactic or preventative measures,
wherein the object is to prevent or slow down (lessen) the targeted
pathologic condition or disorder. A subject in need of treatment
may already have the disorder, or may be prone to have the disorder
or may be in whom the disorder is to be prevented.
[0401] "Chronic" administration refers to administration of the
agent(s) in a continuous mode as opposed to an acute mode, so as to
maintain the initial therapeutic effect (activity) for an extended
period of time. "Intermittent" administration is treatment that is
not consecutively done without interruption, but rather is cyclic
in nature.
[0402] "Mammal" for purposes of treatment refers to any animal
classified as a mammal, including humans, rodents such as rats or
mice, domestic and farm animals, and zoo, sports, or pet animals,
such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits,
etc. Preferably, the mammal is human.
[0403] Administration "in combination with" one or more further
therapeutic agents includes simultaneous (concurrent) and
consecutive administration in any order.
[0404] "Carriers" as used herein include pharmaceutically
acceptable carriers, excipients, or stabilizers which are nontoxic
to the cell or mammal being exposed thereto at the dosages and
concentrations employed. Often the physiologically acceptable
carrier is an aqueous pH buffered solution. Examples of
physiologically acceptable carriers include buffers such as
phosphate, citrate, and other organic acids; antioxidants including
ascorbic acid; low molecular weight (less than about 10 residues)
polypeptide; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, arginine or
lysine; monosaccharides, disaccharides, and other carbohydrates
including glucose, mannose, or dextrins; chelating agents such as
EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming
counterions such as sodium; and/or nonionic surfactants such as
TWEEN.TM., polyethylene glycol (PEG), and PLURONICS.TM..
[0405] By "solid phase" is meant a non-aqueous matrix to which the
antibody of the present invention can adhere. Examples of solid
phases encompassed herein include those formed partially or
entirely of glass (e.g., controlled pore glass), polysaccharides
(e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol
and silicones. Depending on the context, the solid phase can
comprise the well of an assay plate; in others it is a purification
column (e.g., an affinity chromatography column). This term also
includes a discontinuous solid phase of discrete particles, such as
those described in U.S. Pat. No. 4,275,149.
[0406] A "liposome" is a small vesicle composed of various types of
lipids, phospholipids and/or surfactant which is useful for
delivery of a drug (such as a PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide or antibody thereto) to a mammal. The components of the
liposome are commonly arranged in a bilayer formation, similar to
the lipid arrangement of biological membranes.
[0407] A "small molecule" is defined herein to have a molecular
weight below about 500 Daltons.
[0408] An "effective amount" of a PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide, an anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236,
anti-PRO245, anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328,
anti-PRO344, anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731,
anti-PRO732, anti-PRO1003, anti-PRO1104, anti-PRO1151,
anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570,
anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725,
anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102,
anti-PRO10282, anti-PRO61709 or anti-PRO779 antibody, a PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 binding oligopeptide, a PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
binding organic molecule or an agonist or antagonist thereof as
disclosed herein is an amount sufficient to carry out a
specifically stated purpose. An "effective amount" may be
determined empirically and in a routine manner, in relation to the
stated purpose.
[0409] The term "therapeutically effective amount" refers to an
amount of an anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236,
anti-PRO245, anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328,
anti-PRO344, anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731,
anti-PRO732, anti-PRO1003, anti-PRO1104, anti-PRO1151,
anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570,
anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725,
anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102,
anti-PRO10282, anti-PRO61709 or anti-PRO779 antibody, a PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide, a PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
binding oligopeptide, a PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 binding
organic molecule or other drug effective to "treat" a disease or
disorder in a subject or mammal. In the case of cancer, the
therapeutically effective amount of the drug may reduce the number
of cancer cells; reduce the tumor size; inhibit (i.e., slow to some
extent and preferably stop) cancer cell infiltration into
peripheral organs; inhibit (i.e., slow to some extent and
preferably stop) tumor metastasis; inhibit, to some extent, tumor
growth; and/or relieve to some extent one or more of the symptoms
associated with the cancer. See the definition herein of
"treating". To the extent the drug may prevent growth and/or kill
existing cancer cells, it may be cytostatic and/or cytotoxic.
[0410] The phrases "cardiovascular, endothelial and angiogenic
disorder", "cardiovascular, endothelial and angiogenic
dysfunction", "cardiovascular, endothelial or angiogenic disorder"
and "cardiovascular, endothelial or angiogenic dysfunction" are
used interchangeably and refer in part to systemic disorders that
affect vessels, such as diabetes mellitus, as well as diseases of
the vessels themselves, such as of the arteries, capillaries,
veins, and/or lymphatics. This would include indications that
stimulate angiogenesis and/or cardiovascularization, and those that
inhibit angiogenesis and/or cardiovascularization. Such disorders
include, for example, arterial disease, such as atherosclerosis,
hypertension, inflammatory vasculitides, Reynaud's disease and
Reynaud's phenomenon, aneurysms, and arterial restenosis; venous
and lymphatic disorders such as thrombophlebitis, lymphangitis, and
lymphedema; and other vascular disorders such as peripheral
vascular disease, cancer such as vascular tumors, e.g., hemangioma
(capillary and cavernous), glomus tumors, telangiectasia, bacillary
angiomatosis, hemangioendothelioma, angiosarcoma,
haemangiopericytoma, Kaposi's sarcoma, lymphangioma, and
lymphangiosarcoma, tumor angiogenesis, trauma such as wounds,
burns, and other injured tissue, implant fixation, scarring,
ischemia reperfusion injury, rheumatoid arthritis, cerebrovascular
disease, renal diseases such as acute renal failure, or
osteoporosis. This would also include angina, myocardial
infarctions such as acute myocardial infarctions, cardiac
hypertrophy, and heart failure such as CHF.
[0411] "Hypertrophy", as used herein, is defined as an increase in
mass of an organ or structure independent of natural growth that
does not involve tumor formation. Hypertrophy of an organ or tissue
is due either to an increase in the mass of the individual cells
(true hypertrophy), or to an increase in the number of cells making
up the tissue (hyperplasia), or both. Certain organs, such as the
heart, lose the ability to divide shortly after birth. Accordingly,
"cardiac hypertrophy" is defined as an increase in mass of the
heart, which, in adults, is characterized by an increase in myocyte
cell size and contractile protein content without concomitant cell
division. The character of the stress responsible for inciting the
hypertrophy, (e.g., increased preload, increased afterload, loss of
myocytes, as in myocardial infarction, or primary depression of
contractility), appears to play a critical role in determining the
nature of the response. The early stage of cardiac hypertrophy is
usually characterized morphologically by increases in the size of
myofibrils and mitochondria, as well as by enlargement of
mitochondria and nuclei. At this stage, while muscle cells are
larger than normal, cellular organization is largely preserved. At
a more advanced stage of cardiac hypertrophy, there are
preferential increases in the size or number of specific
organelles, such as mitochondria, and new contractile elements are
added in localized areas of the cells, in an irregular manner.
Cells subjected to long-standing hypertrophy show more obvious
disruptions in cellular organization, including markedly enlarged
nuclei with highly lobulated membranes, which displace adjacent
myofibrils and cause breakdown of normal Z-band registration. The
phrase "cardiac hypertrophy" is used to include all stages of the
progression of this condition, characterized by various degrees of
structural damage of the heart muscle, regardless of the underlying
cardiac disorder. Hence, the term also includes physiological
conditions instrumental in the development of cardiac hypertrophy,
such as elevated blood pressure, aortic stenosis, or myocardial
infarction.
[0412] "Heart failure" refers to an abnormality of cardiac function
where the heart does not pump blood at the rate needed for the
requirements of metabolizing tissues. The heart failure can be
caused by a number of factors, including ischemic, congenital,
rheumatic, or idiopathic forms.
[0413] "Congestive heart failure" (CHF) is a progressive pathologic
state where the heart is increasingly unable to supply adequate
cardiac output (the volume of blood pumped by the heart over time)
to deliver the oxygenated blood to peripheral tissues. As CHF
progresses, structural and hemodynamic damages occur. While these
damages have a variety of manifestations, one characteristic
symptom is ventricular hypertrophy. CHF is a common end result of a
number of various cardiac disorders.
[0414] "Myocardial infarction" generally results from
atherosclerosis of the coronary arteries, often with superimposed
coronary thrombosis. It may be divided into two major types:
transmural infarcts, in which myocardial necrosis involves the full
thickness of the ventricular wall, and subendocardial
(nontransmural) infarcts, in which the necrosis involves the
subendocardium, the intramural myocardium, or both, without
extending all the way through the ventricular wall to the
epicardium. Myocardial infarction is known to cause both a change
in hemodynamic effects and an alteration in structure in the
damaged and healthy zones of the heart. Thus, for example,
myocardial infarction reduces the maximum cardiac output and the
stroke volume of the heart. Also associated with myocardial
infarction is a stimulation of the DNA synthesis occurring in the
interstice as well as an increase in the formation of collagen in
the areas of the heart not affected.
[0415] As a result of the increased stress or strain placed on the
heart in prolonged hypertension due, for example, to the increased
total peripheral resistance, cardiac hypertrophy has long been
associated with "hypertension". A characteristic of the ventricle
that becomes hypertrophic as a result of chronic pressure overload
is an impaired diastolic performance. Fouad et al., J. Am. Coll.
Cardiol., 4: 1500-1506 (1984); Smith et al., J. Am. Coll. Cardiol.,
5: 869-874 (1985). A prolonged left ventricular relaxation has been
detected in early essential hypertension, in spite of normal or
supranormal systolic function. Hartford et al., Hypertension, 6:
329-338 (1984). However, there is no close parallelism between
blood pressure levels and cardiac hypertrophy. Although improvement
in left ventricular function in response to antihypertensive
therapy has been reported in humans, patients variously treated
with a diuretic (hydrochlorothiazide), a .beta.-blocker
(propranolol), or a calcium channel blocker (diltiazem), have shown
reversal of left ventricular hypertrophy, without improvement in
diastolic function. Inouye et al., Am. J. Cardiol., 53: 1583-7
(1984).
[0416] Another complex cardiac disease associated with cardiac
hypertrophy is "hypertrophic cardiomyopathy". This condition is
characterized by a great diversity of morphologic, functional, and
clinical features (Maron et al., N. Engl. J. Med., 316: 780-789
(1987); Spirito et al., N. Engl. J. Med., 320: 749-755 (1989);
Louie and Edwards, Prog. Cardiovasc. Dis., 36: 275-308 (1994);
Wigle et al., Circulation, 92: 1680-1692 (1995)), the heterogeneity
of which is accentuated by the fact that it afflicts patients of
all ages. Spirito et al., N. Engl. J. Med., 336: 775-785 (1997).
The causative factors of hypertrophic cardiomyopathy are also
diverse and little understood. In general, mutations in genes
encoding sarcomeric proteins are associated with hypertrophic
cardiomyopathy. Recent data suggest that .beta.-myosin heavy chain
mutations may account for approximately 30 to 40 percent of cases
of familial hypertrophic cardiomyopathy. Watkins et al., N. Engl.
J. Med., 326: 1108-1114 (1992); Schwartz et al, Circulation, 91:
532-540 (1995); Marian and Roberts, Circulation, 92: 1336-1347
(1995); Thierfelder et al., Cell, 77: 701-712 (1994); Watkins et
al., Nat. Gen., 11: 434-437 (1995). Besides .beta.-myosin heavy
chain, other locations of genetic mutations include cardiac
troponin T, alpha topomyosin, cardiac myosin binding protein C,
essential myosin light chain, and regulatory myosin light chain.
See, Malik and Watkins, Curr. Opin. Cardiol., 12: 295-302
(1997).
[0417] Supravalvular "aortic stenosis" is an inherited vascular
disorder characterized by narrowing of the ascending aorta, but
other arteries, including the pulmonary arteries, may also be
affected. Untreated aortic stenosis may lead to increased
intracardiac pressure resulting in myocardial hypertrophy and
eventually heart failure and death. The pathogenesis of this
disorder is not fully understood, but hypertrophy and possibly
hyperplasia of medial smooth muscle are prominent features of this
disorder. It has been reported that molecular variants of the
elastin gene are involved in the development and pathogenesis of
aortic stenosis. U.S. Pat. No. 5,650,282 issued Jul. 22, 1997.
[0418] "Valvular regurgitation" occurs as a result of heart
diseases resulting in disorders of the cardiac valves. Various
diseases, like rheumatic fever, can cause the shrinking or pulling
apart of the valve orifice, while other diseases may result in
endocarditis, an inflammation of the endocardium or lining membrane
of the atrioventricular orifices and operation of the heart.
Defects such as the narrowing of the valve stenosis or the
defective closing of the valve result in an accumulation of blood
in the heart cavity or regurgitation of blood past the valve. If
uncorrected, prolonged valvular stenosis or insufficiency may
result in cardiac hypertrophy and associated damage to the heart
muscle, which may eventually necessitate valve replacement.
[0419] The term "immune related disease" means a disease in which a
component of the immune system of a mammal causes, mediates or
otherwise contributes to a morbidity in the mammal. Also included
are diseases in which stimulation or intervention of the immune
response has an ameliorative effect on progression of the disease.
Included within this term are immune-mediated inflammatory
diseases, non-immune-mediated inflammatory diseases, infectious
diseases, immunodeficiency diseases, neoplasia, etc.
[0420] The term "T cell mediated disease" means a disease in which
T cells directly or indirectly mediate or otherwise contribute to a
morbidity in a mammal. The T cell mediated disease may be
associated with cell mediated effects, lymphokine mediated effects,
etc., and even effects associated with B cells if the B cells are
stimulated, for example, by the lymphokines secreted by T
cells.
[0421] Examples of immune-related and inflammatory diseases, some
of which are immune or T cell mediated, include systemic lupus
erythematosis, rheumatoid arthritis, juvenile chronic arthritis,
spondyloarthropathies, systemic sclerosis (scleroderma), idiopathic
inflammatory myopathies (dermatomyositis, polymyositis), Sjogren's
syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic
anemia (immune pancytopenia, paroxysmal nocturnal hemoglobinuria),
autoimmune thrombocytopenia (idiopathic thrombocytopenic purpura,
immune-mediated thrombocytopenia), thyroiditis (Grave's disease,
Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis, atrophic
thyroiditis), diabetes mellitus, immune-mediated renal disease
(glomerulonephritis, tubulointerstitial nephritis), demyelinating
diseases of the central and peripheral nervous systems such as
multiple sclerosis, idiopathic demyelinating polyneuropathy or
Guillain-Barre syndrome, and chronic inflammatory demyelinating
polyneuropathy, hepatobiliary diseases such as infectious hepatitis
(hepatitis A, B, C, D, E and other non-hepatotropic viruses),
autoimmune chronic active hepatitis, primary biliary cirrhosis,
granulomatous hepatitis, and sclerosing cholangitis, inflammatory
bowel disease (ulcerative colitis: Crohn's disease),
gluten-sensitive enteropathy, and Whipple's disease, autoimmune or
immune-mediated skin diseases including bullous skin diseases,
erythema multiforme and contact dermatitis, psoriasis, allergic
diseases such as asthma, allergic rhinitis, atopic dermatitis, food
hypersensitivity and urticaria, immunologic diseases of the lung
such as eosinophilic pneumonia, idiopathic pulmonary fibrosis and
hypersensitivity pneumonitis, or transplantation associated
diseases including graft rejection and graft-versus-host-disease.
Infectious diseases including viral diseases such as AIDS (HIV
infection), hepatitis A, B, C, D, and E, herpes, etc., bacterial
infections, fungal infections, protozoal infections and parasitic
infections.
[0422] An "autoimmune disease" herein is a disease or disorder
arising from and directed against an individual's own tissues or a
co-segregate or manifestation thereof or resulting condition
therefrom. Examples of autoimmune diseases or disorders include,
but are not limited to arthritis (rheumatoid arthritis, juvenile
rheumatoid arthritis, osteoarthritis, psoriatic arthritis, and
ankylosing spondylitis), psoriasis, dermatitis including atopic
dermatitis; chronic idiopathic urticaria, including chronic
autoimmune urticaria, polymyositis/dermatomyositis, toxic epidermal
necrolysis, systemic scleroderma and sclerosis, responses
associated with inflammatory bowel disease (IBD) (Crohn's disease,
ulcerative colitis), and IBD with co-segregate of pyoderma
gangrenosum, erythema nodosum, primary sclerosing cholangitis,
and/or episcleritis), respiratory distress syndrome, including
adult respiratory distress syndrome (ARDS), meningitis,
IgE-mediated diseases such as anaphylaxis and allergic rhinitis,
encephalitis such as Rasmussen's encephalitis, uveitis, colitis
such as microscopic colitis and collagenous colitis,
glomerulonephritis (GN) such as membranous GN, idiopathic
membranous GN, membranous proliferative GN (MPGN), including Type I
and Type II, and rapidly progressive GN, allergic conditions,
eczema, asthma, conditions involving infiltration of T cells and
chronic inflammatory responses, atherosclerosis, autoimmune
myocarditis, leukocyte adhesion deficiency, systemic lupus
erythematosus (SLE) such as cutaneous SLE, lupus (including
nephritis, cerebritis, pediatric, non-renal, discoid, alopecia),
juvenile onset diabetes, multiple sclerosis (MS) such as
spino-optical MS, allergic encephalomyelitis, immune responses
associated with acute and delayed hypersensitivity mediated by
cytokines and T-lymphocytes, tuberculosis, sarcoidosis,
granulomatosis including Wegener's granulomatosis, agranulocytosis,
vasculitis (including Large Vessel vasculitis (including
Polymyalgia Rheumatica and Giant Cell (Takayasu's) Arteritis),
Medium Vessel vasculitis (including Kawasaki's Disease and
Polyarteritis Nodosa), CNS vasculitis, and ANCA-associated
vasculitis, such as Churg-Strauss vasculitis or syndrome (CSS)),
aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia,
immune hemolytic anemia including autoimmune hemolytic anemia
(AIHA), pernicious anemia, pure red cell aplasia (PRCA), Factor
VIII deficiency, hemophilia A, autoimmune neutropenia,
pancytopenia, leukopenia, diseases involving leukocyte diapedesis,
CNS inflammatory disorders, multiple organ injury syndrome,
myasthenia gravis, antigen-antibody complex mediated diseases,
anti-glomerular basement membrane disease, anti-phospholipid
antibody syndrome, allergic neuritis, Bechet disease, Castleman's
syndrome, Goodpasture's Syndrome, Lambert-Eaton Myasthenic
Syndrome, Reynaud's syndrome, Sjorgen's syndrome, Stevens-Johnson
syndrome, solid organ transplant rejection (including pretreatment
for high panel reactive antibody titers, IgA deposit in tissues,
and rejection arising from renal transplantation, liver
transplantation, intestinal transplantation, cardiac
transplantation, etc.), graft versus host disease (GVHD),
pemphigoid bullous, pemphigus (including vulgaris, foliaceus, and
pemphigus mucus-membrane pemphigoid), autoimmune
polyendocrinopathies, Reiter's disease, stiff-man syndrome, immune
complex nephritis, IgM polyneuropathies or IgM mediated neuropathy,
idiopathic thrombocytopenic purpura (ITP), thrombotic
throbocytopenic purpura (TTP), thrombocytopenia (as developed by
myocardial infarction patients, for example), including autoimmune
thrombocytopenia, autoimmune disease of the testis and ovary
including autoimune orchitis and oophoritis, primary
hypothyroidism; autoimmune endocrine diseases including autoimmune
thyroiditis, chronic thyroiditis (Hashimoto's Thyroiditis),
subacute thyroiditis, idiopathic hypothyroidism, Addison's disease,
Grave's disease, autoimmune polyglandular syndromes (or
polyglandular endocrinopathy syndromes), Type I diabetes also
referred to as insulin-dependent diabetes mellitus (IDDM),
including pediatric IDDM, and Sheehan's syndrome; autoimmune
hepatitis, Lymphoid interstitial pneumonitis (HIV), bronchiolitis
obliterans (non-transplant) vs NSIP, Guillain-Barre Syndrome,
Berger's Disease (IgA nephropathy), primary biliary cirrhosis,
celiac sprue (gluten enteropathy), refractory sprue with
co-segregate dermatitis herpetiformis, cryoglobulinemia,
amylotrophic lateral sclerosis (ALS; Lou Gehrig's disease),
coronary artery disease, autoimmune inner ear disease (AIED),
autoimmune hearing loss, opsoclonus myoclonus syndrome (OMS),
polychondritis such as refractory polychondritis, pulmonary
alveolar proteinosis, amyloidosis, giant cell hepatitis, scleritis,
monoclonal gammopathy of uncertain/unknown significance (MGUS),
peripheral neuropathy, paraneoplastic syndrome, channelopathies
such as epilepsy, migraine, arrhythmia, muscular disorders,
deafness, blindness, periodic paralysis, and channelopathies of the
CNS; autism, inflammatory myopathy, and focal segmental
glomerulosclerosis (FSGS).
[0423] The phrase "anxiety related disorders" refers to disorders
of anxiety, mood, and substance abuse, including but not limited
to: depression, generalized anxiety disorders, attention deficit
disorder, sleep disorder, hyperactivity disorder, obsessive
compulsive disorder, schizophrenia, cognitive disorders,
hyperalgesia and sensory disorders. Such disorders include the mild
to moderate anxiety, anxiety disorder due to a general medical
condition, anxiety disorder not otherwise specified, generalized
anxiety disorder, panic attack, panic disorder with agoraphobia,
panic disorder without agoraphobia, posttraumatic stress disorder,
social phobia, social anxiety, autism, specific phobia,
substance-induced anxiety disorder, acute alcohol withdrawal,
obsessive compulsive disorder, agoraphobia, monopolar disorders,
bipolar disorder I or II, bipolar disorder not otherwise specified,
cyclothymic disorder, depressive disorder, major depressive
disorder, mood disorder, substance-induced mood disorder,
enhancement of cognitive function, loss of cognitive function
associated with but not limited to Alzheimer's disease, stroke, or
traumatic injury to the brain, seizures resulting from disease or
injury including but not limited to epilepsy, learning
disorders/disabilities, cerebral palsy. In addition, anxiety
disorders may apply to personality disorders including but not
limited to the following types: paranoid, antisocial, avoidant
behavior, borderline personality disorders, dependent, histronic,
narcissistic, obsessive-compulsive, schizoid, and schizotypal.
[0424] The term "lipid metabolic disorder" refers to abnormal
clinical chemistry levels of cholesterol and triglycerides, wherein
elevated levels of these lipids is an indication for
atherosclerosis. Additionally, abnormal serum lipid levels may be
an indication of various cardiovascular diseases including
hypertension, stroke, coronary artery diseases, diabetes and/or
obesity.
[0425] The phrase "eye abnormality" refers to such potential
disorders of the eye as they may be related to atherosclerosis or
various ophthalmological abnormalities. Such disorders include but
are not limited to the following: retinal dysplasia, various
retinopathies, restenosis, retinal artery obstruction or occlusion;
retinal degeneration causing secondary atrophy of the retinal
vasculature, retinitis pigmentosa, macular dystrophies, Stargardt's
disease, congenital stationary night blindness, choroideremia,
gyrate atrophy, Leber's congenital amaurosis, retinoschisis
disorders, Wagner's syndrome, Usher syndromes, Zellweger syndrome,
Saldino-Mainzer syndrome, Senior-Loken syndrome, Bardet-Biedl
syndrome, Alport's syndrome, Alstrom's syndrome, Cockayne's
syndrome, dysplaisa spondyloepiphysaria congentia, Flynn-Aird
syndrome, Friedreich ataxia, Hallgren syndrome, Marshall syndrome,
Albers-Schnoberg disease, Refsum's disease, Kearns-Sayre syndrome,
Waardenburg's syndrome, Alagile syndrome, myotonic dystrophy,
olivopontocerebellar atrophy, Pierre-Marie dunsdrome, Stickler
syndrome, carotinemeia, cystinosis, Wolfram syndrome,
Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentia
pigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria,
or mannosidosis. Cataracts are also considered an eye abnormality
and are associated with such systemic diseases as: Human Down's
syndrome, Hallerman-Streiff syndrome, Lowe syndrome, galactosemia,
Marfan syndrome, Trismoy 13-15 condition, Alport syndrome, myotonic
dystrophy, Fabry disease, hypothroidisms, or Conradi syndrome.
Other ocular developmental anomalies include: Aniridia, anterior
segment and dysgenesis syndrome. Cataracts may also occur as a
result of an intraocular infection or inflammation (uveitis).
[0426] A "growth inhibitory amount" of an anti-PRO196, anti-PRO217,
anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258,
anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526,
anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104,
anti-PRO1151, anti-PRO1244, anti-PRO1298, anti-PRO1313,
anti-PRO1570, anti-PRO1886, anti-PRO1891, anti-PRO4409,
anti-PRO5725, anti-PRO5994, anti-PRO6097, anti-PRO7425,
anti-PRO10102, anti-PRO10282, anti-PRO61709 or anti-PRO779
antibody, PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 polypeptide, PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 binding oligopeptide or PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
binding organic molecule is an amount capable of inhibiting the
growth of a cell, especially tumor, e.g., cancer cell, either in
vitro or in vivo. A "growth inhibitory amount" of an anti-PRO196,
anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246,
anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357,
anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003,
anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298,
anti-PRO1313, anti-PRO1570, anti-PRO1886, anti-PRO1891,
anti-PRO4409, anti-PRO5725, anti-PRO5994, anti-PRO6097,
anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709 or
anti-PRO779 antibody, PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide, PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 binding
oligopeptide or PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 binding organic
molecule for purposes of inhibiting neoplastic cell growth may be
determined empirically and in a routine manner.
[0427] A "cytotoxic amount" of an anti-PRO196, anti-PRO217,
anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258,
anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526,
anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104,
anti-PRO1151, anti-PRO1244, anti-PRO1298, anti-PRO1313,
anti-PRO1570, anti-PRO1886, anti-PRO1891, anti-PRO4409,
anti-PRO5725, anti-PRO5994, anti-PRO6097, anti-PRO7425,
anti-PRO10102, anti-PRO10282, anti-PRO61709 or anti-PRO779
antibody, PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 polypeptide, PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 binding oligopeptide or PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
binding organic molecule is an amount capable of causing the
destruction of a cell, especially tumor, e.g., cancer cell, either
in vitro or in vivo. A "cytotoxic amount" of an anti-PRO196,
anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246,
anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357,
anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003,
anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298,
anti-PRO1313, anti-PRO1570, anti-PRO1886, anti-PRO1891,
anti-PRO4409, anti-PRO5725, anti-PRO5994, anti-PRO6097,
anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709 or
anti-PRO779 antibody, PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide, PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 binding
oligopeptide or PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 binding organic
molecule for purposes of inhibiting neoplastic cell growth may be
determined empirically and in a routine manner.
[0428] The term "antibody" is used in the broadest sense and
specifically covers, for example, single anti-PRO196, anti-PRO217,
anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258,
anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526,
anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104,
anti-PRO1151, anti-PRO1244, anti-PRO1298, anti-PRO1313,
anti-PRO1570, anti-PRO1886, anti-PRO1891, anti-PRO4409,
anti-PRO5725, anti-PRO5994, anti-PRO6097, anti-PRO7425,
anti-PRO10102, anti-PRO10282, anti-PRO61709 or anti-PRO779 antibody
monoclonal antibodies (including agonist, antagonist, and
neutralizing antibodies), anti-PRO196, anti-PRO217, anti-PRO231,
anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258, anti-PRO287,
anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526, anti-PRO724,
anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104, anti-PRO1151,
anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570,
anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725,
anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102,
anti-PRO10282, anti-PRO61709 or anti-PRO779 antibody compositions
with polyepitopic specificity, polyclonal antibodies, single chain
anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245,
anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344,
anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732,
anti-PRO1003, anti-PRO1104, anti-PRO1151, anti-PRO1244,
anti-PRO1298, anti-PRO1313, anti-PRO1570, anti-PRO1886,
anti-PRO1891, anti-PRO4409, anti-PRO5725, anti-PRO5994,
anti-PRO6097, anti-PRO7425, anti-PRO10102, anti-PRO10282,
anti-PRO61709 or anti-PRO779 antibodies, and fragments of
anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245,
anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344,
anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732,
anti-PRO1003, anti-PRO1104, anti-PRO1151, anti-PRO1244,
anti-PRO1298, anti-PRO1313, anti-PRO1570, anti-PRO1886,
anti-PRO1891, anti-PRO4409, anti-PRO5725, anti-PRO5994,
anti-PRO6097, anti-PRO7425, anti-PRO10102, anti-PRO10282,
anti-PRO61709 or anti-PRO779 antibodies (see below) as long as they
exhibit the desired biological or immunological activity. The term
"immunoglobulin" (Ig) is used interchangeable with antibody
herein.
[0429] An "isolated antibody" is one which has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials which would interfere with diagnostic or therapeutic uses
for the antibody, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. The invention provides
that the antibody will be purified (1) to greater than 95% by
weight of antibody as determined by the Lowry method, and most
preferably more than 99% by weight, (2) to a degree sufficient to
obtain at least 15 residues of N-terminal or internal amino acid
sequence by use of a spinning cup sequenator, or (3) to homogeneity
by SDS-PAGE under reducing or nonreducing conditions using
Coomassie blue or, preferably, silver stain. Isolated antibody
includes the antibody in situ within recombinant cells since at
least one component of the antibody's natural environment will not
be present. Ordinarily, however, isolated antibody will be prepared
by at least one purification step.
[0430] The basic 4-chain antibody unit is a heterotetrameric
glycoprotein composed of two identical light (L) chains and two
identical heavy (H) chains (an IgM antibody consists of 5 of the
basic heterotetramer unit along with an additional polypeptide
called J chain, and therefore contain 10 antigen binding sites,
while secreted IgA antibodies can polymerize to form polyvalent
assemblages comprising 2-5 of the basic 4-chain units along with J
chain). In the case of IgGs, the 4-chain unit is generally about
150,000 daltons. Each L chain is linked to a H chain by one
covalent disulfide bond, while the two H chains are linked to each
other by one or more disulfide bonds depending on the H chain
isotype. Each H and L chain also has regularly spaced intrachain
disulfide bridges. Each H chain has at the N-terminus, a variable
domain (V.sub.H) followed by three constant domains (C.sub.H) for
each of the .alpha. and .gamma. chains and four C.sub.H domains for
.mu. and .epsilon. isotypes. Each L chain has at the N-terminus, a
variable domain (V.sub.L) followed by a constant domain (C.sub.L)
at its other end. The V.sub.L is aligned with the V.sub.H and the
C.sub.L is aligned with the first constant domain of the heavy
chain (C.sub.H1). Particular amino acid residues are believed to
form an interface between the light chain and heavy chain variable
domains. The pairing of a V.sub.H and V.sub.L together forms a
single antigen-binding site. For the structure and properties of
the different classes of antibodies, see, e.g., Basic and Clinical
Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and
Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, Conn.,
1994, page 71 and Chapter 6.
[0431] The L chain from any vertebrate species can be assigned to
one of two clearly distinct types, called kappa and lambda, based
on the amino acid sequences of their constant domains. Depending on
the amino acid sequence of the constant domain of their heavy
chains (C.sub.H), immunoglobulins can be assigned to different
classes or isotypes. There are five classes of immunoglobulins:
IgA, IgD, IgE, IgG, and IgM, having heavy chains designated
.alpha., .delta., .epsilon., .gamma., and .mu., respectively. The
.gamma. and .alpha. classes are further divided into subclasses on
the basis of relatively minor differences in C.sub.H sequence and
function, e.g., humans express the following subclasses: IgG1,
IgG2, IgG3, IgG4, IgA1, and IgA2.
[0432] The term "variable" refers to the fact that certain segments
of the variable domains differ extensively in sequence among
antibodies. The V domain mediates antigen binding and define
specificity of a particular antibody for its particular antigen.
However, the variability is not evenly distributed across the
110-amino acid span of the variable domains. Instead, the V regions
consist of relatively invariant stretches called framework regions
(FRs) of 15-30 amino acids separated by shorter regions of extreme
variability called "hypervariable regions" that are each 9-12 amino
acids long. The variable domains of native heavy and light chains
each comprise four FRs, largely adopting a .beta.-sheet
configuration, connected by three hypervariable regions, which form
loops connecting, and in some cases forming part of, the
.beta.-sheet structure. The hypervariable regions in each chain are
held together in close proximity by the FRs and, with the
hypervariable regions from the other chain, contribute to the
formation of the antigen-binding site of antibodies (see Kabat et
al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991)). The constant domains are not involved directly in binding
an antibody to an antigen, but exhibit various effector functions,
such as participation of the antibody in antibody dependent
cellular cytotoxicity (ADCC).
[0433] The term "hypervariable region" when used herein refers to
the amino acid residues of an antibody which are responsible for
antigen-binding. The hypervariable region generally comprises amino
acid residues from a "complementarity determining region" or "CDR"
(e.g. around about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3)
in the V.sub.L, and around about 1-35 (H1), 50-65 (H2) and 95-102
(H3) in the V.sub.H; Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991)) and/or those residues
from a "hypervariable loop" (e.g. residues 26-32 (L1), 50-52 (L2)
and 91-96 (L3) in the V.sub.L, and 26-32 (H1), 53-55 (H2) and
96-101 (H3) in the V.sub.H; Chothia and Lesk J. Mol. Biol.
196:901-917 (1987)).
[0434] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic site. Furthermore, in contrast to polyclonal antibody
preparations which include different antibodies directed against
different determinants (epitopes), each monoclonal antibody is
directed against a single determinant on the antigen. In addition
to their specificity, the monoclonal antibodies are advantageous in
that they may be synthesized uncontaminated by other antibodies.
The modifier "monoclonal" is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies useful in the present invention may be
prepared by the hybridoma methodology first described by Kohler et
al., Nature, 256:495 (1975), or may be made using recombinant DNA
methods in bacterial, eukaryotic animal or plant cells (see, e.g.,
U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be
isolated from phage antibody libraries using the techniques
described in Clackson et al., Nature, 352:624-628 (1991) and Marks
et al., J. Mol. Biol., 222:581-597 (1991), for example.
[0435] The monoclonal antibodies herein include "chimeric"
antibodies in which a portion of the heavy and/or light chain is
identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is identical with or homologous to corresponding sequences
in antibodies derived from another species or belonging to another
antibody class or subclass, as well as fragments of such
antibodies, so long as they exhibit the desired biological activity
(see U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl.
Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of
interest herein include "primatized" antibodies comprising variable
domain antigen-binding sequences derived from a non-human primate
(e.g. Old World Monkey, Ape etc), and human constant region
sequences.
[0436] An "intact" antibody is one which comprises an
antigen-binding site as well as a C.sub.L and at least heavy chain
constant domains, C.sub.H1, C.sub.H2 and C.sub.H3. The constant
domains may be native sequence constant domains (e.g. human native
sequence constant domains) or amino acid sequence variant thereof.
Preferably, the intact antibody has one or more effector
functions.
[0437] "Antibody fragments" comprise a portion of an intact
antibody, preferably the antigen binding or variable region of the
intact antibody. Examples of antibody fragments include Fab, Fab',
F(ab').sub.2, and Fv fragments; diabodies; linear antibodies (see
U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng.
8(10): 1057-1062 [1995]); single-chain antibody molecules; and
multispecific antibodies formed from antibody fragments.
[0438] Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, and a residual
"Fc" fragment, a designation reflecting the ability to crystallize
readily. The Fab fragment consists of an entire L chain along with
the variable region domain of the H chain (V.sub.H), and the first
constant domain of one heavy chain (C.sub.H1). Each Fab fragment is
monovalent with respect to antigen binding, i.e., it has a single
antigen-binding site. Pepsin treatment of an antibody yields a
single large F(ab').sub.2 fragment which roughly corresponds to two
disulfide linked Fab fragments having divalent antigen-binding
activity and is still capable of cross-linking antigen. Fab'
fragments differ from Fab fragments by having additional few
residues at the carboxy terminus of the C.sub.H1 domain including
one or more cysteines from the antibody hinge region. Fab'-SH is
the designation herein for Fab' in which the cysteine residue(s) of
the constant domains bear a free thiol group. F(ab').sub.2 antibody
fragments originally were produced as pairs of Fab' fragments which
have hinge cysteines between them. Other chemical couplings of
antibody fragments are also known.
[0439] The Fc fragment comprises the carboxy-terminal portions of
both H chains held together by disulfides. The effector functions
of antibodies are determined by sequences in the Fc region, which
region is also the part recognized by Fc receptors (FcR) found on
certain types of cells.
[0440] "Fv" is the minimum antibody fragment which contains a
complete antigen-recognition and -binding site. This fragment
consists of a dimer of one heavy- and one light-chain variable
region domain in tight, non-covalent association. From the folding
of these two domains emanate six hypervariable loops (3 loops each
from the H and L chain) that contribute the amino acid residues for
antigen binding and confer antigen binding specificity to the
antibody. However, even a single variable domain (or half of an Fv
comprising only three CDRs specific for an antigen) has the ability
to recognize and bind antigen, although at a lower affinity than
the entire binding site.
[0441] "Single-chain Fv" also abbreviated as "sFv" or "scFv" are
antibody fragments that comprise the V.sub.H and V.sub.L antibody
domains connected into a single polypeptide chain. Preferably, the
sFv polypeptide further comprises a polypeptide linker between the
V.sub.H and V.sub.L domains which enables the sFv to form the
desired structure for antigen binding. For a review of sFv, see
Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,
Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315
(1994); Borrebaeck 1995, infra
[0442] The term "diabodies" refers to small antibody fragments
prepared by constructing sFv fragments (see preceding paragraph)
with short linkers (about 5-10 residues) between the V.sub.H and
V.sub.L domains such that inter-chain but not intra-chain pairing
of the V domains is achieved, resulting in a bivalent fragment,
i.e., fragment having two antigen-binding sites. Bispecific
diabodies are heterodimers of two "crossover" sFv fragments in
which the V.sub.H and V.sub.L domains of the two antibodies are
present on different polypeptide chains. Diabodies are described
more fully in, for example, EP 404,097; WO 93/11161; and Hollinger
et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
[0443] "Humanized" forms of non-human (e.g., rodent) antibodies are
chimeric antibodies that contain minimal sequence derived from the
non-human antibody. For the most part, humanized antibodies are
human immunoglobulins (recipient antibody) in which residues from a
hypervariable region of the recipient are replaced by residues from
a hypervariable region of a non-human species (donor antibody) such
as mouse, rat, rabbit or non-human primate having the desired
antibody specificity, affinity, and capability. In some instances,
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies may comprise residues that are not found in
the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin and all or substantially all of the FRs
are those of a human immunoglobulin sequence. The humanized
antibody optionally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see Jones et al., Nature
321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988);
and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).
[0444] A "species-dependent antibody," e.g., a mammalian anti-human
IgE antibody, is an antibody which has a stronger binding affinity
for an antigen from a first mammalian species than it has for a
homologue of that antigen from a second mammalian species.
Normally, the species-dependent antibody "bind specifically" to a
human antigen (i.e., has a binding affinity (Kd) value of no more
than about 1.times.10.sup.-7 M, preferably no more than about
1.times.10.sup.-8 and most preferably no more than about
1.times.10.sup.-9 M) but has a binding affinity for a homologue of
the antigen from a second non-human mammalian species which is at
least about 50 fold, or at least about 500 fold, or at least about
1000 fold, weaker than its binding affinity for the human antigen.
The species-dependent antibody can be of any of the various types
of antibodies as defined above, but preferably is a humanized or
human antibody.
[0445] A "PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 binding oligopeptide" is an
oligopeptide that binds, preferably specifically, to a PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide as described herein. PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 binding oligopeptides may be chemically synthesized using
known oligopeptide synthesis methodology or may be prepared and
purified using recombinant technology. PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
binding oligopeptides usually are or are at least about 5 amino
acids in length, alternatively are or are at least about 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,
43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,
60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,
77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,
94, 95, 96, 97, 98, 99, or 100 amino acids in length or more,
wherein such oligopeptides that are capable of binding, preferably
specifically, to a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide as
described herein. PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 binding
oligopeptides may be identified without undue experimentation using
well known techniques. In this regard, it is noted that techniques
for screening oligopeptide libraries for oligopeptides that are
capable of specifically binding to a polypeptide target are well
known in the art (see, e.g., U.S. Pat. Nos. 5,556,762, 5,750,373,
4,708,871, 4,833,092, 5,223,409, 5,403,484, 5,571,689, 5,663,143;
PCT Publication Nos. WO 84/03506 and WO84/03564; Geysen et al.,
Proc. Natl. Acad. Sci. U.S.A., 81:3998-4002 (1984); Geysen et al.,
Proc. Natl. Acad. Sci. U.S.A., 82:178-182 (1985); Geysen et al., in
Synthetic Peptides as Antigens, 130-149 (1986); Geysen et al., J.
Immunol. Meth., 102:259-274 (1987); Schoofs et al., J. Immunol.,
140:611-616 (1988), Cwirla, S. E. et al. (1990) Proc. Natl. Acad.
Sci. USA, 87:6378; Lowman, H. B. et al. (1991) Biochemistry,
30:10832; Clackson, T. et al. (1991) Nature, 352: 624; Marks, J. D.
et al. (1991), J. Mol. Biol., 222:581; Kang, A. S. et al. (1991)
Proc. Natl. Acad. Sci. USA, 88:8363, and Smith, G. P. (1991)
Current Opin. Biotechnol., 2:668).
[0446] A "PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 binding organic molecule" is
an organic molecule other than an oligopeptide or antibody as
defined herein that binds, preferably specifically, to a PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide as described herein. PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 binding organic molecules may be identified and chemically
synthesized using known methodology (see, e.g., PCT Publication
Nos. WO00/00823 and WO00/39585). PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
binding organic molecules are usually less than about 2000 daltons
in size, alternatively less than about 1500, 750, 500, 250 or 200
daltons in size, wherein such organic molecules that are capable of
binding, preferably specifically, to a PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide as described herein may be identified without undue
experimentation using well known techniques. In this regard, it is
noted that techniques for screening organic molecule libraries for
molecules that are capable of binding to a polypeptide target are
well known in the art (see, e.g., PCT Publication Nos. WO00/00823
and WO00/39585).
[0447] An antibody, oligopeptide or other organic molecule "which
binds" an antigen of interest, e.g. a tumor-associated polypeptide
antigen target, is one that binds the antigen with sufficient
affinity such that the antibody, oligopeptide or other organic
molecule is preferably useful as a diagnostic and/or therapeutic
agent in targeting a cell or tissue expressing the antigen, and
does not significantly cross-react with other proteins. The extent
of binding of the antibody, oligopeptide or other organic molecule
to a "non-target" protein will be less than about 10% of the
binding of the antibody, oligopeptide or other organic molecule to
its particular target protein as determined by fluorescence
activated cell sorting (FACS) analysis or radioimmunoprecipitation
(RIA). With regard to the binding of an antibody, oligopeptide or
other organic molecule to a target molecule, the term "specific
binding" or "specifically binds to" or is "specific for" a
particular polypeptide or an epitope on a particular polypeptide
target means binding that is measurably different from a
non-specific interaction. Specific binding can be measured, for
example, by determining binding of a molecule compared to binding
of a control molecule, which generally is a molecule of similar
structure that does not have binding activity. For example,
specific binding can be determined by competition with a control
molecule that is similar to the target, for example, an excess of
non-labeled target. In this case, specific binding is indicated if
the binding of the labeled target to a probe is competitively
inhibited by excess unlabeled target. The term "specific binding"
or "specifically binds to" or is "specific for" a particular
polypeptide or an epitope on a particular polypeptide target as
used herein can be exhibited, for example, by a molecule having a
Kd for the target of at least about 10.sup.-4 M, alternatively at
least about 10.sup.-5 M, alternatively at least about 10.sup.-6 M,
alternatively at least about 10.sup.-7 M, alternatively at least
about 10.sup.-8 M, alternatively at least about 10.sup.-9 M,
alternatively at least about 10.sup.-10 M, alternatively at least
about 10.sup.-11 M, alternatively at least about 10.sup.-12 M, or
greater. The term "specific binding" refers to binding where a
molecule binds to a particular polypeptide or epitope on a
particular polypeptide without substantially binding to any other
polypeptide or polypeptide epitope.
[0448] An antibody, oligopeptide or other organic molecule that
"inhibits the growth of tumor cells expressing a "PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779" or a "growth inhibitory" antibody, oligopeptide or other
organic molecule is one which results in measurable growth
inhibition of cancer cells expressing or overexpressing the
appropriate PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 polypeptide. The PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide may be a transmembrane polypeptide
expressed on the surface of a cancer cell or may be a polypeptide
that is produced and secreted by a cancer cell. Preferred growth
inhibitory anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236,
anti-PRO245, anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328,
anti-PRO344, anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731,
anti-PRO732, anti-PRO1003, anti-PRO1104, anti-PRO1151,
anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570,
anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725,
anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102,
anti-PRO10282, anti-PRO61709 or anti-PRO779 antibodies,
oligopeptides or organic molecules inhibit growth of PRO196-,
PRO217-, PRO231-, PRO236-, PRO245-, PRO246-, PRO258-, PRO287-,
PRO328-, PRO344-, PRO357-, PRO526-, PRO724-, PRO731-, PRO732-,
PRO1003-, PRO1104-, PRO1151-, PRO1244-, PRO1298-, PRO1313-,
PRO1570-, PRO1886-, PRO1891-, PRO4409-, PRO5725-, PRO5994-,
PRO6097-, PRO7425-, PRO10102-, PRO10282-, PRO61709- or
PRO779-expressing tumor cells by or by greater than 20%, preferably
from about 20% to about 50%, and even more preferably, by or by
greater than 50% (e.g., from about 50% to about 100%) as compared
to the appropriate control, the control typically being tumor cells
not treated with the antibody, oligopeptide or other organic
molecule being tested. Growth inhibition can be measured at an
antibody concentration of about 0.1 to 30 .mu.g/ml or about 0.5 nM
to 200 nM in cell culture, where the growth inhibition is
determined 1-10 days after exposure of the tumor cells to the
antibody. Growth inhibition of tumor cells in vivo can be
determined in various ways. The antibody is growth inhibitory in
vivo if administration of the anti-PRO196, anti-PRO217,
anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258,
anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526,
anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104,
anti-PRO1151, anti-PRO1244, anti-PRO1298, anti-PRO1313,
anti-PRO1570, anti-PRO1886, anti-PRO1891, anti-PRO4409,
anti-PRO5725, anti-PRO5994, anti-PRO6097, anti-PRO7425,
anti-PRO10102, anti-PRO10282, anti-PRO61709 or anti-PRO779 antibody
at about 1 .mu.g/kg to about 100 mg/kg body weight results in
reduction in tumor size or tumor cell proliferation within about 5
days to 3 months from the first administration of the antibody,
preferably within about 5 to 30 days.
[0449] An antibody, oligopeptide or other organic molecule which
"induces apoptosis" is one which induces programmed cell death as
determined by binding of annexin V, fragmentation of DNA, cell
shrinkage, dilation of endoplasmic reticulum, cell fragmentation,
and/or formation of membrane vesicles (called apoptotic bodies).
The cell is usually one which overexpresses a PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 polypeptide. Preferably the cell is a tumor cell, e.g., a
prostate, breast, ovarian, stomach, endometrial, lung, kidney,
colon, bladder cell. Various methods are available for evaluating
the cellular events associated with apoptosis. For example,
phosphatidyl serine (PS) translocation can be measured by annexin
binding; DNA fragmentation can be evaluated through DNA laddering;
and nuclear/chromatin condensation along with DNA fragmentation can
be evaluated by any increase in hypodiploid cells. Preferably, the
antibody, oligopeptide or other organic molecule which induces
apoptosis is one which results in or in about 2 to 50 fold,
preferably in or in about 5 to 50 fold, and most preferably in or
in about 10 to 50 fold, induction of annexin binding relative to
untreated cell in an annexin binding assay.
[0450] Antibody "effector functions" refer to those biological
activities attributable to the Fc region (a native sequence Fc
region or amino acid sequence variant Fc region) of an antibody,
and vary with the antibody isotype. Examples of antibody effector
functions include: C1q binding and complement dependent
cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated
cytotoxicity (ADCC); phagocytosis; down regulation of cell surface
receptors (e.g., B cell receptor); and B cell activation.
[0451] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC"
refers to a form of cytotoxicity in which secreted Ig bound onto Fc
receptors (FcRs) present on certain cytotoxic cells (e.g., Natural
Killer (NK) cells, neutrophils, and macrophages) enable these
cytotoxic effector cells to bind specifically to an antigen-bearing
target cell and subsequently kill the target cell with cytotoxins.
The antibodies "arm" the cytotoxic cells and are absolutely
required for such killing. The primary cells for mediating ADCC, NK
cells, express Fc.gamma.RIII only, whereas monocytes express
Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII. FcR expression on
hematopoietic cells is summarized in Table 3 on page 464 of Ravetch
and Kinet, Annu. Rev. Immunol. 9:457-92 (1991). To assess ADCC
activity of a molecule of interest, an in vitro ADCC assay, such as
that described in U.S. Pat. No. 5,500,362 or 5,821,337 may be
performed. Useful effector cells for such assays include peripheral
blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
Alternatively, or additionally, ADCC activity of the molecule of
interest may be assessed in vivo, e.g., in a animal model such as
that disclosed in Clynes et al. Proc. Natl. Acad. Sci. U.S.A.
95:652-656 (1998).
[0452] "Fc receptor" or "FcR" describes a receptor that binds to
the Fc region of an antibody. The preferred FcR is a native
sequence human FcR. Moreover, a preferred FcR is one which binds an
IgG antibody (a gamma receptor) and includes receptors of the
Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII subclasses, including
allelic variants and alternatively spliced forms of these
receptors. Fc.gamma.RII receptors include Fc.gamma.RIIA (an
"activating receptor") and Fc.gamma.RIIB (an "inhibiting
receptor"), which have similar amino acid sequences that differ
primarily in the cytoplasmic domains thereof. Activating receptor
Fc.gamma.RIIA contains an immunoreceptor tyrosine-based activation
motif (ITAM) in its cytoplasmic domain. Inhibiting receptor
Fc.gamma.RIIB contains an immunoreceptor tyrosine-based inhibition
motif (ITIM) in its cytoplasmic domain. (see review M. in Daeron,
Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in
Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991); Capel et
al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab.
Clin. Med. 126:330-41 (1995). Other FcRs, including those to be
identified in the future, are encompassed by the term "FcR" herein.
The term also includes the neonatal receptor, FcRn, which is
responsible for the transfer of maternal IgGs to the fetus (Guyer
et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol.
24:249 (1994)).
[0453] "Human effector cells" are leukocytes which express one or
more FcRs and perform effector functions. Preferably, the cells
express at least Fc.gamma.RIII and perform ADCC effector function.
Examples of human leukocytes which mediate ADCC include peripheral
blood mononuclear cells (PBMC), natural killer (NK) cells,
monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK
cells being preferred. The effector cells may be isolated from a
native source, e.g., from blood.
[0454] "Complement dependent cytotoxicity" or "CDC" refers to the
lysis of a target cell in the presence of complement. Activation of
the classical complement pathway is initiated by the binding of the
first component of the complement system (C1q) to antibodies (of
the appropriate subclass) which are bound to their cognate antigen.
To assess complement activation, a CDC assay, e.g., as described in
Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be
performed.
[0455] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth. Examples of cancer include but are not
limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
More particular examples of such cancers include squamous cell
cancer, lung cancer (including small-cell lung cancer, non-small
cell lung cancer, adenocarcinoma of the lung, and squamous
carcinoma of the lung), cancer of the peritoneum, hepatocellular
cancer, gastric or stomach cancer (including gastrointestinal
cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian
cancer, liver cancer, bladder cancer, hepatoma, breast cancer,
colon cancer, colorectal cancer, endometrial or uterine carcinoma,
salivary gland carcinoma, kidney or renal cancer, liver cancer,
prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma
and various types of head and neck cancer, as well as B-cell
lymphoma (including low grade/follicular non-Hodgkin's lymphoma
(NHL); small lymphocytic (SL) NHL; intermediate grade/follicular
NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL;
high grade lymphoblastic NHL; high grade small non-cleaved cell
NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related
lymphoma; and Waldenstrom's Macroglobulinemia); chronic lymphocytic
leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell
leukemia; chronic myeloblastic leukemia; and post-transplant
lymphoproliferative disorder (PTLD). Preferably, the cancer
comprises a tumor that expresses an IGF receptor, more preferably
breast cancer, lung cancer, colorectal cancer, or prostate cancer,
and most preferably breast or prostate cancer.
[0456] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer. Examples of chemotherapeutic agents
include alkylating agents such as thiotepa and CYTOXAN.RTM.
cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan
and piposulfan; aziridines such as benzodopa, carboquone,
meturedopa, and uredopa; ethylenimines and methylamelamines
including altretamine, triethylenemelamine,
trietylenephosphoramide, triethiylenethiophosphoramide and
trimethylolomelamine; acetogenins (especially bullatacin and
bullatacinone); a camptothecin (including the synthetic analogue
topotecan); bryostatin; callystatin; CC-1065 (including its
adozelesin, carzelesin and bizelesin synthetic analogues);
cryptophycins (particularly cryptophycin 1 and cryptophycin 8);
dolastatin; duocarmycin (including the synthetic analogues, KW-2189
and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;
spongistatin; nitrogen mustards such as chlorambucil,
chlornaphazine, cholophosphamide, estramustine, ifosfamide,
mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,
novembichin, phenesterine, prednimustine, trofosfamide, uracil
mustard; nitrosureas such as carmustine, chlorozotocin,
fotemustine, lomustine, nimustine, and ranimnustine; antibiotics
such as the enediyne antibiotics (e.g., calicheamicin, especially
calicheamicin gamma1I and calicheamicin omegaI1 (see, e.g., Agnew,
Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including
dynemicin A; bisphosphonates, such as dodronate; an esperamicin; as
well as neocarzinostatin chromophore and related chromoprotein
enediyne antiobiotic chromophores), adacinomysins, actinomycin,
authramycin, azaserine, bleomycins, cactinomycin, carabicin,
caminomycin, carzinophilin, chromomycinis, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,
ADRIAMYCIN.RTM. doxorubicin (including morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and
deoxydoxorubicin), epirubicin, esorubicin, idarubicin,
marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,
nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,
quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin; anti-metabolites such as
methotrexate and 5-fluorouracil (5-FU); folic acid analogues such
as denopterin, methotrexate, pteropterin, trimetrexate; purine
analogs such as fludarabine, 6-mercaptopurine, thiamiprine,
thioguanine; pyrimidine analogs such as ancitabine, azacitidine,
6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine,
enocitabine, floxuridine; androgens such as calusterone,
dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;
defofamine; demecolcine; diaziquone; elformithine; elliptinium
acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea;
lentinan; lonidainine; maytansinoids such as maytansine and
ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine;
pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic
acid; 2-ethylhydrazide; procarbazine; PSK.RTM. polysaccharide
complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;
sizofuran; spirogermanium; tenuazonic acid; triaziquone;
2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2
toxin, verracurin A, roridin A and anguidine); urethan; vindesine;
dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;
gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa;
taxoids, e.g., TAXOL.RTM. paclitaxel (Bristol-Myers Squibb
Oncology, Princeton, N.J.), ABRAXANE.TM. Cremophor-free,
albumin-engineered nanoparticle formulation of paclitaxel (American
Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE.RTM.
doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;
GEMZAR.RTM. gemcitabine; 6-thioguanine; mercaptopurine;
methotrexate; platinum analogs such as cisplatin and carboplatin;
vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;
vincristine; NAVELBINE.RTM. vinorelbine; novantrone; teniposide;
edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11;
topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO);
retinoids such as retinoic acid; capecitabine; and pharmaceutically
acceptable salts, acids or derivatives of any of the above.
[0457] Also included in this definition are anti-hormonal agents
that act to regulate or inhibit hormone action on tumors such as
anti-estrogens and selective estrogen receptor modulators (SERMs),
including, for example, tamoxifen (including NOLVADEX.RTM.
tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen,
trioxifene, keoxifene, LY117018, onapristone, and FARESTON
toremifene; aromatase inhibitors that inhibit the enzyme aromatase,
which regulates estrogen production in the adrenal glands, such as,
for example, 4(5)-imidazoles, aminoglutethimide, MEGASE.RTM.
megestrol acetate, AROMASIN.RTM. exemestane, formestanie,
fadrozole, RIVISOR.RTM. vorozole, FEMARA.RTM. letrozole, and
ARIMIDEX.RTM. anastrozole; and anti-androgens such as flutamide,
nilutamide, bicalutamide, leuprolide, and goserelin; as well as
troxacitabine (a 1,3-dioxolane nucleoside cytosine analog);
antisense oligonucleotides, particularly those which inhibit
expression of genes in signaling pathways implicated in abherant
cell proliferation, such as, for example, PKC-alpha, Ralf and
H-Ras; ribozymes such as a VEGF expression inhibitor (e.g.,
ANGIOZYME.RTM. ribozyme) and a HER2 expression inhibitor; vaccines
such as gene therapy vaccines, for example, ALLOVECTIN.RTM.
vaccine, LEUVECTIN.RTM. vaccine, and VAXID.RTM. vaccine;
PROLEUKIN.RTM. rIL-2; LURTOTECAN.RTM. topoisomerase 1 inhibitor;
ABARELIX.RTM. rmRH; and pharmaceutically acceptable salts, acids or
derivatives of any of the above.
[0458] The terms "cell proliferative disorder" and "proliferative
disorder" refer to disorders that are associated with some degree
of abnormal cell proliferation. In one aspect of the invention, the
cell proliferative disorder is cancer.
[0459] "Tumor", as used herein, refers to all neoplastic cell
growth and proliferation, whether malignant or benign, and all
pre-cancerous and cancerous cells and tissues.
[0460] An antibody, oligopeptide or other organic molecule which
"induces cell death" is one which causes a viable cell to become
nonviable. The cell is one which expresses a PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 polypeptide, preferably a cell that overexpresses a PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide as compared to a normal cell of the
same tissue type. The PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide may be a transmembrane polypeptide expressed on the
surface of a cancer cell or may be a polypeptide that is produced
and secreted by a cancer cell. Preferably, the cell is a cancer
cell, e.g., a breast, ovarian, stomach, endometrial, salivary
gland, lung, kidney, colon, thyroid, pancreatic or bladder cell.
Cell death in vitro may be determined in the absence of complement
and immune effector cells to distinguish cell death induced by
antibody-dependent cell-mediated cytotoxicity (ADCC) or complement
dependent cytotoxicity (CDC). Thus, the assay for cell death may be
performed using heat inactivated serum (i.e., in the absence of
complement) and in the absence of immune effector cells. To
determine whether the antibody, oligopeptide or other organic
molecule is able to induce cell death, loss of membrane integrity
as evaluated by uptake of propidium iodide (PI), trypan blue (see
Moore et al. Cytotechnology 17:1-11 (1995)) or 7AAD can be assessed
relative to untreated cells. Preferred cell death-inducing
antibodies, oligopeptides or other organic molecules are those
which induce PI uptake in the PI uptake assay in BT474 cells.
[0461] As used herein, the term "immunoadhesion" designates
antibody-like molecules which combine the binding specificity of a
heterologous protein (an "adhesion") with the effector functions of
immunoglobulin constant domains. Structurally, the immunoadhesions
comprise a fusion of an amino acid sequence with the desired
binding specificity which is other than the antigen recognition and
binding site of an antibody (i.e., is "heterologous"), and an
immunoglobulin constant domain sequence. The adhesion part of an
immunoadhesion molecule typically is a contiguous amino acid
sequence comprising at least the binding site of a receptor or a
ligand. The immunoglobulin constant domain sequence in the
immunoadhesion may be obtained from any immunoglobulin, such as
IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and
IgA-2), IgE, IgD or IgM.
[0462] The word "label" when used herein refers to a detectable
compound or composition which is conjugated directly or indirectly
to the antibody so as to generate a "labeled" antibody. The label
may be detectable by itself (e.g. radioisotope labels or
fluorescent labels) or, in the case of an enzymatic label, may
catalyze chemical alteration of a substrate compound or composition
which is detectable.
[0463] "Replication-preventing agent" is an agent wherein
replication, function, and/or growth of the cells is inhibited or
prevented, or cells are destroyed, no matter what the mechanism,
such as by apoptosis, angiostasis, cytosis, tumoricide, mytosis
inhibition, blocking cell cycle progression, arresting cell growth,
binding to tumors, acting as cellular mediators, etc. Such agents
include a chemotherapeutic agent, cytotoxic agent, cytokine,
growth-inhibitory agent, or anti-hormonal agent, e.g., an
anti-estrogen compound such as tamoxifen, an anti-progesterone such
as onapristone (see, EP 616 812); or an anti-androgen such as
flutamide, as well as aromidase inhibitors, or a hormonal agent
such as an androgen.
[0464] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents the function of cells and/or
causes destruction of cells. The term is intended to include
radioactive isotopes (e.g., At.sup.211, I.sup.131, I.sup.125,
Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153, Bi.sup.212, P.sup.32
and radioactive isotopes of Lu), chemotherapeutic agents e.g.
methotrexate, adriamicin, vinca alkaloids (vincristine,
vinblastine, etoposide), doxorubicin, melphalan, mitomycin C,
chlorambucil, daunorubicin or other intercalating agents, enzymes
and fragments thereof such as nucleolytic enzymes, antibiotics, and
toxins such as small molecule toxins or enzymatically active toxins
of bacterial, fungal, plant or animal origin, including fragments
and/or variants thereof, and the various antitumor or anticancer
agents disclosed below. Other cytotoxic agents are described below.
A tumoricidal agent causes destruction of tumor cells.
[0465] Preferred cytotoxic agents herein for the specific tumor
types to use in combination with the antagonists herein are as
follows:
[0466] 1. Prostate cancer: androgens, docetaxel, paclitaxel,
estramustine, doxorubicin, mitoxantrone, antibodies to ErbB2
domain(s) such as 2C4 (WO 01/00245; hybridoma ATCC HB-12697), which
binds to a region in the extracellular domain of ErbB2 (e.g., any
one or more residues in the region from about residue 22 to about
residue 584 of ErbB2, inclusive), AVASTIN.TM. anti-vascular
endothelial growth factor (VEGF), TARCEVA.TM. OSI-774 (erlotinib)
(Genenetech and OSI Pharmaceuticals), or other epidermal growth
factor receptor tyrosine kinase inhibitors (EGFR TKI's).
[0467] 2. Stomach cancer: 5-fluorouracil (5FU), XELODA.TM.
capecitabine, methotrexate, etoposide, cisplatin/carboplatin,
pacliitaxel, docetaxel, gemcitabine, doxorubicin, and CPT-11
(camptothcin-11; irinotecan, USA Brand Name: CAMPTOSAR.RTM.).
[0468] 3. Pancreatic cancer: gemcitabine, 5FU, XELODA.TM.
capecitabine, CPT-11, docetaxel, paclitaxel, cisplatin,
carboplatin, TARCEVA.TM. erlotinib, and other EGFR TKI's.
[0469] 4. Colorectal cancer: 5FU, XELODA.TM. capecitabine, CPT-11,
oxaliplatin, AVASTIN.TM. anti-VEGF, TARCEVA.TM. erlotinib and other
EGFR TKI's, and ERBITUX.TM. (formerly known as IMC-C225)
human:murine-chimerized monoclonal antibody that binds to EGFR and
blocks the ability of EGF to initiate receptor activation and
signaling to the tumor.
[0470] 5. Renal cancer: IL-2, interferon alpha, AVASTIN.TM.
anti-VEGF, MEGACE.TM. (Megestrol acetate) progestin, vinblastine,
TARCEVA.TM. erlotinib, and other EGFR TKI's.
[0471] A "growth inhibitory agent" when used herein refers to a
compound or composition which inhibits growth of a cell, especially
a PRO196-, PRO217-, PRO231-, PRO236-, PRO245-, PRO246-, PRO258-,
PRO287-, PRO328-, PRO344-, PRO357-, PRO526-, PRO724-, PRO731-,
PRO732-, PRO1003-, PRO1104-, PRO1151-, PRO1244-, PRO1298-,
PRO1313-, PRO1570-, PRO1886-, PRO1891-, PRO4409-, PRO5725-,
PRO5994-, PRO6097-, PRO7425-, PRO10102-, PRO10282-, PRO61709- or
PRO779-expressing cancer cell, either in vitro or in vivo. Thus,
the growth inhibitory agent may be one which significantly reduces
the percentage of PRO196-, PRO217-, PRO231-, PRO236-, PRO245-,
PRO246-, PRO258-, PRO287-, PRO328-, PRO344-, PRO357-, PRO526-,
PRO724-, PRO731-, PRO732-, PRO1003-, PRO1104-, PRO1151-, PRO1244-,
PRO1298-, PRO1313-, PRO1570-, PRO1886-, PRO1891-, PRO4409-,
PRO5725-, PRO5994-, PRO6097-, PRO7425-, PRO10102-, PRO10282-,
PRO61709- or PRO779-expressing cells in S phase. Examples of growth
inhibitory agents include agents that block cell cycle progression
(at a place other than S phase), such as agents that induce G1
arrest and M-phase arrest. Classical M-phase blockers include the
vincas (vincristine and vinblastine), taxanes, and topoisomerase II
inhibitors such as doxorubicin, epirubicin, daunorubicin,
etoposide, and bleomycin. Those agents that arrest G1 also spill
over into S-phase arrest, for example, DNA alkylating agents such
as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin,
methotrexate, 5-fluorouracil, and ara-C. Further information can be
found in The Molecular Basis of Cancer, Mendelsohn and Israel,
eds., Chapter 1, entitled "Cell cycle regulation, oncogenes, and
antineoplastic drugs" by Murakami et al. (WB Saunders:
Philadelphia, 1995), especially p. 13. The taxanes (paclitaxel and
docetaxel) are anticancer drugs both derived from the yew tree.
Docetaxel (TAXOTERE.RTM., Rhone-Poulenc Rorer), derived from the
European yew, is a semisynthetic analogue of paclitaxel
(TAXOL.RTM., Bristol-Myers Squibb). Paclitaxel and docetaxel
promote the assembly of microtubules from tubulin dimers and
stabilize microtubules by preventing depolymerization, which
results in the inhibition of mitosis in cells.
[0472] "Doxorubicin" is an anthracycline antibiotic. The full
chemical name of doxorubicin is
(8S-cis)-10-[(3-amino-2,3,6-trideoxy-.alpha.-L-lyxo-hexapyranosyl)oxy]-7,-
8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-napht-
hacenedione
[0473] The term "cytokine" is a generic term for proteins released
by one cell population which act on another cell as intercellular
mediators. Examples of such cytokines are lymphokines, monokines,
and traditional polypeptide hormones. Included among the cytokines
are growth hormone such as human growth hormone, N-methionyl human
growth hormone, and bovine growth hormone; parathyroid hormone;
thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein
hormones such as follicle stimulating hormone (FSH), thyroid
stimulating hormone (TSH), and luteinizing hormone (LH); hepatic
growth factor; fibroblast growth factor; prolactin; placental
lactogen; tumor necrosis factor-.alpha. and -.beta.;
mullerian-inhibiting substance; mouse gonadotropin-associated
peptide; inhibin; activin; vascular endothelial growth factor;
integrin; thrombopoietin (TPO); nerve growth factors such as
NGF-.beta.; platelet-growth factor; transforming growth factors
(TGFs) such as TGF-.alpha. and TGF-.beta.; insulin-like growth
factor-I and -II; erythropoietin (EPO); osteoinductive factors;
interferons such as interferon-.alpha., -.beta., and -.gamma.;
colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);
granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);
interleukins (ILs) such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5,
IL-6, IL-7, IL-8, IL-9, IL-11, IL-12; a tumor necrosis factor such
as TNF-.alpha. or TNF-.beta.; and other polypeptide factors
including LIF and kit ligand (KL). As used herein, the term
cytokine includes proteins from natural sources or from recombinant
cell culture and biologically active equivalents of the native
sequence cytokines.
[0474] The term "package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic
products, that contain information about the indications, usage,
dosage, administration, contraindications and/or warnings
concerning the use of such therapeutic products.
[0475] The term "gene" refers to (a) a gene containing at least one
of the DNA sequences disclosed herein; (b) any DNA sequence that
encodes the amino acid sequence encoded by the DNA sequences
disclosed herein and/or; {circle around (c)}) any DNA sequence that
hybridizes to the complement of the coding sequences disclosed
herein. Preferably, the term includes coding as well as noncoding
regions, and preferably includes all sequences necessary for normal
gene expression.
[0476] The term "gene targeting" refers to a type of homologous
recombination that occurs when a fragment of genomic DNA is
introduced into a mammalian cell and that fragment locates and
recombines with endogenous homologous sequences. Gene targeting by
homologous recombination employs recombinant DNA technologies to
replace specific genomic sequences with exogenous DNA of particular
design.
[0477] The term "homologous recombination" refers to the exchange
of DNA fragments between two DNA molecules or chromatids at the
site of homologous nucleotide sequences.
[0478] The term "target gene" (alternatively referred to as "target
gene sequence" or "target DNA sequence") refers to any nucleic acid
molecule, polynucleotide, or gene to be modified by homologous
recombination. The target sequence includes an intact gene, an exon
or intron, a regulatory sequence or any region between genes. The
target gene my comprise a portion of a particular gene or genetic
locus in the individual's genomic DNA.
[0479] "Disruption" of a PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 gene
occurs when a fragment of genomic DNA locates and recombines with
an endogenous homologous sequence wherein the disruption is a
deletion of the native gene or a portion thereof, or a mutation in
the native gene or wherein the disruption is the functional
inactivation of the native gene. Alternatively, sequence
disruptions may be generated by nonspecific insertional
inactivation using a gene trap vector (i.e. non-human transgenic
animals containing and expressing a randomly inserted transgene;
see for example U.S. Pat. No. 6,436,707 issued Aug. 20, 2002).
These sequence disruptions or modifications may include insertions,
missense, frameshift, deletion, or substitutions, or replacements
of DNA sequence, or any combination thereof. Insertions include the
insertion of entire genes, which may be of animal, plant, fungal,
insect, prokaryotic, or viral origin. Disruption, for example, can
alter the normal gene product by inhibiting its production
partially or completely or by enhancing the normal gene product's
activity. Preferably, the disruption is a null disruption, wherein
there is no significant expression of the PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
gene.
[0480] The term "native expression" refers to the expression of the
full-length polypeptide encoded by the PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
gene, at expression levels present in the wild-type mouse. Thus, a
disruption in which there is "no native expression" of the
endogenous PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 gene refers to a partial or
complete reduction of the expression of at least a portion of a
polypeptide encoded by an endogenous PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
gene of a single cell, selected cells, or all of the cells of a
mammal.
[0481] The term "knockout" refers to the disruption of a PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 gene wherein the disruption results in: the
functional inactivation of the native gene; the deletion of the
native gene or a portion thereof; or a mutation in the native
gene.
[0482] The term "knock-in" refers to the replacement of the mouse
ortholog (or other mouse gene) with a human cDNA encoding any of
the specific human PRO196-, PRO217-, PRO231-, PRO236-, PRO245-,
PRO246-, PRO258-, PRO287-, PRO328-, PRO344-, PRO357-, PRO526-,
PRO724-, PRO731-, PRO732-, PRO1003-, PRO1104-, PRO1151-, PRO1244-,
PRO1298-, PRO1313-, PRO1570-, PRO1886-, PRO1891-, PRO4409-,
PRO5725-, PRO5994-, PRO6097-, PRO7425-, PRO10102-, PRO10282-,
PRO61709- or PRO779-encoding genes or variants thereof (ie. the
disruption results in a replacement of a native mouse gene with a
native human gene).
[0483] The term "construct" or "targeting construct" refers to an
artificially assembled DNA segment to be transferred into a target
tissue, cell line or animal. Typically, the targeting construct
will include a gene or a nucleic acid sequence of particular
interest, a marker gene and appropriate control sequences. As
provided herein, the targeting construct comprises a PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 targeting construct. A "PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
targeting construct" includes a DNA sequence homologous to at least
one portion of a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 gene and is capable
of producing a disruption in a PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
gene in a host cell.
[0484] The term "transgenic cell" refers to a cell containing
within its genome a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 gene that has been
disrupted, modified, altered, or replaced completely or partially
by the method of gene targeting.
[0485] The term "transgenic animal" refers to an animal that
contains within its genome a specific gene that has been disrupted
or otherwise modified or mutated by the methods described herein or
methods otherwise well known in the art. Preferably the non-human
transgenic animal is a mammal. More preferably, the mammal is a
rodent such as a rat or mouse. In addition, a "transgenic animal"
may be a heterozygous animal (i.e., one defective allele and one
wild-type allele) or a homozygous animal (i.e., two defective
alleles). An embryo is considered to fall within the definition of
an animal. The provision of an animal includes the provision of an
embryo or foetus in utero, whether by mating or otherwise, and
whether or not the embryo goes to term.
[0486] As used herein, the terms "selective marker" and position
selection marker" refer to a gene encoding a product that enables
only the cells that carry the gene to survive and/or grow under
certain conditions. For example, plant and animal cells that
express the introduced neomycin resistance (Ned) gene are resistant
to the compound G418. Cells that do not carry the Ned gene marker
are killed by G418. Other positive selection markers are known to,
or are within the purview of, those of ordinary skill in the
art.
[0487] The term "modulates" or "modulation" as used herein refers
to the decrease, inhibition, reduction, amelioration, increase or
enhancement of a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 gene function,
expression, activity, or alternatively a phenotype associated with
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 gene.
[0488] The term "ameliorates" or "amelioration" as used herein
refers to a decrease, reduction or elimination of a condition,
disease, disorder, or phenotype, including an abnormality or
symptom.
[0489] The term "abnormality" refers to any disease, disorder,
condition, or phenotype in which PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
is implicated, including pathological conditions and behavioral
observations.
TABLE-US-00001 TABLE 1 /* * * C-C increased from 12 to 15 * Z is
average of EQ * B is average of ND * match with stop is _M;
stop-stop = 0; J (joker) match = 0 */ #define _M -8 /* value of a
match with a stop */ int _day[26][26] = { /* A B C D E F G H I J K
L M N O P Q R S T U V W X Y Z */ /* A */ { 2, 0,-2, 0, 0,-4,
1,-1,-1, 0,-1,-2,-1, 0,_M, 1, 0,-2, 1, 1, 0, 0,-6, 0,-3, 0}, /* B
*/ { 0, 3,-4, 3, 2,-5, 0, 1,-2, 0, 0,-3,-2, 2,_M,-1, 1, 0, 0, 0,
0,-2,-5, 0,-3, 1}, /* C */ {-2,-4,15,-5,-5,-4,-3,-3,-2,
0,-5,-6,-5,-4,_M,-3,-5,-4, 0,-2, 0,-2,-8, 0, 0,-5}, /* D */ { 0,
3,-5, 4, 3,-6, 1, 1,-2, 0, 0,-4,-3, 2,_M,-1, 2,-1, 0, 0, 0,-2,-7,
0,-4, 2}, /* E */ { 0, 2,-5, 3, 4,-5, 0, 1,-2, 0, 0,-3,-2, 1,_M,-1,
2,-1, 0, 0, 0,-2,-7, 0,-4, 3}, /* F */ {-4,-5,-4,-6,-5, 9,-5,-2, 1,
0,-5, 2, 0,-4,_M,-5,-5,-4,-3,-3, 0,-1, 0, 0, 7,-5}, /* G */ { 1,
0,-3, 1, 0,-5, 5,-2,-3, 0,-2,-4,-3, 0,_M,-1,-1,-3, 1, 0, 0,-1,-7,
0,-5, 0}, /* H */ {-1, 1,-3, 1, 1,-2,-2, 6,-2, 0, 0,-2,-2, 2,_M, 0,
3, 2,-1,-1, 0,-2,-3, 0, 0, 2}, /* I */ {-1,-2,-2,-2,-2, 1,-3,-2, 5,
0,-2, 2, 2,-2,_M,-2,-2,-2,-1, 0, 0, 4,-5, 0,-1,-2}, /* J */ { 0, 0,
0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,_M, 0, 0, 0, 0, 0, 0, 0, 0, 0,
0, 0}, /* K */ {-1, 0,-5, 0, 0,-5,-2, 0,-2, 0, 5,-3, 0, 1,_M,-1, 1,
3, 0, 0, 0,-2,-3, 0,-4, 0}, /* L */ {-2,-3,-6,-4,-3, 2,-4,-2, 2,
0,-3, 6, 4,-3,_M,-3,-2,-3,-3,-1, 0, 2,-2, 0,-1,-2}, /* M */
{-1,-2,-5,-3,-2, 0,-3,-2, 2, 0, 0, 4, 6,-2,_M,-2,-1, 0,-2,-1, 0,
2,-4, 0,-2,-1}, /* N */ { 0, 2,-4, 2, 1,-4, 0, 2,-2, 0, 1,-3,-2,
2,_M,-1, 1, 0, 1, 0, 0,-2,-4, 0,-2, 1}, /* O */
{_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,
0,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M}, /* P */ { 1,-1,-3,-1,-1,-5,-1,
0,-2, 0,-1,-3,-2,-1,_M, 6, 0, 0, 1, 0, 0,-1,-6, 0,-5, 0}, /* Q */ {
0, 1,-5, 2, 2,-5,-1, 3,-2, 0, 1,-2,-1, 1,_M, 0, 4, 1,-1,-1,
0,-2,-5, 0,-4, 3}, /* R */ {-2, 0,-4,-1,-1,-4,-3, 2,-2, 0, 3,-3, 0,
0,_M, 0, 1, 6, 0,-1, 0,-2, 2, 0,-4, 0}, /* S */ { 1, 0, 0, 0, 0,-3,
1,-1,-1, 0, 0,-3,-2, 1,_M, 1,-1, 0, 2, 1, 0,-1,-2, 0,-3, 0}, /* T
*/ { 1, 0,-2, 0, 0,-3, 0,-1, 0, 0, 0,-1,-1, 0,_M, 0,-1,-1, 1, 3, 0,
0,-5, 0,-3, 0}, /* U */ { 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,
0,_M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0}, /* V */ {
0,-2,-2,-2,-2,-1,-1,-2, 4, 0,-2, 2, 2,-2,_M,-1,-2,-2,-1, 0, 0,
4,-6, 0,-2,-2}, /* W */ {-6,-5,-8,-7,-7, 0,-7,-3,-5,
0,-3,-2,-4,-4,_M,-6,-5, 2,-2,-5, 0,-6,17, 0, 0,-6}, /* X */ { 0, 0,
0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,_M, 0, 0, 0, 0, 0, 0, 0, 0, 0,
0, 0}, /* Y */ {-3,-3, 0,-4,-4, 7,-5, 0,-1,
0,-4,-1,-2,-2,_M,-5,-4,-4,-3,-3, 0,-2, 0, 0,10,-4}, /* Z */ { 0,
1,-5, 2, 3,-5, 0, 2,-2, 0, 0,-2,-1, 1,_M, 0, 3, 0, 0, 0, 0,-2,-6,
0,-4, 4} }; /* */ #include <stdio.h> #include <ctype.h>
#define MAXJMP 16 /* max jumps in a diag */ #define MAXGAP 24 /*
don't continue to penalize gaps larger than this */ #define JMPS
1024 /* max jmps in an path */ #define MX 4 /* save if there's at
least MX-1 bases since last jmp */ #define DMAT 3 /* value of
matching bases */ #define DMIS 0 /* penalty for mismatched bases */
#define DINS0 8 /* penalty for a gap */ #define DINS1 1 /* penalty
per base */ #define PINS0 8 /* penalty for a gap */ #define PINS1 4
/* penalty per residue */ struct jmp { short n[MAXJMP]; /* size of
jmp (neg for dely) */ unsigned short x[MAXJMP]; /* base no. of jmp
in seq x */ }; /* limits seq to 2{circumflex over ( )}16 -1 */
struct diag { int score; /* score at last jmp */ long offset; /*
offset of prev block */ short ijmp; /* current jmp index */ struct
jmp jp; /* list of jmps */ }; struct path { int spc; /* number of
leading spaces */ short n[JMPS];/* size of jmp (gap) */ int
x[JMPS];/* loc of jmp (last elem before gap) */ }; char *ofile; /*
output file name */ char *namex[2]; /* seq names: getseqs( ) */
char *prog; /* prog name for err msgs */ char *seqx[2]; /* seqs:
getseqs( ) */ int dmax; /* best diag: nw( ) */ int dmax0; /* final
diag */ int dna; /* set if dna: main( ) */ int endgaps; /* set if
penalizing end gaps */ int gapx, gapy; /* total gaps in seqs */ int
len0, len1; /* seq lens */ int ngapx, ngapy; /* total size of gaps
*/ int smax; /* max score: nw( ) */ int *xbm; /* bitmap for
matching */ long offset; /* current offset in jmp file */ struct
diag *dx; /* holds diagonals */ struct path pp[2]; /* holds path
for seqs */ char *calloc( ), *malloc( ), *index( ), *strcpy( );
char *getseq( ), *g_calloc( ); /* Needleman-Wunsch alignment
program * * usage: progs file1 file2 * where file1 and file2 are
two dna or two protein sequences. * The sequences can be in upper-
or lower-case an may contain ambiguity * Any lines beginning with
`;`, `>` or `<` are ignored * Max file length is 65535
(limited by unsigned short x in the jmp struct) * A sequence with
1/3 or more of its elements ACGTU is assumed to be DNA * Output is
in the file "align.out" * * The program may create a tmp file in
/tmp to hold info about traceback. * Original version developed
under BSD 4.3 on a vax 8650 */ #include "nw.h" #include "day.h"
static _dbval[26] = {
1,14,2,13,0,0,4,11,0,0,12,0,3,15,0,0,0,5,6,8,8,7,9,0,10,0 }; static
_pbval[26] = { 1, 2|(1<<(`D`-`A`))|(1<<(`N`-`A`)), 4,
8, 16, 32, 64, 128, 256, 0xFFFFFFF, 1<<10, 1<<11,
1<<12, 1<<13, 1<<14, 1<<15, 1<<16,
1<<17, 1<<18, 1<<19, 1<<20, 1<<21,
1<<22, 1<<23, 1<<24,
1<<25|(1<<(`E`-`A`))|(1<<(`Q`-`A`)) }; main(ac,
av) main int ac; char *av[ ]; { prog = av[0]; if (ac != 3) {
fprintf(stderr,"usage: %s file1 file2\n", prog);
fprintf(stderr,"where file1 and file2 are two dna or two protein
sequences.\n"); fprintf(stderr,"The sequences can be in upper- or
lower-case\n"); fprintf(stderr,"Any lines beginning with `;` or
`<` are ignored\n"); fprintf(stderr,"Output is in the file
\"align.out\"\n"); exit(1); } namex[0] = av[1]; namex[1] = av[2];
seqx[0] = getseq(namex[0], &len0); seqx[1] = getseq(namex[1],
&len1); xbm = (dna)? _dbval : _pbval; endgaps = 0; /* 1 to
penalize endgaps */ ofile = "align.out"; /* output file */ nw( );
/* fill in the matrix, get the possible jmps */ readjmps( ); /* get
the actual jmps */ print( ); /* print stats, alignment */
cleanup(0); /* unlink any tmp files */} /* do the alignment, return
best score: main( ) * dna: values in Fitch and Smith, PNAS, 80,
1382-1386, 1983 * pro: PAM 250 values * When scores are equal, we
prefer mismatches to any gap, prefer * a new gap to extending an
ongoing gap, and prefer a gap in seqx * to a gap in seq y. */ nw( )
nw { char *px, *py; /* seqs and ptrs */ int *ndely, *dely; /* keep
track of dely */ int ndelx, delx; /* keep track of delx */ int
*tmp; /* for swapping row( ), row1 */ int mis; /* score for each
type */ int ins0, ins1; /* insertion penalties */ register id; /*
diagonal index */ register ij; /* jmp index */ register *col0,
*col1; /* score for curr, last row */ register xx, yy; /* index
into seqs */ dx = (struct diag *)g_calloc("to get diags",
len0+len1+1, sizeof(struct diag)); ndely = (int *)g_calloc("to get
ndely", len1+1, sizeof(int)); dely = (int *)g_calloc("to get dely",
len1+1, sizeof(int)); col0 = (int *)g_calloc("to get col0", len1+1,
sizeof(int)); col1 = (int *)g_calloc("to get col1", len1+1,
sizeof(int)); ins0 = (dna)? DINS0 : PINS0; ins1 = (dna)? DINS1 :
PINS1; smax = -10000; if (endgaps) { for (col0[0] = dely[0] =
-ins0, yy = 1; yy <= len1; yy++) { col0[yy] = dely[yy] =
col0[yy-1] - ins1; ndely[yy] = yy; } col0[0] = 0; /* Waterman Bull
Math Biol 84 */ } else for (yy = 1; yy <= len1; yy++) dely[yy] =
-ins0; /* fill in match matrix */ for (px = seqx[0], xx = 1; xx
<= len0; px++, xx++) { /* initialize first entry in col */ if
(endgaps) { if (xx == 1) col1[0] = delx = -(ins0+ins1); else
col1[0] = delx = col0[0] - ins1; ndelx = xx; } else { col1[0] = 0;
delx = -ins0; ndelx = 0; } ...nw for (py = seqx[1], yy = 1; yy
<= len1; py++, yy++) { mis = col0[yy-1]; if (dna) mis +=
(xbm[*px-`A`]&xbm[*py-`A`])? DMAT : DMIS; else mis +=
_day[*px-`A`][*py-`A`]; /* update penalty for del in x seq; * favor
new del over ongong del * ignore MAXGAP if weighting endgaps */ if
(endgaps || ndely[yy] < MAXGAP) { if (col0[yy] - ins0 >=
dely[yy]) { dely[yy] = col0[yy] - (ins0+ins1); ndely[yy] = 1; }
else { dely[yy] -= ins1; ndely[yy]++; } } else { if (col0[yy] -
(ins0+ins1) >= dely[yy]) { dely[yy] = col0[yy] - (ins0+ins1);
ndely[yy] = 1; } else ndely[yy]++; } /* update penalty for del in y
seq; * favor new del over ongong del
*/ if (endgaps || ndelx < MAXGAP) { if (col1[yy-1] - ins0 >=
delx) { delx = col1[yy-1] - (ins0+ins1); ndelx = 1; } else { delx
-= ins1; ndelx++; } } else { if (col1[yy-1] - (ins0+ins1) >=
delx) { delx = col1[yy-1] - (ins0+ins1); ndelx = 1; } else ndelx++;
} /* pick the maximum score; we're favoring * mis over any del and
delx over dely */ ...nw id = xx - yy + len1 - 1; if (mis >= delx
&& mis >= dely[yy]) col1[yy] = mis; else if (delx >=
dely[yy]) { col1[yy] = delx; ij = dx[id].ijmp; if (dx[id].jp.n[0]
&& (!dna || (ndelx >= MAXJMP && xx >
dx[id].jp.x[ij]+MX) || mis > dx[id].score+DINS0)) {
dx[id].ijmp++; if (++ij >= MAXJMP) { writejmps(id); ij =
dx[id].ijmp = 0; dx[id].offset = offset; offset += sizeof(struct
jmp) + sizeof(offset); } } dx[id].jp.n[ij] = ndelx; dx[id].jp.x[ij]
= xx; dx[id].score = delx; } else { col1[yy] = dely[yy]; ij =
dx[id].ijmp; if (dx[id].jp.n[0] && (!dna || (ndely[yy]
>= MAXJMP && xx > dx[id].jp.x[ij]+MX) || mis >
dx[id].score+DINS0)) { dx[id].ijmp++; if (++ij >= MAXJMP) {
writejmps(id); ij = dx[id].ijmp = 0; dx[id].offset = offset; offset
+= sizeof(struct jmp) + sizeof(offset); } } dx[id].jp.n[ij] =
-ndely[yy]; dx[id].jp.x[ij] = xx; dx[id].score = dely[yy]; } if (xx
== len0 && yy < len1) { /* last col */ if (endgaps)
col1[yy] -= ins0+ins1*(len1-yy); if (col1[yy] > smax) { smax =
col1[yy]; dmax = id; } } } if (endgaps && xx < len0)
col1[yy-1] -= ins0+ins1*(len0-xx); if (col1[yy-1] > smax) { smax
= col1[yy-1]; dmax = id; } tmp = col0; col0 = col1; col1 = tmp; }
(void) free((char *)ndely); (void) free((char *)dely); (void)
free((char *)col0); (void) free((char *)col1); } /* * * print( ) --
only routine visible outside this module * * static: * getmat( ) --
trace back best path, count matches: print( ) * pr_align( ) --
print alignment of described in array p[ ]: print( ) * dumpblock( )
-- dump a block of lines with numbers, stars: pr_align( ) * nums( )
-- put out a number line: dumpblock( ) * putline( ) -- put out a
line (name, [num], seq, [num]): dumpblock( ) * stars( ) - -put a
line of stars: dumpblock( ) * stripname( ) -- strip any path and
prefix from a seqname */ #include "nw.h" #define SPC 3 #define
P_LINE 256 /* maximum output line */ #define P_SPC 3 /* space
between name or num and seq */ extern _day[26][26]; int olen; /*
set output line length */ FILE *fx; /* output file */ print( )
print { int lx, ly, firstgap, lastgap; /* overlap */ if ((fx =
fopen(ofile, "w")) == 0) { fprintf(stderr,"%s: can't write %s\n",
prog, ofile); cleanup(1); } fprintf(fx, "<first sequence: %s
(length = %d)\n", namex[0], len0); fprintf(fx, "<second
sequence: %s (length = %d)\n", namex[1], len1); olen = 60; lx =
len0; ly = len1; firstgap = lastgap = 0; if (dmax < len1 - 1) {
/* leading gap in x */ pp[0].spc = firstgap = len1 - dmax - 1; ly
-= pp[0].spc; } else if (dmax > len1 - 1) { /* leading gap in y
*/ pp[1].spc = firstgap = dmax - (len1 - 1); lx -= pp[1].spc; } if
(dmax0 < len0 - 1) { /* trailing gap in x */ lastgap = len0 -
dmax0 -1; lx -= lastgap; } else if (dmax0 > len0 - 1) { /*
trailing gap in y */ lastgap = dmax0 - (len0 - 1); ly -= lastgap; }
getmat(lx, ly, firstgap, lastgap); pr_align( ); } /* * trace back
the best path, count matches */ static getmat(lx, ly, firstgap,
lastgap) getmat int lx, ly; /* "core" (minus endgaps) */ int
firstgap, lastgap; /* leading trailing overlap */ { int nm, i0, i1,
siz0, siz1; char outx[32]; double pct; register n0, n1; register
char *p0, *p1; /* get total matches, score */ i0 = i1 = siz0 = siz1
= 0; p0 = seqx[0] + pp[1].spc; p1 = seqx[1] + pp[0].spc; n0 =
pp[1].spc + 1; n1 = pp[0].spc + 1; nm = 0; while ( *p0 &&
*p1 ) { if (siz0) { p1++; n1++; siz0--; } else if (siz1) { p0++;
n0++; siz1--; } else { if (xbm[*p0-`A`]&xbm[*p1-`A`]) nm++; if
(n0++ == pp[0].x[i0]) siz0 = pp[0].n[i0++]; if (n1++ ==
pp[1].x[i1]) siz1 = pp[1].n[i1++]; p0++; p1++; } } /* pct homology:
* if penalizing endgaps, base is the shorter seq * else, knock off
overhangs and take shorter core */ if (endgaps) lx = (len0 <
len1)? len0 : len1; else lx = (lx < ly)? lx : ly; pct =
100.*(double)nm/(double)lx; fprintf(fx, "\n"); fprintf(fx, "<%d
match%s in an overlap of %d: %.2f percent similarity\n", nm, (nm ==
1)? "" : "es", lx, pct); fprintf(fx, "<gaps in first sequence:
%d", gapx); ...getmat if (gapx) { (void) sprintf(outx, " (%d
%s%s)", ngapx, (dna)? "base":"residue", (ngapx == 1)? "":"s");
fprintf(fx,"%s", outx); fprintf(fx, ", gaps in second sequence:
%d", gapy); if (gapy) { (void) sprintf(outx, " (%d %s%s)", ngapy,
(dna)? "base":"residue", (ngapy == 1)? "":"s"); fprintf(fx,"%s",
outx); } if (dna) fprintf(fx, "\n<score: %d (match = %d,
mismatch = %d, gap penalty = %d + %d per base)\n", smax, DMAT,
DMIS, DINS0, DINS1); else fprintf(fx, "\n<score: %d (Dayhoff PAM
250 matrix, gap penalty = %d + %d per residue)\n", smax, PINS0,
PINS1); if (endgaps) fprintf(fx, "<endgaps penalized. left
endgap: %d %s%s, right endgap: %d %s%s\n", firstgap, (dna)? "base"
: "residue", (firstgap == 1)? "" : "s", lastgap, (dna)? "base" :
"residue", (lastgap == 1)? "" : "s"); else fprintf(fx, "<endgaps
not penalized\n"); } static nm; /* matches in core -- for checking
*/ static lmax; /* lengths of stripped file names */ static ij[2];
/* jmp index for a path */ static nc[2]; /* number at start of
current line */ static ni[2]; /* current elem number -- for gapping
*/ static siz[2]; static char *ps[2]; /* ptr to current element */
static char *po[2]; /* ptr to next output char slot */ static char
out[2][P_LINE]; /* output line */ static char star[P_LINE]; /* set
by stars( ) */ /* * print alignment of described in struct path pp[
] */ static pr_align( ) pr_align { int nn; /* char count */ int
more; register I; for (I = 0, lmax = 0; I < 2; I++) { nn =
stripname(namex[i]); if (nn > lmax) lmax = nn; nc[i] = 1; ni[i]
= 1; siz[i] = ij[i] = 0; ps[i] = seqx[i]; po[i] = out[i]; } for (nn
= nm = 0, more = 1; more; ) { ...pr_align for (I = more = 0; I <
2; I++) { /* * do we have more of this sequence? */ if (!*ps[i])
continue; more++;
if (pp[i].spc) { /* leading space */ *po[i]++ = ` `; pp[i].spc--; }
else if (siz[i]) { /* in a gap */ *po[i]++ = `-`; siz[i]--; } else
{ /* we're putting a seq element */ *po[i] = *ps[i]; if
(islower(*ps[i])) *ps[i] = toupper(*ps[i]); po[i]++; ps[i]++; /* *
are we at next gap for this seq? */ if (ni[i] == pp[i].x[ij[i]]) {
/* * we need to merge all gaps * at this location */ siz[i] =
pp[i].n[ij[i]++]; while (ni[i] == pp[i].x[ij[i]]) siz[i] +=
pp[i].n[ij[i]++]; } ni[i]++; } } if (++nn == olen || !more
&& nn) { dumpblock( ); for (I = 0; I < 2; I++) po[i] =
out[i]; nn = 0; } } } /* * dump a block of lines, including
numbers, stars: pr_align( ) */ static dumpblock( ) dumpblock {
register I; for (I = 0; I < 2; I++) *po[i]-- = `\0`;
...dumpblock (void) putc(`\n`, fx); for (I = 0; I < 2; I++) { if
(*out[i] && (*out[i] != ` ` || *(po[i]) != ` `)) { if (I ==
0) nums(I); if (I == 0 && *out[1]) stars( ); putline(I); if
(I == 0 && *out[1]) fprintf(fx, star); if (I == 1) nums(I);
} } } /* * put out a number line: dumpblock( ) */ static nums(ix)
nums int ix; /* index in out[ ] holding seq line */ { char
nline[P_LINE]; register I, j; register char *pn, *px, *py; for (pn
= nline, I = 0; I < lmax+P_SPC; I++, pn++) *pn = ` `; for (I =
nc[ix], py = out[ix]; *py; py++, pn++) { if (*py == ` ` || *py ==
`-`) *pn = ` `; else { if (I%10 == 0 || (I == 1 && nc[ix]
!= 1)) { j = (I < 0)? -I : I; for (px = pn; j; j /= 10, px--)
*px = j%10 + `0`; if (I < 0) *px = `-`; } else *pn = ` `; I++; }
} *pn = `\0`; nc[ix] = I; for (pn = nline; *pn; pn++) (void)
putc(*pn, fx); (void) putc(`\n`, fx); } /* * put out a line (name,
[num], seq, [num]): dumpblock( ) */ static putline(ix) putline int
ix; { ...putline int I; register char *px; for (px = namex[ix], I =
0; *px && *px != `:`; px++, I++) (void) putc(*px, fx); for
(; I < lmax+P_SPC; I++) (void) putc(` `, fx); /* these count
from 1: * ni[ ] is current element (from 1) * nc[ ] is number at
start of current line */ for (px = out[ix]; *px; px++) (void)
putc(*px&0x7F, fx); (void) putc(`\n`, fx); } /* * put a line of
stars (seqs always in out[0], out[1]): dumpblock( ) */ static
stars( ) stars { int I; register char *p0, *p1, cx, *px; if
(!*out[0] || (*out[0] == ` ` && *(po[0]) == ` `) ||
!*out[1] || (*out[1] == ` ` && *(po[1]) == ` `)) return; px
= star; for (I = lmax+P_SPC; I; I--) *px++ = ` `; for (p0 = out[0],
p1 = out[1]; *p0 && *p1; p0++, p1++) { if (isalpha(*p0)
&& isalpha(*p1)) { if (xbm[*p0-`A`]&xbm[*p1-`A`]) { cx
= `*`; nm++; } else if (!dna && _day[*p0-`A`][*p1-`A`] >
0) cx = `.`; else cx = ` `; } else cx = ` `; *px++ = cx; } *px++ =
`\n`; *px = `\0`; } /* * strip path or prefix from pn, return len:
pr_align( ) */ static stripname(pn) stripname char *pn; /* file
name (may be path) */ { register char *px, *py; py = 0; for (px =
pn; *px; px++) if (*px == `/`) py = px + 1; if (py) (void)
strcpy(pn, py); return(strlen(pn)); } /* * cleanup( ) -- cleanup
any tmp file * getseq( ) -- read in seq, set dna, len, maxlen *
g_calloc( ) -- calloc( ) with error checkin * readjmps( ) -- get
the good jmps, from tmp file if necessary * writejmps( ) -- write a
filled array of jmps to a tmp file: nw( ) */ #include "nw.h"
#include <sys/file.h> char *jname = "/tmp/homgXXXXXX"; /* tmp
file for jmps */ FILE *fj; int cleanup( ); /* cleanup tmp file */
long lseek( ); /* * remove any tmp file if we blow */ cleanup(I)
cleanup int I; { if (fj) (void) unlink(jname); exit(I); } /* *
read, return ptr to seq, set dna, len, maxlen * skip lines starting
with `;`, `<`, or `>` * seq in upper or lower case */ char *
getseq(file, len) getseq char *file; /* file name */ int *len; /*
seq len */ { char line[1024], *pseq; register char *px, *py; int
natgc, tlen; FILE *fp; if ((fp = fopen(file,"r")) == 0) {
fprintf(stderr,"%s: can't read %s\n", prog, file); exit(1); } tlen
= natgc = 0; while (fgets(line, 1024, fp)) { if (*line == `;` ||
*line == `<` || *line == `>`) continue; for (px = line; *px
!= `\n`; px++) if (isupper(*px) || islower(*px)) tlen++; } if
((pseq = malloc((unsigned)(tlen+6))) == 0) { fprintf(stderr,"%s:
malloc( ) failed to get %d bytes for %s\n", prog, tlen+6, file);
exit(1); } pseq[0] = pseq[1] = pseq[2] = pseq[3] = `\0`; ...getseq
py = pseq + 4; *len = tlen; rewind(fp); while (fgets(line, 1024,
fp)) { if (*line == `;` || *line == `<` || *line == `>`)
continue; for (px = line; *px != `\n`; px++) { if (isupper(*px))
*py++ = *px; else if (islower(*px)) *py++ = toupper(*px); if
(index("ATGCU",*(py-1))) natgc++; } } *py++ = `\0`; *py = `\0`;
(void) fclose(fp); dna = natgc > (tlen/3); return(pseq+4); }
char * g_calloc(msg, nx, sz) g_calloc char *msg; /* program,
calling routine */ int nx, sz; /* number and size of elements */ {
char *px, *calloc( ); if ((px = calloc((unsigned)nx, (unsigned)sz))
== 0) { if (*msg) {
fprintf(stderr, "%s: g_calloc( ) failed %s (n=%d, sz=%d)\n", prog,
msg, nx, sz); exit(1); } } return(px); } /* * get final jmps from
dx[ ] or tmp file, set pp[ ], reset dmax: main( ) */ readjmps( )
readjmps { int fd = -1; int siz, i0, i1; register I, j, xx; if (fj)
{ (void) fclose(fj); if ((fd = open(jname, O_RDONLY, 0)) < 0) {
fprintf(stderr, "%s: can't open( ) %s\n", prog, jname); cleanup(1);
} } for (I = i0 = i1 = 0, dmax0 = dmax, xx = len0; ; I++) { while
(1) { for (j = dx[dmax].ijmp; j >= 0 && dx[dmax].jp.x[j]
>= xx; j--) ; ...readjmps if (j < 0 &&
dx[dmax].offset && fj) { (void) lseek(fd, dx[dmax].offset,
0); (void) read(fd, (char *)&dx[dmax].jp, sizeof(struct jmp));
(void) read(fd, (char *)&dx[dmax].offset,
sizeof(dx[dmax].offset)); dx[dmax].ijmp = MAXJMP-1; } else break; }
if (I >= JMPS) { fprintf(stderr, "%s: too many gaps in
alignment\n", prog); cleanup(1); } if (j >= 0) { siz =
dx[dmax].jp.n[j]; xx = dx[dmax].jp.x[j]; dmax += siz; if (siz <
0) { /* gap in second seq */ pp[1].n[i1] = -siz; xx += siz; /* id =
xx - yy + len1 - 1 */ pp[1].x[i1] = xx - dmax + len1 - 1; gapy++;
ngapy -= siz; /* ignore MAXGAP when doing endgaps */ siz = (-siz
< MAXGAP || endgaps)? -siz : MAXGAP; i1++; } else if (siz >
0) { /* gap in first seq */ pp[0].n[i0] = siz; pp[0].x[i0] = xx;
gapx++; ngapx += siz; /* ignore MAXGAP when doing endgaps */ siz =
(siz < MAXGAP || endgaps)? siz : MAXGAP; i0++; } } else break; }
/* reverse the order of jmps */ for (j = 0, i0--; j < i0; j++,
i0--) { I = pp[0].n[j]; pp[0].n[j] = pp[0].n[i0]; pp[0].n[i0] = I;
I = pp[0].x[j]; pp[0].x[j] = pp[0].x[i0]; pp[0].x[i0] = I; } for (j
= 0, i1--; j < i1; j++, i1--) { I = pp[1].n[j]; pp[1].n[j] =
pp[1].n[i1]; pp[1].n[i1] = I; I = pp[1].x[j]; pp[1].x[j] =
pp[1].x[i1]; pp[1].x[i1] = I; } if (fd >= 0) (void) close(fd);
if (fj) { (void) unlink(jname); fj = 0; offset = 0; } } /* * write
a filled jmp struct offset of the prev one (if any): nw( ) */
writejmps(ix) writejmps int ix; { char *mktemp( ); if (!fj) { if
(mktemp(jname) < 0) { fprintf(stderr, "%s: can't mktemp( )
%s\n", prog, jname); cleanup(1); } if ((fj = fopen(jname, "w")) ==
0) { fprintf(stderr, "%s: can't write %s\n", prog, jname); exit(1);
} } (void) fwrite((char *)&dx[ix].jp, sizeof(struct jmp), 1,
fj); (void) fwrite((char *)&dx[ix].offset,
sizeof(dx[ix].offset), 1, fj); }
TABLE-US-00002 TABLE 2 PRO XXXXXXXXXXXXXXX (Length = 15 amino
acids) Comparison XXXXXYYYYYYY (Length = 12 amino acids) Protein %
amino acid sequence identity = (the number of identically matching
amino acid residues between the two polypeptide sequences as
determined by ALIGN-2) divided by (the total number of amino acid
residues of the PRO polypeptide) = 5 divided by 15 = 33.3%
TABLE-US-00003 TABLE 3 PRO XXXXXXXXXX (Length = 10 amino acids)
Comparison XXXXXYYYYYYZZYZ (Length = 15 amino acids) Protein %
amino acid sequence identity = (the number of identically matching
amino acid residues between the two polypeptide sequences as
determined by ALIGN-2) divided by (the total number of amino acid
residues of the PRO polypeptide) = 5 divided by 10 = 50%
TABLE-US-00004 TABLE 4 PRO-DNA NNNNNNNNNNNNNN (Length = 14
nucleotides) Comparison NNNNNNLLLLLLLLLL (Length = 16 nucleotides)
DNA % nucleic acid sequence identity = (the number of identically
matching nucleotides between the two nucleic acid sequences as
determined by ALIGN-2) divided by (the total number of nucleotides
of the PRO-DNA nucleic acid sequence) = 6 divided by 14 = 42.9%
TABLE-US-00005 TABLE 5 PRO-DNA NNNNNNNNNNNN (Length = 12
nucleotides) Comparison DNA NNNNLLLVV (Length = 9 nucleotides) %
nucleic acid sequence identity = (the number of identically
matching nucleotides between the two nucleic acid sequences as
determined by ALIGN-2) divided by (the total number of nucleotides
of the PRO-DNA nucleic acid sequence) = 4 divided by 12 = 33.3%
II. Compositions and Methods of the Invention
[0490] A. Full-Length PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
Polypeptides
[0491] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptides. In particular, cDNAs encoding various PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 polypeptides have been identified and isolated, as disclosed
in further detail in the Examples below. It is noted that proteins
produced in separate expression rounds may be given different PRO
numbers but the UNQ number is unique for any given DNA and the
encoded protein, and will not be changed. However, for sake of
simplicity, in the present specification the protein encoded by the
full length native nucleic acid molecules disclosed herein as well
as all further native homologues and variants included in the
foregoing definition of PRO, will be referred to as "PRO/number",
regardless of their origin or mode of preparation.
[0492] As disclosed in the Examples below, various cDNA clones have
been deposited with the ATCC. The actual nucleotide sequences of
those clones can readily be determined by the skilled artisan by
sequencing of the deposited clone using routine methods in the art.
The predicted amino acid sequence can be determined from the
nucleotide sequence using routine skill. For the PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 polypeptides and encoding nucleic acids described herein,
Applicants have identified what is believed to be the reading frame
best identifiable with the sequence information available at the
time.
[0493] B. PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 Polypeptide Variants
[0494] In addition to the full-length native sequence PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptides described herein, it is
contemplated that PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 variants can be
prepared. PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 variants can be prepared by
introducing appropriate nucleotide changes into the PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 DNA, and/or by synthesis of the desired PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 polypeptide. Those skilled in the art will appreciate that
amino acid changes may alter post-translational processes of the
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide, such as changing the
number or position of glycosylation sites or altering the membrane
anchoring characteristics.
[0495] Variations in the native full-length sequence PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide or in various domains of the PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide described herein, can be made, for
example, using any of the techniques and guidelines for
conservative and non-conservative mutations set forth, for
instance, in U.S. Pat. No. 5,364,934. Variations may be a
substitution, deletion or insertion of one or more codons encoding
the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide that results in a change
in the amino acid sequence of the PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide as compared with the native sequence PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 polypeptide. Optionally the variation is by substitution of
at least one amino acid with any other amino acid in one or more of
the domains of the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide.
Guidance in determining which amino acid residue may be inserted,
substituted or deleted without adversely affecting the desired
activity may be found by comparing the sequence of the PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide with that of homologous known
protein molecules and minimizing the number of amino acid sequence
changes made in regions of high homology. Amino acid substitutions
can be the result of replacing one amino acid with another amino
acid having similar structural and/or chemical properties, such as
the replacement of a leucine with a serine, i.e., conservative
amino acid replacements. Insertions or deletions may optionally be
in the range of about 1 to 5 amino acids. The variation allowed may
be determined by systematically making insertions, deletions or
substitutions of amino acids in the sequence and testing the
resulting variants for activity exhibited by the full-length or
mature native sequence.
[0496] PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 polypeptide fragments are
provided herein. Such fragments may be truncated at the N-terminus
or C-terminus, or may lack internal residues, for example, when
compared with a full length native protein. Certain fragments lack
amino acid residues that are not essential for a desired biological
activity of the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide.
[0497] PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 fragments may be prepared by
any of a number of conventional techniques. Desired peptide
fragments may be chemically synthesized. An alternative approach
involves generating PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 fragments by
enzymatic digestion, e.g., by treating the protein with an enzyme
known to cleave proteins at sites defined by particular amino acid
residues, or by digesting the DNA with suitable restriction enzymes
and isolating the desired fragment. Yet another suitable technique
involves isolating and amplifying a DNA fragment encoding a desired
polypeptide fragment, by polymerase chain reaction (PCR).
Oligonucleotides that define the desired termini of the DNA
fragment are employed at the 5' and 3' primers in the PCR.
Preferably, PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 polypeptide fragments share
at least one biological and/or immunological activity with the
native PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 polypeptide disclosed
herein.
[0498] Conservative substitutions of interest are shown in Table 6
under the heading of preferred substitutions. If such substitutions
result in a change in biological activity, then more substantial
changes, denominated exemplary substitutions in Table 6, or as
further described below in reference to amino acid classes, are
preferably introduced and the products screened.
TABLE-US-00006 TABLE 6 Original Exemplary Preferred Residue
Substitutions Substitutions Ala (A) Val; Leu; Ile Val Arg .RTM.)
Lys; Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu;
Asn Glu Cys .COPYRGT.) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E)
Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I)
Leu; Val; Met; Ala; Leu Phe; Norleucine Leu (L) Norleucine; Ile;
Val; Ile Met; Ala; Phe Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;
Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala
Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y)
Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Leu Ala;
Norleucine
[0499] Substantial modifications in function or immunological
identity of the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide are
accomplished by selecting substitutions that differ significantly
in their effect on maintaining (a) the structure of the polypeptide
backbone in the area of the substitution, for example, as a sheet
or helical conformation, (b) the charge or hydrophobicity of the
molecule at the target site, or (c) the bulk of the side chain.
Naturally occurring residues are divided into groups based on
common side-chain properties:
Amino acids may be grouped according to similarities in the
properties of their side chains (in A. L. Lehninger, in
Biochemistry, second ed., pp. 73-75, Worth Publishers, New York
(1975)): (1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro
(P), Phe (F), Trp (W), Met (M) (2) uncharged polar: Gly (G), Ser
(S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q) (3) acidic: Asp
(D), Glu (E) (4) basic: Lys (K), Arg (R), His (H) Alternatively,
naturally occurring residues may be divided into groups based on
common side-chain properties: (1) hydrophobic: Norleucine, Met,
Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn,
Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues
that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr,
Phe.
[0500] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class. Such substituted
residues also may be introduced into the conservative substitution
sites or, more preferably, into the remaining (non-conserved)
sites.
[0501] The variations can be made using methods known in the art
such as oligonucleotide-mediated (site-directed) mutagenesis,
alanine scanning, and PCR mutagenesis. Site-directed mutagenesis
[Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al.,
Nucl. Acids Res., 10:6487 (1987)], cassette mutagenesis [Wells et
al., Gene, 34:315 (1985)], restriction selection mutagenesis [Wells
et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)] or
other known techniques can be performed on the cloned DNA to
produce the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 variant DNA.
[0502] Scanning amino acid analysis can also be employed to
identify one or more amino acids along a contiguous sequence. Among
the preferred scanning amino acids are relatively small, neutral
amino acids. Such amino acids include alanine, glycine, serine, and
cysteine. Alanine is typically a preferred scanning amino acid
among this group because it eliminates the side-chain beyond the
beta-carbon and is less likely to alter the main-chain conformation
of the variant [Cunningham and Wells, Science, 244: 1081-1085
(1989)]. Alanine is also typically preferred because it is the most
common amino acid. Further, it is frequently found in both buried
and exposed positions [Creighton, The Proteins, (W.H. Freeman &
Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)]. If alanine
substitution does not yield adequate amounts of variant, an
isoteric amino acid can be used.
[0503] C. Modifications of PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287 PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
Polypeptides
[0504] Covalent modifications of PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptides are included within the scope of this invention. One
type of covalent modification includes reacting targeted amino acid
residues of a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide with an
organic derivatizing agent that is capable of reacting with
selected side chains or the N- or C-terminal residues of the
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide. Derivatization with
bifunctional agents is useful, for instance, for crosslinking
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptides to a water-insoluble
support matrix or surface for use in the method for purifying
anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245,
anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344,
anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732,
anti-PRO1003, anti-PRO1104, anti-PRO1151, anti-PRO1244,
anti-PRO1298, anti-PRO1313, anti-PRO1570, anti-PRO1886,
anti-PRO1891, anti-PRO4409, anti-PRO5725, anti-PRO5994,
anti-PRO6097, anti-PRO7425, anti-PRO10102, anti-PRO10282,
anti-PRO61709 or anti-PRO779 antibodies, and vice-versa. Commonly
used crosslinking agents include, e.g.,
1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,
N-hydroxysuccinimide esters, for example, esters with
4-azidosalicylic acid, homobifunctional imidoesters, including
disuccinimidyl esters such as
3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides
such as bis-N-maleimido-1,8-octane and agents such as
methyl-3-[(p-azidophenyl)dithio]propioimidate.
[0505] Other modifications include deamidation of glutaminyl and
asparaginyl residues to the corresponding glutamyl and aspartyl
residues, respectively, hydroxylation of proline and lysine,
phosphorylation of hydroxyl groups of seryl or threonyl residues,
methylation of the .alpha.-amino groups of lysine, arginine, and
histidine side chains [T. E. Creighton, Proteins: Structure and
Molecular Properties, W.H. Freeman & Co., San Francisco, pp.
79-86 (1983)], acetylation of the N-terminal amine, and amidation
of any C-terminal carboxyl group.
[0506] Another type of covalent modification of the PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 polypeptide included within the scope of this invention
comprises altering the native glycosylation pattern of the
polypeptide. "Altering the native glycosylation pattern" is
intended for purposes herein to mean deleting one or more
carbohydrate moieties found in native sequence PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 polypeptides (either by removing the underlying
glycosylation site or by deleting the glycosylation by chemical
and/or enzymatic means), and/or adding one or more glycosylation
sites that are not present in the native sequence PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 polypeptide. In addition, the phrase includes qualitative
changes in the glycosylation of the native proteins, involving a
change in the nature and proportions of the various carbohydrate
moieties present.
[0507] Addition of glycosylation sites to the PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 polypeptide may be accomplished by altering the amino acid
sequence. The alteration may be made, for example, by the addition
of, or substitution by, one or more serine or threonine residues to
the native sequence PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 (for O-linked
glycosylation sites). The PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 amino acid
sequence may optionally be altered through changes at the DNA
level, particularly by mutating the DNA encoding the PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide at preselected bases such that
codons are generated that will translate into the desired amino
acids.
[0508] Another means of increasing the number of carbohydrate
moieties on the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide is by
chemical or enzymatic coupling of glycosides to the polypeptide.
Such methods are described in the art, e.g., in WO 87/05330
published 11 Sep. 1987, and in Aplin and Wriston, CRC Crit. Rev.
Biochem., pp. 259-306 (1981).
[0509] Removal of carbohydrate moieties present on the PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide may be accomplished chemically or
enzymatically or by mutational substitution of codons encoding for
amino acid residues that serve as targets for glycosylation.
Chemical deglycosylation techniques are known in the art and
described, for instance, by Hakimuddin, et al., Arch. Biochem.
Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem., 118:131
(1981). Enzymatic cleavage of carbohydrate moieties on polypeptides
can be achieved by the use of a variety of endo- and
exo-glycosidases as described by Thotakura et al., Meth. Enzymol.,
138:350 (1987).
[0510] Another type of covalent modification of PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 polypeptides comprises linking the PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide to one of a variety of nonproteinaceous polymers, e.g.,
polyethylene glycol (PEG), polypropylene glycol, or
polyoxyalkylenes, in the manner set forth in U.S. Pat. No.
4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or
4,179,337.
[0511] The PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 polypeptides of the present
invention may also be modified in a way to form a chimeric molecule
comprising the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide fused
to another, heterologous polypeptide or amino acid sequence.
[0512] Such a chimeric molecule comprises a fusion of the PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide with a tag polypeptide which
provides an epitope to which an anti-tag antibody can selectively
bind. The epitope tag is generally placed at the amino- or
carboxyl-terminus of the PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide. The presence of such epitope-tagged forms of the
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide can be detected using an
antibody against the tag polypeptide. Also, provision of the
epitope tag enables the PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide to be readily purified by affinity purification using
an anti-tag antibody or another type of affinity matrix that binds
to the epitope tag. Various tag polypeptides and their respective
antibodies are well known in the art. Examples include
poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly)
tags; the flu HA tag polypeptide and its antibody 12CA5 [Field et
al., Mol. Cell. Biol., 8:2159-2165 (1988)]; the c-myc tag and the
8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al.,
Molecular and Cellular Biology, 5:3610-3616 (1985)]; and the Herpes
Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et
al., Protein Engineering, 3(6):547-553 (1990)]. Other tag
polypeptides include the Flag-peptide [Hopp et al., BioTechnology,
6:1204-1210 (1988)]; the KT3 epitope peptide [Martin et al.,
Science, 255:192-194 (1992)]; an .alpha.-tubulin epitope peptide
[Skinner et al., J. Biol. Chem., 266:15163-15166 (1991)]; and the
T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl.
Acad. Sci. USA, 87:6393-6397 (1990)].
[0513] The chimeric molecule may comprise a fusion of the PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide with an immunoglobulin or a
particular region of an immunoglobulin. For a bivalent form of the
chimeric molecule (also referred to as an "immunoadhesin"), such a
fusion could be to the Fc region of an IgG molecule. The Ig fusions
preferably include the substitution of a soluble (transmembrane
domain deleted or inactivated) form of a PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide in place of at least one variable region within an Ig
molecule. In a particularly preferred aspect of the invention, the
immunoglobulin fusion includes the hinge, CH2 and CH3, or the
hinge, CH1, CH2 and CH3 regions of an IgG1 molecule. For the
production of immunoglobulin fusions see also U.S. Pat. No.
5,428,130 issued Jun. 27, 1995.
[0514] D. Preparation of PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
Polypeptides
[0515] The description below relates primarily to production of
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptides by culturing cells
transformed or transfected with a vector containing PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 nucleic acid. It is, of course, contemplated that
alternative methods, which are well known in the art, may be
employed to prepare PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptides. For
instance, the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 sequence, or
portions thereof, may be produced by direct peptide synthesis using
solid-phase techniques [see, e.g., Stewart et al., Solid-Phase
Peptide Synthesis, W.H. Freeman Co., San Francisco, Calif. (1969);
Merrifield, J. Am. Chem. Soc., 85:2149-2154 (1963)]. In vitro
protein synthesis may be performed using manual techniques or by
automation. Automated synthesis may be accomplished, for instance,
using an Applied Biosystems Peptide Synthesizer (Foster City,
Calif.) using manufacturer's instructions. Various portions of the
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide may be chemically
synthesized separately and combined using chemical or enzymatic
methods to produce the full-length PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide.
[0516] 1. Isolation of DNA Encoding PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
Polypeptides
[0517] DNA encoding PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptides may be
obtained from a cDNA library prepared from tissue believed to
possess the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 mRNA and to express it at a
detectable level. Accordingly, human PRO196-, PRO217-, PRO231-,
PRO236-, PRO245-, PRO246-, PRO258-, PRO287-, PRO328-, PRO344-,
PRO357-, PRO526-, PRO724-, PRO731-, PRO732-, PRO1003-, PRO1104-,
PRO1151-, PRO1244-, PRO1298-, PRO1313-, PRO1570-, PRO1886-,
PRO1891-, PRO4409-, PRO5725-, PRO5994-, PRO6097-, PRO7425-,
PRO10102-, PRO10282-, PRO61709- or PRO779-DNA can be conveniently
obtained from a cDNA library prepared from human tissue, such as
described in the Examples. The PRO196-, PRO217-, PRO231-, PRO236-,
PRO245-, PRO246-, PRO258-, PRO287-, PRO328-, PRO344-, PRO357-,
PRO526-, PRO724-, PRO731-, PRO732-, PRO1003-, PRO1104-, PRO1151-,
PRO1244-, PRO1298-, PRO1313-, PRO1570-, PRO1886-, PRO1891-,
PRO4409-, PRO5725-, PRO5994-, PRO6097-, PRO7425-, PRO10102-,
PRO10282-, PRO61709- or PRO779-encoding gene may also be obtained
from a genomic library or by known synthetic procedures (e.g.,
automated nucleic acid synthesis).
[0518] Libraries can be screened with probes (such as antibodies to
the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide or oligonucleotides of at
least about 20-80 bases) designed to identify the gene of interest
or the protein encoded by it. Screening the cDNA or genomic library
with the selected probe may be conducted using standard procedures,
such as described in Sambrook et al., Molecular Cloning: A
Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,
1989). An alternative means to isolate the gene encoding PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 is to use PCR methodology [Sambrook et al.,
supra; Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold
Spring Harbor Laboratory Press, 1995)].
[0519] The Examples below describe techniques for screening a cDNA
library. The oligonucleotide sequences selected as probes should be
of sufficient length and sufficiently unambiguous that false
positives are minimized. The oligonucleotide is preferably labeled
such that it can be detected upon hybridization to DNA in the
library being screened. Methods of labeling are well known in the
art, and include the use of radiolabels like .sup.32P-labeled ATP,
biotinylation or enzyme labeling. Hybridization conditions,
including moderate stringency and high stringency, are provided in
Sambrook et al., supra.
[0520] Sequences identified in such library screening methods can
be compared and aligned to other known sequences deposited and
available in public databases such as GenBank or other private
sequence databases. Sequence identity (at either the amino acid or
nucleotide level) within defined regions of the molecule or across
the full-length sequence can be determined using methods known in
the art and as described herein.
[0521] Nucleic acid having protein coding sequence may be obtained
by screening selected cDNA or genomic libraries using the deduced
amino acid sequence disclosed herein for the first time, and, if
necessary, using conventional primer extension procedures as
described in Sambrook et al., supra, to detect precursors and
processing intermediates of mRNA that may not have been
reverse-transcribed into cDNA.
[0522] 2. Selection and Transformation of Host Cells
[0523] Host cells are transfected or transformed with expression or
cloning vectors described herein for PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide production and cultured in conventional nutrient media
modified as appropriate for inducing promoters, selecting
transformants, or amplifying the genes encoding the desired
sequences. The culture conditions, such as media, temperature, pH
and the like, can be selected by the skilled artisan without undue
experimentation. In general, principles, protocols, and practical
techniques for maximizing the productivity of cell cultures can be
found in Mammalian Cell Biotechnology: a Practical Approach, M.
Butler, ed. (IRL Press, 1991) and Sambrook et al., supra.
[0524] Methods of eukaryotic cell transfection and prokaryotic cell
transformation are known to the ordinarily skilled artisan, for
example, CaCl.sub.2, CaPO.sub.4, liposome-mediated and
electroporation. Depending on the host cell used, transformation is
performed using standard techniques appropriate to such cells. The
calcium treatment employing calcium chloride, as described in
Sambrook et al., supra, or electroporation is generally used for
prokaryotes. Infection with Agrobacterium tumefaciens is used for
transformation of certain plant cells, as described by Shaw et al.,
Gene, 23:315 (1983) and WO 89/05859 published 29 Jun. 1989. For
mammalian cells without such cell walls, the calcium phosphate
precipitation method of Graham and van der Eb, Virology, 52:456-457
(1978) can be employed. General aspects of mammalian cell host
system transfections have been described in U.S. Pat. No.
4,399,216. Transformations into yeast are typically carried out
according to the method of Van Solingen et al., J. Bact., 130:946
(1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76:3829
(1979). However, other methods for introducing DNA into cells, such
as by nuclear microinjection, electroporation, bacterial protoplast
fusion with intact cells, or polycations, e.g., polybrene,
polyornithine, may also be used. For various techniques for
transforming mammalian cells, see Keown et al., Methods in
Enzymology, 185:527-537 (1990) and Mansour et al., Nature,
336:348-352 (1988).
[0525] Suitable host cells for cloning or expressing the DNA in the
vectors herein include prokaryote, yeast, or higher eukaryote
cells. Suitable prokaryotes include but are not limited to
eubacteria, such as Gram-negative or Gram-positive organisms, for
example, Enterobacteriaceae such as E. coli. Various E. coli
strains are publicly available, such as E. coli K12 strain MM294
(ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli strain W3110
(ATCC 27,325) and K5 772 (ATCC 53,635). Other suitable prokaryotic
host cells include Enterobacteriaceae such as Escherichia, e.g., E.
coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g.,
Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and
Shigella, as well as Bacilli such as B. subtilis and B.
licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710
published 12 Apr. 1989), Pseudomonas such as P. aeruginosa, and
Streptomyces. These examples are illustrative rather than limiting.
Strain W3110 is one particularly preferred host or parent host
because it is a common host strain for recombinant DNA product
fermentations. Preferably, the host cell secretes minimal amounts
of proteolytic enzymes. For example, strain W3110 may be modified
to effect a genetic mutation in the genes encoding proteins
endogenous to the host, with examples of such hosts including E.
coli W3110 strain 1A2, which has the complete genotype tonA; E.
coli W3110 strain 9E4, which has the complete genotype tonA ptr3;
E. coli W3110 strain 27C7 (ATCC 55,244), which has the complete
genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT had; E. coli
W3110 strain 37D6, which has the complete genotype tonA ptr3 phoA
E15 (argF-lac)169 degP ompT rbs7 ilvG kan.sup.r; E. coli W3110
strain 40B4, which is strain 37D6 with a non-kanamycin resistant
degP deletion mutation; and an E. coli strain having mutant
periplasmic protease disclosed in U.S. Pat. No. 4,946,783 issued 7
Aug. 1990. Alternatively, in vitro methods of cloning, e.g., PCR or
other nucleic acid polymerase reactions, are suitable.
[0526] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779-encoding vectors. Saccharomyces
cerevisiae is a commonly used lower eukaryotic host microorganism.
Others include Schizosaccharomyces pombe (Beach and Nurse, Nature,
290: 140 [1981]; EP 139,383 published 2 May 1985); Kluyveromyces
hosts (U.S. Pat. No. 4,943,529; Fleer et al., Bio/Technology,
9:968-975 (1991)) such as, e.g., K lactis (MW98-8C, CBS683,
CBS4574; Louvencourt et al., J. Bacteriol., 154(2):737-742 [1983]),
K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K.
wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum
(ATCC 36,906; Van den Berg et al., Bio/Technology, 8:135 (1990)),
K. thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia
pastoris (EP 183,070; Sreekrishna et al., J. Basic Microbiol.,
28:265-278 [1988]); Candida; Trichoderma reesia (EP 244,234);
Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA,
76:5259-5263 [1979]); Schwanniomyces such as Schwanniomyces
occidentalis (EP 394,538 published 31 Oct. 1990); and filamentous
fungi such as, e.g., Neurospora, Penicillium, Tolypocladium (WO
91/00357 published 10 Jan. 1991), and Aspergillus hosts such as A.
nidulans (Ballance et al., Biochem. Biophys. Res. Commun.,
112:284-289 [1983]; Tilburn et al., Gene, 26:205-221 [1983]; Yelton
et al., Proc. Natl. Acad. Sci. USA, 81: 1470-1474 [1984]) and A.
niger (Kelly and Hynes, EMBO J., 4:475-479 [1985]). Methylotropic
yeasts are suitable herein and include, but are not limited to,
yeast capable of growth on methanol selected from the genera
consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces,
Torulopsis, and Rhodotorula. A list of specific species that are
exemplary of this class of yeasts may be found in C. Anthony, The
Biochemistry of Methylotrophs, 269 (1982).
[0527] Suitable host cells for the expression of glycosylated
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptides are derived from
multicellular organisms. Examples of invertebrate cells include
insect cells such as Drosophila S2 and Spodoptera Sf9, as well as
plant cells. Examples of useful mammalian host cell lines include
Chinese hamster ovary (CHO) and COS cells. More specific examples
include monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL
1651); human embryonic kidney line (293 or 293 cells subcloned for
growth in suspension culture, Graham et al., J. Gen Virol., 36:59
(1977)); Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasin,
Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse sertoli cells
(TM4, Mather, Biol. Reprod., 23:243-251 (1980)); human lung cells
(W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); and mouse
mammary tumor (MMT 060562, ATCC CCL51). The selection of the
appropriate host cell is deemed to be within the skill in the
art.
[0528] 3. Selection and Use of a Replicable Vector
[0529] The nucleic acid (e.g., cDNA or genomic DNA) encoding
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptides may be inserted into a
replicable vector for cloning (amplification of the DNA) or for
expression. Various vectors are publicly available. The vector may,
for example, be in the form of a plasmid, cosmid, viral particle,
or phage. The appropriate nucleic acid sequence may be inserted
into the vector by a variety of procedures. In general, DNA is
inserted into an appropriate restriction endonuclease site(s) using
techniques known in the art. Vector components generally include,
but are not limited to, one or more of a signal sequence, an origin
of replication, one or more marker genes, an enhancer element, a
promoter, and a transcription termination sequence. Construction of
suitable vectors containing one or more of these components employs
standard ligation techniques which are known to the skilled
artisan.
[0530] The PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 polypeptide may be produced
recombinantly not only directly, but also as a fusion polypeptide
with a heterologous polypeptide, which may be a signal sequence or
other polypeptide having a specific cleavage site at the N-terminus
of the mature protein or polypeptide. In general, the signal
sequence may be a component of the vector, or it may be a part of
the PRO196-, PRO217-, PRO231-, PRO236-, PRO245-, PRO246-, PRO258-,
PRO287-, PRO328-, PRO344-, PRO357-, PRO526-, PRO724-, PRO731-,
PRO732-, PRO1003-, PRO1104-, PRO1151-, PRO1244-, PRO1298-,
PRO1313-, PRO1570-, PRO1886-, PRO1891-, PRO4409-, PRO5725-,
PRO5994-, PRO6097-, PRO7425-, PRO10102-, PRO10282-, PRO61709- or
PRO779-encoding DNA that is inserted into the vector. The signal
sequence may be a prokaryotic signal sequence selected, for
example, from the group of the alkaline phosphatase, penicillinase,
lpp, or heat-stable enterotoxin II leaders. For yeast secretion the
signal sequence may be, e.g., the yeast invertase leader, alpha
factor leader (including Saccharomyces and Kluyveromyces
.alpha.-factor leaders, the latter described in U.S. Pat. No.
5,010,182), or acid phosphatase leader, the C. albicans
glucoamylase leader (EP 362,179 published 4 Apr. 1990), or the
signal described in WO 90/13646 published 15 Nov. 1990. In
mammalian cell expression, mammalian signal sequences may be used
to direct secretion of the protein, such as signal sequences from
secreted polypeptides of the same or related species, as well as
viral secretory leaders.
[0531] Both expression and cloning vectors contain a nucleic acid
sequence that enables the vector to replicate in one or more
selected host cells. Such sequences are well known for a variety of
bacteria, yeast, and viruses. The origin of replication from the
plasmid pBR322 is suitable for most Gram-negative bacteria, the
2.mu. plasmid origin is suitable for yeast, and various viral
origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for
cloning vectors in mammalian cells.
[0532] Expression and cloning vectors will typically contain a
selection gene, also termed a selectable marker. Typical selection
genes encode proteins that (a) confer resistance to antibiotics or
other toxins, e.g., ampicillin, neomycin, methotrexate, or
tetracycline, (b) complement auxotrophic deficiencies, or (c)
supply critical nutrients not available from complex media, e.g.,
the gene encoding D-alanine racemase for Bacilli.
[0533] An example of suitable selectable markers for mammalian
cells are those that enable the identification of cells competent
to take up the PRO196-, PRO217-, PRO231-, PRO236-, PRO245-,
PRO246-, PRO258-, PRO287-, PRO328-, PRO344-, PRO357-, PRO526-,
PRO724-, PRO731-, PRO732-, PRO1003-, PRO1104-, PRO1151-, PRO1244-,
PRO1298-, PRO1313-, PRO1570-, PRO1886-, PRO1891-, PRO4409-,
PRO5725-, PRO5994-, PRO6097-, PRO7425-, PRO10102-, PRO10282-,
PRO61709- or PRO779-encoding nucleic acid, such as DHFR or
thymidine kinase. An appropriate host cell when wild-type DHFR is
employed is the CHO cell line deficient in DHFR activity, prepared
and propagated as described by Urlaub et al., Proc. Natl. Acad.
Sci. USA, 77:4216 (1980). A suitable selection gene for use in
yeast is the trp1 gene present in the yeast plasmid YRp7
[Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene,
7:141 (1979); Tschemper et al., Gene, 10:157 (1980)]. The trp1 gene
provides a selection marker for a mutant strain of yeast lacking
the ability to grow in tryptophan, for example, ATCC No. 44076 or
PEP4-1 [Jones, Genetics, 85:12 (1977)].
[0534] Expression and cloning vectors usually contain a promoter
operably linked to the PRO196-, PRO217-, PRO231-, PRO236-, PRO245-,
PRO246-, PRO258-, PRO287-, PRO328-, PRO344-, PRO357-, PRO526-,
PRO724-, PRO731-, PRO732-, PRO1003-, PRO1104-, PRO1151-, PRO1244-,
PRO1298-, PRO1313-, PRO1570-, PRO1886-, PRO1891-, PRO4409-,
PRO5725-, PRO5994-, PRO6097-, PRO7425-, PRO10102-, PRO10282-,
PRO61709- or PRO779-encoding nucleic acid sequence to direct mRNA
synthesis. Promoters recognized by a variety of potential host
cells are well known. Promoters suitable for use with prokaryotic
hosts include the .beta.-lactamase and lactose promoter systems
[Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature,
281:544 (1979)], alkaline phosphatase, a tryptophan (trp) promoter
system [Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776], and
hybrid promoters such as the tac promoter [deBoer et al., Proc.
Natl. Acad. Sci. USA, 80:21-25 (1983)]. Promoters for use in
bacterial systems also will contain a Shine-Dalgarno (S.D.)
sequence operably linked to the DNA encoding PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 polypeptides.
[0535] Examples of suitable promoting sequences for use with yeast
hosts include the promoters for 3-phosphoglycerate kinase [Hitzeman
et al., J. Biol. Chem., 255:2073 (1980)] or other glycolytic
enzymes [Hess et al., J. Adv. Enzyme Reg., 7:149 (1968); Holland,
Biochemistry, 17:4900 (1978)], such as enolase,
glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate
decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate
isomerase, phosphoglucose isomerase, and glucokinase.
[0536] Other yeast promoters, which are inducible promoters having
the additional advantage of transcription controlled by growth
conditions, are the promoter regions for alcohol dehydrogenase 2,
isocytochrome C, acid phosphatase, degradative enzymes associated
with nitrogen metabolism, metallothionein,
glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible
for maltose and galactose utilization. Suitable vectors and
promoters for use in yeast expression are further described in EP
73,657.
[0537] PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 transcription from vectors
in mammalian host cells is controlled, for example, by promoters
obtained from the genomes of viruses such as polyoma virus, fowlpox
virus (UK 2,211,504 published 5 Jul. 1989), adenovirus (such as
Adenovirus 2), bovine papilloma virus, avian sarcoma virus,
cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus
40 (SV40), from heterologous mammalian promoters, e.g., the actin
promoter or an immunoglobulin promoter, and from heat-shock
promoters, provided such promoters are compatible with the host
cell systems.
[0538] Transcription of a DNA encoding the PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide by higher eukaryotes may be increased by inserting an
enhancer sequence into the vector. Enhancers are cis-acting
elements of DNA, usually about from 10 to 300 bp, that act on a
promoter to increase its transcription. Many enhancer sequences are
now known from mammalian genes (globin, elastase, albumin,
.alpha.-fetoprotein, and insulin). Typically, however, one will use
an enhancer from a eukaryotic cell virus. Examples include the SV40
enhancer on the late side of the replication origin (bp 100-270),
the cytomegalovirus early promoter enhancer, the polyoma enhancer
on the late side of the replication origin, and adenovirus
enhancers. The enhancer may be spliced into the vector at a
position 5' or 3' to the PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 coding
sequence, but is preferably located at a site 5' from the
promoter.
[0539] Expression vectors used in eukaryotic host cells (yeast,
fungi, insect, plant, animal, human, or nucleated cells from other
multicellular organisms) will also contain sequences necessary for
the termination of transcription and for stabilizing the mRNA. Such
sequences are commonly available from the 5' and, occasionally 3',
untranslated regions of eukaryotic or viral DNAs or cDNAs. These
regions contain nucleotide segments transcribed as polyadenylated
fragments in the untranslated portion of the mRNA encoding PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptides.
[0540] Still other methods, vectors, and host cells suitable for
adaptation to the synthesis of PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptides in recombinant vertebrate cell culture are described
in Gething et al., Nature, 293:620-625 (1981); Mantei et al.,
Nature, 281:40-46 (1979); EP 117,060; and EP 117,058.
[0541] 4. Detecting Gene Amplification/Expression
[0542] Gene amplification and/or expression may be measured in a
sample directly, for example, by conventional Southern blotting,
Northern blotting to quantitate the transcription of mRNA [Thomas,
Proc. Natl. Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA
analysis), or in situ hybridization, using an appropriately labeled
probe, based on the sequences provided herein. Alternatively,
antibodies may be employed that can recognize specific duplexes,
including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes
or DNA-protein duplexes. The antibodies in turn may be labeled and
the assay may be carried out where the duplex is bound to a
surface, so that upon the formation of duplex on the surface, the
presence of antibody bound to the duplex can be detected.
[0543] Gene expression, alternatively, may be measured by
immunological methods, such as immunohistochemical staining of
cells or tissue sections and assay of cell culture or body fluids,
to quantitate directly the expression of gene product. Antibodies
useful for immunohistochemical staining and/or assay of sample
fluids may be either monoclonal or polyclonal, and may be prepared
in any mammal. Conveniently, the antibodies may be prepared against
a native sequence PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide or
against a synthetic peptide based on the DNA sequences provided
herein or against exogenous sequence fused to PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 DNA and encoding a specific antibody epitope.
[0544] 5. Purification of Polypeptide
[0545] Forms of PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptides may be
recovered from culture medium or from host cell lysates. If
membrane-bound, it can be released from the membrane using a
suitable detergent solution (e.g. Triton-X 100) or by enzymatic
cleavage. Cells employed in expression of PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptides can be disrupted by various physical or chemical
means, such as freeze-thaw cycling, sonication, mechanical
disruption, or cell lysing agents.
[0546] It may be desired to purify PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptides from recombinant cell proteins or polypeptides. The
following procedures are exemplary of suitable purification
procedures: by fractionation on an ion-exchange column; ethanol
precipitation; reverse phase HPLC; chromatography on silica or on a
cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE;
ammonium sulfate precipitation; gel filtration using, for example,
Sephadex G-75; protein A Sepharose columns to remove contaminants
such as IgG; and metal chelating columns to bind epitope-tagged
forms of the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide.
Various methods of protein purification may be employed and such
methods are known in the art and described for example in
Deutscher, Methods in Enzymology, 182 (1990); Scopes, Protein
Purification: Principles and Practice, Springer-Verlag, New York
(1982). The purification step(s) selected will depend, for example,
on the nature of the production process used and the particular
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide produced.
[0547] E. Uses for PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 Polypeptides
[0548] Nucleotide sequences (or their complement) encoding PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptides have various applications in the
art of molecular biology, including uses as hybridization probes,
in chromosome and gene mapping and in the generation of anti-sense
RNA and DNA. PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 nucleic acid will
also be useful for the preparation of PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptides by the recombinant techniques described herein.
[0549] The full-length native sequence PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
gene, or portions thereof, may be used as hybridization probes for
a cDNA library to isolate the full-length PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
cDNA or to isolate still other cDNAs (for instance, those encoding
naturally-occurring variants of PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptides or PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptides from
other species) which have a desired sequence identity to the native
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 sequence disclosed herein. Optionally,
the length of the probes will be about 20 to about 50 bases. The
hybridization probes may be derived from at least partially novel
regions of the full length native nucleotide sequence wherein those
regions may be determined without undue experimentation or from
genomic sequences including promoters, enhancer elements and
introns of native sequence PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779. By way of
example, a screening method will comprise isolating the coding
region of the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 gene using the
known DNA sequence to synthesize a selected probe of about 40
bases. Hybridization probes may be labeled by a variety of labels,
including radionucleotides such as .sup.32P or .sup.35S, or
enzymatic labels such as alkaline phosphatase coupled to the probe
via avidin/biotin coupling systems. Labeled probes having a
sequence complementary to that of the PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
gene of the present invention can be used to screen libraries of
human cDNA, genomic DNA or mRNA to determine which members of such
libraries the probe hybridizes to. Hybridization techniques are
described in further detail in the Examples below.
[0550] Any EST sequences disclosed in the present application may
similarly be employed as probes, using the methods disclosed
herein.
[0551] Other useful fragments of the PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
nucleic acids include antisense or sense oligonucleotides
comprising a singe-stranded nucleic acid sequence (either RNA or
DNA) capable of binding to target PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
mRNA (sense) or PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 DNA (antisense)
sequences. Antisense or sense oligonucleotides, according to the
present invention, comprise a fragment of the coding region of
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 DNA. Such a fragment generally
comprises at least about 14 nucleotides, preferably from about 14
to 30 nucleotides. The ability to derive an antisense or a sense
oligonucleotide, based upon a cDNA sequence encoding a given
protein is described in, for example, Stein and Cohen (Cancer Res.
48:2659, 1988) and van der Krol et al. (BioTechniques 6:958,
1988).
[0552] Binding of antisense or sense oligonucleotides to target
nucleic acid sequences results in the formation of duplexes that
block transcription or translation of the target sequence by one of
several means, including enhanced degradation of the duplexes,
premature termination of transcription or translation, or by other
means. The antisense oligonucleotides thus may be used to block
expression of PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779. Antisense or sense
oligonucleotides further comprise oligonucleotides having modified
sugar-phosphodiester backbones (or other sugar linkages, such as
those described in WO 91/06629) and wherein such sugar linkages are
resistant to endogenous nucleases. Such oligonucleotides with
resistant sugar linkages are stable in vivo (i.e., capable of
resisting enzymatic degradation) but retain sequence specificity to
be able to bind to target nucleotide sequences.
[0553] Other examples of sense or antisense oligonucleotides
include those oligonucleotides which are covalently linked to
organic moieties, such as those described in WO 90/10048, and other
moieties that increases affinity of the oligonucleotide for a
target nucleic acid sequence, such as poly-(L-lysine). Further
still, intercalating agents, such as ellipticine, and alkylating
agents or metal complexes may be attached to sense or antisense
oligonucleotides to modify binding specificities of the antisense
or sense oligonucleotide for the target nucleotide sequence.
[0554] Antisense or sense oligonucleotides may be introduced into a
cell containing the target nucleic acid sequence by any gene
transfer method, including, for example, CaPO.sub.4-mediated DNA
transfection, electroporation, or by using gene transfer vectors
such as Epstein-Barr virus. In a preferred procedure, an antisense
or sense oligonucleotide is inserted into a suitable retroviral
vector. A cell containing the target nucleic acid sequence is
contacted with the recombinant retroviral vector, either in vivo or
ex vivo. Suitable retroviral vectors include, but are not limited
to, those derived from the murine retrovirus M-MuLV, N2 (a
retrovirus derived from M-MuLV), or the double copy vectors
designated DCT5A, DCT5B and DCT5C (see WO 90/13641).
[0555] Sense or antisense oligonucleotides also may be introduced
into a cell containing the target nucleotide sequence by formation
of a conjugate with a ligand binding molecule, as described in WO
91/04753. Suitable ligand binding molecules include, but are not
limited to, cell surface receptors, growth factors, other
cytokines, or other ligands that bind to cell surface receptors.
Preferably, conjugation of the ligand binding molecule does not
substantially interfere with the ability of the ligand binding
molecule to bind to its corresponding molecule or receptor, or
block entry of the sense or antisense oligonucleotide or its
conjugated version into the cell.
[0556] Alternatively, a sense or an antisense oligonucleotide may
be introduced into a cell containing the target nucleic acid
sequence by formation of an oligonucleotide-lipid complex, as
described in WO 90/10448. The sense or antisense
oligonucleotide-lipid complex is preferably dissociated within the
cell by an endogenous lipase.
[0557] Antisense or sense RNA or DNA molecules are generally at
least about 5 bases in length, about 10 bases in length, about 15
bases in length, about 20 bases in length, about 25 bases in
length, about 30 bases in length, about 35 bases in length, about
40 bases in length, about 45 bases in length, about 50 bases in
length, about 55 bases in length, about 60 bases in length, about
65 bases in length, about 70 bases in length, about 75 bases in
length, about 80 bases in length, about 85 bases in length, about
90 bases in length, about 95 bases in length, about 100 bases in
length, or more.
[0558] The probes may also be employed in PCR techniques to
generate a pool of sequences for identification of closely related
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 coding sequences.
[0559] Nucleotide sequences encoding a PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide can also be used to construct hybridization probes for
mapping the gene which encodes that PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide and for the genetic analysis of individuals with
genetic disorders. The nucleotide sequences provided herein may be
mapped to a chromosome and specific regions of a chromosome using
known techniques, such as in situ hybridization, linkage analysis
against known chromosomal markers, and hybridization screening with
libraries.
[0560] When the coding sequences for PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
encode a protein which binds to another protein (for example, where
the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 is a receptor), the PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 polypeptide can be used in assays to identify the other
proteins or molecules involved in the binding interaction. By such
methods, inhibitors of the receptor/ligand binding interaction can
be identified. Proteins involved in such binding interactions can
also be used to screen for peptide or small molecule inhibitors or
agonists of the binding interaction. Also, the receptor PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 can be used to isolate correlative ligand(s).
Screening assays can be designed to find lead compounds that mimic
the biological activity of a native PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide or a receptor for PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptides. Such screening assays will include assays amenable to
high-throughput screening of chemical libraries, making them
particularly suitable for identifying small molecule drug
candidates. Small molecules contemplated include synthetic organic
or inorganic compounds. The assays can be performed in a variety of
formats, including protein-protein binding assays, biochemical
screening assays, immunoassays and cell based assays, which are
well characterized in the art.
[0561] Nucleic acids which encode PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptides or its modified forms can also be used to generate
either transgenic animals or "knock out" animals which, in turn,
are useful in the development and screening of therapeutically
useful reagents. A transgenic animal (e.g., a mouse or rat) is an
animal having cells that contain a transgene, which transgene was
introduced into the animal or an ancestor of the animal at a
prenatal, e.g., an embryonic stage. A transgene is a DNA which is
integrated into the genome of a cell from which a transgenic animal
develops. The invention provides cDNA encoding a PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 polypeptide which can be used to clone genomic DNA encoding
a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide in accordance with
established techniques and the genomic sequences used to generate
transgenic animals that contain cells which express DNA encoding
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptides. Any technique known in
the art may be used to introduce a target gene transgene into
animals to produce the founder lines of transgenic animals. Such
techniques include, but are not limited to pronuclear
microinjection (U.S. Pat. Nos. 4,873,191, 4,736,866 and 4,870,009);
retrovirus mediated gene transfer into germ lines (Van der Putten,
et al., Proc. Natl. Acad. Sci., USA, 82:6148-6152 (1985)); gene
targeting in embryonic stem cells (Thompson, et al., Cell,
56:313-321 (1989)); nonspecific insertional inactivation using a
gene trap vector (U.S. Pat. No. 6,436,707); electroporation of
embryos (Lo, Mol. Cell. Biol., 3:1803-1814 (1983)); and
sperm-mediated gene transfer (Lavitrano, et al., Cell, 57:717-723
(1989)); etc. Typically, particular cells would be targeted for a
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 transgene incorporation with
tissue-specific enhancers. Transgenic animals that include a copy
of a transgene encoding a PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide introduced into the germ line of the animal at an
embryonic stage can be used to examine the effect of increased
expression of DNA encoding PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptides. Such animals can be used as tester animals for
reagents thought to confer protection from, for example,
pathological conditions associated with its overexpression. In
accordance with this facet of the invention, an animal is treated
with the reagent and a reduced incidence of the pathological
condition, compared to untreated animals bearing the transgene,
would indicate a potential therapeutic intervention for the
pathological condition. Alternatively, non-human homologues of
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptides can be used to construct
a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 "knock out" animal which has a
defective or altered gene encoding PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
proteins as a result of homologous recombination between the
endogenous gene encoding PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptides and altered genomic DNA encoding PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 polypeptides introduced into an embryonic stem cell of the
animal. Preferably the knock out animal is a mammal. More
preferably, the mammal is a rodent such as a rat or mouse. For
example, cDNA encoding PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptides can be used to clone genomic DNA encoding PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptides in accordance with established
techniques. A portion of the genomic DNA encoding the PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide can be deleted or replaced with
another gene, such as a gene encoding a selectable marker which can
be used to monitor integration. Typically, several kilobases of
unaltered flanking DNA (both at the 5' and 3' ends) are included in
the vector [see e.g., Thomas and Capecchi, Cell, 51:503 (1987) for
a description of homologous recombination vectors]. The vector is
introduced into an embryonic stem cell line (e.g., by
electroporation) and cells in which the introduced DNA has
homologously recombined with the endogenous DNA are selected [see
e.g., Li et al., Cell, 69:915 (1992)]. The selected cells are then
injected into a blastocyst of an animal (e.g., a mouse or rat) to
form aggregation chimeras [see e.g., Bradley, in Teratocarcinomas
and Embryonic Stem Cells: A Practical Approach, E. J. Robertson,
ed. (IRL, Oxford, 1987), pp. 113-152]. A chimeric embryo can then
be implanted into a suitable pseudopregnant female foster animal
and the embryo brought to term to create a "knock out" animal.
Progeny harboring the homologously recombined DNA in their germ
cells can be identified by standard techniques and used to breed
animals in which all cells of the animal contain the homologously
recombined DNA. Knockout animals can be characterized for instance,
for their ability to defend against certain pathological conditions
and for their development of pathological conditions due to absence
of the gene encoding the PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide.
[0562] In addition, knockout mice can be highly informative in the
discovery of gene function and pharmaceutical utility for a drug
target, as well as in the determination of the potential on-target
side effects associated with a given target. Gene function and
physiology are so well conserved between mice and humans, since
they are both mammals and contain similar numbers of genes, which
are highly conserved between the species. It has recently been well
documented, for example, that 98% of genes on mouse chromosome 16
have a human ortholog (Mural et al., Science 296:1661-71
(2002)).
[0563] Although gene targeting in embryonic stem (ES) cells has
enabled the construction of mice with null mutations in many genes
associated with human disease, not all genetic diseases are
attributable to null mutations. One can design valuable mouse
models of human diseases by establishing a method for gene
replacement (knock-in) which will disrupt the mouse locus and
introduce a human counterpart with mutation, Subsequently one can
conduct in vivo drug studies targeting the human protein (Kitamoto
et. Al., Biochemical and Biophysical Res. Commun., 222:742-47
(1996)).
[0564] Nucleic acid encoding the PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptides may also be used in gene therapy. In gene therapy
applications, genes are introduced into cells in order to achieve
in vivo synthesis of a therapeutically effective genetic product,
for example for replacement of a defective gene. "Gene therapy"
includes both conventional gene therapy where a lasting effect is
achieved by a single treatment, and the administration of gene
therapeutic agents, which involves the one time or repeated
administration of a therapeutically effective DNA or mRNA.
Antisense RNAs and DNAs can be used as therapeutic agents for
blocking the expression of certain genes in vivo. It has already
been shown that short antisense oligonucleotides can be imported
into cells where they act as inhibitors, despite their low
intracellular concentrations caused by their restricted uptake by
the cell membrane. (Zamecnik et al., Proc. Natl. Acad. Sci. USA
83:4143-4146 [1986]). The oligonucleotides can be modified to
enhance their uptake, e.g. by substituting their negatively charged
phosphodiester groups by uncharged groups.
[0565] There are a variety of techniques available for introducing
nucleic acids into viable cells. The techniques vary depending upon
whether the nucleic acid is transferred into cultured cells in
vitro, or in vivo in the cells of the intended host. Techniques
suitable for the transfer of nucleic acid into mammalian cells in
vitro include the use of liposomes, electroporation,
microinjection, cell fusion, DEAE-dextran, the calcium phosphate
precipitation method, etc. The currently preferred in vivo gene
transfer techniques include transfection with viral (typically
retroviral) vectors and viral coat protein-liposome mediated
transfection (Dzau et al., Trends in Biotechnology 11, 205-210
[1993]). In some situations it is desirable to provide the nucleic
acid source with an agent that targets the target cells, such as an
antibody specific for a cell surface membrane protein or the target
cell, a ligand for a receptor on the target cell, etc. Where
liposomes are employed, proteins which bind to a cell surface
membrane protein associated with endocytosis may be used for
targeting and/or to facilitate uptake, e.g. capsid proteins or
fragments thereof tropic for a particular cell type, antibodies for
proteins which undergo internalization in cycling, proteins that
target intracellular localization and enhance intracellular
half-life. The technique of receptor-mediated endocytosis is
described, for example, by Wu et al., J. Biol. Chem. 262, 4429-4432
(1987); and Wagner et al., Proc. Natl. Acad. Sci. USA 87, 3410-3414
(1990). For review of gene marking and gene therapy protocols see
Anderson et al., Science 256, 808-813 (1992).
[0566] The PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 polypeptides described
herein may also be employed as molecular weight markers for protein
electrophoresis purposes and the isolated nucleic acid sequences
may be used for recombinantly expressing those markers.
[0567] The nucleic acid molecules encoding the PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 polypeptides or fragments thereof described herein are
useful for chromosome identification. In this regard, there exists
an ongoing need to identify new chromosome markers, since
relatively few chromosome marking reagents, based upon actual
sequence data are presently available. Each PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
nucleic acid molecule of the present invention can be used as a
chromosome marker.
[0568] The PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 polypeptides and nucleic
acid molecules of the present invention may also be used
diagnostically for tissue typing, wherein the PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 polypeptides of the present invention may be differentially
expressed in one tissue as compared to another, preferably in a
diseased tissue as compared to a normal tissue of the same tissue
type. PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 nucleic acid molecules will
find use for generating probes for PCR, Northern analysis, Southern
analysis and Western analysis.
[0569] The PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 polypeptides described
herein may also be employed as therapeutic agents. The PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptides of the present invention can be
formulated according to known methods to prepare pharmaceutically
useful compositions, whereby the PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
product hereof is combined in admixture with a pharmaceutically
acceptable carrier vehicle. Therapeutic formulations are prepared
for storage by mixing the active ingredient having the desired
degree of purity with optional physiologically acceptable carriers,
excipients or stabilizers (Remington's Pharmaceutical Sciences 16th
edition, Osol, A. Ed. (1980)), in the form of lyophilized
formulations or aqueous solutions. Acceptable carriers, excipients
or stabilizers are nontoxic to recipients at the dosages and
concentrations employed, and include buffers such as phosphate,
citrate and other organic acids; antioxidants including ascorbic
acid; low molecular weight (less than about 10 residues)
polypeptides; proteins, such as serum albumin, gelatin or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone,
amino acids such as glycine, glutamine, asparagine, arginine or
lysine; monosaccharides, disaccharides and other carbohydrates
including glucose, mannose, or dextrins; chelating agents such as
EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming
counterions such as sodium; and/or nonionic surfactants such as
TWEEN.TM., PLURONICS.TM. or PEG.
[0570] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes, prior to or following lyophilization
and reconstitution.
[0571] Therapeutic compositions herein generally are placed into a
container having a sterile access port, for example, an intravenous
solution bag or vial having a stopper pierceable by a hypodermic
injection needle.
[0572] The route of administration is in accord with known methods,
e.g. injection or infusion by intravenous, intraperitoneal,
intracerebral, intramuscular, intraocular, intraarterial or
intralesional routes, topical administration, or by sustained
release systems.
[0573] Dosages and desired drug concentrations of pharmaceutical
compositions of the present invention may vary depending on the
particular use envisioned. The determination of the appropriate
dosage or route of administration is well within the skill of an
ordinary physician. Animal experiments provide reliable guidance
for the determination of effective doses for human therapy.
Interspecies scaling of effective doses can be performed following
the principles laid down by Mordenti, J. and Chappell, W. "The use
of interspecies scaling in toxicokinetics" In Toxicokinetics and
New Drug Development, Yacobi et al., Eds., Pergamon Press, New York
1989, pp. 42-96.
[0574] When in vivo administration of a PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide or agonist or antagonist thereof is employed, normal
dosage amounts may vary from about 10 ng/kg to up to 100 mg/kg of
mammal body weight or more per day, preferably about 1 .mu.g/kg/day
to 10 mg/kg/day, depending upon the route of administration.
Guidance as to particular dosages and methods of delivery is
provided in the literature; see, for example, U.S. Pat. No.
4,657,760; 5,206,344; or 5,225,212. It is anticipated that
different formulations will be effective for different treatment
compounds and different disorders, that administration targeting
one organ or tissue, for example, may necessitate delivery in a
manner different from that to another organ or tissue.
[0575] Where sustained-release administration of a PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 polypeptide is desired in a formulation with release
characteristics suitable for the treatment of any disease or
disorder requiring administration of the PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide, microencapsulation of the PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide is contemplated. Microencapsulation of recombinant
proteins for sustained release has been successfully performed with
human growth hormone (rhGH), interferon-(rhIFN-), interleukin-2,
and MN rgp120. Johnson et al., Nat. Med., 2:795-799 (1996); Yasuda,
Biomed. Ther., 27:1221-1223 (1993); Hora et al., Bio/Technology,
8:755-758 (1990); Cleland, "Design and Production of Single
Immunization Vaccines Using Polylactide Polyglycolide Microsphere
Systems," in Vaccine Design: The Subunit and Adjuvant Approach,
Powell and Newman, eds, (Plenum Press: New York, 1995), pp.
439-462; WO 97/03692, WO 96/40072, WO 96/07399; and U.S. Pat. No.
5,654,010.
[0576] The sustained-release formulations of these proteins were
developed using poly-lactic-coglycolic acid (PLGA) polymer due to
its biocompatibility and wide range of biodegradable properties.
The degradation products of PLGA, lactic and glycolic acids, can be
cleared quickly within the human body. Moreover, the degradability
of this polymer can be adjusted from months to years depending on
its molecular weight and composition. Lewis, "Controlled release of
bioactive agents from lactide/glycolide polymer," in: M. Chasin and
R. Langer (Eds.), Biodegradable Polymers as Drug Delivery Systems
(Marcel Dekker: New York, 1990), pp. 1-41.
[0577] This invention encompasses methods of screening compounds to
identify those that mimic the PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide (agonists) or prevent the effect of the PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 polypeptide (antagonists). Agonists that mimic a PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide would be especially valuable
therapeutically in those instances where a negative phenotype is
observed based on findings with the non-human transgenic animal
whose genome comprises a disruption of the gene which encodes for
the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide. Antagonists that prevent
the effects of a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide would
be especially valuable therapeutically in those instances where a
positive phenotype is observed based upon observations with the
non-human transgenic knockout animal. Screening assays for
antagonist drug candidates are designed to identify compounds that
bind or complex with the PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide encoded by the genes identified herein, or otherwise
interfere with the interaction of the encoded polypeptide with
other cellular proteins. Such screening assays will include assays
amenable to high-throughput screening of chemical libraries, making
them particularly suitable for identifying small molecule drug
candidates.
[0578] The assays can be performed in a variety of formats,
including protein-protein binding assays, biochemical screening
assays, immunoassays, and cell-based assays, which are well
characterized in the art.
[0579] All assays for antagonists are common in that they call for
contacting the drug candidate with a PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide encoded by a nucleic acid identified herein under
conditions and for a time sufficient to allow these two components
to interact.
[0580] In binding assays, the interaction is binding and the
complex formed can be isolated or detected in the reaction mixture.
The PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide encoded by the gene
identified herein or the drug candidate is immobilized on a solid
phase, e.g., on a microtiter plate, by covalent or non-covalent
attachments. Non-covalent attachment generally is accomplished by
coating the solid surface with a solution of the PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 polypeptide and drying. Alternatively, an immobilized
antibody, e.g., a monoclonal antibody, specific for the PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide to be immobilized can be used to
anchor it to a solid surface. The assay is performed by adding the
non-immobilized component, which may be labeled by a detectable
label, to the immobilized component, e.g., the coated surface
containing the anchored component. When the reaction is complete,
the non-reacted components are removed, e.g., by washing, and
complexes anchored on the solid surface are detected. When the
originally non-immobilized component carries a detectable label,
the detection of label immobilized on the surface indicates that
complexing occurred. Where the originally non-immobilized component
does not carry a label, complexing can be detected, for example, by
using a labeled antibody specifically binding the immobilized
complex.
[0581] If the candidate compound interacts with but does not bind
to a particular PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide encoded
by a gene identified herein, its interaction with that polypeptide
can be assayed by methods well known for detecting protein-protein
interactions. Such assays include traditional approaches, such as,
e.g., cross-linking, co-immunoprecipitation, and co-purification
through gradients or chromatographic columns. In addition,
protein-protein interactions can be monitored by using a
yeast-based genetic system described by Fields and co-workers
(Fields and Song, Nature (London), 340:245-246 (1989); Chien et
al., Proc. Natl. Acad. Sci. USA, 88:9578-9582 (1991)) as disclosed
by Chevray and Nathans, Proc. Natl. Acad. Sci. USA, 89: 5789-5793
(1991). Many transcriptional activators, such as yeast GAL4,
consist of two physically discrete modular domains, one acting as
the DNA-binding domain, the other one functioning as the
transcription-activation domain. The yeast expression system
described in the foregoing publications (generally referred to as
the "two-hybrid system") takes advantage of this property, and
employs two hybrid proteins, one in which the target protein is
fused to the DNA-binding domain of GAL4, and another, in which
candidate activating proteins are fused to the activation domain.
The expression of a GAL1-lacZ reporter gene under control of a
GAL4-activated promoter depends on reconstitution of GAL4 activity
via protein-protein interaction. Colonies containing interacting
polypeptides are detected with a chromogenic substrate for
.beta.-galactosidase. A complete kit (MATCHMAKER.TM.) for
identifying protein-protein interactions between two specific
proteins using the two-hybrid technique is commercially available
from Clontech. This system can also be extended to map protein
domains involved in specific protein interactions as well as to
pinpoint amino acid residues that are crucial for these
interactions.
[0582] Compounds that interfere with the interaction of a gene
encoding a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 polypeptide identified
herein and other intra- or extracellular components can be tested
as follows: usually a reaction mixture is prepared containing the
product of the gene and the intra- or extracellular component under
conditions and for a time allowing for the interaction and binding
of the two products. To test the ability of a candidate compound to
inhibit binding, the reaction is run in the absence and in the
presence of the test compound. In addition, a placebo may be added
to a third reaction mixture, to serve as positive control. The
binding (complex formation) between the test compound and the
intra- or extracellular component present in the mixture is
monitored as described hereinabove. The formation of a complex in
the control reaction(s) but not in the reaction mixture containing
the test compound indicates that the test compound interferes with
the interaction of the test compound and its reaction partner.
[0583] To assay for antagonists, the PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide may be added to a cell along with the compound to be
screened for a particular activity and the ability of the compound
to inhibit the activity of interest in the presence of the PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide indicates that the compound is an
antagonist to the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide.
Alternatively, antagonists may be detected by combining the PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide and a potential antagonist with
membrane-bound PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide
receptors or recombinant receptors under appropriate conditions for
a competitive inhibition assay. The PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide can be labeled, such as by radioactivity, such that the
number of PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 polypeptide molecules bound
to the receptor can be used to determine the effectiveness of the
potential antagonist. The gene encoding the receptor can be
identified by numerous methods known to those of skill in the art,
for example, ligand panning and FACS sorting. Coligan et al.,
Current Protocols in Immun., 1(2): Chapter 5 (1991). Preferably,
expression cloning is employed wherein polyadenylated RNA is
prepared from a cell responsive to the PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide and a cDNA library created from this RNA is divided
into pools and used to transfect COS cells or other cells that are
not responsive to the PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide. Transfected cells that are grown on glass slides are
exposed to labeled PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide. The
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide can be labeled by a
variety of means including iodination or inclusion of a recognition
site for a site-specific protein kinase. Following fixation and
incubation, the slides are subjected to autoradiographic analysis.
Positive pools are identified and sub-pools are prepared and
re-transfected using an interactive sub-pooling and re-screening
process, eventually yielding a single clone that encodes the
putative receptor.
[0584] As an alternative approach for receptor identification, the
labeled PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 polypeptide can be
photoaffinity-linked with cell membrane or extract preparations
that express the receptor molecule. Cross-linked material is
resolved by PAGE and exposed to X-ray film. The labeled complex
containing the receptor can be excised, resolved into peptide
fragments, and subjected to protein micro-sequencing. The amino
acid sequence obtained from micro-sequencing would be used to
design a set of degenerate oligonucleotide probes to screen a cDNA
library to identify the gene encoding the putative receptor.
[0585] Another approach in assessing the effect of an antagonist to
a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide, would be administering a
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 antagonist to a wild-type mouse in
order to mimic a known knockout phenotype. Thus, one would
initially knockout the PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 gene of
interest and observe the resultant phenotype as a consequence of
knocking out or disrupting the PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
gene. Subsequently, one could then assess the effectiveness of an
antagonist to the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide by
administering an antagonist to the PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide to a wild-type mouse. An effective antagonist would be
expected to mimic the phenotypic effect that was initially observed
in the knockout animal.
[0586] Likewise, one could assess the effect of an agonist to a
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide, by administering a
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 agonist to a non-human transgenic
mouse in order to ameliorate a known negative knockout phenotype.
Thus, one would initially knockout the PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
gene of interest and observe the resultant phenotype as a
consequence of knocking out or disrupting the PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 gene. Subsequently, one could then assess the effectiveness
of an agonist to the PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide by administering an agonist to the PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 polypeptide to a the non-human transgenic mouse. An
effective agonist would be expected to ameliorate the negative
phenotypic effect that was initially observed in the knockout
animal.
[0587] In another assay for antagonists, mammalian cells or a
membrane preparation expressing the receptor would be incubated
with a labeled PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide in the
presence of the candidate compound. The ability of the compound to
enhance or block this interaction could then be measured.
[0588] More specific examples of potential antagonists include an
oligonucleotide that binds to the fusions of immunoglobulin with
the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide, and, in particular,
antibodies including, without limitation, poly- and monoclonal
antibodies and antibody fragments, single-chain antibodies,
anti-idiotypic antibodies, and chimeric or humanized versions of
such antibodies or fragments, as well as human antibodies and
antibody fragments. Alternatively, a potential antagonist may be a
closely related protein, for example, a mutated form of the PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide that recognizes the receptor but
imparts no effect, thereby competitively inhibiting the action of
the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide.
[0589] Another potential PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide antagonist is an antisense RNA or DNA construct
prepared using antisense technology, where, e.g., an antisense RNA
or DNA molecule acts to block directly the translation of mRNA by
hybridizing to targeted mRNA and preventing protein translation.
Antisense technology can be used to control gene expression through
triple-helix formation or antisense DNA or RNA, both of which
methods are based on binding of a polynucleotide to DNA or RNA. For
example, the 5' coding portion of the polynucleotide sequence,
which encodes the mature PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptides herein, is used to design an antisense RNA
oligonucleotide of from about 10 to 40 base pairs in length. A DNA
oligonucleotide is designed to be complementary to a region of the
gene involved in transcription (triple helix--see Lee et al., Nucl.
Acids Res., 6:3073 (1979); Cooney et al., Science, 241: 456 (1988);
Dervan et al., Science, 251:1360 (1991)), thereby preventing
transcription and the production of the PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide. The antisense RNA oligonucleotide hybridizes to the
mRNA in vivo and blocks translation of the mRNA molecule into the
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide (antisense--Okano,
Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense
Inhibitors of Gene Expression (CRC Press: Boca Raton, Fla., 1988).
The oligonucleotides described above can also be delivered to cells
such that the antisense RNA or DNA may be expressed in vivo to
inhibit production of the PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide. When antisense DNA is used, oligodeoxyribonucleotides
derived from the translation-initiation site, e.g., between about
-10 and +10 positions of the target gene nucleotide sequence, are
preferred.
[0590] Potential antagonists include small molecules that bind to
the active site, the receptor binding site, or growth factor or
other relevant binding site of the PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide, thereby blocking the normal biological activity of the
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide. Examples of small
molecules include, but are not limited to, small peptides or
peptide-like molecules, preferably soluble peptides, and synthetic
non-peptidyl organic or inorganic compounds.
[0591] Ribozymes are enzymatic RNA molecules capable of catalyzing
the specific cleavage of RNA. Ribozymes act by sequence-specific
hybridization to the complementary target RNA, followed by
endonucleolytic cleavage. Specific ribozyme cleavage sites within a
potential RNA target can be identified by known techniques. For
further details see, e.g., Rossi, Current Biology, 4:469-471
(1994), and PCT publication No. WO 97/33551 (published Sep. 18,
1997).
[0592] Nucleic acid molecules in triple-helix formation used to
inhibit transcription should be single-stranded and composed of
deoxynucleotides. The base composition of these oligonucleotides is
designed such that it promotes triple-helix formation via Hoogsteen
base-pairing rules, which generally require sizeable stretches of
purines or pyrimidines on one strand of a duplex. For further
details see, e.g., PCT publication No. WO 97/33551, supra.
[0593] These small molecules can be identified by any one or more
of the screening assays discussed hereinabove and/or by any other
screening techniques well known for those skilled in the art.
[0594] Diagnostic and therapeutic uses of the herein disclosed
molecules may also be based upon the positive functional assay hits
disclosed and described below.
[0595] F. Anti-PRO196, Anti-PRO217, Anti-PRO231, Anti-PRO236,
Anti-PRO245, Anti-PRO246, Anti-PRO258, Anti-PRO287, Anti-PRO328,
Anti-PRO344, Anti-PRO357, Anti-PRO526, Anti-PRO724, Anti-PRO731,
Anti-PRO732, Anti-PRO1003, Anti-PRO1104, Anti-PRO1151,
Anti-PRO1244, Anti-PRO1298, Anti-PRO1313, Anti-PRO1570,
Anti-PRO1886, Anti-PRO1891, Anti-PRO4409, Anti-PRO5725,
Anti-PRO5994, Anti-PRO6097, Anti-PRO7425, Anti-PRO10102,
Anti-PRO10282, Anti-PRO61709 or Anti-PRO779 Antibodies
[0596] The present invention provides anti-PRO196, anti-PRO217,
anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258,
anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526,
anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104,
anti-PRO1151, anti-PRO1244, anti-PRO1298, anti-PRO1313,
anti-PRO1570, anti-PRO1886, anti-PRO1891, anti-PRO4409,
anti-PRO5725, anti-PRO5994, anti-PRO6097, anti-PRO7425,
anti-PRO10102, anti-PRO10282, anti-PRO61709 or anti-PRO779
antibodies which may find use herein as therapeutic and/or
diagnostic agents. Exemplary antibodies include polyclonal,
monoclonal, humanized, bispecific, and heteroconjugate
antibodies.
[0597] 1. Polyclonal Antibodies
[0598] Polyclonal antibodies are preferably raised in animals by
multiple subcutaneous (sc) or intraperitoneal (ip) injections of
the relevant antigen and an adjuvant. It may be useful to conjugate
the relevant antigen (especially when synthetic peptides are used)
to a protein that is immunogenic in the species to be immunized.
For example, the antigen can be conjugated to keyhole limpet
hemocyanin (KLH), serum albumin, bovine thyroglobulin, or soybean
trypsin inhibitor, using a bifunctional or derivatizing agent,
e.g., maleimidobenzoyl sulfosuccinimide ester (conjugation through
cysteine residues), N-hydroxysuccinimide (through lysine residues),
glutaraldehyde, succinic anhydride, SOCl.sub.2, or
R.sup.1N.dbd.C.dbd.NR, where R and R.sup.1 are different alkyl
groups.
[0599] Animals are immunized against the antigen, immunogenic
conjugates, or derivatives by combining, e.g., 100 .mu.g or 5 .mu.g
of the protein or conjugate (for rabbits or mice, respectively)
with 3 volumes of Freund's complete adjuvant and injecting the
solution intradermally at multiple sites. One month later, the
animals are boosted with 1/5 to 1/10 the original amount of peptide
or conjugate in Freund's complete adjuvant by subcutaneous
injection at multiple sites. Seven to 14 days later, the animals
are bled and the serum is assayed for antibody titer. Animals are
boosted until the titer plateaus. Conjugates also can be made in
recombinant cell culture as protein fusions. Also, aggregating
agents such as alum are suitably used to enhance the immune
response.
[0600] 2. Monoclonal Antibodies
[0601] Monoclonal antibodies may be made using the hybridoma method
first described by Kohler et al., Nature, 256:495 (1975), or may be
made by recombinant DNA methods (U.S. Pat. No. 4,816,567).
[0602] In the hybridoma method, a mouse or other appropriate host
animal, such as a hamster, is immunized as described above to
elicit lymphocytes that produce or are capable of producing
antibodies that will specifically bind to the protein used for
immunization. Alternatively, lymphocytes may be immunized in vitro.
After immunization, lymphocytes are isolated and then fused with a
myeloma cell line using a suitable fusing agent, such as
polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal
Antibodies: Principles and Practice, pp. 59-103 (Academic Press,
1986)).
[0603] The hybridoma cells thus prepared are seeded and grown in a
suitable culture medium which medium preferably contains one or
more substances that inhibit the growth or survival of the unfused,
parental myeloma cells (also referred to as fusion partner). For
example, if the parental myeloma cells lack the enzyme hypoxanthine
guanine phosphoribosyl transferase (HGPRT or HPRT), the selective
culture medium for the hybridomas typically will include
hypoxanthine, aminopterin, and thymidine (HAT medium), which
substances prevent the growth of HGPRT-deficient cells.
[0604] Preferred fusion partner myeloma cells are those that fuse
efficiently, support stable high-level production of antibody by
the selected antibody-producing cells, and are sensitive to a
selective medium that selects against the unfused parental cells.
Preferred myeloma cell lines are murine myeloma lines, such as
those derived from MOPC-21 and MPC-11 mouse tumors available from
the Salk Institute Cell Distribution Center, San Diego, Calif. USA,
and SP-2 and derivatives e.g., X63-Ag8-653 cells available from the
American Type Culture Collection, Manassas, Va., USA. Human myeloma
and mouse-human heteromyeloma cell lines also have been described
for the production of human monoclonal antibodies (Kozbor, J.
Immunol., 133:3001 (1984); and Brodeur et al., Monoclonal Antibody
Production Techniques and Applications, pp. 51-63 (Marcel Dekker,
Inc., New York, 1987)).
[0605] Culture medium in which hybridoma cells are growing is
assayed for production of monoclonal antibodies directed against
the antigen. Preferably, the binding specificity of monoclonal
antibodies produced by hybridoma cells is determined by
immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunosorbent assay
(ELISA).
[0606] The binding affinity of the monoclonal antibody can, for
example, be determined by the Scatchard analysis described in
Munson et al., Anal. Biochem., 107:220 (1980).
[0607] Once hybridoma cells that produce antibodies of the desired
specificity, affinity, and/or activity are identified, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods (Goding, Monoclonal Antibodies: Principles and
Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture
media for this purpose include, for example, D-MEM or RPMI-1640
medium. In addition, the hybridoma cells may be grown in vivo as
ascites tumors in an animal e.g., by i.p. injection of the cells
into mice.
[0608] The monoclonal antibodies secreted by the subclones are
suitably separated from the culture medium, ascites fluid, or serum
by conventional antibody purification procedures such as, for
example, affinity chromatography (e.g., using protein A or protein
G-Sepharose) or ion-exchange chromatography, hydroxylapatite
chromatography, gel electrophoresis, dialysis, etc.
[0609] DNA encoding the monoclonal antibodies is readily isolated
and sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of murine antibodies).
The hybridoma cells serve as a preferred source of such DNA. Once
isolated, the DNA may be placed into expression vectors, which are
then transfected into host cells such as E. coli cells, simian COS
cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do
not otherwise produce antibody protein, to obtain the synthesis of
monoclonal antibodies in the recombinant host cells. Review
articles on recombinant expression in bacteria of DNA encoding the
antibody include Skerra et al., Curr. Opinion in Immunol.,
5:256-262 (1993) and Pluckthun, Immunol. Revs. 130:151-188
(1992).
[0610] Monoclonal antibodies or antibody fragments can be isolated
from antibody phage libraries generated using the techniques
described in McCafferty et al., Nature, 348:552-554 (1990).
Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J.
Mol. Biol., 222:581-597 (1991) describe the isolation of murine and
human antibodies, respectively, using phage libraries. Subsequent
publications describe the production of high affinity (nM range)
human antibodies by chain shuffling (Marks et al., Bio/Technology,
10:779-783 (1992)), as well as combinatorial infection and in vivo
recombination as a strategy for constructing very large phage
libraries (Waterhouse et al., Nuc. Acids. Res. 21:2265-2266
(1993)). Thus, these techniques are viable alternatives to
traditional monoclonal antibody hybridoma techniques for isolation
of monoclonal antibodies.
[0611] The DNA that encodes the antibody may be modified to produce
chimeric or fusion antibody polypeptides, for example, by
substituting human heavy chain and light chain constant domain
(C.sub.H and C.sub.L) sequences for the homologous murine sequences
(U.S. Pat. No. 4,816,567; and Morrison, et al., Proc. Natl. Acad.
Sci. USA, 81:6851 (1984)), or by fusing the immunoglobulin coding
sequence with all or part of the coding sequence for a
non-immunoglobulin polypeptide (heterologous polypeptide). The
non-immunoglobulin polypeptide sequences can substitute for the
constant domains of an antibody, or they are substituted for the
variable domains of one antigen-combining site of an antibody to
create a chimeric bivalent antibody comprising one
antigen-combining site having specificity for an antigen and
another antigen-combining site having specificity for a different
antigen.
[0612] 3. Human and Humanized Antibodies
[0613] The anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236,
anti-PRO245, anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328,
anti-PRO344, anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731,
anti-PRO732, anti-PRO1003, anti-PRO1104, anti-PRO1151,
anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570,
anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725,
anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102,
anti-PRO10282, anti-PRO61709 or anti-PRO779 antibodies of the
invention may further comprise humanized antibodies or human
antibodies. Humanized forms of non-human (e.g., murine) antibodies
are chimeric immunoglobulins, immunoglobulin chains or fragments
thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other
antigen-binding subsequences of antibodies) which contain minimal
sequence derived from non-human immunoglobulin. Humanized
antibodies include human immunoglobulins (recipient antibody) in
which residues from a complementary determining region (CDR) of the
recipient are replaced by residues from a CDR of a non-human
species (donor antibody) such as mouse, rat or rabbit having the
desired specificity, affinity and capacity. In some instances, Fv
framework residues of the human immunoglobulin are replaced by
corresponding non-human residues. Humanized antibodies may also
comprise residues which are found neither in the recipient antibody
nor in the imported CDR or framework sequences. In general, the
humanized antibody will comprise substantially all of at least one,
and typically two, variable domains, in which all or substantially
all of the CDR regions correspond to those of a non-human
immunoglobulin and all or substantially all of the FR regions are
those of a human immunoglobulin consensus sequence. The humanized
antibody optimally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann
et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct.
Biol., 2:593-596 (1992)].
[0614] Methods for humanizing non-human antibodies are well known
in the art. Generally, a humanized antibody has one or more amino
acid residues introduced into it from a source which is non-human.
These non-human amino acid residues are often referred to as
"import" residues, which are typically taken from an "import"
variable domain. Humanization can be essentially performed
following the method of Winter and co-workers [Jones et al.,
Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327
(1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by
substituting rodent CDRs or CDR sequences for the corresponding
sequences of a human antibody. Accordingly, such "humanized"
antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567),
wherein substantially less than an intact human variable domain has
been substituted by the corresponding sequence from a non-human
species. In practice, humanized antibodies are typically human
antibodies in which some CDR residues and possibly some FR residues
are substituted by residues from analogous sites in rodent
antibodies.
[0615] The choice of human variable domains, both light and heavy,
to be used in making the humanized antibodies is very important to
reduce antigenicity and HAMA response (human anti-mouse antibody)
when the antibody is intended for human therapeutic use. According
to the so-called "best-fit" method, the sequence of the variable
domain of a rodent antibody is screened against the entire library
of known human variable domain sequences. The human V domain
sequence which is closest to that of the rodent is identified and
the human framework region (FR) within it accepted for the
humanized antibody (Sims et al., J. Immunol. 151:2296 (1993);
Chothia et al., J. Mol. Biol., 196:901 (1987)). Another method uses
a particular framework region derived from the consensus sequence
of all human antibodies of a particular subgroup of light or heavy
chains. The same framework may be used for several different
humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA,
89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993)).
[0616] It is further important that antibodies be humanized with
retention of high binding affinity for the antigen and other
favorable biological properties. To achieve this goal, according to
a preferred method, humanized antibodies are prepared by a process
of analysis of the parental sequences and various conceptual
humanized products using three-dimensional models of the parental
and humanized sequences. Three-dimensional immunoglobulin models
are commonly available and are familiar to those skilled in the
art. Computer programs are available which illustrate and display
probable three-dimensional conformational structures of selected
candidate immunoglobulin sequences. Inspection of these displays
permits analysis of the likely role of the residues in the
functioning of the candidate immunoglobulin sequence, i.e., the
analysis of residues that influence the ability of the candidate
immunoglobulin to bind its antigen. In this way, FR residues can be
selected and combined from the recipient and import sequences so
that the desired antibody characteristic, such as increased
affinity for the target antigen(s), is achieved. In general, the
hypervariable region residues are directly and most substantially
involved in influencing antigen binding.
[0617] Various forms of a humanized anti-PRO196, anti-PRO217,
anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258,
anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526,
anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104,
anti-PRO1151, anti-PRO1244, anti-PRO1298, anti-PRO1313,
anti-PRO1570, anti-PRO1886, anti-PRO1891, anti-PRO4409,
anti-PRO5725, anti-PRO5994, anti-PRO6097, anti-PRO7425,
anti-PRO10102, anti-PRO10282, anti-PRO61709 or anti-PRO779 antibody
are contemplated. For example, the humanized antibody may be an
antibody fragment, such as a Fab, which is optionally conjugated
with one or more cytotoxic agent(s) in order to generate an
immunoconjugate. Alternatively, the humanized antibody may be an
intact antibody, such as an intact IgG1 antibody.
[0618] As an alternative to humanization, human antibodies can be
generated. For example, it is now possible to produce transgenic
animals (e.g., mice) that are capable, upon immunization, of
producing a full repertoire of human antibodies in the absence of
endogenous immunoglobulin production. For example, it has been
described that the homozygous deletion of the antibody heavy-chain
joining region (J.sub.H) gene in chimeric and germ-line mutant mice
results in complete inhibition of endogenous antibody production.
Transfer of the human germ-line immunoglobulin gene array into such
germ-line mutant mice will result in the production of human
antibodies upon antigen challenge. See, e.g., Jakobovits et al.,
Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al.,
Nature, 362:255-258 (1993); Bruggemann et al., Year in Immuno. 7:33
(1993); U.S. Pat. Nos. 5,545,806, 5,569,825, 5,591,669 (all of
GenPharm); 5,545,807; and WO 97/17852.
[0619] Alternatively, phage display technology (McCafferty et al.,
Nature 348:552-553 [1990]) can be used to produce human antibodies
and antibody fragments in vitro, from immunoglobulin variable (V)
domain gene repertoires from unimmunized donors. According to this
technique, antibody V domain genes are cloned in-frame into either
a major or minor coat protein gene of a filamentous bacteriophage,
such as M13 or fd, and displayed as functional antibody fragments
on the surface of the phage particle. Because the filamentous
particle contains a single-stranded DNA copy of the phage genome,
selections based on the functional properties of the antibody also
result in selection of the gene encoding the antibody exhibiting
those properties. Thus, the phage mimics some of the properties of
the B-cell. Phage display can be performed in a variety of formats,
reviewed in, e.g., Johnson, Kevin S, and Chiswell, David J.,
Current Opinion in Structural Biology 3:564-571 (1993). Several
sources of V-gene segments can be used for phage display. Clackson
et al., Nature, 352:624-628 (1991) isolated a diverse array of
anti-oxazolone antibodies from a small random combinatorial library
of V genes derived from the spleens of immunized mice. A repertoire
of V genes from unimmunized human donors can be constructed and
antibodies to a diverse array of antigens (including self-antigens)
can be isolated essentially following the techniques described by
Marks et al., J. Mol. Biol. 222:581-597 (1991), or Griffith et al.,
EMBO J. 12:725-734 (1993). See, also, U.S. Pat. Nos. 5,565,332 and
5,573,905.
[0620] As discussed above, human antibodies may also be generated
by in vitro activated B cells (see U.S. Pat. Nos. 5,567,610 and
5,229,275).
[0621] 4. Antibody Fragments
[0622] In certain circumstances there are advantages of using
antibody fragments, rather than whole antibodies. The smaller size
of the fragments allows for rapid clearance, and may lead to
improved access to solid tumors.
[0623] Various techniques have been developed for the production of
antibody fragments. Traditionally, these fragments were derived via
proteolytic digestion of intact antibodies (see, e.g., Morimoto et
al., Journal of Biochemical and Biophysical Methods 24:107-117
(1992); and Brennan et al., Science, 229:81 (1985)). However, these
fragments can now be produced directly by recombinant host cells.
Fab, Fv and ScFv antibody fragments can all be expressed in and
secreted from E. coli, thus allowing the facile production of large
amounts of these fragments. Antibody fragments can be isolated from
the antibody phage libraries discussed above. Alternatively,
Fab'-SH fragments can be directly recovered from E. coli and
chemically coupled to form F(ab').sub.2 fragments (Carter et al.,
Bio/Technology 10:163-167 (1992)). According to another approach,
F(ab').sub.2 fragments can be isolated directly from recombinant
host cell culture. Fab and F(ab').sub.2 fragment with increased in
vivo half-life comprising a salvage receptor binding epitope
residues are described in U.S. Pat. No. 5,869,046. Other techniques
for the production of antibody fragments will be apparent to the
skilled practitioner. The antibody of choice is a single chain Fv
fragment (scFv). See WO 93/16185; U.S. Pat. No. 5,571,894; and U.S.
Pat. No. 5,587,458. Fv and sFv are the only species with intact
combining sites that are devoid of constant regions; thus, they are
suitable for reduced nonspecific binding during in vivo use. sFv
fusion proteins may be constructed to yield fusion of an effector
protein at either the amino or the carboxy terminus of an sFv. See
Antibody Engineering, ed. Borrebaeck, supra. The antibody fragment
may also be a "linear antibody", e.g., as described in U.S. Pat.
No. 5,641,870 for example. Such linear antibody fragments may be
monospecific or bispecific.
[0624] 5. Bispecific Antibodies
[0625] Bispecific antibodies are antibodies that have binding
specificities for at least two different epitopes. Exemplary
bispecific antibodies may bind to two different epitopes of a
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 protein as described herein. Other
such antibodies may combine a PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
binding site with a binding site for another protein.
Alternatively, an anti-PRO196, anti-PRO217, anti-PRO231,
anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258, anti-PRO287,
anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526, anti-PRO724,
anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104, anti-PRO1151,
anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570,
anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725,
anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102,
anti-PRO10282, anti-PRO61709 or anti-PRO779 arm may be combined
with an arm which binds to a triggering molecule on a leukocyte
such as a T-cell receptor molecule (e.g. CD3), or Fc receptors for
IgG (Fc.gamma.R), such as Fc.gamma.RI (CD64), Fc.gamma.RII (CD32)
and Fc.gamma.RIII (CD16), so as to focus and localize cellular
defense mechanisms to the PRO196-, PRO217-, PRO231-, PRO236-,
PRO245-, PRO246-, PRO258-, PRO287-, PRO328-, PRO344-, PRO357-,
PRO526-, PRO724-, PRO731-, PRO732-, PRO1003-, PRO1104-, PRO1151-,
PRO1244-, PRO1298-, PRO1313-, PRO1570-, PRO1886-, PRO1891-,
PRO4409-, PRO5725-, PRO5994-, PRO6097-, PRO7425-, PRO10102-,
PRO10282-, PRO61709- or PRO779-expressing cell. Bispecific
antibodies may also be used to localize cytotoxic agents to cells
which express a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide. These
antibodies possess a PRO196-, PRO217-, PRO231-, PRO236-, PRO245-,
PRO246-, PRO258-, PRO287-, PRO328-, PRO344-, PRO357-, PRO526-,
PRO724-, PRO731-, PRO732-, PRO1003-, PRO1104-, PRO1151-, PRO1244-,
PRO1298-, PRO1313-, PRO1570-, PRO1886-, PRO1891-, PRO4409-,
PRO5725-, PRO5994-, PRO6097-, PRO7425-, PRO10102-, PRO10282-,
PRO61709- or PRO779-binding arm and an arm which binds the
cytotoxic agent (e.g., saporin, anti-interferon-.alpha., vinca
alkaloid, ricin A chain, methotrexate or radioactive isotope
hapten). Bispecific antibodies can be prepared as full length
antibodies or antibody fragments (e.g., F(ab').sub.2 bispecific
antibodies).
[0626] WO 96/16673 describes a bispecific
anti-ErbB2/anti-Fc.gamma.RIII antibody and U.S. Pat. No. 5,837,234
discloses a bispecific anti-ErbB2/anti-Fc.gamma.RI antibody. A
bispecific anti-ErbB2/Fc.alpha. antibody is shown in WO98/02463.
U.S. Pat. No. 5,821,337 teaches a bispecific anti-ErbB2/anti-CD3
antibody.
[0627] Methods for making bispecific antibodies are known in the
art. Traditional production of full length bispecific antibodies is
based on the co-expression of two immunoglobulin heavy chain-light
chain pairs, where the two chains have different specificities
(Millstein et al., Nature 305:537-539 (1983)). Because of the
random assortment of immunoglobulin heavy and light chains, these
hybridomas (quadromas) produce a potential mixture of 10 different
antibody molecules, of which only one has the correct bispecific
structure. Purification of the correct molecule, which is usually
done by affinity chromatography steps, is rather cumbersome, and
the product yields are low. Similar procedures are disclosed in WO
93/08829, and in Traunecker et al., EMBO J. 10:3655-3659
(1991).
[0628] According to a different approach, antibody variable domains
with the desired binding specificity (antibody-antigen combining
sites) are fused to immunoglobulin constant domain sequences.
Preferably, the fusion is with an Ig heavy chain constant domain,
comprising at least part of the hinge, C.sub.H2, and C.sub.H3
regions. It is preferred to have the first heavy-chain constant
region (C.sub.H1) containing the site necessary for light chain
bonding, present in at least one of the fusions. DNAs encoding the
immunoglobulin heavy chain fusions and, if desired, the
immunoglobulin light chain, are inserted into separate expression
vectors, and are co-transfected into a suitable host cell. This
provides for greater flexibility in adjusting the mutual
proportions of the three polypeptide fragments when unequal ratios
of the three polypeptide chains used in the construction provide
the optimum yield of the desired bispecific antibody. It is,
however, possible to insert the coding sequences for two or all
three polypeptide chains into a single expression vector when the
expression of at least two polypeptide chains in equal ratios
results in high yields or when the ratios have no significant
affect on the yield of the desired chain combination.
[0629] The invention provides bispecific antibodies which are
composed of a hybrid immunoglobulin heavy chain with a first
binding specificity in one arm, and a hybrid immunoglobulin heavy
chain-light chain pair (providing a second binding specificity) in
the other arm. It was found that this asymmetric structure
facilitates the separation of the desired bispecific compound from
unwanted immunoglobulin chain combinations, as the presence of an
immunoglobulin light chain in only one half of the bispecific
molecule provides for a facile way of separation. This approach is
disclosed in WO 94/04690. For further details of generating
bispecific antibodies see, for example, Suresh et al., Methods in
Enzymology 121:210 (1986).
[0630] According to another approach described in U.S. Pat. No.
5,731,168, the interface between a pair of antibody molecules can
be engineered to maximize the percentage of heterodimers which are
recovered from recombinant cell culture. The preferred interface
comprises at least a part of the C.sub.H3 domain. In this method,
one or more small amino acid side chains from the interface of the
first antibody molecule are replaced with larger side chains (e.g.,
tyrosine or tryptophan). Compensatory "cavities" of identical or
similar size to the large side chain(s) are created on the
interface of the second antibody molecule by replacing large amino
acid side chains with smaller ones (e.g., alanine or threonine).
This provides a mechanism for increasing the yield of the
heterodimer over other unwanted end-products such as
homodimers.
[0631] Bispecific antibodies include cross-linked or
"heteroconjugate" antibodies. For example, one of the antibodies in
the heteroconjugate can be coupled to avidin, the other to biotin.
Such antibodies have, for example, been proposed to target immune
system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for
treatment of HIV infection (WO 91/00360, WO 92/200373, and EP
03089). Heteroconjugate antibodies may be made using any convenient
cross-linking methods. Suitable cross-linking agents are well known
in the art, and are disclosed in U.S. Pat. No. 4,676,980, along
with a number of cross-linking techniques.
[0632] Techniques for generating bispecific antibodies from
antibody fragments have also been described in the literature. For
example, bispecific antibodies can be prepared using chemical
linkage. Brennan et al., Science 229:81 (1985) describe a procedure
wherein intact antibodies are proteolytically cleaved to generate
F(ab').sub.2 fragments. These fragments are reduced in the presence
of the dithiol complexing agent, sodium arsenite, to stabilize
vicinal dithiols and prevent intermolecular disulfide formation.
The Fab' fragments generated are then converted to
thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB
derivatives is then reconverted to the Fab'-thiol by reduction with
mercaptoethylamine and is mixed with an equimolar amount of the
other Fab'-TNB derivative to form the bispecific antibody. The
bispecific antibodies produced can be used as agents for the
selective immobilization of enzymes.
[0633] Recent progress has facilitated the direct recovery of
Fab'-SH fragments from E. coli, which can be chemically coupled to
form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:
217-225 (1992) describe the production of a fully humanized
bispecific antibody F(ab').sub.2 molecule. Each Fab' fragment was
separately secreted from E. coli and subjected to directed chemical
coupling in vitro to form the bispecific antibody. The bispecific
antibody thus formed was able to bind to cells overexpressing the
ErbB2 receptor and normal human T cells, as well as trigger the
lytic activity of human cytotoxic lymphocytes against human breast
tumor targets. Various techniques for making and isolating
bispecific antibody fragments directly from recombinant cell
culture have also been described. For example, bispecific
antibodies have been produced using leucine zippers. Kostelny et
al., J. Immunol. 148(5):1547-1553 (1992). The leucine zipper
peptides from the Fos and Jun proteins were linked to the Fab'
portions of two different antibodies by gene fusion. The antibody
homodimers were reduced at the hinge region to form monomers and
then re-oxidized to form the antibody heterodimers. This method can
also be utilized for the production of antibody homodimers. The
"diabody" technology described by Hollinger et al., Proc. Natl.
Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative
mechanism for making bispecific antibody fragments. The fragments
comprise a V.sub.H connected to a V.sub.L by a linker which is too
short to allow pairing between the two domains on the same chain.
Accordingly, the V.sub.H and V.sub.L domains of one fragment are
forced to pair with the complementary V.sub.L and V.sub.H domains
of another fragment, thereby forming two antigen-binding sites.
Another strategy for making bispecific antibody fragments by the
use of single-chain Fv (sFv) dimers has also been reported. See
Gruber et al., J. Immunol., 152:5368 (1994).
[0634] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. Tutt et al.,
J. Immunol. 147:60 (1991).
[0635] 6. Heteroconjugate Antibodies
[0636] Heteroconjugate antibodies are also within the scope of the
present invention. Heteroconjugate antibodies are composed of two
covalently joined antibodies. Such antibodies have, for example,
been proposed to target immune system cells to unwanted cells [U.S.
Pat. No. 4,676,980], and for treatment of HIV infection [WO
91/00360; WO 92/200373; EP 03089]. It is contemplated that the
antibodies may be prepared in vitro using known methods in
synthetic protein chemistry, including those involving crosslinking
agents. For example, immunotoxins may be constructed using a
disulfide exchange reaction or by forming a thioether bond.
Examples of suitable reagents for this purpose include
iminothiolate and methyl-4-mercaptobutyrimidate and those
disclosed, for example, in U.S. Pat. No. 4,676,980.
[0637] 7. Multivalent Antibodies
[0638] A multivalent antibody may be internalized (and/or
catabolized) faster than a bivalent antibody by a cell expressing
an antigen to which the antibodies bind. The antibodies of the
present invention can be multivalent antibodies (which are other
than of the IgM class) with three or more antigen binding sites
(e.g. tetravalent antibodies), which can be readily produced by
recombinant expression of nucleic acid encoding the polypeptide
chains of the antibody. The multivalent antibody can comprise a
dimerization domain and three or more antigen binding sites. The
preferred dimerization domain comprises (or consists of) an Fc
region or a hinge region. In this scenario, the antibody will
comprise an Fc region and three or more antigen binding sites
amino-terminal to the Fc region. The preferred multivalent antibody
herein comprises (or consists of) three to about eight, but
preferably four, antigen binding sites. The multivalent antibody
comprises at least one polypeptide chain (and preferably two
polypeptide chains), wherein the polypeptide chain(s) comprise two
or more variable domains. For instance, the polypeptide chain(s)
may comprise VD1-(X1).sub.n-VD2-(X2).sub.n-Fc, wherein VD1 is a
first variable domain, VD2 is a second variable domain, Fc is one
polypeptide chain of an Fc region, X1 and X2 represent an amino
acid or polypeptide, and n is 0 or 1. For instance, the polypeptide
chain(s) may comprise: VH-CH1-flexible linker-VH-CH1-Fc region
chain; or VH-CH1-VH-CH1-Fc region chain. The multivalent antibody
herein preferably further comprises at least two (and preferably
four) light chain variable domain polypeptides. The multivalent
antibody herein may, for instance, comprise from about two to about
eight light chain variable domain polypeptides. The light chain
variable domain polypeptides contemplated here comprise a light
chain variable domain and, optionally, further comprise a CL
domain.
[0639] 8. Effector Function Engineering
[0640] It may be desirable to modify the antibody of the invention
with respect to effector function, e.g., so as to enhance
antigen-dependent cell-mediated cytotoxicity (ADCC) and/or
complement dependent cytotoxicity (CDC) of the antibody. This may
be achieved by introducing one or more amino acid substitutions in
an Fc region of the antibody. Alternatively or additionally,
cysteine residue(s) may be introduced in the Fc region, thereby
allowing interchain disulfide bond formation in this region. The
homodimeric antibody thus generated may have improved
internalization capability and/or increased complement-mediated
cell killing and antibody-dependent cellular cytotoxicity (ADCC).
See Caron et al., J. Exp Med. 176:1191-1195 (1992) and Shopes, B.
J. Immunol. 148:2918-2922 (1992). Homodimeric antibodies with
enhanced anti-tumor activity may also be prepared using
heterobifunctional cross-linkers as described in Wolff et al.,
Cancer Research 53:2560-2565 (1993). Alternatively, an antibody can
be engineered which has dual Fc regions and may thereby have
enhanced complement lysis and ADCC capabilities. See Stevenson et
al., Anti-Cancer Drug Design 3:219-230 (1989). To increase the
serum half life of the antibody, one may incorporate a salvage
receptor binding epitope into the antibody (especially an antibody
fragment) as described in U.S. Pat. No. 5,739,277, for example. As
used herein, the term "salvage receptor binding epitope" refers to
an epitope of the Fc region of an IgG molecule (e.g., IgG.sub.1,
IgG.sub.2, IgG.sub.3, or IgG.sub.4) that is responsible for
increasing the in vivo serum half-life of the IgG molecule.
[0641] 9. Immunoconjugates
[0642] The invention also pertains to immunoconjugates comprising
an antibody conjugated to a cytotoxic agent such as a
chemotherapeutic agent, a growth inhibitory agent, a toxin (e.g.,
an enzymatically active toxin of bacterial, fungal, plant, or
animal origin, or fragments thereof), or a radioactive isotope
(i.e., a radioconjugate).
[0643] Chemotherapeutic agents useful in the generation of such
immunoconjugates have been described above. Enzymatically active
toxins and fragments thereof that can be used include diphtheria A
chain, nonbinding active fragments of diphtheria toxin, exotoxin A
chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain,
modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin
proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S),
momordica charantia inhibitor, curcin, crotin, sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin, and the tricothecenes. A variety of
radionuclides are available for the production of radioconjugated
antibodies. Examples include .sup.212Bi, .sup.131I, .sup.131In,
.sup.90Y, and .sup.186Re. Conjugates of the antibody and cytotoxic
agent are made using a variety of bifunctional protein-coupling
agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate
(SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters
(such as dimethyl adipimidate HCL), active esters (such as
disuccinimidyl suberate), aldehydes (such as glutareldehyde),
bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine),
bis-diazonium derivatives (such as
bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as
tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such
as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin
immunotoxin can be prepared as described in Vitetta et al.,
Science, 238: 1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026.
[0644] Conjugates of an antibody and one or more small molecule
toxins, such as a calicheamicin, maytansinoids, a trichothene, and
CC1065, and the derivatives of these toxins that have toxin
activity, are also contemplated herein.
Maytansine and Maytansinoids
[0645] The invention provides an anti-PRO196, anti-PRO217,
anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258,
anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526,
anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104,
anti-PRO1151, anti-PRO1244, anti-PRO1298, anti-PRO1313,
anti-PRO1570, anti-PRO1886, anti-PRO1891, anti-PRO4409,
anti-PRO5725, anti-PRO5994, anti-PRO6097, anti-PRO7425,
anti-PRO10102, anti-PRO10282, anti-PRO61709 or anti-PRO779 antibody
(full length or fragments) which is conjugated to one or more
maytansinoid molecules.
[0646] Maytansinoids are mitototic inhibitors which act by
inhibiting tubulin polymerization. Maytansine was first isolated
from the east African shrub Maytenus serrata (U.S. Pat. No.
3,896,111). Subsequently, it was discovered that certain microbes
also produce maytansinoids, such as maytansinol and C-3 maytansinol
esters (U.S. Pat. No. 4,151,042). Synthetic maytansinol and
derivatives and analogues thereof are disclosed, for example, in
U.S. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608;
4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428;
4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650;
4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533, the
disclosures of which are hereby expressly incorporated by
reference.
Maytansinoid-Antibody Conjugates
[0647] In an attempt to improve their therapeutic index, maytansine
and maytansinoids have been conjugated to antibodies specifically
binding to tumor cell antigens. Immunoconjugates containing
maytansinoids and their therapeutic use are disclosed, for example,
in U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425
235 B1, the disclosures of which are hereby expressly incorporated
by reference. Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623
(1996) described immunoconjugates comprising a maytansinoid
designated DM1 linked to the monoclonal antibody C242 directed
against human colorectal cancer. The conjugate was found to be
highly cytotoxic towards cultured colon cancer cells, and showed
antitumor activity in an in vivo tumor growth assay. Chari et al.,
Cancer Research 52:127-131 (1992) describe immunoconjugates in
which a maytansinoid was conjugated via a disulfide linker to the
murine antibody A7 binding to an antigen on human colon cancer cell
lines, or to another murine monoclonal antibody TA.1 that binds the
HER-2/neu oncogene. The cytotoxicity of the TA.1-maytansonoid
conjugate was tested in vitro on the human breast cancer cell line
SK-BR-3, which expresses 3.times.10.sup.5 HER-2 surface antigens
per cell. The drug conjugate achieved a degree of cytotoxicity
similar to the free maytansonid drug, which could be increased by
increasing the number of maytansinoid molecules per antibody
molecule. The A7-maytansinoid conjugate showed low systemic
cytotoxicity in mice.
Anti-PRO196, Anti-PRO217, Anti-PRO231, Anti-PRO236, Anti-PRO245,
Anti-PRO246, Anti-PRO258, Anti-PRO287, Anti-PRO328, Anti-PRO344,
Anti-PRO357, Anti-PRO526, Anti-PRO724, Anti-PRO731, Anti-PRO732,
Anti-PRO1003, Anti-PRO1104, Anti-PRO1151, Anti-PRO1244,
Anti-PRO1298, Anti-PRO1313, Anti-PRO1570, Anti-PRO1886,
Anti-PRO1891, Anti-PRO4409, Anti-PRO5725, Anti-PRO5994,
Anti-PRO6097, Anti-PRO7425, Anti-PRO10102, Anti-PRO10282,
Anti-PRO61709 or Anti-PRO779 Antibody-Maytansinoid Conjugates
(Immunoconjugates)
[0648] Anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236,
anti-PRO245, anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328,
anti-PRO344, anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731,
anti-PRO732, anti-PRO1003, anti-PRO1104, anti-PRO1151,
anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570,
anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725,
anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102,
anti-PRO10282, anti-PRO61709 or anti-PRO779 antibody-maytansinoid
conjugates are prepared by chemically linking an anti-PRO196,
anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246,
anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357,
anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003,
anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298,
anti-PRO1313, anti-PRO1570, anti-PRO1886, anti-PRO1891,
anti-PRO4409, anti-PRO5725, anti-PRO5994, anti-PRO6097,
anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709 or
anti-PRO779 antibody to a maytansinoid molecule without
significantly diminishing the biological activity of either the
antibody or the maytansinoid molecule. An average of 3-4
maytansinoid molecules conjugated per antibody molecule has shown
efficacy in enhancing cytotoxicity of target cells without
negatively affecting the function or solubility of the antibody,
although even one molecule of toxin/antibody would be expected to
enhance cytotoxicity over the use of naked antibody. Maytansinoids
are well known in the art and can be synthesized by known
techniques or isolated from natural sources. Suitable maytansinoids
are disclosed, for example, in U.S. Pat. No. 5,208,020 and in the
other patents and nonpatent publications referred to hereinabove.
Preferred maytansinoids are maytansinol and maytansinol analogues
modified in the aromatic ring or at other positions of the
maytansinol molecule, such as various maytansinol esters.
[0649] There are many linking groups known in the art for making
antibody-maytansinoid conjugates, including, for example, those
disclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, and
Chari et al., Cancer Research 52:127-131 (1992). The linking groups
include disufide groups, thioether groups, acid labile groups,
photolabile groups, peptidase labile groups, or esterase labile
groups, as disclosed in the above-identified patents, disulfide and
thioether groups being preferred.
[0650] Conjugates of the antibody and maytansinoid may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCL), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutareldehyde), bis-azido compounds
(such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as toluene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).
Particularly preferred coupling agents include
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Carlsson et
al., Biochem. J. 173:723-737 [1978]) and
N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for a
disulfide linkage.
[0651] The linker may be attached to the maytansinoid molecule at
various positions, depending on the type of the link. For example,
an ester linkage may be formed by reaction with a hydroxyl group
using conventional coupling techniques. The reaction may occur at
the C-3 position having a hydroxyl group, the C-14 position
modified with hyrdoxymethyl, the C-15 position modified with a
hydroxyl group, and the C-20 position having a hydroxyl group. The
linkage is formed at the C-3 position of maytansinol or a
maytansinol analogue.
Calicheamicin
[0652] Another immunoconjugate of interest comprises an
anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245,
anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344,
anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732,
anti-PRO1003, anti-PRO1104, anti-PRO1151, anti-PRO1244,
anti-PRO1298, anti-PRO1313, anti-PRO1570, anti-PRO1886,
anti-PRO1891, anti-PRO4409, anti-PRO5725, anti-PRO5994,
anti-PRO6097, anti-PRO7425, anti-PRO10102, anti-PRO10282,
anti-PRO61709 or anti-PRO779 antibody conjugated to one or more
calicheamicin molecules. The calicheamicin family of antibiotics
are capable of producing double-stranded DNA breaks at
sub-picomolar concentrations. For the preparation of conjugates of
the calicheamicin family, see U.S. Pat. Nos. 5,712,374, 5,714,586,
5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, 5,877,296
(all to American Cyanamid Company). Structural analogues of
calicheamicin which may be used include, but are not limited to,
.gamma..sub.1.sup.1, .alpha..sub.2.sup.1, .alpha..sub.3.sup.1,
N-acetyl-.gamma..sub.1.sup.1, PSAG and .theta..sup.1.sub.1 (Hinman
et al., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer
Research 58:2925-2928 (1998) and the aforementioned U.S. patents to
American Cyanamid). Another anti-tumor drug that the antibody can
be conjugated is QFA which is an antifolate. Both calicheamicin and
QFA have intracellular sites of action and do not readily cross the
plasma membrane. Therefore, cellular uptake of these agents through
antibody mediated internalization greatly enhances their cytotoxic
effects.
Other Cytotoxic Agents
[0653] Other antitumor agents that can be conjugated to the
anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245,
anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344,
anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732,
anti-PRO1003, anti-PRO1104, anti-PRO1151, anti-PRO1244,
anti-PRO1298, anti-PRO1313, anti-PRO1570, anti-PRO1886,
anti-PRO1891, anti-PRO4409, anti-PRO5725, anti-PRO5994,
anti-PRO6097, anti-PRO7425, anti-PRO10102, anti-PRO10282,
anti-PRO61709 or anti-PRO779 antibodies of the invention include
BCNU, streptozoicin, vincristine and 5-fluorouracil, the family of
agents known collectively LL-E33288 complex described in U.S. Pat.
Nos. 5,053,394, 5,770,710, as well as esperamicins (U.S. Pat. No.
5,877,296).
[0654] Enzymatically active toxins and fragments thereof which can
be used include diphtheria A chain, nonbinding active fragments of
diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),
ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin,
Aleurites fordii proteins, dianthin proteins, Phytolaca americana
proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor,
curcin, crotin, sapaonaria officinalis inhibitor, gelonin,
mitogellin, restrictocin, phenomycin, enomycin and the
tricothecenes. See, for example, WO 93/21232 published Oct. 28,
1993.
[0655] The present invention further contemplates an
immunoconjugate formed between an antibody and a compound with
nucleolytic activity (e.g., a ribonuclease or a DNA endonuclease
such as a deoxyribonuclease; DNase).
[0656] For selective destruction of the tumor, the antibody may
comprise a highly radioactive atom. A variety of radioactive
isotopes are available for the production of radioconjugated
anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245,
anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344,
anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732,
anti-PRO1003, anti-PRO1104, anti-PRO1151, anti-PRO1244,
anti-PRO1298, anti-PRO1313, anti-PRO1570, anti-PRO1886,
anti-PRO1891, anti-PRO4409, anti-PRO5725, anti-PRO5994,
anti-PRO6097, anti-PRO7425, anti-PRO10102, anti-PRO10282,
anti-PRO61709 or anti-PRO779 antibodies. Examples include
At.sup.211, I.sup.131, I.sup.125, Y.sup.90, Re.sup.186, Re.sup.188,
Sm.sup.153, Bi.sup.212, P.sup.32, Pb.sup.212 and radioactive
isotopes of Lu. When the conjugate is used for diagnosis, it may
comprise a radioactive atom for scintigraphic studies, for example
tc.sup.99m or I.sup.123, or a spin label for nuclear magnetic
resonance (NMR) imaging (also known as magnetic resonance imaging,
mri), such as iodine-123 again, iodine-131, indium-111,
fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium,
manganese or iron.
[0657] The radio- or other labels may be incorporated in the
conjugate in known ways. For example, the peptide may be
biosynthesized or may be synthesized by chemical amino acid
synthesis using suitable amino acid precursors involving, for
example, fluorine-19 in place of hydrogen. Labels such as
tc.sup.99m or I.sup.123, Re.sup.186, Re.sup.188 and In.sup.111 can
be attached via a cysteine residue in the peptide. Yttrium-90 can
be attached via a lysine residue. The IODOGEN method (Fraker et al
(1978) Biochem. Biophys. Res. Commun. 80: 49-57 can be used to
incorporate iodine-123. "Monoclonal Antibodies in
Immunoscintigraphy" (Chatal, CRC Press 1989) describes other
methods in detail.
[0658] Conjugates of the antibody and cytotoxic agent may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCL), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutareldehyde), bis-azido compounds
(such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026. The linker may be
a "cleavable linker" facilitating release of the cytotoxic drug in
the cell. For example, an acid-labile linker, peptidase-sensitive
linker, photolabile linker, dimethyl linker or disulfide-containing
linker (Chari et al., Cancer Research 52:127-131 (1992); U.S. Pat.
No. 5,208,020) may be used.
[0659] Alternatively, a fusion protein comprising the anti-PRO196,
anti-PRO217, anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246,
anti-PRO258, anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357,
anti-PRO526, anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003,
anti-PRO1104, anti-PRO1151, anti-PRO1244, anti-PRO1298,
anti-PRO1313, anti-PRO1570, anti-PRO1886, anti-PRO1891,
anti-PRO4409, anti-PRO5725, anti-PRO5994, anti-PRO6097,
anti-PRO7425, anti-PRO10102, anti-PRO10282, anti-PRO61709 or
anti-PRO779 antibody and cytotoxic agent may be made, e.g., by
recombinant techniques or peptide synthesis. The length of DNA may
comprise respective regions encoding the two portions of the
conjugate either adjacent one another or separated by a region
encoding a linker peptide which does not destroy the desired
properties of the conjugate.
[0660] The invention provides that the antibody may be conjugated
to a "receptor" (such streptavidin) for utilization in tumor
pre-targeting wherein the antibody-receptor conjugate is
administered to the patient, followed by removal of unbound
conjugate from the circulation using a clearing agent and then
administration of a "ligand" (e.g., avidin) which is conjugated to
a cytotoxic agent (e.g., a radionucleotide).
[0661] 10. Immunoliposomes
[0662] The anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236,
anti-PRO245, anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328,
anti-PRO344, anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731,
anti-PRO732, anti-PRO1003, anti-PRO1104, anti-PRO1151,
anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570,
anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725,
anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102,
anti-PRO10282, anti-PRO61709 or anti-PRO779 antibodies disclosed
herein may also be formulated as immunoliposomes. A "liposome" is a
small vesicle composed of various types of lipids, phospholipids
and/or surfactant which is useful for delivery of a drug to a
mammal. The components of the liposome are commonly arranged in a
bilayer formation, similar to the lipid arrangement of biological
membranes. Liposomes containing the antibody are prepared by
methods known in the art, such as described in Epstein et al.,
Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al., Proc.
Natl. Acad. Sci. USA 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and
4,544,545; and WO97/38731 published Oct. 23, 1997. Liposomes with
enhanced circulation time are disclosed in U.S. Pat. No.
5,013,556.
[0663] Particularly useful liposomes can be generated by the
reverse phase evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter. Fab' fragments of the antibody of the present invention
can be conjugated to the liposomes as described in Martin et al.,
J. Biol. Chem. 257:286-288 (1982) via a disulfide interchange
reaction. A chemotherapeutic agent is optionally contained within
the liposome. See Gabizon et al., J. National Cancer Inst.
81(19):1484 (1989).
[0664] 11. Pharmaceutical Compositions of Antibodies
[0665] Antibodies specifically binding a PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide identified herein, as well as other molecules
identified by the screening assays disclosed hereinbefore, can be
administered for the treatment of various disorders in the form of
pharmaceutical compositions.
[0666] If the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide is
intracellular and whole antibodies are used as inhibitors,
internalizing antibodies are preferred. However, lipofections or
liposomes can also be used to deliver the antibody, or an antibody
fragment, into cells. Where antibody fragments are used, the
smallest inhibitory fragment that specifically binds to the binding
domain of the target protein is preferred. For example, based upon
the variable-region sequences of an antibody, peptide molecules can
be designed that retain the ability to bind the target protein
sequence. Such peptides can be synthesized chemically and/or
produced by recombinant DNA technology. See, e.g., Marasco et al.,
Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993). The formulation
herein may also contain more than one active compound as necessary
for the particular indication being treated, preferably those with
complementary activities that do not adversely affect each other.
Alternatively, or in addition, the composition may comprise an
agent that enhances its function, such as, for example, a cytotoxic
agent, cytokine, chemotherapeutic agent, or growth-inhibitory
agent. Such molecules are suitably present in combination in
amounts that are effective for the purpose intended.
[0667] The active ingredients may also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacylate)
microcapsules, respectively, in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles, and nanocapsules) or in macroemulsions. Such
techniques are disclosed in Remington's Pharmaceutical Sciences,
supra.
[0668] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0669] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g., films, or
microcapsules. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and .gamma. ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins
for shorter time periods. When encapsulated antibodies remain in
the body for a long time, they may denature or aggregate as a
result of exposure to moisture at 37.degree. C., resulting in a
loss of biological activity and possible changes in immunogenicity.
Rational strategies can be devised for stabilization depending on
the mechanism involved. For example, if the aggregation mechanism
is discovered to be intermolecular S--S bond formation through
thio-disulfide interchange, stabilization may be achieved by
modifying sulfhydryl residues, lyophilizing from acidic solutions,
controlling moisture content, using appropriate additives, and
developing specific polymer matrix compositions.
G. Uses for Anti-PRO196, Anti-PRO217, Anti-PRO231, Anti-PRO236,
Anti-PRO245, Anti-PRO246, Anti-PRO258, Anti-PRO287, Anti-PRO328,
Anti-PRO344, Anti-PRO357, Anti-PRO526, Anti-PRO724, Anti-PRO731,
Anti-PRO732, Anti-PRO1003, Anti-PRO1104, Anti-PRO1151,
Anti-PRO1244, Anti-PRO1298, Anti-PRO1313, Anti-PRO1570,
Anti-PRO1886, Anti-PRO1891, Anti-PRO4409, Anti-PRO5725,
Anti-PRO5994, Anti-PRO6097, Anti-PRO7425, Anti-PRO10102,
Anti-PRO10282, Anti-PRO61709 or Anti-PRO779 Antibodies
[0670] The anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236,
anti-PRO245, anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328,
anti-PRO344, anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731,
anti-PRO732, anti-PRO1003, anti-PRO1104, anti-PRO1151,
anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570,
anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725,
anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102,
anti-PRO10282, anti-PRO61709 or anti-PRO779 antibodies of the
invention have various therapeutic and/or diagnostic utilities for
a neurological disorder; a cardiovascular, endothelial or
angiogenic disorder; an immunological disorder; an oncological
disorder; an embryonic developmental disorder or lethality, or a
metabolic abnormality. For example, anti-PRO196, anti-PRO217,
anti-PRO231, anti-PRO236, anti-PRO245, anti-PRO246, anti-PRO258,
anti-PRO287, anti-PRO328, anti-PRO344, anti-PRO357, anti-PRO526,
anti-PRO724, anti-PRO731, anti-PRO732, anti-PRO1003, anti-PRO1104,
anti-PRO1151, anti-PRO1244, anti-PRO1298, anti-PRO1313,
anti-PRO1570, anti-PRO1886, anti-PRO1891, anti-PRO4409,
anti-PRO5725, anti-PRO5994, anti-PRO6097, anti-PRO7425,
anti-PRO10102, anti-PRO10282, anti-PRO61709 or anti-PRO779
antibodies may be used in diagnostic assays for PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779, e.g., detecting its expression (and in some cases,
differential expression) in specific cells, tissues, or serum.
Various diagnostic assay techniques known in the art may be used,
such as competitive binding assays, direct or indirect sandwich
assays and immunoprecipitation assays conducted in either
heterogeneous or homogeneous phases [Zola, Monoclonal Antibodies: A
Manual of Techniques, CRC Press, Inc. (1987) pp. 147-158]. The
antibodies used in the diagnostic assays can be labeled with a
detectable moiety. The detectable moiety should be capable of
producing, either directly or indirectly, a detectable signal. For
example, the detectable moiety may be a radioisotope, such as
.sup.3H, .sup.14C, .sup.32P, .sup.35S, or .sup.125I, a fluorescent
or chemiluminescent compound, such as fluorescein isothiocyanate,
rhodamine, or luciferin, or an enzyme, such as alkaline
phosphatase, beta-galactosidase or horseradish peroxidase. Any
method known in the art for conjugating the antibody to the
detectable moiety may be employed, including those methods
described by Hunter et al., Nature, 144:945 (1962); David et al.,
Biochemistry, 13:1014 (1974); Pain et al., J. Immunol. Meth.,
40:219 (1981); and Nygren, J. Histochem. and Cytochem., 30:407
(1982).
[0671] Anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236,
anti-PRO245, anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328,
anti-PRO344, anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731,
anti-PRO732, anti-PRO1003, anti-PRO1104, anti-PRO1151,
anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570,
anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725,
anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102,
anti-PRO10282, anti-PRO61709 or anti-PRO779 antibodies also are
useful for the affinity purification of PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptides from recombinant cell culture or natural sources. In
this process, the antibodies against PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptides are immobilized on a suitable support, such a Sephadex
resin or filter paper, using methods well known in the art. The
immobilized antibody then is contacted with a sample containing the
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide to be purified, and
thereafter the support is washed with a suitable solvent that will
remove substantially all the material in the sample except the
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide, which is bound to the
immobilized antibody. Finally, the support is washed with another
suitable solvent that will release the PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide from the antibody.
[0672] The following examples are offered for illustrative purposes
only, and are not intended to limit the scope of the present
invention in any way.
[0673] All patent and literature references cited in the present
specification are hereby incorporated by reference in their
entirety.
EXAMPLES
[0674] Commercially available reagents referred to in the examples
were used according to manufacturer's instructions unless otherwise
indicated. The source of those cells identified in the following
examples, and throughout the specification, by ATCC accession
numbers is the American Type Culture Collection, Manassas, Va.
Example 1
Extracellular Domain Homology Screening to Identify Novel
Polypeptides and cDNA Encoding Therefor
[0675] The extracellular domain (ECD) sequences (including the
secretion signal sequence, if any) from about 950 known secreted
proteins from the Swiss-Prot public database were used to search
EST databases. The EST databases included public databases (e.g.,
Dayhoff, GenBank), and proprietary databases (e.g. LIFESEQ.TM.,
Incyte Pharmaceuticals, Palo Alto, Calif.). The search was
performed using the computer program BLAST or BLAST-2 (Altschul et
al., Methods in Enzymology, 266:460-480 (1996)) as a comparison of
the ECD protein sequences to a 6 frame translation of the EST
sequences. Those comparisons with a BLAST score of 70 (or in some
cases 90) or greater that did not encode known proteins were
clustered and assembled into consensus DNA sequences with the
program "phrap" (Phil Green, University of Washington, Seattle,
Wash.).
[0676] Using this extracellular domain homology screen, consensus
DNA sequences were assembled relative to the other identified EST
sequences using phrap. In addition, the consensus DNA sequences
obtained were often (but not always) extended using repeated cycles
of BLAST or BLAST-2 and phrap to extend the consensus sequence as
far as possible using the sources of EST sequences discussed
above.
[0677] Based upon the consensus sequences obtained as described
above, oligonucleotides were then synthesized and used to identify
by PCR a cDNA library that contained the sequence of interest and
for use as probes to isolate a clone of the full-length coding
sequence for a PRO polypeptide. Forward and reverse PCR primers
generally range from 20 to 30 nucleotides and are often designed to
give a PCR product of about 100-1000 bp in length. The probe
sequences are typically 40-55 bp in length. In some cases,
additional oligonucleotides are synthesized when the consensus
sequence is greater than about 1-1.5 kbp. In order to screen
several libraries for a full-length clone, DNA from the libraries
was screened by PCR amplification, as per Ausubel et al., Current
Protocols in Molecular Biology, with the PCR primer pair. A
positive library was then used to isolate clones encoding the gene
of interest using the probe oligonucleotide and one of the primer
pairs.
[0678] The cDNA libraries used to isolate the cDNA clones were
constructed by standard methods using commercially available
reagents such as those from Invitrogen, San Diego, Calif. The cDNA
was primed with oligo dT containing a NotI site, linked with blunt
to SalI hemikinased adaptors, cleaved with NotI, sized
appropriately by gel electrophoresis, and cloned in a defined
orientation into a suitable cloning vector (such as pRKB or pRKD;
pRK5B is a precursor of pRK5D that does not contain the SfiI site;
see, Holmes et al., Science, 253:1278-1280 (1991)) in the unique
XhoI and NotI sites.
Example 2
Isolation of cDNA Clones by Amylase Screening
[0679] 1. Preparation of Oligo dT Primed cDNA Library
[0680] mRNA was isolated from a human tissue of interest using
reagents and protocols from Invitrogen, San Diego, Calif. (Fast
Track 2). This RNA was used to generate an oligo dT primed cDNA
library in the vector pRK5D using reagents and protocols from Life
Technologies, Gaithersburg, Md. (Super Script Plasmid System). In
this procedure, the double stranded cDNA was sized to greater than
1000 bp and the SalI/NotI linkered cDNA was cloned into XhoI/NotI
cleaved vector. pRK5D is a cloning vector that has an sp6
transcription initiation site followed by an SfiI restriction
enzyme site preceding the XhoI/NotI cDNA cloning sites.
[0681] 2. Preparation of Random Primed cDNA Library
[0682] A secondary cDNA library was generated in order to
preferentially represent the 5' ends of the primary cDNA clones.
Sp6 RNA was generated from the primary library (described above),
and this RNA was used to generate a random primed cDNA library in
the vector pSST-AMY.0 using reagents and protocols from Life
Technologies (Super Script Plasmid System, referenced above). In
this procedure the double stranded cDNA was sized to 500-1000 bp,
linkered with blunt to NotI adaptors, cleaved with SfiI, and cloned
into SfiI/NotI cleaved vector. pSST-AMY.0 is a cloning vector that
has a yeast alcohol dehydrogenase promoter preceding the cDNA
cloning sites and the mouse amylase sequence (the mature sequence
without the secretion signal) followed by the yeast alcohol
dehydrogenase terminator, after the cloning sites. Thus, cDNAs
cloned into this vector that are fused in frame with amylase
sequence will lead to the secretion of amylase from appropriately
transfected yeast colonies.
[0683] 3. Transformation and Detection
[0684] DNA from the library described in paragraph 2 above was
chilled on ice to which was added electrocompetent DH10B bacteria
(Life Technologies, 20 ml). The bacteria and vector mixture was
then electroporated as recommended by the manufacturer.
Subsequently, SOC media (Life Technologies, 1 ml) was added and the
mixture was incubated at 37.degree. C. for 30 minutes. The
transformants were then plated onto 20 standard 150 mm LB plates
containing ampicillin and incubated for 16 hours (37.degree. C.).
Positive colonies were scraped off the plates and the DNA was
isolated from the bacterial pellet using standard protocols, e.g.
CsCl-gradient. The purified DNA was then carried on to the yeast
protocols below.
[0685] The yeast methods were divided into three categories: (1)
Transformation of yeast with the plasmid/cDNA combined vector; (2)
Detection and isolation of yeast clones secreting amylase; and (3)
PCR amplification of the insert directly from the yeast colony and
purification of the DNA for sequencing and further analysis.
[0686] The yeast strain used was HD56-5A (ATCC-90785). This strain
has the following genotype: MAT alpha, ura3-52, leu2-3, leu2-112,
his3-11, his3-15, MAL.sup.+, SUC.sup.+, GAL.sup.+. Preferably,
yeast mutants can be employed that have deficient
post-translational pathways. Such mutants may have translocation
deficient alleles in sec71, sec72, sec62, with truncated sec71
being most preferred. Alternatively, antagonists (including
antisense nucleotides and/or ligands) which interfere with the
normal operation of these genes, other proteins implicated in this
post translation pathway (e.g., SEC61p, SEC72p, SEC62p, SEC63p,
TDJ1p or SSA1p-4p) or the complex formation of these proteins may
also be preferably employed in combination with the
amylase-expressing yeast.
[0687] Transformation was performed based on the protocol outlined
by Gietz et al., Nucl. Acid. Res., 20:1425 (1992). Transformed
cells were then inoculated from agar into YEPD complex media broth
(100 ml) and grown overnight at 30.degree. C. The YEPD broth was
prepared as described in Kaiser et al., Methods in Yeast Genetics,
Cold Spring Harbor Press, Cold Spring Harbor, N.Y., p. 207 (1994).
The overnight culture was then diluted to about 2.times.10.sup.6
cells/ml (approx. OD.sub.600=0.1) into fresh YEPD broth (500 ml)
and regrown to 1.times.10.sup.7 cells/ml (approx.
OD.sub.600=0.4-0.5).
[0688] The cells were then harvested and prepared for
transformation by transfer into GS3 rotor bottles in a Sorval GS3
rotor at 5,000 rpm for 5 minutes, the supernatant discarded, and
then resuspended into sterile water, and centrifuged again in 50 ml
falcon tubes at 3,500 rpm in a Beckman GS-6KR centrifuge. The
supernatant was discarded and the cells were subsequently washed
with LiAc/TE (10 ml, 10 mM Tris-HCl, 1 mM EDTA pH 7.5, 100 mM
Li.sub.2OOCCH.sub.3), and resuspended into LiAc/TE (2.5 ml).
[0689] Transformation took place by mixing the prepared cells (100
.mu.l) with freshly denatured single stranded salmon testes DNA
(Lofstrand Labs, Gaithersburg, Md.) and transforming DNA (1 .mu.g,
vol. <10 .mu.l) in microfuge tubes. The mixture was mixed
briefly by vortexing, then 40% PEG/TE (600 .mu.l, 40% polyethylene
glycol-4000, 10 mM Tris-HCl, 1 mM EDTA, 100 mM Li.sub.2OOCCH.sub.3,
pH 7.5) was added. This mixture was gently mixed and incubated at
30.degree. C. while agitating for 30 minutes. The cells were then
heat shocked at 42.degree. C. for 15 minutes, and the reaction
vessel centrifuged in a microfuge at 12,000 rpm for 5-10 seconds,
decanted and resuspended into TE (500 .mu.l, 10 mM Tris-HCl, 1 mM
EDTA pH 7.5) followed by recentrifugation. The cells were then
diluted into TE (1 ml) and aliquots (200 .mu.l) were spread onto
the selective media previously prepared in 150 mm growth plates
(VWR).
[0690] Alternatively, instead of multiple small reactions, the
transformation was performed using a single, large scale reaction,
wherein reagent amounts were scaled up accordingly.
[0691] The selective media used was a synthetic complete dextrose
agar lacking uracil (SCD-Ura) prepared as described in Kaiser et
al., Methods in Yeast Genetics, Cold Spring Harbor Press, Cold
Spring Harbor, N.Y., p. 208-210 (1994). Transformants were grown at
30.degree. C. for 2-3 days.
[0692] The detection of colonies secreting amylase was performed by
including red starch in the selective growth media. Starch was
coupled to the red dye (Reactive Red-120, Sigma) as per the
procedure described by Biely et al., Anal. Biochem., 172:176-179
(1988). The coupled starch was incorporated into the SCD-Ura agar
plates at a final concentration of 0.15% (w/v), and was buffered
with potassium phosphate to a pH of 7.0 (50-100 mM final
concentration).
[0693] The positive colonies were picked and streaked across fresh
selective media (onto 150 mm plates) in order to obtain well
isolated and identifiable single colonies. Well isolated single
colonies positive for amylase secretion were detected by direct
incorporation of red starch into buffered SCD-Ura agar. Positive
colonies were determined by their ability to break down starch
resulting in a clear halo around the positive colony visualized
directly.
[0694] 4. Isolation of DNA by PCR Amplification
[0695] When a positive colony was isolated, a portion of it was
picked by a toothpick and diluted into sterile water (30 .mu.l) in
a 96 well plate. At this time, the positive colonies were either
frozen and stored for subsequent analysis or immediately amplified.
An aliquot of cells (5 .mu.l) was used as a template for the PCR
reaction in a 25 .mu.l volume containing: 0.5 .mu.l Klentaq
(Clontech, Palo Alto, Calif.); 4.0 .mu.l 10 mM dNTP's (Perkin
Elmer-Cetus); 2.5 .mu.l Kentaq buffer (Clontech); 0.25 .mu.l
forward oligo 1; 0.25 .mu.l reverse oligo 2; 12.5 .mu.l distilled
water. The sequence of the forward oligonucleotide 1 was:
TABLE-US-00007 (SEQ ID NO: 67)
5'-TGTAAAACGACGGCCAGTTAAATAGACCTGCAATTATTAATCT-3'
The sequence of reverse oligonucleotide 2 was:
TABLE-US-00008 (SEQ ID NO: 68)
5'-CAGGAAACAGCTATGACCACCTGCACACCTGCAAATCCATT-3'
PCR was then performed as follows:
TABLE-US-00009 a. Denature 92.degree. C., 5 minutes b. 3 cycles of:
Denature 92.degree. C., 30 seconds Anneal 59.degree. C., 30 seconds
Extend 72.degree. C., 60 seconds c. 3 cycles of: Denature
92.degree. C., 30 seconds Anneal 57.degree. C., 30 seconds Extend
72.degree. C., 60 seconds d. 25 cycles of: Denature 92.degree. C.,
30 seconds Anneal 55.degree. C., 30 seconds Extend 72.degree. C.,
60 seconds e. Hold 4.degree. C.
[0696] The underlined regions of the oligonucleotides annealed to
the ADH promoter region and the amylase region, respectively, and
amplified a 307 bp region from vector pSST-AMY.0 when no insert was
present. Typically, the first 18 nucleotides of the 5' end of these
oligonucleotides contained annealing sites for the sequencing
primers. Thus, the total product of the PCR reaction from an empty
vector was 343 bp. However, signal sequence-fused cDNA resulted in
considerably longer nucleotide sequences.
[0697] Following the PCR, an aliquot of the reaction (5 .mu.l) was
examined by agarose gel electrophoresis in a 1% agarose gel using a
Tris-Borate-EDTA (TBE) buffering system as described by Sambrook et
al., supra. Clones resulting in a single strong PCR product larger
than 400 bp were further analyzed by DNA sequencing after
purification with a 96 Qiaquick PCR clean-up column (Qiagen Inc.,
Chatsworth, Calif.).
Example 3
Isolation of cDNA Clones Using Signal Algorithm Analysis
[0698] Various polypeptide-encoding nucleic acid sequences were
identified by applying a proprietary signal sequence finding
algorithm developed by Genentech, Inc. (South San Francisco,
Calif.) upon ESTs as well as clustered and assembled EST fragments
from public (e.g., GenBank) and/or private (LIFESEQ.RTM., Incyte
Pharmaceuticals, Inc., Palo Alto, Calif.) databases. The signal
sequence algorithm computes a secretion signal score based on the
character of the DNA nucleotides surrounding the first and
optionally the second methionine codon(s) (ATG) at the 5'-end of
the sequence or sequence fragment under consideration. The
nucleotides following the first ATG must code for at least 35
unambiguous amino acids without any stop codons. If the first ATG
has the required amino acids, the second is not examined. If
neither meets the requirement, the candidate sequence is not
scored. In order to determine whether the EST sequence contains an
authentic signal sequence, the DNA and corresponding amino acid
sequences surrounding the ATG codon are scored using a set of seven
sensors (evaluation parameters) known to be associated with
secretion signals. Use of this algorithm resulted in the
identification of numerous polypeptide-encoding nucleic acid
sequences.
[0699] Using the techniques described in Examples 1 to 3 above,
numerous full-length cDNA clones were identified as encoding
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO6097, PRO7425, PRO10102, PRO10282, or
PRO779 polypeptides as disclosed herein. These cDNAs were then
deposited under the terms of the Budapest Treaty with the American
Type Culture Collection, 10801 University Blvd., Manassas, Va.
20110-2209, USA (ATCC) as shown in Table 7 below. In addition, the
sequence of DNA98591 encoding PRO5994 polypeptides was identified
from GenBank accession no.: AF048700; the sequence of DNA347767
encoding PRO61709 polypeptides was identified from GenBank
accession no.: AB029000.
TABLE-US-00010 TABLE 7 Material ATCC Dep. No. Deposit Date
DNA22779-1130 209280 Sep. 18, 1997 DNA33094-1131 209256 Sep. 16,
1997 DNA34434-1139 209252 Sep. 16, 1997 DNA35599-1168 209373 Oct.
16, 1997 DNA35638-1141 209265 Sep. 16, 1997 DNA35639-1172 209396
Oct. 17, 1997 DNA35918-1174 209402 Oct. 17, 1997 DNA39969-1185
209400 Oct. 17, 1997 DNA40587-1231 209438 Nov. 7, 1997
DNA40592-1242 209492 Nov. 21, 1997 DNA44804-1248 209527 Dec. 10,
1997 DNA44184-1319 209704 Mar. 26, 1998 DNA49631-1328 209806 Apr.
28, 1998 DNA48331-1329 209715 Mar. 31, 1998 DNA48334-1435 209924
Jun. 2, 1998 DNA58846-1409 209957 Jun. 9, 1998 DNA59616-1465 209991
Jun. 16, 1998 DNA44694-1500 203114 Aug. 11, 1998 DNA64883-1526
203253 Sep. 9, 1998 DNA66511-1563 203228 Sep. 15, 1998
DNA64966-1575 203575 Jan. 12, 1999 DNA68885-1678 203311 Oct. 6,
1998 DNA80796-2523 203555 Dec. 22, 1998 DNA76788-2526 203551 Dec.
22, 1998 DNA88004-2575 203890 Mar. 30, 1999 DNA92265-2669 PTA-256
Jun. 22, 1999 DNA107701-2711 PTA-487 Aug. 3, 1999 DNA108792-2753
PTA-617 Aug. 31, 1999 DNA129542-2808 PTA-1178 Jan. 11, 2000
DNA148380-2827 PTA-1181 Jan. 11, 2000 DNA58801-1052 55820 Sep. 5,
1996
[0700] These deposits were made under the provisions of the
Budapest Treaty on the International Recognition of the Deposit of
Microorganisms for the Purpose of Patent Procedure and the
Regulations thereunder (Budapest Treaty). This assures maintenance
of a viable culture of the deposit for 30 years from the date of
deposit. The deposits will be made available by ATCC under the
terms of the Budapest Treaty, and subject to an agreement between
Genentech, Inc. and ATCC, which assures permanent and unrestricted
availability of the progeny of the culture of the deposit to the
public upon issuance of the pertinent U.S. patent or upon laying
open to the public of any U.S. or foreign patent application,
whichever comes first, and assures availability of the progeny to
one determined by the U.S. Commissioner of Patents and Trademarks
to be entitled thereto according to 35 USC .sctn.122 and the
Commissioner's rules pursuant thereto (including 37 CFR .sctn.1.14
with particular reference to 886 OG 638).
[0701] The assignee of the present application has agreed that if a
culture of the materials on deposit should die or be lost or
destroyed when cultivated under suitable conditions, the materials
will be promptly replaced on notification with another of the same.
Availability of the deposited material is not to be construed as a
license to practice the invention in contravention of the rights
granted under the authority of any government in accordance with
its patent laws.
Example 4
Isolation of cDNA Clones Encoding Human PRO196 (NL1) Polypeptides
[UNQ170]
[0702] NL1 was identified by screening the GenBank database using
the computer program BLAST (Altshul et al., Methods in Enzymology
266:460-480 (1996)). The NL1 sequence shows homology with known
expressed sequence tag (EST) sequences T35448, T11442, and W77823.
None of the known EST sequences have been identified as full length
sequences, or described as ligands associate 1d with the TIE
receptors.
[0703] Following its identification, NL1 was cloned from a human
fetal lung library prepared from mRNA purchased from Clontech, Inc.
(Palo Alto, Calif., USA), catalog #6528-1, following the
manufacturer's instructions.
[0704] The library was ligated into pRK5B vector, which is a
precursor of pRK5D that does not contain the SfiI site; see, Holmes
et al., Science, 253:1278-1280 (1991). pRK5D, in turn, is a
derivative of pRK5 (EP 307,247, published 15 Mar. 1989), with minor
differences within the polylinker sequence. The library was
screened by hybridization with synthetic oligonucleotide
probes:
TABLE-US-00011 NL1.5-1 SEQ. ID. NO: 69
5'-GCTGACGAACCAAGGCAACTACAAACTCCTGGT NL1.3-1 SEQ. ID. NO: 70
5'-TGCGGCCGGACCAGTCCTCCATGGTCACCAGGAGTTTGTAG NL1.3-2 SEQ. ID. NO:
71 5'-GGTGGTGAACTGCTTGCCGTTGTGCCATGTAAA
based on the ESTs found in the GenBank database. cDNA sequences
were sequenced in their entireties.
[0705] The nucleotide and amino acid sequences of NL1 are shown in
FIG. 1 (SEQ. ID. NO:1; DNA22779-1130) and FIG. 2 (SEQ. ID. NO: 2;
PRO196), respectively.
[0706] NL1 shows a 23% sequence identity with both the TIE1 and the
TIE2 ligand.
[0707] A clone of NL1 (herein designated DNA22779-1130) was
deposited with the American Type Culture Collection (ATCC), 12301
Parklawn Drive, Rockville, Md. 20852, on 18 Sep. 1997, under the
terms of the Budapest Treaty, and has been assigned the deposit
number ATCC 209280.
[0708] NL1 has been mapped to chromosome 9, band arm q13-q21.
Example 5
Isolation of cDNA Clones Encoding Human PRO217 Polypeptides
[UNQ191]
[0709] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described in Example 1 above. This
consensus sequence is designated herein as DNA28760. Based on the
assembled DNA28760 consensus sequence, oligonucleotides were
synthesized: 1) to identify by PCR a cDNA library that contained
the sequence of interest, and 2) for use as probes to isolate a
clone of the full-length coding sequence for PRO217.
[0710] A pair of PCR primers (forward and reverse) were
synthesized:
TABLE-US-00012 forward PCR primer: 5'-AAAGACGCATCTGCGAGTGTCC-3'
(SEQ ID NO: 72) reverse PCR primer: 5'-TGCTGATTTCACACTGCTCTCCC-3'
(SEQ ID NO: 73)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the DNA28760 consensus sequence which had the
following nucleotide sequence:
TABLE-US-00013 hybridization probe: (SEQ ID NO: 74)
5'-CCCACGATGTATGAATGGTGGACTTTGTGTGACTCCTGGTTTCTG CATC-3'
[0711] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones encoding the PRO217 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human fetal
lung tissue.
[0712] DNA sequencing of the isolated clones isolated as described
above gave the full-length DNA sequence for DNA33094-1131 [FIG. 3,
SEQ ID NO:3]; and the derived protein sequence for PRO217.
[0713] The entire coding sequence of DNA33094-1131 is included in
FIG. 3 (SEQ ID NO:3). Clone DNA33094-1131 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 146-148, and an apparent stop codon at
nucleotide positions 1283-1285. The predicted polypeptide precursor
is 379 amino acids long with a molecular weight of approximately
41,528 daltons and an estimated pI of about 7.97. Analysis of the
full-length PRO217 sequence shown in FIG. 4 (SEQ ID NO:4) evidences
the presence of a variety of important polypeptide domains, wherein
the locations given for those important polypeptide domains are
approximate as described above. Analysis of the full-length PRO217
polypeptide shown in FIG. 4 evidences the presence of the
following: a signal peptide from about amino acid 1 to about amino
acid 28; N-glycosylation sites from about amino acid 88 to about
amino acid 92, and from about amino acid 245 to about amino acid
249; a tyrosine kinase phosphorylation site from about amino acid
370 to about amino acid 378; N-myristoylation sites from about
amino acid 184 to about amino acid 190, from about amino acid 185
to about amino acid 191, from about amino acid 189 to about amino
acid 195, and from about amino acid 315 to about amino acid 321; an
ATP/GTP-binding site motif A (P-loop) from about amino acid 285 to
about amino acid 293; and EGF-like domain cysteine pattern
signatures from about amino acid 198 to about amino acid 210, from
about amino acid 230 to about amino acid 242, from about amino acid
262 to about amino acid 274, from about amino acid 294 to about
amino acid 306, and from about amino acid 326 to about amino acid
338. Clone DNA33094-1131 has been deposited with the ATCC on Sep.
16, 1997 and is assigned ATCC deposit no. 209256.
[0714] Based on a BLAST and FastA sequence alignment analysis of
the full-length sequence shown in FIG. 4 (SEQ ID NO:4), PRO217
appears to be a novel EGF-like homologue.
Example 6
Isolation of cDNA Clones Encoding Human PRO231 Polypeptides
[UNQ205]
[0715] A consensus DNA sequence was assembled relative to the other
identified EST sequences as described in Example 1 above, wherein
the consensus sequence was designated herein as DNA30933. Based on
the DNA30933 consensus sequence, oligonucleotides were synthesized
to identify by PCR a cDNA library that contained the sequence of
interest and for use as probes to isolate a clone of the
full-length coding sequence for PRO231.
[0716] Three PCR primers (two forward and one reverse) were
synthesized:
TABLE-US-00014 forward PCR primer 1 5'-CCAACTACCAAAGCTGCTGGAGCC-3'
(SEQ ID NO: 75) forward PCR primer 2 5'-GCAGCTCTATTACCACGGGAAGGA-3'
(SEQ ID NO: 76) reverse PCR primer 5'-TCCTTCCCGTGGTAATAGAGCTGC-3'
(SEQ ID NO: 77)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA30933 sequence which had the
following nucleotide sequence
TABLE-US-00015 hybridization probe (SEQ ID NO: 78)
5'-GGCAGAGAACCAGAGGCCGGAGGAGACTGCCTCTTTACAGCCAGG-3'
[0717] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pairs identified above. A
positive library was then used to isolate clones encoding the
PRO231 gene using the probe oligonucleotide and one of the PCR
primers.
[0718] RNA for construction of the cDNA libraries was isolated from
human fetal liver tissue.
[0719] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO231 [herein designated as
DNA34434-1139] and the derived protein sequence for PRO231.
[0720] The entire nucleotide sequence of DNA34434-1139 is shown in
FIG. 5 (SEQ ID NO:5). Clone DNA34434-1139 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 173-175 and ending at the stop codon at
nucleotide positions 1457-1459 (FIG. 5; SEQ ID NO:5). The predicted
polypeptide precursor is 428 amino acids long (FIG. 6; SEQ ID
NO:6). Clone DNA34434-1139 has been deposited with ATCC on Sep. 16,
1997 and is assigned ATCC deposit no. ATCC 209252.
[0721] Analysis of the amino acid sequence of the full-length
PRO231 suggests that it possesses 30% and 31% amino acid identity
with the human and rat prostatic acid phosphatase precursor
proteins, respectively.
Example 7
Isolation of cDNA Clones Encoding Human PRO236 Polypeptides
[UNQ210]
[0722] Consensus DNA sequences were assembled relative to other EST
sequences using pinup as described in Example 1 above. This
consensus sequences is herein designated DNA30901. Based on the
DNA30901 consensus sequence, oligonucleotides were synthesized: 1)
to identify by PCR a cDNA library that contained the sequence of
interest, and 2) for use as probes to isolate a clone of the
full-length coding sequence for PRO236. Based upon the DNA30901
consensus sequence, a pair of PCR primers (forward and reverse)
were synthesized:
TABLE-US-00016 forward PCR primer 5'-TGGCTACTCCAAGACCCTGGCATG-3'
(SEQ ID NO: 79) reverse PCR primer 5'-TGGACAAATCCCCTTGCTCAGCCC-3'
(SEQ ID NO: 80)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA30901 sequence which had the
following nucleotide sequence
TABLE-US-00017 hybridization probe (SEQ ID NO: 81)
5'-GGGCTTCACCGAAGCAGTGGACCTTTATTTTGACCACCTGATGTC CAGGG-3'
[0723] In order to screen several libraries for a source of
full-length clones, DNA from the libraries was screened by PCR
amplification with the PCR primer pairs identified above. Positive
libraries were then used to isolate clones encoding the PRO236 gene
using the probe oligonucleotides and one of the PCR primers.
[0724] RNA for construction of the cDNA libraries was isolated from
human fetal lung tissue for PRO236.
[0725] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO236 [herein designated as
DNA35599-1168] (SEQ ID NO:7), the derived protein sequence for
PRO236.
[0726] The entire nucleotide sequence of DNA35599-1168 is shown in
FIG. 7 (SEQ ID NO:7). Clone DNA35599-1168 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 69-71 and ending at the stop codon at
nucleotide positions 1977-1979 (FIG. 7; SEQ ID NO:7). The predicted
polypeptide precursor is 636 amino acids long (FIG. 8; SEQ ID
NO:8). Clone DNA35599-1168 has been deposited with ATCC on Oct. 16,
1997 and is assigned ATCC deposit no. ATCC 209373.
[0727] Analysis of the amino acid sequence of the full-length
PRO236 polypeptide suggests that portions of this polypeptide
possesses significant homology to beta-galactosidase proteins
derived from various sources, thereby indicating that PRO236 may be
a novel beta-galactosidase homolog.
Example 8
Isolation of cDNA Clones Encoding Human PRO245 Polypeptides
[UNO219]
[0728] A consensus DNA sequence was assembled relative to the other
identified EST sequences as described in Example 1 above, wherein
the consensus sequence is designated herein as DNA30954.
[0729] Based on the DNA30954 consensus sequence, oligonucleotides
were synthesized to identify by PCR a cDNA library that contained
the sequence of interest and for use as probes to isolate a clone
of the full-length coding sequence for PRO245.
[0730] A pair of PCR primers (forward and reverse) were
synthesized:
TABLE-US-00018 forward PCR primer 5'-ATCGTTGTGAAGTTAGTGCCCC-3' (SEQ
ID NO: 82) reverse PCR primer 5'-ACCTGCGATATCCAACAGAATTG-3' (SEQ ID
NO: 83)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA30954 sequence which had the
following nucleotide sequence
TABLE-US-00019 hybridization probe (SEQ ID NO: 84)
5'-GGAAGAGGATACAGTCACTCTGGAAGTATTAGTGGCTCCAGCAG TTCC-3'
[0731] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones encoding the PRO245 gene
using the probe oligonucleotide and one of the PCR primers.
[0732] RNA for construction of the cDNA libraries was isolated from
human fetal liver tissue. DNA sequencing of the clones isolated as
described above gave the full-length DNA sequence for PRO245
[herein designated as DNA35638-1141] and the derived protein
sequence for PRO245.
[0733] The entire nucleotide sequence of DNA35638-1141 is shown in
FIG. 9 (SEQ ID NO:9). Clone DNA35638-1141 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 89-91 and ending at the stop codon at
nucleotide positions 1025-1027 (FIG. 9; SEQ ID NO:9). The predicted
polypeptide precursor is 312 amino acids long (FIG. 10; SEQ ID
NO:10). Clone DNA35638-1141 has been deposited with ATCC on Sep.
16, 1997 and is assigned ATCC deposit no. ATCC 209265.
[0734] Analysis of the amino acid sequence of the full-length
PRO245 suggests that a portion of it possesses 60% amino acid
identity with the human c-myb protein and, therefore, may be a new
member of the transmembrane protein receptor tyrosine kinase
family.
Example 9
Isolation of cDNA Clones Encoding Human PRO246 Polypeptides
[UNQ220]
[0735] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described in Example 1 above. This
consensus sequence is herein designated DNA30955. Based on the
DNA30955 consensus sequence, oligonucleotides were synthesized: 1)
to identify by PCR a cDNA library that contained the sequence of
interest, and 2) for use as probes to isolate a clone of the
full-length coding sequence for PRO246.
[0736] A pair of PCR primers (forward and reverse) were
synthesized:
TABLE-US-00020 forward PCR primer 5'-AGGGTCTCCAGGAGAAAGACTC-3' (SEQ
ID NO: 85) reverse PCR primer 5'-ATTGTGGGCCTTGCAGACATAGAC-3' (SEQ
ID NO: 86)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA30955 sequence which had the
following nucleotide sequence
TABLE-US-00021 hybridization probe (SEQ ID NO: 87)
5'-GGCCACAGCATCAAAACCTTAGAACTCAATGTACTGGTTCCTCCA GCTCC-3'
[0737] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones encoding the PRO246 gene
using the probe oligonucleotide and one of the PCR primers.
[0738] RNA for construction of the cDNA libraries was isolated from
human fetal liver tissue. DNA sequencing of the clones isolated as
described above gave the full-length DNA sequence for PRO246
[herein designated as DNA35639-1172] (SEQ ID NO:11) and the derived
protein sequence for PRO246.
[0739] The entire nucleotide sequence of DNA35639-1172 is shown in
FIG. 11 (SEQ ID NO:11). Clone DNA35639-1172 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 126-128 and ending at the stop codon at
nucleotide positions 1296-1298 (FIG. 11). The predicted polypeptide
precursor is 390 amino acids long (FIG. 12; SEQ ID NO:12). Clone
DNA35639-1172 has been deposited with ATCC on Oct. 17, 1997 and is
assigned ATCC deposit no. ATCC 209396.
[0740] Analysis of the amino acid sequence of the full-length
PRO246 polypeptide suggests that it possess significant homology to
the human cell surface protein HCAR, thereby indicating that PRO246
may be a novel cell surface virus receptor.
Example 10
Isolation of cDNA Clones Encoding Human PRO258 Polypeptides
[UNQ225]
[0741] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described in Example 1 above. This
consensus sequence is herein designated DNA28746.
[0742] Based on the DNA28746 consensus sequence, oligonucleotides
were synthesized: 1) to identify by PCR a cDNA library that
contained the sequence of interest, and 2) for use as probes to
isolate a clone of the full-length coding sequence for PRO258.
[0743] PCR primers (forward and reverse) were synthesized:
TABLE-US-00022 forward PCR primer 5'-GCTAGGAATTCCACAGAAGCCC-3' (SEQ
ID NO: 88) reverse PCR primer 5'-AACCTGGAATGTCACCGAGCTG-3' (SEQ ID
NO: 89) reverse PCR primer 5'-CCTAGCACAGTGACGAGGGACTTGGC-3' (SEQ ID
NO: 90)
Additionally, synthetic oligonucleotide hybridization probes were
constructed from the consensus DNA28740 sequence which had the
following nucleotide sequence:
TABLE-US-00023 hybridization probe (SEQ ID NO: 91)
5'-AAGACACAGCCACCCTAAACTGTCAGTCTTCTGGGAGCAAGCCTG CAGCC-3' (SEQ ID
NO: 92) 5'-GCCCTGGCAGACGAGGGCGAGTACACCTGCTCAATCTTCACTATG
CCTGT-3'
[0744] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones encoding the PRO258 gene
using the probe oligonucleotide and one of the PCR primers.
[0745] RNA for construction of the cDNA libraries was isolated from
human fetal lung tissue. DNA sequencing of the clones isolated as
described above gave the full-length DNA sequence for PRO258
[herein designated as DNA35918-1174] (SEQ ID NO:13) and the derived
protein sequence for PRO258.
[0746] The entire nucleotide sequence of DNA35918-1174 is shown in
FIG. 13 (SEQ ID NO:13). Clone DNA35918-1174 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 147-149 of SEQ ID NO:13 and ending at the stop
codon after nucleotide position 1340 of SEQ ID NO:13 (FIG. 13). The
predicted polypeptide precursor is 398 amino acids long (FIG. 14;
SEQ ID NO:14). Clone DNA35918-1174 has been deposited with ATCC on
Oct. 17, 1997 and is assigned ATCC deposit no. ATCC 209402.
[0747] Analysis of the amino acid sequence of the full-length
PRO258 polypeptide suggests that portions of it possess significant
homology to the CRTAM and the poliovirus receptor and have an Ig
domain, thereby indicating that PRO258 is a new member of the Ig
superfamily.
Example 11
Isolation of cDNA Clones Encoding Human PRO287 Polypeptides
[UNQ250]
[0748] A consensus DNA sequence encoding PRO287 was assembled
relative to the other identified EST sequences as described in
Example 1 above, wherein the consensus sequence is designated
herein as DNA28728. Based on the DNA28728 consensus sequence,
oligonucleotides were synthesized to identify by PCR a cDNA library
that contained the sequence of interest and for use as probes to
isolate a clone of the full-length coding sequence for PRO287.
[0749] A pair of PCR primers (forward and reverse) were
synthesized:
TABLE-US-00024 forward PCR primer 5'-CCGATTCATAGACCTCGAGAGT-3' (SEQ
ID NO: 93) reverse PCR primer 5'-GTCAAGGAGTCCTCCACAATAC-3' (SEQ ID
NO: 94)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA28728 sequence which had the
following nucleotide sequence
TABLE-US-00025 hybridization probe (SEQ ID NO: 95)
5'-GTGTACAATGGCCATGCCAATGGCCAGCGCATTGGCCGCTTC TGT-3'
[0750] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones encoding the PRO287 gene
using the probe oligonucleotide and one of the PCR primers.
[0751] RNA for construction of the cDNA libraries was isolated from
human fetal kidney tissue.
[0752] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO287 [herein designated as
DNA39969-1185, SEQ ID NO:15] and the derived protein sequence for
PRO287.
[0753] The entire nucleotide sequence of DNA39969-1185 is shown in
FIG. 15 (SEQ ID NO:15). Clone DNA39969-1185 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 307-309 and ending at the stop codon at
nucleotide positions 1552-1554 (FIG. 15; SEQ ID NO:15). The
predicted polypeptide precursor is 415 amino acids long (FIG. 16;
SEQ ID NO:16). Clone DNA39969-1185 has been deposited with ATCC on
Oct. 17, 1997 and is assigned ATCC deposit no. ATCC 209400.
[0754] Analysis of the amino acid sequence of the full-length
PRO287 suggests that it may possess one or more procollagen
C-proteinase enhancer protein precursor or procollagen C-proteinase
enhancer protein-like domains. Based on a BLAST and FastA sequence
alignment analysis of the full-length sequence, PRO287 shows
nucleic acid sequence identity to procollagen C-proteinase enhancer
protein precursor and procollagen C-proteinase enhancer protein (47
and 54%, respectively).
Example 12
Isolation of cDNA Clones Encoding Human PRO328 Polypeptides
[UNQ289]
[0755] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described in Example 1 above. This
consensus sequence is herein designated DNA35615. Based on the
DNA35615 consensus sequence, oligonucleotides were synthesized: 1)
to identify by PCR a cDNA library that contained the sequence of
interest, and 2) for use as probes to isolate a clone of the
full-length coding sequence for PRO328.
[0756] Forward and reverse PCR primers were synthesized:
TABLE-US-00026 forward PCR primer 5'-TCCTGCAGTTTCCTGATGC-3' (SEQ ID
NO: 96) reverse PCR primer 5'-CTCATATTGCACACCAGTAATTCG-3' (SEQ ID
NO: 97)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA35615 sequence which had the
following nucleotide sequence
TABLE-US-00027 hybridization probe (SEQ ID NO: 98)
5'-ATGAGGAGAAACGTTTGATGGTGGAGCTGCACAACCTCTACCGGG-3'
[0757] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones encoding the PRO328 gene
using the probe oligonucleotide and one of the PCR primers.
[0758] RNA for construction of the cDNA libraries was isolated from
human fetal kidney tissue.
[0759] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO328 [herein designated as
DNA40587-1231] (SEQ ID NO:17) and the derived protein sequence for
PRO328.
[0760] The entire nucleotide sequence of DNA40587-1231 is shown in
FIG. 17 (SEQ ID NO:17). Clone DNA40587-1231 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 15-17 and ending at the stop codon at
nucleotide positions 1404-1406 (FIG. 17). The predicted polypeptide
precursor is 463 amino acids long (FIG. 18; SEQ ID NO:18). Clone
DNA40587-1231 has been deposited with ATCC on Nov. 7, 1997 and is
assigned ATCC deposit no. ATCC 209438.
[0761] Analysis of the amino acid sequence of the full-length
PRO328 polypeptide suggests that portions of it possess significant
homology to the human glioblastoma protein and to the cysteine rich
secretory protein thereby indicating that PRO328 may be a novel
glioblastoma protein or cysteine rich secretory protein.
Example 13
Isolation of cDNA Clones Encoding Human PRO344 Polypeptides
[UNQ303]
[0762] A consensus DNA sequence was assembled relative to other EST
sequences as described in Example 1 above. This consensus sequence
is herein designated DNA34398. Based on the DNA34398 consensus
sequence, oligonucleotides were synthesized: 1) to identify by PCR
a cDNA library that contained the sequence of interest, and 2) for
use as probes to isolate a clone of the full-length coding sequence
for PRO344.
[0763] Based on the DNA34398 consensus sequence, forward and
reverse PCR primers were synthesized as follows:
TABLE-US-00028 forward PCR primer(3439811)
5'-TACAGGCCCAGTCAGGACCAGGGG-3' (SEQ ID NO: 99) forward PCR
primer(34398.f2) 5'-AGCCAGCCTCGCTCTCGG-3' (SEQ ID NO: 100) forward
PCR primer(34398.f3) 5'-GTCTGCGATCAGGTCTGG-3' (SEQ ID NO: 101)
reverse PCR primer(34398.r1) 5'-GAAAGAGGCAATGGATTCGC-3' (SEQ ID NO:
102) reverse PCR primer(34398.r2) 5'-GACTTACACTTGCCAGCACAGCAC-3'
(SEQ ID NO: 103)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the DNA34398 consensus sequence which had the
following nucleotide sequence
TABLE-US-00029 hybridization probe (34398.p1) (SEQ ID NO: 104)
5'-GGAGCACCACCAACTGGAGGGTCCGGAGTAGCGAGCGCCCCGAAG-3'
[0764] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with one of the PCR primer pairs identified above. A
positive library was then used to isolate clones encoding the
PRO344 genes using the probe oligonucleotide and one of the PCR
primers. RNA for construction of the cDNA libraries was isolated
from human fetal kidney tissue.
[0765] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO344 [herein designated as
DNA40592-1242] (SEQ ID NO:19) and the derived protein sequence for
PRO344.
[0766] The entire nucleotide sequence of DNA40592-1242 is shown in
FIG. 19 (SEQ ID NO:19). Clone DNA40592-1242 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 227-229 and ending at the stop codon at
nucleotide positions 956-958 (FIG. 19). The predicted polypeptide
precursor is 243 amino acids long (FIG. 20; SEQ ID NO:20).
Important regions of the native PRO344 amino acid sequence include
the signal peptide, the start of the mature protein, and two
potential N-myristoylation sites as shown in FIG. 20. Clone
DNA40592-1242 has been deposited with the ATCC on Nov. 21, 1997 and
is assigned ATCC deposit no. ATCC 209492.
[0767] Analysis of the amino acid sequence of the full-length
PRO344 polypeptides suggests that portions of them possess
significant homology to various human and murine complement
proteins, thereby indicating that PRO344 may be a novel complement
protein.
Example 14
Isolation of cDNA Clones Encoding Human PRO357 Polypeptides
[UNQ314]
[0768] The sequence expression tag clone no. "2452972" by Incyte
Pharmaceuticals, Palo Alto, Calif. was used to begin a data base
search. The extracellular domain (ECD) sequences (including the
secretion signal, if any) of from about 950 known secreted proteins
from the Swiss-Prot public protein database were used to search
expressed sequence tag (EST) databases which overlapped with a
portion of Incyte EST clone no. "2452972". The EST databases
included public EST databases (e.g., GenBank) and a proprietary EST
DNA database (LIFESEQ.RTM., Incyte Pharmaceuticals, Palo Alto,
Calif.). The search was performed using the computer program BLAST
or BLAST2 (Altshul et al., Methods in Enzymology 266:460-480
(1996)) as a comparison of the ECD protein sequences to a 6 frame
translation of the EST sequence. Those comparisons resulting in a
BLAST score of 70 (or in some cases 90) or greater that did not
encode known proteins were clustered and assembled into consensus
DNA sequences with the program "phrap" (Phil Green, University of
Washington, Seattle, Wash.).
[0769] A consensus DNA sequence was then assembled relative to
other EST sequences using phrap. This consensus sequence is herein
designated DNA37162. In this case, the consensus DNA sequence was
extended using repeated cycles of BLAST and phrap to extend the
consensus sequence as far as possible using the sources of EST
sequences discussed above.
[0770] Based on the DNA37162 consensus sequence, oligonucleotides
were synthesized: 1) to identify by PCR a cDNA library that
contained the sequence of interest, and 2) for use as probes to
isolate a clone of the full-length coding sequence for PRO357.
Forward and reverse PCR primers generally range from 20 to 30
nucleotides and are often designed to give a PCR product of about
100-1000 bp in length. The probe sequences are typically 40-55 bp
in length. In some cases, additional oligonucleotides are
synthesized when the consensus sequence is greater than about 1-1.5
kbp. In order to screen several libraries for a full-length clone,
DNA from the libraries was screened by PCR amplification, as ber
Ausubel et al., Current Protocols in Molecular Biology, with the
PCR primer pair. A positive library was then used to isolate clones
encoding the gene of interest using the probe oligonucleotide and
one of the primer pairs.
[0771] PCR primers were synthesized as follows:
TABLE-US-00030 forward primer 1: 5'-CCCTCCACTGCCCCACCGACTG-3'; (SEQ
ID NO: 105) reverse primer 1: 5'-CGGTTCTGGGGACGTTAGGGCTCG-3'; (SEQ
ID NO: 106) and forward primer 2: 5'-CTGCCCACCGTCCACCTGCCTCAAT-3'.
(SEQ ID NO: 107)
Additionally, two synthetic oligonucleotide hybridization probes
were constructed from the consensus DNA37162 sequence which had the
following nucleotide sequences:
TABLE-US-00031 hybridization probe 1: (SEQ ID NO: 108)
5'-AGGACTGCCCACCGTCCACCTGCCTCAATGGGGGCACATGCCA CC-3'; and
hybridization probe 2: (SEQ ID NO: 109)
5'-ACGCAAAGCCCTACATCTAAGCCAGAGAGAGACAGGGCAGCTG GG-3'.
[0772] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with a PCR primer pair identified above. A positive
library was then used to isolate clones encoding the PRO357 gene
using the probe oligonucleotide and one of the PCR primers.
[0773] RNA for construction of the cDNA libraries was isolated from
human fetal liver tissue. The cDNA libraries used to isolate the
cDNA clones were constructed by standard methods using commercially
available reagents such as those from Invitrogen, San Diego, Calif.
The cDNA was primed with olio dT containing a NotI site, linked
with blunt to SalI hemikinased adaptors, cleaved with NotI, sized
appropriately by gel electrophoresis, and cloned in a defined
orientation into a suitable cloning vector (such as pRKB or pRKD;
pRK5B is a precursor of pRK5D that does not contain the SfiI site;
see, Holmes et al., Science, 253:1278-1280 (1991)) in the unique
XhoI and NotI sites.
[0774] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO357 [herein designated as
DNA44804-1248] (SEQ ID NO:21) and the derived protein sequence for
PRO357.
[0775] The entire nucleotide sequence of DNA44804-1248 is shown in
FIG. 21 (SEQ ID NO:21). Clone DNA44804-1248 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 137-139 and ending at the stop codon at
nucleotide positions 1931-1933 (FIG. 21). The predicted polypeptide
precursor is 598 amino acids long (FIG. 22; SEQ ID NO:22). Clone
DNA44804-1248 has been deposited with ATCC on Dec. 10, 1997 and is
assigned ATCC deposit no. ATCC 209527.
[0776] Analysis of the amino acid sequence of the full-length
PRO357 polypeptide therefore suggests that portions of it possess
significant homology to ALS, thereby indicating that PRO357 may be
a novel leucine rich repeat protein related to ALS.
Example 15
Isolation of cDNA Clones Encoding Human PRO526 Polypeptides
[UNQ330]
[0777] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA39626. Based on
the DNA39626 consensus sequence, oligonucleotides were synthesized:
1) to identify by PCR a cDNA library that contained the sequence of
interest, and 2) for use as probes to isolate a clone of the
full-length coding sequence for PRO526.
[0778] A pair of PCR primers (forward and reverse) were
synthesized:
TABLE-US-00032 forward PCR primer 5'-TGGCTGCCCTGCAGTACCTCTACC-3';
(SEQ ID NO: 110) reverse PCR primer 5'-CCCTGCAGGTCATTGGCAGCTAGG-3'.
(SEQ ID NO: 111)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the DNA39626 consensus sequence which had the
following nucleotide sequence:
TABLE-US-00033 hybridization probe (SEQ ID NO: 112)
5'-AGGCACTGCCTGATGACACCTTCCGCGACCTGGGCAACCTCAC AC-3'.
[0779] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones encoding the PRO526 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human fetal
liver tissue (LIB228).
[0780] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO526 [herein designated as
UNQ330 (DNA44184-1319)] (SEQ ID NO:23) and the derived protein
sequence for PRO526.
[0781] The entire nucleotide sequence of UNQ330 (DNA44184-1319) is
shown in FIG. 23 (SEQ ID NO:23). Clone UNQ330 (DNA44184-1319)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 514-516 and ending at the
stop codon at nucleotide positions 1933-1935 (FIG. 23). The
predicted polypeptide precursor is 473 amino acids long (FIG. 24;
SEQ ID NO:24). The full-length PRO526 protein shown in FIG. 24 has
an estimated molecular weight of about 50,708 daltons and a pI of
about 9.28. Clone UNQ330 (DNA44184-1319) has been deposited with
the ATCC on Mar. 26, 1998 under ATCC accession no: 209704. It is
understood that the clone contains the actual sequence, whereas the
sequences presented herein are representative based on current
sequencing techniques.
[0782] Analysis of the amino acid sequence of the full-length
PRO526 polypeptide suggests that portions of it possess significant
homology to the leucine repeat rich proteins including ALS, SLIT,
carboxypeptidase and platelet glycoprotein V thereby indicating
that PRO526 is a novel protein which is involved in protein-protein
interactions.
[0783] Still analyzing SEQ ID NO:24, the signal peptide sequence is
at about amino acids 1-26. A leucine zipper pattern is at about
amino acids 135-156. A glycosaminoglycan attachment is at about
amino acids 436-439. N-glycosylation sites are at about amino acids
82-85, 179-182, 237-240 and 423-426. A von Willebrand factor (VWF)
type C domain(s) is found at about amino acids 411-425. The skilled
artisan can understand which nucleotides correspond to these amino
acids based on the sequences provided herein.
Example 16
Isolation of cDNA Clones Encoding Human PRO724 Polypeptides
[UNQ389]
[0784] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA35603. Based on
the DNA35603 consensus sequence, oligonucleotides were synthesized:
1) to identify by PCR a cDNA library that contained the sequence of
interest, and 2) for use as probes to isolate a clone of the
full-length coding sequence for PRO724.
[0785] Pairs of PCR primers (forward and reverse) were
synthesized:
TABLE-US-00034 forward PCR primer 1 5'-GGCTGTCACTGTGGAGACAC-3' (SEQ
ID NO: 113) forward PCR primer 2 5'-GCAAGGTCATTACAGCTG-3' (SEQ ID
NO: 114) reverse PCR primer 1 5'-AGAACATAGGAGCAGTCCCACTC-3' (SEQ ID
NO: 115) reverse PCR primer 2 5'-TGCCTGCTGCTGCACAATCTCAG-3' (SEQ ID
NO: 116)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA35603 sequence which had the
following nucleotide sequence
TABLE-US-00035 hybridization probe (SEQ ID NO: 117)
5'-GGCTATTGCTTGCCTTGGGACAGACCCTGTGGCTTAGGCTCTGGC-3'
[0786] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pairs identified above. A
positive library was then used to isolate clones encoding the
PRO724 gene using the probe oligonucleotide and one of the PCR
primers. RNA for construction of the cDNA libraries was isolated
from human fetal lung tissue (LIB26).
[0787] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO724 [herein designated as
UNQ389 (DNA49631-1328)] (SEQ ID NO:25) and the derived protein
sequence for PRO724.
[0788] The entire nucleotide sequence of UNQ389 (DNA49631-1328) is
shown in FIG. 25 (SEQ ID NO:25). Clone UNQ389 (DNA49631-1328)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 546-548 and ending at the
stop codon at nucleotide positions 2685-2687 (FIG. 25). The
predicted polypeptide precursor is 713 amino acids long (FIG. 26;
SEQ ID NO:26). The full-length PRO724 protein shown in FIG. 26 has
an estimated molecular weight of about 76,193 daltons and a pI of
about 5.42. Analysis of the full-length PRO724 amino acid sequence
shown in FIG. 26 (SEQ ID NO:26) evidences the presence of the
following: a signal peptide from about amino acid 1 to about amino
acid 16, a transmembrane domain from about amino acid 442 to about
amino acid 462 and LDL receptor class A domain regions from about
amino acid 152 to about amino acid 171, about amino acid 331 to
about amino acid 350, about amino acid 374 to about amino acid 393
and about amino acid 411 to about amino acid 430. Clone UNQ389
(DNA49631-1328) has been deposited with ATCC on Apr. 28, 1998 and
is assigned ATCC deposit no. 209806.
[0789] Analysis of the amino acid sequence of the full-length
PRO724 polypeptide suggests that it possesses significant sequence
similarity to the human LDL receptor protein, thereby indicating
that PRO724 may be a novel LDL receptor homolog. More specifically,
an analysis of the Dayhoff database (version 35.45 SwissProt 35)
evidenced significant homology between the PRO724 amino acid
sequence and the following Dayhoff sequences, P_R48547,
MMAM2R.sub.--1, LRP2_RAT, P_R60517, P_R47861, P_R05533,
A44513.sub.--1, A30363, P_R74692 and LMLIPOPHO.sub.--1.
Example 17
Isolation of cDNA Clones Encoding Human PRO731 Polypeptides
[UNQ395]
[0790] A database was used to search expressed sequence tag (EST)
databases. The EST database used herein was the proprietary EST DNA
database LIFESEQ.RTM., of Incyte Pharmaceuticals, Palo Alto, Calif.
Incyte clone 2581326 was herein identified and termed DNA42801.
Based on the DNA42801 sequence, oligonucleotides were synthesized:
1) to identify by PCR a cDNA library that contained the sequence of
interest, and 2) for use as probes to isolate a clone of the
full-length coding sequence for PRO731.
[0791] A pair of PCR primers (forward and reverse) were
synthesized:
TABLE-US-00036 forward PCR primer 5'-GTAAGCACATGCCTCCAGAGGTGC-3';
(SEQ ID NO: 118) reverse PCR primer 5'-GTGACGTGGATGCTTGGGATGTTG-3'.
(SEQ ID NO: 119)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the DNA42801 sequence which had the following
nucleotide sequence:
TABLE-US-00037 hybridization probe (SEQ ID NO: 120)
5'-TGGACACCTTCAGTATTGATGCCAAGACAGGCCAGGTCATTCTGC GTCGA-3'.
[0792] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones encoding the PRO731 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human bone
marrow tissue (LIB255). The cDNA libraries used to isolate the cDNA
clones were constructed by standard methods using commercially
available reagents such as those from Invitrogen, San Diego, Calif.
The cDNA was primed with olio dT containing a NotI site, linked
with blunt to SalI hemikinased adaptors, cleaved with NotI, sized
appropriately by gel electrophoresis, and cloned in a defined
orientation into a suitable cloning vector (such as pRKB or pRKD;
pRK5B is a precursor of pRK5D that does not contain the SfiI site;
see, Holmes et al., Science, 253:1278-1280 (1991)) in the unique
XhoI and NotI sites.
[0793] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO731 [herein designated as
UNQ395 (DNA48331-1329)] (SEQ ID NO:27) and the derived protein
sequence for PRO731.
[0794] The entire nucleotide sequence of UNQ395 (DNA48331-1329) is
shown in FIG. 27 (SEQ ID NO:27). Clone UNQ395 (DNA48331-1329)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 329-331 and ending at the
stop codon at nucleotide positions 3881-3883 (FIG. 27). The
predicted polypeptide precursor is 1184 amino acids long (FIG. 28;
SEQ ID NO:28). The full-length PRO731 protein shown in FIG. 28 has
an estimated molecular weight of about 129,022 daltons and a pI of
about 5.2. Clone UNQ395 (DNA48331-1329) was deposited with the ATCC
on Mar. 31, 1998 under ATCC accession no: 209715. Regarding the
sequence, it is understood that the deposited clone contains the
correct sequence, and the sequences provided herein are based on
known sequencing techniques.
[0795] Analysis of the amino acid sequence of the full-length
PRO731 polypeptide suggests that portions of it possess significant
identity and similarity to members of the protocadherin family,
thereby indicating that PRO731 may be a novel protocadherin.
[0796] Still analyzing the amino acid sequence of SEQ ID NO:28, the
putative signal peptide is at about amino acids 1-13 of SEQ ID
NO:28. The transmembrane domain is at amino acids 719-739 of SEQ ID
NO:28. The N-glycosylation of SEQ ID NO:28 are as follows: 415-418,
582-586, 659-662, 662-665, and 857-860. The cadherin extracellular
repeated domain signatures are at about amino acids (of SEQ ID
NO:28): 123-133, 232-242, 340-350, 448-458, and 553-563. The
corresponding nucleotides can be routinely determined given the
sequences provided herein.
Example 18
Isolation of cDNA Clones Encoding Human PRO732 Polypeptides
[UNQ0396]
[0797] A yeast screening assay was employed to identify cDNA clones
that encoded potential secreted proteins. Use of this yeast
screening assay allowed identification of a single cDNA clone whose
sequence (herein designated as DNA42580). The DNA42580 sequence was
then compared to a variety of known EST sequences to identify
homologies. The EST databases employed included public EST
databases (e.g., GenBank) and a proprietary EST DNA database
(LIFESEQ.RTM., Incyte Pharmaceuticals, Palo Alto, Calif.). The
search was performed using the computer program BLAST or BLAST2
(Altshul et al., Methods in Enzymology 266:460-480 (1996)) as a
comparison to a 6 frame translation of the EST sequence. Those
comparisons resulting in a BLAST score of 70 (or in some cases 90)
or greater that did not encode known proteins were clustered and
assembled into consensus DNA sequences with the program "phrap"
(Phil Green, University of Washington, Seattle, Wash.).
[0798] Using the above analysis, a consensus DNA sequence was
assembled relative to other EST sequences using phrap. This
consensus sequence is herein designated consen01. Proprietary
Genentech EST sequences were employed in the consensus assembly and
they are herein designated DNA20239, DNA38050 and DNA40683.
[0799] Based on the consen01 sequence, oligonucleotides were
synthesized: 1) to identify by PCR a cDNA library that contained
the sequence of interest, and 2) for use as probes to isolate a
clone of the full-length coding sequence for PRO732. Forward and
reverse PCR primers generally range from 20 to 30 nucleotides and
are often designed to give a PCR product of about 100-1000 bp in
length. The probe sequences are typically 40-55 bp in length. In
some cases, additional oligonucleotides are synthesized when the
consensus sequence is greater than about 1-1.5 kbp. In order to
screen several libraries for a full-length clone, DNA from the
libraries was screened by PCR amplification, as per Ausubel et al.,
Current Protocols in Molecular Biology, with the PCR primer pair. A
positive library was then used to isolate clones encoding the gene
of interest using the probe oligonucleotide and one of the primer
pairs.
[0800] PCR primers (forward and reverse) were synthesized:
TABLE-US-00038 forward PCR primer 5'-ATGTTTGTGTGGAAGTGCCCCG-3' (SEQ
ID NO: 121) forward PCR primer 5'-GTCAACATGCTCCTCTGC-3' (SEQ ID NO:
122) reverse PCR primer 5'-AATCCATTGTGCACTGCAGCTCTAGG-3' (SEQ ID
NO: 123) reverse PCR primer 5'-GAGCATGCCACCACTGGACTGAC-3' (SEQ ID
NO: 124)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA44143 sequence which had the
following nucleotide sequence
TABLE-US-00039 hybridization probe (SEQ ID NO: 125)
5'-GCCGATGCTGTCCTAGTGGAAACAACTCCACTGTAACTAGATTGA TCTATGCAC-3'
[0801] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pairs identified above. A
positive library was then used to isolate clones encoding the
PRO732 gene using the probe oligonucleotide and one of the PCR
primers.
[0802] RNA for construction of the cDNA libraries was isolated from
human fetal lung tissue (LIB26). The cDNA libraries used to isolate
the cDNA clones were constructed by standard methods using
commercially available reagents such as those from Invitrogen, San
Diego, Calif. The cDNA was primed with olio dT containing a NotI
site, linked with blunt to SalI hemikinased adaptors, cleaved with
NotI, sized appropriately by gel electrophoresis, and cloned in a
defined orientation into a suitable cloning vector (such as pRKB or
pRKD; pRK5B is a precursor of pRK5D that does not contain the SfiI
site; see, Holmes et al., Science, 253:1278-1280 (1991)) in the
unique XhoI and NotI sites.
[0803] A full length clone was identified that contained a single
open reading frame with an apparent translational initiation site
at nucleotide positions 88-90 and ending at the stop codon found at
nucleotide positions 1447-1449 (FIG. 29, SEQ ID NO:29). The
predicted polypeptide precursor is 453 amino acids long, has a
calculated molecular weight of approximately 50,419 daltons and an
estimated pI of approximately 5.78. Analysis of the full-length
PRO732 sequence shown in FIG. 30 (SEQ ID NO:30) evidences the
presence of the following: a signal peptide from about amino acid 1
to about amino acid 28, transmembrane domains from about amino acid
37 to about amino acid 57, from about amino acid 93 to about amino
acid 109, from about amino acid 126 to about amino acid 148, from
about amino acid 151 to about amino acid 172, from about amino acid
197 to about amino acid 215, from about amino acid 231 to about
amino acid 245, from about amino acid 260 to about amino acid 279,
from about amino acid 315 to about amino acid 333, from about amino
acid 384 to about amino acid 403 and from about amino acid 422 to
about amino acid 447, potential N-glycosylation sites from about
amino acid 33 to about amino acid 36, from about amino acid 34 to
about amino acid 37, from about amino acid 179 to about amino acid
183, from about amino acid 298 to about amino acid 301, from about
amino acid 337 to about amino acid 340 and from about amino acid
406 to about amino acid 409, an amino acid block having homology to
the MIP family of proteins from about amino acid 119 to about amino
acid 149 and an amino acid block having homology to DNA/RNA
non-specific endonuclease proteins from about amino acid 279 to
about amino acid 286. Clone DNA48334-1435 has been deposited with
ATCC on Jun. 2, 1998 and is assigned ATCC deposit no. 209924.
[0804] Analysis of the amino acid sequence of the full-length
PRO732 polypeptide suggests that it possesses significant sequence
similarity to the Diff33 protein, thereby indicating that PRO732
may be a novel Diff33 homolog. More specifically, an analysis of
the Dayhoff database (version 35.45 SwissProt 35) evidenced
significant homology between the PRO732 amino acid sequence and the
following Dayhoff sequences, HS179M20.sub.--2, MUSTETU.sub.--1,
CER11H6.sub.--2, RATDRP.sub.--1, S51256, E69226, AE000869.sub.--1,
JC4120, CYB_PARTE and P_R50619.
Example 19
Isolation of cDNA Clones Encoding Human PRO1003 Polypeptides
[UNQ487]
[0805] Use of the signal sequence algorithm described in Example 3
above allowed identification of a single Incyte EST cluster
sequence designated herein as 43055. This sequence was then
compared to a variety of EST databases which included public EST
databases (e.g., GenBank) and a proprietary EST DNA database
(LIFESEQ.RTM., Incyte Pharmaceuticals, Palo Alto, Calif.) to
identify existing homologies. The homology search was performed
using the computer program BLAST or BLAST2 (Altshul et al., Methods
in Enzymology 266:460-480 (1996)). Those comparisons resulting in a
BLAST score of 70 (or in some cases 90) or greater that did not
encode known proteins were clustered and assembled into a consensus
DNA sequence with the program "phrap" (Phil Green, University of
Washington, Seattle, Wash.). The consensus sequence obtained
therefrom is herein designated consen01.
[0806] In light of an observed sequence homology between the
consensus sequence and an EST sequence encompassed within the
Incyte EST clone no. 2849382, the Incyte EST clone 2849382 was
purchased and the cDNA insert was obtained and sequenced. It was
found that this insert encoded a full-length protein. The sequence
of this cDNA insert is shown in FIG. 31.
[0807] The entire nucleotide sequence of DNA58846-1409 is shown in
FIG. 31 (SEQ ID NO:31). Clone DNA58846-1409 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 41-43 and ending at the stop codon at
nucleotide positions 293-295 (FIG. 31). The predicted polypeptide
precursor is 84 amino acids long (FIG. 32; SEQ ID NO:32). The
full-length PRO1003 protein shown in FIG. 32 has an estimated
molecular weight of about 9,408 daltons and a pI of about 9.28.
Analysis of the full-length PRO1003 sequence shown in FIG. 32 (SEQ
ID NO:32) evidences the presence of a signal peptide at amino acids
1 to about 24, and a cAMP- and cGMP-dependent protein kinase
phosphorylation site at about amino acids 58 to about 61. Analysis
of the amino acid sequence of the full-length PRO1003 polypeptide
using the Dayhoff database (version 35.45 SwissProt 35) evidenced
homology between the PRO1003 amino acid sequence and the following
Dayhoff sequences: AOPCZA363.sub.--3, SRTX_ATREN, A48298,
MHVJHMS.sub.--1, VGL2_CVMJH, DHDHTC2.sub.--2, CORT_RAT, TAL6_HUMAN,
P_W14123, and DVUFI.sub.--2.
Example 20
Isolation of cDNA Clones Encoding Human PRO1104 Polypeptides
[UNQ547]
[0808] Use of the signal sequence algorithm described in Example 3
above allowed identification of an EST cluster sequence from the
Incyte database. This EST cluster sequence was then compared to a
variety of expressed sequence tag (EST) databases which included
public EST databases (e.g., GenBank) and a proprietary EST DNA
database (Lifeseq.RTM., Incyte Pharmaceuticals, Palo Alto, Calif.)
to identify existing homologies. The homology search was performed
using the computer program BLAST or BLAST2 (Altshul et al., Methods
in Enzymology 266:460-480 (1996)). Those comparisons resulting in a
BLAST score of 70 (or in some cases 90) or greater that did not
encode known proteins were clustered and assembled into a consensus
DNA sequence with the program "phrap" (Phil Green, University of
Washington, Seattle, Wash.). The consensus sequence obtained
therefrom is herein designated DNA56446.
[0809] In light of an observed sequence homology between the
DNA56446 sequence and an EST sequence encompassed within the Incyte
EST clone no. 2837496, the Incyte EST clone 2837496 was purchased
and the cDNA insert was obtained and sequenced. It was found that
this insert encoded a full-length protein. The sequence of this
cDNA insert is shown in FIG. 33 and is herein designated as
DNA59616-1465.
[0810] The entire nucleotide sequence of DNA59616-1465 is shown in
FIG. 33 (SEQ ID NO:33). Clone DNA59616-1465 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 109-111 and ending at the stop codon at
nucleotide positions 1132-1134 of SEQ ID NO:33 (FIG. 33). The
predicted polypeptide precursor is 341 amino acids long (FIG. 34;
SEQ ID NO:34). The full-length PRO1104 protein shown in FIG. 34 has
an estimated molecular weight of about 36,769 daltons and a pI of
about 9.03. Clone DNA59616-1465 has been deposited with ATCC on
Jun. 16, 1998 under ATCC accession no: 209991. It is understood
that the deposited clone has the actual nucleic acid sequence and
that the sequences provided herein are based on known sequencing
techniques.
[0811] Analyzing FIG. 34, a signal peptide is at about amino acids
1-22 of SEQ ID NO:34. N-myristoylation sites are at about amino
acids 41-46, 110-115, 133-138, 167-172 and 179-184 of SEQ ID
NO:34.
Example 21
Isolation of cDNA Clones Encoding Human PRO1151 Polypeptides
[UNQ581]
[0812] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described in Example 1 above. This
consensus sequence is herein designated DNA40665. Based on the
DNA40665 consensus sequence, oligonucleotides were synthesized: 1)
to identify by PCR a cDNA library that contained the sequence of
interest, and 2) for use as probes to isolate a clone of the
full-length coding sequence for PRO1151.
[0813] PCR primers (forward and reverse) were synthesized:
TABLE-US-00040 forward PCR primer 5'-CCAGACGCTGCTCTTCGAAAGGGTC-3'
(SEQ ID NO: 126) reverse PCR primer 5'-GGTCCCCGTAGGCCAGGTCCAGC-3'
(SEQ ID NO: 127)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA40665 sequence which had the
following nucleotide sequence
TABLE-US-00041 hybridization probe (SEQ ID NO: 128)
5'-CTACTTCTTCAGCCTCAATGTGCACAGCTGGAATTACAAGGAGAC GTACG-3'
[0814] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones encoding the PRO1151 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human fetal
kidney tissue.
[0815] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO1151 (designated herein as
DNA44694-1500 [FIG. 35, SEQ ID NO:35]; and the derived protein
sequence for PRO1151.
[0816] The entire nucleotide sequence of DNA44694-1500 is shown in
FIG. 35 (SEQ ID NO:35). Clone DNA44694-1500 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 272-274 and ending at the stop codon at
nucleotide positions 1049-1051 (FIG. 35). The predicted polypeptide
precursor is 259 amino acids long (FIG. 36; SEQ ID NO:36). The
full-length PRO1151 protein shown in FIG. 36 has an estimated
molecular weight of about 28,770 daltons and a pI of about 6.12.
Analysis of the full-length PRO1151 sequence shown in FIG. 36 (SEQ
ID NO:36) evidences the presence of the following: a signal peptide
from about amino acid 1 to about amino acid 20, a potential
N-glycosylation site from about amino acid 72 to about amino acid
75 and amino acid sequence blocks having homology to C1q
domain-containing proteins from about amino acid 144 to about amino
acid 178, from about amino acid 78 to about amino acid 111 and from
about amino acid 84 to about amino acid 117. Clone UNQ581
(DNA44694-1500) has been deposited with ATCC on Aug. 11, 1998 and
is assigned ATCC deposit no. 203114.
[0817] An analysis of the Dayhoff database (version 35.45 SwissProt
35), using a WU-BLAST-2 sequence alignment analysis of the
full-length sequence shown in FIG. 36 (SEQ ID NO:36), evidenced
significant homology between the PRO1151 amino acid sequence and
the following Dayhoff sequences: ACR3_HUMAN, HP25_TAMAS,
HUMC1QB2.sub.--1, P_R99306, CA1F_HUMAN, JX0369, CA24_HUMAN, S32436,
P_R28916 and CA54_HUMAN.
Example 22
Isolation of cDNA Clones Encoding Human PRO1244 Polypeptides
[UNQ628]
[0818] Use of the signal sequence algorithm described in Example 3
above allowed identification of an EST cluster sequence from the
LIFESEQ.RTM. database, designated cluster no. 7874. This EST
cluster sequence was then compared to a variety of expressed
sequence tag (EST) databases which included public EST databases
(e.g., GenBank) and a proprietary EST DNA databases (LIFESEQ.RTM.,
Incyte Pharmaceuticals, Palo Alto, Calif.; Genentech, South San
Francisco, Calif.) to identify existing homologies. One or more of
the ESTs was derived from a library constructed from tissue of the
corpus cavernosum. The homology search was performed using the
computer program BLAST or BLAST2 (Altshul et al., Methods in
Enzymology 266:460-480 (1996)). Those comparisons resulting in a
BLAST score of 70 (or in some cases 90) or greater that did not
encode known proteins were clustered and assembled into a consensus
DNA sequence with the program "phrap" (Phil Green, University of
Washington, Seattle, Wash.). The consensus sequence obtained
therefrom is herein designated "DNA56011".
[0819] In light of the sequence homology between the DNA56011
sequence and an EST sequence contained within Incyte EST No.
3202349, the EST clone no. 3202349 was purchased and the cDNA
insert was obtained and sequenced. The sequence of this cDNA insert
is shown in FIG. 37 (SEQ ID NO:37) and is herein designated
"DNA64883-1526".
[0820] The full length clone shown in FIG. 37 contained a single
open reading frame with an apparent translational initiation site
at nucleotide positions 9-11 and ending at the stop codon found at
nucleotide positions 1014-1016 (FIG. 37; SEQ ID NO:37). The
predicted polypeptide precursor (FIG. 38, SEQ ID NO:38) is 335
amino acids long. PRO1244 has a calculated molecular weight of
approximately 38,037 daltons and an estimated pI of approximately
9.87. Other features include a signal peptide at about amino acids
1-29; transmembrane domains at about amino acids 183-205, 217-237,
271-287, and 301-321; potential N-glycosylation sites at about
amino acids 71-74, and 215-218; and a cell attachment sequence at
about amino acids 150-152.
[0821] An analysis of the Dayhoff database (version 35.45 SwissProt
35), using a WU-BLAST2 sequence alignment analysis of the
full-length sequence shown in FIG. 38 (SEQ ID NO:38), revealed
homology between the PRO1244 amino acid sequence and the following
Dayhoff sequences: AF008554.sub.--1, P.sub.--485334, G02297,
HUMN33S11.sub.--1, HUMN33S10.sub.--1, YO13_CAEEL, GEN13255, S49758,
E70107, and ERP5_MEDSA.
[0822] Clone DNA64883-1526 was deposited with the ATCC on Sep. 9,
1998, and is assigned ATCC deposit no. 203253.
Example 23
Isolation of cDNA Clones Encoding Human PRO1298 Polypeptides
[UNQ666]
[0823] Use of the signal sequence algorithm described in Example 3
above allowed identification of an EST cluster sequence from an
Incyte database. This EST cluster sequence was then compared to a
variety of expressed sequence tag (EST) databases which included
public EST databases (e.g., GenBank) and a proprietary EST DNA
database (LIFESEQ.RTM., Incyte Pharmaceuticals, Palo Alto, Calif.)
to identify existing homologies. One or more of the ESTs was
derived from a diseased prostate tissue library. The homology
search was performed using the computer program BLAST or BLAST2
(Altshul et al., Methods in Enzymology 266:460-480 (1996)). Those
comparisons resulting in a BLAST score of 70 (or in some cases 90)
or greater that did not encode known proteins were clustered and
assembled into a consensus DNA sequence with the program "phrap"
(Phil Green, University of Washington, Seattle, Wash.). The
consensus sequence obtained therefrom is herein designated
DNA56389.
[0824] In light of the sequence homology between the DNA56389
sequence and an EST sequence contained within an Incyte EST within
the assembly from with the consensus sequence was derived, Incyte
clone 3355717 was purchased and the cDNA insert was obtained and
sequenced. The sequence of this cDNA insert is shown in FIG. 39 and
is herein designated as DNA66511-1563.
[0825] The full length clone shown in FIG. 39 contained a single
open reading frame with an apparent translational initiation site
at nucleotide positions 94-96 and ending at the stop codon found at
nucleotide positions 1063-1065 (FIG. 39; SEQ ID NO:39). The
predicted polypeptide precursor (FIG. 40, SEQ ID NO:40) is 323
amino acids long. The signal peptide is at about amino acids 1-15
of SEQ ID NO:40. PRO1298 has a calculated molecular weight of
approximately 37,017 daltons and an estimated pI of approximately
8.83. Clone DNA66511-1563 was deposited with the ATCC on Sep. 15,
1998 and is assigned ATCC deposit no. 203228.
[0826] An analysis of the Dayhoff database (version 35.45 SwissProt
35), using a WU-BLAST2 sequence alignment analysis of the
full-length sequence shown in FIG. 40 (SEQ ID NO:40), revealed
sequence identity between the PRO1298 amino acid sequence and the
following Dayhoff sequences (data incorporated herein): ALG2_YEAST,
CAPM_STAAU, C69098, C69255, SUS2_MAIZE, A69143, S74778,
AB009527.sub.--13, AF050103.sub.--2 and BBA224769.sub.--1.
Example 24
Isolation of cDNA Clones Encoding Human PRO1313 Polypeptides
[UNQ679]
[0827] The extracellular domain (ECD) sequences (including the
secretion signal sequence, if any) from about 950 known secreted
proteins from the Swiss-Prot public database were used to search
EST databases. The EST databases included public EST databases
(e.g., GenBank), a proprietary EST database (LIFESEQ.RTM., Incyte
Pharmaceuticals, Palo Alto, Calif.), and proprietary ESTs from
Genentech. The search was performed using the computer program
BLAST or BLAST2 [Altschul et al., Methods in Enzymology,
266:460-480 (1996)] as a comparison of the ECD protein sequences to
a 6 frame translation of the EST sequences. Those comparisons
resulting in a BLAST score of 70 (or in some cases, 90) or greater
that did not encode known proteins were clustered and assembled
into consensus DNA sequences with the program "phrap" (Phil Green,
University of Washington, Seattle, Wash.).
[0828] A consensus DNA sequence was assembled relative to other EST
sequences using phrap. This consensus sequence is herein designated
DNA64876. Based on the DNA64876 consensus sequence and upon a
search for sequence homology with a proprietary Genentech EST
sequence designated as DNA57711, a Merck/Washington University EST
sequence designated R80613 was found to have significant homology
with DNA64876 and DNA57711. Therefore, the Merck/Washington
University EST clone no. R80613 was purchased and the insert
thereof obtained and sequence, thereby giving rise to the
DNA64966-1575 sequence shown in FIG. 41.
[0829] DNA sequencing of the R80613 insert obtained as described
above gave the full-length DNA sequence for PRO1313 (designated
herein as DNA64966-1575 [FIG. 41, SEQ ID NO: 41]; (UNQ679) and the
derived protein sequence for PRO1313.
[0830] The entire nucleotide sequence of UNQ679 (DNA64966-1575) is
shown in FIG. 41 (SEQ ID NO:41). Clone UNQ679 (DNA64966-1575)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 115-117 and ending at the
stop codon at nucleotide positions 1036-1038 (FIG. 41). The
predicted polypeptide precursor is 307 amino acids long (FIG. 42;
SEQ ID NO:42). The full-length PRO1313 protein shown in FIG. 42 has
an estimated molecular weight of about 35,098 daltons and a pI of
about 8.11. Analysis of the full-length PRO1313 sequence shown in
FIG. 42 (SEQ ID NO:42) evidences the presence of the following: a
signal peptide from about amino acid 1 to about amino acid 15,
transmembrane domains from about amino acid 134 to about amino acid
157, from about amino acid 169 to about amino acid 189, from about
amino acid 230 to about amino acid 248 and from about amino acid
272 to about amino acid 285, potential N-glycosylation sites from
about amino acid 34 to about amino acid 37, from about amino acid
135 to about amino acid 138 and from about amino acid 203 to about
amino acid 206 and ATP/GTP binding site motif A from about amino
acid 53 to about amino acid 60. Clone UNQ679 (DNA64966-1575) has
been deposited with ATCC on Jan. 12, 1999 and is assigned ATCC
deposit no. 203575.
[0831] An analysis of the Dayhoff database (version 35.45 SwissProt
35), using a WU-BLAST2 sequence alignment analysis of the
full-length sequence shown in FIG. 42 (SEQ ID NO:42), evidenced
significant homology between the PRO1313 amino acid sequence and
the following Dayhoff sequences: CELT27A1.sub.--3, CEF09C6.sub.--7,
U93688.sub.--9, H64896, YDCX_ECOLI and RNU06101.sub.--1.
Example 25
Isolation of cDNA Clones Encoding Human PRO1570 Polypeptides
[UNQ776]
[0832] A consensus DNA sequence encoding PRO1570 was assembled
relative to other EST sequences using phrap as described in Example
1 above to form an assembly. This consensus sequence is designated
herein as "DNA65415". Based on the DNA65415 consensus sequence and
other discoveries and information provided herein, the clone
including Incyte EST 3232285 (from a uterine/colon cancer tissue
library) was purchased and sequenced in full which gave SEQ ID
NO:43.
[0833] The entire coding sequence of PRO1570 is included in FIG. 43
(SEQ ID NO:43). Clone DNA68885-1678 contains a single open reading
frame with an apparent translational initiation site at nucleotide
positions 210-212 and an apparent stop codon at nucleotide
positions 1506-1508 of SEQ ID NO:43. The predicted polypeptide
precursor is 432 amino acids long. FIG. 44 (SEQ ID NO:44) shows a
number of motifs. Clone DNA68885-1678 has been deposited with the
ATCC on Oct. 6, 1998 and is assigned ATCC deposit no. 203311. The
full-length PRO1570 protein shown in FIG. 44 has an estimated
molecular weight of about 47,644 daltons and a pI of about
5.18.
[0834] An analysis of the Dayhoff database (version 35.45 SwissProt
35), using a WU-BLAST2 sequence alignment analysis of the
full-length sequence shown in FIG. 44 (SEQ ID NO:44), revealed
sequence identity between the PRO1570 amino acid sequence and the
following Dayhoff sequences (incorporated herein): P_W22986,
TMS2_HUMAN, HEPS_HUMAN, P_R89435, AB002134.sub.--1, KAL_MOUSE,
ACRO_HUMAN, GEN12917, AF045649.sub.--1, and P_W34285.
Example 26
Isolation of cDNA Clones Encoding Human PRO1886 Polypeptides
[UNQ870]
[0835] An initial DNA sequence was identified using a yeast screen,
in a human aortic endothelial cDNA library that preferentially
represents the 5' ends of the primary cDNA clones. This sequence
was compared to ESTs from public databases (e.g., GenBank,
Merck/Wash U.), and a proprietary EST database (LIFESEQ.RTM.,
Incyte Pharmaceuticals, Palo Alto, Calif.), using the computer
program BLAST or BLAST2 [Altschul et al., Methods in Enzymology,
266:460-480 (1996)]. The ESTs were clustered and assembled into a
consensus DNA sequence using the computer program "phrap" (Phil
Green, University of Washington, Seattle, Wash.;
http://bozeman.mbt.washington.edu/phrap.docs/phrap.html). This
consensus sequence is designated herein as "DNA78722". Other novel
sequences were identified in the alignment of sequences which
formed DNA78722. Based on the DNA78722 consensus sequence,
oligonucleotides were synthesized for use as probes to isolate a
clone of the full-length coding sequence for PRO1886 from a human
aortic endothelial cells cDNA library.
[0836] The full length DNA80796-2523 clone shown in FIG. 45
contained a single open reading frame with an apparent
translational initiation site at nucleotide positions 73-75 and
ending at the stop codon found at nucleotide positions 1022-1025
(FIG. 45; SEQ ID NO:45). The predicted polypeptide precursor (FIG.
46, SEQ ID NO:46) is 316 amino acids long. Other features are
indicated in FIG. 46. PRO1886 has a calculated molecular weight of
approximately 36045 daltons and an estimated pI of approximately
8.18. Clone DNA80796-2523 (UNQ870), designated as DNA80796-2523 has
been deposited with the ATCC on Dec. 22, 1998 and is assigned ATCC
deposit no. 203555.
[0837] An analysis of the Dayhoff database (version 35.45 SwissProt
35), using a WU-BLAST2 sequence alignment analysis of the
full-length sequence shown in FIG. 46 (SEQ ID NO:46), revealed
sequence identity between the PRO1886 amino acid sequence and the
following Dayhoff sequences: CELT26A8.sub.--2 and S43230.
Example 27
Isolation of cDNA Clones Encoding Human PRO1891 Polypeptides
[UNQ873]
[0838] The extracellular domain (ECD) sequences (including the
secretion signal sequence, if any) from about 950 known secreted
proteins from the Swiss-Prot public database were used to search
EST databases. The EST databases included public EST databases
(e.g., GenBank), and a proprietary EST database (LIFESEQ.RTM.,
Incyte Pharmaceuticals, Palo Alto, Calif.). The search was
performed using the computer program BLAST or BLAST2 [Altschul et
al., Methods in Enzymology, 266:460-480 (1996)] as a comparison of
the ECD protein sequences to a 6 frame translation of the EST
sequences. Those comparisons resulting in a BLAST score of 70 (or
in some cases, 90) or greater that did not encode known proteins
were clustered and assembled into consensus DNA sequences with the
program "phrap" (Phil Green, University of Washington, Seattle,
Wash.). A consensus DNA sequence encoding PRO1891 was assembled
relative to other EST sequences using phrap. This consensus
sequence is designated herein "DNA44813"
[0839] Based on the DNA44813 consensus sequence, oligonucleotides
were synthesized: 1) to identify by PCR a cDNA library that
contained the sequence of interest, and 2) for use as probes to
isolate a clone of the full-length coding sequence for PRO1891.
Forward and reverse PCR primers generally range from 20 to 30
nucleotides and are often designed to give a PCR product of about
100-1000 bp in length. The probe sequences are typically 40-55 bp
in length. In some cases, additional oligonucleotides are
synthesized when the consensus sequence is greater than about 1-1.5
kbp. In order to screen several libraries for a full-length clone,
DNA from the libraries was screened by PCR amplification, as per
Ausubel et al., Current Protocols in Molecular Biology, supra, with
the PCR primer pair. A positive library was then used to isolate
clones encoding the gene of interest using the probe
oligonucleotide and one of the primer pairs.
[0840] PCR primers (forward and reverse) were synthesized:
TABLE-US-00042 forward PCR primers: GCTGCTTTGCTCACAACTGCTCGC,
(44813.f1; SEQ ID NO: 129) CATGACACCTTCCTGCTG (44813.f2; SEQ ID NO:
130) and CAGCCATGGGTGACTGTGACCTCC (44813.f3; SEQ ID NO: 131)
reverse PCR primers: CTCCTGGGAGTCGGTAGCAACACC, (44813.r1; SEQ ID
NO: 132) GGGAGGTCACAGTCACCC (44813.r2; SEQ ID NO: 133) and
GGCTGGGCTTTCCACCCTGGCAC. (44813.r3; SEQ ID NO: 134)
[0841] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA44813 sequence which
had the following nucleotide sequence:
TABLE-US-00043 hybridization probe: (44813.p1; SEQ ID NO: 135)
CAGCCATGGGTGACTGTGACCTCCCTGAGTTTTGCACGGG.
[0842] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones encoding the PRO1891 gene
using the probe oligonucleotide and one of the PCR primers.
[0843] RNA for construction of the cDNA libraries was isolated
human bone marrow. The cDNA libraries used to isolate the cDNA
clones were constructed by standard methods using commercially
available reagents such as those from Invitrogen, San Diego, Calif.
The cDNA was primed with olio dT containing a NotI site, linked
with blunt to SalI hemikinased adaptors, cleaved with NotI, sized
appropriately by gel electrophoresis, and cloned in a defined
orientation into a suitable cloning vector (such as pRKB or pRKD;
pRK5B is a precursor of pRK5D that does not contain the SfiI site;
see, Holmes et al., Science, 253:1278-1280 (1991)) in the unique
XhoI and NotI sites.
[0844] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO1891, designated herein as
"DNA76788-2526"(FIG. 47; SEQ ID NO:47), and the derived protein
sequence for PRO1891.
[0845] The entire coding sequence of PRO1891 is shown in FIG. 47
(SEQ ID NO:47). Clone DNA76788-2526 contains a single open reading
frame with an apparent translational initiation site at nucleotide
positions 114-116, and an apparent stop codon at nucleotide
positions 2553-2555. The predicted polypeptide precursor is 813
amino acids long. The full-length PRO1891 protein shown in FIG. 48
(SEQ ID NO:48) has an estimated molecular weight of about 87,739
daltons and a pI of about 6.94. Additional features include a
signal peptide at about amino acids 1-27; a transmembrane domain at
about amino acids 702-720; potential N-glycosylation sites at about
amino acids 109-112, 145-148, 231-234, 276-279, and 448-451; a
tyrosine kinase phosphorylation site at about amino acids 236-243;
potential N-myristoylation sites at about amino acids 29-34,
285-190, 195-200, 308-313, 318-323, 326-331, 338-343, 370-375,
400-405, 402-407, 454-459, 504-509, 510-515, 517-522, 580-585,
601-606, 661-666, 687-692, 717-722, and 719-724; an amidation site
at about amino acids 200-203; and a neutral zinc metallopeptidases,
zinc-binding region signature at about amino acids 342-351.
[0846] An analysis of the Dayhoff database (version 35.45 SwissProt
35), using a WU-BLAST2 sequence alignment analysis of the
full-length sequence shown in FIG. 48 (SEQ ID NO:48), revealed
significant homology between the PRO1891 amino acid sequence and
the following Dayhoff sequences: XLU66003.sub.--1, P_W25716,
AF023477.sub.--1, P_W01825, P_R99801, P_W25722, P_W44120, P_R67759,
AF029899.sub.--1, and P_W14772.
[0847] Clone DNA76788 (UNQ873), designated as DNA76788-2526 was
deposited with the ATCC on Dec. 22, 1998 and is assigned ATCC
deposit no. 203551.
Example 28
Isolation of cDNA Clones Encoding Human PRO4409 Polypeptides
[UNQ1934]
[0848] DNA88004-2575 was identified by applying a proprietary
signal sequence finding algorithm developed by Genentech, Inc.
(South San Francisco, Calif.) upon ESTs as well as clustered and
assembled EST fragments from public (e.g., GenBank) and/or private
(LIFESEQ.RTM., Incyte Pharmaceuticals, Inc., Palo Alto, Calif.)
databases. The signal sequence algorithm computes a secretion
signal score based on the character of the DNA nucleotides
surrounding the first and optionally the second methionine codon(s)
(ATG) at the 5'-end of the sequence or sequence fragment under
consideration. The nucleotides following the first ATG must code
for at least 35 unambiguous amino acids without any stop codons. If
the first ATG has the required amino acids, the second is not
examined. If neither meets the requirement, the candidate sequence
is not scored. In order to determine whether the EST sequence
contains an authentic signal sequence, the DNA and corresponding
amino acid sequences surrounding the ATG codon are scored using a
set of seven sensors (evaluation parameters) known to be associated
with secretion signals.
[0849] Use of the above described signal sequence algorithm allowed
identification of an EST cluster sequence from the Incyte database.
This EST cluster sequence was then compared to a variety of
expressed sequence tag (EST) databases which included public EST
databases (e.g., GenBank) and a proprietary EST DNA database
(LIFESEQ.RTM., Incyte Pharmaceuticals, Palo Alto, Calif.) to
identify existing homologies. The homology search was performed
using the computer program BLAST or BLAST2 (Altshul et al., Methods
in Enzymology 266:460-480 (1996)). Those comparisons resulting in a
BLAST score of 70 (or in some cases 90) or greater that did not
encode known proteins were clustered and assembled into a consensus
DNA sequence with the program "phrap" (Phil Green, University of
Washington, Seattle, Wash. The consensus sequence obtained
therefrom is herein designated DNA79305. In light of DNA79305, a
human brain library cDNA library was screened with the following
two primers to identify DNA88004-2575: 5'GAGCTGAAGTCAGCCTTTGAG3'
(SEQ ID NO:136, forward) and 5'CTCTGCAGAAGTCTCGTTCC3' (SEQ ID
NO:137, reverse).
[0850] The full length clone shown in FIG. 49 contained a single
open reading frame with an apparent translational initiation site
at nucleotide positions 337-339 and ending at the stop codon found
at nucleotide positions 1171-1173 (FIG. 49; SEQ ID NO:49). The
predicted polypeptide precursor (FIG. 50, SEQ ID NO:50) is 278
amino acids long. PRO4409 has a calculated molecular weight of
approximately 30748 daltons and an estimated pI of approximately
5.47.
[0851] An analysis of the Dayhoff database (version 35.45 SwissProt
35), using a WU-BLAST2 sequence alignment analysis of the
full-length sequence shown in FIG. 50 (SEQ ID NO:50), revealed
homology between the PRO4409 amino acid sequence and the following
Dayhoff sequences (sequences nad related text incorporated herein):
HGS_RF300, HSU80744.sub.--1, CEC11H1.sub.--7, CEVK04G11.sub.--2,
HGS_RF177, CEF09E8.sub.--2, AF034802.sub.--1, P_R51227, I46014 and
CYL2_BOVIN.
[0852] Clone DNA88004-2575 (UNQ1934), designated as DNA88004-2575
was deposited with the ATCC on Mar. 30, 1999 and is assigned ATCC
deposit no. 203890.
Example 29
Isolation of cDNA Clones Encoding Human PRO5725 Polypeptides
[UNQ2446]
[0853] An expressed sequence tag (EST) DNA database (LIFESEQ.RTM.,
Incyte Pharmaceuticals, Palo Alto, Calif.) was searched and an EST
was identified which showed homology to Neuritin. EST clone no.
3705684 was then purchased from LIFESEQ.RTM., Incyte
Pharmaceuticals, Palo Alto, Calif. and the cDNA insert of that
clone was obtained and sequenced in entirety.
[0854] The entire nucleotide sequence of the clone, designated
herein as DNA92265-2669, is shown in FIG. 51 (SEQ ID NO: 51). The
DNA92265-2669 clone contains a single open reading frame with an
apparent translational initiation site at nucleotide positions
27-29 and a stop signal at nucleotide positions 522-524 (FIG. 51,
SEQ ID NO:51). The predicted polypeptide precursor is 165 amino
acids long, has a calculated molecular weight of approximately
17,786 daltons and an estimated pI of approximately 8.43. Analysis
of the full-length PRO5725 sequence shown in FIG. 52 (SEQ ID NO:52)
evidences the presence of a variety of important polypeptide
domains as shown in FIG. 52, wherein the locations given for those
important polypeptide domains are approximate as described above.
Clone DNA92265-2669 has been deposited with ATCC on Jun. 22, 1999
and is assigned ATCC deposit no. PTA-256.
[0855] An analysis of the Dayhoff database (version 35.45 SwissProt
35), using the ALIGN-2 sequence alignment analysis of the
full-length sequence shown in FIG. 52 (SEQ ID NO:52), evidenced
sequence identity between the PRO5725 amino acid sequence and the
following Dayhoff sequences: RNU88958.sub.--1; P_W37859; P_W37858;
JC6305; HGS_RE778; HGS_RE777; P_W27652; P_W44088; HGS_RE776; and
HGS_RE425.
Example 30
Isolation of cDNA Clones Encoding Human PRO6097 Polypeptides
[UNQ2545]
[0856] 1. Preparation of Oligo dT Primed cDNA Library
[0857] mRNA was isolated from human SK-Lu-1 adenocarcinoma cell
line tissue using reagents and protocols from Invitrogen, San
Diego, Calif. (Fast Track 2). This RNA was used to generate an
oligo dT primed cDNA library in the vector pRK5D using reagents and
protocols from Life Technologies, Gaithersburg, Md. (Super Script
Plasmid System). In this procedure, the double stranded cDNA was
sized to greater than 1000 bp and the SalI/NotI linkered cDNA was
cloned into XhoI/NotI cleaved vector. pRK5D is a cloning vector
that has an sp6 transcription initiation site followed by an SfiI
restriction enzyme site preceding the XhoI/NotI cDNA cloning
sites.
[0858] 2. Preparation of Random Primed cDNA Library
[0859] A secondary cDNA library was generated in order to
preferentially represent the 5' ends of the primary cDNA clones.
Sp6 RNA was generated from the primary library (described above),
and this RNA was used to generate a random primed cDNA library in
the vector pSST-AMY.0 using reagents and protocols from Life
Technologies (Super Script Plasmid System, referenced above). In
this procedure the double stranded cDNA was sized to 500-1000 bp,
linkered with blunt to NotI adaptors, cleaved with SfiI, and cloned
into SfiI/NotI cleaved vector. pSST-AMY.0 is a cloning vector that
has a yeast alcohol dehydrogenase promoter preceding the cDNA
cloning sites and the mouse amylase sequence (the mature sequence
without the secretion signal) followed by the yeast alcohol
dehydrogenase terminator, after the cloning sites. Thus, cDNAs
cloned into this vector that are fused in frame with the amylase
sequence will lead to the secretion of amylase from appropriately
transfected yeast colonies.
[0860] 3. Transformation and Detection
[0861] DNA from the library described in paragraph 2 above was
chilled on ice to which was added electrocompetent DH10B bacteria
(Life Technologies, 20 ml). The bacteria and vector mixture was
then electroporated as recommended by the manufacturer.
Subsequently, SOC media (Life Technologies, 1 ml) was added and the
mixture was incubated at 37.degree. C. for 30 minutes. The
transformants were then plated onto 20 standard 150 mm LB plates
containing ampicillin and incubated for 16 hours (37.degree. C.).
Positive colonies were scraped off the plates and the DNA was
isolated from the bacterial pellet using standard protocols, e.g.
CsCl-gradient. The purified DNA was then carried on to the yeast
protocols below.
[0862] The yeast methods were divided into three categories: (1)
Transformation of yeast with the plasmid/cDNA combined vector; (2)
Detection and isolation of yeast clones secreting amylase; and (3)
PCR amplification of the insert directly from the yeast colony and
purification of the DNA for sequencing and further analysis.
[0863] The yeast strain used was HD56-5A (ATCC-90785). This strain
has the following genotype: MAT alpha, ura3-52, leu2-3, leu2-112,
his3-11, his3-15, MAL.sup.+, SUC.sup.+, GAL.sup.+. Preferably,
yeast mutants can be employed that have deficient
post-translational pathways. Such mutants may have translocation
deficient alleles in sec71, sec72, sec62, with truncated sec71
being most preferred. Alternatively, antagonists (including
antisense nucleotides and/or ligands) which interfere with the
normal operation of these genes, other proteins implicated in this
post translation pathway (e.g., SEC61p, SEC72p, SEC62p, SEC63p,
TDJ1p or SSA1p-4p) or the complex formation of these proteins may
also be preferably employed in combination with the
amylase-expressing yeast.
[0864] Transformation was performed based on the protocol outlined
by Gietz et al., Nucl. Acid. Res., 20:1425 (1992). Transformed
cells were then inoculated from agar into YEPD complex media broth
(100 ml) and grown overnight at 30.degree. C. The YEPD broth was
prepared as described in Kaiser et al., Methods in Yeast Genetics,
Cold Spring Harbor Press, Cold Spring Harbor, N.Y., p. 207 (1994).
The overnight culture was then diluted to about 2.times.10.sup.6
cells/ml (approx. OD.sub.600=0.1) into fresh YEPD broth (500 ml)
and regrown to 1.times.10.sup.7 cells/ml (approx.
OD.sub.600=0.4-0.5).
[0865] The cells were then harvested and prepared for
transformation by transfer into GS3 rotor bottles in a Sorval GS3
rotor at 5,000 rpm for 5 minutes, the supernatant discarded, and
then resuspended into sterile water, and centrifuged again in 50 ml
falcon tubes at 3,500 rpm in a Beckman GS-6KR centrifuge. The
supernatant was discarded and the cells were subsequently washed
with LiAc/TE (10 ml, 10 mM Tris-HCl, 1 mM EDTA pH 7.5, 100 mM
Li.sub.2OOCCH.sub.3), and resuspended into LiAc/TE (2.5 ml).
[0866] Transformation took place by mixing the prepared cells (100
.mu.l) with freshly denatured single stranded salmon testes DNA
(Lofstrand Labs, Gaithersburg, Md.) and transforming DNA (1 .mu.g,
vol. <10 .mu.l) in microfuge tubes. The mixture was mixed
briefly by vortexing, then 40% PEG/TE (600 .mu.l, 40% polyethylene
glycol-4000, 10 mM Tris-HCl, 1 mM EDTA, 100 mM Li.sub.2OOCCH.sub.3,
pH 7.5) was added. This mixture was gently mixed and incubated at
30.degree. C. while agitating for 30 minutes. The cells were then
heat shocked at 42.degree. C. for 15 minutes, and the reaction
vessel centrifuged in a microfuge at 12,000 rpm for 5-10 seconds,
decanted and resuspended into TE (500 .mu.l, 10 mM Tris-HCl, 1 mM
EDTA pH 7.5) followed by recentrifugation. The cells were then
diluted into TE (1 ml) and aliquots (200 .mu.l) were spread onto
the selective media previously prepared in 150 mm growth plates
(VWR).
[0867] Alternatively, instead of multiple small reactions, the
transformation was performed using a single, large scale reaction,
wherein reagent amounts were scaled up accordingly.
[0868] The selective media used was a synthetic complete dextrose
agar lacking uracil (SCD-Ura) prepared as described in Kaiser et
al., Methods in Yeast Genetics, Cold Spring Harbor Press, Cold
Spring Harbor, N.Y., p. 208-210 (1994). Transformants were grown at
30.degree. C. for 2-3 days.
[0869] The detection of colonies secreting amylase was performed by
including red starch in the selective growth media. Starch was
coupled to the red dye (Reactive Red-120, Sigma) as per the
procedure described by Biely et al., Anal. Biochem., 172:176-179
(1988). The coupled starch was incorporated into the SCD-Ura agar
plates at a final concentration of 0.15% (w/v), and was buffered
with potassium phosphate to a pH of 7.0 (50-100 mM final
concentration).
[0870] The positive colonies were picked and streaked across fresh
selective media (onto 150 mm plates) in order to obtain well
isolated and identifiable single colonies. Well isolated single
colonies positive for amylase secretion were detected by direct
incorporation of red starch into buffered SCD-Ura agar. Positive
colonies were determined by their ability to break down starch
resulting in a clear halo around the positive colony visualized
directly.
[0871] 4. Isolation of DNA by PCR Amplification
[0872] When a positive colony was isolated, a portion of it was
picked by a toothpick and diluted into sterile water (30 .mu.l) in
a 96 well plate. At this time, the positive colonies were either
frozen and stored for subsequent analysis or immediately amplified.
An aliquot of cells (5 .mu.l) was used as a template for the PCR
reaction in a 25 .mu.l volume containing: 0.5 .mu.l Klentaq
(Clontech, Palo Alto, Calif.); 4.0 .mu.l 10 mM dNTP's (Perkin
Elmer-Cetus); 2.5 .mu.l Klentaq buffer (Clontech); 0.25 .mu.l
forward oligo 1; 0.25 .mu.l reverse oligo 2; 12.5 .mu.l distilled
water. The sequence of the forward oligonucleotide 1 was:
TABLE-US-00044 (SEQ ID NO: 67)
5'-TGTAAAACGACGGCCAGTTAAATAGACCTGCAATTATTAATCT-3'
The sequence of reverse oligonucleotide 2 was:
TABLE-US-00045 (SEQ ID NO: 68)
5'-CAGGAAACAGCTATGACCACCTGCACACCTGCAAATCCATT-3'
[0873] PCR was then performed as follows:
TABLE-US-00046 a. Denature 92.degree. C., 5 minutes b. 3 cycles of:
Denature 92.degree. C., 30 seconds Anneal 59.degree. C., 30 seconds
Extend 72.degree. C., 60 seconds c. 3 cycles of: Denature
92.degree. C., 30 seconds Anneal 57.degree. C., 30 seconds Extend
72.degree. C., 60 seconds d. 25 cycles of: Denature 92.degree. C.,
30 seconds Anneal 55.degree. C., 30 seconds Extend 72.degree. C.,
60 seconds e. Hold 4.degree. C.
[0874] The underlined regions of the oligonucleotides disclosed
above annealed to the ADH promoter region and the amylase region,
respectively, and amplified a 307 bp region from vector pSST-AMY.0
when no insert was present. Typically, the first 18 nucleotides of
the 5' end of these oligonucleotides contained annealing sites for
the sequencing primers. Thus, the total product of the PCR reaction
from an empty vector was 343 bp. However, signal sequence-fused
cDNA resulted in considerably longer nucleotide sequences.
[0875] Following the PCR, an aliquot of the reaction (5 .mu.l) was
examined by agarose gel electrophoresis in a 1% agarose gel using a
Tris-Borate-EDTA (TBE) buffering system as described by Sambrook et
al., supra. Clones resulting in a single strong PCR product larger
than 400 bp were further analyzed by DNA sequencing after
purification with a 96 Qiaquick PCR clean-up column (Qiagen Inc.,
Chatsworth, Calif.).
[0876] 5. Identification of Full-Length Clone
[0877] A cDNA sequence isolated in the above screen is herein
designated DNA84712. Probes were then generated from the sequence
of the DNA84712 molecule and used to screen a human SK-Lu-1
adenocarcinoma cell line library (247) prepared as described in
paragraph 1 above. The cloning vector was pRK5B (pRK5B is a
precursor of pRK5D that does not contain the SfiI site; see, Holmes
et al., Science, 253:1278-1280 (1991)), and the cDNA size cut was
less than 2800 bp. The oligonucleotides probes were synthesized: 1)
to identify by PCR a cDNA library that contained the sequence of
interest, and 2) for use as probes to isolate a clone of the
full-length coding sequence for PRO6097. Forward and reverse PCR
primers generally range from 20 to 30 nucleotides and are often
designed to give a PCR product of about 100-1000 bp in length. The
probe sequences are typically 40-55 bp in length. In order to
screen several libraries for a full-length clone, DNA from the
libraries was screened by PCR amplification, as per Ausubel et al.,
Current Protocols in Molecular Biology, supra, with the PCR primer
pair. A positive library was then used to isolate clones encoding
the gene of interest using the probe oligonucleotide and one of the
primer pairs.
[0878] The oligonucleotide probes employed were as follows:
TABLE-US-00047 forward PCR primer (SEQ ID NO: 138)
5'-CTGACCGGTCCGCTCATGG-3' reverse PCR primer (SEQ ID NO: 139)
5'-CAGCATGCTTTCCGCGAAGTC-3' hybridization probe (SEQ ID NO: 140)
5'-GGCAGGAAGGCCAGGGGTGCTGAGTTCTTCACCTCCTTTTAGAC TG-3'
[0879] A full length clone was identified that contained a single
open reading frame with an apparent translational initiation site
at nucleotide positions 158-160 and a stop signal at nucleotide
positions 1727-1729 (FIG. 55, SEQ ID NO: 55). The predicted
polypeptide precursor is 523 amino acids long, has a calculated
molecular weight of approximately 58,887 daltons and an estimated
pI of approximately 9.57. Analysis of the full-length PRO6097
sequence shown in FIG. 56 (SEQ ID NO: 56) evidences the presence of
a variety of important polypeptide domains as shown in FIG. 56,
wherein the locations given for those important polypeptide domains
are approximate as described above. Clone DNA107701-2711 has been
deposited with ATCC on Aug. 3, 1999 and is assigned ATCC Deposit
No. PTA-487.
[0880] An analysis of the Dayhoff database (version 35.45 SwissProt
35), using the ALIGN-2 sequence alignment analysis of the
full-length sequence shown in FIG. 56 (SEQ ID NO: 56), evidenced
sequence identity between the PRO6097 amino acid sequence and the
following Dayhoff sequences: YMB8_YEAST; S49759; ATF10N7.sub.--5;
SPBC405.sub.--3; S69718; H69798; D71226; U95370.sub.--5; A69780;
B69461.
Example 31
Isolation of cDNA Clones Encoding Human PRO7425 Polypeptides
[UNQ2966]
[0881] The extracellular domain (ECD) sequences (including the
secretion signal sequence, if any) from about 950 known secreted
proteins from the Swiss-Prot public database were used to search
EST databases. The EST databases included (1) public EST databases
(e.g., Merck/Washington University), (2) a proprietary EST database
(LIFESEQ.RTM., Incyte Pharmaceuticals, Palo Alto, Calif.), (3) a
proprietary EST database from Genentech. The search was performed
using the computer program BLAST or BLAST2 [Altschul et al.,
Methods in Enzymology, 266:460-480 (1996)] as a comparison of the
ECD protein sequences to a 6 frame translation of the EST
sequences. Those comparisons resulting in a BLAST score of 70 (or
in some cases, 90) or greater that did not encode known proteins
were clustered and assembled into consensus DNA sequences with the
program "phrap" (Phil Green, University of Washington, Seattle,
Wash.).
[0882] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described above. This consensus sequence
is herein designated DNA86620. In some cases, the DNA86620
consensus sequence derives from an intermediate consensus DNA
sequence which was extended using repeated cycles of BLAST and
phrap to extend that intermediate consensus sequence as far as
possible using the sources of EST sequences discussed above.
[0883] Based on the DNA86620 consensus sequence, and in light of an
observed sequence homology between the DNA86620 sequence and an EST
sequence encompassed within clone no. 4797137 from the
LIFESEQ.RTM., Incyte Pharmaceuticals, Palo Alto, Calif. database,
clone no. 4797137 was purchased and the cDNA insert was obtained
and sequenced. It was found herein that that cDNA insert encoded a
full-length protein. The sequence of this cDNA insert is shown in
FIG. 57 and is herein designated as DNA108792-2753.
[0884] The full length clone identified above contained a single
open reading frame with an apparent translational initiation site
at nucleotide positions 3-5 and a stop signal at nucleotide
positions 708-710 (FIG. 57, SEQ ID NO: 57). The predicted
polypeptide precursor is 235 amino acids long, has a calculated
molecular weight of approximately 25989 daltons and an estimated pI
of approximately 8.32. Analysis of the full-length PRO7425 sequence
shown in FIG. 58 (SEQ ID NO: 58) evidences the presence of a
variety of important polypeptide domains as shown in FIG. 58,
wherein the locations given for those important polypeptide domains
are approximate as described above. Clone DNA108792-2753 has been
deposited with ATCC on Aug. 31, 1999 and is assigned ATCC Deposit
No. PTA-617.
[0885] An analysis of the Dayhoff database (version 35.45 SwissProt
35), using the ALIGN-2 sequence alignment analysis of the
full-length sequence shown in FIG. 58 (SEQ ID NO: 58), evidenced
sequence identity between the PRO7425 amino acid sequence and the
following Dayhoff sequences: P_Y11831; P_Y11619; MYP0_HUMAN;
MYP0_MOUSE; HSPMPO2.sub.--1; AF087020.sub.--1; GEN13751;
AF007783.sub.--1; P_W14146; XLU43330.sub.--1.
Example 32
Isolation of cDNA Clones Encoding Human PRO10102 Polypeptides
[UNQ3103]
[0886] DNA129542-2808 was identified by applying a proprietary
signal sequence finding algorithm developed by Genentech, Inc.
(South San Francisco, Calif.) upon ESTs as well as clustered and
assembled EST fragments from public (e.g., GenBank) and/or private
(LIFESEQ.RTM., Incyte Pharmaceuticals, Inc., Palo Alto, Calif.)
databases. The signal sequence algorithm computes a secretion
signal score based on the character of the DNA nucleotides
surrounding the first and optionally the second methionine codon(s)
(ATG) at the 5'-end of the sequence or sequence fragment under
consideration. The nucleotides following the first ATG must code
for at least 35 unambiguous amino acids without any stop codons. If
the first ATG has the required amino acids, the second is not
examined. If neither meets the requirement, the candidate sequence
is not scored. In order to determine whether the EST sequence
contains an authentic signal sequence, the DNA and corresponding
amino acid sequences surrounding the ATG codon are scored using a
set of seven sensors (evaluation parameters) known to be associated
with secretion signals.
[0887] Use of the above described signal sequence algorithm allowed
identification of an EST cluster sequence from the LIFESEQ.RTM.
(Incyte Pharmaceuticals, Inc., Palo Alto, Calif.) database,
designated herein as 166950H1. This EST cluster sequence was then
compared to a variety of expressed sequence tag (EST) databases
which included public EST databases (e.g., GenBank) and a
proprietary EST DNA database (LIFESEQ.RTM., Incyte Pharmaceuticals,
Palo Alto, Calif.) to identify existing homologies. The homology
search was performed using the computer program BLAST or BLAST2
(Altshul et al., Methods in Enzymology 266:460-480 (1996)). Those
comparisons resulting in a BLAST score of 70 (or in some cases 90)
or greater that did not encode known proteins were clustered and
assembled into a consensus DNA sequence with the program "phrap"
(Phil Green, University of Washington, Seattle, Wash.). The
consensus sequence obtained therefrom is herein designated
DNA112560.
[0888] In light of an observed sequence homology between the
DNA112560 sequence and an EST sequence encompassed within clone no.
166950 from LIFESEQ.RTM. (Incyte Pharmaceuticals, Inc., Palo Alto,
Calif.) database, clone no. 166950 was purchased and the cDNA
insert was obtained and sequenced. It was found herein that that
cDNA insert encoded a full-length protein. The sequence of this
cDNA insert is shown in FIG. 59 and is herein designated as
DNA129542-2808.
[0889] Clone DNA129542-2808 contains a single open reading frame
with an apparent translational initiation site at nucleotide
positions 58-60 and ending at the stop codon at nucleotide
positions 1786-1788 (FIG. 59; SEQ ID NO:59). The predicted
polypeptide precursor is 576 amino acids long (FIG. 60; SEQ ID
NO:60). The full-length PRO10102 protein shown in FIG. 60 has an
estimated molecular weight of about 62128 daltons and a pI of about
7.41. Analysis of the full-length PRO10102 sequence shown in FIG.
60 (SEQ ID NO:60) evidences the presence of a variety of important
polypeptide domains as shown in FIG. 60, wherein the locations
given for those important polypeptide domains are approximate as
described above. Clone DNA129542-2808 has been deposited with ATCC
on Feb. 23, 2000 and is assigned ATCC deposit no. PTA-1405.
[0890] An analysis of the Dayhoff database (version 35.45 SwissProt
35), using the ALIGN-2 sequence alignment analysis of the
full-length sequence shown in FIG. 60 (SEQ ID NO:60), evidenced
sequence identity between the PRO10102 amino acid sequence and the
following Dayhoff sequences: rTNFSF3L.sub.--1, P_Y00771,
AC007785.sub.--2, AF076483.sub.--1, P_W23722, P_W37837, P_Y00770
and AB016605.sub.--1. The PRO10102 polypeptide is much longer than
the sequences with which it shares homology. For example, it has
378 amino acid residues at the N-terminal end that are not
homologous to rTNFSF3L.sub.--1 and P_Y00771.
Example 33
Isolation of cDNA Clones Encoding Human PRO10282 Polypeptides
[UNQ3126]
[0891] A cDNA clone (DNA148380-2827) encoding a native human
PRO10282 polypeptide was identified using a yeast screen, in a
human cDNA library that preferentially represents the 5' ends of
the primary cDNA clones.
Clone DNA148380-2827-1 contains a single open reading frame with an
apparent translational initiation site at nucleotide positions
49-51 and ending at the stop codon at nucleotide positions
2050-2052 (FIG. 61; SEQ ID NO:61). The predicted polypeptide
precursor is 667 amino acids long (FIG. 62; SEQ ID NO:62). The
full-length PRO10282 protein shown in FIG. 62 has an estimated
molecular weight of about 73502 daltons and a pI of about 9.26.
Analysis of the full-length PRO10282 sequence shown in FIG. 62 (SEQ
ID NO:62) evidences the presence of a variety of important
polypeptide domains as shown in FIG. 62, wherein the locations
given for those important polypeptide domains are approximate as
described above. Clone DNA148380-2827 has been deposited with ATCC
on Jan. 11, 2000 and is assigned ATCC deposit no. PTA-1181.
[0892] An analysis of the Dayhoff database (version 35.45 SwissProt
35), using the ALIGN-2 sequence alignment analysis of the
full-length sequence shown in FIG. 62 (SEQ ID NO:62), evidenced
sequence identity between the PRO10282 amino acid sequence and the
following Dayhoff sequences: AF062476, P_W88559 and HGS_RE259.
Example 34
Isolation of cDNA Clones Encoding Human PRO779 Polypeptides
[UNQ455]
[0893] Human fetal heart and human fetal lung lgt10 bacteriophage
cDNA libraries (both purchased from Clontech) were screened by
hybridization with synthetic oligonucleotide probes based on an EST
(GenBank locus W71984), which showed some degree of homology to the
intracellular domain (ICD) of human TNFR1 and CD95. W71984 is a 523
bp EST, which in its -1 reading frame has 27 identities to a 43
amino acid long sequence in the ICD of human TNFR1. The
oligonucleotide probes used in the screening were 27 and 25 bp
long, respectively, with the following sequences:
TABLE-US-00048 5'-GGCGCTCTGGTGGCCCTTGCAGAAGCC-3' (SEQ ID NO: 141)
5'-TTCGGCCGAGAAGTTGAGAAATGTC-3' (SEQ ID NO: 142)
[0894] Hybridization was done with a 1:1 mixture of the two probes
overnight at room temperature in buffer containing 20% formamide,
5.times.SSC, 10% dextran sulfate, 0.1% NaPiPO4) 0.05 M NaPO4, 0.05
mg salmon sperm DNA, and 0.1% sodium dodecyl sulfate (SDS),
followed consecutively by one wash at room temperature in
6.times.SSC, two washes at 37 C in 1.times.SSC/0.1% SDS, two washes
at 37 C in 0.5.times.SSC/0.1% SDS, and two washes at 37 C in
0.2.times.SSC/0.1% SDS. One positive clone from each of the fetal
heart (FH20A.57) and fetal lung (FL8A.53) libraries were confirmed
to be specific by PCR using the respective above hybridization
probes as primers. Single phage plaques containing each of the
positive clones were isolated by limiting dilution and the DNA was
purified using a Wizard lambda prep DNA purification kit
(Promega).
[0895] The cDNA inserts were excised from the lambda vector arms by
digestion with EcoRI, gel-purified, and subcloned into pRK5 that
was predigested with EcoRI. The clones were then sequenced in
entirety.
[0896] Clone (FH20A.57) DNA58801-1052 (also referred to as Apo 3
clone FH20.57 deposited as ATCC 55820, as indicated below) contains
a single open reading frame with an apparent translational
initiation site at nucleotide positions 103-105 and ending at the
stop codon found at nucleotide positions 1354-1356 [FIG. 65, SEQ ID
NO:65]. The predicted polypeptide precursor is 417 amino acids long
(FIG. 66; SEQ ID NO:66). The full-length PRO779 protein shown in
FIG. 66 has an estimated molecular weight of about 45,000 daltons
and a pI of about 6.40. Analysis of the full-length PRO779 sequence
shown in FIG. 66 (SEQ ID NO:66) evidences the presence of a variety
of important polypeptide domains, wherein the locations given for
those important polypeptide domains are approximate as described
above. Analysis of the full-length PRO779 sequence shown in FIG. 66
evidences the presence of the following: a signal peptide from
about amino acid 1 to about amino acid 24; a transmembrane domain
from about amino acid 199 to about amino acid 219; N-glycosylation
sites from about amino acid 67 to about amino acid 71 and from
about amino acid 106 to about amino acid 110; a cAMP- and
cGMP-dependent protein kinase phosphorylation site from about amino
acid 157 to about amino acid 161; a tyrosine kinase phosphorylation
site from about amino acid 370 to about amino acid 377;
N-myristoylation sites from about amino acid 44 to about amino acid
50, from about amino acid 50 to about amino acid 56, from about
amino acid 66 to about amino acid 72, from about amino acid 116 to
about amino acid 122, from about amino acid 217 to about amino acid
223, from about amino acid 355 to about amino acid 361, from about
amino acid 391 to about amino acid 397, and from about amino acid
401 to about amino acid 407; and a prokaryotic membrane lipoprotein
lipid attachment site from about amino acid 177 to about amino acid
188. Clone DNA58801-1052 has been deposited with ATCC on Sep. 5,
1996 and is assigned ATCC deposit no. 55820.
[0897] The ECD contains 4 cysteine-rich repeats which resemble the
corresponding regions of human TNFR1 (4 repeats), of human CD95 (3
repeats) and of the other known TNFR family members. The ICD
contains a death domain sequence that resembles the death domains
found in the ICD of TNFR1 and CD95 and in the cytoplasmic death
signaling proteins such as human FADD/MORT1, TRADD, RIP, and
Drosophila Reaper. Both globally and in individual regions, PRO779
(Apo 3) is more closely related to TNFR1 than to CD95; the
respective amino acid identities are 29.3% and 22.8% overall, 28.2%
and 24.7% in the ECD, 31.6% and 18.3% in the ICD, and 47.5% and 20%
in the death domain.
Example 35
Generation and Analysis of Mice Comprising PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
Gene Disruptions
[0898] To investigate the role of PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptides, disruptions in PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
genes were produced by homologous recombination or retroviral
insertion techniques. Specifically, transgenic mice comprising
disruptions in PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 genes (i.e.,
knockout mice) were created by either gene targeting or gene
trapping. Mutations were confirmed by southern blot analysis to
confirm correct targeting on both the 5' and 3' ends. Gene-specific
genotyping was also performed by genomic PCR to confirm the loss of
the endogenous native transcript as demonstrated by RT-PCR using
primers that anneal to exons flanking the site of insertion.
Targeting vectors were electroporated into 129 strain ES cells and
targeted clones were identified. Targeted clones were microinjected
into host blastocysts to produce chimeras. Chimeras were bred with
C57 animals to produce F1 heterozygotes. Heterozygotes were
intercrossed to produce F2 wild-type, heterozygote and homozygote
cohorts which were used for phenotypic analysis. Rarely, if not
enough F1 heterozygotes were produced, the F1 hets were bred to
wild-type C57 mice to produce sufficient heterozygotes to breed for
cohorts to be analyzed for a phenotype. All phenotypic analysis was
performed from 12-16 weeks after birth.
Overall Summary of Results
[0899] 35.1. Generation and Analysis of Mice Comprising
DNA22779-1130 (UNQ170) Gene Disruptions
[0900] In these knockout experiments, the gene encoding PRO196
polypeptides (designated as DNA22779-1130) (UNQ170) was disrupted.
The gene specific information for these studies is as follows: the
mutated mouse gene corresponds to nucleotide reference:
NM.sub.--011923 or ACCESSION: NM.sub.--011923 NID: gi 6753119 ref
NM.sub.--011923.1 Mus musculus angiopoietin-like 2 (Angptl2);
protein reference: Q9R045 or ACCESSION: Q9R045NID: Mus musculus
(Mouse). ANGIOPOIETIN-RELATED PROTEIN 2 PRECURSOR
(ANGIOPOIETIN-LIKE 2). MOUSESPTRNRDB; the human gene sequence
reference: NM.sub.--012098 or ACCESSION: NM.sub.--012098 NID: gi
6912235 ref NM.sub.--012098.1 Homo sapiens angiopoietin-like 2
(ANGPTL2); the human protein sequence corresponds to reference:
Q9UKU9 or ACCESSION: Q9UKU9 NID: Homo sapiens (Human).
ANGIOPOIETIN-RELATED PROTEIN 2 PRECURSOR (ANGIOPOIETIN-LIKE 2).
HUMANSPTRNRDB.
[0901] The mouse gene of interest is angiopoietin-like 2 (Angptl2),
ortholog of human ANGPTL2. Aliases include angiopoietin related
protein 2 (Arp2), HARP, and MGC8889. Angptl2 is a secreted
glycoprotein hormone expressed in vascular endothelial cells,
vascular smooth muscle, heart, small intestine, spleen, stomach,
colon, ovary, adrenal gland, skeletal muscle, and prostate.
Although Angptl2 is structurally similar to the angiopoietin family
of hormones, Angptl2 does not bind to angiopoietin receptors Tie1
and Tie2. Angptl2 induces sprouting in endothelial cells, which is
consistent with the role of angiopoietins in blood vessel formation
(Kim et al., J Biol Chem, 274(37):26523-8 (1999)).
[0902] Targeted or gene trap mutations were generated in strain
129SvEv.sup.Brd-derived embryonic stem (ES) cells. The chimeric
mice were bred to C57BL/6J albino mice to generate F1 heterozygous
animals. These progeny were intercrossed to generate F2 wild type,
heterozygous, and homozygous mutant progeny. On rare occasions, for
example when very few F1 mice were obtained from the chimera, F1
heterozygous mice were crossed to 129SvEv.sup.Brd/C57 hybrid mice
to yield additional heterozygous animals for the intercross to
generate the F2 mice.
Level I phenotypic analysis is performed on mice from this
generation.
TABLE-US-00049 wt het hom Total Observed 23 32 23 78 Expected 19.5
39 19.5 78 Chi-Sq. = 2.51 Significance = 0.28467 (hom/n) = 0.29
Avg. Litter Size = 8
Mutation Type: Homologous Recombination (standard). Coding exon 1
was targeted (NCBI accession NM.sub.--011923.1). Wild-type
expression of the target gene was detected in embryonic stem (ES)
cells and in all 13 adult tissue samples tested by RT-PCR, except
skeletal muscle, bone, and adipose. Disruption of the target gene
was confirmed by Southern hybridization analysis.
[0903] 35.1.1. Phenotypic Analysis (for Disrupted Gene:
DNA22779-1130 (UNQ170)
[0904] (a) Overall Phenotypic Summary:
[0905] Mutation of the gene encoding the ortholog of human
angiopoietin-like 2 (ANGPTL2) resulted in decreased mean serum
MCP-1, TNF-alpha, and IL-6 responses to LPS challenge in (-/-)
mice. The (-/-) mice also exhibited significant growth retardation
marked by decreased total tissue mass, total body fat, decreased
body weight and length as well as decreased mean vertebral
trabecular bone measurements. Gene disruption was confirmed by
Southern blot.
[0906] (b) Immunology Phenotypic Analysis
[0907] Immune related and inflammatory diseases are the
manifestation or consequence of fairly complex, often multiple
interconnected biological pathways which in normal physiology are
critical to respond to insult or injury, initiate repair from
insult or injury, and mount innate and acquired defense against
foreign organisms. Disease or pathology occurs when these normal
physiological pathways cause additional insult or injury either as
directly related to the intensity of the response, as a consequence
of abnormal regulation or excessive stimulation, as a reaction to
self, or as a combination of these.
[0908] Though the genesis of these diseases often involves
multistep pathways and often multiple different biological
systems/pathways, intervention at critical points in one or more of
these pathways can have an ameliorative or therapeutic effect.
Therapeutic intervention can occur by either antagonism of a
detrimental process/pathway or stimulation of a beneficial
process/pathway.
[0909] T lymphocytes (T cells) are an important component of a
mammalian immune response. T cells recognize antigens which are
associated with a self-molecule encoded by genes within the major
histocompatibility complex (MHC). The antigen may be displayed
together with MHC molecules on the surface of antigen presenting
cells, virus infected cells, cancer cells, grafts, etc. The T cell
system eliminates these altered cells which pose a health threat to
the host mammal. T cells include helper T cells and cytotoxic T
cells. Helper T cells proliferate extensively following recognition
of an antigen-MHC complex on an antigen presenting cell. Helper T
cells also secrete a variety of cytokines, i.e., lymphokines, which
play a central role in the activation of B cells, cytotoxic T cells
and a variety of other cells which participate in the immune
response.
[0910] In many immune responses, inflammatory cells infiltrate the
site of injury or infection. The migrating cells may be
neutrophilic, eosinophilic, monocytic or lymphocytic as can be
determined by histological examination of the affected tissues.
Current Protocols in Immunology, ed. John E. Coligan, 1994, John
Wiley & Sons, Inc.
[0911] Many immune related diseases are known and have been
extensively studied. Such diseases include immune-mediated
inflammatory diseases (such as rheumatoid arthritis, immune
mediated renal disease, hepatobiliary diseases, inflammatory bowel
disease (IBD), psoriasis, and asthma), non-immune-mediated
inflammatory diseases, infectious diseases, immunodeficiency
diseases, neoplasia, and graft rejection, etc. In the area of
immunology, targets were identified for the treatment of
inflammation and inflammatory disorders.
[0912] In the area of immunology, targets have been identified
herein for the treatment of inflammation and inflammatory
disorders. Immune related diseases, in one instance, could be
treated by suppressing the immune response. Using neutralizing
antibodies that inhibit molecules having immune stimulatory
activity would be beneficial in the treatment of immune-mediated
and inflammatory diseases. Molecules which inhibit the immune
response can be utilized (proteins directly or via the use of
antibody agonists) to inhibit the immune response and thus
ameliorate immune related disease.
[0913] The following test was performed:
[0914] Acute Phase Response:
[0915] Test Description: Bacterial lipopolysaccharide (LPS) is an
endotoxin, and as such is a potent inducer of an acute phase
response and systemic inflammation. The Level I LPS mice were
injected intraperitoneally (i.p.) with a sublethal dose of LPS in
200 .mu.L sterile saline using a 26 gauge needle. The doses were
based on the average weight of the mice tested at 1 .mu.g/g body
weight 3 hours after injection; a 100 ul blood sample was then
taken and analyzed for the presence of TNFa, MCP-1, and IL-6 on the
FACSCalibur instrument.
[0916] Results:
[0917] The (-/-) mice exhibited a decreased mean serum MCP-1, IL-6
and TNF-alpha response to LPS challenge when compared with their
(+/+) littermates and the historical mean.
[0918] Analyzed wt/het/hom: 6/4/10
[0919] In summary, the LPS endotoxin challenge demonstrated that
knockout mice deficient in the gene encoding PRO196 polypeptides
exhibit immunological abnormalities when compared with their
wild-type littermates. In particular, the mutant mice exhibited a
decreased ability to elicit an immunological response (MCP-1,
TNF-alpha and IL-6 production) when challenged with the LPS
endotoxin indicating a deficiency in the proinflammatory response.
IL-6 contributes to the later stages of B cell activation. In
addition, IL-6 plays a critical role in inducing the acute phase
response and systemic inflammation. This suggests that PRO196
polypeptides or agonists thereof would stimulate the immune system
and would find utility in the cases wherein this effect would be
beneficial to the individual such as in the case of leukemia, and
other types of cancer, and in immunocompromised patients, such as
AIDS sufferers. Accordingly, inhibitors or antagonists to PRO196
polypeptides would play a role in inhibiting the immune response
and would be useful candidates for suppressing harmful immune
responses, e.g. in the case of graft rejection or graft-versus-host
diseases.
[0920] (c) Bone Metabolism & Body Diagnostics
[0921] (1) Tissue Mass & Lean Body Mass Measurements--Dexa
[0922] Dexa Analysis--Test Description:
[0923] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. Dual Energy X-ray
Absorptiometry (DEXA) has been used successfully to identify
changes in total tissue mass (TTM).
[0924] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PIXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI,
i.e., whole body, vertebrae, and both femurs).
[0925] Body Measurements (Body Length & Weight):
[0926] Body Measurements: A measurement of body length and weight
was performed at approximately 16 weeks of age.
[0927] Results:
[0928] The (-/-) mice exhibited decreased mean body weight and mean
body length (at least two (2) standard deviations (SD) below normal
especially in the first eight (8) weeks) when compared with their
gender-matched (+/+) littermates and the historical means.
[0929] Analyzed wt/het/hom: 23/32/23
[0930] (2) Bone Metabolism: Radiology Phenotypic Analysis
[0931] In the area of bone metabolism, targets were identified
herein for the treatment of arthritis, osteoporosis, osteopenia and
osteopetrosis as well as identifying targets that promote bone
healing. Tests included:
[0932] DEXA for measurement of bone mineral density on femur and
vertebra
[0933] MicroCT for very high resolution and very high sensitivity
measurements of bone mineral density for both trabecular and
cortical bone.
[0934] Dexa Analysis--Test Description:
[0935] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. Dual Energy X-ray
Absorptiometry (DEXA) has been used successfully to identify
changes in bone. Anesthetized animals were examined and bone
mineral content (BMC), BMC/LBM ratios, volumetric bone mineral
density (vBMD), total body BMD, femur BMD and vertebra BMD were
measured.
[0936] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PIXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI)
[i.e., whole body, vertebrae, and both femurs].
[0937] Bone MicroCT Analysis:
[0938] Procedure: MicroCT was also used to get very sensitive
measurements of BMD. One vertebra and 1 femur were taken from a
cohort of 4 wild type and 8 homozygous mice. Measurements were
taken of lumbar 5 vertebra trabecular bone volume, trabecular
thickness, connectivity density and midshaft femur total bone area
and cortical thickness. The .mu.CT40 scans provided detailed
information on bone mass and architecture. Multiple bones were
placed into sample holders and scanned automatically. Instrument
software was used to select regions of interest for analysis.
Trabecular bone parameters were analyzed in the fifth lumbar
vertebrae (LV5) at 16 micrometer resolution and cortical bone
parameters were analyzed in the femur midshaft at a resolution of
20 micrometers.
[0939] Results:
[0940] DEXA: The (-/-) mice exhibited decreased mean total tissue
mass and total body fat when compared with their gender-matched
(+/+) littermates and the historical means.
[0941] Micro-CT: The (-/-) mice exhibited a decreased mean
vertebral trabecular bone volume, thickness, and connectivity
density when compared with their gender-matched (+/+) littermates
and the historical means.
Analyzed wt/het/hom: 4/4/10
[0942] Summary
[0943] These results demonstrate that knockout mutant mice exhibit
abnormal bone metabolism with significant bone loss similar to
osteoporosis characterized by decrease in bone mass with decreased
density and possibly fragility leading to bone fractures. Thus, it
appears that PRO196 polypeptides or agonists thereof play a role in
maintaining bone homeostasis. In addition, PRO196 or its encoding
gene would be useful in bone healing or useful for the treatment of
osteoarthritis or osteoporosis; whereas antagonists to PRO196 or
its encoding gene would lead to abnormal or pathological bone
disorders including inflammatory diseases associated with abnormal
bone metabolism such as arthritis, osteoporosis, and
osteopenia.
[0944] The (-/-) mice analyzed by DEXA exhibited significant growth
retardation marked by a notably decreased total tissue mass and
total body fat as well as reduction in body weight and length when
compared with their (+/+) littermates. These results may be due to
problems associated with the hypothalamic-pituitary axis which can
affect bone growth. This in conjunction with the observations of
decreased bone measurements suggest a tissue wasting condition such
as cachexia. Thus, PRO196 polypeptides or agonists thereof would be
useful in the treatment of bone disorders but would also be useful
for treating growth disorders or for the prevention of cachexia or
other tissue wasting diseases.
[0945] 35.2. Generation and Analysis of Mice Comprising
DNA33094-1131 (UNQ191) Gene Disruptions
[0946] In these knockout experiments, the gene encoding PRO217
polypeptides (designated as DNA33094-1131) (UNQ191) was disrupted.
The gene specific information for these studies is as follows: the
mutated mouse gene corresponds to nucleotide reference:
NM.sub.--011915 or ACCESSION: NM.sub.--011915 NID: 6755998 Mus
musculus Mus musculus Wnt inhibitory factor 1 (Wif1); protein
reference: Q9WUA1 or ACCESSION: Q9WUA1 NID: Mus musculus (Mouse).
WNT INHIBITORY FACTOR 1 PRECURSOR (WIF-1). MOUSESPTRNRDB; the human
gene sequence reference: NM.sub.--007191 or ACCESSION:
NM.sub.--007191 NID: 18379354 Homo sapiens Homo sapiens WNT
inhibitory factor 1 (WIF1); the human protein sequence corresponds
to reference: Q9Y5W5 or ACCESSION: Q9Y5W5 NID: Homo sapiens
(Human). Wnt inhibitory factor 1 precursor (WIF-1).
HUMANSPTRNRDB.
[0947] The mouse gene of interest is Wif1 (Wnt inhibitory factor
1), ortholog of human WIF1. Aliases include WIF-1 and Wnt
inhibitory factor-1. WIF1 is a secreted protein expressed during
embryonic development that binds with Wnt proteins, disrupting
activation of their cognate receptors. Wnt proteins are
extracellular signaling molecules involved in developmental
processes (Hseih et al., Nature, 398(6726):431-6 (1999). WIF1 is
down-regulated in prostate, breast, lung, and bladder cancer but
up-regulated in colon adenocarcinoma cell lines, suggesting that
alterations in WIF1 expression may be related to tumorigenesis in
these tissues (Wissmann et al.,
J Pathol, 201(2):204-12 (2003); Cebrat et al., Cancer Lett,
206(1):107-13 (2004)).
[0948] Targeted or gene trap mutations were generated in strain
129SvEv.sup.Brd-derived embryonic stem (ES) cells. The chimeric
mice were bred to C57BL/6J albino mice to generate F1 heterozygous
animals. These progeny were intercrossed to generate F2 wild type,
heterozygous, and homozygous mutant progeny. On rare occasions, for
example when very few F1 mice were obtained from the chimera, F1
heterozygous mice were crossed to 129SvEv.sup.Brd/C57 hybrid mice
to yield additional heterozygous animals for the intercross to
generate the F2 mice.
Level I phenotypic analysis is performed on mice from this
generation.
TABLE-US-00050 wt het hom Total Observed 24 35 20 79 Expected 19.75
39.5 19.75 79 Chi-Sq. = 1.43 Significance = 0.48910 (hom/n) = 0.25
Avg. Litter Size = 8
Mutation Type: Homologous Recombination (standard) Coding exon 1
was targeted (NCBI accession NM.sub.--011915.1). Wild-type
expression of the target gene was detected in brain, spinal cord,
eye, and heart among the 13 adult tissue samples tested by RT-PCR.
Disruption of the target gene was confirmed by Southern
hybridization analysis.
[0949] 35.2.1. Phenotypic Analysis (for Disrupted Gene:
DNA33094-1131 (UNQ191)
[0950] (a) Overall Phenotypic Summary:
[0951] Mutation of the gene encoding the ortholog of human Wnt
inhibitory factor 1 (WIF1) resulted in growth retardation
accompanied by decreased bone measurements in (-/-) mice. In
addition, mutant (-/-) mice exhibited an increased IgG2a response
to an ovalbumin challenge. Gene disruption was confirmed by
Southern blot.
[0952] (b) Immunology Phenotypic Analysis
[0953] Immune related and inflammatory diseases are the
manifestation or consequence of fairly complex, often multiple
interconnected biological pathways which in normal physiology are
critical to respond to insult or injury, initiate repair from
insult or injury, and mount innate and acquired defense against
foreign organisms. Disease or pathology occurs when these normal
physiological pathways cause additional insult or injury either as
directly related to the intensity of the response, as a consequence
of abnormal regulation or excessive stimulation, as a reaction to
self, or as a combination of these.
[0954] Though the genesis of these diseases often involves
multistep pathways and often multiple different biological
systems/pathways, intervention at critical points in one or more of
these pathways can have an ameliorative or therapeutic effect.
Therapeutic intervention can occur by either antagonism of a
detrimental process/pathway or stimulation of a beneficial
process/pathway.
[0955] T lymphocytes (T cells) are an important component of a
mammalian immune response. T cells recognize antigens which are
associated with a self-molecule encoded by genes within the major
histocompatibility complex (MHC). The antigen may be displayed
together with MHC molecules on the surface of antigen presenting
cells, virus infected cells, cancer cells, grafts, etc. The T cell
system eliminates these altered cells which pose a health threat to
the host mammal. T cells include helper T cells and cytotoxic T
cells. Helper T cells proliferate extensively following recognition
of an antigen-MHC complex on an antigen presenting cell. Helper T
cells also secrete a variety of cytokines, i.e., lymphokines, which
play a central role in the activation of B cells, cytotoxic T cells
and a variety of other cells which participate in the immune
response.
[0956] In many immune responses, inflammatory cells infiltrate the
site of injury or infection. The migrating cells may be
neutrophilic, eosinophilic, monocytic or lymphocytic as can be
determined by histological examination of the affected tissues.
Current Protocols in Immunology, ed. John E. Coligan, 1994, John
Wiley & Sons, Inc.
[0957] Many immune related diseases are known and have been
extensively studied. Such diseases include immune-mediated
inflammatory diseases (such as rheumatoid arthritis, immune
mediated renal disease, hepatobiliary diseases, inflammatory bowel
disease (IBD), psoriasis, and asthma), non-immune-mediated
inflammatory diseases, infectious diseases, immunodeficiency
diseases, neoplasia, and graft rejection, etc. In the area of
immunology, targets were identified for the treatment of
inflammation and inflammatory disorders.
[0958] In the area of immunology, targets have been identified
herein for the treatment of inflammation and inflammatory
disorders. Immune related diseases, in one instance, could be
treated by suppressing the immune response. Using neutralizing
antibodies that inhibit molecules having immune stimulatory
activity would be beneficial in the treatment of immune-mediated
and inflammatory diseases. Molecules which inhibit the immune
response can be utilized (proteins directly or via the use of
antibody agonists) to inhibit the immune response and thus
ameliorate immune related disease.
[0959] The following test was performed:
[0960] Ovalbumin Challenge
[0961] Procedure: This assay was carried out on 7 wild types and 8
homozygotes. Chicken ovalbumin (OVA) is a T-cell dependent antigen,
which is commonly used as a model protein for studying
antigen-specific immune responses in mice. OVA is non-toxic and
inert and therefore will not cause harm to the animals even if no
immune response is induced. The murine immune response to OVA has
been well characterized, to the extent that the immuno-dominant
peptides for eliciting T cell responses have been identified.
Anti-OVA antibodies are detectable 8 to 10 days after immunization
using enzyme-linked immunosorbent assay (ELIZA), and determination
of different isotypes of antibodies gives further information on
the complex processes that may lead to a deficient response in
genetically engineered mice.
[0962] As noted above, this protocol assesses the ability of mice
to raise an antigen-specific immune response. Animals were injected
IP with 50 mg of chicken ovalbumin emulsified in Complete Freund's
Adjuvant and 14 days later the serum titer of anti-ovalbumin
antibodies (IgM, IgG1 and IgG2 subclasses) was measured. The amount
of OVA-specific antibody in the serum sample is proportional to the
Optical Density (OD) value generated by an instrument that scans a
96-well sample plate. Data was collected for a set of serial
dilutions of each serum sample.
Analyzed wt/het/hom: 8/4/9
[0963] Results of this Challenge:
[0964] The (-/-) mice exhibited an increased mean serum IgG2a
response to ovalbumin challenge when compared with their (+/+)
littermates and the historical means. Thus, these knockout mice
exhibited an increased ability to elicit an OVA-specific antibody
response to the T-cell dependent OVA antigen or a positive
immunological phenotype (proinflammatory response). Thus, the gene
encoding PRO217 polypeptides would be expected to lead to
inhibition of the proinflammatory response which could be caused by
Th, B or plasma cell defects.
[0965] In summary, the ovalbumin challenge studies indicate that
knockout mice deficient in the gene encoding PRO217 polypeptides
exhibit immunological abnormalities when compared with their
wild-type littermates. In particular, the mutant mice exhibited an
increased ability to elicit an immunological response when
challenged with the T-cell dependent OVA antigen. Thus, inhibitors
or antagonists of PRO217 polypeptides would be useful for
stimulating the immune system (such as T cell proliferation) and
would find utility in the cases wherein this effect would be
beneficial to the individual such as in the case of leukemia, and
other types of cancer, and in immunocompromised patients, such as
AIDS sufferers. Accordingly, PRO217 polypeptides or agonists
thereof would be useful for inhibiting the immune response and thus
would be useful candidates for suppressing harmful immune
responses, e.g. in the case of graft rejection or graft-versus-host
diseases.
[0966] (c) Bone Metabolism & Body Diagnostics
[0967] (1) Tissue Mass & Lean Body Mass Measurements--Dexa
[0968] Dexa Analysis--Test Description:
[0969] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. Dual Energy X-ray
Absorptiometry (DEXA) has been used successfully to identify
changes in total tissue mass (TTM).
[0970] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PIXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI,
i.e., whole body, vertebrae, and both femurs).
[0971] Body Measurements (Body Length & Weight):
[0972] Body Measurements: A measurement of body length and weight
was performed at approximately 16 weeks of age.
[0973] Results:
[0974] The (-/-) mice exhibited decreased mean body weight and mean
body length (one (1) standard deviation (SD) below historic mean in
weight and two (2) standard deviations (SD) below historical mean
in length) when compared with their gender-matched (+/+)
littermates and the historical means.
[0975] Heart Rate: The heart rate decreased 1-2 standard deviations
relative to the historic controls in both male and female knockout
(-/-) mice.
[0976] Fertility: The single male (-/-) mouse (M-151) available for
analysis was infertile.
[0977] (2) Bone Metabolism: Radiology Phenotypic Analysis
[0978] In the area of bone metabolism, targets were identified
herein for the treatment of arthritis, osteoporosis, osteopenia and
osteopetrosis as well as identifying targets that promote bone
healing. Tests included:
[0979] DEXA for measurement of bone mineral density on femur and
vertebra
[0980] MicroCT for very high resolution and very high sensitivity
measurements of bone mineral density for both trabecular and
cortical bone.
[0981] Dexa Analysis--Test Description:
[0982] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. Dual Energy X-ray
Absorptiometry (DEXA) has been used successfully to identify
changes in bone. Anesthetized animals were examined and bone
mineral content (BMC), BMC/LBM ratios, volumetric bone mineral
density (vBMD), total body BMD, femur BMD and vertebra BMD were
measured.
[0983] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PIXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI)
[i.e., whole body, vertebrae, and both femurs].
[0984] Results:
[0985] The (-/-) mice exhibited decreased mean total tissue mass
and lean body mass and decreased mean bone mineral content and bone
mineral content when compared with their gender-matched (+/+)
littermates and the historical means. Analyzed wt/het/hom:
4/4/8
[0986] Summary
[0987] The (-/-) mice analyzed by DEXA exhibited notably decreased
total tissue mass and lean body mass as well as decreased bone
measurements when compared with their (+/+) littermates, suggestive
of growth retardation in these mutants. This in conjunction with
the observations of decreased body weight and length suggest a
tissue wasting condition such as cachexia or other growth related
disorders. Thus, PRO217 polypeptides or agonists thereof would be
useful in the treatment or prevention of growth disorders including
cachexia or other tissue wasting diseases.
[0988] 35.3. Generation and Analysis of Mice Comprising
DNA34434-1139 (UNQ205) Gene Disruptions
[0989] In these knockout experiments, the gene encoding PRO231
polypeptides (designated as DNA34434-1139) (UNQ205) was disrupted.
The gene specific information for these studies is as follows: the
mutated mouse gene corresponds to nucleotide reference:
NM.sub.--019800 or ACCESSION: NM.sub.--019800 NID: 9790058 Mus
musculus Mus musculus acid phosphatase 6, lysophosphatidic (Acp6);
protein reference: Q9JMG5 or ACCESSION: Q9JMG5 NID: Mus musculus
(Mouse). MPACPL1. MOUSESPTRNRDB; the human gene sequence reference:
NM.sub.--016361 or ACCESSION: NM.sub.--016361 NID: 21359910 Homo
sapiens Homo sapiens LPAP for lysophosphatidic acid phosphatase
(LOC51205); the human protein sequence corresponds to reference:
Q9NPH0 or ACCESSION: Q9NPH0 NID: Homo sapiens (Human). HPACPL1
(CDNA FLJ20650 FIS, CLONE KAT01962) (LPAP FOR LYSOPHOSPHATIDIC ACID
PHOSPHATASE) (LYSOPHOSPHATIDIC ACID PHOSPHATASE PRECURSOR).
HUMANSPTRNRDB.
[0990] The targeted mouse gene is Acp6 (acid phosphatase 6,
lysophosphatidic), which is the ortholog of human ACP6. Aliases
include: ACPL1; mPACPL1; 5730559A09Rik; acid phosphatase like 1;
LPAP; PACPL1; and likely ortholog of mouse acid phosphatase 6,
lysophosphatidic. ACP6 is a cytoplasmic or mitochondrial enzyme
that catalyzes the hydrolysis of lysophosphatidic acid to
monoacylglycerol and phosphate. Expression of ACP6 is detected in a
wide variety of tissues but is particularly high in kidney, heart,
small intestine, muscle, and liver. The enzyme also has been
detected in the cytosolic fraction of brain homogenates and in
interstitial cells of Cajal (ICC), which function as pacemakers and
mediators of motor neurotransmission in gastrointestinal smooth
muscle. ACP6 may play a role in lipid metabolism (Hiroyama and
Takenawa, Biochem J, 336 (Pt 2): 483-9 (1998); Hiroyama and
Takenawa, J Biol Chem, 274 (41): 29172-80 (1999); Takayama et al.,
Gut 50(6):790-6 (2002)).
Genetics Information:
TABLE-US-00051 [0991] wt het hom Total Observed 19 39 23 81
Expected 20.25 40.5 20.25 81 Chi-Sq. = 0.51 Significance = 0.77640
(hom/n) = 0.28 Avg. Litter Size = 8
Mutation Type: Retroviral Insertion (OST).
[0992] Retroviral insertion occurred in the intron between coding
exons 1 and 2 (Accession: NM.sub.--019800.1). Wild-type expression
of the target gene was detected in all 13 adult tissue samples
tested by RT-PCR, except skeletal muscle, bone, and adipose. RT-PCR
analysis revealed that the transcript was absent in the (-/-) mouse
analyzed (M-46). Disruption of the target gene was confirmed by
Inverse PCR.
[0993] 35.3.1. Phenotypic Analysis (for Disrupted Gene:
DNA34434-1139 (UNQ205)
[0994] (a) Overall Phenotypic Summary:
[0995] Mutation of the gene encoding the ortholog of human acid
phosphatase 6, lysophosphatidic (ACP6) resulted in the observation
of an increased mean platelet count in the (-/-) mutant mice
compared with the wild-type (+/+) littermates. In addition, the
(-/-) mutant mice appeared to have decreased total body fat (% and
gram) as well as decreased total tissue mass indicative of tissue
wasting diseases. Transcript was absent by RT-PCR.
[0996] (b) Immunology Phenotypic Analysis
[0997] Immune related and inflammatory diseases are the
manifestation or consequence of fairly complex, often multiple
interconnected biological pathways which in normal physiology are
critical to respond to insult or injury, initiate repair from
insult or injury, and mount innate and acquired defense against
foreign organisms. Disease or pathology occurs when these normal
physiological pathways cause additional insult or injury either as
directly related to the intensity of the response, as a consequence
of abnormal regulation or excessive stimulation, as a reaction to
self, or as a combination of these.
[0998] Though the genesis of these diseases often involves
multistep pathways and often multiple different biological
systems/pathways, intervention at critical points in one or more of
these pathways can have an ameliorative or therapeutic effect.
Therapeutic intervention can occur by either antagonism of a
detrimental process/pathway or stimulation of a beneficial
process/pathway.
[0999] T lymphocytes (T cells) are an important component of a
mammalian immune response. T cells recognize antigens which are
associated with a self-molecule encoded by genes within the major
histocompatibility complex (MHC). The antigen may be displayed
together with MHC molecules on the surface of antigen presenting
cells, virus infected cells, cancer cells, grafts, etc. The T cell
system eliminates these altered cells which pose a health threat to
the host mammal. T cells include helper T cells and cytotoxic T
cells. Helper T cells proliferate extensively following recognition
of an antigen-MHC complex on an antigen presenting cell. Helper T
cells also secrete a variety of cytokines, i.e., lymphokines, which
play a central role in the activation of B cells, cytotoxic T cells
and a variety of other cells which participate in the immune
response.
[1000] In many immune responses, inflammatory cells infiltrate the
site of injury or infection. The migrating cells may be
neutrophilic, eosinophilic, monocytic or lymphocytic as can be
determined by histological examination of the affected tissues.
Current Protocols in Immunology, ed. John E. Coligan, 1994, John
Wiley & Sons, Inc.
[1001] Many immune related diseases are known and have been
extensively studied. Such diseases include immune-mediated
inflammatory diseases (such as rheumatoid arthritis, immune
mediated renal disease, hepatobiliary diseases, inflammatory bowel
disease (IBD), psoriasis, and asthma), non-immune-mediated
inflammatory diseases, infectious diseases, immunodeficiency
diseases, neoplasia, and graft rejection, etc. In the area of
immunology, targets were identified for the treatment of
inflammation and inflammatory disorders.
[1002] In the area of immunology, targets have been identified
herein for the treatment of inflammation and inflammatory
disorders. Immune related diseases, in one instance, could be
treated by suppressing the immune response. Using neutralizing
antibodies that inhibit molecules having immune stimulatory
activity would be beneficial in the treatment of immune-mediated
and inflammatory diseases. Molecules which inhibit the immune
response can be utilized (proteins directly or via the use of
antibody agonists) to inhibit the immune response and thus
ameliorate immune related disease.
[1003] The following test was performed:
[1004] Hematology Analysis:
[1005] Test Description: Blood tests are carried out by Abbott's
Cell-Dyn 3500R, an automated hematology analyzer. Some of its
features include a five-part WBC differential. `Patient` reports
can cover over 22 parameters in all.
[1006] Results:
[1007] The (-/-) mice exhibited an increased mean platelet count
when compared with their (+/+) littermates and the historical mean.
Analyzed wt/het/hom: 7/5/8
[1008] Thus, mutant mice deficient in the DNA34434-1139 gene
resulted in a phenotype related to coagulation disorders. In this
regard, inhibitors or antagonists of PRO231 polypeptides would be
useful in treating disorders related to abnormal blood coagulation
such as hemophilia.
[1009] (c) Bone Metabolism & Body Diagnostics: Radiology
Phenotypic Analysis
[1010] In the area of bone metabolism, targets were identified
herein for the treatment of arthritis, osteoporosis, osteopenia and
osteopetrosis as well as identifying targets that promote bone
healing. Tests included:
[1011] DEXA for measurement of bone mineral density on femur and
vertebra
[1012] MicroCT for very high resolution and very high sensitivity
measurements of bone mineral density for both trabecular and
cortical bone.
[1013] Dexa Analysis--Test Description:
[1014] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. Dual Energy X-ray
Absorptiometry (DEXA) has been used successfully to identify
changes in bone. Anesthetized animals were examined and bone
mineral content (BMC), BMC/LBM ratios, volumetric bone mineral
density (vBMD), total body BMD, femur BMD and vertebra BMD were
measured.
[1015] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PIXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI)
[i.e., whole body, vertebrae, and both femurs].
[1016] Results:
[1017] DEXA: The (-/-) mice exhibited a decreased total body fat (%
and g) and total tissue mass when compared with their
gender-matched (+/+) littermates and the historical means.
[1018] Summary
[1019] These results demonstrate that knockout mutant mice exhibit
abnormal body measurements with significant decreased body fat and
mass suggestive of tissue wasting diseases. The (-/-) mice analyzed
by DEXA exhibited notably decreased total tissue mass and lean body
mass when compared with their (+/+) littermates, suggestive of
growth retardation in these mutants. Thus, PRO231 polypeptides or
agonists thereof would be useful for treating growth disorders or
for the prevention of cachexia or other tissue wasting
diseases.
[1020] 35.4. Generation and Analysis of Mice Comprising
DNA35599-1168 (UNQ210) Gene Disruptions
[1021] In these knockout experiments, the gene encoding PRO236
polypeptides (designated as DNA35599-1168) (UNQ210) was disrupted.
The gene specific information for these studies is as follows: the
mutated mouse gene corresponds to nucleotide reference:
NM.sub.--153803 or ACCESSION: NM.sub.--153803 NID: gi 24418924 ref
NM.sub.--153803.1 Mus musculus hypothetical protein MGC47419
(MGC47419); protein reference: Q8CFT1 or ACCESSION: Q8CFT1 NID: Mus
musculus (Mouse) Similar to RIKEN cDNA 4921509F24 gene; the human
gene sequence reference: NM.sub.--138342 or ACCESSION:
NM.sub.--138342 NID: gi 24308391 ref NM.sub.--138342.1 Homo sapiens
hypothetical protein BC008326 (LOC89944); the human protein
sequence corresponds to reference: Q81W92 or ACCESSION: Q81W92 NID:
Homo sapiens (Human). Hypothetical protein BC008326.
[1022] The mouse gene of interest is "cDNA sequence BC038479"
(BC038479), ortholog of human "hypothetical protein BC008326."
Aliases include hypothetical protein MGC47419.
[1023] BC038479 is a putative lysosomal enzyme that contains a
glycosyl hydrolase family 35 domain. Proteins with this domain
include beta-galactosidase, a lysosomal enzyme that catalyzes the
cleavage of the terminal galactose from gangliosides,
glycoproteins, and glycosaminoglycans (Pfam accession PF01301).
[1024] Targeted or gene trap mutations were generated in strain
129SvEv.sup.Brd-derived embryonic stem (ES) cells. The chimeric
mice were bred to C57BL/6J albino mice to generate F1 heterozygous
animals. These progeny were intercrossed to generate F2 wild type,
heterozygous, and homozygous mutant progeny. On rare occasions, for
example when very few F1 mice were obtained from the chimera, F1
heterozygous mice were crossed to 129SvEv.sup.Brd/C57 hybrid mice
to yield additional heterozygous animals for the intercross to
generate the F2 mice.
Level I phenotypic analysis is performed on mice from this
generation.
TABLE-US-00052 wt het hom Total Observed 22 25 26 73 Expected 18.25
36.5 18.25 73 Chi-Sq. = 7.68 Significance = 0.02144 (hom/n) = 0.36
Avg. Litter Size = 7
Mutation Type: Homologous Recombination (standard) Coding exon 1
was targeted (NCBI accession NM.sub.--153803.1). Wild-type
expression of the target gene was detected in embryonic stem (ES)
cells and in all 13 adult tissue samples tested by RT-PCR.
Disruption of the target gene was confirmed by Southern
hybridization analysis.
[1025] 35.4.1. Phenotypic Analysis (for Disrupted Gene:
DNA35599-1168 (UNQ210)
[1026] (a) Overall Phenotypic Summary:
[1027] Mutation of the gene encoding the ortholog of a hypothetical
human protein (BC008326) resulted in decreased mean white blood
cell (WBC) and lymphocyte counts and a decreased mean percentage of
natural killer cells in (-/-) mice. The (-/-) mice also exhibited
impaired glucose tolerance. In addition, the (-/-) mutant mice
exhibited abnormal bone measurements indicative of osteoporosis.
Gene disruption was confirmed by Southern blot.
[1028] (b) Immunology Phenotypic Analysis
[1029] Immune related and inflammatory diseases are the
manifestation or consequence of fairly complex, often multiple
interconnected biological pathways which in normal physiology are
critical to respond to insult or injury, initiate repair from
insult or injury, and mount innate and acquired defense against
foreign organisms. Disease or pathology occurs when these normal
physiological pathways cause additional insult or injury either as
directly related to the intensity of the response, as a consequence
of abnormal regulation or excessive stimulation, as a reaction to
self, or as a combination of these.
[1030] Though the genesis of these diseases often involves
multistep pathways and often multiple different biological
systems/pathways, intervention at critical points in one or more of
these pathways can have an ameliorative or therapeutic effect.
Therapeutic intervention can occur by either antagonism of a
detrimental process/pathway or stimulation of a beneficial
process/pathway.
[1031] T lymphocytes (T cells) are an important component of a
mammalian immune response. T cells recognize antigens which are
associated with a self-molecule encoded by genes within the major
histocompatibility complex (MHC). The antigen may be displayed
together with MHC molecules on the surface of antigen presenting
cells, virus infected cells, cancer cells, grafts, etc. The T cell
system eliminates these altered cells which pose a health threat to
the host mammal. T cells include helper T cells and cytotoxic T
cells. Helper T cells proliferate extensively following recognition
of an antigen-MHC complex on an antigen presenting cell. Helper T
cells also secrete a variety of cytokines, i.e., lymphokines, which
play a central role in the activation of B cells, cytotoxic T cells
and a variety of other cells which participate in the immune
response.
[1032] In many immune responses, inflammatory cells infiltrate the
site of injury or infection. The migrating cells may be
neutrophilic, eosinophilic, monocytic or lymphocytic as can be
determined by histological examination of the affected tissues.
Current Protocols in Immunology, ed. John E. Coligan, 1994, John
Wiley & Sons, Inc.
[1033] Many immune related diseases are known and have been
extensively studied. Such diseases include immune-mediated
inflammatory diseases (such as rheumatoid arthritis, immune
mediated renal disease, hepatobiliary diseases, inflammatory bowel
disease (IBD), psoriasis, and asthma), non-immune-mediated
inflammatory diseases, infectious diseases, immunodeficiency
diseases, neoplasia, and graft rejection, etc. In the area of
immunology, targets were identified for the treatment of
inflammation and inflammatory disorders.
[1034] In the area of immunology, targets have been identified
herein for the treatment of inflammation and inflammatory
disorders. Immune related diseases, in one instance, could be
treated by suppressing the immune response. Using neutralizing
antibodies that inhibit molecules having immune stimulatory
activity would be beneficial in the treatment of immune-mediated
and inflammatory diseases. Molecules which inhibit the immune
response can be utilized (proteins directly or via the use of
antibody agonists) to inhibit the immune response and thus
ameliorate immune related disease.
[1035] The following tests were performed:
[1036] (1) Flourescence-Activated Cell-Sorting (FACS) Analysis
[1037] Procedure:
[1038] FACS analysis of immune cell composition from peripheral
blood was performed including CD4, CD8 and T cell receptor to
evaluate T lymphocytes, CD19 for B lymphocytes, CD45 as a leukocyte
marker and pan NK for natural killer cells. The FACS analysis was
carried out on 2 wild type and 6 homozygous mice and included cells
derived from thymus, spleen, bone marrow and lymph node.
[1039] In these studies, analyzed cells were isolated from thymus,
peripheral blood, spleen, bone marrow and lymph nodes. Flow
cytometry was designed to determine the relative proportions of CD4
and CD8 positive T cells, B cells, NK cells and monocytes in the
mononuclear cell population. A Becton-Dickinson FACSCalibur 3-laser
FACS machine was used to assess immune status. For Phenotypic
Assays and Screening, this machine records CD4+/CD8-, CD8+/CD4-,
NK, B cell and monocyte numbers in addition to the CD4+/CD8+ ratio.
The mononuclear cell profile was derived by staining a single
sample of lysed peripheral blood from each mouse with a panel of
six lineage-specific antibodies: CD45 PerCP, anti-TCRb APC, CD4 PE,
CD8 FITC, pan-NK PE, and CD19 FITC. The two FITC and PE labeled
antibodies stain mutually exclusive cell types. The samples were
analyzed using a Becton Dickinson FACSCalibur flow cytometer with
CellQuest software.
[1040] (2) Hematology Analysis:
[1041] Test Description: Blood tests are carried out by Abbott's
Cell-Dyn 3500R, an automated hematology analyzer. Some of its
features include a five-part WBC differential. `Patient` reports
can cover over 22 parameters in all.
[1042] Results:
[1043] The (-/-) mice exhibited decreased mean total white blood
cell and absolute lymphocyte counts when compared with their (+/+)
littermates and the historical means.
[1044] FACS analysis also demonstrated that (-/-) mice exhibited a
decreased mean percentage of natural killer cells when compared
with their (+/+) littermates and the historical mean.
[1045] Analyzed wt/het/hom: 7/5/8
[1046] In summary, the hematology and FACS results indicate that
the homozygous mutant mice have an impaired immune system,
especially in view of the decreased total white blood cell count
and absolute lymphocyte counts. Furthermore, the decreased mean
percentage of natural killer cells is an additional indicator of a
negative phenotype associated with knocking out the DNA35599-1168
gene which encodes PRO236 polypeptides. Natural killer cells are
the first line of defense to viral infection since these cells have
been implicated in viral immunity and in defense against tumors.
Natural killer cells or NK cells act as effectors in
antibody-dependent cell-mediated cytotoxicity and have been
identified by their ability to kill certain lymphoid tumor cell
lines in vitro without the need for prior immunization or
activation. However, their known function in host defense is in the
early phases of infection with several intracellular pathogens,
particularly herpes viruses. Thus, PRO236 polypeptides and agonists
thereof would be important for a healthy immune system and would be
useful in stimulating the immune system especially against viral
infections.
[1047] (c) Bone Metabolism & Body Diagnostics: Bone Metabolism:
Radiology Phenotypic Analysis
[1048] In the area of bone metabolism, targets were identified
herein for the treatment of arthritis, osteoporosis, osteopenia and
osteopetrosis as well as identifying targets that promote bone
healing. Tests included:
[1049] DEXA for measurement of bone mineral density on femur and
vertebra
[1050] MicroCT for very high resolution and very high sensitivity
measurements of bone mineral density for both trabecular and
cortical bone.
[1051] Dexa Analysis--Test Description:
[1052] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. Dual Energy X-ray
Absorptiometry (DEXA) has been used successfully to identify
changes in bone. Anesthetized animals were examined and bone
mineral content (BMC), BMC/LBM ratios, volumetric bone mineral
density (vBMD), total body BMD, femur BMD and vertebra BMD were
measured.
[1053] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PIXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI)
[i.e., whole body, vertebrae, and both femurs].
[1054] Bone MicroCT Analysis:
[1055] Procedure: MicroCT was also used to get very sensitive
measurements of BMD. One vertebra and 1 femur were taken from a
cohort of 4 wild type and 8 homozygous mice. Measurements were
taken of lumbar 5 vertebra trabecular bone volume, trabecular
thickness, connectivity density and midshaft femur total bone area
and cortical thickness. The .mu.CT40 scans provided detailed
information on bone mass and architecture. Multiple bones were
placed into sample holders and scanned automatically. Instrument
software was used to select regions of interest for analysis.
Trabecular bone parameters were analyzed in the fifth lumbar
vertebrae (LV5) at 16 micrometer resolution and cortical bone
parameters were analyzed in the femur midshaft at a resolution of
20 micrometers.
[1056] Results:
[1057] Micro-CT: The (-/-) mice exhibited decreased bone
measurements in the 5.sup.th lumbar vertebrae (decreased mean
vertebral trabecular bone volume and thickness) when compared with
their gender-matched (+/+) littermates and the historical
means.
[1058] Summary
[1059] These results demonstrate that knockout mutant mice exhibit
abnormal bone metabolism with significant bone loss similar to
osteoporosis characterized by decrease in bone mass with decreased
density and possibly fragility leading to bone fractures. Thus, it
appears that PRO236 polypeptides or agonists thereof play a role in
maintaining bone homeostasis. In addition, PRO236 or its encoding
gene would be useful in bone healing or useful for the treatment of
arthritis or osteoporosis; whereas antagonists to PRO236 or its
encoding gene would lead to abnormal or pathological bone disorders
including inflammatory diseases associated with abnormal bone
metabolism such as arthritis, osteoporosis, and osteopenia.
[1060] (d) Phenotypic Analysis: Metabolism-Blood Chemistry/Glucose
Tolerance
[1061] In the area of metabolism, targets may be identified for the
treatment of diabetes. Blood chemistry phenotypic analysis includes
blood glucose measurements. The COBAS Integra 400 (mfr: Roche) was
used for running blood chemistry tests on the mice. In the area of
metabolism, targets may be identified for the treatment of
diabetes. Blood chemistry phenotypic analysis includes glucose
tolerance tests to measure insulin sensitivity and changes in
glucose metabolism. Abnormal glucose tolerance test results may
indicate but may not be limited to the following disorders or
conditions: Diabetes Type 1 and Type 2, Syndrome X, various
cardiovascular diseases and/or obesity.
[1062] Procedure: A cohort of 2 wild type and 4 homozygote mice
were used in this assay. The glucose tolerance test is the standard
for defining impaired glucose homeostasis in mammals. Glucose
tolerance tests were performed using a Lifescan glucometer. Animals
were injected IP at 2 g/kg with D-glucose delivered as a 20%
solution and blood glucose levels were measured at 0, 30, 60 and 90
minutes after injection. Analyzed wt/het/hom: 4/4/8
[1063] Results:
[1064] During the glucose tolerance test the (-/-) mice exhibited
impaired glucose tolerance when compared with their gender-matched
(+/+) littermates and the historical means.
[1065] These studies indicated that (-/-) mice exhibit a decreased
glucose tolerance in the presence of normal fasting glucose at all
3 intervals tested when compared with their gender-matched (+/+)
littermates and the historical means. Thus, knockout mice exhibited
the phenotypic pattern of an impaired glucose homeostasis. In light
of this observation, PRO236 polypeptides (or agonists thereof) or
its encoding gene would play be useful in the treatment of impaired
glucose homeostasis and/or various cardiovascular diseases,
including the treatment of diabetes.
[1066] 35.5. Generation and Analysis of Mice Comprising
DNA35638-1141 (UNQ219) Gene Disruptions
[1067] In these knockout experiments, the gene encoding PRO245
polypeptides (designated as DNA35638-1141) (UNQ219) was disrupted.
The gene specific information for these studies is as follows: the
mutated mouse gene corresponds to nucleotide reference:
NM.sub.--023844 or Mus musculus RIKEN cDNA 1110002N23 gene
(1110002N23Rik); protein reference: Q9JI59 or VASCULAR ENDOTHELIAL
JUNCTION-ASSOCIATED MOLECULE (JUNCTIONAL ADHESION MOLECULE-3)
(2410030G21RIK PROTEIN); the human gene sequence reference:
NM.sub.--021219 or ACCESSION: NM.sub.--021219 NID: gi 21704284 ref
NM.sub.--021219.2 Homo sapiens junctional adhesion molecule 2
(JAM2); the human protein sequence corresponds to reference: P57087
or ACCESSION: P57087 NID: Homo sapiens (Human). JUNCTIONAL ADHESION
MOLECULE 2 PRECURSOR (VASCULAR ENDOTHELIAL JUNCTION-ASSOCIATED
MOLECULE) (VE-JAM) HUMANSPTRNRDB.
[1068] The disrupted mouse gene is Jam2 (junction adhesion molecule
2), the ortholog of human JAM2. Aliases include VEJAM, VE-JAM,
JAM-2, Jcam2, C21orf43, chromosome 21 open reading frame 43,
vascular endothelial junction-associated molecule, and junction
cell adhesion molecule 2.
[1069] JAM2, an Ig superfamily member, is a membrane-lodged
extracellular protein. JAM2 is specifically localized at junctions
of lymphatic endothelial cells and high endothelial venules within
lymph nodes and Peyer patches (Johnson-Leger et al., Blood,
100(7):2479-86 (2002)). JAM2 binds T, NK, and dendritic cells via
interactions with alpha4-beta1 integrin and JAM3 (Liang et al., J
Immunol, 168(4):1618-26 (2002)). JAM2 apparently plays a central
role in tight junction formation, transendothelial and lymphocyte
migration, and the establishment of cell polarity in endothelial
tissue (Ebnet et al., J Cell Sci 116(Pt 19):3879-91 (2003),
Aurrand-Lions et al., Cells Tissues Organs, 172(3):152-60
(2002)).
Genetics Information:
TABLE-US-00053 [1070] wt het hom Total Observed 18 32 22 72
Expected 18 36 18 72 Chi-Sq. = 1.33 Significance = 0.51342 (hom/n)
= 0.31 Avg. Litter Size = 7
Mutation Type: Retroviral Insertion (OST)
[1071] Retroviral insertion occurred between coding exons 3 and 4
(Accession: NM.sub.--023844). Wild-type expression of the target
gene was detected in embryonic stem (ES) cells and in all 13 adult
tissue samples tested by RT-PCR, except adipose. RT-PCR analysis
revealed that the transcript was absent in the (-/-) mouse analyzed
(F-58).
[1072] 35.5.1. Phenotypic Analysis (for Disrupted Gene:
DNA35638-1141 (UNQ219)
[1073] (a) Overall Phenotypic Summary:
[1074] Mutation of the gene encoding the ortholog of human junction
adhesion molecule 2 (JAM2) resulted in an increased anxiety-related
response in (-/-) mice. In addition, mutant (-/-) mice demonstrated
a decreased ovalbumin response compared with their (+/+) wild-type
littermates. Also, the (-/-) mutant mice showed opthamological
abnormalities associated with an increase in retinal artery
tortuosity. Transcript was absent by RT-PCR.
[1075] (b) Immunology Phenotypic Analysis
[1076] Immune related and inflammatory diseases are the
manifestation or consequence of fairly complex, often multiple
interconnected biological pathways which in normal physiology are
critical to respond to insult or injury, initiate repair from
insult or injury, and mount innate and acquired defense against
foreign organisms. Disease or pathology occurs when these normal
physiological pathways cause additional insult or injury either as
directly related to the intensity of the response, as a consequence
of abnormal regulation or excessive stimulation, as a reaction to
self, or as a combination of these.
[1077] Though the genesis of these diseases often involves
multistep pathways and often multiple different biological
systems/pathways, intervention at critical points in one or more of
these pathways can have an ameliorative or therapeutic effect.
Therapeutic intervention can occur by either antagonism of a
detrimental process/pathway or stimulation of a beneficial
process/pathway.
[1078] T lymphocytes (T cells) are an important component of a
mammalian immune response. T cells recognize antigens which are
associated with a self-molecule encoded by genes within the major
histocompatibility complex (MHC). The antigen may be displayed
together with MHC molecules on the surface of antigen presenting
cells, virus infected cells, cancer cells, grafts, etc. The T cell
system eliminates these altered cells which pose a health threat to
the host mammal. T cells include helper T cells and cytotoxic T
cells. Helper T cells proliferate extensively following recognition
of an antigen-MHC complex on an antigen presenting cell. Helper T
cells also secrete a variety of cytokines, i.e., lymphokines, which
play a central role in the activation of B cells, cytotoxic T cells
and a variety of other cells which participate in the immune
response.
[1079] In many immune responses, inflammatory cells infiltrate the
site of injury or infection. The migrating cells may be
neutrophilic, eosinophilic, monocytic or lymphocytic as can be
determined by histological examination of the affected tissues.
Current Protocols in Immunology, ed. John E. Coligan, 1994, John
Wiley & Sons, Inc.
[1080] Many immune related diseases are known and have been
extensively studied. Such diseases include immune-mediated
inflammatory diseases (such as rheumatoid arthritis, immune
mediated renal disease, hepatobiliary diseases, inflammatory bowel
disease (IBD), psoriasis, and asthma), non-immune-mediated
inflammatory diseases, infectious diseases, immunodeficiency
diseases, neoplasia, and graft rejection, etc. In the area of
immunology, targets were identified for the treatment of
inflammation and inflammatory disorders.
[1081] In the area of immunology, targets have been identified
herein for the treatment of inflammation and inflammatory
disorders. Immune related diseases, in one instance, could be
treated by suppressing the immune response. Using neutralizing
antibodies that inhibit molecules having immune stimulatory
activity would be beneficial in the treatment of immune-mediated
and inflammatory diseases. Molecules which inhibit the immune
response can be utilized (proteins directly or via the use of
antibody agonists) to inhibit the immune response and thus
ameliorate immune related disease.
[1082] The following test was performed:
[1083] Ovalbumin Challenge
[1084] Procedure: This assay was carried out on 7 wild types and 8
homozygotes. Chicken ovalbumin (OVA) is a T-cell dependent antigen,
which is commonly used as a model protein for studying
antigen-specific immune responses in mice. OVA is non-toxic and
inert and therefore will not cause harm to the animals even if no
immune response is induced. The murine immune response to OVA has
been well characterized, to the extent that the immuno-dominant
peptides for eliciting T cell responses have been identified.
Anti-OVA antibodies are detectable 8 to 10 days after immunization
using enzyme-linked immunosorbent assay (ELIZA), and determination
of different isotypes of antibodies gives further information on
the complex processes that may lead to a deficient response in
genetically engineered mice.
[1085] As noted above, this protocol assesses the ability of mice
to raise an antigen-specific immune response. Animals were injected
IP with 50 mg of chicken ovalbumin emulsified in Complete Freund's
Adjuvant and 14 days later the serum titer of anti-ovalbumin
antibodies (IgM, IgG1 and IgG2 subclasses) was measured. The amount
of OVA-specific antibody in the serum sample is proportional to the
Optical Density (OD) value generated by an instrument that scans a
96-well sample plate. Data was collected for a set of serial
dilutions of each serum sample.
Analyzed wt/het/hom: 8/4/9
[1086] Results of this Challenge:
[1087] The (-/-) mice exhibited a decreased mean serum IgG2a
response to ovalbumin challenge when compared with their (+/+)
littermates and the historical means. Thus, these knockout mice
exhibited a decreased ability to elicit an OVA-specific antibody
response to the T-cell dependent OVA antigen.
[1088] In summary, the ovalbumin challenge studies indicate that
knockout mice deficient in the gene encoding PRO245 polypeptides
exhibit immunological abnormalities when compared with their
wild-type littermates. In particular, the mutant mice exhibited a
decreased ability to elicit an immunological response when
challenged with the T-cell dependent OVA antigen. Thus, PRO245
polypeptides or agonists thereof, would be useful for stimulating
the immune system (such as T cell proliferation) and would find
utility in the cases wherein this effect would be beneficial to the
individual such as in the case of leukemia, and other types of
cancer, and in immunocompromised patients, such as AIDS sufferers.
Accordingly, inhibitors (antagonists) of PRO245 polypeptides would
be useful for inhibiting the immune response and thus would be
useful candidates for suppressing harmful immune responses, e.g. in
the case of graft rejection or graft-versus-host diseases.
[1089] (c) Phenotypic Analysis: CNS/Neurology
[1090] In the area of neurology, analysis focused herein on
identifying in vivo validated targets for the treatment of
neurological and psychiatric disorders including depression,
generalized anxiety disorders, attention deficit hyperactivity
disorder, obsessive compulsive disorder, schizophrenia, cognitive
disorders, hyperalgesia and sensory disorders. Neurological
disorders include the category defined as "anxiety disorders" which
include but are not limited to: mild to moderate anxiety, anxiety
disorder due to a general medical condition, anxiety disorder not
otherwise specified, generalized anxiety disorder, panic attack,
panic disorder with agoraphobia, panic disorder without
agoraphobia, posttraumatic stress disorder, social phobia, specific
phobia, substance-induced anxiety disorder, acute alcohol
withdrawal, obsessive compulsive disorder, agoraphobia, bipolar
disorder I or II, bipolar disorder not otherwise specified,
cyclothymic disorder, depressive disorder, major depressive
disorder, mood disorder, substance-induced mood disorder. In
addition, anxiety disorders may apply to personality disorders
including but not limited to the following types: paranoid,
antisocial, avoidant behavior, borderline personality disorders,
dependent, histronic, narcissistic, obsessive-compulsive, schizoid,
and schizotypal.
[1091] Procedure:
[1092] Behavioral screens were performed on a cohort of 4 wild
type, 4 heterozygous and 8 homozygous mutant mice. All behavioral
tests were done between 12 and 16 weeks of age unless reduced
viability necessitates earlier testing. These tests included open
field to measure anxiety, activity levels and exploration.
[1093] Open Field Test:
[1094] Several targets of known drugs have exhibited phenotypes in
the open field test. These include knockouts of the seratonin
transporter, the dopamine transporter (Giros et al., Nature. 1996
Feb. 15; 379(6566):606-12), and the GABA receptor (Homanics et al.,
Proc Natl Acad Sci USA. 1997 Apr. 15; 94(8):4143-8). An automated
open-field assay was customized to address changes related to
affective state and exploratory patterns related to learning.
First, the field (40.times.40 cm) was selected to be relatively
large for a mouse, thus designed to pick up changes in locomotor
activity associated with exploration. In addition, there were 4
holes in the floor to allow for nose-poking, an activity
specifically related to exploration. Several factors were also
designed to heighten the affective state associated with this test.
The open-field test is the first experimental procedure in which
the mice are tested, and the measurements that were taken were the
subjects' first experience with the chamber. In addition, the
open-field was brightly lit. All these factors will heighten the
natural anxiety associated with novel and open spaces. The pattern
and extent of exploratory activity, and especially the
center-to-total distance traveled ratio, may then be able to
discern changes related to susceptibility to anxiety or depression.
A large arena (40 cm.times.40 cm, VersaMax animal activity
monitoring system from AccuScan Instruments) with infrared beams at
three different levels was used to record rearing, hole poke, and
locomotor activity. The animal was placed in the center and its
activity was measured for 20 minutes. Data from this test was
analyzed in five, 4-minute intervals. The total distance traveled
(cm), vertical movement number (rearing), number of hole pokes, and
the center to total distance ratio were recorded.
[1095] The propensity for mice to exhibit normal habituation
responses to a novel environment is assessed by determining the
overall change in their horizontal locomotor activity across the 5
time intervals. This calculated slope of the change in activity
over time is determined using normalized, rather than absolute,
total distance traveled. The slope is determined from the
regression line through the normalized activity at each of the 5
time intervals. Normal habituation is represented by a negative
slope value. Analyzed wt/het/hom: 5/4/8
[1096] Results:
[1097] The (-/-) mice exhibited a decreased median sum
time-in-center during open field testing when compared with their
gender-matched (+/+) littermates and the historical mean,
suggesting an increased anxiety-like response in the mutants. As
noted above, a notable difference was observed during open field
activity testing. The (-/-) mice exhibited a decreased median sum
time in the center area when compared with their gender-matched
(+/+) littermates. This type of behavior is consistent with an
increased anxiety like response. Thus, the knockout mice
demonstrated a phenotype consistent with anxiety related disorders
which are associated with mild to moderate anxiety, anxiety due to
a general medical condition, and/or bipolar disorders;
hyperactivity; sensory disorders; obsessive-compulsive disorders,
schizophrenia or a paranoid personality. Thus, PRO245 polypeptides
or agonists thereof would be useful in the treatment of such
neurological disorders or the amelioration of the symptoms
associated with anxiety disorders.
[1098] (d) Cardiovascular Phenotypic Analysis:
[1099] In the area of cardiovascular biology, phenotypic testing
was performed to identify potential targets for the treatment of
cardiovascular, endothelial or angiogenic disorders. One such
phenotypic test included optic fundus photography and angiography
to determine the retinal arteriovenous ratio (A/V ratio) in order
to flag various eye abnormalities. An abnormal A/V ratio signals
such systemic diseases or disorders that may be related to the
vascular disease of hypertension (and any disease that causes
hypertension, e.g. atherosclerosis), diabetes or other ocular
diseases corresponding to ophthalmological disorders. Such eye
abnormalities may include but are not limited to the following:
retinal abnormality is retinal dysplasia, various retinopathies,
restenosis, retinal artery obstruction or occlusion; retinal
degeneration causing secondary atrophy of the retinal vasculature,
retinitis pigmentosa, macular dystrophies, Stargardt's disease,
congenital stationary night blindness, choroideremia, gyrate
atrophy, Leber's congenital amaurosis, retinoschisis disorders,
Wagner's syndrome, Usher syndromes, Zellweger syndrome,
Saldino-Mainzer syndrome, Senior-Loken syndrome, Bardet-Biedl
syndrome, Alport's syndrome, Alstom's syndrome, Cockayne's
syndrome, dysplaisa spondyloepiphysaria congentia, Flynn-Aird
syndrome, Friedreich ataxia, Hallgren syndrome, Marshall syndrome,
Albers-Schnoberg disease, Refsum's disease, Kearns-Sayre syndrome,
Waardenburg's syndrome, Alagile syndrome, myotonic dystrophy,
olivopontocerebellar atrophy, Pierre-Marie dunsdrome, Stickler
syndrome, carotinemeia, cystinosis, Wolfram syndrome,
Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentia
pigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria,
or mannosidosis.
[1100] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. Optic fundus photography was
performed on conscious animals using a Kowa Genesis small animal
fundus camera modified according to Hawes and coauthors (Hawes et
al., 1999 Molecular Vision 1999; 5:22). Intra-peritoneal injection
of fluorescein permitted the acquisition of direct light fundus
images and fluorescent angiograms for each examination. In addition
to direct ophthalmological changes, this test can detect retinal
changes associated with systemic diseases such as diabetes and
atherosclerosis or other retinal abnormalities. Pictures were
provided of the optic fundus under normal light. The angiographic
pictures allowed examination of the arteries and veins of the eye.
In addition an artery to vein (A/V) ratio was determined for the
eye.
[1101] Ophthalmology analysis was performed on generated F2 wild
type, heterozygous, and homozygous mutant progeny using the
protocol described above. Specifically, the A/V ratio was measured
and calculated according to the fundus images with Kowa COMIT+
software. This test takes color photographs through a dilated
pupil: the images help in detecting and classifying many diseases.
The artery to vein ratio (A/V) is the ratio of the artery diameter
to the vein diameter (measured before the bifurcation of the
vessels). Many diseases will influence the ratio, i.e., diabetes,
cardiovascular disorders, papilledema, optic atrophy or other eye
abnormalities such as retinal degeneration (known as retinitis
pigmentosa) or retinal dysplasia, vision problems or blindness.
Thus, phenotypic observations which result in an increased
artery-to-vein ratio in homozygous (-/-) and heterozygous (+/-)
mutant progeny compared to wild-type (+/+) littermates would be
indicative of such pathological conditions.
[1102] Results:
[1103] Pathological microscopic observations showed an increase in
retinal artery tortuosity in the (-/-) mice analyzed. In addition,
corneal inflammation and ulceration was also noted in the (-/-)
mutant mice. Analyzed wt/het/hom: 0/1/4
[1104] In summary, in this study, (-/-) mice showed opthamological
abnormalities which would lead to attenuated retinal vessels and
possibly retinal degeneration when compared with their (+/+)
littermates. In summary, by knocking out the gene identified as
DNA35638-1141 encoding PRO245 polypeptides, homozygous mutant
progeny exhibit phenotypes which are associated with retinal artery
abnormalities. Such detected retinal changes are most commonly
associated with cardiovascular systemic diseases or disorders that
may be related to the vascular disease of hypertension (and any
disease that causes hypertension, e.g. atherosclerosis), diabetes
or other ocular diseases corresponding to ophthalmological
disorders such as retinal degeneration. Thus, antagonists of PRO245
encoding genes would lead to similar pathological retinal changes,
whereas agonists would be useful as therapeutic agents in the
treatment of hypertension, atherosclerosis or other opthamological
disorders including retinal degeneration and diseases associated
with this condition (as indicated above).
[1105] 35.6. Generation and Analysis of Mice Comprising
DNA35639-1172 (UNQ220) Gene Disruptions
[1106] In these knockout experiments, the gene encoding PRO246
polypeptides (designated as DNA35639-1172) (UNQ220) was disrupted.
The gene specific information for these studies is as follows: the
mutated mouse gene corresponds to nucleotide reference:
NM.sub.--027102 OR ACCESSION: NM.sub.--027102 NID: 22094994 Mus
musculus Mus musculus endothelial cell-selective adhesion molecule;
protein reference: Q925F2 or ACCESSION: Q925F2 NID: Mus musculus
(Mouse). ENDOTHELIAL CELL-SELECTIVE ADHESION MOLECULE.
MOUSESPTRNRDB; the human gene sequence reference: NM.sub.--138961
or ACCESSION: NM.sub.--138961 NID: 20452463 Homo sapiens Homo
sapiens similar to endothelial cell-selective adhesion molecule;
the human protein sequence corresponds to reference: Q96AP7 or
ACCESSION: Q96AP7 NID: Homo sapiens (Human). Hypothetical protein
PLACE1000456.
[1107] The disrupted mouse gene is Esam1 (endothelial cell-specific
adhesion molecule), ortholog of human ESAM. Aliases include W117m,
2310008D05Rik, and HUEL (C4orf1)-interacting protein.
[1108] ESAM is a cell adhesion molecule of the immunoglobulin
receptor family expressed at tight junctions on endothelial cells.
ESAM is likely to play a role in inter endothelial cell adhesion
(Hirata et al., J Biol Chem, 276(19):16223-31 (2001); Nasdala et
al, J Biol Chem, 277(18):16294-303 (2002)). Ishida and colleagues,
[J Biol Chem, 278(36):34598-604 (2003)] showed that tumor volume of
ESAM homozygous null mice was notably smaller than that of
wild-type mice. By matrigel plug assay, Ishida and colleagues also
showed that neovascularization was notably lower in ESAM homozygous
null mice than in wild-type mice. They concluded that ESAM played a
role in pathological angiogenic processes such as tumor growth.
[1109] Targeted or gene trap mutations were generated in strain
129SvEv.sup.Brd-derived embryonic stem (ES) cells. The chimeric
mice were bred to C57BL/6J albino mice to generate F1 heterozygous
animals. These progeny were intercrossed to generate F2 wild type,
heterozygous, and homozygous mutant progeny. On rare occasions, for
example when very few F1 mice were obtained from the chimera, F1
heterozygous mice were crossed to 129SvEv.sup.Brd/C57 hybrid mice
to yield additional heterozygous animals for the intercross to
generate the F2 mice.
Level I phenotypic analysis is performed on mice from this
generation.
TABLE-US-00054 wt het hom Total Observed 18 26 15 59 Expected 14.75
29.5 14.75 59 Chi-Sq. = 1.14 Significance = 0.56677 (hom/n) = 0.25
Avg. Litter Size = 7
Mutation Type: Retroviral Insertion (OST)
[1110] Retroviral insertion occurred in the intron between coding
exons 2 and 3 (accession: NM.sub.--027102.1). Wild-type expression
of the target gene was detected in embryonic stem (ES) cells and in
all 13 adult tissue samples tested by RT-PCR, except adipose.
RT-PCR analysis revealed that the transcript was absent in the
(-/-) mouse analyzed (M-156). Disruption of the target gene was
confirmed by Inverse PCR.
[1111] 35.6.1. Phenotypic Analysis (for Disrupted gene:
DNA35639-1172 (UNQ220)
[1112] (a) Overall Phenotypic Summary:
[1113] Mutation of the gene encoding the ortholog of human
endothelial cell-specific adhesion molecule (ESAM) resulted in an
increased mean serum glucose levels and a decreased or impaired
glucose tolerance. In addition, (-/-) mice exhibited a decreased
heart rate (one standard deviation below the historic mean).
Transcript was absent by RT-PCR.
[1114] (b) Phenotypic Analysis: Metabolism-Blood Chemistry/Glucose
Tolerance
[1115] In the area of metabolism, targets may be identified for the
treatment of diabetes. Blood chemistry phenotypic analysis includes
blood glucose measurements. The COBAS Integra 400 (mfr: Roche) was
used for running blood chemistry tests on the mice. In the area of
metabolism, targets may be identified for the treatment of
diabetes. Blood chemistry phenotypic analysis includes glucose
tolerance tests to measure insulin sensitivity and changes in
glucose metabolism. Abnormal glucose tolerance test results may
indicate but may not be limited to the following disorders or
conditions: Diabetes Type 1 and Type 2, Syndrome X, various
cardiovascular diseases and/or obesity.
[1116] Procedure: A cohort of 2 wild type and 4 homozygous mice
were used in this assay. The glucose tolerance test is the standard
for defining impaired glucose homeostasis in mammals. Glucose
tolerance tests were performed using a Lifescan glucometer. Animals
were injected IP at 2 g/kg with D-glucose delivered as a 20%
solution and blood glucose levels were measured at 0, 30, 60 and 90
minutes after injection.
[1117] Results:
[1118] The (-/-) mice exhibited an increased mean serum glucose
level [two (2) standard deviations (SD) above the historic mean]
when compared with their gender-matched (+/+) littermates and the
historical mean. In addition, the (-/-) mice exhibited impaired
glucose tolerance when compared with their gender-matched (+/+)
littermates and the historical means.
[1119] Analyzed wt/het/hom: 4/4/8
[1120] These studies indicated that (-/-) mice exhibit a decreased
or impaired glucose tolerance in the presence of normal fasting
glucose at all 3 intervals tested when compared with their
gender-matched (+/+) littermates and the historical means. Thus,
knockout mutant mice exhibited the phenotypic pattern of an
impaired glucose homeostasis, and therefor PRO246 polypeptides (or
agonists thereof) or its encoding gene would be useful in the
treatment of conditions associated with an impaired glucose
homeostasis and/or various cardiovascular diseases, including
diabetes.
[1121] 35.7. Generation and Analysis of Mice Comprising
DNA35918-1174 (UNQ225) Gene Disruptions
[1122] In these knockout experiments, the gene encoding PRO258
polypeptides (designated as DNA35918-1174) (UNQ225) was disrupted.
The gene specific information for these studies is as follows: the
mutated mouse gene corresponds to nucleotide reference:
NM.sub.--053199 or Mus musculus immunoglobulin superfamily, member
4B (Igsf4b); protein reference: Q99N28 or ACCESSION: Q99N28 NID:
Mus musculus (Mouse). NECTIN-LIKE PROTEIN 1. MOUSESPTRNRDB; the
human gene sequence reference: NM.sub.--021189 or Homo sapiens
immunoglobulin superfamily, member 4B (IGSF4B); the human protein
sequence corresponds to reference: Q9UJP1 or ACCESSION: Q9UJP1 NID:
Homo sapiens (Human). BK134P22.1 (NOVEL PROTEIN SIMILAR TO MOUSE
IMMUNOSUPERFAMILY PROTEIN BL2) (NECTIN-LIKE PROTEIN 1).
HUMANSPTRNRDB.
[1123] The mouse gene of interest is Igsf4b (immunoglobulin
superfamily, member 4B), ortholog of human IGSF4B. Aliases include
BIgR, Necl1, Tsll1, FLJ10698, nectin-like 1, TSLC1-like 1,
nectin-like protein 1, and brain immunoglobulin receptor
precursor.
[1124] IGSF4B is a type I plasma membrane protein expressed
primarily in neurogenic cells that likely functions as a cell
adhesion molecule. The protein contains a signal peptide, three
immunoglobulin-like domains (Pfam accession PF00047), a
transmembrane segment, and a short cytoplasmic C terminus. IGSF4B
is structurally similar to TSLC1, a tumor suppressor in human
non-small cell lung carcinomas, and IGSF4B expression is lost or
markedly reduced in many glioma cell lines, suggesting that IGSF4B
may also function as a tumor suppressor (Fukuhara et al., Oncogene,
20(38):5401-7 (2001); Shingai et al, J Biol Chem, 278(37):35421-7
(2003); Fukami et al, Gene 323:11-8 (2003)).
[1125] Targeted or gene trap mutations were generated in strain
129SvEv.sup.Brd-derived embryonic stem (ES) cells. The chimeric
mice were bred to C57BL/6J albino mice to generate F1 heterozygous
animals. These progeny were intercrossed to generate F2 wild type,
heterozygous, and homozygous mutant progeny. On rare occasions, for
example when very few F1 mice were obtained from the chimera, F1
heterozygous mice were crossed to 129SvEv.sup.Brd/C57 hybrid mice
to yield additional heterozygous animals for the intercross to
generate the F2 mice.
Level I phenotypic analysis is performed on mice from this
generation.
TABLE-US-00055 wt het hom Total Observed 17 30 22 69 Expected 17.25
34.5 17.25 69 Chi-Sq. = 1.90 Significance = 0.38702 (hom/n) = 0.32
Avg. Litter Size = 7
Mutation Type: Homologous Recombination (standard) Coding exon 1
was targeted (NCBI accession NM.sub.--053199.2). Wild-type
expression of the target gene was detected in all 13 adult tissue
samples tested by RT-PCR, except brain and eye. Disruption of the
target gene was confirmed by Southern hybridization analysis.
[1126] 35.7.1. Phenotypic Analysis (for Disrupted Gene:
DNA35918-1174 (UNQ225)
[1127] (a) Overall Phenotypic Summary:
[1128] Mutation of the gene encoding the ortholog of human
immunoglobulin superfamily, member 4B (IGSF4B) resulted in numerous
immunological abnormalities in (-/-) mice. The (-/-) mice also
exhibited enhanced motor coordination. However, circadian rhythm
testing showed decreased ambulation for the (-/-) mice. In
addition, the mutant (-/-) mice showed a significant increase in
mean serum cholesterol levels and mean serum glucose levels.
MicroCT observations indicated decreased 5.sup.th lumbar bone
measurements. Gene disruption was confirmed by Southern blot.
[1129] (b) Immunology Phenotypic Analysis
[1130] Immune related and inflammatory diseases are the
manifestation or consequence of fairly complex, often multiple
interconnected biological pathways which in normal physiology are
critical to respond to insult or injury, initiate repair from
insult or injury, and mount innate and acquired defense against
foreign organisms. Disease or pathology occurs when these normal
physiological pathways cause additional insult or injury either as
directly related to the intensity of the response, as a consequence
of abnormal regulation or excessive stimulation, as a reaction to
self, or as a combination of these.
[1131] Though the genesis of these diseases often involves
multistep pathways and often multiple different biological
systems/pathways, intervention at critical points in one or more of
these pathways can have an ameliorative or therapeutic effect.
Therapeutic intervention can occur by either antagonism of a
detrimental process/pathway or stimulation of a beneficial
process/pathway.
[1132] T lymphocytes (T cells) are an important component of a
mammalian immune response. T cells recognize antigens which are
associated with a self-molecule encoded by genes within the major
histocompatibility complex (MHC). The antigen may be displayed
together with MHC molecules on the surface of antigen presenting
cells, virus infected cells, cancer cells, grafts, etc. The T cell
system eliminates these altered cells which pose a health threat to
the host mammal. T cells include helper T cells and cytotoxic T
cells. Helper T cells proliferate extensively following recognition
of an antigen-MHC complex on an antigen presenting cell. Helper T
cells also secrete a variety of cytokines, i.e., lymphokines, which
play a central role in the activation of B cells, cytotoxic T cells
and a variety of other cells which participate in the immune
response.
[1133] In many immune responses, inflammatory cells infiltrate the
site of injury or infection. The migrating cells may be
neutrophilic, eosinophilic, monocytic or lymphocytic as can be
determined by histological examination of the affected tissues.
Current Protocols in Immunology, ed. John E. Coligan, 1994, John
Wiley & Sons, Inc.
[1134] Many immune related diseases are known and have been
extensively studied. Such diseases include immune-mediated
inflammatory diseases (such as rheumatoid arthritis, immune
mediated renal disease, hepatobiliary diseases, inflammatory bowel
disease (IBD), psoriasis, and asthma), non-immune-mediated
inflammatory diseases, infectious diseases, immunodeficiency
diseases, neoplasia, and graft rejection, etc. In the area of
immunology, targets were identified for the treatment of
inflammation and inflammatory disorders.
[1135] In the area of immunology, targets have been identified
herein for the treatment of inflammation and inflammatory
disorders. Immune related diseases, in one instance, could be
treated by suppressing the immune response. Using neutralizing
antibodies that inhibit molecules having immune stimulatory
activity would be beneficial in the treatment of immune-mediated
and inflammatory diseases. Molecules which inhibit the immune
response can be utilized (proteins directly or via the use of
antibody agonists) to inhibit the immune response and thus
ameliorate immune related disease.
[1136] The following tests were performed:
[1137] Hematology Analysis:
[1138] Test Description: Blood tests are carried out by Abbott's
Cell-Dyn 3500R, an automated hematology analyzer. Some of its
features include a five-part WBC differential. `Patient` reports
can cover over 22 parameters in all.
[1139] Results:
[1140] The (-/-) mice exhibited decreased mean total white blood
cell, absolute lymphocyte, and absolute monocyte counts when
compared with their (+/+) littermates and the historical means.
Analyzed wt/het/hom: 7/4/8
[1141] Flourescence-Activated Cell-Sorting (FACS) Analysis
[1142] Procedure:
[1143] FACS analysis of immune cell composition from peripheral
blood was performed including CD4, CD8 and T cell receptor to
evaluate T lymphocytes, CD19 for B lymphocytes, CD45 as a leukocyte
marker and pan NK for natural killer cells. The FACS analysis was
carried out on 2 wild type and 6 homozygous mice and included cells
derived from thymus, spleen, bone marrow and lymph node.
[1144] In these studies, analyzed cells were isolated from thymus,
peripheral blood, spleen, bone marrow and lymph nodes. Flow
cytometry was designed to determine the relative proportions of CD4
and CD8 positive T cells, B cells, NK cells and monocytes in the
mononuclear cell population. A Becton-Dickinson FACSCalibur 3-laser
FACS machine was used to assess immune status. For Phenotypic
Assays and Screening, this machine records CD4+/CD8-, CD8+/CD4-,
NK, B cell and monocyte numbers in addition to the CD4+/CD8+ ratio.
The mononuclear cell profile was derived by staining a single
sample of lysed peripheral blood from each mouse with a panel of
six lineage-specific antibodies: CD45 PerCP, anti-TCRb APC, CD4 PE,
CD8 FITC, pan-NK PE, and CD19 FITC. The two FITC and PE labeled
antibodies stain mutually exclusive cell types. The samples were
analyzed using a Becton Dickinson FACSCalibur flow cytometer with
CellQuest software.
[1145] Results:
[1146] The (-/-) mice exhibited an increased mean percentage of CD4
cells and a decreased mean percentage of B cells when compared with
their (+/+) littermates and the historical means.
Analyzed wt/het/hom: 7/4/8
[1147] Ovalbumin Challenge
[1148] Procedure: This assay was carried out on 7 wild types and 8
homozygotes. Chicken ovalbumin (OVA) is a T-cell dependent antigen,
which is commonly used as a model protein for studying
antigen-specific immune responses in mice. OVA is non-toxic and
inert and therefore will not cause harm to the animals even if no
immune response is induced. The murine immune response to OVA has
been well characterized, to the extent that the immuno-dominant
peptides for eliciting T cell responses have been identified.
Anti-OVA antibodies are detectable 8 to 10 days after immunization
using enzyme-linked immunosorbent assay (ELIZA), and determination
of different isotypes of antibodies gives further information on
the complex processes that may lead to a deficient response in
genetically engineered mice.
[1149] As noted above, this protocol assesses the ability of mice
to raise an antigen-specific immune response. Animals were injected
IP with 50 mg of chicken ovalbumin emulsified in Complete Freund's
Adjuvant and 14 days later the serum titer of anti-ovalbumin
antibodies (IgM, IgG1 and IgG2 subclasses) was measured. The amount
of OVA-specific antibody in the serum sample is proportional to the
Optical Density (OD) value generated by an instrument that scans a
96-well sample plate. Data was collected for a set of serial
dilutions of each serum sample. Analyzed wt/het/hom: 7/4/8
[1150] Results of this challenge: The (-/-) mice exhibited a
decreased (virtually absent) mean serum IgG2a response to the
ovalbumin challenge when compared with their (+/+) littermates.
Thus, these knockout mice exhibited an decreased ability to elicit
an OVA-specific antibody response to the T-cell dependent OVA
antigen more than likely due to a defect in Th cells. PRO258
polypeptides or agonists thereof would therefore be expected to
stimulate the immune system and would find utility in the cases
wherein this effect would be beneficial to the individual such as
in the case of leukemia, and other types of cancer, and in
immunocompromised patients, such as AIDS sufferers. Accordingly,
inhibitors or antagonists of PRO258 polypeptides would play a role
in inhibiting the immune response and would be useful candidates
for suppressing harmful immune responses, e.g. in the case of graft
rejection or graft-versus-host diseases.
[1151] Overall Summary of Immunological Observations
[1152] In summary, the ovalbumin challenge studies, hematology and
FACS results indicate that the homozygous mutant mice have an
impaired immune system, especially in view of the decreased total
white blood cell count and absolute lymphocyte and monocyte counts
as well as an inability to elicit an OVA-specific antibody
response. These studies all serve as an indicator of a negative
phenotype associated with knocking out the DNA35918-1174 gene which
encodes PRO258 polypeptides. Thus, PRO258 polypeptides and agonists
thereof would be important for a healthy immune system and would be
useful in stimulating or inducing the immune system's protective
functions.
[1153] (c) Phenotypic Analysis: CNS/Neurology
[1154] In the area of neurology, analysis focused herein on
identifying in vivo validated targets for the treatment of
neurological and psychiatric disorders including depression,
generalized anxiety disorders, attention deficit hyperactivity
disorder, obsessive compulsive disorder, schizophrenia, cognitive
disorders, hyperalgesia and sensory disorders. Neurological
disorders include the category defined as "anxiety disorders" which
include but are not limited to: mild to moderate anxiety, anxiety
disorder due to a general medical condition, anxiety disorder not
otherwise specified, generalized anxiety disorder, panic attack,
panic disorder with agoraphobia, panic disorder without
agoraphobia, posttraumatic stress disorder, social phobia, specific
phobia, substance-induced anxiety disorder, acute alcohol
withdrawal, obsessive compulsive disorder, agoraphobia, bipolar
disorder I or II, bipolar disorder not otherwise specified,
cyclothymic disorder, depressive disorder, major depressive
disorder, mood disorder, substance-induced mood disorder. In
addition, anxiety disorders may apply to personality disorders
including but not limited to the following types: paranoid,
antisocial, avoidant behavior, borderline personality disorders,
dependent, histronic, narcissistic, obsessive-compulsive, schizoid,
and schizotypal.
[1155] Procedure:
[1156] Behavioral screens were performed on a cohort of 4 wild
type, 4 heterozygous and 8 homozygous mutant mice. All behavioral
tests were done between 12 and 16 weeks of age unless reduced
viability necessitates earlier testing. These tests included open
field to measure anxiety, activity levels and exploration.
[1157] Circadian Test Description:
[1158] Female mice are individually housed at 4 pm on the first day
of testing in 48.2 cm.times.26.5 cm home cages and administered
food and water ad libitum. Animals are exposed to a 12-hour
light/dark cycle with lights turning on at 7 am and turning off at
7 pm. The system software records the number of beam interruptions
caused by the animal's movements, with beam breaks automatically
divided into ambulations. Activity is recorded in 60, one-hour
intervals during the three-day test. Data generated are displayed
by median activity levels recorded for each hour (circadian rhythm)
and median total activity during each light/dark cycle (locomotor
activity) over the three-day testing period.
[1159] Results:
[1160] The (-/-) mice exhibited decreased ambulatory counts during
the 1-hour habituation period of home-cage activity testing when
compared with their gender-matched (+/+) littermates and the
historical mean. These results are indicative of a suppression of
circadian rhythm especially since there was a marked decrease in
the dark period relative to the littermate controls.
[1161] Analyzed wt/het/hom: 6/4/12
[1162] Inverted Screen Testing:
[1163] Behavioral screens were performed on a cohort of 4 wild
type, 4 heterozygous and 8 homozygous mutant mice. All behavioral
tests were done between 12 and 16 weeks of age unless reduced
viability necessitates earlier testing. These tests included open
field to measure anxiety, activity levels and exploration.
[1164] Inverted Screen Test Data:
[1165] The Inverted Screen is used to measure motor
strength/coordination. Untrained mice were placed individually on
top of a square (7.5 cm.times.7.5 cm) wire screen which was mounted
horizontally on a metal rod. The rod was then rotated 180 degrees
so that the mice were on the bottom of the screens. The following
behavioral responses were recorded over a 1 min testing session:
fell off, did not climb, and climbed up.
TABLE-US-00056 Ratio Ratio Genotype Fell Down % Climbed up % +/+ (n
= 4) 0/4 0 0/4 0 +/- (n = 4) 0/4 0 4/4 100* -/- (n = 8) 1/8 13 7/8
88* *coding indicates a notable difference.
A motor strength deficit is apparent when there is a 50% point
difference between (-/-) or (+/-) mice and (+/+) mice for the fell
down response. 0/8 or 1/8 (-/-) or (+/-) mice not climbing
indicates impaired motor coordination. 7/8 or 8/8(-/-) or (+/-)
mice climbing up indicates enhanced motor coordination.
[1166] Results:
[1167] The Inverted Screen Test is designed to measure basic
sensory & motor observations: Enhanced motor coordination was
observed in the (-/-) mice during inverted screen testing when 7/8
(-/-) mice climbed up the screen, whereas 0/4 (+/+) mice climbed
up.
Analyzed wt/het/hom: 6/4/12
[1168] (d) Phenotypic Analysis: Cardiology
[1169] In the area of cardiovascular biology, targets were
identified herein for the treatment of hypertension,
atherosclerosis, heart failure, stroke, various coronary artery
diseases, dyslipidemias such as high cholesterol
(hypercholesterolemia) and elevated serum triglycerides
(hypertriglyceridemia), diabetes and/or obesity. The phenotypic
tests included the measurement of serum cholesterol and
triglycerides.
[1170] Blood Lipids
[1171] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. High cholesterol levels and
increased triglyceride blood levels are recognized risk factors in
the development of cardiovascular disease and/or diabetes.
Measuring blood lipids facilitates the finding of biological
switches that regulate blood lipid levels Inhibition of factors
which elevate blood lipid levels may be useful for reducing the
risk for cardiovascular disease. In these blood chemistry tests,
cholesterol measurements were recorded using the COBAS Integra 400
(mfr: Roche). Analyzed wt/het/hom: 4/4/8
[1172] Results:
[1173] (1) The (-/-) mice exhibited an increased mean serum
cholesterol level when compared with their gender-matched (+/+)
littermates and the historical mean. The mean serum cholesterol
values were significantly above the normal range. (2) The (-/-)
mice also exhibited a significantly increased mean serum glucose
level. During the glucose tolerance test, the (-/-) mice exhibited
an increased mean fasting serum glucose level when compared with
their gender-matched (+/+) littermates and the historical mean.
[1174] In summary, these knockout mutant mice exhibited a negative
phenotype with regards to lipid metabolism. Thus, mutant mice
deficient in the PRO258 gene can serve as a model for treatment of
cardiovascular disease. PRO258 polypeptides, agonists thereof or
the encoding gene for PRO258 would be useful in regulating blood
lipids and in particular in maintaining normal cholesterol
metabolism. Thus, such agents would be useful in the treatment of
such cardiovascular diseases associated with dyslipidemia as:
hypertension, atherosclerosis, heart failure, stroke, various
coronary artery diseases, and/or obesity.
[1175] In concurrence, knockout mice exhibited the phenotypic
pattern of an impaired glucose homeostasis with elevated levels of
fasting serum glucose indicative of diabetes or a pre-diabetic
condition. Based on these results, PRO258 (or agonists thereof) or
its encoding gene would be useful in the treatment of an impaired
glucose metabolism and/or diabetes.
[1176] (e) Bone Metabolism & Body Diagnostics: Bone Metabolism:
Radiology Phenotypic Analysis
[1177] In the area of bone metabolism, targets were identified
herein for the treatment of arthritis, osteoporosis, osteopenia and
osteopetrosis as well as identifying targets that promote bone
healing. Tests included:
[1178] DEXA for measurement of bone mineral density on femur and
vertebra
[1179] MicroCT for very high resolution and very high sensitivity
measurements of bone mineral density for both trabecular and
cortical bone.
[1180] Dexa Analysis--Test Description:
[1181] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. Dual Energy X-ray
Absorptiometry (DEXA) has been used successfully to identify
changes in bone. Anesthetized animals were examined and bone
mineral content (BMC), BMC/LBM ratios, volumetric bone mineral
density (vBMD), total body BMD, femur BMD and vertebra BMD were
measured.
[1182] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PIXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI)
[i.e., whole body, vertebrae, and both femurs].
[1183] Bone MicroCT Analysis:
[1184] Procedure: MicroCT was also used to get very sensitive
measurements of BMD. One vertebra and 1 femur were taken from a
cohort of 4 wild type and 8 homozygous mice. Measurements were
taken of lumbar 5 vertebra trabecular bone volume, trabecular
thickness, connectivity density and midshaft femur total bone area
and cortical thickness. The .mu.CT40 scans provided detailed
information on bone mass and architecture. Multiple bones were
placed into sample holders and scanned automatically. Instrument
software was used to select regions of interest for analysis.
Trabecular bone parameters were analyzed in the fifth lumbar
vertebrae (LV5) at 16 micrometer resolution and cortical bone
parameters were analyzed in the femur midshaft at a resolution of
20 micrometers.
[1185] Results:
[1186] Micro-CT: The (-/-) mice exhibited decreased bone
measurements in the 5.sup.th lumbar vertebrae (decreased mean
vertebral trabecular bone volume, number, thickness, and
connectivity density) when compared with their gender-matched (+/+)
littermates and the historical means. The midshaft femur showed a
decreased total area. In addition, the mutant (-/-) mice exhibited
a decreased heart rate which was two (2) standard deviations below
the mean.
[1187] Summary
[1188] These results demonstrate that knockout mutant mice exhibit
abnormal bone metabolism with significant bone loss similar to
osteoporosis characterized by decrease in bone mass with decreased
density and possibly fragility leading to bone fractures. Thus, it
appears that PRO258 polypeptides or agonists thereof play a role in
maintaining bone homeostasis. In addition, PRO258 or its encoding
gene would be useful in bone healing or useful for the treatment of
arthritis or osteoporosis; whereas antagonists to PRO258 or its
encoding gene would lead to abnormal or pathological bone disorders
including inflammatory diseases associated with abnormal bone
metabolism such as arthritis, osteoporosis, and osteopenia.
[1189] 35.8. Generation and Analysis of Mice Comprising
DNA39969-1185 (UNQ250) Gene Disruptions
[1190] In these knockout experiments, the gene encoding PRO287
polypeptides (designated as DNA39969-1185) (UNQ250) was disrupted.
The gene specific information for these studies is as follows: the
mutated mouse gene corresponds to nucleotide reference:
NM.sub.--029620 or ACCESSION: NM.sub.--029620 NID: gi 22095010 ref
NM.sub.--029620.1 Mus musculus procollagen C-endopeptidase enhancer
2 (Pcolce2); protein reference: NP.sub.--083896 or ACCESSION:
NP.sub.--083896 NID: gi 22095011 ref NP.sub.--083896.1
(NM.sub.--029620) procollagen C-endopeptidase enhancer 2 [Mus
musculus]; the human gene sequence reference: NM.sub.--013363 or
ACCESSION: NM.sub.--013363 NID: gi 16904386 ref NM.sub.--013363.2
Homo sapiens procollagen C-endopeptidase enhancer 2 (PCOLCE2); the
human protein sequence corresponds to reference: NP.sub.--037495 or
ACCESSION: NP.sub.--037495 NID: gi 7019483 ref NP.sub.--037495.1
(NM.sub.--013363) procollagen C-endopeptidase enhancer 2 [Homo
sapiens].
[1191] The mouse gene of interest is Pcolce2 (procollagen
C-endopeptidase enhancer 2), ortholog of human PCOLCE2. Aliases
include PCPE2.
[1192] PCOLCE2 is a secreted glycoprotein that binds with the C
terminus of type I procollagen, enhancing cleavage of procollagen
by C-proteinases such as bone morphogenic protein-1. Trabecular
meshwork, lungs, heart, brain, liver, skeletal muscle, kidney,
pancreas, and placenta express PCOLCE2 mRNA; however, PCOLCE2
protein is detected mainly in trabecular network. PCOLCE2 is likely
to play a role in cartilage formation in different tissues during
development (Steiglitz et al., J Biol Chem, 277(51):49820-30
(2002); Xu et al., Genomics, 66(3):264-73 (2000)).
Genetics Information:
TABLE-US-00057 [1193] wt het hom Total Observed 25 39 18 82
Expected 20.5 41 20.5 82 Chi-Sq. = 1.39 Significance = 0.49901
(hom/n) = 0.22 Avg. Litter Size = 8
Mutation Type: Retroviral Insertion (OST)
[1194] Retroviral insertion occurred in the intron between coding
exons 1 and 2 (NCBI accession NM.sub.--029620.1). Wild-type
expression of the target gene was detected in brain, spinal cord,
eye, thymus, spleen, and lung among the 13 adult tissue samples
tested by RT-PCR. RT-PCR analysis revealed that the transcript was
absent in the (-/-) mouse analyzed (M-201).
[1195] 35.8.1. Phenotypic Analysis (for Disrupted Gene:
DNA39969-1185 (UNQ250)
[1196] (a) Overall Phenotypic Summary:
[1197] Mutation of the gene encoding the ortholog of human
procollagen C-endopeptidase enhancer 2 (PCOLCE2) resulted in growth
retardation and decreased bone measurements in (-/-) mice.
Increased uric acid levels and decreased serum phosphate levels
were also observed in the mutant (-/-) mice. Transcript was absent
by RT-PCR.
[1198] (b) Bone Metabolism & Body Diagnostics
[1199] (1) Tissue Mass & Lean Body Mass Measurements--Dexa
[1200] Dexa Analysis--Test Description:
[1201] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. Dual Energy X-ray
Absorptiometry (DEXA) has been used successfully to identify
changes in total tissue mass (TTM).
[1202] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PIXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI,
i.e., whole body, vertebrae, and both femurs).
[1203] Body Measurements (Body Length & Weight):
[1204] Body Measurements: A measurement of body length and weight
was performed at approximately 16 weeks of age.
[1205] Results:
[1206] The (-/-) mice exhibited decreased mean body weight and mean
body length when compared with their gender-matched (+/+)
littermates and the historical means. Analyzed wt/het/hom:
37/54/28
[1207] (2) Bone Metabolism: Radiology Phenotypic Analysis
[1208] In the area of bone metabolism, targets were identified
herein for the treatment of arthritis, osteoporosis, osteopenia and
osteopetrosis as well as identifying targets that promote bone
healing. Tests included:
[1209] DEXA for measurement of bone mineral density on femur and
vertebra
[1210] MicroCT for very high resolution and very high sensitivity
measurements of bone mineral density for both trabecular and
cortical bone.
[1211] Dexa Analysis--Test Description:
[1212] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. Dual Energy X-ray
Absorptiometry (DEXA) has been used successfully to identify
changes in bone. Anesthetized animals were examined and bone
mineral content (BMC), BMC/LBM ratios, volumetric bone mineral
density (vBMD), total body BMD, femur BMD and vertebra BMD were
measured.
[1213] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PIXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI)
[i.e., whole body, vertebrae, and both femurs].
[1214] Bone MicroCT Analysis:
[1215] Procedure: MicroCT was also used to get very sensitive
measurements of BMD. One vertebra and 1 femur were taken from a
cohort of 4 wild type and 8 homozygous mice. Measurements were
taken of lumbar 5 vertebra trabecular bone volume, trabecular
thickness, connectivity density and midshaft femur total bone area
and cortical thickness. The .mu.CT40 scans provided detailed
information on bone mass and architecture. Multiple bones were
placed into sample holders and scanned automatically. Instrument
software was used to select regions of interest for analysis.
Trabecular bone parameters were analyzed in the fifth lumbar
vertebrae (LV5) at 16 micrometer resolution and cortical bone
parameters were analyzed in the femur midshaft at a resolution of
20 micrometers.
[1216] Results:
[1217] DEXA: The (-/-) mice exhibited decreased mean total tissue
mass and lean body mass when compared with their gender-matched
(+/+) littermates and the historical means. These mutant mice also
exhibited decreased mean bone mineral content and bone mineral
density.
[1218] Micro-CT: The (-/-) mice exhibited decreased mean vertebral
trabecular bone volume, number, thickness, and connectivity density
and decreased mean femoral mid-shaft cross-sectional area when
compared with their gender-matched (+/+) littermates and the
historical means. Analyzed wt/het/hom: 4/4/8
[1219] In addition, the (-/-) mice had a significant decrease in
body fat.
[1220] Summary
[1221] These results demonstrate that knockout mutant mice exhibit
abnormal bone metabolism with significant bone loss similar to
osteoporosis characterized by decrease in bone mass with decreased
density and possibly fragility leading to bone fractures. This in
conjunction with blood chemistry analysis (see below) wherein
increased uric acid levels and decreased serum phosphate levels
were observed. Thus, it appears that PRO287 or agonists thereof
would be useful in maintaining bone homeostasis. In addition,
PRO287 or its encoding gene would be important in bone healing or
for the treatment of arthritis or osteoporosis; whereas antagonists
to PRO287 would lead to abnormal or pathological bone disorders
including inflammatory diseases associated with abnormal bone
metabolism including arthritis, osteoporosis, and osteopenia. In
addition, the (-/-) mice analyzed by DEXA exhibited notably
decreased total tissue mass and lean body mass wand decreased body
fat when compared with their (+/+) littermates, suggestive of
growth retardation in these mutants. This in conjunction with the
observations of decreased body weight and length suggest a tissue
wasting condition such as cachexia or other growth disorder. Thus,
PRO287 polypeptides or agonists thereof would be useful in the
treatment or prevention of growth disorders such as cachexia and/or
other tissue wasting diseases.
[1222] (c) Blood Chemistry--Uric Acid and Serum Phosphate
Levels
[1223] Blood chemistry analysis was performed using the COBAS
Integra 400 (mfr: Roche) in its clinical settings for running blood
chemistry tests on mice.
[1224] Results: Mutant (-/-) mice exhibited increased uric acid
levels as well as decreased serum phosphate levels compared to
their control wild-type littermates.
[1225] 35.9. Generation and Analysis of Mice Comprising
DNA40587-1231 (UNQ289) Gene Disruptions
[1226] In these knockout experiments, the gene encoding PRO328
polypeptides (designated as DNA40587-1231) (UNQ289) was disrupted.
The gene specific information for these studies is as follows: the
mutated mouse gene corresponds to nucleotide reference:
NM.sub.--023734 or ACCESSION: NM.sub.--023734 NID: 12963802 Mus
musculus Mus musculus RIKEN cDNA 1200009H11 gene (1200009H11Rik);
protein reference: Q9ET66 or ACCESSION: Q9ET66 NID: Mus musculus
(Mouse). CYSTEINE-RICH PROTEASE INHIBITOR. MOUSESPTRNRDB; the human
gene sequence reference: NM.sub.--153370 or Homo sapiens protease
inhibitor 16 (PI16); the human protein sequence corresponds to
reference: Q8NBK0 or ACCESSION: Q8NBK0 NID: Homo sapiens (Human).
Hypothetical protein PLACE1010482.
[1227] The mouse gene of interest encodes a hypothetical secreted
protein, which is the ortholog of human PI16 (protease inhibitor
16).
[1228] PI16 contains a signal peptide, an SCP-like extracellular
protein (SCP) domain, and several internal repeats. SCP domains are
found in extracellular proteins from many different species.
Examples include insect venom allergens, mammalian testis-specific
proteins, and plant pathogenesis-related proteins (Pfam accession
PF00188). PI16 is a homolog of protease inhibitor 15 (PI15), which
also contains an SCP domain and functions as a secreted serine
protease (trypsin) inhibitor (Yamakawa et al., Biochim Biophys
Acta, 1395(2):202-8 (1998)). The function of PI16 has not been
demonstrated experimentally.
Genetics Information:
TABLE-US-00058 [1229] wt het hom Total Observed 16 45 22 83
Expected 20.75 41.5 20.75 83 Chi-Sq. = 1.46 Significance = 0.48243
(hom/n) = 0.27 Avg. Litter Size = 8
Mutation Type: Retroviral Insertion (OST)
[1230] Retroviral insertion occurred in the intron between coding
exons 2 and 3 (NCBI accession NM.sub.--023734.2). Wild-type
expression of the target gene was detected in embryonic stem (ES)
cells and in all 13 adult tissue samples tested by RT-PCR. RT-PCR
analysis revealed that the transcript was absent in the (-/-) mouse
analyzed (M-97).
[1231] 35.9.1. Phenotypic Analysis (for Disrupted Gene:
DNA40587-1231 (UNQ289)
[1232] (a) Overall Phenotypic Summary:
[1233] Mutation of the gene encoding the ortholog of human protease
inhibitor 16 (PI16) resulted in a immunological phenotype in (-/-)
mice. An enhanced glucose tolerance was also observed during the
glucose tolerance testing for the (-/-) mice. Changes in lumbar 5
vertebrae were also noted (decreased measurements compared to
littermate controls). Transcript was absent by RT-PCR.
[1234] (b) Immunology Phenotypic Analysis
[1235] Immune related and inflammatory diseases are the
manifestation or consequence of fairly complex, often multiple
interconnected biological pathways which in normal physiology are
critical to respond to insult or injury, initiate repair from
insult or injury, and mount innate and acquired defense against
foreign organisms. Disease or pathology occurs when these normal
physiological pathways cause additional insult or injury either as
directly related to the intensity of the response, as a consequence
of abnormal regulation or excessive stimulation, as a reaction to
self, or as a combination of these.
[1236] Though the genesis of these diseases often involves
multistep pathways and often multiple different biological
systems/pathways, intervention at critical points in one or more of
these pathways can have an ameliorative or therapeutic effect.
Therapeutic intervention can occur by either antagonism of a
detrimental process/pathway or stimulation of a beneficial
process/pathway.
[1237] T lymphocytes (T cells) are an important component of a
mammalian immune response. T cells recognize antigens which are
associated with a self-molecule encoded by genes within the major
histocompatibility complex (MHC). The antigen may be displayed
together with MHC molecules on the surface of antigen presenting
cells, virus infected cells, cancer cells, grafts, etc. The T cell
system eliminates these altered cells which pose a health threat to
the host mammal. T cells include helper T cells and cytotoxic T
cells. Helper T cells proliferate extensively following recognition
of an antigen-MHC complex on an antigen presenting cell. Helper T
cells also secrete a variety of cytokines, i.e., lymphokines, which
play a central role in the activation of B cells, cytotoxic T cells
and a variety of other cells which participate in the immune
response.
[1238] In many immune responses, inflammatory cells infiltrate the
site of injury or infection. The migrating cells may be
neutrophilic, eosinophilic, monocytic or lymphocytic as can be
determined by histological examination of the affected tissues.
Current Protocols in Immunology, ed. John E. Coligan, 1994, John
Wiley & Sons, Inc.
[1239] Many immune related diseases are known and have been
extensively studied. Such diseases include immune-mediated
inflammatory diseases (such as rheumatoid arthritis, immune
mediated renal disease, hepatobiliary diseases, inflammatory bowel
disease (IBD), psoriasis, and asthma), non-immune-mediated
inflammatory diseases, infectious diseases, immunodeficiency
diseases, neoplasia, and graft rejection, etc. In the area of
immunology, targets were identified for the treatment of
inflammation and inflammatory disorders.
[1240] In the area of immunology, targets have been identified
herein for the treatment of inflammation and inflammatory
disorders. Immune related diseases, in one instance, could be
treated by suppressing the immune response. Using neutralizing
antibodies that inhibit molecules having immune stimulatory
activity would be beneficial in the treatment of immune-mediated
and inflammatory diseases. Molecules which inhibit the immune
response can be utilized (proteins directly or via the use of
antibody agonists) to inhibit the immune response and thus
ameliorate immune related disease.
[1241] The following tests were performed:
[1242] Hematology Analysis:
[1243] Test Description: Blood tests are carried out by Abbott's
Cell-Dyn 3500R, an automated hematology analyzer. Some of its
features include a five-part WBC differential. `Patient` reports
can cover over 22 parameters in all.
[1244] Results:
[1245] Hematology:
[1246] The (-/-) mice exhibited an increased mean red cell
distribution width when compared with their (+/+) littermates and
the historical mean.
Analyzed wt/het/hom: 9/4/14
[1247] Flourescence-Activated Cell-Sorting (FACS) Analysis/Tissue
Specific FACS
[1248] Procedure:
[1249] FACS analysis of immune cell composition from peripheral
blood was performed including CD4, CD8 and T cell receptor to
evaluate T lymphocytes, CD19 for B lymphocytes, CD45 as a leukocyte
marker and pan NK for natural killer cells. The FACS analysis was
carried out on 2 wild type and 6 homozygous mice and included cells
derived from thymus, spleen, bone marrow and lymph node.
[1250] In these studies, analyzed cells were isolated from thymus,
peripheral blood, spleen, bone marrow and lymph nodes. Flow
cytometry was designed to determine the relative proportions of CD4
and CD8 positive T cells, B cells, NK cells and monocytes in the
mononuclear cell population. A Becton-Dickinson FACSCalibur 3-laser
FACS machine was used to assess immune status. For Phenotypic
Assays and Screening, this machine records CD4+/CD8-, CD8+/CD4-,
NK, B cell and monocyte numbers in addition to the CD4+/CD8+ ratio.
The mononuclear cell profile was derived by staining a single
sample of lysed peripheral blood from each mouse with a panel of
six lineage-specific antibodies: CD45 PerCP, anti-TCRb APC, CD4 PE,
CD8 FITC, pan-NK PE, and CD19 FITC. The two FITC and PE labeled
antibodies stain mutually exclusive cell types. The samples were
analyzed using a Becton Dickinson FACSCalibur flow cytometer with
CellQuest software.
[1251] Results:
[1252] Tissue Specific FACS: The (-/-) mice exhibited a decreased
mean percentage of CD21Hi CD23Med cells in the spleen and lymph
nodes. Increased mean percentages of CD25+ cells in both spleen and
lymph nodes were also noted in the (-/-) mice.
Analyzed wt/het/hom: 9/4/14
[1253] In summary, knocking out DNA40587-1231 (the gene which
encodes PRO328 polypeptides) resulted in a a decrease in a subset
of B cells--marginal zone B cells--that contain a pool of memory
cells and participate in fast immune responses. Thus, antagonists
or inhibitors of PRO328 polypeptides would be expected to
demonstrate the same phenotype. PRO328 polypeptides would be useful
in the development or production of marginal zone B cells useful
for participating in fast immune responses.
[1254] (c) Phenotypic Analysis: Metabolism-Blood Chemistry/Glucose
Tolerance
[1255] In the area of metabolism, targets may be identified for the
treatment of diabetes. Blood chemistry phenotypic analysis includes
blood glucose measurements. The COBAS Integra 400 (mfr: Roche) was
used for running blood chemistry tests on the mice. In the area of
metabolism, targets may be identified for the treatment of
diabetes. Blood chemistry phenotypic analysis includes glucose
tolerance tests to measure insulin sensitivity and changes in
glucose metabolism. Abnormal glucose tolerance test results may
indicate but may not be limited to the following disorders or
conditions: Diabetes Type 1 and Type 2, Syndrome X, various
cardiovascular diseases and/or obesity.
[1256] Procedure: A cohort of 2 wild type and 4 homozygous mice
were used in this assay. The glucose tolerance test is the standard
for defining impaired glucose homeostasis in mammals. Glucose
tolerance tests were performed using a Lifescan glucometer. Animals
were injected IP at 2 g/kg with D-glucose delivered as a 20%
solution and blood glucose levels were measured at 0, 30, 60 and 90
minutes after injection. Analyzed wt/het/hom: 4/4/8
[1257] Results:
[1258] The (-/-) mice exhibited an enhanced glucose tolerance when
compared with their gender-matched (+/+) littermates and the
historical means. Thus, knockout mice exhibited the opposite
phenotypic pattern of an impaired glucose homeostasis, and as such
antagonists to PRO328 or its encoding gene would be useful in the
treatment of impaired glucose homeostasis and diseases associated
with abnormal glucose metabolism.
[1259] (d) Bone Metabolism & Body Diagnostics: Bone Metabolism:
Radiology Phenotypic Analysis
[1260] In the area of bone metabolism, targets were identified
herein for the treatment of arthritis, osteoporosis, osteopenia and
osteopetrosis as well as identifying targets that promote bone
healing. Tests included:
[1261] DEXA for measurement of bone mineral density on femur and
vertebra
[1262] MicroCT for very high resolution and very high sensitivity
measurements of bone mineral density for both trabecular and
cortical bone.
[1263] Dexa Analysis--Test Description:
[1264] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. Dual Energy X-ray
Absorptiometry (DEXA) has been used successfully to identify
changes in bone. Anesthetized animals were examined and bone
mineral content (BMC), BMC/LBM ratios, volumetric bone mineral
density (vBMD), total body BMD, femur BMD and vertebra BMD were
measured.
[1265] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PIXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI)
[i.e., whole body, vertebrae, and both femurs].
[1266] Bone MicroCT Analysis:
[1267] Procedure: MicroCT was also used to get very sensitive
measurements of BMD. One vertebra and 1 femur were taken from a
cohort of 4 wild type and 8 homozygous mice. Measurements were
taken of lumbar 5 vertebra trabecular bone volume, trabecular
thickness, connectivity density and midshaft femur total bone area
and cortical thickness. The .mu.CT40 scans provided detailed
information on bone mass and architecture. Multiple bones were
placed into sample holders and scanned automatically. Instrument
software was used to select regions of interest for analysis.
Trabecular bone parameters were analyzed in the fifth lumbar
vertebrae (LV5) at 16 micrometer resolution and cortical bone
parameters were analyzed in the femur midshaft at a resolution of
20 micrometers.
[1268] Results:
[1269] Micro-CT: The (-/-) mice exhibited decreased mean vertebral
trabecular bone volume, number, thickness, and connectivity density
and decreased mean femoral mid-shaft cross-sectional area when
compared with their gender-matched (+/+) littermates and the
historical means. Analyzed wt/het/hom: 4/4/8
[1270] These results demonstrate that knockout mutant mice exhibit
abnormal bone metabolism with significant bone loss similar to
osteoporosis characterized by decrease in bone mass with decreased
density and possibly fragility leading to bone fractures. Thus, it
appears that PRO328 or agonists thereof would be useful in
maintaining bone homeostasis. In addition, PRO328 or its encoding
gene would be important in bone healing or for the treatment of
arthritis or osteoporosis; whereas antagonists to PRO328 would lead
to abnormal or pathological bone disorders including inflammatory
diseases associated with abnormal bone metabolism including
arthritis, osteoporosis, and osteopenia.
[1271] 35.10. Generation and Analysis of Mice Comprising
DNA40592-1242 (UNQ303) Gene Disruptions
[1272] In these knockout experiments, the gene encoding PRO344
polypeptides (designated as DNA40592-1242) (UNQ303) was disrupted.
The gene specific information for these studies is as follows: the
mutated mouse gene corresponds to nucleotide reference: BC023068 or
Mus musculus C1q and tumor necrosis factor related protein 5;
protein reference: Q8R002 or ACCESSION: Q8R002NID: Mus musculus
(Mouse). Similar to DKFZP586B0621 protein (Hypothetical protein);
the human gene sequence reference: NM.sub.--015645 or Homo sapiens
C1q and tumor necrosis factor related protein 5 (C1QTNF5); the
human protein sequence corresponds to reference: Q9BXJ0 or
ACCESSION: Q9BXJ0 NID: Homo sapiens (Human). Complement C1q tumor
necrosis factor-related protein 5 precursor.
[1273] The disrupted mouse gene is C1qtnf5 (C1q and tumor necrosis
factor related protein 5), which is the ortholog of human C1QTNF5.
Aliases include CTRP5 and complement-c1q tumor necrosis
factor-related protein 5.
[1274] C1QTNF5 is a hypothetical secreted protein, containing a
signal peptide, a collagen triple helix repeat (Pfam accession
PF01391), and a complement component C1q domain (SMART accession
SM00110). C1q domains are globular structures found in many
collagens and in the C1 enzyme complex that activates the serum
complement system. The fold of this domain is similar to that of
tumor necrosis factor. The molecular function and biological role
of C1QTNF5 is not known.
Genetics Information:
TABLE-US-00059 [1275] wt het hom Total Observed 19 31 16 66
Expected 16.5 33 16.5 66 Chi-Sq. = 0.52 Significance = 0.77292
(hom/n) = 0.24 Avg. Litter Size = 7
Mutation Type: Retroviral Insertion (OST)
[1276] Retroviral insertion occurred between coding exons 1 and 2
(Accession: NM.sub.--145613.2). Wild-type expression of the target
gene was detected in brain, spinal cord, eye, lung, kidney,
skeletal muscle, and heart among the 13 adult tissue samples tested
by RT-PCR. The larger bands are unspliced gene-specific
transcripts. PCR analysis revealed that the transcript was absent
in the (-/-) mouse analyzed (M-131).
[1277] 35.10.1. Phenotypic Analysis (for Disrupted Gene:
DNA40592-1242 (UNQ303)
[1278] (a) Overall Phenotypic Summary:
[1279] Mutation of the gene encoding the ortholog of human C1q and
tumor necrosis factor related protein 5 (C1QTNF5) resulted in
retinal degeneration in (-/-) mice. In addition, the (-/-) mice
exhibited abnormal bone measurements with decreased measurements in
lumbar 5. The knockout mice also showed a significant decrease in
total body fat and showed increased uric acid levels. Transcript
was absent by RT-PCR.
[1280] (b) Cardiovascular Phenotypic Analysis:
[1281] In the area of cardiovascular biology, phenotypic testing
was performed to identify potential targets for the treatment of
cardiovascular, endothelial or angiogenic disorders. One such
phenotypic test included optic fundus photography and angiography
to determine the retinal arteriovenous ratio (A/V ratio) in order
to flag various eye abnormalities. An abnormal A/V ratio signals
such systemic diseases or disorders that may be related to the
vascular disease of hypertension (and any disease that causes
hypertension, e.g. atherosclerosis), diabetes or other ocular
diseases corresponding to ophthalmological disorders. Such eye
abnormalities may include but are not limited to the following:
retinal abnormality is retinal dysplasia, various retinopathies,
restenosis, retinal artery obstruction or occlusion; retinal
degeneration causing secondary atrophy of the retinal vasculature,
retinitis pigmentosa, macular dystrophies, Stargardt's disease,
congenital stationary night blindness, choroideremia, gyrate
atrophy, Leber's congenital amaurosis, retinoschisis disorders,
Wagner's syndrome, Usher syndromes, Zellweger syndrome,
Saldino-Mainzer syndrome, Senior-Loken syndrome, Bardet-Biedl
syndrome, Alport's syndrome, Alstom's syndrome, Cockayne's
syndrome, dysplaisa spondyloepiphysaria congentia, Flynn-Aird
syndrome, Friedreich ataxia, Hallgren syndrome, Marshall syndrome,
Albers-Schnoberg disease, Refsum's disease, Kearns-Sayre syndrome,
Waardenburg's syndrome, Alagile syndrome, myotonic dystrophy,
olivopontocerebellar atrophy, Pierre-Marie dunsdrome, Stickler
syndrome, carotinemeia, cystinosis, Wolfram syndrome,
Bassen-Kornzweig syndrome, abetalipoproteinemia, incontinentia
pigmenti, Batten's disease, mucopolysaccharidoses, homocystinuria,
or mannosidosis.
[1282] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. Optic fundus photography was
performed on conscious animals using a Kowa Genesis small animal
fundus camera modified according to Hawes and coauthors (Hawes et
al., 1999 Molecular Vision 1999; 5:22). Intra-peritoneal injection
of fluorescein permitted the acquisition of direct light fundus
images and fluorescent angiograms for each examination. In addition
to direct ophthalmological changes, this test can detect retinal
changes associated with systemic diseases such as diabetes and
atherosclerosis or other retinal abnormalities. Pictures were
provided of the optic fundus under normal light. The angiographic
pictures allowed examination of the arteries and veins of the eye.
In addition an artery to vein (A/V) ratio was determined for the
eye.
[1283] Ophthalmology analysis was performed on generated F2 wild
type, heterozygous, and homozygous mutant progeny using the
protocol described above. Specifically, the A/V ratio was measured
and calculated according to the fundus images with Kowa COMIT+
software. This test takes color photographs through a dilated
pupil: the images help in detecting and classifying many diseases.
The artery to vein ratio (A/V) is the ratio of the artery diameter
to the vein diameter (measured before the bifurcation of the
vessels). Many diseases will influence the ratio, i.e., diabetes,
cardiovascular disorders, papilledema, optic atrophy or other eye
abnormalities such as retinal degeneration (known as retinitis
pigmentosa) or retinal dysplasia, vision problems or blindness.
Thus, phenotypic observations which result in an increased
artery-to-vein ratio in homozygous (-/-) and heterozygous (+/-)
mutant progeny compared to wild-type (+/+) littermates would be
indicative of such pathological conditions.
[1284] Results:
[1285] Fundus: Of the 7 non-albino (-/-) mice analyzed, 6 (F-134,
F-155, F-158, M-164, M-171, and F-183) exhibited multiple white
spots covering the entire retina with attenuated retinal arteries,
suggesting retinal degeneration in the mutants similar to flecked
retinal disease in humans. Analyzed wt/het/hom: 4/4/8
[1286] Pathological microscopic observations showed signs of
retinal degeneration in the 4 (-/-) mice analyzed (M-173, M-188,
F-197, and F-199). Histological changes were noted wherein the
retina showed increased apoptotic cells in the outer nuclear layer.
Gene expression analysis could not be performed for this project.
In summary, in this study, optic fundus photography showed that
(-/-) mice exhibited signs of severe retinal degeneration, namely
notably attenuated retinal vessels when compared with their (+/+)
littermates. Angiograms demonstrated that the mutant (-/-) mice
showed attenuated retinal vessels with micro aneurysms. Likewise,
microscopic observations showed bilateral retinal degeneration in
the mutant (-/-) mice. In summary, by knocking out the gene
identified as DNA40592-1242 encoding PRO344 polypeptides,
homozygous mutant progeny exhibit phenotypes which are associated
with retinal degeneration. Such detected retinal changes are most
commonly associated with cardiovascular systemic diseases or
disorders that may be related to the vascular disease of
hypertension (and any disease that causes hypertension, e.g.
atherosclerosis), diabetes or other ocular diseases corresponding
to ophthalmological disorders such as retinal degeneration. Thus,
antagonists of PRO344 encoding genes would lead to similar
pathological retinal changes, whereas agonists would be useful as
therapeutic agents in the treatment of hypertension,
atherosclerosis or other opthamological disorders including retinal
degeneration and diseases associated with this condition (as
indicated above).
[1287] (c) Bone Metabolism & Body Diagnostics: Bone Metabolism:
Radiology Phenotypic Analysis
[1288] In the area of bone metabolism, targets were identified
herein for the treatment of arthritis, osteoporosis, osteopenia and
osteopetrosis as well as identifying targets that promote bone
healing. Tests included:
[1289] DEXA for measurement of bone mineral density on femur and
vertebra
[1290] MicroCT for very high resolution and very high sensitivity
measurements of bone mineral density for both trabecular and
cortical bone.
[1291] Dexa Analysis--Test Description:
[1292] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. Dual Energy X-ray
Absorptiometry (DEXA) has been used successfully to identify
changes in bone. Anesthetized animals were examined and bone
mineral content (BMC), BMC/LBM ratios, volumetric bone mineral
density (vBMD), total body BMD, femur BMD and vertebra BMD were
measured.
[1293] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PIXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI)
[i.e., whole body, vertebrae, and both femurs].
[1294] Bone MicroCT Analysis:
[1295] Procedure: MicroCT was also used to get very sensitive
measurements of BMD. One vertebra and 1 femur were taken from a
cohort of 4 wild type and 8 homozygous mice. Measurements were
taken of lumbar 5 vertebra trabecular bone volume, trabecular
thickness, connectivity density and midshaft femur total bone area
and cortical thickness. The .mu.CT40 scans provided detailed
information on bone mass and architecture. Multiple bones were
placed into sample holders and scanned automatically. Instrument
software was used to select regions of interest for analysis.
Trabecular bone parameters were analyzed in the fifth lumbar
vertebrae (LV5) at 16 micrometer resolution and cortical bone
parameters were analyzed in the femur midshaft at a resolution of
20 micrometers.
[1296] Results:
[1297] DEXA: The (-/-) mutant mice showed a significant decrease in
total body fat content compared to their littermate controls. In
addition, uric acid levels were elevated in the (-/-) mutant mice
(as observed in blood chemistry analysis).
[1298] Micro-CT: The (-/-) mice exhibited decreased mean vertebral
trabecular bone volume, number, thickness, and connectivity density
and decreased mean femoral mid-shaft cross-sectional area when
compared with their gender-matched (+/+) littermates and the
historical means. Analyzed wt/het/hom: 4/4/8
[1299] These results demonstrate that knockout mutant mice exhibit
abnormal bone metabolism with significant bone loss similar to
osteoporosis characterized by decrease in bone mass with decreased
density and possibly fragility leading to bone fractures. Thus, it
appears that PRO344 or agonists thereof would be useful in
maintaining bone homeostasis. In addition, PRO344 or its encoding
gene would be important in bone healing or for the treatment of
arthritis or osteoporosis; whereas antagonists to PRO344 would lead
to abnormal or pathological bone disorders including inflammatory
diseases associated with abnormal bone metabolism including
osteoarthritis, osteoporosis, and osteopenia. In addition, the
decrease in total body fat in the (-/-) mice was indicative of a
tissue wasting disease.
[1300] 35.11. Generation and Analysis of Mice Comprising
DNA44804-1248 (UNQ314) Gene Disruptions
[1301] In these knockout experiments, the gene encoding PRO357
polypeptides (designated as DNA44804-1248) (UNQ312) was disrupted.
The gene specific information for these studies is as follows: the
mutated mouse gene corresponds to nucleotide reference:
NM.sub.--139307 or Mus musculus Slit-like 2 (Drosophila) (Slit12);
protein reference: NP.sub.--647468 or Slit-like 2 [Mus musculus];
the human gene sequence reference: BC013767 or ACCESSION: BC013767
NID: 15489338 Homo sapiens Homo sapiens, Similar to RIKEN cDNA
2610528G05 gene, clone IMAGE: 3875837; the human protein sequence
corresponds to reference: Q96CX1 or ACCESSION: Q96CX1 NID: Homo
sapiens (Human). SIMILAR TO RIKEN cDNA 2610528G05 GENE (FRAGMENT).
HUMANSPTRNRDB.
[1302] The disrupted mouse gene is Slit12 (Slit-like 2
[Drosophila]), the ortholog of human hypothetical protein
LOC114990. Aliases include hypothetical protein BC013767 and
2610528G05Rik.
[1303] Slit-type proteins (e.g., SLIT1) are extracellular proteins
that play a critical role in developmental processes, especially
nervous and endocrine system formation. Slit-type proteins are
thought to assist in cellular movement (Piper and Little,
Bioessays, 25(1):32-8 (2003)). Thus, by homology, LOC114990 is
likely secreted or mostly extracellular and assists in
developmental processes.
Genetics Information:
TABLE-US-00060 [1304] wt het hom Total Observed 15 33 13 61
Expected 15.25 30.5 15.25 61 Chi-Sq. = 0.54 Significance = 0.76300
(hom/n) = 0.21 Avg. Litter Size = 4
Mutation Type: Retroviral Insertion (OST)
[1305] Retroviral insertion disrupted the gene in coding exon 2
(Accession: NM.sub.--139307). Wild-type expression of the target
gene was detected in embryonic stem (ES) cells and, among the 13
adult tissue samples tested by RT-PCR, in kidney, skeletal muscle,
and heart. RT-PCR analysis revealed that the transcript was absent
in the (-/-) mouse analyzed (F-59). Disruption of the target gene
was confirmed by Inverse PCR.
[1306] 35.11.1. Phenotypic Analysis (for Disrupted Gene:
DNA44804-1248 (UNQ314)
[1307] (a) Overall Phenotypic Summary:
[1308] Mutation of the gene encoding the ortholog of a human
hypothetical protein (BC013767) resulted in an increased mean bone
mineral content, volumetric bone mineral density and bone mineral
density in total body, femur and vertebrae. Several (-/-) mice were
smaller than their (+/+) littermates and died early (by 19 days
after birth). The (+/-) mice also showed diminished size and died
early. Male (-/-) mice exhibited infertility. The knockout mice
showed increase in serum triglycerides, ketone bodies and glucose
levels and had elevated blood pressure. The (-/-) mice also showed
increased bone measurements and increased total body fat.
Transcript was absent by RT-PCR.
[1309] (b) Bone Metabolism & Body Diagnostics
[1310] (1) Tissue Mass & Lean Body Mass Measurements--Dexa
[1311] Dexa Analysis--Test Description:
[1312] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. Dual Energy X-ray
Absorptiometry (DEXA) has been used successfully to identify
changes in total tissue mass (TTM).
[1313] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PIXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI,
i.e., whole body, vertebrae, and both femurs).
[1314] Body Measurements (Body Length & Weight):
[1315] Body Measurements: A measurement of body length and weight
was performed at approximately 16 weeks of age.
[1316] Results:
[1317] Several (-/-) mice were smaller than their (+/+)
littermates, and 2 died by 19 days after birth. The remaining (-/-)
mice seemed healthy. A number of (+/-) mice were also small,
exhibited reduced organ weights and died early. The 2 male (-/-)
mice available for analysis exhibited increased mean systolic blood
pressure when compared with their gender-matched (+/+) littermates
and the historical mean. Analyzed wt/het/hom: 14/27/10
[1318] Fertility: The male (-/-) mouse analyzed (M-106) was
infertile.
[1319] Pathology: Microscopic observations for the single (-/-)
mouse available for analysis (M-106) showed a sero purulent exudate
from the middle ear on one side. Additionally, a cyst was noted on
the testis along with degeneration unilaterally. Analyzed
wt/het/hom: 0/2/1
[1320] (2) Bone Metabolism: Radiology Phenotypic Analysis
[1321] In the area of bone metabolism, targets were identified
herein for the treatment of arthritis, osteoporosis, osteopenia and
osteopetrosis as well as identifying targets that promote bone
healing. Tests included:
[1322] DEXA for measurement of bone mineral density on femur and
vertebra
[1323] MicroCT for very high resolution and very high sensitivity
measurements of bone mineral density for both trabecular and
cortical bone.
[1324] Dexa Analysis--Test Description:
[1325] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. Dual Energy X-ray
Absorptiometry (DEXA) has been used successfully to identify
changes in bone. Anesthetized animals were examined and bone
mineral content (BMC), BMC/LBM ratios, volumetric bone mineral
density (vBMD), total body BMD, femur BMD and vertebra BMD were
measured.
[1326] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PIXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI)
[i.e., whole body, vertebrae, and both femurs].
[1327] Bone MicroCT Analysis:
[1328] Procedure: MicroCT was also used to get very sensitive
measurements of BMD. One vertebra and 1 femur were taken from a
cohort of 4 wild type and 8 homozygous mice. Measurements were
taken of lumbar 5 vertebra trabecular bone volume, trabecular
thickness, connectivity density and midshaft femur total bone area
and cortical thickness. The .mu.CT40 scans provided detailed
information on bone mass and architecture. Multiple bones were
placed into sample holders and scanned automatically. Instrument
software was used to select regions of interest for analysis.
Trabecular bone parameters were analyzed in the fifth lumbar
vertebrae (LV5) at 16 micrometer resolution and cortical bone
parameters were analyzed in the femur midshaft at a resolution of
20 micrometers.
[1329] Results:
[1330] DEXA: The (-/-) mice exhibited increased mean bone mineral
content, volumetric bone mineral density, and bone mineral density
in total body, femur, and vertebrae when compared with their
gender-matched (+/+) littermates and the historical means. The
mutant mice also showed increased total body fat content and a
corresponding increase in blood triglycerides indicative of
dyslipidemia. Analyzed wt/het/hom: 4/8/8
[1331] MicroCT: The (-/-) mice increased lumbar 5 vertebrae
measurements compared to their wild-type littermates.
[1332] Summary
[1333] Several (-/-) mice showed signs of growth retardation since
several mutant mice were much smaller than their (+/+) littermates.
In addition, the heterozygous (+/-) mice showed signs of decreased
growth and reduced viability being small and not surviving as long
as their (+/+) littermates. Furthermore, the (-/-) mice exhibited
increased mean bone mineral content, volumetric bone mineral
density and total body and femoral bone mineral density when
compared with their gender-matched (+/+) littermates. These results
indicate that the knockout mutants not only showed signs of growth
deficiencies and reduced viability but also exhibited bone
abnormalities associated with such bone diseases as osteopetrosis.
Osteopetrosis is a condition characterized by abnormal thickening
and hardening of bone and abnormal fragility of the bones. As such,
PRO357 polypeptides or agonists thereof would be important for
normal growth and normal bone metabolism and would be useful for
the treatment of osteopetrosis or other related bone disorders. In
addition, the male (-/-) mice showed signs of infertility and
degenerate testes. Thus, PRO357 polypeptides or agonists thereof
would be useful in the prevention and/or treatment of such bone
disorders and also would play a role in maintaining normal growth
and development.
[1334] (c) Phenotypic Analysis: Cardiology
[1335] In the area of cardiovascular biology, targets were
identified herein for the treatment of hypertension,
atherosclerosis, heart failure, stroke, various coronary artery
diseases, dyslipidemias such as high cholesterol
(hypercholesterolemia) and elevated serum triglycerides
(hypertriglyceridemia), diabetes and/or obesity. The phenotypic
tests included the measurement of serum cholesterol and
triglycerides.
[1336] Blood Lipids
[1337] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. High cholesterol levels and
increased triglyceride blood levels are recognized risk factors in
the development of cardiovascular disease and/or diabetes.
Measuring blood lipids facilitates the finding of biological
switches that regulate blood lipid levels Inhibition of factors
which elevate blood lipid levels may be useful for reducing the
risk for cardiovascular disease. In these blood chemistry tests,
cholesterol measurements were recorded using the COBAS Integra 400
(mfr: Roche).
[1338] Results:
[1339] The (-/-) mice exhibited increased mean serum triglyceride,
ketone bodies and glucose levels when compared with their
gender-matched (+/+) littermates and the historical means. In
addition, the (-/-) mice exhibited elevated blood pressure.
[1340] As summarized above, the (-/-) mice exhibited notably
increased triglyceride levels when compared with their
gender-matched (+/+) littermates and the historical means for the
male (+/+) mice. In addition, the increased mean serum glucose
levels suggesting diabetes. The elevated mean systolic blood
pressure along with the blood chemistry is indicative of
hypertension or other cardiovascular disease. Thus, mutant mice
deficient in the PRO357 gene can serve as a model for
cardiovascular disease including diabetes. PRO357 polypeptides or
its encoding gene would be useful in regulating normal blood lipid
levels such as triglycerides and/or blood sugars. Thus, PRO357
polypeptides or agonists thereof would be useful in the treatment
of such cardiovascular diseases as hypertension, atherosclerosis,
heart failure, stroke, various coronary diseases,
hypercholesterolemia, hypertriglyceridemia, diabetes and/or
obesity.
[1341] 35.12. Generation and Analysis of Mice Comprising
DNA44184-1319 (UNQ330) Gene Disruptions
[1342] In these knockout experiments, the gene encoding PRO526
polypeptides (designated as DNA44184-1319) (UNQ330) was disrupted.
The gene specific information for these studies is as follows: the
mutated mouse gene corresponds to nucleotide reference:
NM.sub.--022982 or ACCESSION: NM.sub.--022982 NID: gi 12667793 ref
NM.sub.--022982.1 Mus musculus reticulon 4 receptor (Rtn4r);
protein reference: Q99PI8 or Reticulon 4 receptor precursor (Nogo
receptor) (NgR) (Nogo-66 receptor); the human gene sequence
reference: NM.sub.--023004 or Homo sapiens reticulon 4 receptor
(RTN4R); the human protein sequence corresponds to reference:
Q9BZR6 or Reticulon 4 receptor precursor (Nogo receptor) (NgR)
(Nogo-66 receptor) (UNQ330/PRO526).
[1343] The mouse gene of interest is Rtn4r (reticulon 4 receptor),
ortholog of human RTN4R. Aliases include NgR, NOGOR, nogo receptor,
and Nogo-66 receptor.
[1344] RTN4R is a glycosylphosphatidylinositol-anchored
extracellular protein that functions as a receptor for reticulon 4
(RTN4; also known as NOGO), oligodendrocyte myelin glycoprotein
(OMG), and myelin-associated glycoprotein (MAG), which mediate
inhibition of axonal growth (Fournier et al., Nature,
409(6818):341-6 (2001); Wang, Koprivica et al., Nature,
417(6892):941-4 (2002); Liu et al., Science, 297(5584):1190-3
(2002)). Upon binding with these ligands, RTN4R associates with
p75, a transmembrane protein and receptor of the neurotrophin
growth factor family, and LINGO-1, a nervous system-specific
transmembrane protein. This association transduces the signal to
the interior of the cell (Wang, Kim et al., Nature, 420(6911):74-8
(2002); Wong et al., Nat Neurosci, 5(12):1302-8 (2002); Mi et al.,
Nat Neurosci, 7(3):221-8 (2004)). RTN4R is involved in axonal
guidance and nervous system development. Moreover, RTN4R and its
signaling components are targets for inhibitors that enable axonal
regeneration following injury to the central nervous system (Fisher
et al., J Neurosci, 24(7):1646-51 (2004); Song et al., J Neurosci,
24(2):542-6 (2004); Lee et al., Nat Rev Drug Discov, 2(11):872-8
(2003)).
[1345] Targeted or gene trap mutations were generated in strain
129SvEv.sup.Brd-derived embryonic stem (ES) cells. The chimeric
mice were bred to C57BL/6J albino mice to generate F1 heterozygous
animals. These progeny were intercrossed to generate F2 wild type,
heterozygous, and homozygous mutant progeny. On rare occasions, for
example when very few F1 mice were obtained from the chimera, F1
heterozygous mice were crossed to 129SvEv.sup.Brd/C57 hybrid mice
to yield additional heterozygous animals for the intercross to
generate the F2 mice.
Level I phenotypic analysis is performed on mice from this
generation.
TABLE-US-00061 wt het hom Total Observed 20 36 22 78 Expected 19.5
39 19.5 78 Chi-Sq. = 0.56 Significance = 0.75424 (hom/n) = 0.28
Avg. Litter Size = 8
Mutation Type: Homologous Recombination (standard) Coding exon 2
was targeted (NCBI accession NM.sub.--022982.1). Wild-type
expression of the target gene was detected in embryonic stem (ES)
cells and in all 13 adult tissue samples tested by RT-PCR.
Disruption of the target gene was confirmed by Southern
hybridization analysis.
[1346] 35.12.1. Phenotypic Analysis (for Disrupted Gene:
DNA44184-1319 (UNQ330)
[1347] (a) Overall Phenotypic Summary:
[1348] Mutation of the gene encoding the ortholog of human
reticulon 4 receptor (RTN4R) resulted in enhanced glucose tolerance
and decreased mean serum cholesterol, triglycerides and glucose
levels in (-/-) mice. The male (-/-) mutant mice exhibited an
increased lean body mass, increased bone mineral density, and
increased bone mineral content in total body, femurs and vertebrae,
as well as increased mean body weight. The mutant mice also
exhibited a highly elevated TNF-alpha and IL-6 response to the LPS
challenge. Gene disruption was confirmed by Southern blot.
[1349] (b) Immunology Phenotypic Analysis
[1350] Immune related and inflammatory diseases are the
manifestation or consequence of fairly complex, often multiple
interconnected biological pathways which in normal physiology are
critical to respond to insult or injury, initiate repair from
insult or injury, and mount innate and acquired defense against
foreign organisms. Disease or pathology occurs when these normal
physiological pathways cause additional insult or injury either as
directly related to the intensity of the response, as a consequence
of abnormal regulation or excessive stimulation, as a reaction to
self, or as a combination of these.
[1351] Though the genesis of these diseases often involves
multistep pathways and often multiple different biological
systems/pathways, intervention at critical points in one or more of
these pathways can have an ameliorative or therapeutic effect.
Therapeutic intervention can occur by either antagonism of a
detrimental process/pathway or stimulation of a beneficial
process/pathway.
[1352] T lymphocytes (T cells) are an important component of a
mammalian immune response. T cells recognize antigens which are
associated with a self-molecule encoded by genes within the major
histocompatibility complex (MHC). The antigen may be displayed
together with MHC molecules on the surface of antigen presenting
cells, virus infected cells, cancer cells, grafts, etc. The T cell
system eliminates these altered cells which pose a health threat to
the host mammal. T cells include helper T cells and cytotoxic T
cells. Helper T cells proliferate extensively following recognition
of an antigen-MHC complex on an antigen presenting cell. Helper T
cells also secrete a variety of cytokines, i.e., lymphokines, which
play a central role in the activation of B cells, cytotoxic T cells
and a variety of other cells which participate in the immune
response.
[1353] In many immune responses, inflammatory cells infiltrate the
site of injury or infection. The migrating cells may be
neutrophilic, eosinophilic, monocytic or lymphocytic as can be
determined by histological examination of the affected tissues.
Current Protocols in Immunology, ed. John E. Coligan, 1994, John
Wiley & Sons, Inc.
[1354] Many immune related diseases are known and have been
extensively studied. Such diseases include immune-mediated
inflammatory diseases (such as rheumatoid arthritis, immune
mediated renal disease, hepatobiliary diseases, inflammatory bowel
disease (IBD), psoriasis, and asthma), non-immune-mediated
inflammatory diseases, infectious diseases, immunodeficiency
diseases, neoplasia, and graft rejection, etc. In the area of
immunology, targets were identified for the treatment of
inflammation and inflammatory disorders.
[1355] In the area of immunology, targets have been identified
herein for the treatment of inflammation and inflammatory
disorders. Immune related diseases, in one instance, could be
treated by suppressing the immune response. Using neutralizing
antibodies that inhibit molecules having immune stimulatory
activity would be beneficial in the treatment of immune-mediated
and inflammatory diseases. Molecules which inhibit the immune
response can be utilized (proteins directly or via the use of
antibody agonists) to inhibit the immune response and thus
ameliorate immune related disease.
[1356] The following test was performed:
[1357] Acute Phase Response:
[1358] Test Description: Bacterial lipopolysaccharide (LPS) is an
endotoxin, and as such is a potent inducer of an acute phase
response and systemic inflammation. The Level I LPS mice were
injected intraperitoneally (i.p.) with a sublethal dose of LPS in
200 .mu.L sterile saline using a 26 gauge needle. The doses were
based on the average weight of the mice tested at 1 .mu.g/g body
weight 3 hours after injection; a 100 .mu.l blood sample was then
taken and analyzed for the presence of TNFa, MCP-1, and IL-6 on the
FACSCalibur instrument.
[1359] Results:
[1360] The (-/-) mice exhibited a highly elevated mean serum IL-6
and TNF-alpha response to LPS challenge when compared with their
(+/+) littermates and the historical mean.
[1361] In summary, the LPS endotoxin challenge demonstrated that
knockout mice deficient in the gene encoding PRO526 polypeptides
exhibit immunological abnormalities when compared with their
wild-type littermates. In particular, the mutant mice exhibited an
increased ability to elicit an immunological response (TNF-alpha
and IL-6 production) when challenged with the LPS endotoxin
indicating a strong proinflammatory response. IL-6 and TNF-alpha
contribute to the later stages of B cell activation. In addition,
IL-6 plays a critical role in inducing the acute phase response and
systemic inflammation. This suggests that inhibitors or antagonists
of PRO526 polypeptides or its encoding gene would stimulate the
immune system and would find utility in the cases wherein this
effect would be beneficial to the individual such as in the case of
leukemia, and other types of cancer, and in immuno-compromised
patients, such as AIDS sufferers. Accordingly, PRO526 polypeptides
or agonists thereof thereof would play a role in inhibiting the
immune response and would be useful candidates for suppressing
harmful immune responses, e.g. in the case of graft rejection or
graft-versus-host diseases.
[1362] (c) Bone Metabolism & Body Diagnostics
[1363] (1) Tissue Mass & Lean Body Mass Measurements--Dexa
[1364] Dexa Analysis--Test Description:
[1365] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. Dual Energy X-ray
Absorptiometry (DEXA) has been used successfully to identify
changes in total tissue mass (TTM).
[1366] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PIXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI,
i.e., whole body, vertebrae, and both femurs).
[1367] Body Measurements (Body Length & Weight):
[1368] Body Measurements: A measurement of body length and weight
was performed at approximately 16 weeks of age.
[1369] Results:
[1370] The mutant (-/-) mice exhibited a significant increased body
weight compared to their wild-type littermate controls.
[1371] (2) Bone Metabolism: Radiology Phenotypic Analysis
[1372] In the area of bone metabolism, targets were identified
herein for the treatment of arthritis, osteoporosis, osteopenia and
osteopetrosis as well as identifying targets that promote bone
healing. Tests included:
[1373] DEXA for measurement of bone mineral density on femur and
vertebra
[1374] MicroCT for very high resolution and very high sensitivity
measurements of bone mineral density for both trabecular and
cortical bone.
[1375] Dexa Analysis--Test Description:
[1376] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. Dual Energy X-ray
Absorptiometry (DEXA) has been used successfully to identify
changes in bone. Anesthetized animals were examined and bone
mineral content (BMC), BMC/LBM ratios, volumetric bone mineral
density (vBMD), total body BMD, femur BMD and vertebra BMD were
measured.
[1377] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PIXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI)
[i.e., whole body, vertebrae, and both femurs].
[1378] Bone MicroCT Analysis:
[1379] Procedure: MicroCT was also used to get very sensitive
measurements of BMD. One vertebra and 1 femur were taken from a
cohort of 4 wild type and 8 homozygous mice. Measurements were
taken of lumbar 5 vertebra trabecular bone volume, trabecular
thickness, connectivity density and midshaft femur total bone area
and cortical thickness. The .mu.CT40 scans provided detailed
information on bone mass and architecture. Multiple bones were
placed into sample holders and scanned automatically. Instrument
software was used to select regions of interest for analysis.
Trabecular bone parameters were analyzed in the fifth lumbar
vertebrae (LV5) at 16 micrometer resolution and cortical bone
parameters were analyzed in the femur midshaft at a resolution of
20 micrometers.
[1380] Results:
[1381] DEXA: The male (-/-) mice exhibited increased mean lean body
mass and increased mean bone mineral content and bone mineral
density in total body, femurs, and vertebrae when compared with
their gender-matched (+/+) littermates and the historical
means.
[1382] MicroCT: The (-/-) mice increased trabecular connectivity
density compared to their wild-type littermates and the historical
mean.
[1383] Summary
[1384] In summary, the male (-/-) mice exhibited increased mean
lean body mass, bone mineral content, and total body and femoral
bone mineral density when compared with their gender-matched (+/+)
littermates. These results indicate that the knockout mutant
phenotype may be associated with such bone abnormalities as
osteopetrosis. Osteopetrosis is a condition characterized by
abnormal thickening and hardening of bone and abnormal fragility of
the bones. As such, PRO526 polypeptides or agonists thereof would
be beneficial for the treatment of osteopetrosis or other osteo
diseases. On the other hand, inhibitors or antagonists of PRO526
polypeptides would be useful in bone healing. Analyzed wt/het/hom:
4/4/8
[1385] (d) Phenotypic Analysis: Cardiology
[1386] In the area of cardiovascular biology, targets were
identified herein for the treatment of hypertension,
atherosclerosis, heart failure, stroke, various coronary artery
diseases, dyslipidemias such as high cholesterol
(hypercholesterolemia) and elevated serum triglycerides
(hypertriglyceridemia), diabetes and/or obesity. The phenotypic
tests included the measurement of serum cholesterol and
triglycerides.
[1387] Blood Lipids
[1388] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. High cholesterol levels and
increased triglyceride blood levels are recognized risk factors in
the development of cardiovascular disease and/or diabetes.
Measuring blood lipids facilitates the finding of biological
switches that regulate blood lipid levels Inhibition of factors
which elevate blood lipid levels may be useful for reducing the
risk for cardiovascular disease. In these blood chemistry tests,
cholesterol measurements were recorded using the COBAS Integra 400
(mfr: Roche).
[1389] Results:
[1390] The (-/-) mice exhibited decreased mean serum cholesterol,
triglyceride and glucose levels when compared with their
gender-matched (+/+) littermates and the historical mean. In
summary, these knockout mutant mice exhibited a positive phenotype
with regards to lipid and/or glucose metabolism. Thus, mutant mice
deficient in the PRO526 gene can serve as a model for treatment of
cardiovascular disease. Antagonists to PRO526 or its encoding gene
would be useful in regulating blood lipids and in particular in
maintaining normal cholesterol metabolism. Such inhibitors or
antagonists to PRO526 polypeptides would be useful in the treatment
of such cardiovascular diseases associated with dyslipidemia as:
hypertension, atherosclerosis, heart failure, stroke, various
coronary artery diseases, obesity and/or diabetes.
[1391] (e) Phenotypic Analysis: Metabolism-Blood Chemistry/Glucose
Tolerance
[1392] In the area of metabolism, targets may be identified for the
treatment of diabetes. Blood chemistry phenotypic analysis includes
blood glucose measurements. The COBAS Integra 400 (mfr: Roche) was
used for running blood chemistry tests on the mice. In the area of
metabolism, targets may be identified for the treatment of
diabetes. Blood chemistry phenotypic analysis includes glucose
tolerance tests to measure insulin sensitivity and changes in
glucose metabolism. Abnormal glucose tolerance test results may
indicate but may not be limited to the following disorders or
conditions: Diabetes Type 1 and Type 2, Syndrome X, various
cardiovascular diseases and/or obesity.
[1393] Procedure: A cohort of 2 wild type and 4 homozygous mice
were used in this assay. The glucose tolerance test is the standard
for defining impaired glucose homeostasis in mammals. Glucose
tolerance tests were performed using a Lifescan glucometer. Animals
were injected IP at 2 g/kg with D-glucose delivered as a 20%
solution and blood glucose levels were measured at 0, 30, 60 and 90
minutes after injection. Analyzed wt/het/hom: 4/4/8
[1394] Results:
[1395] The (-/-) mice exhibited an enhanced glucose tolerance when
compared with their gender-matched (+/+) littermates and the
historical means. Thus, knockout mice exhibited the opposite
phenotypic pattern of an impaired glucose homeostasis, and as such
antagonists to PRO526 or its encoding gene would be useful in the
treatment of impaired glucose homeostasis and diseases associated
with abnormal glucose metabolism.
[1396] 35.13. Generation and Analysis of Mice Comprising
DNA49631-1328 (UNQ389) Gene Disruptions
[1397] In these knockout experiments, the gene encoding PRO724
polypeptides (designated as DNA49631-1328) (UNQ389) was disrupted.
The gene specific information for these studies is as follows: the
mutated mouse gene corresponds to nucleotide reference:
NM.sub.--022993 or ACCESSION: NM.sub.--022993 NID: 12667805 Mus
musculus Mus musculus low-density lipoprotein receptor-related
protein 10 (Lrp10); protein reference: Q9EPE8 or ACCESSION: Q9EPE8
NID: Mus musculus (Mouse). LOW-DENSITY LIPOPROTEIN RECEPTOR-RELATED
PROTEIN 9. MOUSESPTRNRDB; the human gene sequence reference:
NM.sub.--014045 or ACCESSION: NM.sub.--014045 NID: 13027587 Homo
sapiens Homo sapiens DKFZP564C1940 protein (DKFZP564C1940); the
human protein sequence corresponds to reference: Q86T02. ACCESSION:
Q86T02 NID: Homo sapiens (Human). Human full-length cDNA clone
CSODK002YO06 of HeLa cells of Homo sapiens (Human).
[1398] The mouse gene of interest is Lrp10 (low-density lipoprotein
receptor-related protein 10), ortholog of human LRP10. Aliases
include Lrp9, MGC8675, and DKFZP564C1940.
[1399] LRP10 is a predicted type I plasma membrane protein
expressed primarily in liver that mediates the uptake of
apolipoprotein E-enriched beta-VLDL in vitro and, thus, likely
functions as a receptor for low-density lipoproteins in vivo. In
addition to liver, LRP10 is also found in kidney and brain, with
particularly high levels occurring in vascular walls. LRP10 may
play a role in apoE-containing lipoprotein uptake (Sugiyama et al.,
Biochemistry, 39(51):15817-25 (2000)).
[1400] Targeted or gene trap mutations were generated in strain
129SvEv.sup.Brd-derived embryonic stem (ES) cells. The chimeric
mice were bred to C57BL/6J albino mice to generate F1 heterozygous
animals. These progeny were intercrossed to generate F2 wild type,
heterozygous, and homozygous mutant progeny. On rare occasions, for
example when very few F1 mice were obtained from the chimera, F1
heterozygous mice were crossed to 129SvEv.sup.Brd/C57 hybrid mice
to yield additional heterozygous animals for the intercross to
generate the F2 mice.
Level I phenotypic analysis is performed on mice from this
generation.
TABLE-US-00062 wt het hom Total Observed 17 29 17 63 Expected 15.75
31.5 15.75 63 Chi-Sq. = 0.40 Significance = 0.82003 (hom/n) = 0.27
Avg. Litter Size = 6
Mutation Type: Homologous Recombination (standard) Coding exons 3
through 7 were targeted (NCBI accession NM.sub.--022993.2).
Wild-type expression of the target gene was detected in embryonic
stem (ES) cells and in all 13 adult tissue samples tested by
RT-PCR. Disruption of the target gene was confirmed by Southern
hybridization analysis.
[1401] 35.13.1. Phenotypic Analysis (for Disrupted Gene:
DNA49631-1328 (UNQ389)
[1402] (a) Overall Phenotypic Summary:
[1403] Mutation of the gene encoding the ortholog of human
low-density lipoprotein receptor-related protein 10 (LRP10)
resulted in a dramatically decreased skin fibroblast proliferation
rate in (-/-) mice. In addition, (-/-) mice exhibited increased
bone measurements. An impaired glucose tolerance was also noted for
the mutant mice. Also, the mutant (-/-) mice exhibited
immunological abnormalities marked by decreased levels of
eosinophils and monocytes but also increased levels of CD8+ cells.
Gene disruption was confirmed by Southern blot.
[1404] (b) Immunology Phenotypic Analysis
[1405] Immune related and inflammatory diseases are the
manifestation or consequence of fairly complex, often multiple
interconnected biological pathways which in normal physiology are
critical to respond to insult or injury, initiate repair from
insult or injury, and mount innate and acquired defense against
foreign organisms. Disease or pathology occurs when these normal
physiological pathways cause additional insult or injury either as
directly related to the intensity of the response, as a consequence
of abnormal regulation or excessive stimulation, as a reaction to
self, or as a combination of these.
[1406] Though the genesis of these diseases often involves
multistep pathways and often multiple different biological
systems/pathways, intervention at critical points in one or more of
these pathways can have an ameliorative or therapeutic effect.
Therapeutic intervention can occur by either antagonism of a
detrimental process/pathway or stimulation of a beneficial
process/pathway.
[1407] T lymphocytes (T cells) are an important component of a
mammalian immune response. T cells recognize antigens which are
associated with a self-molecule encoded by genes within the major
histocompatibility complex (MHC). The antigen may be displayed
together with MHC molecules on the surface of antigen presenting
cells, virus infected cells, cancer cells, grafts, etc. The T cell
system eliminates these altered cells which pose a health threat to
the host mammal. T cells include helper T cells and cytotoxic T
cells. Helper T cells proliferate extensively following recognition
of an antigen-MHC complex on an antigen presenting cell. Helper T
cells also secrete a variety of cytokines, i.e., lymphokines, which
play a central role in the activation of B cells, cytotoxic T cells
and a variety of other cells which participate in the immune
response.
[1408] In many immune responses, inflammatory cells infiltrate the
site of injury or infection. The migrating cells may be
neutrophilic, eosinophilic, monocytic or lymphocytic as can be
determined by histological examination of the affected tissues.
Current Protocols in Immunology, ed. John E. Coligan, 1994, John
Wiley & Sons, Inc.
[1409] Many immune related diseases are known and have been
extensively studied. Such diseases include immune-mediated
inflammatory diseases (such as rheumatoid arthritis, immune
mediated renal disease, hepatobiliary diseases, inflammatory bowel
disease (IBD), psoriasis, and asthma), non-immune-mediated
inflammatory diseases, infectious diseases, immunodeficiency
diseases, neoplasia, and graft rejection, etc. In the area of
immunology, targets were identified for the treatment of
inflammation and inflammatory disorders.
[1410] In the area of immunology, targets have been identified
herein for the treatment of inflammation and inflammatory
disorders. Immune related diseases, in one instance, could be
treated by suppressing the immune response. Using neutralizing
antibodies that inhibit molecules having immune stimulatory
activity would be beneficial in the treatment of immune-mediated
and inflammatory diseases. Molecules which inhibit the immune
response can be utilized (proteins directly or via the use of
antibody agonists) to inhibit the immune response and thus
ameliorate immune related disease.
[1411] The following tests were performed:
[1412] (1) Flourescence-Activated Cell-Sorting (FACS) Analysis
[1413] Procedure:
[1414] FACS analysis of immune cell composition from peripheral
blood was performed including CD4, CD8 and T cell receptor to
evaluate T lymphocytes, CD19 for B lymphocytes, CD45 as a leukocyte
marker and pan NK for natural killer cells. The FACS analysis was
carried out on 2 wild type and 6 homozygous mice and included cells
derived from thymus, spleen, bone marrow and lymph node.
[1415] In these studies, analyzed cells were isolated from thymus,
peripheral blood, spleen, bone marrow and lymph nodes. Flow
cytometry was designed to determine the relative proportions of CD4
and CD8 positive T cells, B cells, NK cells and monocytes in the
mononuclear cell population. A Becton-Dickinson FACSCalibur 3-laser
FACS machine was used to assess immune status. For Phenotypic
Assays and Screening, this machine records CD4+/CD8-, CD8+/CD4-,
NK, B cell and monocyte numbers in addition to the CD4+/CD8+ ratio.
The mononuclear cell profile was derived by staining a single
sample of lysed peripheral blood from each mouse with a panel of
six lineage-specific antibodies: CD45 PerCP, anti-TCRb APC, CD4 PE,
CD8 FITC, pan-NK PE, and CD19 FITC. The two FITC and PE labeled
antibodies stain mutually exclusive cell types. The samples were
analyzed using a Becton Dickinson FACSCalibur flow cytometer with
CellQuest software.
[1416] (2) Hematology Analysis:
[1417] Test Description: Blood tests are carried out by Abbott's
Cell-Dyn 3500R, an automated hematology analyzer. Some of its
features include a five-part WBC differential. `Patient` reports
can cover over 22 parameters in all.
[1418] Results:
[1419] The (-/-) mice exhibited decreased eosinophils and monocytes
when compared with their (+/+) littermates and the historical
means.
[1420] FACS analysis also demonstrated that (-/-) mice exhibit an
increased mean percentage of CD8+ cells when compared with their
(+/+) littermates and the historical mean.
Analyzed wt/het/hom: 7/5/8
[1421] In summary, the hematology and FACS results indicate that
the homozygous mutant mice exhibited decreased eosinophil and
monocyte counts compared to their littermate controls indicating
low levels of precursors of macrophages. However, the (-/-) mutant
mice also showed an increased percentage of CD8+ cells. CD8+
molecules are the co-receptor molecules which cooperate with the
T-cell receptor in antigen recognition and in particular
specifically bind only to the invariant parts of the MHC class I
molecule. During antigen recognition, the CD8+ molecules associate
on the T-cell surface with components of the T-cell receptor to
form the cytotoxic CD8+ T-cell. Thus, inhibitors or antagonists of
PRO724 polypeptides would be important in the T-cell mediated
response involving the MHC class I pathway and would be beneficial
in those instances wherein cytotoxic T cells are required in host
defense against cytosolic pathogens. In contrast, PRO724
polypeptides or agonists thereof, would be expected to mimic a
negative phenotype resulting in a deficiency in the mean percentage
of CD8+ cells and therefore an MHC class I deficiency would result.
One such disease model occurs when there is an almost complete
absence of cell-surface MHC class I molecules. Patients with this
condition have normal levels of mRNA encoding MHC class I molecules
and normal levels of production of MHC class I proteins. However,
these individuals are immunodeficient, specifically owing to the
lack of CD8+ T cells. This results in a severe immunodeficiency
disease wherein the response to nearly all pathogens is critically
suppressed.
[1422] (c) Bone Metabolism: Radiology Phenotypic Analysis
[1423] In the area of bone metabolism, targets were identified
herein for the treatment of arthritis, osteoporosis, osteopenia and
osteopetrosis as well as identifying targets that promote bone
healing. Tests included:
[1424] DEXA for measurement of bone mineral density on femur and
vertebra
[1425] MicroCT for very high resolution and very high sensitivity
measurements of bone mineral density for both trabecular and
cortical bone.
[1426] Dexa Analysis--Test Description:
[1427] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. Dual Energy X-ray
Absorptiometry (DEXA) has been used successfully to identify
changes in bone. Anesthetized animals were examined and bone
mineral content (BMC), BMC/LBM ratios, volumetric bone mineral
density (vBMD), total body BMD, femur BMD and vertebra BMD were
measured.
[1428] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PIXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI)
[i.e., whole body, vertebrae, and both femurs].
[1429] Bone MicroCT Analysis:
[1430] Procedure: MicroCT was also used to get very sensitive
measurements of BMD. One vertebra and 1 femur were taken from a
cohort of 4 wild type and 8 homozygous mice. Measurements were
taken of lumbar 5 vertebra trabecular bone volume, trabecular
thickness, connectivity density and midshaft femur total bone area
and cortical thickness. The .mu.CT40 scans provided detailed
information on bone mass and architecture. Multiple bones were
placed into sample holders and scanned automatically. Instrument
software was used to select regions of interest for analysis.
Trabecular bone parameters were analyzed in the fifth lumbar
vertebrae (LV5) at 16 micrometer resolution and cortical bone
parameters were analyzed in the femur midshaft at a resolution of
20 micrometers.
[1431] Results:
[1432] DEXA: The (-/-) mice exhibited increased mean bone mineral
density when compared with their gender-matched (+/+) littermates
and the historical means.
[1433] Micro-CT: The (-/-) mice exhibited increased mean vertebral
trabecular bone volume, number, and connectivity density when
compared with their gender-matched (+/+) littermates and the
historical means.
[1434] Analyzed wt/het/hom: 4/4/9
[1435] In summary, the (-/-) mice exhibited increased mean bone
mineral content, and total body and femoral bone mineral density
when compared with their gender-matched (+/+) littermates. These
results indicate that the knockout mutant phenotype is associated
with bone abnormalities such as osteopetrosis. Osteopetrosis is a
condition characterized by abnormal thickening and hardening of
bone and abnormal fragility of the bones. As such, PRO724
polypeptides or agonists thereof would be beneficial for the
treatment of osteopetrosis or other osteo diseases. On the other
hand, inhibitors or antagonists of PRO724 polypeptides would be
useful in bone healing.
[1436] (d) Oncology Phenotypic Analysis
[1437] In the area of oncology, targets were identified herein for
the treatment of solid tumors, lymphomas and leukemia.
[1438] Adult Skin Cell Proliferation:
[1439] Procedure: Skin cells were isolated from 16 week old animals
(2 wild type and 4 homozygotes). These were developed into primary
fibroblast cultures and the fibroblast proliferation rates were
measured in a strictly controlled protocol. The ability of this
assay to detect hyper-proliferative and hypo-proliferative
phenotypes has been demonstrated with p53 and Ku80. Proliferation
was measured using Brdu incorporation.
[1440] Specifically, in these studies the skin fibroblast
proliferation assay was used. An increase in the number of cells in
a standardized culture was used as a measure of relative
proliferative capacity. Primary fibroblasts were established from
skin biopsies taken from wild type and mutant mice. Duplicate or
triplicate cultures of 0.05 million cells were plated and allowed
to grow for six days. At the end of the culture period, the number
of cells present in the culture was determined using a electronic
particle counter.
[1441] Results:
[1442] The (-/-) mice exhibited a dramatically decreased mean skin
fibroblast proliferation rate when compared with their
gender-matched (+/+) littermates and the historical mean. Thus,
homozygous mutant mice demonstrated a hypo-proliferative phenotype.
As suggested by these observations, antagonists of a PRO724
polypeptide or its encoding gene would be useful in decreasing
abnormal cell proliferation such as tumor cell growth.
[1443] (e) Phenotypic Analysis: Metabolism-Blood Chemistry/Glucose
Tolerance
[1444] In the area of metabolism, targets may be identified for the
treatment of diabetes. Blood chemistry phenotypic analysis includes
blood glucose measurements. The COBAS Integra 400 (mfr: Roche) was
used for running blood chemistry tests on the mice. In the area of
metabolism, targets may be identified for the treatment of
diabetes. Blood chemistry phenotypic analysis includes glucose
tolerance tests to measure insulin sensitivity and changes in
glucose metabolism. Abnormal glucose tolerance test results may
indicate but may not be limited to the following disorders or
conditions: Diabetes Type 1 and Type 2, Syndrome X, various
cardiovascular diseases and/or obesity.
[1445] Procedure: A cohort of 2 wild type and 4 homozygote mice
were used in this assay. The glucose tolerance test is the standard
for defining impaired glucose homeostasis in mammals. Glucose
tolerance tests were performed using a Lifescan glucometer. Animals
were injected IP at 2 g/kg with D-glucose delivered as a 20%
solution and blood glucose levels were measured at 0, 30, 60 and 90
minutes after injection.
[1446] Results:
[1447] The (-/-) mice exhibited an increased mean serum glucose
level when compared with their gender-matched (+/+) littermates and
the historical mean. In addition, the (-/-) mice exhibited impaired
glucose tolerance when compared with their gender-matched (+/+)
littermates and the historical means.
[1448] Analyzed wt/het/hom: 4/4/8
[1449] These studies indicated that (-/-) mice exhibit a decreased
glucose tolerance in the presence of normal fasting glucose at all
3 intervals tested when compared with their gender-matched (+/+)
littermates and the historical means. Thus, knockout mutant mice
exhibited the phenotypic pattern of an impaired glucose
homeostasis, and therefor PRO724 polypeptides (or agonists thereof)
or its encoding gene would be useful in the treatment of impaired
glucose homeostasis and/or various cardiovascular diseases,
including diabetes.
[1450] 35.14. Generation and Analysis of Mice Comprising
DNA48331-1329 (UNQ395) Gene Disruptions
[1451] In these knockout experiments, the gene encoding PRO731
polypeptides (designated as DNA48331-1329) (UNQ395) was disrupted.
The gene specific information for these studies is as follows: the
mutated mouse gene corresponds to nucleotide reference:
NM.sub.--017378 or ACCESSION: NM.sub.--017378 NID: 8393915 Mus
musculus Mus musculus protocadherin 12 (Pcdh12); protein reference:
O55134 or ACCESSION: O55134 NID: Mus musculus (Mouse). VASCULAR
CADHERIN-2. MOUSESPTRNRDB; the human gene sequence reference:
NM.sub.--016580 or ACCESSION: NM.sub.--016580 NID: 14589925 Homo
sapiens Homo sapiens protocadherin 12 (PCDH12); the human protein
sequence corresponds to reference: Q9NPG4 or ACCESSION: Q9NPG4 NID:
Homo sapiens (Human). Protocadherin 12 precursor (Vascular
cadherin-2) (Vascular endothelial cadherin-2) (VE-cadherin-2)
(VE-cad-2). HUMANSPTRNRDB.
[1452] The mouse gene of interest is Pcdh12 (protocadherin 12),
ortholog of human PCDH12. Aliases include Pcdh14, VE-cad-2,
VE-cadherin-2, VECAD2, protocadherin 14, and vascular endothelial
cadherin-2.
[1453] PCDH12 is a type I membrane protein expressed in vascular
endothelium that likely functions as a cadherin family cell
adhesion molecule. The protein consists of an extracellular domain
containing six cadherin repeats, a transmembrane segment, and a
cytoplasmic C terminus. Unlike vascular endothelial cadherin-1,
PCDH12 neither interacts with catenins nor affects cell migration
or growth. PCDH12 promotes homotypic calcium-dependent adhesion and
aggregation clusters at intercellular junctions (Telo et al., J
Biol Chem 273(28):17565-72 (1998); Wu and Maniatis, Proc Natl Acad
Sci USA, 97(7):3124-9 (2000)).
[1454] Targeted or gene trap mutations were generated in strain
129SvEv.sup.Brd-derived embryonic stem (ES) cells. The chimeric
mice were bred to C57BL/6J albino mice to generate F1 heterozygous
animals. These progeny were intercrossed to generate F2 wild type,
heterozygous, and homozygous mutant progeny. On rare occasions, for
example when very few F1 mice were obtained from the chimera, F1
heterozygous mice were crossed to 129SvEv.sup.Brd/C57 hybrid mice
to yield additional heterozygous animals for the intercross to
generate the F2 mice.
Level I phenotypic analysis is performed on mice from this
generation.
TABLE-US-00063 wt het hom Total Observed 20 40 19 79 Expected 19.75
39.5 19.75 79 Chi-Sq. = 0.04 Significance = 0.98119 (hom/n) = 0.24
Avg. Litter Size = 8
Mutation Type: Homologous Recombination (standard) Coding exon 1
was targeted (NCBI accession NM.sub.--017378.1). Wild-type
expression of the target gene was detected in all 13 adult tissue
samples tested by RT-PCR, except stomach, small intestine, and
colon. Disruption of the target gene was confirmed by Southern
hybridization analysis.
[1455] 35.14.1. Phenotypic Analysis (for Disrupted Gene:
DNA48331-1329 (UNQ395)
[1456] (a) Overall Phenotypic Summary:
[1457] Mutation of the gene encoding the ortholog of human
protocadherin 12 (PCDH12) resulted in larger (-/-) mice, exhibiting
increased body weight, total tissue mass, and lean body mass as
well increased bone related measurements. The (-/-) mice exhibited
increased organ weight, total body fat and total fat mass. In
addition, the (-/-) mice exhibited a decreased percentage of
natural killer cells and blood eosinophils. Gene disruption was
confirmed by Southern blot.
[1458] (b) Immunology Phenotypic Analysis
[1459] Immune related and inflammatory diseases are the
manifestation or consequence of fairly complex, often multiple
interconnected biological pathways which in normal physiology are
critical to respond to insult or injury, initiate repair from
insult or injury, and mount innate and acquired defense against
foreign organisms. Disease or pathology occurs when these normal
physiological pathways cause additional insult or injury either as
directly related to the intensity of the response, as a consequence
of abnormal regulation or excessive stimulation, as a reaction to
self, or as a combination of these.
[1460] Though the genesis of these diseases often involves
multistep pathways and often multiple different biological
systems/pathways, intervention at critical points in one or more of
these pathways can have an ameliorative or therapeutic effect.
Therapeutic intervention can occur by either antagonism of a
detrimental process/pathway or stimulation of a beneficial
process/pathway.
[1461] T lymphocytes (T cells) are an important component of a
mammalian immune response. T cells recognize antigens which are
associated with a self-molecule encoded by genes within the major
histocompatibility complex (MHC). The antigen may be displayed
together with MHC molecules on the surface of antigen presenting
cells, virus infected cells, cancer cells, grafts, etc. The T cell
system eliminates these altered cells which pose a health threat to
the host mammal. T cells include helper T cells and cytotoxic T
cells. Helper T cells proliferate extensively following recognition
of an antigen-MHC complex on an antigen presenting cell. Helper T
cells also secrete a variety of cytokines, i.e., lymphokines, which
play a central role in the activation of B cells, cytotoxic T cells
and a variety of other cells which participate in the immune
response.
[1462] In many immune responses, inflammatory cells infiltrate the
site of injury or infection. The migrating cells may be
neutrophilic, eosinophilic, monocytic or lymphocytic as can be
determined by histological examination of the affected tissues.
Current Protocols in Immunology, ed. John E. Coligan, 1994, John
Wiley & Sons, Inc.
[1463] Many immune related diseases are known and have been
extensively studied. Such diseases include immune-mediated
inflammatory diseases (such as rheumatoid arthritis, immune
mediated renal disease, hepatobiliary diseases, inflammatory bowel
disease (IBD), psoriasis, and asthma), non-immune-mediated
inflammatory diseases, infectious diseases, immunodeficiency
diseases, neoplasia, and graft rejection, etc. In the area of
immunology, targets were identified for the treatment of
inflammation and inflammatory disorders.
[1464] In the area of immunology, targets have been identified
herein for the treatment of inflammation and inflammatory
disorders. Immune related diseases, in one instance, could be
treated by suppressing the immune response. Using neutralizing
antibodies that inhibit molecules having immune stimulatory
activity would be beneficial in the treatment of immune-mediated
and inflammatory diseases. Molecules which inhibit the immune
response can be utilized (proteins directly or via the use of
antibody agonists) to inhibit the immune response and thus
ameliorate immune related disease.
[1465] Flourescence-Activated Cell-Sorting (FACS) Analysis
[1466] Procedure:
[1467] FACS analysis of immune cell composition from peripheral
blood was performed including CD4, CD8 and T cell receptor to
evaluate T lymphocytes, CD19 for B lymphocytes, CD45 as a leukocyte
marker and pan NK for natural killer cells. The FACS analysis was
carried out on 2 wild type and 6 homozygous mice and included cells
derived from thymus, spleen, bone marrow and lymph node.
[1468] In these studies, analyzed cells were isolated from thymus,
peripheral blood, spleen, bone marrow and lymph nodes. Flow
cytometry was designed to determine the relative proportions of CD4
and CD8 positive T cells, B cells, NK cells and monocytes in the
mononuclear cell population. A Becton-Dickinson FACSCalibur 3-laser
FACS machine was used to assess immune status. For Phenotypic
Assays and Screening, this machine records CD4+/CD8-, CD8+/CD4-,
NK, B cell and monocyte numbers in addition to the CD4+/CD8+ ratio.
The mononuclear cell profile was derived by staining a single
sample of lysed peripheral blood from each mouse with a panel of
six lineage-specific antibodies: CD45 PerCP, anti-TCRb APC, CD4 PE,
CD8 FITC, pan-NK PE, and CD19 FITC. The two FITC and PE labeled
antibodies stain mutually exclusive cell types. The samples were
analyzed using a Becton Dickinson FACSCalibur flow cytometer with
CellQuest software.
[1469] Results:
[1470] FACS: The (-/-) mice exhibited a decreased mean percentage
of natural killer cells and blood eosinophils when compared with
their (+/+) littermates and the historical mean. Analyzed
wt/het/hom: 7/4/8
[1471] In summary, the FACS results indicate that the homozygous
mutant mice have an impaired immune system, especially in view of
the decreased mean percentage of natural killer cells which is an
indicator of a negative phenotype associated with knocking out the
DNA48331-1329 gene which encodes PRO731 polypeptides. Natural
killer cells are the first line of defense to viral infection since
these cells have been implicated in viral immunity and in defense
against tumors. Natural killer cells or NK cells act as effectors
in antibody-dependent cell-mediated cytotoxicity and have been
identified by their ability to kill certain lymphoid tumor cell
lines in vitro without the need for prior immunization or
activation. However, their known function in host defense is in the
early phases of infection with several intracellular pathogens,
particularly herpes viruses. Thus, PRO731 polypeptides and agonists
thereof would be important for a healthy immune system and would be
useful in stimulating the immune system particularly during viral
infections.
[1472] (c) Bone Metabolism & Body Diagnostics
[1473] (1) Tissue Mass & Lean Body Mass Measurements--Dexa
[1474] Dexa Analysis--Test Description:
[1475] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. Dual Energy X-ray
Absorptiometry (DEXA) has been used successfully to identify
changes in total tissue mass (TTM).
[1476] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PIXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI,
i.e., whole body, vertebrae, and both femurs).
[1477] Body Measurements (Body Length & Weight):
[1478] Body Measurements: A measurement of body length and weight
was performed at approximately 16 weeks of age.
[1479] Results:
[1480] The (-/-) mice exhibited increased mean body weight when
compared with their gender-matched (+/+) littermates and the
historical means. Organ weights were also significantly increased.
Analyzed wt/het/hom: 28/58/33
[1481] Pathology: Microscopic observations revealed apoptosis of
the olfactory neuro epithelial cells in 3/6 (-/-) and 1/2 (+/+)
mice.
[1482] Gene Expression: LacZ activity was not detected in the panel
of tissues by immuno histochemical analysis. Analyzed wt/het/hom:
4/2/13
[1483] (2) Bone Metabolism: Radiology Phenotypic Analysis
[1484] In the area of bone metabolism, targets were identified
herein for the treatment of arthritis, osteoporosis, osteopenia and
osteopetrosis as well as identifying targets that promote bone
healing. Tests included:
[1485] DEXA for measurement of bone mineral density on femur and
vertebra
[1486] MicroCT for very high resolution and very high sensitivity
measurements of bone mineral density for both trabecular and
cortical bone.
[1487] Dexa Analysis--Test Description:
[1488] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. Dual Energy X-ray
Absorptiometry (DEXA) has been used successfully to identify
changes in bone. Anesthetized animals were examined and bone
mineral content (BMC), BMC/LBM ratios, volumetric bone mineral
density (vBMD), total body BMD, femur BMD and vertebra BMD were
measured.
[1489] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PIXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI)
[i.e., whole body, vertebrae, and both femurs].
[1490] Bone MicroCT Analysis:
[1491] Procedure: MicroCT was also used to get very sensitive
measurements of BMD. One vertebra and 1 femur were taken from a
cohort of 4 wild type and 8 homozygous mice. Measurements were
taken of lumbar 5 vertebra trabecular bone volume, trabecular
thickness, connectivity density and midshaft femur total bone area
and cortical thickness. The .mu.CT40 scans provided detailed
information on bone mass and architecture. Multiple bones were
placed into sample holders and scanned automatically. Instrument
software was used to select regions of interest for analysis.
Trabecular bone parameters were analyzed in the fifth lumbar
vertebrae (LV5) at 16 micrometer resolution and cortical bone
parameters were analyzed in the femur midshaft at a resolution of
20 micrometers.
[1492] Results:
[1493] DEXA: The (-/-) mice exhibited increased mean total tissue
mass, lean body mass, total body bone mineral density, and
volumetric bone mineral density when compared with their
gender-matched (+/+) littermates and the historical means. In
addition, the (-/-) mice exhibited increased mean percent total
body fat and total fat mass.
[1494] Micro-CT: The (-/-) mice exhibited increased mean vertebral
trabecular bone volume, number and connectivity density and
increased mean femoral mid-shaft cortical thickness and
cross-sectional area when compared with their gender-matched (+/+)
littermates and the historical means.
Analyzed wt/het/hom: 4/4/8
[1495] In summary, the (-/-) mice exhibited increased body weight,
increased body fat, increased mean lean body mass, increased bone
mineral content, and increased total body and femoral bone mineral
density when compared with their gender-matched (+/+) littermates.
The observations of an increased body weight, body fat and mean
lean body mass in the (-/-) mutant mice suggests an obesity
phenotype. These data suggest that the DNA48331-1329 gene encoding
PRO731 polypeptides serves to negatively regulate proliferation and
growth (cell/organ size) and would be important for cellular
survival. In addition, the abnormal bone measurements indicate that
the knockout mutant phenotype is associated with bone abnormalities
such as osteopetrosis. Osteopetrosis is a condition characterized
by abnormal thickening and hardening of bone and abnormal fragility
of the bones. As such, PRO731 polypeptides or agonists thereof
would be beneficial for the treatment of osteopetrosis or other
osteo diseases. On the other hand, inhibitors or antagonists of
PRO731 polypeptides would be useful in bone healing.
[1496] 38.15. Generation and Analysis of Mice Comprising
DNA48334-1435 (UNQ396) Gene Disruptions
[1497] In these knockout experiments, the gene encoding PRO732
polypeptides (designated as DNA48334-1435) (UNQ396) was disrupted.
The gene specific information for these studies is as follows: the
mutated mouse gene corresponds to nucleotide reference:
NM.sub.--019760 or ACCESSION: NM.sub.--019760 NID: gi 9790268 ref
NM.sub.--019760.1 Mus musculus tumor differentially expressed 1,
like (Tde1l); protein reference: Q9QZI8 or ACCESSION: Q9QZI8 NID:
Mus musculus (Mouse). Tumor differentially expressed 1 protein like
(Membrane protein TMS-2). MOUSESPTRNRDB; the human gene sequence
reference: NM.sub.--020755 or ACCESSION: NM.sub.--020755 NID: gi
24308212 ref NM.sub.--020755.1 Homo sapiens likely ortholog of
mouse tumor differentially expressed 1, like (TDE1L); the human
protein sequence corresponds to reference: Q9NRX5 or ACCESSION:
Q9NRX5 NID: Homo sapiens (Human). Tumor differentially expressed 1
protein like. HUMANSPTRNRDB.
[1498] The mouse gene of interest is Tde2 (tumor differentially
expressed 2), ortholog of human TDE2. Aliases include Tms2, TMS-2,
Tde1l, AIGP2, membrane protein TMS-2, and tumor differentially
expressed 1-like.
TDE2 is a putative integral plasma membrane protein expressed in
neurons of the central nervous system (Grossman et al., J Exp Biol
203 Pt 3:447-57 (2000)) and in several other tissues, such as
bladder, kidney, and muscle (Player et al, Int J Cancer,
107(2):238-43 (2003)). The protein consists of several
transmembrane segments contained within a "TMS membrane
protein/tumor differentially expressed protein (TDE)" domain. This
domain is found in several other proteins, constituting a family
that is differentially expressed in various tumor and cell lines
(Pfam accession PF03348). For example, TDE2 expression tends to be
higher in non small cell lung cancers than in adjacent normal
tissue. In contrast, TDE2 expression in lung tumors tends to be
lower than that in adjacent nonmalignant bronchiole epithelium
(Player et al., Int J Cancer, 107(2):238-43 (2003)). The function
of this protein is not known.
Genetics Information:
TABLE-US-00064 [1499] wt het hom Total Observed 13 32 9 54 Expected
13.5 27 13.5 54 Chi-Sq. = 2.44 Significance = 0.29457 (hom/n) =
0.17 Avg. Litter Size = 6
Mutation Type: Retroviral Insertion (OST)
[1500] Retroviral insertion occurred in the intron between coding
exons 1 and 2 (NCBI accession NM.sub.--019760.1). Wild-type
expression of the target gene was detected in all 13 adult tissue
samples tested by RT-PCR, except bone. RT-PCR analysis revealed
that the transcript was absent in the (-/-) mouse analyzed (M-133).
Disruption of the target gene was confirmed by Inverse PCR.
[1501] 35.15.1. Phenotypic Analysis (for Disrupted Gene:
DNA48334-1435 (UNQ396)
[1502] (a) Overall Phenotypic Summary:
[1503] Mutation of the gene encoding the ortholog of human tumor
differentially expressed 2 (TDE2) resulted in growth retardation as
well as bone abnormalities (decreased bone measurements) in (-/-)
mice. The (-/-) mice also exhibited an increased mean serum glucose
level and impaired glucose tolerance. Hydrocephalus was noted in
several of the (-/-) mutant mice. In addition, open field testing
revealed increased activity during open field testing in the mutant
(-/-) mice. Transcript was absent by RT-PCR.
[1504] (b) Bone Metabolism & Body Diagnostics
[1505] (1) Tissue Mass & Lean Body Mass Measurements--Dexa
[1506] Dexa Analysis--Test Description:
[1507] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. Dual Energy X-ray
Absorptiometry (DEXA) has been used successfully to identify
changes in total tissue mass (TTM).
[1508] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PIXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI,
i.e., whole body, vertebrae, and both femurs).
[1509] Body Measurements (Body Length & Weight):
[1510] Body Measurements: A measurement of body length and weight
was performed at approximately 16 weeks of age.
[1511] Results:
[1512] Both the male and female (-/-) mice exhibited decreased mean
body weight and mean body length when compared with their
gender-matched (+/+) littermates and the historical means. The
difference was more notable in the males. Analyzed wt/het/hom:
20/45/10
[1513] Pathology/Microscopic Observations: Hydrocephalus was noted
in several (-/-) mice available for analysis.
[1514] Gene Expression: LacZ activity was not detected in the panel
of tissues by immunohistochemical analysis. Analyzed wt/het/hom:
2/2/6
[1515] (2) Bone Metabolism: Radiology Phenotypic Analysis
[1516] In the area of bone metabolism, targets were identified
herein for the treatment of arthritis, osteoporosis, osteopenia and
osteopetrosis as well as identifying targets that promote bone
healing. Tests included:
[1517] DEXA for measurement of bone mineral density on femur and
vertebra
[1518] MicroCT for very high resolution and very high sensitivity
measurements of bone mineral density for both trabecular and
cortical bone.
[1519] Dexa Analysis--Test Description:
[1520] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. Dual Energy X-ray
Absorptiometry (DEXA) has been used successfully to identify
changes in bone. Anesthetized animals were examined and bone
mineral content (BMC), BMC/LBM ratios, volumetric bone mineral
density (vBMD), total body BMD, femur BMD and vertebra BMD were
measured.
[1521] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PIXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI)
[i.e., whole body, vertebrae, and both femurs].
[1522] Bone MicroCT Analysis:
[1523] Procedure: MicroCT was also used to get very sensitive
measurements of BMD. One vertebra and 1 femur were taken from a
cohort of 4 wild type and 8 homozygous mice. Measurements were
taken of lumbar 5 vertebra trabecular bone volume, trabecular
thickness, connectivity density and midshaft femur total bone area
and cortical thickness. The .mu.CT40 scans provided detailed
information on bone mass and architecture. Multiple bones were
placed into sample holders and scanned automatically. Instrument
software was used to select regions of interest for analysis.
Trabecular bone parameters were analyzed in the fifth lumbar
vertebrae (LV5) at 16 micrometer resolution and cortical bone
parameters were analyzed in the femur midshaft at a resolution of
20 micrometers.
[1524] Results:
[1525] DEXA: The (-/-) mice exhibited decreased mean total tissue
mass and lean body mass when compared with their gender-matched
(+/+) littermates and the historical means. These mutant animals
also exhibited decreased mean bone mineral content and bone mineral
density-related measurements.
[1526] Micro-CT: The (-/-) mice exhibited decreased mean vertebral
trabecular bone volume, number, thickness, and connectivity density
and decreased mean femoral mid-shaft cortical thickness when
compared with their gender-matched (+/+) littermates and the
historical means.
[1527] Analyzed wt/het/hom: 4/4/8
[1528] Summary
[1529] These results demonstrate that knockout mutant mice exhibit
abnormal bone metabolism with significant bone loss similar to
osteoporosis characterized by decrease in bone mass with decreased
density and possibly fragility leading to bone fractures. Thus, it
appears that PRO732 or agonists thereof would play a role in
maintaining bone homeostasis. In addition, PRO732 or its encoding
gene would be useful for maintaining bone homeostasis and for bone
healing or for the treatment of arthritis or osteoporosis; whereas
antagonists to PRO732 or its encoding gene would lead to abnormal
or pathological bone disorders including inflammatory diseases
associated with abnormal bone metabolism including arthritis,
osteoporosis, and osteopenia.
[1530] The (-/-) mice analyzed by DEXA exhibited notably decreased
total tissue mass and lean body mass when compared with their (+/+)
littermates, suggestive of growth retardation in these mutants.
This in conjunction with the observations of abnormal bone
measurements suggest a tissue wasting condition or other growth
related disorders such as cachexia. Thus, PRO732 polypeptides or
agonists thereof would be useful in the treatment of bone disorders
but also would be useful for the prevention of growth related
disorders such as cachexia and/or other tissue wasting
diseases.
[1531] (c) Phenotypic Analysis: Metabolism-Blood Chemistry/Glucose
Tolerance
[1532] In the area of metabolism, targets may be identified for the
treatment of diabetes. Blood chemistry phenotypic analysis includes
blood glucose measurements. The COBAS Integra 400 (mfr: Roche) was
used for running blood chemistry tests on the mice. In the area of
metabolism, targets may be identified for the treatment of
diabetes. Blood chemistry phenotypic analysis includes glucose
tolerance tests to measure insulin sensitivity and changes in
glucose metabolism. Abnormal glucose tolerance test results may
indicate but may not be limited to the following disorders or
conditions: Diabetes Type 1 and Type 2, Syndrome X, various
cardiovascular diseases and/or obesity.
[1533] Procedure: A cohort of 2 wild type and 4 homozygous mice
were used in this assay. The glucose tolerance test is the standard
for defining impaired glucose homeostasis in mammals. Glucose
tolerance tests were performed using a Lifescan glucometer. Animals
were injected IP at 2 g/kg with D-glucose delivered as a 20%
solution and blood glucose levels were measured at 0, 30, 60 and 90
minutes after injection.
[1534] Results:
[1535] Blood Chemistry: The (-/-) mice exhibited an increased mean
serum glucose level when compared with their gender-matched (+/+)
littermates and the historical mean. In the glucose tolerance test:
4 (-/-) mice analyzed, 2 (M-157 and M-167), exhibited increased
fasting serum glucose levels when compared with their
gender-matched (+/+) littermates and the historical mean. Analyzed
wt/het/hom: 4/5/8
[1536] These studies indicated that (-/-) mice exhibit a decreased
glucose tolerance in the presence of normal fasting glucose at all
3 intervals tested when compared with their gender-matched (+/+)
littermates and the historical means. Thus, knockout mice exhibited
the phenotypic pattern of an impaired glucose homeostasis, and
therefor PRO732 polypeptides (or agonists thereof) or its encoding
gene would be useful in the treatment of impaired glucose
homeostasis.
[1537] (d) Phenotypic Analysis: CNS/Neurology
[1538] In the area of neurology, analysis focused herein on
identifying in vivo validated targets for the treatment of
neurological and psychiatric disorders including depression,
generalized anxiety disorders, attention deficit hyperactivity
disorder, obsessive compulsive disorder, schizophrenia, cognitive
disorders, hyperalgesia and sensory disorders. Neurological
disorders include the category defined as "anxiety disorders" which
include but are not limited to: mild to moderate anxiety, anxiety
disorder due to a general medical condition, anxiety disorder not
otherwise specified, generalized anxiety disorder, panic attack,
panic disorder with agoraphobia, panic disorder without
agoraphobia, posttraumatic stress disorder, social phobia, specific
phobia, substance-induced anxiety disorder, acute alcohol
withdrawal, obsessive compulsive disorder, agoraphobia, bipolar
disorder I or II, bipolar disorder not otherwise specified,
cyclothymic disorder, depressive disorder, major depressive
disorder, mood disorder, substance-induced mood disorder. In
addition, anxiety disorders may apply to personality disorders
including but not limited to the following types: paranoid,
antisocial, avoidant behavior, borderline personality disorders,
dependent, histronic, narcissistic, obsessive-compulsive, schizoid,
and schizotypal.
[1539] Procedure:
[1540] Behavioral screens were performed on a cohort of 4 wild
type, 4 heterozygous and 8 homozygous mutant mice. All behavioral
tests were done between 12 and 16 weeks of age unless reduced
viability necessitates earlier testing. These tests included open
field to measure anxiety, activity levels and exploration.
[1541] Open Field Test:
[1542] Several targets of known drugs have exhibited phenotypes in
the open field test. These include knockouts of the seratonin
transporter, the dopamine transporter (Giros et al., Nature. 1996
Feb. 15; 379(6566):606-12), and the GABA receptor (Homanics et al.,
Proc Natl Acad Sci USA. 1997 Apr. 15; 94(8):4143-8). An automated
open-field assay was customized to address changes related to
affective state and exploratory patterns related to learning First,
the field (40.times.40 cm) was selected to be relatively large for
a mouse, thus designed to pick up changes in locomotor activity
associated with exploration. In addition, there were 4 holes in the
floor to allow for nose-poking, an activity specifically related to
exploration. Several factors were also designed to heighten the
affective state associated with this test. The open-field test is
the first experimental procedure in which the mice are tested, and
the measurements that were taken were the subjects' first
experience with the chamber. In addition, the open-field was
brightly lit. All these factors will heighten the natural anxiety
associated with novel and open spaces. The pattern and extent of
exploratory activity, and especially the center-to-total distance
traveled ratio, may then be able to discern changes related to
susceptibility to anxiety or depression. A large arena (40
cm.times.40 cm, VersaMax animal activity monitoring system from
AccuScan Instruments) with infrared beams at three different levels
was used to record rearing, hole poke, and locomotor activity. The
animal was placed in the center and its activity was measured for
20 minutes. Data from this test was analyzed in five, 4-minute
intervals. The total distance traveled (cm), vertical movement
number (rearing), number of hole pokes, and the center to total
distance ratio were recorded.
[1543] The propensity for mice to exhibit normal habituation
responses to a novel environment is assessed by determining the
overall change in their horizontal locomotor activity across the 5
time intervals. This calculated slope of the change in activity
over time is determined using normalized, rather than absolute,
total distance traveled. The slope is determined from the
regression line through the normalized activity at each of the 5
time intervals. Normal habituation is represented by a negative
slope value. Analyzed wt/het/hom: 5/4/8
[1544] Results:
[1545] The (-/-) mice exhibited a decreased median sum
time-in-center during open field testing when compared with their
gender-matched (+/+) littermates and the historical mean,
suggesting an increased anxiety-like response in the mutants. As
noted above, a notable difference was observed during open field
activity testing. The (-/-) mice exhibited a decreased median sum
time in the center area when compared with their gender-matched
(+/+) littermates. This type of behavior is consistent with an
increased anxiety like response. Thus, the knockout mice
demonstrated a phenotype consistent with anxiety related disorders
which are associated with mild to moderate anxiety, anxiety due to
a general medical condition, and/or bipolar disorders;
hyperactivity; sensory disorders; obsessive-compulsive disorders,
schizophrenia or a paranoid personality. Thus, PRO732 polypeptides
or agonists thereof would be useful in the treatment of such
neurological disorders or the amelioration of the symptoms
associated with anxiety disorders.
[1546] 35.16. Generation and Analysis of Mice Comprising
DNA58846-1409 (UN0487) Gene Disruptions
[1547] In these knockout experiments, the gene encoding PRO1003
polypeptides (designated as DNA58846-1409) (UNQ487) was disrupted.
The gene specific information for these studies is as follows: the
mutated mouse gene corresponds to nucleotide reference:
NM.sub.--032541 or ACCESSION: NM.sub.--032541 NID: gi 14211541 ref
NM.sub.--032541.1 Mus musculus hepcidin antimicrobial peptide
(Hamp); protein reference: Q9EQ21 or ACCESSION: Q9EQ21 NID: Mus
musculus (Mouse). PROHEPCIDIN (HEPCIDIN ANTIMICROBIAL PEPTIDE).
MOUSESPTRNRDB; the human gene sequence reference: NM.sub.--021175
or ACCESSION: NM.sub.--021175 NID: gi 10863972 ref
NM.sub.--021175.1 Homo sapiens hepcidin antimicrobial peptide
(HAMP); the human protein sequence corresponds to reference: P81172
or ACCESSION: P81172 NID: Homo sapiens (Human). ANTIMICROBIAL
PEPTIDE HEPCIDIN PRECURSOR (LIVER-EXPRESSED ANTIMICROBIAL PEPTIDE)
(LEAP-1) (PUTATIVE LIVER TUMOR REGRESSOR) (PLTR) [CONTAINS:
HEPCIDIN 25 (HEPC25); HEPCIDIN 20 (HEPC20)]. HUMANSPTRNRDB.
[1548] The disrupted mouse gene is hepcidin antimicrobial peptide
(Hamp), ortholog of human HAMP. Aliases include HEPC1, HEPC, HFE2,
LEAP1, LEAP-1, and liver-expressed antimicrobial peptide.
[1549] HAMP is a protein secreted mainly from liver that functions
as an iron regulatory hormone and mediator of innate immunity The
84-amino acid protein contains a signal peptide, a propeptide, and
a 25-amino acid hepcidin core at the C-terminus. The positively
charged hydrophilic residues and the hydrophobic residues of the
mature 25 amino acid hepcidin core peptide are spatially separated,
enabling the peptide to disrupt microbial membranes (Ganz Tomas.,
Blood 102(3):783-8 (2003)).
[1550] HAMP is involved in iron homeostasis. Increases in iron load
increase HAMP expression, resulting in decreased dietary iron
absorption, transplacental iron transport, and iron mobilization
from splenic and hepatic macrophages. Mutations in the HAMP gene
can cause juvenile hereditary hemochromatosis, leading to iron
overload, cirrhosis, cardiomyopathy, arthritis, and endocrine
failure. HAMP has strong antimicrobial activity against bacteria
and some fungi. However, individuals lacking active HAMP still have
the ability to prevent infection, suggesting that the role of HAMP
as an antimicrobial is not critical (Roetto et al., Nat Genet,
33(1):21-2 (2003)). Inflammation upregulates HAMP, possibly
resulting in iron disorders and anemia in response to inflammatory
diseases such as infectious disease, osteoarticular diseases, and
malignancies (Ganz Tomas, Blood, 102(3):783-8 (2003)).
[1551] Nicolas and colleagues, Proc Natl Acad Sci USA,
98(15):8780-5 (2001) showed that HAMP-deficient mice (caused by
knockout of a transcription factor gene that regulates HAMP
expression) develop multivisceral iron overload. In a mouse model
of hemochromatosis, Nicolas and colleagues, Nat Genet, 34(1):97-101
(2003) showed that iron accumulation normally occurring in these
mice was inhibited by overexpression of HAMP. In wild-type mice,
Nicolas and colleagues, Proc Natl Acad Sci USA, 99(7):4596-601
(2002) showed that HAMP overexpression produced mice with pale skin
that died a few hours after birth. These animals had decreased body
iron levels and presented severe microcytic hypochromic anemia.
[1552] Targeted or gene trap mutations were generated in strain
129SvEv.sup.Brd-derived embryonic stem (ES) cells. The chimeric
mice were bred to C57BL/6J albino mice to generate F1 heterozygous
animals. These progeny were intercrossed to generate F2 wild type,
heterozygous, and homozygous mutant progeny. On rare occasions, for
example when very few F1 mice were obtained from the chimera, F1
heterozygous mice were crossed to 129SvEv.sup.Brd/C57 hybrid mice
to yield additional heterozygous animals for the intercross to
generate the F2 mice.
Level I phenotypic analysis is performed on mice from this
generation
TABLE-US-00065 wt het hom Total Observed 20 33 24 77 Expected 19.25
38.5 19.25 77 Chi-Sq. = 1.99 Significance = 0.37028 (hom/n) = 0.31
Avg. Litter Size = 8
Mutation Type: Homologous Recombination (standard) Coding exons 1
and 2 were targeted (NCBI accession NM.sub.--032541.1). Wild-type
expression of the target gene was detected in embryonic stem (ES)
cells and, in all 13 adult tissues samples tested by RT-PCR, except
liver, heart and adipose. Disruption of the target gene was
confirmed by Southern hybridization analysis.
35.16.1. Phenotypic Analysis (for Disrupted Gene: DNA58846-1409
(UNQ487)
[1553] (a) Overall Phenotypic Summary:
[1554] Mutation of the gene encoding the ortholog of human hepcidin
antimicrobial peptide (HAMP) resulted in enhanced glucose tolerance
in (-/-) mice. The knockout mice (-/-) also showed elevated levels
of serum uric acid. Gene disruption was confirmed by Southern
blot.
[1555] (b) Phenotypic Analysis: Metabolism-Blood Chemistry/Glucose
Tolerance
[1556] In the area of metabolism, targets may be identified for the
treatment of diabetes. Blood chemistry phenotypic analysis includes
blood glucose measurements. The COBAS Integra 400 (mfr: Roche) was
used for running blood chemistry tests on the mice. In the area of
metabolism, targets may be identified for the treatment of
diabetes. Blood chemistry phenotypic analysis includes glucose
tolerance tests to measure insulin sensitivity and changes in
glucose metabolism. Abnormal glucose tolerance test results may
indicate but may not be limited to the following disorders or
conditions: Diabetes Type 1 and Type 2, Syndrome X, various
cardiovascular diseases and/or obesity.
[1557] Procedure: A cohort of 2 wild type and 4 homozygous mice
were used in this assay. The glucose tolerance test is the standard
for defining impaired glucose homeostasis in mammals. Glucose
tolerance tests were performed using a Lifescan glucometer. Animals
were injected IP at 2 g/kg with D-glucose delivered as a 20%
solution and blood glucose levels were measured at 0, 30, 60 and 90
minutes after injection. Analyzed wt/het/horn: 4/4/9
[1558] Results:
[1559] The (-/-) mice exhibited enhanced glucose tolerance and a
significant decrease in mean serum glucose when compared with their
gender-matched (+/+) littermates and the historical means. Thus,
knockout mice exhibited the opposite phenotypic pattern of an
impaired glucose homeostasis, and as such antagonists to PRO1003 or
its encoding gene would be useful in the treatment of impaired
glucose homeostasis and/or any associated metabolic disorder. In
addition, the knockout mutant mice (-/-) exhibited elevated serum
uric acid levels (greater than 2 SD above the historical mean).
However, there were no other indices of renal compromise.
[1560] 35.17. Generation and Analysis of Mice Comprising
DNA59616-1465 (UNQ547) Gene Disruptions
[1561] In these knockout experiments, the gene encoding PRO1104
polypeptides (designated as DNA59616-1465) (UNQ547) was disrupted.
The gene specific information for these studies is as follows: the
mutated mouse gene corresponds to nucleotide reference:
XM.sub.--131066 or Mus musculus similar to hypothetical protein
FLJ20519 (LOC229588); protein reference: XP.sub.--131066 or similar
to hypothetical protein FLJ20519 [Mus musculus]; the human gene
sequence reference: NM.sub.--017860 or Homo sapiens hypothetical
protein FLJ20519 (FLJ20519); the human protein sequence corresponds
to reference: NP.sub.--060330 or hypothetical protein FLJ20519
[Homo sapiens].
The disrupted mouse gene is a hypothetical protein (interim name,
LOC229588), which is orthologous to human hypothetical protein
FLJ20519.
[1562] A signal peptide-like region is predicted at the N-terminus
through bioinformatic analysis. Overall, the protein is predicted
to be secreted or to reside in the plasma membrane.
Genetics Information:
TABLE-US-00066 [1563] wt het hom Total Observed 8 19 20 47 Expected
11.75 23.5 11.75 47 Chi-Sq. = 7.85 Significance = 0.01973 (hom/n) =
0.43 Avg. Litter Size = 7
Mutation Type: Retroviral Insertion (OST)
[1564] Retroviral insertion occurred in the first coding exon
(Accession: XM.sub.--131066). Wild-type expression of the target
gene was detected in embryonic stem (ES) cells and, among the 13
adult tissue samples tested by RT-PCR, in spinal cord, eye, thymus,
and lung. RT-PCR analysis revealed that the transcript was absent
in the (-/-) mouse analyzed (M-104). Disruption of the target gene
was confirmed by Inverse PCR.
[1565] 35.17.1. Phenotypic Analysis (for Disrupted Gene:
DNA59616-1465 (UNQ547)
[1566] (a) Overall Phenotypic Summary:
[1567] Mutation of the gene encoding the ortholog of a human
hypothetical protein (FLJ2519) resulted in an increased body weight
and length measurements as well as increased body mass and femoral
bone measurements. The ovalbumin (OVA) challenge resulted in
reduced anti-OVA titers in the homozygous knockout mice. In
addition, the (-/-) mice showed an enhanced glucose tolerance
compared with their wild-type littermates. Transcript was absent by
RT-PCR.
[1568] (b) Immunology Phenotypic Analysis
[1569] Immune related and inflammatory diseases are the
manifestation or consequence of fairly complex, often multiple
interconnected biological pathways which in normal physiology are
critical to respond to insult or injury, initiate repair from
insult or injury, and mount innate and acquired defense against
foreign organisms. Disease or pathology occurs when these normal
physiological pathways cause additional insult or injury either as
directly related to the intensity of the response, as a consequence
of abnormal regulation or excessive stimulation, as a reaction to
self, or as a combination of these.
[1570] Though the genesis of these diseases often involves
multistep pathways and often multiple different biological
systems/pathways, intervention at critical points in one or more of
these pathways can have an ameliorative or therapeutic effect.
Therapeutic intervention can occur by either antagonism of a
detrimental process/pathway or stimulation of a beneficial
process/pathway.
[1571] T lymphocytes (T cells) are an important component of a
mammalian immune response. T cells recognize antigens which are
associated with a self-molecule encoded by genes within the major
histocompatibility complex (MHC). The antigen may be displayed
together with MHC molecules on the surface of antigen presenting
cells, virus infected cells, cancer cells, grafts, etc. The T cell
system eliminates these altered cells which pose a health threat to
the host mammal. T cells include helper T cells and cytotoxic T
cells. Helper T cells proliferate extensively following recognition
of an antigen-MHC complex on an antigen presenting cell. Helper T
cells also secrete a variety of cytokines, i.e., lymphokines, which
play a central role in the activation of B cells, cytotoxic T cells
and a variety of other cells which participate in the immune
response.
[1572] In many immune responses, inflammatory cells infiltrate the
site of injury or infection. The migrating cells may be
neutrophilic, eosinophilic, monocytic or lymphocytic as can be
determined by histological examination of the affected tissues.
Current Protocols in Immunology, ed. John E. Coligan, 1994, John
Wiley & Sons, Inc.
[1573] Many immune related diseases are known and have been
extensively studied. Such diseases include immune-mediated
inflammatory diseases (such as rheumatoid arthritis, immune
mediated renal disease, hepatobiliary diseases, inflammatory bowel
disease (IBD), psoriasis, and asthma), non-immune-mediated
inflammatory diseases, infectious diseases, immunodeficiency
diseases, neoplasia, and graft rejection, etc. In the area of
immunology, targets were identified for the treatment of
inflammation and inflammatory disorders.
[1574] In the area of immunology, targets have been identified
herein for the treatment of inflammation and inflammatory
disorders. Immune related diseases, in one instance, could be
treated by suppressing the immune response. Using neutralizing
antibodies that inhibit molecules having immune stimulatory
activity would be beneficial in the treatment of immune-mediated
and inflammatory diseases. Molecules which inhibit the immune
response can be utilized (proteins directly or via the use of
antibody agonists) to inhibit the immune response and thus
ameliorate immune related disease.
[1575] The following test was performed:
[1576] Ovalbumin Challenge
[1577] Procedure: This assay was carried out on 7 wild types and 8
homozygotes. Chicken ovalbumin (OVA) is a T-cell dependent antigen,
which is commonly used as a model protein for studying
antigen-specific immune responses in mice. OVA is non-toxic and
inert and therefore will not cause harm to the animals even if no
immune response is induced. The murine immune response to OVA has
been well characterized, to the extent that the immuno-dominant
peptides for eliciting T cell responses have been identified.
Anti-OVA antibodies are detectable 8 to 10 days after immunization
using enzyme-linked immunosorbent assay (ELIZA), and determination
of different isotypes of antibodies gives further information on
the complex processes that may lead to a deficient response in
genetically engineered mice.
[1578] As noted above, this protocol assesses the ability of mice
to raise an antigen-specific immune response. Animals were injected
IP with 50 mg of chicken ovalbumin emulsified in Complete Freund's
Adjuvant and 14 days later the serum titer of anti-ovalbumin
antibodies (IgM, IgG1 and IgG2 subclasses) was measured. The amount
of OVA-specific antibody in the serum sample is proportional to the
Optical Density (OD) value generated by an instrument that scans a
96-well sample plate. Data was collected for a set of serial
dilutions of each serum sample. Analyzed wt/het/hom: 7/4/8
[1579] Results of this challenge: The (-/-) mice exhibited a
decreased mean serum IgG2a response to the ovalbumin challenge when
compared with their (+/+) littermates. Thus, these knockout mice
exhibited an decreased ability to elicit an OVA-specific antibody
response to the T-cell dependent OVA antigen more than likely due
to a defect in Th cells. PRO1104 polypeptides or agonists thereof
would therefore be expected to stimulate the immune system and
would find utility in the cases wherein this effect would be
beneficial to the individual such as in the case of leukemia, and
other types of cancer, and in immunocompromised patients, such as
AIDS sufferers. Accordingly, inhibitors or antagonists of PRO1104
polypeptides would play a role in inhibiting the immune response
and would be useful candidates for suppressing harmful immune
responses, e.g. in the case of graft rejection or graft-versus-host
diseases.
[1580] (c) Bone Metabolism & Body Diagnostics
[1581] (1) Tissue Mass & Lean Body Mass Measurements--Dexa
[1582] Dexa Analysis--Test Description:
[1583] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. Dual Energy X-ray
Absorptiometry (DEXA) has been used successfully to identify
changes in total tissue mass (TTM).
[1584] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PIXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI,
i.e., whole body, vertebrae, and both femurs).
[1585] Body Measurements (Body Length & Weight):
[1586] Body Measurements: A measurement of body length and weight
was performed at approximately 16 weeks of age.
[1587] Results:
[1588] The (-/-) mice exhibited increased mean body weight and
increased mean body length (by at least one (1) standard deviation
heavier than controls before 8 weeks of age) when compared with
their gender-matched (+/+) littermates and the historical mean.
Analyzed wt/het/hom: 15/22/16
[1589] (2) Bone Metabolism: Radiology Phenotypic Analysis
[1590] In the area of bone metabolism, targets were identified
herein for the treatment of arthritis, osteoporosis, osteopenia and
osteopetrosis as well as identifying targets that promote bone
healing. Tests included:
[1591] DEXA for measurement of bone mineral density on femur and
vertebra
[1592] MicroCT for very high resolution and very high sensitivity
measurements of bone mineral density for both trabecular and
cortical bone.
[1593] Dexa Analysis--Test Description:
[1594] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. Dual Energy X-ray
Absorptiometry (DEXA) has been used successfully to identify
changes in bone. Anesthetized animals were examined and bone
mineral content (BMC), BMC/LBM ratios, volumetric bone mineral
density (vBMD), total body BMD, femur BMD and vertebra BMD were
measured.
[1595] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PIXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI)
[i.e., whole body, vertebrae, and both femurs].
[1596] Bone MicroCT Analysis:
[1597] Procedure: MicroCT was also used to get very sensitive
measurements of BMD. One vertebra and 1 femur were taken from a
cohort of 4 wild type and 8 homozygous mice. Measurements were
taken of lumbar 5 vertebra trabecular bone volume, trabecular
thickness, connectivity density and midshaft femur total bone area
and cortical thickness. The .mu.CT40 scans provided detailed
information on bone mass and architecture. Multiple bones were
placed into sample holders and scanned automatically. Instrument
software was used to select regions of interest for analysis.
Trabecular bone parameters were analyzed in the fifth lumbar
vertebrae (LV5) at 16 micrometer resolution and cortical bone
parameters were analyzed in the femur midshaft at a resolution of
20 micrometers.
[1598] Results:
[1599] DEXA: The (-/-) mice exhibited increased mean total tissue
mass and lean body mass when compared with their gender-matched
(+/+) littermates and the historical means.
[1600] Micro-CT: The (-/-) mice exhibited increased mean femoral
mid-shaft cross-sectional area when compared with their
gender-matched (+/+) littermates and the historical means.
[1601] Analyzed wt/het/hom: 4/4/8
[1602] In summary, the (-/-) mice exhibited increased body weight
and length, increased mean total tissue mass and lean body mass and
increased bone cross-sectional measurements when compared with
their gender-matched (+/+) littermates. These observations suggests
an obesity and/or growth disorder type phenotype. In addition, the
mutant (-/-) mice exhibited an abnormal bone development. Thus,
PRO1104 polypeptides or agonists thereof, would be useful for
normal growth and bone development and would play a role in the
treatment of related growth or metabolic disorders associated with
obesity and/or bone disorders.
[1603] (d) Phenotypic Analysis: Metabolism-Blood Chemistry/Glucose
Tolerance
[1604] In the area of metabolism, targets may be identified for the
treatment of diabetes. Blood chemistry phenotypic analysis includes
blood glucose measurements. The COBAS Integra 400 (mfr: Roche) was
used for running blood chemistry tests on the mice. In the area of
metabolism, targets may be identified for the treatment of
diabetes. Blood chemistry phenotypic analysis includes glucose
tolerance tests to measure insulin sensitivity and changes in
glucose metabolism. Abnormal glucose tolerance test results may
indicate but may not be limited to the following disorders or
conditions: Diabetes Type 1 and Type 2, Syndrome X, various
cardiovascular diseases and/or obesity.
[1605] Procedure: A cohort of 2 wild type and 4 homozygous mice
were used in this assay. The glucose tolerance test is the standard
for defining impaired glucose homeostasis in mammals. Glucose
tolerance tests were performed using a Lifescan glucometer. Animals
were injected IP at 2 g/kg with D-glucose delivered as a 20%
solution and blood glucose levels were measured at 0, 30, 60 and 90
minutes after injection. Analyzed wt/het/horn: 4/4/9
[1606] Results:
[1607] The (-/-) mice exhibited enhanced glucose tolerance and a
significant decrease in mean serum glucose when compared with their
gender-matched (+/+) littermates and the historical means. Thus,
knockout mice exhibited the opposite phenotypic pattern of an
impaired glucose homeostasis, and as such antagonists to PRO1104 or
its encoding gene would be useful in the treatment of impaired
glucose homeostasis and/or any associated metabolic disorder.
[1608] 35.18. Generation and Analysis of Mice Comprising
DNA44694-1500 (UNQ581) Gene Disruptions
[1609] In these knockout experiments, the gene encoding PRO1151
polypeptides (designated as DNA44694-1500) (UNQ581) was disrupted.
The gene specific information for these studies is as follows: the
mutated mouse gene corresponds to nucleotide reference:
NM.sub.--028331 or Mus musculus C1q and tumor necrosis factor
related protein 6 (C1qtnf6); protein reference: Q8BKR0 or
ACCESSION: Q8BKR0 NID: Mus musculus (Mouse). Weakly similar to
complement-C1Q tumor necrosis factor-related protein; the human
gene sequence reference: NM.sub.--031910 or Homo sapiens C1q and
tumor necrosis factor related protein 6 (C1QTNF6), transcript
variant 1; the human protein sequence corresponds to reference:
NP.sub.--114116 or C1q and tumor necrosis factor related protein 6;
complement-c1q tumor necrosis factor-related protein 6 [Homo
sapiens] gi|32967300|ref|NP.sub.--872292.1| C1q and tumor necrosis
factor related protein 6; complement-c1q tumor necrosis
factor-related protein 6 [Homo sapiens] gi|13274531|gb|AAK17966.1|
complement-c1q tumor necrosis factor-related protein [Homo
sapiens].
[1610] The mouse gene of interest is C1qtnf6 (C1q and tumor
necrosis factor related protein 6), ortholog of human C1QTNF6.
Aliases include CTRP6, ZACRP6, and complement-c1q tumor necrosis
factor-related protein 6.
[1611] C1QTNF6 is a putative secreted protein, consisting of a
signal peptide, a collagen triple helix repeat, and a "complement
component C1q" domain. Collagen triple helix repeats are most often
found in collagens, which are generally extracellular structural
proteins (Pfam accession PF01391). C1q domains are found in many
collagens and in the C1q subunit of C1 enzyme complex, which
activates the serum complement system. C1q and tumor necrosis
factor fold similarly (SMART accession SM00110). The function of
this protein is not known.
[1612] Targeted or gene trap mutations were generated in strain
129SvEv.sup.Brd-derived embryonic stem (ES) cells. The chimeric
mice were bred to C57BL/6J albino mice to generate F1 heterozygous
animals. These progeny were intercrossed to generate F2 wild type,
heterozygous, and homozygous mutant progeny. On rare occasions, for
example when very few F1 mice were obtained from the chimera, F1
heterozygous mice were crossed to 129SvEv.sup.Brd/C57 hybrid mice
to yield additional heterozygous animals for the intercross to
generate the F2 mice.
Level I phenotypic analysis is performed on mice from this
generation
TABLE-US-00067 wt het hom Total Observed 18 25 13 56 Expected 14 28
14 56 Chi-Sq. = 1.54 Significance = 0.46401 (hom/n) = 0.23 Avg.
Litter Size = 8
Mutation Type: Homologous Recombination (standard) Coding exon 2
was targeted (NCBI accession NM.sub.--028331.2). Wild-type
expression of the target gene was detected in embryonic stem (ES)
cells and in all 13 adult tissue samples tested by RT-PCR, except
skeletal muscle. Disruption of the target gene was confirmed by
Southern hybridization analysis.
[1613] 35.18.1. Phenotypic Analysis (for Disrupted Gene:
DNA44694-1500 (UNQ581)
[1614] (a) Overall Phenotypic Summary:
[1615] Mutation of the gene encoding the ortholog of human C1q and
tumor necrosis factor related protein 6 (C1QTNF6) resulted in
decreased bone mineral density measurements. Gene disruption was
confirmed by Southern blot.
[1616] (b) Bone Metabolism: Radiology Phenotypic Analysis
[1617] In the area of bone metabolism, targets were identified
herein for the treatment of arthritis, osteoporosis, osteopenia and
osteopetrosis as well as identifying targets that promote bone
healing. Tests included:
[1618] DEXA for measurement of bone mineral density on femur and
vertebra
[1619] MicroCT for very high resolution and very high sensitivity
measurements of bone mineral density for both trabecular and
cortical bone.
[1620] Dexa Analysis--Test Description:
[1621] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. Dual Energy X-ray
Absorptiometry (DEXA) has been used successfully to identify
changes in bone. Anesthetized animals were examined and bone
mineral content (BMC), BMC/LBM ratios, volumetric bone mineral
density (vBMD), total body BMD, femur BMD and vertebra BMD were
measured.
[1622] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PIXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI)
[i.e., whole body, vertebrae, and both femurs].
[1623] Bone MicroCT Analysis:
[1624] Procedure: MicroCT was also used to get very sensitive
measurements of BMD. One vertebra and 1 femur were taken from a
cohort of 4 wild type and 8 homozygous mice. Measurements were
taken of lumbar 5 vertebra trabecular bone volume, trabecular
thickness, connectivity density and midshaft femur total bone area
and cortical thickness. The .mu.CT40 scans provided detailed
information on bone mass and architecture. Multiple bones were
placed into sample holders and scanned automatically. Instrument
software was used to select regions of interest for analysis.
Trabecular bone parameters were analyzed in the fifth lumbar
vertebrae (LV5) at 16 micrometer resolution and cortical bone
parameters were analyzed in the femur midshaft at a resolution of
20 micrometers.
[1625] Results:
[1626] DEXA: The (-/-) mice exhibited decreased mean bone mineral
density in total body and femur when compared with their
gender-matched (+/+) littermates and the historical means.
[1627] Micro-CT: The (-/-) mice exhibited decreased mean femoral
midshaft cortical thickness when compared with their gender-matched
(+/+) littermates and the historical means.
Analyzed wt/het/hom: 5/4/8
[1628] These results demonstrate that knockout mutant mice exhibit
abnormal bone metabolism with significant bone loss similar to
osteoporosis characterized by decrease in bone mass with decreased
density and possibly fragility leading to bone fractures. Thus, it
appears that PRO1151 or agonists thereof would be useful in
maintaining bone homeostasis. In addition, PRO1151 or its encoding
gene would be useful in bone healing or for the treatment of
arthritis or osteoporosis; whereas antagonists to PRO1151 or its
encoding gene would lead to abnormal or pathological bone disorders
including inflammatory diseases associated with abnormal bone
metabolism including arthritis, osteoporosis, and osteopenia.
[1629] 35.19. Generation and Analysis of Mice Comprising
DNA64883-1526 (UNQ628) Gene Disruptions
[1630] In these knockout experiments, the gene encoding PRO1244
polypeptides (designated as DNA64883-1526) (UNQ628) was disrupted.
The gene specific information for these studies is as follows: the
mutated mouse gene corresponds to nucleotide reference:
NM.sub.--025952 or Mus musculus RIKEN cDNA 2610529C04 gene
(2610529C04Rik); protein reference: NP.sub.--080228 or
implantation-associated protein [Mus musculus]; the human gene
sequence reference: NM.sub.--032121 or Homo sapiens
implantation-associated protein (DKFZp564K142); the human protein
sequence corresponds to reference: NP.sub.--115497 or
implantation-associated protein [Homo sapiens].
[1631] The disrupted mouse gene encodes a hypothetical protein,
which is the ortholog of human FLJ14726. Aliases include
2610529C04Rik, IAG2, 2410001C15Rik, PRO0756, DKFZp564K142,
implantation-associated protein, and implantation-associated
uterine protein.
[1632] FLJ14726 contains an OST3/OST6 motif (Pfam PF04756)
suggesting that it might be an oligosaccharide transferase and
might be located in the endoplasmic reticulum. Proteins with
similar function have been described and studied in yeast (Knauer
and Lehle, J Biol Chem, 274(24):17249-56 (1999).
This project is X-linked, hemizygotes have no notable
phenotype.
Summary of X-linked Gene Distribution by Sex and Genotype
[1633] (Only the agouti pups from the male chimeras are
included.)
TABLE-US-00068 Summary of X-linked Gene Distributions for Sex by
Genotype Agouti F1 F1a Progeny (M chimera .times. wt) Progeny (F
het .times. wt) Sex wt het Sex wt het hemi M 24 0 M 32 n/a 23 F 16
11 F 18 18 n/a
[1634] Targeted or gene trap mutations were generated in strain
129SvEv.sup.Brd-derived embryonic stem (ES) cells. The chimeric
mice were bred to C57BL/6J albino mice to generate F1 heterozygous
animals. These progeny were intercrossed to generate F2 wild type,
heterozygous, and homozygous mutant progeny. On rare occasions, for
example when very few F1 mice were obtained from the chimera, F1
heterozygous mice were crossed to 129SvEv.sup.Brd/C57 hybrid mice
to yield additional heterozygous animals for the intercross to
generate the F2 mice.
Level I phenotypic analysis is performed on mice from this
generation.
TABLE-US-00069 wt het hom Total Observed 22 18 30 70 Expected 17.5
35 17.5 70 Chi-Sq. = 18.34 Significance = 0.00010 (hom/n) = 0.43
Avg. Litter Size = 7
Mutation Type: Retroviral Insertion (OST)
[1635] Retroviral insertion occurred between coding exons 1 and 2
(Accession: NM.sub.--025952). Wild-type expression of the target
gene was detected in all 13 adult tissue samples tested by RT-PCR,
except skeletal muscle, bone, and adipose. RT-PCR analysis revealed
that the transcript was absent in the (0/-) mouse analyzed
(M-97).
[1636] 35.19.1. Phenotypic Analysis (for Disrupted Gene:
DNA64883-1526 (UNQ628)
[1637] (a) Overall Phenotypic Summary:
[1638] Mutation of the gene encoding the ortholog of human
implantation-associated protein (DKFZp564K142) protein resulted in
an increased mean serum glucose level. Transcript was absent by
RT-PCR.
[1639] (b) Phenotypic Analysis: Metabolism-Blood Chemistry/Glucose
Tolerance
[1640] In the area of metabolism, targets may be identified for the
treatment of diabetes. Blood chemistry phenotypic analysis includes
blood glucose measurements. The COBAS Integra 400 (mfr: Roche) was
used for running blood chemistry tests on the mice. In the area of
metabolism, targets may be identified for the treatment of
diabetes. Blood chemistry phenotypic analysis includes glucose
tolerance tests to measure insulin sensitivity and changes in
glucose metabolism. Abnormal glucose tolerance test results may
indicate but may not be limited to the following disorders or
conditions: Diabetes Type 1 and Type 2, Syndrome X, various
cardiovascular diseases and/or obesity.
[1641] Procedure: A cohort of 2 wild type and 4 homozygous mice
were used in this assay. The glucose tolerance test is the standard
for defining impaired glucose homeostasis in mammals. Glucose
tolerance tests were performed using a Lifescan glucometer. Animals
were injected IP at 2 g/kg with D-glucose delivered as a 20%
solution and blood glucose levels were measured at 0, 30, 60 and 90
minutes after injection. Analyzed wt/het/horn: 4/4/12
[1642] Results:
[1643] Blood Chemistry: The (-/-) mice exhibited an increased mean
serum glucose level when compared with their gender-matched (+/+)
littermates and the historical mean.
[1644] Thus, knockout mice exhibited the phenotypic pattern of an
impaired glucose homeostasis, and as such PRO1244 polypeptides or
agonists thereof, would be useful in the treatment of impaired
glucose homeostasis and/or any associated metabolic disease.
[1645] 35.20. Generation and Analysis of Mice Comprising
DNA66511-1563 (UNQ666) Gene Disruptions
[1646] In these knockout experiments, the gene encoding PRO1298
polypeptides (designated as DNA66511-1563) (UNQ666) was disrupted.
The gene specific information for these studies is as follows: the
mutated mouse gene corresponds to nucleotide reference:
NM.sub.--019998 or ACCESSION: NM.sub.--019998 NID: 9910439 Mus
musculus Mus musculus RIKEN cDNA 1300013N08 gene (1300013N08Rik);
protein reference: Q9JJA8 or Q9JJA8 Q9JJA8 BRAIN cDNA, CLONE
MNCB-5081; the human gene sequence reference: NM.sub.--033087 or
ACCESSION: NM.sub.--033087 NID: 14861835 Homo sapiens Homo sapiens
hypothetical protein FLJ14511 (FLJ14511); the human protein
sequence corresponds to reference: Q9H553 or Q9H553 Q9H553 BA13B9.1
NOVEL PROTEIN SIMILAR TO A GLYCOS.
[1647] The mouse gene of interest is Alg2 (asparagine-linked
glycosylation 2 homolog [yeast, alpha-1,3-mannosyltransferase]),
ortholog of human ALG2. Aliases include ALPG2, CDGIi, MNCb-5081,
hALPG2, FLJ14511, homolog of yeast ALG2, and
GDP-Man:Man(1)GlcNAc(2)-PP-dolichol mannosyltransferase.
[1648] ALG2 is an alpha 1,3-mannosyltransferase located in the
lumen of the endoplasmic reticulum that catalyzes the formation of
Manalpha1,3-ManGlcNAc2-PP dolichol from Man1GlcNAc2-PP-dolichol and
GDP-mannose. ALG2 appears to play an important role in glycoprotein
biosynthesis. Loss-of-function mutations in ALG2 cause congenital
disorder of glycosylation type Ii. Individuals with this mutation
are normal at birth but develop a multisystemic disorder in the
first year of life. The disorder includes mental retardation,
seizures, coloboma of the iris, hypomyelination, hepatomegaly, and
coagulation abnormalities (Thiel et al., J Biol Chem,
278(25):22498-505 (2003)).
[1649] Targeted or gene trap mutations were generated in strain
129SvEv.sup.Brd-derived embryonic stem (ES) cells. The chimeric
mice were bred to C57BL/6J albino mice to generate F1 heterozygous
animals. These progeny were intercrossed to generate F2 wild type,
heterozygous, and homozygous mutant progeny. On rare occasions, for
example when very few F1 mice were obtained from the chimera, F1
heterozygous mice were crossed to 129SvEv.sup.Brd/C57 hybrid mice
to yield additional heterozygous animals for the intercross to
generate the F2 mice.
TABLE-US-00070 wt het hom Total Observed 10 37 0 47 Expected 11.75
23.5 11.75 47 Chi-Sq. = 19.77 Significance = 0.00005 (hom/n) = 0.00
Avg. Litter Size = 5
Mutation Type: Homologous Recombination (standard) Coding exon 2
was targeted (NCBI accession NM.sub.--019998.2). Wild-type
expression of the target gene was detected in embryonic stem (ES)
cells and in all 13 adult tissue samples tested by RT-PCR, except
lung, bone, and adipose. Disruption of the target gene was
confirmed by Southern hybridization analysis.
[1650] 35.20.1. Phenotypic Analysis (for Disrupted Gene:
DNA66511-1563 (UNQ666)
[1651] (a) Overall Phenotypic Summary:
[1652] Mutation of the gene encoding the ortholog of human
asparagine-linked glycosylation 2 homolog (ALG2) resulted in
lethality of (-/-) mutants. The (+/-) mice exhibited a decreased
skin fibroblast proliferation rate. The heterozygous (+/-) mice
exhibited an increased total tissue mass, increased fat %, and
increased BMC/LBM ration, as well as increased trabecular bone
measurements. Gene disruption was confirmed by Southern blot.
[1653] Discussion Related to Embryonic Developmental Abnormality of
Lethality:
[1654] Embryonic lethality in knockout mice usually results from
various serious developmental problems including but not limited to
neuro-degenerative diseases, angiogenic disorders, inflammatory
diseases, or where the gene/protein has an important role in basic
cell signaling processes in many cell types. In addition, embryonic
lethals are useful as potential cancer models. Likewise, the
corresponding heterozygous (+/-) mutant animals are particularly
useful when they exhibit a phenotype and/or a pathology report
which reveals highly informative clues as to the function of the
knocked-out gene. For instance, EPO knockout animals were embryonic
lethals, but the pathology reports on the embryos showed a profound
lack of RBCs.
[1655] (b) Pathology
[1656] Microscopic Observations: Not tested due to embryonic
lethality. At 12.5 days, 40 embryos were observed: 28 (+/-)
embryos, 6 (+/+) embryos, 4 resorption moles, and 2
inconclusive.
[1657] Gene Expression: LacZ activity was not detected in the panel
of tissues by immunohistochemical analysis.
[1658] (c) Oncology Phenotypic Analysis
[1659] In the area of oncology, targets were identified herein for
the treatment of solid tumors, lymphomas and leukemia.
[1660] Adult Skin Cell Proliferation:
[1661] Procedure: Skin cells were isolated from 16 week old animals
(2 wild type and 4 heterozygotes). These were developed into
primary fibroblast cultures and the fibroblast proliferation rates
were measured in a strictly controlled protocol. The ability of
this assay to detect hyper-proliferative and hypo-proliferative
phenotypes has been demonstrated with p53 and Ku80. Proliferation
was measured using Brdu incorporation.
[1662] Specifically, in these studies the skin fibroblast
proliferation assay was used. An increase in the number of cells in
a standardized culture was used as a measure of relative
proliferative capacity. Primary fibroblasts were established from
skin biopsies taken from wild type and mutant mice. Duplicate or
triplicate cultures of 0.05 million cells were plated and allowed
to grow for six days. At the end of the culture period, the number
of cells present in the culture was determined using a electronic
particle counter.
[1663] Results:
[1664] The (+/-) mice exhibited a decreased mean skin fibroblast
proliferation rate when compared with their gender-matched (+/+)
littermates and the historical mean. Thus, heterozygous mutant mice
demonstrated a hypo-proliferative phenotype. As suggested by these
observations, antagonists of a PRO1298 polypeptide or its encoding
gene would be useful in the treatment of diseases associated with
abnormal cell proliferation.
[1665] (d) Bone Metabolism & Body Diagnostics: Bone Metabolism:
Radiology Phenotypic Analysis
[1666] In the area of bone metabolism, targets were identified
herein for the treatment of arthritis, osteoporosis, osteopenia and
osteopetrosis as well as identifying targets that promote bone
healing. Tests included:
[1667] DEXA for measurement of bone mineral density on femur and
vertebra
[1668] MicroCT for very high resolution and very high sensitivity
measurements of bone mineral density for both trabecular and
cortical bone.
[1669] Dexa Analysis--Test Description:
[1670] Procedure: Dual Energy X-ray Absorptiometry (DEXA) has been
used successfully to identify changes in bone. Anesthetized animals
were examined and bone mineral content (BMC), BMC/LBM ratios,
volumetric bone mineral density (vBMD), total body BMD, femur BMD
and vertebra BMD were measured.
[1671] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PIXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI)
[i.e., whole body, vertebrae, and both femurs].
[1672] Bone MicroCT Analysis:
[1673] Procedure: MicroCT was also used to get very sensitive
measurements of BMD. One vertebra and 1 femur were taken from a
cohort of 4 wild type and 8 heterozygous mice. Measurements were
taken of lumbar 5 vertebra trabecular bone volume, trabecular
thickness, connectivity density and midshaft femur total bone area
and cortical thickness. The .mu.CT40 scans provided detailed
information on bone mass and architecture. Multiple bones were
placed into sample holders and scanned automatically. Instrument
software was used to select regions of interest for analysis.
Trabecular bone parameters were analyzed in the fifth lumbar
vertebrae (LV5) at 16 micrometer resolution and cortical bone
parameters were analyzed in the femur midshaft at a resolution of
20 micrometers.
[1674] Results:
[1675] DEXA: The (+/-) mice exhibited increased mean total tissue
mass and increased total body fat (% and gram), and an increased
BMC/LBM ratio when compared with their gender-matched (+/+)
littermates and the historical means.
[1676] Micro-CT: The (+/-) mice exhibited increased trabecular bone
volume, number, thickness, and connectivity density as well as mean
femoral mid-shaft cortical thickness area when compared with their
gender-matched (+/+) littermates and the historical means.
[1677] In summary, the (+/-) mice exhibited increased mean total
tissue mass and total body fat and increased trabecular bone and
femoral mid-shaft bone measurements when compared with their
gender-matched (+/+) littermates. These observations suggest an
obesity and/or growth disorder type phenotype. In addition, the
mutant (+/-) mice exhibited an abnormal bone development. Thus,
PRO1298 polypeptides or agonists thereof, would be useful for
normal growth and bone development and would play a role in the
treatment of related growth or metabolic disorders associated with
obesity and/or bone disorders.
[1678] 35.21. Generation and Analysis of Mice Comprising
DNA64966-1575 (UNQ679) Gene Disruptions
[1679] In these knockout experiments, the gene encoding PRO1313
polypeptides (designated as DNA64966-1575) (UNQ679) was disrupted.
The gene specific information for these studies is as follows: the
mutated mouse gene corresponds to nucleotide reference:
NM.sub.--175187 or Mus musculus RIKEN cDNA 2810446P07 gene; protein
reference: NP.sub.--780396 or RIKEN cDNA 2810446P07 [Mus musculus];
the human gene sequence reference: NM.sub.--153354 or Homo sapiens
hypothetical protein MGC33214 (MGC33214); the human protein
sequence corresponds to reference: NP.sub.--699185 or hypothetical
protein MGC33214 [Homo sapiens].
[1680] The mouse gene of interest encodes a hypothetical membrane
protein (2810446P07Rik), which is the ortholog of a human
hypothetical membrane protein (MGC33214).
[1681] MGC33214 is likely to be a membrane protein, containing
seven transmembrane domains. The function of this protein is
currently unknown. MGC33214 is most similar to other hypothetical
proteins from a wide variety of species, including a very large
(5722 amino acids) rat hypothetical protein annotated as "similar
to ATP binding cassette transporter A13." Bioinformatic analysis
suggests that the protein may be located on the plasma membrane;
however, endoplasmic reticulum and nuclear membrane are also
possible cell locations.
Genetics Information:
TABLE-US-00071 [1682] wt het hom Total Observed 22 41 8 71 Expected
17.75 35.5 17.75 71 Chi-Sq. = 7.23 Significance = 0.02698 (hom/n) =
0.11 Avg. Litter Size = 8
Mutation Type: Retroviral Insertion (OST)
[1683] Retroviral insertion occurred in the intron between coding
exons 1 and 2 (NCBI accession number NM.sub.--175187.3). Wild-type
expression of the target gene was detected in embryonic stem (ES)
cells and in all 13 adult tissue samples tested by RT-PCR. Due to
lethality, transcript expression analysis was not performed.
Disruption of the target gene was confirmed by Inverse PCR.
[1684] 35.21.1. Phenotypic Analysis (for Disrupted Gene:
DNA64966-1575 (UNQ679)
[1685] (a) Overall Phenotypic Summary:
[1686] Mutation of the gene encoding the ortholog of a human
hypothetical membrane protein (MGC33214) resulted in lethality of
(-/-) mutants. No notable phenotype was observed for the (+/-)
mice. All (-/-) pups were dead at the time of genotyping.
[1687] Discussion Related to Embryonic Developmental Abnormality of
Lethality:
[1688] Embryonic lethality in knockout mice usually results from
various serious developmental problems including but not limited to
neuro-degenerative diseases, angiogenic disorders, inflammatory
diseases, or where the gene/protein has an important role in basic
cell signaling processes in many cell types. In addition, embryonic
lethals are useful as potential cancer models. Likewise, the
corresponding heterozygous (+/-) mutant animals are particularly
useful when they exhibit a phenotype and/or a pathology report
which reveals highly informative clues as to the function of the
knocked-out gene. For instance, EPO knockout animals were embryonic
lethals, but the pathology reports on the embryos showed a profound
lack of RBCs.
[1689] (b) Embryonic Expression Studies
[1690] In situ hybridization (ISH) studies:
[1691] Using a probe made to base pairs 969-1407 from the
initiating ATG of NM.sub.--175187 (exons 10-12), specific and
ubiquitous staining was observed in the embryonic placenta of (+/+)
mice at E6.5d, E7.5d, E9.5d, E10.5d, E11.5d and E12.5D. Yet another
probe showed UNQ679 staining specific to the dorsal midline
(ubiquitous staining) (probe was made to base pairs 1317-1888 from
the initiating ATG of NM.sub.--175187 (exon 12 to start of 3' UTR).
On the other hand, UNQ679 homozygous (-/-) mice were shown to be
anemic and much smaller compared to the wild-type (+/+) control
mice at E13.5d. Similar results were noted at E18.5d. A placental
defect was notably displayed in the mutant (-/-) mice at 14.5d
compared to the wild-type embryos at E14.5d. In addition, the head
and eyes of the UNQ679 (-/-) embryos at E16.5d were improperly
formed. UNQ679 (-/-) mutant embryos were smaller, anemic and have
malformed hearts at E15.5d. See EXAMPLE 44 for ISH protocol. Thus,
UNQ679 is essential for placenta development and normal
development. The observed anemia in the mutant (-/-) embryos at
E13.5d was more than likely a result of the defective placental
development.
[1692] 35.22. Generation and Analysis of Mice Comprising
DNA68885-1678 (UNQ776) Gene Disruptions
[1693] In these knockout experiments, the gene encoding PRO1570
polypeptides (designated as DNA68885-1678) (UNQ776) was disrupted.
The gene specific information for these studies is as follows: the
mutated mouse gene corresponds to nucleotide reference:
NM.sub.--145403 or ACCESSION: NM.sub.--145403 NID: 21703805 Mus
musculus Mus musculus similar to Transmembrane protease, serine 4
(Membrane-type serine protease 2) (MT-SP2) (LOC214523); protein
reference: Q8VCA5 or ACCESSION: Q8VCA5 NID: Mus musculus (Mouse).
SIMILAR TO TRANSMEMBRANE PROTEASE, SERINE 4. MOUSESPTRNRDB; the
human gene sequence reference: NM.sub.--019894 or ACCESSION:
NM.sub.--019894 NID: 15451939 Homo sapiens Homo sapiens
transmembrane protease, serine 4 (TMPRSS4); the human protein
sequence corresponds to reference: Q9NRS4 or ACCESSION: Q9NRS4 NID:
Homo sapiens (Human). TRANSMEMBRANE PROTEASE, SERINE 4 (EC
3.4.21.-) (MEMBRANE-TYPE SERINE PROTEASE 2) (MT-SP2).
HUMANSPTRNRDB.
[1694] The mouse gene of interest is Tmprss4 (transmembrane
protease, serine 4), ortholog of human TMPRSS4. Aliases include
membrane-type serine protease 2, MT-SP2, transmembrane serine
protease 3, and TMPRSS3.
[1695] TMPRSS4 is a type II membrane protein that is likely to
function as a serine protease of the chymotrypsin family. The
protein contains a signal anchor and an extracellular trypsin-like
serine protease domain. TMPRSS4 is overexpressed in pancreatic
cancer but not in normal pancreas, suggesting that TMPRSS4 may be
involved in metastasis (Wallrapp et al., Cancer Res, 60(10):2602-6
(2000); Gress et al., Genes Chromosomes Cancer, 19(2):97-103
(1997)). TMPRSS4 may regulate renal sodium transport by activating
the epithelial sodium channel (ENaC) expressed in collecting
tubules (Vuagniaux et al., J Gen Physiol, 120(2):191-201
(2002)).
[1696] Targeted or gene trap mutations were generated in strain
129SvEv.sup.Brd-derived embryonic stem (ES) cells. The chimeric
mice were bred to C57BL/6J albino mice to generate F1 heterozygous
animals. These progeny were intercrossed to generate F2 wild type,
heterozygous, and homozygous mutant progeny. On rare occasions, for
example when very few F1 mice were obtained from the chimera, F1
heterozygous mice were crossed to 129SvEv.sup.Brd/C57 hybrid mice
to yield additional heterozygous animals for the intercross to
generate the F2 mice.
Level I phenotypic analysis is performed on mice from this
generation
TABLE-US-00072 wt het hom Total Observed 24 33 14 71 Expected 17.75
35.5 17.75 71 Chi-Sq. = 3.17 Significance = 0.20505 (hom/n) = 0.20
Avg. Litter Size = 8
Mutation Type: Homologous Recombination (standard) Coding exons 3
through 5 were targeted (NCBI accession NM.sub.--145403.1).
Wild-type expression of the target gene was detected in all 13
adult tissue samples tested by RT-PCR, except spinal cord, thymus,
bone, and adipose. Disruption of the target gene was confirmed by
Southern hybridization analysis.
[1697] 35.22.1. Phenotypic Analysis (for Disrupted Gene:
DNA68885-16778 (UNQ776)
[1698] (a) Overall Phenotypic Summary:
[1699] Mutation of the gene encoding the ortholog of human
transmembrane protease, serine 4 (TMPRSS4) resulted in an increased
anxiety-related response in (-/-) mice. The (-/-) mice also
exhibited an increased skin fibroblast proliferation rate. Mutant
(-/-) mice also exhibited increased mean serum triglyceride levels,
and increased mean serum glucose levels with an impaired glucose
tolerance. An increased response to the LPS challenge was also
observed in the (-/-) mice. Gene disruption was confirmed by
Southern blot.
[1700] (b) Immunology Phenotypic Analysis
[1701] Immune related and inflammatory diseases are the
manifestation or consequence of fairly complex, often multiple
interconnected biological pathways which in normal physiology are
critical to respond to insult or injury, initiate repair from
insult or injury, and mount innate and acquired defense against
foreign organisms. Disease or pathology occurs when these normal
physiological pathways cause additional insult or injury either as
directly related to the intensity of the response, as a consequence
of abnormal regulation or excessive stimulation, as a reaction to
self, or as a combination of these.
[1702] Though the genesis of these diseases often involves
multistep pathways and often multiple different biological
systems/pathways, intervention at critical points in one or more of
these pathways can have an ameliorative or therapeutic effect.
Therapeutic intervention can occur by either antagonism of a
detrimental process/pathway or stimulation of a beneficial
process/pathway.
[1703] T lymphocytes (T cells) are an important component of a
mammalian immune response. T cells recognize antigens which are
associated with a self-molecule encoded by genes within the major
histocompatibility complex (MHC). The antigen may be displayed
together with MHC molecules on the surface of antigen presenting
cells, virus infected cells, cancer cells, grafts, etc. The T cell
system eliminates these altered cells which pose a health threat to
the host mammal. T cells include helper T cells and cytotoxic T
cells. Helper T cells proliferate extensively following recognition
of an antigen-MHC complex on an antigen presenting cell. Helper T
cells also secrete a variety of cytokines, i.e., lymphokines, which
play a central role in the activation of B cells, cytotoxic T cells
and a variety of other cells which participate in the immune
response.
[1704] In many immune responses, inflammatory cells infiltrate the
site of injury or infection. The migrating cells may be
neutrophilic, eosinophilic, monocytic or lymphocytic as can be
determined by histological examination of the affected tissues.
Current Protocols in Immunology, ed. John E. Coligan, 1994, John
Wiley & Sons, Inc.
[1705] Many immune related diseases are known and have been
extensively studied. Such diseases include immune-mediated
inflammatory diseases (such as rheumatoid arthritis, immune
mediated renal disease, hepatobiliary diseases, inflammatory bowel
disease (IBD), psoriasis, and asthma), non-immune-mediated
inflammatory diseases, infectious diseases, immunodeficiency
diseases, neoplasia, and graft rejection, etc. In the area of
immunology, targets were identified for the treatment of
inflammation and inflammatory disorders.
[1706] In the area of immunology, targets have been identified
herein for the treatment of inflammation and inflammatory
disorders. Immune related diseases, in one instance, could be
treated by suppressing the immune response. Using neutralizing
antibodies that inhibit molecules having immune stimulatory
activity would be beneficial in the treatment of immune-mediated
and inflammatory diseases. Molecules which inhibit the immune
response can be utilized (proteins directly or via the use of
antibody agonists) to inhibit the immune response and thus
ameliorate immune related disease.
[1707] The following test was performed:
[1708] Acute Phase Response:
[1709] Test Description: Bacterial lipopolysaccharide (LPS) is an
endotoxin, and as such is a potent inducer of an acute phase
response and systemic inflammation. The Level I LPS mice were
injected intraperitoneally (i.p.) with a sublethal dose of LPS in
200 .mu.L sterile saline using a 26 gauge needle. The doses were
based on the average weight of the mice tested at 1 .mu.g/g body
weight 3 hours after injection; a 100 ul blood sample was then
taken and analyzed for the presence of TNFa, MCP-1, and IL-6 on the
FACSCalibur instrument.
[1710] Results:
[1711] The (-/-) mice exhibited an increased mean serum MCP-1
response to LPS challenge when compared with their (+/+)
littermates and the historical mean.
[1712] Analyzed wt/het/hom: 7/4/11
[1713] In summary, the LPS endotoxin challenge results indicate
that knockout mice deficient in the gene encoding PRO1570
polypeptides exhibit immunological abnormalities when compared with
their wild-type littermates. In one instance, the mutant mice
exhibited an increased ability to elicit an immunological response
(MCP-1 production) when challenged with the LPS endotoxin
indicating a proinflammatory response. MCP-1 contributes to the
later stages of B cell activation. In addition, MCP-1 plays a
critical role in inducing the actue phase response and systemic
inflammation. This finding suggests that inhibitors or antagonists
to PRO1570 polypeptides would stimulate the immune system and would
find utility in the cases wherein this effect would be beneficial
to the individual such as in the case of leukemia, and other types
of cancer, and in immunocompromised patients, such as AIDS
sufferers. Accordingly, PRO1570 polypeptides or agonists thereof
would be useful in inhibiting the immune response and would be
useful candidates for suppressing harmful immune responses, e.g. in
the case of graft rejection or graft-versus-host diseases.
[1714] (c) Phenotypic Analysis: CNS/Neurology
[1715] In the area of neurology, analysis focused herein on
identifying in vivo validated targets for the treatment of
neurological and psychiatric disorders including depression,
generalized anxiety disorders, attention deficit hyperactivity
disorder, obsessive compulsive disorder, schizophrenia, cognitive
disorders, hyperalgesia and sensory disorders. Neurological
disorders include the category defined as "anxiety disorders" which
include but are not limited to: mild to moderate anxiety, anxiety
disorder due to a general medical condition, anxiety disorder not
otherwise specified, generalized anxiety disorder, panic attack,
panic disorder with agoraphobia, panic disorder without
agoraphobia, posttraumatic stress disorder, social phobia, specific
phobia, substance-induced anxiety disorder, acute alcohol
withdrawal, obsessive compulsive disorder, agoraphobia, bipolar
disorder I or II, bipolar disorder not otherwise specified,
cyclothymic disorder, depressive disorder, major depressive
disorder, mood disorder, substance-induced mood disorder. In
addition, anxiety disorders may apply to personality disorders
including but not limited to the following types: paranoid,
antisocial, avoidant behavior, borderline personality disorders,
dependent, histronic, narcissistic, obsessive-compulsive, schizoid,
and schizotypal.
[1716] Procedure:
[1717] Behavioral screens were performed on a cohort of 4 wild
type, 4 heterozygous and 8 homozygous mutant mice. All behavioral
tests were done between 12 and 16 weeks of age unless reduced
viability necessitates earlier testing. These tests included open
field to measure anxiety, activity levels and exploration.
[1718] Open Field Test:
[1719] Several targets of known drugs have exhibited phenotypes in
the open field test. These include knockouts of the seratonin
transporter, the dopamine transporter (Giros et al., Nature. 1996
Feb. 15; 379(6566):606-12), and the GABA receptor (Homanics et al.,
Proc Natl Acad Sci USA. 1997 Apr. 15; 94(8):4143-8). An automated
open-field assay was customized to address changes related to
affective state and exploratory patterns related to learning First,
the field (40.times.40 cm) was selected to be relatively large for
a mouse, thus designed to pick up changes in locomotor activity
associated with exploration. In addition, there were 4 holes in the
floor to allow for nose-poking, an activity specifically related to
exploration. Several factors were also designed to heighten the
affective state associated with this test. The open-field test is
the first experimental procedure in which the mice are tested, and
the measurements that were taken were the subjects' first
experience with the chamber. In addition, the open-field was
brightly lit. All these factors will heighten the natural anxiety
associated with novel and open spaces. The pattern and extent of
exploratory activity, and especially the center-to-total distance
traveled ratio, may then be able to discern changes related to
susceptibility to anxiety or depression. A large arena (40
cm.times.40 cm, VersaMax animal activity monitoring system from
AccuScan Instruments) with infrared beams at three different levels
was used to record rearing, hole poke, and locomotor activity. The
animal was placed in the center and its activity was measured for
20 minutes. Data from this test was analyzed in five, 4-minute
intervals. The total distance traveled (cm), vertical movement
number (rearing), number of hole pokes, and the center to total
distance ratio were recorded.
[1720] The propensity for mice to exhibit normal habituation
responses to a novel environment is assessed by determining the
overall change in their horizontal locomotor activity across the 5
time intervals. This calculated slope of the change in activity
over time is determined using normalized, rather than absolute,
total distance traveled. The slope is determined from the
regression line through the normalized activity at each of the 5
time intervals. Normal habituation is represented by a negative
slope value. Analyzed wt/het/hom: 4/4/8
[1721] Results:
[1722] The (-/-) mice exhibited decreased median sum time-in-center
and hole poke activity during open field testing when compared with
their gender-matched (+/+) littermates and the historical mean,
suggesting an increased anxiety-like response in the mutants.
[1723] As noted above, a notable difference was observed during
open field activity testing. The (-/-) mice exhibited a decreased
median sum time in the center area when compared with their
gender-matched (+/+) littermates. This type of behavior is
consistent with an increased anxiety like response. Thus, the
knockout mice demonstrated a phenotype consistent with anxiety
related disorders which are associated with mild to moderate
anxiety, anxiety due to a general medical condition, and/or bipolar
disorders; hyperactivity; sensory disorders; obsessive-compulsive
disorders, schizophrenia or a paranoid personality. Thus, PRO1570
polypeptides or agonists thereof would be useful in the treatment
of such neurological disorders or the amelioration of the symptoms
associated with anxiety disorders.
[1724] (d) Oncology Phenotypic Analysis
[1725] In the area of oncology, targets were identified herein for
the treatment of solid tumors, lymphomas and leukemia.
[1726] Adult Skin Cell Proliferation:
[1727] Procedure: Skin cells were isolated from 16 week old animals
(2 wild type and 4 homozygotes). These were developed into primary
fibroblast cultures and the fibroblast proliferation rates were
measured in a strictly controlled protocol. The ability of this
assay to detect hyper-proliferative and hypo-proliferative
phenotypes has been demonstrated with p53 and Ku80. Proliferation
was measured using Brdu incorporation.
[1728] Specifically, in these studies the skin fibroblast
proliferation assay was used. An increase in the number of cells in
a standardized culture was used as a measure of relative
proliferative capacity. Primary fibroblasts were established from
skin biopsies taken from wild type and mutant mice. Duplicate or
triplicate cultures of 0.05 million cells were plated and allowed
to grow for six days. At the end of the culture period, the number
of cells present in the culture was determined using a electronic
particle counter.
[1729] Results:
[1730] The (-/-) mice exhibited an increased mean skin fibroblast
proliferation rate when compared with their gender-matched (+/+)
littermates and the historical mean. Thus, homozygous mutant mice
demonstrated a hyper-proliferative phenotype. As suggested by these
observations, PRO1570 polypeptides or agonists thereof would be
useful in decreasing abnormal cell proliferation such as tumor cell
growth.
[1731] (e) Phenotypic Analysis: Cardiology
[1732] In the area of cardiovascular biology, targets were
identified herein for the treatment of hypertension,
atherosclerosis, heart failure, stroke, various coronary artery
diseases, dyslipidemias such as high cholesterol
(hypercholesterolemia) and elevated serum triglycerides
(hypertriglyceridemia), diabetes and/or obesity. The phenotypic
tests included the measurement of serum cholesterol and
triglycerides.
[1733] Blood Lipids
[1734] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. High cholesterol levels and
increased triglyceride blood levels are recognized risk factors in
the development of cardiovascular disease and/or diabetes.
Measuring blood lipids facilitates the finding of biological
switches that regulate blood lipid levels Inhibition of factors
which elevate blood lipid levels may be useful for reducing the
risk for cardiovascular disease. In these blood chemistry tests,
cholesterol measurements were recorded using the COBAS Integra 400
(mfr: Roche).
[1735] Results:
[1736] The (-/-) mice exhibited increased mean serum triglyceride
and glucose levels when compared with their gender-matched (+/+)
littermates and the historical means.
[1737] As summarized above, the (-/-) mice exhibited notably
increased triglyceride levels when compared with their
gender-matched (+/+) littermates and the historical means for the
male (+/+) mice. In addition, the increased mean serum glucose
levels suggesting diabetes. Thus, mutant mice deficient in the
PRO1570 gene can serve as a model for cardiovascular disease
including diabetes. PRO1570 polypeptides or its encoding gene would
be useful in regulating normal blood lipid levels such as
triglycerides. Thus, PRO1570 polypeptides or agonists thereof would
be useful in the treatment of such cardiovascular diseases as
hypertension, atherosclerosis, heart failure, stroke, various
coronary diseases, hypercholesterolemia, hypertriglyceridemia,
diabetes and/or obesity.
[1738] 35.23. Generation and Analysis of Mice Comprising
DNA80796-2523 (UNQ870) Gene Disruptions
[1739] In these knockout experiments, the gene encoding PRO1886
polypeptides (designated as DNA80796-2523) (UNQ870) was disrupted.
The gene specific information for these studies is as follows: the
mutated mouse gene corresponds to nucleotide reference:
XM.sub.--134593 or Mus musculus hypothetical protein 4932417I16
(4932417I16); protein reference: XP.sub.--134593 or similar to
predicted CDS, putative protein of bilaterial origin (41193) [Mus
musculus]; the human gene sequence reference: AK025820 or Homo
sapiens cDNA: FLJ22167 fis, clone HRC00584; the human protein
sequence corresponds to reference: BAB15244 or unnamed protein
product [Homo sapiens].
[1740] The disrupted mouse gene encodes a hypothetical protein
(4932417I16Rik), which is orthologous to human hypothetical protein
FLJ22167. Aliases include hypothetical protein 4932417I16
(murine).
[1741] Bioinformatic analyses of FLJ22167 indicate that it is a
transmembrane protein, possibly located in the plasma membrane or
the endoplasmic reticulum. In human, some FLJ22167 transcripts
overlap those of an adjacent gene (CHST5), but such overlap is not
observed in mouse. The overlap has sometimes led to
misidentification of FLJ22167 as a possible enzyme (isoform of
CHST5). No critically evaluated information suggests that FLJ22167
is an enzyme.
Genetics Information:
TABLE-US-00073 [1742] wt het hom Total Observed 29 28 0 57 Expected
14.25 28.5 14.25 57 Chi-Sq. = 29.53 Significance = 0.00000 (hom/n)
= 0.00 Avg. Litter Size = 6
Mutation Type: Retroviral Insertion (OST)
[1743] Retroviral insertion occurred within the intron proceeding
coding exon 1 (NCBI Accession AK030046). Wild-type expression of
the target gene was detected in embryonic stem (ES) cells and in
all 13 adult tissue samples tested by RT-PCR. Due to lethality,
transcript expression analysis was not performed. Disruption of the
target gene was confirmed by Inverse PCR.
[1744] 35.23.1. Phenotypic Analysis (for Disrupted Gene:
DNA80796-2523 (UNQ870)
[1745] (a) Overall Phenotypic Summary:
[1746] Mutation of the gene encoding the ortholog of a human
hypothetical protein (FLJ22167) resulted in lethality of (-/-)
mutants. One fourth (1/4) of the UNQ870 pups make it to birth. The
(+/-) mice exhibited increased total body fat (% and gram) and
increased mean total tissue mass. In addition, a reduced fibroblast
proliferation was observed in the (+/-) mice.
[1747] Discussion Related to Embryonic Developmental Abnormality of
Lethality:
[1748] Embryonic lethality in knockout mice usually results from
various serious developmental problems including but not limited to
neuro-degenerative diseases, angiogenic disorders, inflammatory
diseases, or where the gene/protein has an important role in basic
cell signaling processes in many cell types. In addition, embryonic
lethals are useful as potential cancer models. Likewise, the
corresponding heterozygous (+/-) mutant animals are particularly
useful when they exhibit a phenotype and/or a pathology report
which reveals highly informative clues as to the function of the
knocked-out gene. For instance, EPO knockout animals were embryonic
lethals, but the pathology reports on the embryos showed a profound
lack of RBCs.
[1749] (b) Further Expression and Embryonic Observations
[1750] UNQ 870 is upregulated in endometrial adenocarcinoma (See
EXAMPLES 41 and 42 for protocol). Microscopic observations showed
one third (1/3) of the heterozygous (+/-) mice to have
hydronephrosis of the right kidney.
[1751] The UNQ870 homozygous (-/-) embryos showed heart defects at
E15.5d.
[1752] Three fourths (3/4) of UNQ870 homozygous (-/-) embryos are
anemic and have abnormal eyes at E14.5d. One fourth (1/4) of the
UNQ870 (-/-) embryos are anemic and have eyes at E15.5d but no
eyelid closure. Normal eyelid development results in an eyelid
primordia at E12.5d, a fused eyelid between 15.5d-16.5d and eyes
open at P12-14d. There is a parallel between eyelid closure and
wound healing (progressive events run in parallel). Also other
factors such as FGF8 expression, neurogenin 2 expression, Gli3 and
Dlx2 expression, as well as EyaI expression all appear to be
altered in the (-/-) mutant embryos. [FGF8 expression is reduced in
UNQ870 (-/-) at E10.5d; reduced staining in the telencephalic
vescicle and nasal placode in UNQ870 (-/-). Neurogenin 2 expression
is reduced in UNQ870 (-/-) at E10.5d; reduced staining in
telencephalon of UNQ870 (-/-) or reduced or missing ventral
staining in UNQ870 (-/-). Gli3 expression is altered in UNQ870
(-/-) at E10.5d; (reduced staining in telencephalon, branchial
arches, and optic vesicles). Dlx2 expression is altered in UNQ870
(-/-) at E9.5d; tighter domain of staining at AER of limb bud in
wild-type (+/+). EyaI expression is altered in UNQ870 (-/-) at
E9.5d; reduced staining in branchial arches and otic vesicles. See
EXAMPLE 44 for ISH protocol.
[1753] Pax6 knockouts are similar to UNQ870 knockouts. Small eye
(Sey) is a semidominant mutation in the Pax6 gene. Homozygotes
(-/-) result in the complete lack of eyes and nasal primordia. On
the basis of comparative mapping studies and on phenotypic
similarities, Sey has been suggested to be homologous to congenital
aniridia (lack of iris) in humans.
[1754] (c) Bone Metabolism & Body Diagnostics
[1755] (1) Tissue Mass & Lean Body Mass Measurements--Dexa
[1756] Dexa Analysis--Test Description:
[1757] Procedure: Dual Energy X-ray Absorptiometry (DEXA) has been
used successfully to identify changes in total tissue mass
(TTM).
[1758] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PIXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI,
i.e., whole body, vertebrae, and both femurs).
[1759] Body Measurements (Body Length & Weight):
[1760] Body Measurements: A measurement of body length and weight
was performed at approximately 16 weeks of age.
[1761] Results:
[1762] The (+/-) mice exhibited increased mean body weight when
compared with their gender-matched (+/+) littermates and the
historical means.
[1763] (2) Bone Metabolism: Radiology Phenotypic Analysis
[1764] In the area of bone metabolism, targets were identified
herein for the treatment of arthritis, osteoporosis, osteopenia and
osteopetrosis as well as identifying targets that promote bone
healing. Tests included:
[1765] DEXA for measurement of bone mineral density on femur and
vertebra
[1766] MicroCT for very high resolution and very high sensitivity
measurements of bone mineral density for both trabecular and
cortical bone.
[1767] Dexa Analysis--Test Description:
[1768] Procedure: Dual Energy X-ray Absorptiometry (DEXA) has been
used successfully to identify changes in bone. Anesthetized animals
were examined and bone mineral content (BMC), BMC/LBM ratios,
volumetric bone mineral density (vBMD), total body BMD, femur BMD
and vertebra BMD were measured.
[1769] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PIXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI)
[i.e., whole body, vertebrae, and both femurs].
[1770] Results:
[1771] DEXA: The (+/-) mice exhibited increased mean total tissue
mass, percent total body fat, and total fat mass when compared with
their gender-matched (+/+) littermates and the historical
means.
[1772] These studies suggest that mutant (+/-) non-human transgenic
animals exhibit a negative phenotype that may be associated with
obesity. Thus, PRO1886 polypeptides or agonists thereof are
essential for normal growth and metabolic processes and especially
would be important in the prevention and/or treatment of
obesity.
[1773] (d) Oncology Phenotypic Analysis
[1774] In the area of oncology, targets were identified herein for
the treatment of solid tumors, lymphomas and leukemia.
[1775] Adult Skin Cell Proliferation:
[1776] Procedure: Skin cells were isolated from 16 week old animals
(2 wild type and 4 heterozygotes). These were developed into
primary fibroblast cultures and the fibroblast proliferation rates
were measured in a strictly controlled protocol. The ability of
this assay to detect hyper-proliferative and hypo-proliferative
phenotypes has been demonstrated with p53 and Ku80. Proliferation
was measured using Brdu incorporation.
[1777] Specifically, in these studies the skin fibroblast
proliferation assay was used. An increase in the number of cells in
a standardized culture was used as a measure of relative
proliferative capacity. Primary fibroblasts were established from
skin biopsies taken from wild type and (+/-) mutant mice. Duplicate
or triplicate cultures of 0.05 million cells were plated and
allowed to grow for six days. At the end of the culture period, the
number of cells present in the culture was determined using a
electronic particle counter.
[1778] Results:
[1779] The (+/-) mice exhibited a decreased mean skin fibroblast
proliferation rate when compared with their gender-matched (+/+)
littermates and the historical mean. Thus, heterozygous mutant mice
demonstrated a hypo-proliferative phenotype. As suggested by these
observations, antagonists of a PRO1886 polypeptide or its encoding
gene would be useful in decreasing abnormal cell proliferation such
as tumor cell growth.
[1780] 35.24. Generation and Analysis of Mice Comprising
DNA76788-2526 (UNQ873) Gene Disruptions
[1781] In these knockout experiments, the gene encoding PRO1891
polypeptides (designated as DNA76788-2526) (UNQ873) was disrupted.
The gene specific information for these studies is as follows: the
mutated mouse gene corresponds to nucleotide reference:
NM.sub.--033615 or ACCESSION: NM.sub.--033615 NID: gi 23956237 ref
NM.sub.--033615.1 Mus musculus a disintegrin and metalloprotease
domain 33 (Adam33); protein reference: Q923W9 or ACCESSION: Q923W9
NID: Mus musculus (Mouse). ADAM 33 PRECURSOR (EC 3.4.24.) (A
DISINTEGRIN AND METALLOPROTEINASE DOMAIN 33). MOUSESPTRNRDB; the
human gene sequence reference: NM.sub.--025220 or ACCESSION:
NM.sub.--025220 NID: 18252044 Homo sapiens Homo sapiens a
disintegrin and metalloproteinase domain 33 (ADAM33); the human
protein sequence corresponds to reference: Q9BZ11 or ACCESSION:
Q9BZ11 NID: Homo sapiens (Human). ADAM 33 PRECURSOR (EC 3.4.24.-)
(A DISINTEGRIN AND METALLOPROTEINASE DOMAIN 33). HUMANSPTRNRDB.
[1782] The mouse gene of interest is Adam33 (a disintegrin and
metalloprotease domain 33), ortholog of human ADAM33. Aliases
include Adam1, metalloprotease disintegrin, and disintegrin and
reprolysin metalloproteinase family protein.
[1783] ADAM33 is a type I integral membrane protein that likely
functions as a zinc metalloprotease and cell adhesion molecule. The
protein consists of a large extracellular domain, a transmembrane
segment, and a short cytoplasmic C-terminus. The large
extracellular domain contains a propeptide, a zinc metalloprotease
catalytic domain, a disintegrin domain, a cysteine rich region, and
an EGF-like domain. The propeptide is cleaved to form the mature
protein. ADAM33 is widely expressed, with particularly high
expression in placenta, lung, spleen, and veins. Expression of
ADAM33 appears to be absent in liver (Yoshinaka et al., Gene,
282(1-2):227-36 (2002); Van Eerdewegh et al., Nature,
418(6896):426-30 (2002); Garlisi et al., Biochem Biophys Res
Commun, 301(1):35-43 (2003)). The biological role of ADAM33 and
ADAM family members in general are not clearly known; however, they
have been implicated in processes such as fertilization,
neurogenesis, myogenesis, embryonic TGF-alpha release, and the
inflammatory response (Primakoff and Myles, Trends Genet,
16(2):83-7 (2000)).
[1784] ADAM33 has been implicated in asthma and bronchial hyper
responsiveness. The protease is expressed in human lung fibroblasts
and bronchial smooth muscle, which play a central role in airway
remodeling. Damage to airway epithelial cells by activated T cells
leads to smooth muscle hyperplasia, fibroblast proliferation,
increased matrix deposition, and conversion of bronchial smooth
muscle from the quiescent, contractile type to the proliferative,
synthetic type. Alterations in ADAM33 activity or expression may
underlie abnormalities in airway remodeling in bronchial hyper
responsiveness (Van Eerdewegh et al., Nature, 418(6896):426-30
(2002)).
[1785] Targeted or gene trap mutations were generated in strain
129SvEv.sup.Brd-derived embryonic stem (ES) cells. The chimeric
mice were bred to C57BL/6J albino mice to generate F1 heterozygous
animals. These progeny were intercrossed to generate F2 wild type,
heterozygous, and homozygous mutant progeny. On rare occasions, for
example when very few F1 mice were obtained from the chimera, F1
heterozygous mice were crossed to 129SvEv.sup.Brd/C57 hybrid mice
to yield additional heterozygous animals for the intercross to
generate the F2 mice.
Level I phenotypic analysis is performed on mice from this
generation
TABLE-US-00074 wt het hom Total Observed 15 48 21 84 Expected 21 42
21 84 Chi-Sq. = 2.57 Significance = 0.27645 (hom/n) = 0.25 Avg.
Litter Size = 9
Mutation Type: Homologous Recombination (standard) Coding exons 12
through 16 were targeted (NCBI accession NM.sub.--033615.1).
Wild-type expression of the target gene was detected in all 13
adult tissue samples tested by RT-PCR, except adipose. Disruption
of the target gene was confirmed by Southern hybridization
analysis.
[1786] 35.24.1. Phenotypic Analysis (for Disrupted Gene:
DNA76788-2526 (UNQ873)
[1787] (a) Overall Phenotypic Summary:
[1788] Mutation of the gene encoding the ortholog of human a
disintegrin and metalloprotease domain 33 (ADAM33) resulted in an
increased IgG1 response to ovalbumin challenge in (-/-) mice. The
(-/-) mice exhibited increased serum triglycerides, increased mean
serum insulin and decreased bone density measurements. In addition,
glucose tolerance testing suggested insulin resistance.
Immunological abnormalities were also observed in the (-/-) mice.
Gene disruption was confirmed by Southern blot.
[1789] (b) Immunology Phenotypic Analysis
[1790] Immune related and inflammatory diseases are the
manifestation or consequence of fairly complex, often multiple
interconnected biological pathways which in normal physiology are
critical to respond to insult or injury, initiate repair from
insult or injury, and mount innate and acquired defense against
foreign organisms. Disease or pathology occurs when these normal
physiological pathways cause additional insult or injury either as
directly related to the intensity of the response, as a consequence
of abnormal regulation or excessive stimulation, as a reaction to
self, or as a combination of these.
[1791] Though the genesis of these diseases often involves
multistep pathways and often multiple different biological
systems/pathways, intervention at critical points in one or more of
these pathways can have an ameliorative or therapeutic effect.
Therapeutic intervention can occur by either antagonism of a
detrimental process/pathway or stimulation of a beneficial
process/pathway.
[1792] T lymphocytes (T cells) are an important component of a
mammalian immune response. T cells recognize antigens which are
associated with a self-molecule encoded by genes within the major
histocompatibility complex (MHC). The antigen may be displayed
together with MHC molecules on the surface of antigen presenting
cells, virus infected cells, cancer cells, grafts, etc. The T cell
system eliminates these altered cells which pose a health threat to
the host mammal. T cells include helper T cells and cytotoxic T
cells. Helper T cells proliferate extensively following recognition
of an antigen-MHC complex on an antigen presenting cell. Helper T
cells also secrete a variety of cytokines, i.e., lymphokines, which
play a central role in the activation of B cells, cytotoxic T cells
and a variety of other cells which participate in the immune
response.
[1793] In many immune responses, inflammatory cells infiltrate the
site of injury or infection. The migrating cells may be
neutrophilic, eosinophilic, monocytic or lymphocytic as can be
determined by histological examination of the affected tissues.
Current Protocols in Immunology, ed. John E. Coligan, 1994, John
Wiley & Sons, Inc.
[1794] Many immune related diseases are known and have been
extensively studied. Such diseases include immune-mediated
inflammatory diseases (such as rheumatoid arthritis, immune
mediated renal disease, hepatobiliary diseases, inflammatory bowel
disease (IBD), psoriasis, and asthma), non-immune-mediated
inflammatory diseases, infectious diseases, immunodeficiency
diseases, neoplasia, and graft rejection, etc. In the area of
immunology, targets were identified for the treatment of
inflammation and inflammatory disorders.
[1795] In the area of immunology, targets have been identified
herein for the treatment of inflammation and inflammatory
disorders. Immune related diseases, in one instance, could be
treated by suppressing the immune response. Using neutralizing
antibodies that inhibit molecules having immune stimulatory
activity would be beneficial in the treatment of immune-mediated
and inflammatory diseases. Molecules which inhibit the immune
response can be utilized (proteins directly or via the use of
antibody agonists) to inhibit the immune response and thus
ameliorate immune related disease.
[1796] The following test was performed:
[1797] Ovalbumin Challenge
[1798] Procedure: This assay was carried out on 7 wild types and 8
homozygotes. Chicken ovalbumin (OVA) is a T-cell dependent antigen,
which is commonly used as a model protein for studying
antigen-specific immune responses in mice. OVA is non-toxic and
inert and therefore will not cause harm to the animals even if no
immune response is induced. The murine immune response to OVA has
been well characterized, to the extent that the immunodominant
peptides for eliciting T cell responses have been identified.
Anti-OVA antibodies are detectable 8 to 10 days after immunization
using enzyme-linked immunosorbent assay (ELIZA), and determination
of different isotypes of antibodies gives further information on
the complex processes that may lead to a deficient response in
genetically engineered mice.
[1799] As noted above, this protocol assesses the ability of mice
to raise an antigen-specific immune response. Animals were injected
IP with 50 mg of chicken ovalbumin emulsified in Complete Freund's
Adjuvant and 14 days later the serum titer of anti-ovalbumin
antibodies (IgM, IgG1 and IgG2 subclasses) was measured. The amount
of OVA-specific antibody in the serum sample is proportional to the
Optical Density (OD) value generated by an instrument that scans a
96-well sample plate. Data was collected for a set of serial
dilutions of each serum sample. Analyzed wt/het/hom: 9/4/14
[1800] Results of this challenge: The (-/-) mice exhibited an
increased mean serum IgG1 response to the ovalbumin challenge when
compared with their (+/+) littermates. Thus, these knockout mice
exhibited an increased ability to elicit an OVA-specific antibody
response to the T-cell dependent OVA antigen. Inhibitors
(antagonists) of PRO1891 polypeptides would be expected to also
stimulate the immune system and would find utility in the cases
wherein this effect would be beneficial to the individual such as
in the case of leukemia, and other types of cancer, and in
immunocompromised patients, such as AIDS sufferers. Accordingly,
PRO1891 polypeptides or agonists thereof would be useful in
inhibiting the immune response and would be useful candidates for
suppressing harmful immune responses, e.g. in the case of graft
rejection or graft-versus-host diseases.
[1801] (c) Bone Metabolism: Radiology Phenotypic Analysis
[1802] In the area of bone metabolism, targets were identified
herein for the treatment of arthritis, osteoporosis, osteopenia and
osteopetrosis as well as identifying targets that promote bone
healing. Tests included:
[1803] DEXA for measurement of bone mineral density on femur and
vertebra
[1804] MicroCT for very high resolution and very high sensitivity
measurements of bone mineral density for both trabecular and
cortical bone.
[1805] Dexa Analysis--Test Description:
[1806] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. Dual Energy X-ray
Absorptiometry (DEXA) has been used successfully to identify
changes in bone. Anesthetized animals were examined and bone
mineral content (BMC), BMC/LBM ratios, volumetric bone mineral
density (vBMD), total body BMD, femur BMD and vertebra BMD were
measured.
[1807] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PIXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI)
[i.e., whole body, vertebrae, and both femurs].
[1808] Bone MicroCT Analysis:
[1809] Procedure: MicroCT was also used to get very sensitive
measurements of BMD. One vertebra and 1 femur were taken from a
cohort of 4 wild type and 8 homozygous mice. Measurements were
taken of lumbar 5 vertebra trabecular bone volume, trabecular
thickness, connectivity density and midshaft femur total bone area
and cortical thickness. The .mu.CT40 scans provided detailed
information on bone mass and architecture. Multiple bones were
placed into sample holders and scanned automatically. Instrument
software was used to select regions of interest for analysis.
Trabecular bone parameters were analyzed in the fifth lumbar
vertebrae (LV5) at 16 micrometer resolution and cortical bone
parameters were analyzed in the femur midshaft at a resolution of
20 micrometers.
[1810] Results:
[1811] DEXA: Both the male and female (-/-) mice exhibited
decreased mean bone mineral content, bone mineral density, and
volumetric bone mineral density when compared with their
gender-matched (+/+) littermates and the historical means.
[1812] Micro-CT: The (-/-) mice exhibited decreased mean vertebral
trabecular bone volume, number, thickness, and connectivity density
when compared with their gender-matched (+/+) littermate and the
historical means.
Analyzed wt/het/hom: 4/4/8
[1813] These results demonstrate that knockout mutant mice exhibit
abnormal bone metabolism with significant bone loss similar to
osteoporosis characterized by decrease in bone mass with decreased
density and possibly fragility leading to bone fractures. Thus, it
appears that PRO1891 or agonists thereof would be useful in
maintaining bone homeostasis. In addition, PRO1891 or its encoding
gene would be useful in bone healing or for the treatment of
arthritis or osteoporosis; whereas antagonists to PRO1891 or its
encoding gene would lead to abnormal or pathological bone disorders
including inflammatory diseases associated with abnormal bone
metabolism including arthritis, osteoporosis, and osteopenia.
[1814] (d) Phenotypic Analysis: Cardiology
[1815] In the area of cardiovascular biology, targets were
identified herein for the treatment of hypertension,
atherosclerosis, heart failure, stroke, various coronary artery
diseases, dyslipidemias such as high cholesterol
(hypercholesterolemia) and elevated serum triglycerides
(hypertriglyceridemia), diabetes and/or obesity. The phenotypic
tests included the measurement of serum cholesterol and
triglycerides.
[1816] Blood Lipids
[1817] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. High cholesterol levels and
increased triglyceride blood levels are recognized risk factors in
the development of cardiovascular disease and/or diabetes.
Measuring blood lipids facilitates the finding of biological
switches that regulate blood lipid levels Inhibition of factors
which elevate blood lipid levels may be useful for reducing the
risk for cardiovascular disease. In these blood chemistry tests,
cholesterol measurements were recorded using the COBAS Integra 400
(mfr: Roche).
[1818] Results:
[1819] The (-/-) mice exhibited an increased mean serum
triglyceride level when compared with their gender-matched (+/+)
littermates and the historical mean.
[1820] As summarized above, the (-/-) mice exhibited notably
increased triglyceride levels when compared with their
gender-matched (+/+) littermates and the historical means for the
male (+/+) mice. Thus, mutant mice deficient in the PRO1891 gene
can serve as a model for cardiovascular disease. PRO1891
polypeptides or its encoding gene would be useful in regulating
blood lipids such as triglycerides. Thus, PRO1891 polypeptides or
agonists thereof would be useful in the treatment of such
cardiovascular diseases as hypertension, atherosclerosis, heart
failure, stroke, various coronary diseases, hypercholesterolemia,
hypertriglyceridemia, diabetes and/or obesity.
[1821] (e) Phenotypic Analysis: Metabolism-Blood Chemistry/Glucose
Tolerance
[1822] In the area of metabolism, targets may be identified for the
treatment of diabetes. Blood chemistry phenotypic analysis includes
blood glucose measurements. The COBAS Integra 400 (mfr: Roche) was
used for running blood chemistry tests on the mice. In the area of
metabolism, targets may be identified for the treatment of
diabetes. Blood chemistry phenotypic analysis includes glucose
tolerance tests to measure insulin sensitivity and changes in
glucose metabolism. Abnormal glucose tolerance test results may
indicate but may not be limited to the following disorders or
conditions: Diabetes Type 1 and Type 2, Syndrome X, various
cardiovascular diseases and/or obesity.
[1823] Procedure: A cohort of 2 wild type and 4 homozygous mice
were used in this assay. The glucose tolerance test is the standard
for defining impaired glucose homeostasis in mammals. Glucose
tolerance tests were performed using a Lifescan glucometer. Animals
were injected IP at 2 g/kg with D-glucose delivered as a 20%
solution and blood glucose levels were measured at 0, 30, 60 and 90
minutes after injection. Analyzed wt/het/hom: 4/4/8
[1824] Insulin Data:
[1825] Test Description Lexicon Genetics uses the Cobra II Series
Auto-Gamma Counting System in its clinical settings for running
quantitative Insulin assays on mice.
[1826] Results:
[1827] The (-/-) mice exhibited an increased mean serum insulin
level when compared with their gender-matched (+/+) littermates and
the historical mean.
[1828] However, blood chemistry testing showed (-/-) mice
exhibiting an increased mean serum glucose level when compared with
their gender-matched (+/+) littermates and the historical mean.
During the glucose tolerance test, the (-/-) mice exhibited an
increased mean fasting serum glucose level when compared with their
gender-matched (+/+) littermates and the historical mean.
[1829] Thus, knockout mice exhibited the phenotypic pattern of an
impaired glucose homeostasis with elevated levels of fasting serum
glucose indicative of diabetes or a pre-diabetic condition even in
the presence of increased insulin levels. Based on these results,
PRO1891 (or agonists thereof) or its encoding gene would be useful
in the treatment of an impaired glucose metabolism (marked by
insulin resistance) and/or diabetes.
[1830] 35.25. Generation and Analysis of Mice Comprising
DNA88004-2575 (UNQ1934) Gene Disruptions
[1831] In these knockout experiments, the gene encoding PRO4409
polypeptides (designated as DNA88004-2575) (UNQ1934) was disrupted.
The gene specific information for these studies is as follows: the
mutated mouse gene corresponds to nucleotide reference:
NM.sub.--028065 or ACCESSION: NM.sub.--028065 NID: gi 21312509 ref
NM.sub.--028065.1 Mus musculus RIKEN cDNA 1600025D17 gene
(1600025D17Rik); protein reference: Q9DAU1 or ACCESSION: Q9DAU1
NID: Mus musculus (Mouse). 1600025D17Rik protein (Putative retinoic
acid-regulated protein) (RIKEN cDNA 1600025D17 gene).
MOUSESPTRNRDB; the human gene sequence reference: NM.sub.--006586
or Homo sapiens trinucleotide repeat containing 5 (TNRC5); the
human protein sequence corresponds to reference: Q9BT09 or
ACCESSION: Q9BT09 NID: Homo sapiens (Human). Hypothetical protein
(DJ475N16.1) (CTG4A). HUMANSPTRNRDB.
[1832] The mouse gene of interest encodes a hypothetical protein
(1600025D17Rik), which is the ortholog of human protein TNRC5
(trinucleotide repeat containing 5). Aliases include CAG repeat
containing expanded repeat domain and CAG/CTG 5.
[1833] TNRC5 is a hypothetical protein containing CAG repeats,
which are likely to be involved in specific diseases, most with
neuropsychiatric features (Margolis et al., Hum Genet,
100(1):114-22 (1997)). The protein contains a signal peptide but no
other identifiable domains. The cell location of TNRC5 is
ambiguous; bioinformatic analysis suggests that the protein may be
secreted or located in the endoplasmic reticulum. TNRC5 gene
transcription is likely to be under the control of retinoids, which
play an important role in development and physiology (Glozak et
al., Mol Endocrinol, 17(1):27-41 (2003)).
Genetics Information:
TABLE-US-00075 [1834] wt het hom Total Observed 20 37 10 67
Expected 16.75 33.5 16.75 67 Chi-Sq. = 3.72 Significance = 0.15595
(hom/n) = 0.15 Avg. Litter Size = 8
Mutation Type: Retroviral Insertion (OST)
[1835] Retroviral insertion occurred in the intron between coding
exons 1 and 2 (NCBI accession NM.sub.--028065.2). Wild-type
expression of the target gene was detected in embryonic stem (ES)
cells and, among the 13 adult tissue samples tested by RT-PCR, in
kidney; stomach, small intestine, and colon; and adipose. Due to
reduced viability, transcript expression analysis was not
performed. Disruption of the target gene was confirmed by Inverse
PCR.
[1836] 35.25.1. Phenotypic Analysis (for Disrupted Gene:
DNA88004-2575 (UNQ1934)
[1837] (a) Overall Phenotypic Summary:
[1838] Mutation of the gene encoding the ortholog of human
trinucleotide repeat containing 5 (TNRC5) resulted in small (-/-)
mice that failed to thrive. UNQ1934 was shown to be ubiquitously
expressed between 7.5d-12.5d of embryo development in wild-type
pups. The (-/-) mutants were euthanized or transferred to necropsy
by 3 weeks of age. 40% fewer than expected homozygotes were present
at genotyping. Heterozygous (+/-) mice exhibited decreased total
tissue mass and fat (% and gram) compared to their wild-type
littermate controls and the historical mean.
[1839] Discussion Related to Embryonic Developmental Abnormality of
Lethality:
[1840] Embryonic lethality in knockout mice usually results from
various serious developmental problems including but not limited to
neuro-degenerative diseases, angiogenic disorders, inflammatory
diseases, or where the gene/protein has an important role in basic
cell signaling processes in many cell types. In addition, embryonic
lethals are useful as potential cancer models. Likewise, the
corresponding heterozygous (+/-) mutant animals are particularly
useful when they exhibit a phenotype and/or a pathology report
which reveals highly informative clues as to the function of the
knocked-out gene. For instance, EPO knockout animals were embryonic
lethals, but the pathology reports on the embryos showed a profound
lack of RBCs.
[1841] (b) Body Diagnostics--Tissue Mass & Lean Body Mass
Measurements--Dexa
[1842] (1) Dexa Analysis--Test Description:
[1843] Procedure: Dual Energy X-ray Absorptiometry (DEXA) has been
used successfully to identify changes in total tissue mass
(TTM).
[1844] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PIXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI,
i.e., whole body, vertebrae, and both femurs).
[1845] Body Measurements (Body Length & Weight):
[1846] Body Measurements: A measurement of body length and weight
was performed at approximately 16 weeks of age.
[1847] Results:
[1848] General Observations: The (-/-) mice were small, failed to
thrive, and were euthanized or sent to pathology for analysis by 3
weeks of age.
[1849] Weight: The (-/-) mice exhibited decreased mean body weight
at the 2 week measurement when compared with their gender-matched
(+/+) littermates and the historical means.
[1850] Pathology:
[1851] Microscopic Observations: Though the (-/-) mice were smaller
than their (+/+) littermates and died before 4 weeks of age, no
histopathological lesions were observed that explained the early
mortality.
[1852] Gene Expression: Expression of the neo transcript was not
detected in the panel of tissues analyzed by in situ
hybridization.
[1853] Summary
[1854] The (-/-) mice analyzed exhibited notably decreased
viability shown by a failure to survive past four weeks of age when
compared with their (+/+) littermates. The (-/-) mice were quite
small in size and showed a notable decrease in body weight
suggestive of growth retardation in these mutants. Although
pathology observations failed to reveal any histopathological
lesions, the negative phenotype is indicative of a tissue wasting
condition with severe growth retardation. Thus, PRO4409
polypeptides or agonists thereof must be essential for normal
growth and/or growth metabolism and therefore would be useful in
the treatment or prevention of growth disorders such as cachexia or
other tissue wasting diseases.
[1855] (2) Bone Metabolism: Radiology Phenotypic Analysis
[1856] In the area of bone metabolism, targets were identified
herein for the treatment of arthritis, osteoporosis, osteopenia and
osteopetrosis as well as identifying targets that promote bone
healing. Tests included:
[1857] DEXA for measurement of bone mineral density on femur and
vertebra
[1858] MicroCT for very high resolution and very high sensitivity
measurements of bone mineral density for both trabecular and
cortical bone.
[1859] Dexa Analysis--Test Description:
[1860] Procedure: Dual Energy X-ray Absorptiometry (DEXA) has been
used successfully to identify changes in bone. Anesthetized animals
were examined and bone mineral content (BMC), BMC/LBM ratios,
volumetric bone mineral density (vBMD), total body BMD, femur BMD
and vertebra BMD were measured.
[1861] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PIXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI)
[i.e., whole body, vertebrae, and both femurs].
[1862] Results:
[1863] DEXA: The (+/-) mice exhibited decreased mean total tissue
mass, percent total body fat, and total fat mass when compared with
their gender-matched (+/+) littermates and the historical
means.
[1864] These studies suggest that mutant (+/-) non-human transgenic
animals exhibit a negative phenotype that may be associated with
tissue wasting diseases. Thus, PRO4409 polypeptides or agonists
thereof are essential for normal growth and metabolic processes and
especially would be important in the prevention and/or treatment of
growth disorders and/or tissue wasting diseases.
[1865] 35.26. Generation and Analysis of Mice Comprising
DNA92265-2669 (UNQ2446) Gene Disruptions
[1866] In these knockout experiments, the gene encoding PRO5725
polypeptides (designated as DNA92265-2669) (UNQ2446) was disrupted.
The gene specific information for these studies is as follows: the
mutated mouse gene corresponds to nucleotide reference:
NM.sub.--175024 or Mus musculus RIKEN cDNA G630049C14 gene
(G630049C14Rik); protein reference: Q8BN06 or ACCESSION: Q8BN06
NID: Mus musculus (Mouse). Hypothetical protein; the human gene
sequence reference: NM.sub.--198443 or Homo sapiens MRCC2446
(UNQ2446); the human protein sequence corresponds to reference:
NP.sub.--940845 or MRCC2446 [Homo sapiens]
gi|37182683|gb|AAQ89142.1| MRCC2446 [Homo sapiens].
[1867] The targeted mouse gene encodes a hypothetical protein
(G630049C14Rik), which is the ortholog of hypothetical human
protein MRCC2446. The human gene is also known as UNQ2446.
[1868] The human and mouse genes encode putative secreted proteins.
The mouse hypothetical protein contains a signal peptide, a
C-terminal transmembrane segment, and no other identifiable
domains. The human protein contains a signal peptide and no other
identifiable domains. Both proteins appear to be distantly similar
to neuritin, an extracellular protein anchored on the plasma
membrane of neurons that is likely to be involved in neuritogenesis
(Naeve et al., Proc Natl Acad Sci USA, 94(6):2648-53 (1997)).
Genetics Information:
TABLE-US-00076 [1869] wt het hom Total Observed 20 30 15 65
Expected 16.25 32.5 16.25 65 Chi-Sq. = 1.15 Significance = 0.56162
(hom/n) = 0.23 Avg. Litter Size = 7
Mutation Type: Retroviral Insertion (OST)
[1870] Retroviral insertion occurred in coding exon 2 (Accession:
NM.sub.--175024.1). Wild-type expression of the target gene was
detected in embryonic stem (ES) cells and in all 13 adult tissue
samples tested by RT-PCR, except skeletal muscle. RT-PCR analysis
revealed that the transcript was absent in the (-/-) mouse analyzed
(M-121).
[1871] 35.26.1. Phenotypic Analysis (for Disrupted Gene:
DNA92265-2669 (UNQ2446)
[1872] (a) Overall Phenotypic Summary:
[1873] Mutation of the gene encoding the ortholog of a hypothetical
human protein resulted in the observation of decreased body weight,
decreased lean body mass, bone mineral density and bone mineral
content as well as decreased lumbar 5 measurements. Transcript was
absent by RT-PCR.
[1874] (b) Body Diagnostics--Tissue Mass & Lean Body Mass
Measurements--Dexa
[1875] Dexa Analysis--Test Description:
[1876] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. Dual Energy X-ray
Absorptiometry (DEXA) has been used successfully to identify
changes in total tissue mass (TTM).
[1877] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PIXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI,
i.e., whole body, vertebrae, and both femurs).
[1878] Body Measurements (Body Length & Weight):
[1879] Body Measurements: A measurement of body length and weight
was performed at approximately 16 weeks of age.
[1880] Results:
[1881] The male and female (-/-) mice exhibited decreased mean body
weight when compared with their gender-matched (+/+) littermates
and the historical means. Analyzed wt/het/hom: 28/36/22
[1882] The (-/-) mice were quite small in size and showed a notable
decrease in body weight suggestive of growth retardation in these
mutants. Although pathology observations failed to reveal any
histopathological lesions, the negative phenotype suggested growth
retardation. Thus, PRO5725 polypeptides or agonists thereof must be
essential for normal growth and/or growth metabolism and therefore
would be useful in the treatment or prevention of growth related
disorders.
[1883] (2) Bone Metabolism: Radiology Phenotypic Analysis
[1884] In the area of bone metabolism, targets were identified
herein for the treatment of arthritis, osteoporosis, osteopenia and
osteopetrosis as well as identifying targets that promote bone
healing. Tests included:
[1885] DEXA for measurement of bone mineral density on femur and
vertebra
[1886] MicroCT for very high resolution and very high sensitivity
measurements of bone mineral density for both trabecular and
cortical bone.
[1887] Dexa Analysis--Test Description:
[1888] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. Dual Energy X-ray
Absorptiometry (DEXA) has been used successfully to identify
changes in bone. Anesthetized animals were examined and bone
mineral content (BMC), BMC/LBM ratios, volumetric bone mineral
density (vBMD), total body BMD, femur BMD and vertebra BMD were
measured.
[1889] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PIXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI)
[i.e., whole body, vertebrae, and both femurs].
[1890] Bone MicroCT Analysis:
[1891] Procedure: MicroCT was also used to get very sensitive
measurements of BMD. One vertebra and 1 femur were taken from a
cohort of 4 wild type and 8 homozygous mice. Measurements were
taken of lumbar 5 vertebra trabecular bone volume, trabecular
thickness, connectivity density and midshaft femur total bone area
and cortical thickness. The .mu.CT40 scans provided detailed
information on bone mass and architecture. Multiple bones were
placed into sample holders and scanned automatically. Instrument
software was used to select regions of interest for analysis.
Trabecular bone parameters were analyzed in the fifth lumbar
vertebrae (LV5) at 16 micrometer resolution and cortical bone
parameters were analyzed in the femur midshaft at a resolution of
20 micrometers.
[1892] Results:
[1893] DEXA: The (-/-) mice exhibited decreased mean lean body
mass, bone mineral content and bone mineral density when compared
with their gender-matched (+/+) littermates and the historical
means.
[1894] Micro-CT: The (-/-) mice exhibited decreased mean vertebral
trabecular bone volume, number, thickness, and connectivity density
and decreased mean femoral mid-shaft cortical thickness when
compared with their gender-matched (+/+) littermates and the
historical means.
[1895] Analyzed wt/het/hom: 4/4/8
[1896] Summary
[1897] These results demonstrate that knockout mutant mice exhibit
abnormal bone metabolism with significant bone loss similar to
osteoporosis characterized by decrease in bone mass with decreased
density and possibly fragility leading to bone fractures. Thus, it
appears that PRO5725 or agonists thereof would play a role in
maintaining bone homeostasis. In addition, PRO5725 or its encoding
gene would be useful for maintaining bone homeostasis and for bone
healing or for the treatment of arthritis or osteoporosis; whereas
antagonists to PRO5725 or its encoding gene would lead to abnormal
or pathological bone disorders including inflammatory diseases
associated with abnormal bone metabolism including arthritis,
osteoporosis, and osteopenia.
[1898] The (-/-) mice analyzed by DEXA exhibited notably decreased
lean body mass when compared with their (+/+) littermates,
suggestive of growth retardation in these mutants. This in
conjunction with the observations of abnormal bone measurements
suggest a tissue wasting condition or other growth related
disorders such as cachexia. Thus, PRO5725 polypeptides or agonists
thereof would be useful in the treatment of bone disorders but also
would be useful for the prevention of growth related disorders such
as cachexia and/or other tissue wasting diseases.
[1899] 35.27. Generation and Analysis of Mice Comprising DNA98591
(UNQ2506) Gene Disruptions
[1900] In these knockout experiments, the gene encoding PRO5994
polypeptides (designated as DNA98591 (UNQ2506) was disrupted. The
gene specific information for these studies is as follows: the
mutated mouse gene corresponds to nucleotide reference:
NM.sub.--011463 or Mus musculus serine protease inhibitor, Kazal
type 4 (Spink4); protein reference: 035679 or ACCESSION: O35679
NID: Mus musculus (Mouse). SERINE PROTEASE INHIBITOR KAZAL-TYPE 4
PRECURSOR (PEPTIDE PEC-60 HOMOLOG) (MPGC60 PROTEIN). MOUSESPTRNRDB;
the human gene sequence reference: NM.sub.--014471 or ACCESSION:
NM.sub.--014471 NID: gi 7657452 ref NM.sub.--014471.1 Homo sapiens
serine protease inhibitor, Kazal type 4 (SPINK4); the human protein
sequence corresponds to reference: O60575 or ACCESSION: O60575 NID:
Homo sapiens (Human). SERINE PROTEASE INHIBITOR KAZAL-TYPE 4
PRECURSOR (PEPTIDE PEC-60 HOMOLOG). HUMANSPTRNRDB.
[1901] The mouse gene of interest is Spink4 (serine protease
inhibitor, Kazal type 4), ortholog of human SPINK4. Aliases include
MPGC60, PEC-60, and gastrointestinal peptide.
[1902] SPINK4 is a putative secreted serine protease inhibitor
expressed predominantly in the intestinal tract (Krause et al.,
Differentiation, 63(5):285-94 (1998)). SPINK4 is the apparent human
ortholog of porcine PEC-60 (SwissProt P37109), which has been shown
to inhibit glucose-induced insulin secretion. PEC-60 has been
isolated from brains of rat and pig (Norberg et al., Cell Mol Life
Sci, 60(2):378-81 (2003)).
[1903] Targeted or gene trap mutations were generated in strain
129SvEv.sup.Brd-derived embryonic stem (ES) cells. The chimeric
mice were bred to C57BL/6J albino mice to generate F1 heterozygous
animals. These progeny were intercrossed to generate F2 wild type,
heterozygous, and homozygous mutant progeny. On rare occasions, for
example when very few F1 mice were obtained from the chimera, F1
heterozygous mice were crossed to 129SvEv.sup.Brd/C57 hybrid mice
to yield additional heterozygous animals for the intercross to
generate the F2 mice.
Level I phenotypic analysis is performed on mice from this
generation
TABLE-US-00077 wt het hom Total Observed 21 41 27 89 Expected 22.25
44.5 22.25 89 Chi-Sq. = 1.36 Significance = 0.50673 (hom/n) = 0.30
Avg. Litter Size = 9
Mutation Type: Homologous Recombination (standard) Coding exon 1
was targeted (NCBI accession NM.sub.--011463.1). Wild-type
expression of the target gene was detected in all 13 adult tissue
samples tested by RT-PCR, except brain, lung, and skeletal muscle.
Disruption of the target gene was confirmed by Southern
hybridization analysis.
[1904] 35.27.1. Phenotypic Analysis (for Disrupted Gene: DNA98591
(UNQ2506)
[1905] (a) Overall Phenotypic Summary:
[1906] Mutation of the gene encoding the ortholog of human serine
protease inhibitor, Kazal type 4 (SPINK4) resulted in decreased
body weight and length as well as decreased tissue mass
measurements. In addition, the (-/-) mice exhibit lymphoid
hyperplasia and tissue inflammation at an increased incidence. Gene
disruption was confirmed by Southern blot.
[1907] (b) Bone Metabolism & Body Diagnostics
[1908] (1) Tissue Mass & Lean Body Mass Measurements--Dexa
[1909] Dexa Analysis--Test Description:
[1910] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. Dual Energy X-ray
Absorptiometry (DEXA) has been used successfully to identify
changes in total tissue mass (TTM).
[1911] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PIXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI,
i.e., whole body, vertebrae, and both femurs).
[1912] Body Measurements (Body Length & Weight):
[1913] Body Measurements: A measurement of body length and weight
was performed at approximately 16 weeks of age.
[1914] Results:
[1915] General Observations: The agouti (-/-) mouse is much smaller
than its black (+/+) littermate.
[1916] The (-/-) mice exhibited decreased mean body weight and
decreased body length when compared with the historical means.
[1917] (2) Bone Metabolism: Radiology Phenotypic Analysis
[1918] In the area of bone metabolism, targets were identified
herein for the treatment of arthritis, osteoporosis, osteopenia and
osteopetrosis as well as identifying targets that promote bone
healing. Tests included:
[1919] DEXA for measurement of bone mineral density on femur and
vertebra
[1920] MicroCT for very high resolution and very high sensitivity
measurements of bone mineral density for both trabecular and
cortical bone.
[1921] Dexa Analysis--Test Description:
[1922] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. Dual Energy X-ray
Absorptiometry (DEXA) has been used successfully to identify
changes in bone. Anesthetized animals were examined and bone
mineral content (BMC), BMC/LBM ratios, volumetric bone mineral
density (vBMD), total body BMD, femur BMD and vertebra BMD were
measured.
[1923] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PIXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI)
[i.e., whole body, vertebrae, and both femurs].
[1924] Results:
[1925] DEXA: The (-/-) mice exhibited decreased mean total tissue
mass and lean body mass when compared with their gender-matched
(+/+) littermates and the historical means.
Analyzed wt/het/hom: 4/4/8
[1926] Summary
[1927] These results demonstrate that the knockout mutant (-/-)
mice analyzed by DEXA were quite small in size and exhibited a
decrease in body weight and length as well as a decrease in total
tissue, lean body mass suggestive of growth retardation in these
mutants. Thus, PRO5994 polypeptides or agonists thereof must be
essential for normal growth and/or growth metabolism and therefore
would be useful in the treatment or prevention of growth disorders,
cachexia or other tissue wasting diseases.
[1928] (c) Pathological Observations
[1929] The knockout (-/-) mice exhibited lymphoid hyperplasia and
tissue inflammation at an increased incidence.
[1930] 35.28. Generation and Analysis of Mice Comprising
DNA107701-2711 (UNQ2545) Gene Disruptions
[1931] In these knockout experiments, the gene encoding PRO6097
polypeptides (designated as DNA107701-2711) (UNQ2545) was
disrupted. The gene specific information for these studies is as
follows: the mutated mouse gene corresponds to nucleotide
reference: NM.sub.--028787 or Mus musculus solute carrier family
35, member F5 (Slc35f5); protein reference: Q9DBK9 or Q9DBK9 Q9DBK9
1300003P13RIK PROTEIN; the human gene sequence reference:
NM.sub.--025181 or Homo sapiens solute carrier family 35, member F5
(SLC35F5); the human protein sequence corresponds to reference:
Q8WV83 or ACCESSION: Q8WV83 NID: Homo sapiens (Human). Similar to
RIKEN cDNA 1300003P13 gene (NS5ATP3).
[1932] The mouse gene of interest is Slc35f5 (solute carrier family
35, member F5), ortholog of human SLC35F5. Aliases include
1300003P13Rik and FLJ22004.
[1933] SLC35F5 is a predicted integral plasma membrane protein of
unknown function, containing 10 transmembrane segments. Most of the
transmembrane segments are contained within a domain that has weak
similarity to that found in carbohydrate/phosphate translocators
(KOG 4313) and drug permeases (COG0697) (Marchler-Bauer et al.,
Nucleic Acids Res, 31 (1): 383-7 (2003).
[1934] Targeted or gene trap mutations were generated in strain
129SvEv.sup.Brd-derived embryonic stem (ES) cells. The chimeric
mice were bred to C57BL/6J albino mice to generate F1 heterozygous
animals. These progeny were intercrossed to generate F2 wild type,
heterozygous, and homozygous mutant progeny. On rare occasions, for
example when very few F1 mice were obtained from the chimera, F1
heterozygous mice were crossed to 129SvEv.sup.Brd/C57 hybrid mice
to yield additional heterozygous animals for the intercross to
generate the F2 mice.
Level I phenotypic analysis is performed on mice from this
generation
TABLE-US-00078 wt het hom Total Observed 17 35 12 64 Expected 16 32
16 64 Chi-Sq. = 1.34 Significance = 0.51075 (hom/n) = 0.19 Avg.
Litter Size = 7
Mutation Type: Homologous Recombination (standard) Coding exons 1
through 3 were targeted (NCBI accession NM.sub.--028787.2).
Wild-type expression of the target gene was detected in embryonic
stem (ES) cells and in all 13 adult tissue samples tested by
RT-PCR, except bone. Disruption of the target gene was confirmed by
Southern hybridization analysis.
[1935] 35.28.1. Phenotypic Analysis (for Disrupted Gene:
DNA107701-2711 (UNQ2545)
[1936] (a) Overall Phenotypic Summary:
[1937] Mutation of the gene encoding the ortholog of human solute
carrier family 35, member F5 (SLC35F5) resulted in larger (-/-)
mice, exhibiting increased body weight, total tissue mass, lean
body mass and bone measurements. In addition, the mutant knockout
mice exhibited a decreased or reduced IgG2a response to an
ovalbumin challenge. Also, additional immunological abnormalities
were exhibited by observing an increased percentage of T cells,
with decreased natural killer cells and B cells and an increased
percentage of CD4 T cells in the mutant (-/-) mice compared with
their littermate controls. Gene disruption was confirmed by
Southern blot.
[1938] (b) Immunology Phenotypic Analysis
[1939] Immune related and inflammatory diseases are the
manifestation or consequence of fairly complex, often multiple
interconnected biological pathways which in normal physiology are
critical to respond to insult or injury, initiate repair from
insult or injury, and mount innate and acquired defense against
foreign organisms. Disease or pathology occurs when these normal
physiological pathways cause additional insult or injury either as
directly related to the intensity of the response, as a consequence
of abnormal regulation or excessive stimulation, as a reaction to
self, or as a combination of these.
[1940] Though the genesis of these diseases often involves
multistep pathways and often multiple different biological
systems/pathways, intervention at critical points in one or more of
these pathways can have an ameliorative or therapeutic effect.
Therapeutic intervention can occur by either antagonism of a
detrimental process/pathway or stimulation of a beneficial
process/pathway.
[1941] T lymphocytes (T cells) are an important component of a
mammalian immune response. T cells recognize antigens which are
associated with a self-molecule encoded by genes within the major
histocompatibility complex (MHC). The antigen may be displayed
together with MHC molecules on the surface of antigen presenting
cells, virus infected cells, cancer cells, grafts, etc. The T cell
system eliminates these altered cells which pose a health threat to
the host mammal. T cells include helper T cells and cytotoxic T
cells. Helper T cells proliferate extensively following recognition
of an antigen-MHC complex on an antigen presenting cell. Helper T
cells also secrete a variety of cytokines, i.e., lymphokines, which
play a central role in the activation of B cells, cytotoxic T cells
and a variety of other cells which participate in the immune
response.
[1942] In many immune responses, inflammatory cells infiltrate the
site of injury or infection. The migrating cells may be
neutrophilic, eosinophilic, monocytic or lymphocytic as can be
determined by histological examination of the affected tissues.
Current Protocols in Immunology, ed. John E. Coligan, 1994, John
Wiley & Sons, Inc.
[1943] Many immune related diseases are known and have been
extensively studied. Such diseases include immune-mediated
inflammatory diseases (such as rheumatoid arthritis, immune
mediated renal disease, hepatobiliary diseases, inflammatory bowel
disease (IBD), psoriasis, and asthma), non-immune-mediated
inflammatory diseases, infectious diseases, immunodeficiency
diseases, neoplasia, and graft rejection, etc. In the area of
immunology, targets were identified for the treatment of
inflammation and inflammatory disorders.
[1944] In the area of immunology, targets have been identified
herein for the treatment of inflammation and inflammatory
disorders. Immune related diseases, in one instance, could be
treated by suppressing the immune response. Using neutralizing
antibodies that inhibit molecules having immune stimulatory
activity would be beneficial in the treatment of immune-mediated
and inflammatory diseases. Molecules which inhibit the immune
response can be utilized (proteins directly or via the use of
antibody agonists) to inhibit the immune response and thus
ameliorate immune related disease.
[1945] The following tests were performed:
[1946] Ovalbumin Challenge
[1947] Procedure: This assay was carried out on 7 wild types and 8
homozygotes. Chicken ovalbumin (OVA) is a T-cell dependent antigen,
which is commonly used as a model protein for studying
antigen-specific immune responses in mice. OVA is non-toxic and
inert and therefore will not cause harm to the animals even if no
immune response is induced. The murine immune response to OVA has
been well characterized, to the extent that the immunodominant
peptides for eliciting T cell responses have been identified.
Anti-OVA antibodies are detectable 8 to 10 days after immunization
using enzyme-linked immunosorbent assay (ELIZA), and determination
of different isotypes of antibodies gives further information on
the complex processes that may lead to a deficient response in
genetically engineered mice.
[1948] As noted above, this protocol assesses the ability of mice
to raise an antigen-specific immune response. Animals were injected
IP with 50 mg of chicken ovalbumin emulsified in Complete Freund's
Adjuvant and 14 days later the serum titer of anti-ovalbumin
antibodies (IgM, IgG1 and IgG2 subclasses) was measured. The amount
of OVA-specific antibody in the serum sample is proportional to the
Optical Density (OD) value generated by an instrument that scans a
96-well sample plate. Data was collected for a set of serial
dilutions of each serum sample.
Analyzed wt/het/hom: 8/4/9
[1949] Results of this Challenge:
[1950] The (-/-) mice exhibited a decreased mean serum IgG2a
response to ovalbumin challenge when compared with their (+/+)
littermates and the historical means. Thus, these knockout mice
exhibited a decreased ability to elicit an OVA-specific antibody
response to the T-cell dependent OVA antigen.
[1951] In summary, the ovalbumin challenge studies indicate that
knockout mice deficient in the gene encoding PRO6097 polypeptides
exhibit immunological abnormalities when compared with their
wild-type littermates. In particular, the mutant mice exhibited a
decreased ability to elicit an immunological response when
challenged with the T-cell dependent OVA antigen. Thus, PRO6097
polypeptides or agonists thereof, would be useful for stimulating
the immune system (such as T cell proliferation) and would find
utility in the cases wherein this effect would be beneficial to the
individual such as in the case of leukemia, and other types of
cancer, and in immunocompromised patients, such as AIDS sufferers.
Accordingly, inhibitors (antagonists) of PRO6097 polypeptides would
be useful for inhibiting the immune response and thus would be
useful candidates for suppressing harmful immune responses, e.g. in
the case of graft rejection or graft-versus-host diseases.
[1952] Flourescence-Activated Cell-Sorting (FACS) Analysis
[1953] Procedure:
[1954] FACS analysis of immune cell composition from peripheral
blood was performed including CD4, CD8 and T cell receptor to
evaluate T lymphocytes, CD19 for B lymphocytes, CD45 as a leukocyte
marker and pan NK for natural killer cells. The FACS analysis was
carried out on 2 wild type and 6 homozygous mice and included cells
derived from thymus, spleen, bone marrow and lymph node.
[1955] In these studies, analyzed cells were isolated from thymus,
peripheral blood, spleen, bone marrow and lymph nodes. Flow
cytometry was designed to determine the relative proportions of CD4
and CD8 positive T cells, B cells, NK cells and monocytes in the
mononuclear cell population. A Becton-Dickinson FACSCalibur 3-laser
FACS machine was used to assess immune status. For Phenotypic
Assays and Screening, this machine records CD4+/CD8-, CD8+/CD4-,
NK, B cell and monocyte numbers in addition to the CD4+/CD8+ ratio.
The mononuclear cell profile was derived by staining a single
sample of lysed peripheral blood from each mouse with a panel of
six lineage-specific antibodies: CD45 PerCP, anti-TCRb APC, CD4 PE,
CD8 FITC, pan-NK PE, and CD19 FITC. The two FITC and PE labeled
antibodies stain mutually exclusive cell types. The samples were
analyzed using a Becton Dickinson FACSCalibur flow cytometer with
CellQuest software.
[1956] Results:
[1957] FACS: The (-/-) mice exhibited an increased percentage of T
cells (increased % of CD4 T cells) with a decreased mean percentage
of natural killer cells and B cells when compared with their (+/+)
littermates and the historical mean. Analyzed wt/het/hom: 7/4/8
[1958] In summary, the FACS results indicate that the homozygous
mutant mice demonstrate immunological abnormalities marked by
increased T cell population but a decreased mean percentage of
natural killer cells and B cells. [Natural killer cells are the
first line of defense to viral infection since these cells have
been implicated in viral immunity and in defense against tumors].
Natural killer cells or NK cells act as effectors in
antibody-dependent cell-mediated cytotoxicity and have been
identified by their ability to kill certain lymphoid tumor cell
lines in vitro without the need for prior immunization or
activation. However, their known function in host defense is in the
early phases of infection with several intracellular pathogens,
particularly herpes viruses]. On the otherhand, by knocking out the
gene which encodes PRO6097 polypeptides a beneficial effect is
shown by the increase in the T cell population. Thus, PRO6097
polypeptides or the gene encoding PRO6097 appears to act as a
negative regulator of T cell proliferation. An opposite effect was
shown for the B cell and natural killer cell population.
[1959] (c) Bone Metabolism & Body Diagnostics
[1960] (1) Tissue Mass & Lean Body Mass Measurements--Dexa
[1961] Dexa Analysis--Test Description:
[1962] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. Dual Energy X-ray
Absorptiometry (DEXA) has been used successfully to identify
changes in total tissue mass (TTM).
[1963] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PIXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI,
i.e., whole body, vertebrae, and both femurs).
[1964] Body Measurements (Body Length & Weight):
[1965] Body Measurements: A measurement of body length and weight
was performed at approximately 16 weeks of age.
[1966] Results:
[1967] The (-/-) mice exhibited increased mean body weight when
compared with their gender-matched (+/+) littermates and the
historical mean. Analyzed wt/het/hom: 21/40/15
[1968] (2) Bone Metabolism: Radiology Phenotypic Analysis
[1969] In the area of bone metabolism, targets were identified
herein for the treatment of arthritis, osteoporosis, osteopenia and
osteopetrosis as well as identifying targets that promote bone
healing. Tests included:
[1970] DEXA for measurement of bone mineral density on femur and
vertebra
[1971] MicroCT for very high resolution and very high sensitivity
measurements of bone mineral density for both trabecular and
cortical bone.
[1972] Dexa Analysis--Test Description:
[1973] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. Dual Energy X-ray
Absorptiometry (DEXA) has been used successfully to identify
changes in bone. Anesthetized animals were examined and bone
mineral content (BMC), BMC/LBM ratios, volumetric bone mineral
density (vBMD), total body BMD, femur BMD and vertebra BMD were
measured.
[1974] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PIXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI)
[i.e., whole body, vertebrae, and both femurs].
[1975] Bone MicroCT Analysis:
[1976] Procedure: MicroCT was also used to get very sensitive
measurements of BMD. One vertebra and 1 femur were taken from a
cohort of 4 wild type and 8 homozygous mice. Measurements were
taken of lumbar 5 vertebra trabecular bone volume, trabecular
thickness, connectivity density and midshaft femur total bone area
and cortical thickness. The .mu.CT40 scans provided detailed
information on bone mass and architecture. Multiple bones were
placed into sample holders and scanned automatically. Instrument
software was used to select regions of interest for analysis.
Trabecular bone parameters were analyzed in the fifth lumbar
vertebrae (LV5) at 16 micrometer resolution and cortical bone
parameters were analyzed in the femur midshaft at a resolution of
20 micrometers.
[1977] Results:
[1978] DEXA: The (-/-) mice exhibited increased mean total tissue
mass, lean body mass, bone mineral content, and bone mineral
density in total body and femur when compared with their
gender-matched (+/+) littermates and the historical means. Analyzed
wt/het/hom: 4/4/8
[1979] MicroCT: The knockouts (-/-) exhibited an increased
trabecular bone volume, number, and connectivity density compared
with their littermate controls.
[1980] In summary, the (-/-) mice exhibited increased body weight
and length, increased mean total tissue mass and lean body mass and
increased bone mineral density measurements and trabecular bone
measurements when compared with their gender-matched (+/+)
littermates. The heterozygous mice (+/-) exhibited similar bone
effects but the increased measurement values were between the
wild-type and homozygous values. These observations suggest an
obesity and/or growth disorder type phenotype. In addition, the
mutant (-/-) mice exhibited abnormal bone measurements. Thus,
PRO6097 polypeptides or agonists thereof, would be useful for
normal growth and bone development and would play a role in the
treatment of related growth or metabolic disorders associated with
obesity and/or bone disorders such as osteopetrosis.
[1981] 35.29. Generation and Analysis of Mice Comprising
DNA108792-2753 (UNQ2966) Gene Disruptions
[1982] In these knockout experiments, the gene encoding PRO7425
polypeptides (designated as DNA108792-2753) (UNQ2966) was
disrupted. The gene specific information for these studies is as
follows: the mutated mouse gene corresponds to nucleotide
reference: NM.sub.--176993 or Mus musculus RIKEN cDNA A530065I17
gene (A530065I17Rik); protein reference: NP.sub.--795967 or RIKEN
cDNA A530065I17 gene [Mus musculus]; the human gene sequence
reference: NM.sub.--198275 or Homo sapiens hypothetical protein
LOC196264 (LOC196264); the human protein sequence corresponds to
reference: NP.sub.--938016 or hypothetical protein LOC196264 [Homo
sapiens].
[1983] The disrupted mouse gene is a hypothetical protein (interim
name, A530065I17Rik), which is the ortholog of human hypothetical
protein FLJ38080. Aliases for the human locus include LOC196264 and
QQRG2966.
[1984] Analysis of FLJ38080 indicates that two transmembrane
domains (1 near each end) flank a central Igv-type motif (InterPro
IPR0003596). Such features suggest the protein may be partially or
completely extracellular, and perhaps participates in signaling or
protein-protein interactions.
Genetics Information:
TABLE-US-00079 [1985] wt het hom Total Observed 16 32 20 68
Expected 17 34 17 68 Chi-Sq. = 0.71 Significance = 0.70262 (hom/n)
= 0.29 Avg. Litter Size = 8
Mutation Type: Retroviral Insertion (OST)
[1986] Retroviral insertion occurred in the intron between coding
exons 2 and 3 (NCBI accession BQ713326). Wild-type expression of
the target gene was detected in all 13 adult tissues samples tested
by RT-PCR, except thymus, lung, skeletal muscle and adipose. RT-PCR
analysis revealed that the transcript was absent in the (-/-) mouse
analyzed (M-75).
[1987] 35.29.1. Phenotypic Analysis (for Disrupted Gene:
DNA108792-7425 (UNQ2966)
[1988] (a) Overall Phenotypic Summary:
[1989] Mutation of the gene encoding the ortholog of a human
hypothetical protein (FLJ38080) resulted in sebaceous gland
hyperplasia and severe growth retardation in (-/-) mice. In
addition, the (-/-) mice exhibited decreased mean body mass-related
measurements and decreased bone measurements. The (-/-) mice also
exhibited severe depletion of body fat depots and enlarged kidneys.
Lower insulin levels were also observed in the (-/-) mice as well
as abnormal changes in alkaline phosphatase (most likely due to
decreased bone mineralization) and mean serum ALT (alanine amino
transaminase) suggestive of liver disease. Furthermore, the mutant
(-/-) mice exhibited severe hypoactivity in the circadian rhythm
test. Transcript was absent by RT-PCR.
[1990] (b) Phenotypic Analysis: CNS/Neurology
[1991] In the area of neurology, analysis focused herein on
identifying in vivo validated targets for the treatment of
neurological and psychiatric disorders including depression,
generalized anxiety disorders, attention deficit hyperactivity
disorder, obsessive compulsive disorder, schizophrenia, cognitive
disorders, hyperalgesia and sensory disorders. Neurological
disorders include the category defined as "anxiety disorders" which
include but are not limited to: mild to moderate anxiety, anxiety
disorder due to a general medical condition, anxiety disorder not
otherwise specified, generalized anxiety disorder, panic attack,
panic disorder with agoraphobia, panic disorder without
agoraphobia, posttraumatic stress disorder, social phobia, specific
phobia, substance-induced anxiety disorder, acute alcohol
withdrawal, obsessive compulsive disorder, agoraphobia, bipolar
disorder I or II, bipolar disorder not otherwise specified,
cyclothymic disorder, depressive disorder, major depressive
disorder, mood disorder, substance-induced mood disorder. In
addition, anxiety disorders may apply to personality disorders
including but not limited to the following types: paranoid,
antisocial, avoidant behavior, borderline personality disorders,
dependent, histronic, narcissistic, obsessive-compulsive, schizoid,
and schizotypal.
[1992] Procedure:
[1993] Behavioral screens were performed on a cohort of 4 wild
type, 4 heterozygous and 8 homozygous mutant mice. All behavioral
tests were done between 12 and 16 weeks of age unless reduced
viability necessitates earlier testing. These tests included open
field to measure anxiety, activity levels and exploration.
[1994] Open Field Test:
[1995] Several targets of known drugs have exhibited phenotypes in
the open field test. These include knockouts of the seratonin
transporter, the dopamine transporter (Giros et al., Nature. 1996
Feb. 15; 379(6566):606-12), and the GABA receptor (Homanics et al.,
Proc Natl Acad Sci USA. 1997 Apr. 15; 94(8):4143-8). An automated
open-field assay was customized to address changes related to
affective state and exploratory patterns related to learning First,
the field (40.times.40 cm) was selected to be relatively large for
a mouse, thus designed to pick up changes in locomotor activity
associated with exploration. In addition, there were 4 holes in the
floor to allow for nose-poking, an activity specifically related to
exploration. Several factors were also designed to heighten the
affective state associated with this test. The open-field test is
the first experimental procedure in which the mice are tested, and
the measurements that were taken were the subjects' first
experience with the chamber. In addition, the open-field was
brightly lit. All these factors will heighten the natural anxiety
associated with novel and open spaces. The pattern and extent of
exploratory activity, and especially the center-to-total distance
traveled ratio, may then be able to discern changes related to
susceptibility to anxiety or depression. A large arena (40
cm.times.40 cm, VersaMax animal activity monitoring system from
AccuScan Instruments) with infrared beams at three different levels
was used to record rearing, hole poke, and locomotor activity. The
animal was placed in the center and its activity was measured for
20 minutes. Data from this test was analyzed in five, 4-minute
intervals. The total distance traveled (cm), vertical movement
number (rearing), number of hole pokes, and the center to total
distance ratio were recorded.
[1996] The propensity for mice to exhibit normal habituation
responses to a novel environment is assessed by determining the
overall change in their horizontal locomotor activity across the 5
time intervals. This calculated slope of the change in activity
over time is determined using normalized, rather than absolute,
total distance traveled. The slope is determined from the
regression line through the normalized activity at each of the 5
time intervals. Normal habituation is represented by a negative
slope value.
[1997] Results:
[1998] The (-/-) mice exhibited a decreased normalized slope during
open field activity testing when compared with their (+/+)
littermates, suggesting an abnormal habituation response to a novel
environment in the mutants.
[1999] Circadian Test Description:
[2000] Female mice are individually housed at 4 pm on the first day
of testing in 48.2 cm.times.26.5 cm home cages and administered
food and water ad libitum. Animals are exposed to a 12-hour
light/dark cycle with lights turning on at 7 am and turning off at
7 pm. The system software records the number of beam interruptions
caused by the animal's movements, with beam breaks automatically
divided into ambulations. Activity is recorded in 60, one-hour
intervals during the three-day test. Data generated are displayed
by median activity levels recorded for each hour (circadian rhythm)
and median total activity during each light/dark cycle (locomotor
activity) over the three-day testing period. Thus, the mutant (-/-)
mice exhibited severe hypoactivity. Analyzed wt/het/hom: 4/4/8
[2001] Results:
[2002] The (-/-) mice exhibited abnormal activity during the 1-hour
habituation, the 12-hour habituation, and all light and dark
periods of home cage activity testing when compared with their
gender-matched (+/+) littermates and the historical means. The
(-/-) mice exhibited no diurnal alternations during the 3-day
testing period.
[2003] As summarized above, notable differences were observed
during home-cage activity testing. The (-/-) mice exhibited
decreased ambulatory counts during the day 2 light period when
compared with their (+/+) littermates. In addition, the homozygous
(-/-) mice exhibited decreased light-to-dark and light-to-total
activity ratios when compared with their (+/+) littermates,
suggesting an abnormal circadian rhythms response in the mutants.
These results are consistent with the findings during open field
testing and indicate that the homozygous mutant mice exhibit
circadian rhythms which are usually associated with lethargy or
depressive disorders. Thus, PRO7425 polypeptides or its encoded
gene would be useful in the treatment of such neurological
disorders including depressive disorders or other decreased
anxiety-like symptoms.
[2004] Functional Observational Battery (FOB) Test
[2005] The FOB is a series of situations applied to the animal to
determine gross sensory and motor deficits. A subset of tests from
the Irwin neurological screen that evaluates gross neurological
function is used. In general, short-duration, tactile, olfactory,
and visual stimuli are applied to the animal to determine their
ability to detect and respond normally. These simple tests take
approximately 10 minutes and the mouse is returned to its home cage
at the end of testing.
[2006] Results:
Basic Sensory & Motor Observations: All 8 (-/-) mice exhibited
thinning fur with bald patches during the functional observational
battery testing when compared with their (+/+) littermates.
[2007] (c) Bone Metabolism & Body Diagnostics
[2008] (1) Tissue Mass & Lean Body Mass Measurements--Dexa
[2009] Dexa Analysis--Test Description:
[2010] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. Dual Energy X-ray
Absorptiometry (DEXA) has been used successfully to identify
changes in total tissue mass (TTM).
[2011] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PIXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI,
i.e., whole body, vertebrae, and both femurs).
[2012] Body Measurements (Body Length & Weight):
[2013] Body Measurements: A measurement of body length and weight
was performed at approximately 16 weeks of age.
[2014] Results:
[2015] General Observations: The (-/-) mice are much smaller with
oily thinning fur with bald patches and exhibited darker, stickier
feces than their (+/+) littermates.
[2016] Both the male and female (-/-) mice exhibited decreased mean
body weight and decreased mean body length when compared with their
gender-matched (+/+) littermates and the historical means.
[2017] The (-/-) mice also exhibited a decreased mean heart rate
when compared with their gender-matched (+/+) littermates and the
historical mean.
Analyzed wt/het/hom: 12/23/17
[2018] Pathology:
[2019] Microscopic Observations The (-/-) mice exhibited diffuse,
but enhanced sebaceous gland hyperplasia. The skin lesions in the
mutants varied in severity from one area to another and were
generally not associated with inflammation. The majority of
sebaceous glands were active and hyperplastic, even in areas
containing anagenic hair follicles.
[2020] Gene Expression: Expression of the neo transcript was not
detected in the panel of tissues analyzed by in situ hybridization.
Analyzed wt/het/hom: 2/1/6
[2021] (2) Bone Metabolism: Radiology Phenotypic Analysis
[2022] In the area of bone metabolism, targets were identified
herein for the treatment of arthritis, osteoporosis, osteopenia and
osteopetrosis as well as identifying targets that promote bone
healing. Tests included:
[2023] DEXA for measurement of bone mineral density on femur and
vertebra
[2024] MicroCT for very high resolution and very high sensitivity
measurements of bone mineral density for both trabecular and
cortical bone.
[2025] Dexa Analysis--Test Description:
[2026] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. Dual Energy X-ray
Absorptiometry (DEXA) has been used successfully to identify
changes in bone. Anesthetized animals were examined and bone
mineral content (BMC), BMC/LBM ratios, volumetric bone mineral
density (vBMD), total body BMD, femur BMD and vertebra BMD were
measured.
[2027] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PIXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI)
[i.e., whole body, vertebrae, and both femurs].
[2028] Bone MicroCT Analysis:
[2029] Procedure: MicroCT was also used to get very sensitive
measurements of BMD. One vertebra and 1 femur were taken from a
cohort of 4 wild type and 8 homozygous mice. Measurements were
taken of lumbar 5 vertebra trabecular bone volume, trabecular
thickness, connectivity density and midshaft femur total bone area
and cortical thickness. The .mu.CT40 scans provided detailed
information on bone mass and architecture. Multiple bones were
placed into sample holders and scanned automatically. Instrument
software was used to select regions of interest for analysis.
Trabecular bone parameters were analyzed in the fifth lumbar
vertebrae (LV5) at 16 micrometer resolution and cortical bone
parameters were analyzed in the femur midshaft at a resolution of
20 micrometers.
[2030] CAT-Scan Protocol:
[2031] Mice were injected with a CT contrast agent, Omnipaque 300
(Nycomed Amershan, 300 mg of iodine per ml, 0.25 ml per animal, or
2.50-3.75 g iodine/kg of body weight) intraperitoneally. After
resting in the cage for .about.10 minutes, the mouse was then
sedated by intraperitoneal injection of Avertin (1.25%
2,2,2,-tribromoethanol, 20 ml/kg body weight). A CAT-scan was
performed using a MicroCAT scanner (ImTek, Inc.) with the
anesthetized animal lying prone on the test bed. Three dimensional
images were reconstructed by the Feldkamp algorithm in a cluster of
workstations using an ImTek 3D RECON software.
[2032] Results:
[2033] DEXA: Both the male and female (-/-) mice exhibited
decreased mean body mass-related measurements (total tissue mass,
lean body mass, total fat mass, and percent total body fat) and
decreased mean bone mineral-related measurements (bone mineral
content, volumetric bone mineral density, and bone mineral density
in total body, vertebrae, and femur) when compared with their
gender-matched (+/+) littermates and the historical means,
suggesting severe growth retardation in the mutants.
[2034] Micro-CT: The (-/-) mice exhibited decreased mean vertebral
trabecular bone volume, number, thickness, and connective density
and decreased mean femoral mid-shaft cortical thickness and
cross-sectional area when compared with their gender-matched (+/+)
littermates and the historical means.
[2035] These results demonstrate that knockout mutant mice exhibit
abnormal bone metabolism with significant bone loss similar to
osteoporosis characterized by decrease in bone mass with decreased
density and possibly fragility leading to bone fractures. Thus, it
appears that PRO7425 or agonists thereof would be useful in
maintaining bone homeostasis. In addition, PRO7425 or its encoding
gene would be useful in bone healing or for the treatment of
arthritis or osteoporosis; whereas antagonists to PRO7425 or its
encoding gene would lead to abnormal or pathological bone disorders
including inflammatory diseases associated with abnormal bone
metabolism including arthritis, osteoporosis, and osteopenia.
[2036] CAT-Scan: All 3 (-/-) mice available for testing exhibited
generally decreased body size and severe depletion of abdominal and
subcutaneous body fat depots. Bilaterally enlarged kidneys were
noted in 2 mutants (M-83 and M-121) when compared with their (+/+)
and (+/-) littermates of normal body size. However, no obvious
lesion was observed in the kidneys. Analyzed wt/het/hom: 4/4/9
[2037] Thus in summary, the (-/-) mice analyzed by DEXA were quite
small in size and exhibited a notable decrease in body weight and
length as well as a notable depletion of body fat depots, decrease
in total tissue, lean body mass and decreased bone mineral content
and density suggestive of growth retardation in these mutants.
These observations are consistent with a tissue wasting condition
and/or growth retardation. Thus, PRO7425 polypeptides or agonists
thereof must be essential for normal growth and/or growth
metabolism and therefore would be useful in the treatment or
prevention of growth disorders, cachexia or other tissue wasting
diseases.
[2038] (d) Phenotypic Analysis: Metabolism-Blood Chemistry
[2039] In the area of metabolism, targets may be identified for the
treatment of diabetes. Blood chemistry phenotypic analysis includes
measuring serum insulin levels as an indicator of changes in
glucose metabolism. Abnormal glucose metabolism can be related to
the following disorders or conditions: Diabetes Type 1 and Type 2,
Syndrome X, various cardiovascular diseases and/or obesity.
[2040] Insulin Data:
[2041] Test Description: Lexicon Genetics uses the Cobra II Series
Auto-Gamma Counting System in its clinical settings for running
quantitative Insulin assays on mice.
[2042] Results:
[2043] The (-/-) mice exhibited a decreased mean serum insulin
level when compared with their gender-matched (+/+) littermates and
the historical mean. In addition, other Blood Chemistry analysis
indicated that the mutant (-/-) mice showed increased serum
alkaline phosphatase and mean serum ALT (alanine amino
transaminase) suggestive of liver disease. Ketones were also noted
in the urine of the mutant mice. These results indicate a diabetic
phenotype.
[2044] 35.30. Generation and Analysis of Mice Comprising
DNA129542-2808 (UNQ3103) Gene Disruptions
[2045] In these knockout experiments, the gene encoding PRO10102
polypeptides (designated as DNA129542-2808) (UNQ3103) was
disrupted. The gene specific information for these studies is as
follows: the mutated mouse gene corresponds to nucleotide
reference: NM.sub.--021319 or Mus musculus peptidoglycan
recognition protein-like (Pglyrpl-pending); protein reference:
NP.sub.--067294 or peptidoglycan recognition protein-like; TAG-like
[Mus musculus]; the human gene sequence reference: NM.sub.--052890
or Homo sapiens peptidoglycan recognition protein L precursor
(PGRP-L); the human protein sequence corresponds to reference:
NP.sub.--443122 or peptidoglycan recognition protein L precursor
[Homo sapiens].
[2046] The targeted mouse gene is peptidoglycan recognition
protein-like (Pglyrpl-pending), ortholog of human peptidoglycan
recognition protein L precursor (PGRP-L). Aliases include TAGL,
tagL, PGRP-L, TAGL-beta, tagl-beta, TAGL-alpha, tagL-alpha,
PGLYRPL, TAGL-like, and TAG-like.
[2047] PGRP-L is an evolutionarily conserved
N-acetylmuramoyl-L-alanine amidase expressed in liver that cleaves
the lactylamide bond in bacterial cell wall peptidoglycan (Gelius
et al., Biochem Biophys Res Commun, 306(4):988-94 (2003)).
Bioinformatic analysis of PGRP-L suggests that the protein is
secreted, containing a signal peptide and a C-terminal
"N-acetylmuramoyl-L-alanine amidase" domain (Pfam 01510). However,
PGRP-L may be an integral membrane protein (Kibardin et al., J Mol
Biol, 326(2):467-74 (2003)).
[2048] Variants lacking the C-terminal amidase catalytic domain are
still able to bind with gram-positive bacteria, gram-negative
bacteria, and peptidoglycan, indicating that the catalytic and
peptidoglycan-binding domains are separate (Kibardin et al, 2003).
PGRP-L is likely to play a role in innate immunity, recognizing and
degrading bacteria in liver (Gelius et al., Biochem Biophys Res
Commun, 306(4):988-94 (2003); Kibardin et al., J Mol Biol,
326(2):467-74 (2003)).
[2049] Targeted or gene trap mutations were generated in strain
129SvEv.sup.Brd-derived embryonic stem (ES) cells. The chimeric
mice were bred to C57BL/6J albino mice to generate F1 heterozygous
animals. These progeny were intercrossed to generate F2 wild type,
heterozygous, and homozygous mutant progeny. On rare occasions, for
example when very few F1 mice were obtained from the chimera, F1
heterozygous mice were crossed to 129SvEv.sup.Brd/C57 hybrid mice
to yield additional heterozygous animals for the intercross to
generate the F2 mice.
Level I phenotypic analysis is performed on mice from this
generation
TABLE-US-00080 wt het hom Total Observed 17 35 16 68 Expected 17 34
17 68 Chi-Sq. = 0.09 Significance = 0.95684 (hom/n) = 0.24 Avg.
Litter Size = 7
Mutation Type: Homologous Recombination (standard) Exons 1 and 2
were targeted (NCBI NM.sub.--021319). Wild-type expression of the
target gene was detected in embryonic stem (ES) cells and in all 13
adult tissue samples tested by RT-PCR, except kidney, testis, and
bone. Disruption of the target gene was confirmed by Southern
hybridization analysis.
[2050] 35.30.1. Phenotypic Analysis (for Disrupted Gene:
DNA129542-2808 (UNQ3103)
[2051] (a) Overall Phenotypic Summary:
[2052] Mutation of the gene encoding the ortholog of human
peptidoglycan recognition protein L precursor (PGRP-L) resulted in
a decreased anxiety-related response in female (-/-) mice. However,
the male (-/-) mice did not exhibit the same response. There was a
strong trend towards stress-induced hyperthermia noted for the
mutant (-/-) mice. These observations show an anxiety-related
phenotype but a liver specific expression pattern does not suggest
these behavioral effects. Gene disruption was confirmed by Southern
blot.
[2053] (b) Expression Pattern
[2054] UNQ3103 shows a very specific expression in the liver (see
EXAMPLE 41 for protocol)
[2055] (c) Phenotypic Analysis: CNS/Neurology
[2056] In the area of neurology, analysis focused herein on
identifying in vivo validated targets for the treatment of
neurological and psychiatric disorders including depression,
generalized anxiety disorders, attention deficit hyperactivity
disorder, obsessive compulsive disorder, schizophrenia, cognitive
disorders, hyperalgesia and sensory disorders. Neurological
disorders include the category defined as "anxiety disorders" which
include but are not limited to: mild to moderate anxiety, anxiety
disorder due to a general medical condition, anxiety disorder not
otherwise specified, generalized anxiety disorder, panic attack,
panic disorder with agoraphobia, panic disorder without
agoraphobia, posttraumatic stress disorder, social phobia, specific
phobia, substance-induced anxiety disorder, acute alcohol
withdrawal, obsessive compulsive disorder, agoraphobia, bipolar
disorder I or II, bipolar disorder not otherwise specified,
cyclothymic disorder, depressive disorder, major depressive
disorder, mood disorder, substance-induced mood disorder. In
addition, anxiety disorders may apply to personality disorders
including but not limited to the following types: paranoid,
antisocial, avoidant behavior, borderline personality disorders,
dependent, histronic, narcissistic, obsessive-compulsive, schizoid,
and schizotypal.
[2057] Procedure:
[2058] Behavioral screens were performed on a cohort of 4 wild
type, 4 heterozygous and 8 homozygous mutant mice. All behavioral
tests were done between 12 and 16 weeks of age unless reduced
viability necessitates earlier testing. These tests included open
field to measure anxiety, activity levels and exploration.
[2059] Open Field Test:
[2060] Several targets of known drugs have exhibited phenotypes in
the open field test. These include knockouts of the seratonin
transporter, the dopamine transporter (Giros et al., Nature. 1996
Feb. 15; 379(6566):606-12), and the GABA receptor (Homanics et al.,
Proc Natl Acad Sci USA. 1997 Apr. 15; 94(8):4143-8). An automated
open-field assay was customized to address changes related to
affective state and exploratory patterns related to learning First,
the field (40.times.40 cm) was selected to be relatively large for
a mouse, thus designed to pick up changes in locomotor activity
associated with exploration. In addition, there were 4 holes in the
floor to allow for nose-poking, an activity specifically related to
exploration. Several factors were also designed to heighten the
affective state associated with this test. The open-field test is
the first experimental procedure in which the mice are tested, and
the measurements that were taken were the subjects' first
experience with the chamber. In addition, the open-field was
brightly lit. All these factors will heighten the natural anxiety
associated with novel and open spaces. The pattern and extent of
exploratory activity, and especially the center-to-total distance
traveled ratio, may then be able to discern changes related to
susceptibility to anxiety or depression. A large arena (40
cm.times.40 cm, VersaMax animal activity monitoring system from
AccuScan Instruments) with infrared beams at three different levels
was used to record rearing, hole poke, and locomotor activity. The
animal was placed in the center and its activity was measured for
20 minutes. Data from this test was analyzed in five, 4-minute
intervals. The total distance traveled (cm), vertical movement
number (rearing), number of hole pokes, and the center to total
distance ratio were recorded.
[2061] The propensity for mice to exhibit normal habituation
responses to a novel environment is assessed by determining the
overall change in their horizontal locomotor activity across the 5
time intervals. This calculated slope of the change in activity
over time is determined using normalized, rather than absolute,
total distance traveled. The slope is determined from the
regression line through the normalized activity at each of the 5
time intervals. Normal habituation is represented by a negative
slope value.
[2062] Results:
[2063] A difference was found during open field testing. The female
(-/-) mice exhibited an increased median sum time in center when
compared with their gender-matched (+/+) littermates and the
historical mean, suggesting a decreased anxiety-like response in
the mutants. Male (-/-) mice did not exhibit the same response.
[2064] As noted above, a difference was observed during open field
activity testing. The female (-/-) mice exhibited an increased
median sum time in the center area when compared with their
gender-matched (+/+) littermates, which is indicative of a
decreased anxiety-like response in the mutants. Thus, knockout mice
demonstrated a phenotype consistent with depressive disorders,
schizophrenia and/or bipolar disorders. Thus, PRO10102 polypeptides
and agonists thereof would be useful for the treatment or
amelioration of the symptoms associated with depressive
disorders.
[2065] Functional Observational Battery (FOB) Test
[2066] The FOB is a series of situations applied to the animal to
determine gross sensory and motor deficits. A subset of tests from
the Irwin neurological screen that evaluates gross neurological
function is used. In general, short-duration, tactile, olfactory,
and visual stimuli are applied to the animal to determine their
ability to detect and respond normally. These simple tests take
approximately 10 minutes and the mouse is returned to its home cage
at the end of testing.
[2067] Results:
[2068] The mutant (-/-) mice showed a strong trend towards
increased stress induced hyperthermia (three-fourths 3/4 (-/-) mice
tested showed increases two standard deviations (2 SD) above the
historical mean).
[2069] 35.31. Generation and Analysis of Mice Comprising
DNA148380-2827 (UNQ3126) Gene Disruptions
[2070] In these knockout experiments, the gene encoding PRO10282
polypeptides (designated as DNA148380-2827) (UNQ3126) was
disrupted. The gene specific information for these studies is as
follows: the mutated mouse gene corresponds to nucleotide
reference: NM.sub.--009291 or ACCESSION: NM.sub.--009291 NID: gi
6678170 ref NM.sub.--009291.1 Mus musculus stimulated by retinoic
acid gene 6 (Stra6); protein reference: 070491 or ACCESSION: O70491
NID: Mus musculus (Mouse). Retinoic acid-responsive protein.
MOUSESPTRNRDB; the human gene sequence reference: NM.sub.--022369
or ACCESSION: NM.sub.--022369 NID: gi 21314699 ref
NM.sub.--022369.2 Homo sapiens hypothetical protein FLJ12541
similar to Stra6 (FLJ12541); the human protein sequence corresponds
to reference: Q9BX79 or ACCESSION: Q9BX79 NID: Homo sapiens
(Human). STRA6 isoform 1. HUMANSPTRNRDB.
[2071] The mouse gene of interest is Stra6 (stimulated by retinoic
acid gene 6), ortholog of human "stimulated by retinoic acid gene
6." Aliases include FLJ12541.
[2072] Stra6 is a likely integral plasma membrane protein induced
by retinoic acid. The protein contains eight transmembrane segments
but no other discernable domain. Stra6 is often located at
blood-organ barriers and may function as a component of a transport
apparatus. Stra6 is expressed in Sertoli cells during specific
stages of spermatocyte development (Bouillet et al., Mech Dev,
63(2):173-86 (1997)) and in developing mouse limbs (Chazaud et al.,
Dev Genet, 19(1):66-73 (1996)). Wnt-1 potentiates retinoid-induced
Stra6 expression, and Stra6 mRNA may be overexpressed in human
colorectal cancers as well as other tumors driven by activation of
the Wnt-1/beta-catenin pathway (Szeto et al., Cancer Res,
61(10):4197-205 (2001); Tice et al., J Biol Chem, 277(16):14329-35
(2002)).
Genetics Information:
TABLE-US-00081 [2073] wt het hom Total Observed 16 34 19 69
Expected 17.25 34.5 17.25 69 Chi-Sq. = 0.28 Significance = 0.87138
(hom/n) = 0.28 Avg. Litter Size = 7
Mutation Type: Retroviral Insertion (OST)
[2074] Retroviral insertion occurred in the intron between coding
exons 1 and 2 (NCBI accession NM.sub.--009291.1). Wild-type
expression of the target gene was detected in brain, eye, thymus,
and lung among the 13 adult tissue samples tested by RT-PCR. RT-PCR
analysis revealed that the transcript was absent in the (-/-) mouse
analyzed (M-76). Disruption of the target gene was confirmed by
Inverse PCR.
[2075] 35.31.1. Phenotypic Analysis (for Disrupted Gene:
DNA148380-2827 (UNQ3126)
[2076] (a) Overall Phenotypic Summary:
[2077] Mutation of the gene encoding the ortholog of human putative
"stimulated by retinoic acid gene 6" (STRA6) resulted in growth
retardation and decreased bone measurements in (-/-) mice. An
increased level of mean serum glucose was also observed in (-/-)
mice. Transcript was absent by RT-PCR.
[2078] (b) Bone Metabolism & Body Diagnostics
[2079] (1) Tissue Mass & Lean Body Mass Measurements--Dexa
[2080] Dexa Analysis--Test Description:
[2081] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. Dual Energy X-ray
Absorptiometry (DEXA) has been used successfully to identify
changes in total tissue mass (TTM).
[2082] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PIXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI,
i.e., whole body, vertebrae, and both femurs).
[2083] Body Measurements (Body Length & Weight):
[2084] Body Measurements: A measurement of body length and weight
was performed at approximately 16 weeks of age.
[2085] Results:
[2086] The (-/-) mice exhibited decreased mean body weight and
decreased mean body weight when compared with their gender-matched
(+/+) littermates and the historical means. Analyzed wt/het/hom:
16/37/19
[2087] (2) Bone Metabolism: Radiology Phenotypic Analysis
[2088] In the area of bone metabolism, targets were identified
herein for the treatment of arthritis, osteoporosis, osteopenia and
osteopetrosis as well as identifying targets that promote bone
healing. Tests included:
[2089] DEXA for measurement of bone mineral density on femur and
vertebra
[2090] MicroCT for very high resolution and very high sensitivity
measurements of bone mineral density for both trabecular and
cortical bone.
[2091] Dexa Analysis--Test Description:
[2092] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. Dual Energy X-ray
Absorptiometry (DEXA) has been used successfully to identify
changes in bone. Anesthetized animals were examined and bone
mineral content (BMC), BMC/LBM ratios, volumetric bone mineral
density (vBMD), total body BMD, femur BMD and vertebra BMD were
measured.
[2093] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PIXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI)
[i.e., whole body, vertebrae, and both femurs].
[2094] Bone MicroCT Analysis:
[2095] Procedure: MicroCT was also used to get very sensitive
measurements of BMD. One vertebra and 1 femur were taken from a
cohort of 4 wild type and 8 homozygous mice. Measurements were
taken of lumbar 5 vertebra trabecular bone volume, trabecular
thickness, connectivity density and midshaft femur total bone area
and cortical thickness. The .mu.CT40 scans provided detailed
information on bone mass and architecture. Multiple bones were
placed into sample holders and scanned automatically. Instrument
software was used to select regions of interest for analysis.
Trabecular bone parameters were analyzed in the fifth lumbar
vertebrae (LV5) at 16 micrometer resolution and cortical bone
parameters were analyzed in the femur midshaft at a resolution of
20 micrometers.
[2096] Results:
[2097] DEXA: The (-/-) mice exhibited decreased mean total tissue
mass and lean body mass when compared with their gender-matched
(+/+) littermates and the historical means. In addition to these
changes noted, fat (% and gram), bone mineral density and bone
mineral content were all lower relative to their wild-type
littermates consistent with the decreased size of the (-/-)
mice.
[2098] Micro-CT: The (-/-) mice exhibited decreased mean vertebral
trabecular bone volume, number, thickness, and connective density
and decreased mean femoral mid-shaft cortical thickness and
cross-sectional area when compared with their gender-matched (+/+)
littermates and the historical means.
[2099] These results demonstrate that knockout mutant mice exhibit
abnormal bone metabolism with significant bone loss similar to
osteoporosis characterized by decrease in bone mass with decreased
density and possibly fragility leading to bone fractures. Thus, it
appears that PRO10282 or agonists thereof would be useful in
maintaining bone homeostasis. In addition, PRO10282 or its encoding
gene would be useful in bone healing or for the treatment of
arthritis or osteoporosis; whereas antagonists to PRO10282 or its
encoding gene would lead to abnormal or pathological bone disorders
including inflammatory diseases associated with abnormal bone
metabolism including arthritis, osteoporosis, and osteopenia.
[2100] Thus in summary, the (-/-) mice analyzed by DEXA were quite
small in size and exhibited a notable decrease in body weight and
length as well as a notable depletion of body fat depots, decrease
in total tissue mass, lean body mass and decreased bone mineral
content and density suggestive of growth retardation in these
mutants. Thus, PRO10282 polypeptides or agonists thereof must be
essential for normal growth and/or growth metabolism and therefore
would be useful in the treatment or prevention of growth disorders,
cachexia or other tissue wasting diseases.
[2101] (c) Phenotypic Analysis: Metabolism-Blood Chemistry/Glucose
Tolerance
[2102] In the area of metabolism, targets may be identified for the
treatment of diabetes. Blood chemistry phenotypic analysis includes
blood glucose measurements. The COBAS Integra 400 (mfr: Roche) was
used for running blood chemistry tests on the mice. In the area of
metabolism, targets may be identified for the treatment of
diabetes. Blood chemistry phenotypic analysis includes glucose
tolerance tests to measure insulin sensitivity and changes in
glucose metabolism. Abnormal glucose tolerance test results may
indicate but may not be limited to the following disorders or
conditions: Diabetes Type 1 and Type 2, Syndrome X, various
cardiovascular diseases and/or obesity.
[2103] Procedure: A cohort of 2 wild type and 4 homozygous mice
were used in this assay. The glucose tolerance test is the standard
for defining impaired glucose homeostasis in mammals. Glucose
tolerance tests were performed using a Lifescan glucometer. Animals
were injected IP at 2 g/kg with D-glucose delivered as a 20%
solution and blood glucose levels were measured at 0, 30, 60 and 90
minutes after injection. Analyzed wt/het/hom: 4/4/8
[2104] Results:
[2105] Blood Chemistry The (-/-) mice exhibited an increased mean
serum glucose level when compared with their gender-matched (+/+)
littermates and the historical mean. During the glucose tolerance
test, the (-/-) mice exhibited an increased mean fasting serum
glucose level when compared with their gender-matched (+/+)
littermates and the historical mean.
[2106] Thus, knockout mice exhibited the phenotypic pattern of an
impaired glucose homeostasis with elevated levels of fasting serum
glucose indicative of diabetes or a pre-diabetic condition. Based
on these results, PRO10282 (or agonists thereof) or its encoding
gene would be useful in the treatment of an impaired glucose
metabolism and/or diabetes.
[2107] 35.32. Generation and Analysis of Mice Comprising DNA347767
(UNQ14964) Gene Disruptions
[2108] In these knockout experiments, the gene encoding PRO61709
polypeptides (designated as DNA347767 (UNQ14964) was disrupted. The
gene specific information for these studies is as follows: the
mutated mouse gene corresponds to nucleotide reference:
NM.sub.--172294 or Mus musculus sulfatase 1 (Sulf1); protein
reference: Q8K007 or ACCESSION: Q8K007 NID: Mus musculus (Mouse).
Extracellular sulfatase Sulf-1 precursor (EC 3.1.6.-) (MSulf-1);
the human gene sequence reference: NM.sub.--015170 or Homo sapiens
sulfatase 1 (SULF1); the human protein sequence corresponds to
reference: Q81WU6 or Extracellular sulfatase Sulf-1 precursor
(HSulf-1) gi|27356932|gb|AAM76860.1| extracellular sulfatase SULF-1
[Homo sapiens].
[2109] The disrupted mouse gene is Sulf1 (sulfatase 1), ortholog of
human SULF1. Aliases include AW121680, extracellular sulfatase
SULF-1, SULF-1, HSULF-1, KIAA1077, and sulfatase FP.
[2110] SULF1 is a secreted arylsulfatase with the ability to remove
sulfate from glucosamine within subregions of heparin. This action
on heparin is likely to inhibit signaling by heparin-dependent
growth factors, thereby inhibiting cell proliferation and
facilitating apoptosis (Morimoto-Tomita, et al., J Biol Chem,
277(51):49175-85 (2002); Lai et al., J Biol Chem, 278(25):23107-17
(2003); Lai et al., Gastroenterology, 126(1):231-48 (2004)).
Down-regulation of SULF1 may be a mechanism by which certain types
of cancer cells enhance growth factor signaling (Lai et al., J Biol
Chem, 278(25):23107-17 (2003)).
[2111] Targeted or gene trap mutations were generated in strain
129SvEv.sup.Brd-derived embryonic stem (ES) cells. The chimeric
mice were bred to C57BL/6J albino mice to generate F1 heterozygous
animals. These progeny were intercrossed to generate F2 wild type,
heterozygous, and homozygous mutant progeny. On rare occasions, for
example when very few F1 mice were obtained from the chimera, F1
heterozygous mice were crossed to 129SvEv.sup.Brd/C57 hybrid mice
to yield additional heterozygous animals for the intercross to
generate the F2 mice.
Level I phenotypic analysis is performed on mice from this
generation
TABLE-US-00082 wt het hom Total Observed 31 44 19 94 Expected 23.5
47 23.5 94 Chi-Sq. = 3.45 Significance = 0.17846 (hom/n) = 0.20
Avg. Litter Size = 9
Mutation Type: Retroviral Insertion (OST)
[2112] Retroviral insertion occurred in the intron preceding coding
exon 1 (NCBI accession NM.sub.--172294.1). Wild-type expression of
the target gene was detected in embryonic stem (ES) cells and in
all 13 adult tissue samples tested by RT-PCR. RT-PCR analysis
revealed that the transcript was absent in the (-/-) mouse analyzed
(M-208).
[2113] 35.32.1. Phenotypic Analysis (for Disrupted Gene: DNA347767
(UNQ14964)
[2114] (a) Overall Phenotypic Summary:
[2115] Mutation of the gene encoding the ortholog of human
sulfatase 1 (SULF1) resulted in a decreased IL-6, TNF alpha and
MCP-1 response to LPS challenge in (-/-) mice. Transcript was
absent by RT-PCR.
[2116] (b) Immunology Phenotypic Analysis
[2117] Immune related and inflammatory diseases are the
manifestation or consequence of fairly complex, often multiple
interconnected biological pathways which in normal physiology are
critical to respond to insult or injury, initiate repair from
insult or injury, and mount innate and acquired defense against
foreign organisms. Disease or pathology occurs when these normal
physiological pathways cause additional insult or injury either as
directly related to the intensity of the response, as a consequence
of abnormal regulation or excessive stimulation, as a reaction to
self, or as a combination of these.
[2118] Though the genesis of these diseases often involves
multistep pathways and often multiple different biological
systems/pathways, intervention at critical points in one or more of
these pathways can have an ameliorative or therapeutic effect.
Therapeutic intervention can occur by either antagonism of a
detrimental process/pathway or stimulation of a beneficial
process/pathway.
[2119] T lymphocytes (T cells) are an important component of a
mammalian immune response. T cells recognize antigens which are
associated with a self-molecule encoded by genes within the major
histocompatibility complex (MHC). The antigen may be displayed
together with MHC molecules on the surface of antigen presenting
cells, virus infected cells, cancer cells, grafts, etc. The T cell
system eliminates these altered cells which pose a health threat to
the host mammal. T cells include helper T cells and cytotoxic T
cells. Helper T cells proliferate extensively following recognition
of an antigen-MHC complex on an antigen presenting cell. Helper T
cells also secrete a variety of cytokines, i.e., lymphokines, which
play a central role in the activation of B cells, cytotoxic T cells
and a variety of other cells which participate in the immune
response.
[2120] In many immune responses, inflammatory cells infiltrate the
site of injury or infection. The migrating cells may be
neutrophilic, eosinophilic, monocytic or lymphocytic as can be
determined by histological examination of the affected tissues.
Current Protocols in Immunology, ed. John E. Coligan, 1994, John
Wiley & Sons, Inc.
[2121] Many immune related diseases are known and have been
extensively studied. Such diseases include immune-mediated
inflammatory diseases (such as rheumatoid arthritis, immune
mediated renal disease, hepatobiliary diseases, inflammatory bowel
disease (IBD), psoriasis, and asthma), non-immune-mediated
inflammatory diseases, infectious diseases, immunodeficiency
diseases, neoplasia, and graft rejection, etc. In the area of
immunology, targets were identified for the treatment of
inflammation and inflammatory disorders.
[2122] In the area of immunology, targets have been identified
herein for the treatment of inflammation and inflammatory
disorders. Immune related diseases, in one instance, could be
treated by suppressing the immune response. Using neutralizing
antibodies that inhibit molecules having immune stimulatory
activity would be beneficial in the treatment of immune-mediated
and inflammatory diseases. Molecules which inhibit the immune
response can be utilized (proteins directly or via the use of
antibody agonists) to inhibit the immune response and thus
ameliorate immune related disease.
[2123] The following test was performed:
[2124] Acute Phase Response:
[2125] Test Description: Bacterial lipopolysaccharide (LPS) is an
endotoxin, and as such is a potent inducer of an acute phase
response and systemic inflammation. The Level I LPS mice were
injected intraperitoneally (i.p.) with a sublethal dose of LPS in
200 .mu.L sterile saline using a 26 gauge needle. The doses were
based on the average weight of the mice tested at 1 .mu.g/g body
weight 3 hours after injection; a 100 ul blood sample was then
taken and analyzed for the presence of TNFa, MCP-1, and IL-6 on the
FACSCalibur instrument.
[2126] Results:
[2127] The (-/-) mice exhibited a decreased mean serum IL-6
response (as well as TNF-alpha and MCP-1) to LPS challenge when
compared with their (+/+) littermates. Analyzed wt/het/hom:
8/4/8
[2128] In summary, the LPS endotoxin challenge demonstrated that
knockout mice deficient in the gene encoding PRO61709 polypeptides
exhibit immunological abnormalities when compared with their
wild-type littermates. In particular, the mutant mice exhibited a
decreased ability to elicit an immunological response (TNF-alpha,
MCP-1 and IL-6 production) when challenged with the LPS endotoxin
indicating a proinflammatory response. IL-6, MCP-1 and TNF alpha
contribute to the later stages of B cell activation. In addition,
they play a critical role in inducing the acute phase response and
systemic inflammation. This suggests that PRO61709 polypeptides or
agonists thereof would stimulate the immune system and would find
utility in the cases wherein this effect would be beneficial to the
individual such as in the case of leukemia, and other types of
cancer, and in immunocompromised patients, such as AIDS sufferers.
Accordingly, inhibitors or antagonists to PRO61709 polypeptides
thereof would be useful in inhibiting the immune response and would
be useful candidates for suppressing harmful immune responses, e.g.
in the case of graft rejection or graft-versus-host diseases.
[2129] 35.33. Generation and Analysis of Mice Comprising
DNA58801-1052 (UNQ455) Gene Disruptions
[2130] In these knockout experiments, the gene encoding PRO779
polypeptides (designated as DNA58801-1052 (UNQ455) was disrupted.
The gene specific information for these studies is as follows: the
mutated mouse gene corresponds to nucleotide reference:
NM.sub.--033042 or ACCESSION: NM.sub.--033042 NID: 14719437 Mus
musculus Mus musculus tumor necrosis factor receptor superfamily,
member 12 (Tnfrsf12); protein reference: Q99MM1 or Q99MM1 Q99MM1
WSL-1-LIKE PROTEIN; the human gene sequence reference:
NM.sub.--003790 or ACCESSION: NM.sub.--003790 NID: 4507568 Homo
sapiens Homo sapiens tumor necrosis factor receptor superfamily,
member 12 (translocating chain-association membrane protein)
(TNFRSF12); the human protein sequence corresponds to reference:
Q93038 or TR12_HUMAN Q93038 WSL-1 PROTEIN PRECURSOR
APOPTOSIS-MEDI.
[2131] The mouse gene of interest is Tnfrsf25 (tumor necrosis
factor receptor superfamily, member 25), ortholog of human
TNFRSF25. Aliases include DR3, TR3, Wsl, DDR3, LARD, APO-3, TRAMP,
WSL-1, WSL-LR, Tnfrsf12, WSL-1 protein, death receptor beta, death
domain receptor 3, apoptosis inducing receptor, apoptosis-mediating
receptor, death domain receptor 3 soluble form, lymphocyte
associated receptor of death, translocating chain-association
membrane protein, and tumor necrosis factor receptor superfamily
member 12. TNFRSF25 is a type I plasma membrane protein expressed
in thymocytes and lymphocytes that functions as a receptor for the
ligands TNFSF12 (tumor necrosis factor (ligand) superfamily, member
12) and TNFSF15 (tumor necrosis factor (ligand) superfamily, member
15). Activation of TNFRSF25 can stimulate apoptosis or, conversely,
promote T cell expansion by a nuclear factor kappa-B-mediated
process (Chinnaiyan et al, Science 274(5289):990-2 (1996); Kitson
et al, Nature 384(6607):372-5 (1996); Migone et al, Immunity
16(3):479-92 (2002); Wen et al, J Biol Chem 278(40):39251-8
(2003)). TNFRSF25 appears to play a role in lymphocyte homeostasis.
Wang et al., Mol Cell Biol 21(10):3451-61 (2001) showed that
negative selection and anti-CD3-induced apoptosis are impaired in
TNFRSF25 homozygous null mice but not in wild-type mice, suggesting
that TNFRSF25 plays a non redundant role in removal of
self-reactive T cells in the thymus.
[2132] Targeted or gene trap mutations were generated in strain
129SvEv.sup.Brd-derived embryonic stem (ES) cells. The chimeric
mice were bred to C57BL/6J albino mice to generate F1 heterozygous
animals. These progeny were intercrossed to generate F2 wild type,
heterozygous, and homozygous mutant progeny. On rare occasions, for
example when very few F1 mice were obtained from the chimera, F1
heterozygous mice were crossed to 129SvEv.sup.Brd/C57 hybrid mice
to yield additional heterozygous animals for the intercross to
generate the F2 mice.
Level I phenotypic analysis is performed on mice from this
generation
TABLE-US-00083 wt het hom Total Observed 21 42 21 84 Expected 21 42
21 84 Chi-Sq. = 0.00 Significance = 1.00000 (hom/n) = 0.25 Avg.
Litter Size = 8
Mutation Type: Homologous Recombination (standard) Coding exons 1
through 5 were targeted (NCBI accession NM.sub.--033042.2).
Wild-type expression of the target gene was detected in embryonic
stem (ES) cells and in all 13 adult tissue samples tested by
RT-PCR. RT-PCR analysis revealed that the transcript was absent in
the (-/-) mouse analyzed (M-208).
[2133] 35.33.1. Phenotypic Analysis (for Disrupted Gene:
DNA58801-1052 (UNQ455)
[2134] (a) Overall Phenotypic Summary:
[2135] Mutation of the gene encoding the ortholog of human tumor
necrosis factor receptor superfamily, member 25 (TNFRSF25) resulted
in decreased lumbar 5 vertebra measurements in the (-/-) mice
compared to the wild-type littermate controls and historical means.
In addition, the mutant knockout mice exhibited an enhanced IgG
responses to an ovalbumin challenge.
[2136] (b) Immunology Phenotypic Analysis
[2137] Immune related and inflammatory diseases are the
manifestation or consequence of fairly complex, often multiple
interconnected biological pathways which in normal physiology are
critical to respond to insult or injury, initiate repair from
insult or injury, and mount innate and acquired defense against
foreign organisms. Disease or pathology occurs when these normal
physiological pathways cause additional insult or injury either as
directly related to the intensity of the response, as a consequence
of abnormal regulation or excessive stimulation, as a reaction to
self, or as a combination of these.
[2138] Though the genesis of these diseases often involves
multistep pathways and often multiple different biological
systems/pathways, intervention at critical points in one or more of
these pathways can have an ameliorative or therapeutic effect.
Therapeutic intervention can occur by either antagonism of a
detrimental process/pathway or stimulation of a beneficial
process/pathway.
[2139] T lymphocytes (T cells) are an important component of a
mammalian immune response. T cells recognize antigens which are
associated with a self-molecule encoded by genes within the major
histocompatibility complex (MHC). The antigen may be displayed
together with MHC molecules on the surface of antigen presenting
cells, virus infected cells, cancer cells, grafts, etc. The T cell
system eliminates these altered cells which pose a health threat to
the host mammal. T cells include helper T cells and cytotoxic T
cells. Helper T cells proliferate extensively following recognition
of an antigen-MHC complex on an antigen presenting cell. Helper T
cells also secrete a variety of cytokines, i.e., lymphokines, which
play a central role in the activation of B cells, cytotoxic T cells
and a variety of other cells which participate in the immune
response.
[2140] In many immune responses, inflammatory cells infiltrate the
site of injury or infection. The migrating cells may be
neutrophilic, eosinophilic, monocytic or lymphocytic as can be
determined by histological examination of the affected tissues.
Current Protocols in Immunology, ed. John E. Coligan, 1994, John
Wiley & Sons, Inc.
[2141] Many immune related diseases are known and have been
extensively studied. Such diseases include immune-mediated
inflammatory diseases (such as rheumatoid arthritis, immune
mediated renal disease, hepatobiliary diseases, inflammatory bowel
disease (IBD), psoriasis, and asthma), non-immune-mediated
inflammatory diseases, infectious diseases, immunodeficiency
diseases, neoplasia, and graft rejection, etc. In the area of
immunology, targets were identified for the treatment of
inflammation and inflammatory disorders.
[2142] In the area of immunology, targets have been identified
herein for the treatment of inflammation and inflammatory
disorders. Immune related diseases, in one instance, could be
treated by suppressing the immune response. Using neutralizing
antibodies that inhibit molecules having immune stimulatory
activity would be beneficial in the treatment of immune-mediated
and inflammatory diseases. Molecules which inhibit the immune
response can be utilized (proteins directly or via the use of
antibody agonists) to inhibit the immune response and thus
ameliorate immune related disease.
[2143] The following test was performed:
[2144] Ovalbumin Challenge
[2145] Procedure: This assay was carried out on 7 wild types and 8
homozygotes. Chicken ovalbumin (OVA) is a T-cell dependent antigen,
which is commonly used as a model protein for studying
antigen-specific immune responses in mice. OVA is non-toxic and
inert and therefore will not cause harm to the animals even if no
immune response is induced. The murine immune response to OVA has
been well characterized, to the extent that the immunodominant
peptides for eliciting T cell responses have been identified.
Anti-OVA antibodies are detectable 8 to 10 days after immunization
using enzyme-linked immunosorbent assay (ELIZA), and determination
of different isotypes of antibodies gives further information on
the complex processes that may lead to a deficient response in
genetically engineered mice.
[2146] As noted above, this protocol assesses the ability of mice
to raise an antigen-specific immune response. Animals were injected
IP with 50 mg of chicken ovalbumin emulsified in Complete Freund's
Adjuvant and 14 days later the serum titer of anti-ovalbumin
antibodies (IgM, IgG1 and IgG2 subclasses) was measured. The amount
of OVA-specific antibody in the serum sample is proportional to the
Optical Density (OD) value generated by an instrument that scans a
96-well sample plate. Data was collected for a set of serial
dilutions of each serum sample.
Analyzed wt/het/hom: 8/4/9
[2147] Results of this Challenge:
[2148] The (-/-) mice exhibited an increased mean serum IgG2a
response to ovalbumin challenge when compared with their (+/+)
littermates and the historical means. Thus, these knockout mice
exhibited an increased ability to elicit an OVA-specific antibody
response to the T-cell dependent OVA antigen.
[2149] In summary, the ovalbumin challenge studies indicate that
knockout mice deficient in the gene encoding PRO779 polypeptides
exhibit immunological abnormalities when compared with their
wild-type littermates. In particular, the mutant mice exhibited an
increased ability to elicit an immunological response when
challenged with the T-cell dependent OVA antigen. Thus, inhibitors
or antagonists of PRO779 polypeptides would be useful for
stimulating the immune system (such as T cell proliferation) and
would find utility in the cases wherein this effect would be
beneficial to the individual such as in the case of leukemia, and
other types of cancer, and in immunocompromised patients, such as
AIDS sufferers. Accordingly, PRO779 polypeptides or agonists
thereof would be useful for inhibiting the immune response and thus
would be useful candidates for suppressing harmful immune
responses, e.g. in the case of graft rejection or graft-versus-host
diseases.
[2150] (c) Bone Metabolism & Body Diagnostics: Radiology
Phenotypic Analysis
[2151] In the area of bone metabolism, targets were identified
herein for the treatment of arthritis, osteoporosis, osteopenia and
osteopetrosis as well as identifying targets that promote bone
healing. Tests included:
[2152] DEXA for measurement of bone mineral density on femur and
vertebra
[2153] MicroCT for very high resolution and very high sensitivity
measurements of bone mineral density for both trabecular and
cortical bone.
[2154] Dexa Analysis--Test Description:
[2155] Procedure: A cohort of 4 wild type, 4 heterozygotes and 8
homozygotes were tested in this assay. Dual Energy X-ray
Absorptiometry (DEXA) has been used successfully to identify
changes in bone. Anesthetized animals were examined and bone
mineral content (BMC), BMC/LBM ratios, volumetric bone mineral
density (vBMD), total body BMD, femur BMD and vertebra BMD were
measured.
[2156] The mouse was anesthetized by intraperitoneal injection of
Avertin (1.25% 2,2,2,-tribromoethanol, 20 ml/kg body weight), body
length and weight were measured, and then the mouse was placed in a
prone position on the platform of the PIXImus.TM. Densitometer
(Lunar Inc.) for a DEXA scan. Using Lunar PIXImus software, the
bone mineral density (BMD) and fat composition (% fat) and total
tissue mass (TTM) were determined in the regions of interest (ROI)
[i.e., whole body, vertebrae, and both femurs].
[2157] Bone MicroCT Analysis:
[2158] Procedure: MicroCT was also used to get very sensitive
measurements of BMD. One vertebra and 1 femur were taken from a
cohort of 4 wild type and 8 homozygous mice. Measurements were
taken of lumbar 5 vertebra trabecular bone volume, trabecular
thickness, connectivity density and midshaft femur total bone area
and cortical thickness. The .mu.CT40 scans provided detailed
information on bone mass and architecture. Multiple bones were
placed into sample holders and scanned automatically. Instrument
software was used to select regions of interest for analysis.
Trabecular bone parameters were analyzed in the fifth lumbar
vertebrae (LV5) at 16 micrometer resolution and cortical bone
parameters were analyzed in the femur midshaft at a resolution of
20 micrometers.
[2159] Results:
[2160] Micro-CT: The (-/-) mice exhibited a decreased lumbar 5
vertebra measurements in bone volume, trabecular number and
connectivity density when compared with their gender-matched (+/+)
littermates and the historical means. These results demonstrate
that knockout mutant mice exhibit abnormal bone metabolism with
significant bone loss similar to osteoporosis characterized by
decrease in bone mass with decreased density and possibly fragility
leading to bone fractures. Thus, it appears that PRO779
polypeptides or agonists thereof play a role in maintaining bone
homeostasis. In addition, PRO779 or its encoding gene would be
useful in maintaining bone homeostasis and would be important in
bone healing or useful for the treatment of osteoarthritis or
osteoporosis; whereas antagonists to PRO779 or its encoding gene
would lead to abnormal or pathological bone disorders including
inflammatory diseases associated with abnormal bone metabolism
including arthritis, osteoporosis, and osteopenia.
Example 36
Use of PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 as a hybridization probe
[2161] The following method describes use of a nucleotide sequence
encoding a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 polypeptide as a
hybridization probe.
[2162] DNA comprising the coding sequence of full-length or mature
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptides as disclosed herein is
employed as a probe to screen for homologous DNAs (such as those
encoding naturally-occurring variants of PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptides) in human tissue cDNA libraries or human tissue
genomic libraries.
[2163] Hybridization and washing of filters containing either
library DNAs is performed under the following high stringency
conditions. Hybridization of radiolabeled PRO196-, PRO217-,
PRO231-, PRO236-, PRO245-, PRO246-, PRO258-, PRO287-, PRO328-,
PRO344-, PRO357-, PRO526-, PRO724-, PRO731-, PRO732-, PRO1003-,
PRO1104-, PRO1151-, PRO1244-, PRO1298-, PRO1313-, PRO1570-,
PRO1886-, PRO1891-, PRO4409-, PRO5725-, PRO5994-, PRO6097-,
PRO7425-, PRO10102-, PRO10282-, PRO61709- or PRO779-derived probe
to the filters is performed in a solution of 50% formamide,
5.times.SSC, 0.1% SDS, 0.1% sodium pyrophosphate, 50 mM sodium
phosphate, pH 6.8, 2.times.Denhardt's solution, and 10% dextran
sulfate at 42.degree. C. for 20 hours. Washing of the filters is
performed in an aqueous solution of 0.1.times.SSC and 0.1% SDS at
42.degree. C.
[2164] DNAs having a desired sequence identity with the DNA
encoding full-length native sequence PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptides can then be identified using standard techniques known
in the art.
Example 37
Expression of PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 in E. coli
[2165] This example illustrates preparation of an unglycosylated
form of PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 polypeptides by recombinant
expression in E. coli.
[2166] The DNA sequence encoding a PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide is initially amplified using selected PCR primers. The
primers should contain restriction enzyme sites which correspond to
the restriction enzyme sites on the selected expression vector. A
variety of expression vectors may be employed. An example of a
suitable vector is pBR322 (derived from E. coli; see Bolivar et
al., Gene, 2:95 (1977)) which contains genes for ampicillin and
tetracycline resistance. The vector is digested with restriction
enzyme and dephosphorylated. The PCR amplified sequences are then
ligated into the vector. The vector will preferably include
sequences which encode for an antibiotic resistance gene, a trp
promoter, a polyhis leader (including the first six STII codons,
polyhis sequence, and enterokinase cleavage site), the PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 coding region, lambda transcriptional
terminator, and an argU gene.
[2167] The ligation mixture is then used to transform a selected E.
coli strain using the methods described in Sambrook et al., supra.
Transformants are identified by their ability to grow on LB plates
and antibiotic resistant colonies are then selected. Plasmid DNA
can be isolated and confirmed by restriction analysis and DNA
sequencing.
[2168] Selected clones can be grown overnight in liquid culture
medium such as LB broth supplemented with antibiotics. The
overnight culture may subsequently be used to inoculate a larger
scale culture. The cells are then grown to a desired optical
density, during which the expression promoter is turned on.
[2169] After culturing the cells for several more hours, the cells
can be harvested by centrifugation. The cell pellet obtained by the
centrifugation can be solubilized using various agents known in the
art, and the solubilized PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 protein
can then be purified using a metal chelating column under
conditions that allow tight binding of the protein.
[2170] PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 may be expressed in E. coli
in a poly-His tagged form, using the following procedure. The DNA
encoding PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 is initially amplified using
selected PCR primers. The primers will contain restriction enzyme
sites which correspond to the restriction enzyme sites on the
selected expression vector, and other useful sequences providing
for efficient and reliable translation initiation, rapid
purification on a metal chelation column, and proteolytic removal
with enterokinase. The PCR-amplified, poly-His tagged sequences are
then ligated into an expression vector, which is used to transform
an E. coli host based on strain 52 (W3110 fuhA(tonA) lon galE
rpoHts(htpRts) clpP(lacIq). Transformants are first grown in LB
containing 50 mg/ml carbenicillin at 30.degree. C. with shaking
until an O.D.600 of 3-5 is reached. Cultures are then diluted
50-100 fold into CRAP media (prepared by mixing 3.57 g
(NH.sub.4).sub.2SO.sub.4, 0.71 g sodium citrate.2H2O, 1.07 g KCl,
5.36 g Difco yeast extract, 5.36 g Sheffield hycase SF in 500 mL
water, as well as 110 mM MPOS, pH 7.3, 0.55% (w/v) glucose and 7 mM
MgSO.sub.4) and grown for approximately 20-30 hours at 30.degree.
C. with shaking. Samples are removed to verify expression by
SDS-PAGE analysis, and the bulk culture is centrifuged to pellet
the cells. Cell pellets are frozen until purification and
refolding.
[2171] E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets)
is resuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH
8 buffer. Solid sodium sulfite and sodium tetrathionate is added to
make final concentrations of 0.1M and 0.02 M, respectively, and the
solution is stirred overnight at 4.degree. C. This step results in
a denatured protein with all cysteine residues blocked by
sulfitolization. The solution is centrifuged at 40,000 rpm in a
Beckman Ultracentifuge for 30 min. The supernatant is diluted with
3-5 volumes of metal chelate column buffer (6 M guanidine, 20 mM
Tris, pH 7.4) and filtered through 0.22 micron filters to clarify.
The clarified extract is loaded onto a 5 ml Qiagen Ni-NTA metal
chelate column equilibrated in the metal chelate column buffer. The
column is washed with additional buffer containing 50 mM imidazole
(Calbiochem, Utrol grade), pH 7.4. The protein is eluted with
buffer containing 250 mM imidazole. Fractions containing the
desired protein are pooled and stored at 4.degree. C. Protein
concentration is estimated by its absorbance at 280 nm using the
calculated extinction coefficient based on its amino acid
sequence.
[2172] The proteins are refolded by diluting the sample slowly into
freshly prepared refolding buffer consisting of: 20 mM Tris, pH
8.6, 0.3 M NaCl, 2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM
EDTA. Refolding volumes are chosen so that the final protein
concentration is between 50 to 100 micrograms/ml. The refolding
solution is stirred gently at 4.degree. C. for 12-36 hours. The
refolding reaction is quenched by the addition of TFA to a final
concentration of 0.4% (pH of approximately 3). Before further
purification of the protein, the solution is filtered through a
0.22 micron filter and acetonitrile is added to 2-10% final
concentration. The refolded protein is chromatographed on a Poros
R1/H reversed phase column using a mobile buffer of 0.1% TFA with
elution with a gradient of acetonitrile from 10 to 80%. Aliquots of
fractions with A280 absorbance are analyzed on SDS polyacrylamide
gels and fractions containing homogeneous refolded protein are
pooled. Generally, the properly refolded species of most proteins
are eluted at the lowest concentrations of acetonitrile since those
species are the most compact with their hydrophobic interiors
shielded from interaction with the reversed phase resin. Aggregated
species are usually eluted at higher acetonitrile concentrations.
In addition to resolving misfolded forms of proteins from the
desired form, the reversed phase step also removes endotoxin from
the samples.
[2173] Fractions containing the desired folded PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 polypeptide are pooled and the acetonitrile removed using a
gentle stream of nitrogen directed at the solution. Proteins are
formulated into 20 mM Hepes, pH 6.8 with 0.14 M sodium chloride and
4% mannitol by dialysis or by gel filtration using G25 Superfine
(Pharmacia) resins equilibrated in the formulation buffer and
sterile filtered.
Example 38
Expression of PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 in mammalian
cells
[2174] This example illustrates preparation of a potentially
glycosylated form of a PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide by recombinant expression in mammalian cells.
[2175] The vector, pRK5 (see EP 307,247, published Mar. 15, 1989),
is employed as the expression vector. Optionally, the PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 DNA is ligated into pRK5 with selected
restriction enzymes to allow insertion of the PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 DNA using ligation methods such as described in Sambrook et
al., supra. The resulting vector is called pRK5-PRO196,
pRK5-PRO217, pRK5-PRO231, pRK5-PRO236, pRK5-PRO245, pRK5-PRO246,
pRK5-PRO258, pRK5-PRO287, pRK5-PRO328, pRK5-PRO344, pRK5-PRO357,
pRK5-PRO526, pRK5-PRO724, pRK5-PRO731, pRK5-PRO732, pRK5-PRO1003,
pRK5-PRO1104, pRK5-PRO1151, pRK5-PRO1244, pRK5-PRO1298,
pRK5-PRO1313, pRK5-PRO1570, pRK5-PRO1886, pRK5-PRO1891,
pRK5-PRO4409, pRK5-PRO5725, pRK5-PRO5994, pRK5-PRO6097,
pRK5-PRO7425, pRK5-PRO10102, pRK5-PRO10282, pRK5-PRO61709 or
pRK5-PRO779.
[2176] The selected host cells may be 293 cells. Human 293 cells
(ATCC CCL 1573) are grown to confluence in tissue culture plates in
medium such as DMEM supplemented with fetal calf serum and
optionally, nutrient components and/or antibiotics. About 10 .mu.g
pRK5-PRO196, pRK5-PRO217, pRK5-PRO231, pRK5-PRO236, pRK5-PRO245,
pRK5-PRO246, pRK5-PRO258, pRK5-PRO287, pRK5-PRO328, pRK5-PRO344,
pRK5-PRO357, pRK5-PRO526, pRK5-PRO724, pRK5-PRO731, pRK5-PRO732,
pRK5-PRO1003, pRK5-PRO1104, pRK5-PRO1151, pRK5-PRO1244,
pRK5-PRO1298, pRK5-PRO1313, pRK5-PRO1570, pRK5-PRO1886,
pRK5-PRO1891, pRK5-PRO4409, pRK5-PRO5725, pRK5-PRO5994,
pRK5-PRO6097, pRK5-PRO7425, pRK5-PRO10102, pRK5-PRO10282,
pRK5-PRO61709 or pRK5-PRO779 DNA is mixed with about 1 .mu.g DNA
encoding the VA RNA gene [Thimmappaya et al., Cell, 31:543 (1982)]
and dissolved in 500 .mu.l of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 M
CaCl.sub.2. To this mixture is added, dropwise, 500 .mu.l of 50 mM
HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO.sub.4, and a precipitate
is allowed to form for 10 minutes at 25.degree. C. The precipitate
is suspended and added to the 293 cells and allowed to settle for
about four hours at 37.degree. C. The culture medium is aspirated
off and 2 ml of 20% glycerol in PBS is added for 30 seconds. The
293 cells are then washed with serum free medium, fresh medium is
added and the cells are incubated for about 5 days.
[2177] Approximately 24 hours after the transfections, the culture
medium is removed and replaced with culture medium (alone) or
culture medium containing 200 .mu.Ci/ml .sup.35S-cysteine and 200
.mu.Ci/ml .sup.35S-methionine. After a 12 hour incubation, the
conditioned medium is collected, concentrated on a spin filter, and
loaded onto a 15% SDS gel. The processed gel may be dried and
exposed to film for a selected period of time to reveal the
presence of PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 polypeptides. The cultures
containing transfected cells may undergo further incubation (in
serum free medium) and the medium is tested in selected
bioassays.
[2178] In an alternative technique, PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
may be introduced into 293 cells transiently using the dextran
sulfate method described by Somparyrac et al., Proc. Natl. Acad.
Sci., 12:7575 (1981). 293 cells are grown to maximal density in a
spinner flask and 700 .mu.g pRK5-PRO196, pRK5-PRO217, pRK5-PRO231,
pRK5-PRO236, pRK5-PRO245, pRK5-PRO246, pRK5-PRO258, pRK5-PRO287,
pRK5-PRO328, pRK5-PRO344, pRK5-PRO357, pRK5-PRO526, pRK5-PRO724,
pRK5-PRO731, pRK5-PRO732, pRK5-PRO1003, pRK5-PRO1104, pRK5-PRO1151,
pRK5-PRO1244, pRK5-PRO1298, pRK5-PRO1313, pRK5-PRO1570,
pRK5-PRO1886, pRK5-PRO1891, pRK5-PRO4409, pRK5-PRO5725,
pRK5-PRO5994, pRK5-PRO6097, pRK5-PRO7425, pRK5-PRO10102,
pRK5-PRO10282, pRK5-PRO61709 or pRK5-PRO779 DNA is added. The cells
are first concentrated from the spinner flask by centrifugation and
washed with PBS. The DNA-dextran precipitate is incubated on the
cell pellet for four hours. The cells are treated with 20% glycerol
for 90 seconds, washed with tissue culture medium, and
re-introduced into the spinner flask containing tissue culture
medium, 5 .mu.g/ml bovine insulin and 0.1 .mu.g/ml bovine
transferrin. After about four days, the conditioned media is
centrifuged and filtered to remove cells and debris. The sample
containing expressed PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 can then
be concentrated and purified by any selected method, such as
dialysis and/or column chromatography.
[2179] PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 can be expressed in CHO
cells. The pRK5-PRO196, pRK5-PRO217, pRK5-PRO231, pRK5-PRO236,
pRK5-PRO245, pRK5-PRO246, pRK5-PRO258, pRK5-PRO287, pRK5-PRO328,
pRK5-PRO344, pRK5-PRO357, pRK5-PRO526, pRK5-PRO724, pRK5-PRO731,
pRK5-PRO732, pRK5-PRO1003, pRK5-PRO1104, pRK5-PRO1151,
pRK5-PRO1244, pRK5-PRO1298, pRK5-PRO1313, pRK5-PRO1570,
pRK5-PRO1886, pRK5-PRO1891, pRK5-PRO4409, pRK5-PRO5725,
pRK5-PRO5994, pRK5-PRO6097, pRK5-PRO7425, pRK5-PRO10102,
pRK5-PRO10282, pRK5-PRO61709 or pRK5-PRO779 can be transfected into
CHO cells using known reagents such as CaPO.sub.4 or DEAE-dextran.
As described above, the cell cultures can be incubated, and the
medium replaced with culture medium (alone) or medium containing a
radiolabel such as .sup.35S-methionine. After determining the
presence of PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 polypeptide, the culture
medium may be replaced with serum free medium. Preferably, the
cultures are incubated for about 6 days, and then the conditioned
medium is harvested. The medium containing the expressed PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 can then be concentrated and purified by any
selected method.
[2180] Epitope-tagged PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 may also
be expressed in host CHO cells. The PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
may be subcloned out of the pRK5 vector. The subclone insert can
undergo PCR to fuse in frame with a selected epitope tag such as a
poly-his tag into a Baculovirus expression vector. The poly-his
tagged PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 insert can then be subcloned
into a SV40 driven vector containing a selection marker such as
DHFR for selection of stable clones. Finally, the CHO cells can be
transfected (as described above) with the SV40 driven vector.
Labeling may be performed, as described above, to verify
expression. The culture medium containing the expressed poly-His
tagged PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 can then be concentrated and
purified by any selected method, such as by Ni.sup.2+-chelate
affinity chromatography.
[2181] PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 may also be expressed in CHO
and/or COS cells by a transient expression procedure or in CHO
cells by another stable expression procedure.
[2182] Stable expression in CHO cells is performed using the
following procedure. The proteins are expressed as an IgG construct
(immunoadhesin), in which the coding sequences for the soluble
forms (e.g. extracellular domains) of the respective proteins are
fused to an IgG1 constant region sequence containing the hinge, CH2
and CH2 domains and/or is a poly-His tagged form.
[2183] Following PCR amplification, the respective DNAs are
subcloned in a CHO expression vector using standard techniques as
described in Ausubel et al., Current Protocols of Molecular
Biology, Unit 3.16, John Wiley and Sons (1997). CHO expression
vectors are constructed to have compatible restriction sites 5' and
3' of the DNA of interest to allow the convenient shuttling of
cDNA's. The vector used expression in CHO cells is as described in
Lucas et al., Nucl. Acids Res. 24:9 (1774-1779 (1996), and uses the
SV40 early promoter/enhancer to drive expression of the cDNA of
interest and dihydrofolate reductase (DHFR). DHFR expression
permits selection for stable maintenance of the plasmid following
transfection.
[2184] Twelve micrograms of the desired plasmid DNA is introduced
into approximately 10 million CHO cells using commercially
available transfection reagents Superfect.RTM. (Qiagen),
Dosper.RTM. or Fugene.RTM. (Boehringer Mannheim). The cells are
grown as described in Lucas et al., supra. Approximately
3.times.10.sup.7 cells are frozen in an ampule for further growth
and production as described below.
[2185] The ampules containing the plasmid DNA are thawed by
placement into water bath and mixed by vortexing. The contents are
pipetted into a centrifuge tube containing 10 mLs of media and
centrifuged at 1000 rpm for 5 minutes. The supernatant is aspirated
and the cells are resuspended in 10 mL of selective media (0.2
.mu.m filtered PS20 with 5% 0.2 .mu.m diafiltered fetal bovine
serum). The cells are then aliquoted into a 100 mL spinner
containing 90 mL of selective media. After 1-2 days, the cells are
transferred into a 250 mL spinner filled with 150 mL selective
growth medium and incubated at 37.degree. C. After another 2-3
days, 250 mL, 500 mL and 2000 mL spinners are seeded with
3.times.10.sup.5 cells/mL. The cell media is exchanged with fresh
media by centrifugation and resuspension in production medium.
Although any suitable CHO media may be employed, a production
medium described in U.S. Pat. No. 5,122,469, issued Jun. 16, 1992
may actually be used. A 3 L production spinner is seeded at
1.2.times.10.sup.6 cells/mL. On day 0, the cell number pH ie
determined On day 1, the spinner is sampled and sparging with
filtered air is commenced. On day 2, the spinner is sampled, the
temperature shifted to 33.degree. C., and 30 mL of 500 g/L glucose
and 0.6 mL of 10% antifoam (e.g., 35% polydimethylsiloxane
emulsion, Dow Corning 365 Medical Grade Emulsion) taken. Throughout
the production, the pH is adjusted as necessary to keep it at
around 7.2. After 10 days, or until the viability dropped below
70%, the cell culture is harvested by centrifugation and filtering
through a 0.22 .mu.m filter. The filtrate was either stored at
4.degree. C. or immediately loaded onto columns for
purification.
[2186] For the poly-His tagged constructs, the proteins are
purified using a Ni-NTA column (Qiagen). Before purification,
imidazole is added to the conditioned media to a concentration of 5
mM. The conditioned media is pumped onto a 6 ml Ni-NTA column
equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl
and 5 mM imidazole at a flow rate of 4-5 ml/min at 4.degree. C.
After loading, the column is washed with additional equilibration
buffer and the protein eluted with equilibration buffer containing
0.25 M imidazole. The highly purified protein is subsequently
desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl
and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia)
column and stored at -80.degree. C.
[2187] Immunoadhesin (Fc-containing) constructs are purified from
the conditioned media as follows. The conditioned medium is pumped
onto a 5 ml Protein A column (Pharmacia) which had been
equilibrated in 20 mM Na phosphate buffer, pH 6.8. After loading,
the column is washed extensively with equilibration buffer before
elution with 100 mM citric acid, pH 3.5. The eluted protein is
immediately neutralized by collecting 1 ml fractions into tubes
containing 275 .mu.L of 1 M Tris buffer, pH 9. The highly purified
protein is subsequently desalted into storage buffer as described
above for the poly-His tagged proteins. The homogeneity is assessed
by SDS polyacrylamide gels and by N-terminal amino acid sequencing
by Edman degradation.
Example 39
Expression of PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 in Yeast
[2188] The following method describes recombinant expression of
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 in yeast.
[2189] First, yeast expression vectors are constructed for
intracellular production or secretion of PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
from the ADH2/GAPDH promoter. DNA encoding PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
and the promoter is inserted into suitable restriction enzyme sites
in the selected plasmid to direct intracellular expression of
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779. For secretion, DNA encoding PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 can be cloned into the selected plasmid,
together with DNA encoding the ADH2/GAPDH promoter, a native
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 signal peptide or other mammalian
signal peptide, or, for example, a yeast alpha-factor or invertase
secretory signal/leader sequence, and linker sequences (if needed)
for expression of PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779.
[2190] Yeast cells, such as yeast strain AB110, can then be
transformed with the expression plasmids described above and
cultured in selected fermentation media. The transformed yeast
supernatants can be analyzed by precipitation with 10%
trichloroacetic acid and separation by SDS-PAGE, followed by
staining of the gels with Coomassie Blue stain.
[2191] Recombinant PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 can subsequently be
isolated and purified by removing the yeast cells from the
fermentation medium by centrifugation and then concentrating the
medium using selected cartridge filters. The concentrate containing
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 may further be purified using selected
column chromatography resins.
Example 40
Expression of PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 in
Baculovirus-Infected Insect Cells
[2192] The following method describes recombinant expression of
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 in Baculovirus-infected insect
cells.
[2193] The sequence coding for PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
is fused upstream of an epitope tag contained within a baculovirus
expression vector. Such epitope tags include poly-his tags and
immunoglobulin tags (like Fc regions of IgG). A variety of plasmids
may be employed, including plasmids derived from commercially
available plasmids such as pVL1393 (Novagen). Briefly, the sequence
encoding PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 or the desired portion of
the coding sequence of PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 such as
the sequence encoding the extracellular domain of a transmembrane
protein or the sequence encoding the mature protein if the protein
is extracellular is amplified by PCR with primers complementary to
the 5' and 3' regions. The 5' primer may incorporate flanking
(selected) restriction enzyme sites. The product is then digested
with those selected restriction enzymes and subcloned into the
expression vector.
[2194] Recombinant baculovirus is generated by co-transfecting the
above plasmid and BaculoGold.TM. virus DNA (Pharmingen) into
Spodoptera frugiperda ("Sf9") cells (ATCC CRL 1711) using
lipofectin (commercially available from GIBCO-BRL). After 4-5 days
of incubation at 28.degree. C., the released viruses are harvested
and used for further amplifications. Viral infection and protein
expression are performed as described by O'Reilley et al.,
Baculovirus expression vectors: A Laboratory Manual, Oxford: Oxford
University Press (1994).
[2195] Expressed poly-his tagged PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
can then be purified, for example, by Ni.sup.2+-chelate affinity
chromatography as follows. Extracts are prepared from recombinant
virus-infected Sf9 cells as described by Rupert et al., Nature,
362:175-179 (1993). Briefly, Sf9 cells are washed, resuspended in
sonication buffer (25 mL Hepes, pH 7.9; 12.5 mM MgCl.sub.2; 0.1 mM
EDTA; 10% glycerol; 0.1% NP-40; 0.4 M KCl), and sonicated twice for
20 seconds on ice. The sonicates are cleared by centrifugation, and
the supernatant is diluted 50-fold in loading buffer (50 mM
phosphate, 300 mM NaCl, 10% glycerol, pH 7.8) and filtered through
a 0.45 .mu.m filter. A Ni.sup.2+-NTA agarose column (commercially
available from Qiagen) is prepared with a bed volume of 5 mL,
washed with 25 mL of water and equilibrated with 25 mL of loading
buffer. The filtered cell extract is loaded onto the column at 0.5
mL per minute. The column is washed to baseline A.sub.280 with
loading buffer, at which point fraction collection is started.
Next, the column is washed with a secondary wash buffer (50 mM
phosphate; 300 mM NaCl, 10% glycerol, pH 6.0), which elutes
nonspecifically bound protein. After reaching A.sub.280 baseline
again, the column is developed with a 0 to 500 mM Imidazole
gradient in the secondary wash buffer. One mL fractions are
collected and analyzed by SDS-PAGE and silver staining or Western
blot with Ni.sup.2+-NTA-conjugated to alkaline phosphatase
(Qiagen). Fractions containing the eluted His.sub.10-tagged PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 are pooled and dialyzed against loading
buffer.
[2196] Alternatively, purification of the IgG tagged (or Fc tagged)
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 can be performed using known
chromatography techniques, including for instance, Protein A or
protein G column chromatography.
Example 41
Tissue Expression Profiling Using GeneExpress.RTM.
[2197] A proprietary database containing gene expression
information (GeneExpress.RTM., Gene Logic Inc., Gaithersburg, Md.)
was analyzed in an attempt to identify polypeptides (and their
encoding nucleic acids) whose expression is significantly
upregulated in a particular tumor tissue(s) of interest as compared
to other tumor(s) and/or normal tissues. Specifically, analysis of
the GeneExpress.RTM. database was conducted using either software
available through Gene Logic Inc., Gaithersburg, Md., for use with
the GeneExpress.RTM. database or with proprietary software written
and developed at Genentech, Inc. for use with the GeneExpress.RTM.
database. The rating of positive hits in the analysis is based upon
several criteria including, for example, tissue specificity, tumor
specificity and expression level in normal essential and/or normal
proliferating tissues. The following is a list of molecules whose
tissue expression profile as determined from an analysis of the
GeneExpress.RTM. database evidences high tissue expression and
significant upregulation of expression in a specific tumor or
tumors as compared to other tumor(s) and/or normal tissues and
optionally relatively low expression in normal essential and/or
normal proliferating tissues. Tissue expression profiling was
performed on several UNQ genes the results of which are disclosed
in Example 35.
Example 42
Microarray Analysis to Detect Upregulation of UNQ Genes in
Cancerous Tumors
[2198] Nucleic acid microarrays, often containing thousands of gene
sequences, are useful for identifying differentially expressed
genes in diseased tissues as compared to their normal counterparts.
Using nucleic acid microarrays, test and control mRNA samples from
test and control tissue samples are reverse transcribed and labeled
to generate cDNA probes. The cDNA probes are then hybridized to an
array of nucleic acids immobilized on a solid support. The array is
configured such that the sequence and position of each member of
the array is known. For example, a selection of genes known to be
expressed in certain disease states may be arrayed on a solid
support. Hybridization of a labeled probe with a particular array
member indicates that the sample from which the probe was derived
expresses that gene. If the hybridization signal of a probe from a
test (disease tissue) sample is greater than hybridization signal
of a probe from a control (normal tissue) sample, the gene or genes
overexpressed in the disease tissue are identified. The implication
of this result is that an overexpressed protein in a diseased
tissue is useful not only as a diagnostic marker for the presence
of the disease condition, but also as a therapeutic target for
treatment of the disease condition.
[2199] The methodology of hybridization of nucleic acids and
microarray technology is well known in the art. In one example, the
specific preparation of nucleic acids for hybridization and probes,
slides, and hybridization conditions are all detailed in PCT Patent
Application Serial No. PCT/US01/10482, filed on Mar. 30, 2001 and
which is herein incorporated by reference.
[2200] In the present example, cancerous tumors derived from
various human tissues were studied for upregulated gene expression
relative to cancerous tumors from different tissue types and/or
non-cancerous human tissues in an attempt to identify those
polypeptides which are overexpressed in a particular cancerous
tumor(s). In certain experiments, cancerous human tumor tissue and
non-cancerous human tumor tissue of the same tissue type (often
from the same patient) were obtained and analyzed for UNQ
polypeptide expression. Additionally, cancerous human tumor tissue
from any of a variety of different human tumors was obtained and
compared to a "universal" epithelial control sample which was
prepared by pooling non-cancerous human tissues of epithelial
origin, including liver, kidney, and lung. mRNA isolated from the
pooled tissues represents a mixture of expressed gene products from
these different tissues. Microarray hybridization experiments using
the pooled control samples generated a linear plot in a 2-color
analysis. The slope of the line generated in a 2-color analysis was
then used to normalize the ratios of (test:control detection)
within each experiment. The normalized ratios from various
experiments were then compared and used to identify clustering of
gene expression. Thus, the pooled "universal control" sample not
only allowed effective relative gene expression determinations in a
simple 2-sample comparison, it also allowed multi-sample
comparisons across several experiments.
[2201] In the present experiments, nucleic acid probes derived from
the herein described UNQ polypeptide-encoding nucleic acid
sequences were used in the creation of the microarray and RNA from
various tumor tissues were used for the hybridization thereto.
Below is shown the results of these experiments, demonstrating that
various UNQ polypeptides of the present invention are significantly
overexpressed in various human tumor tissues as compared to their
normal counterpart tissue(s). Moreover, all of the molecules shown
below are significantly overexpressed in their specific tumor
tissue(s) as compared to in the "universal" epithelial control. As
described above, these data demonstrate that the UNQ polypeptides
of the present invention are useful not only as diagnostic markers
for the presence of one or more cancerous tumors, but also serve as
therapeutic targets for the treatment of those tumors. Microarray
analysis was performed on several UNQ genes the results of which
are disclosed in Example 35.
Example 43
Quantitative Analysis of UNQ mRNA Expression
[2202] In this assay, a 5' nuclease assay (for example, TaqMan0)
and real-time quantitative PCR (for example, ABI Prizm 7700
Sequence Detection System.RTM. (Perkin Elmer, Applied Biosystems
Division, Foster City, Calif.)), were used to find genes that are
significantly overexpressed in a cancerous tumor or tumors as
compared to other cancerous tumors or normal non-cancerous tissue.
The 5' nuclease assay reaction is a fluorescent PCR-based technique
which makes use of the 5' exonuclease activity of Taq DNA
polymerase enzyme to monitor gene expression in real time. Two
oligonucleotide primers (whose sequences are based upon the gene or
EST sequence of interest) are used to generate an amplicon typical
of a PCR reaction. A third oligonucleotide, or probe, is designed
to detect nucleotide sequence located between the two PCR primers.
The probe is non-extendible by Taq DNA polymerase enzyme, and is
labeled with a reporter fluorescent dye and a quencher fluorescent
dye. Any laser-induced emission from the reporter dye is quenched
by the quenching dye when the two dyes are located close together
as they are on the probe. During the PCR amplification reaction,
the Taq DNA polymerase enzyme cleaves the probe in a
template-dependent manner. The resultant probe fragments
disassociate in solution, and signal from the released reporter dye
is free from the quenching effect of the second fluorophore. One
molecule of reporter dye is liberated for each new molecule
synthesized, and detection of the unquenched reporter dye provides
the basis for quantitative interpretation of the data.
[2203] The 5' nuclease procedure is run on a real-time quantitative
PCR device such as the ABI Prism 7700.TM. Sequence Detection. The
system consists of a thermocycler, laser, charge-coupled device
(CCD) camera and computer. The system amplifies samples in a
96-well format on a thermocycler. During amplification,
laser-induced fluorescent signal is collected in real-time through
fiber optics cables for all 96 wells, and detected at the CCD. The
system includes software for running the instrument and for
analyzing the data.
[2204] The starting material for the screen was mRNA isolated from
a variety of different cancerous tissues. The mRNA is quantitated
precisely, e.g., fluorometrically. As a negative control, RNA was
isolated from various normal tissues of the same tissue type as the
cancerous tissues being tested.
[2205] 5' nuclease assay data are initially expressed as Ct, or the
threshold cycle. This is defined as the cycle at which the reporter
signal accumulates above the background level of fluorescence. The
ACt values are used as quantitative measurement of the relative
number of starting copies of a particular target sequence in a
nucleic acid sample when comparing cancer mRNA results to normal
human mRNA results. As one Ct unit corresponds to 1 PCR cycle or
approximately a 2-fold relative increase relative to normal, two
units corresponds to a 4-fold relative increase, 3 units
corresponds to an 8-fold relative increase and so on, one can
quantitatively measure the relative fold increase in mRNA
expression between two or more different tissues. Using this
technique, the molecules have been identified as being
significantly overexpressed in a particular tumor(s) as compared to
their normal non-cancerous counterpart tissue(s) (from both the
same and different tissue donors) and thus, represent excellent
polypeptide targets for the diagnosis and therapy of cancer in
mammals. Specific results for a UNQ gene are disclosed in Example
35.
Example 44
In Situ Hybridization
[2206] In situ hybridization is a powerful and versatile technique
for the detection and localization of nucleic acid sequences within
cell or tissue preparations. It may be useful, for example, to
identify sites of gene expression, analyze the tissue distribution
of transcription, identify and localize viral infection, follow
changes in specific mRNA synthesis and aid in chromosome
mapping.
[2207] In situ hybridization was performed following an optimized
version of the protocol by Lu and Gillett, Cell Vision 1:169-176
(1994), using PCR-generated .sup.33P-labeled riboprobes. Briefly,
formalin-fixed, paraffin-embedded human tissues were sectioned,
deparaffinized, deproteinated in proteinase K (20/ml) for 15
minutes at 37.degree. C., and further processed for in situ
hybridization as described by Lu and Gillett, supra. A [.sup.33-P]
UTP-labeled antisense riboprobe was generated from a PCR product
and hybridized at 55.degree. C. overnight. The slides were dipped
in Kodak NTB2 nuclear track emulsion and exposed for 4 weeks.
.sup.33P-Riboprobe Synthesis
[2208] 6.0 .mu.l (125 mCi) of .sup.33P-UTP (Amersham BF 1002,
SA<2000 Ci/mmol) were speed vac dried. To each tube containing
dried .sup.33P-UTP, the following ingredients were added:
[2209] 2.0 .mu.l 5.times. transcription buffer
[2210] 1.0 .mu.l DTT (100 mM)
[2211] 2.0 .mu.l NTP mix (2.5 mM: 10.mu.; each of 10 mM GTP, CTP
& ATP+10 .mu.l H.sub.2O)
[2212] 1.0 .mu.l UTP (50 .mu.M)
[2213] 1.0 .mu.l Rnasin
[2214] 1.0 .mu.l DNA template (1 .mu.g)
[2215] 1.0 .mu.l H.sub.2O
[2216] 1.0 .mu.l RNA polymerase (for PCR products T3=AS, T7=S,
usually)
[2217] The tubes were incubated at 37.degree. C. for one hour. 1.0
.mu.l RQ1 DNase were added, followed by incubation at 37.degree. C.
for 15 minutes. 90 .mu.l TE (10 mM Tris pH 7.6/1 mM EDTA pH 8.0)
were added, and the mixture was pipetted onto DE81 paper. The
remaining solution was loaded in a Microcon-50 ultrafiltration
unit, and spun using program 10 (6 minutes). The filtration unit
was inverted over a second tube and spun using program 2 (3
minutes). After the final recovery spin, 100 .mu.l TE were added. 1
.mu.l of the final product was pipetted on DE81 paper and counted
in 6 ml of Biofluor II.
[2218] The probe was run on a TBE/urea gel. 1-3 .mu.l of the probe
or 5 .mu.l of RNA Mrk III were added to 3 .mu.l of loading buffer.
After heating on a 95.degree. C. heat block for three minutes, the
probe was immediately placed on ice. The wells of gel were flushed,
the sample loaded, and run at 180-250 volts for 45 minutes. The gel
was wrapped in saran wrap and exposed to XAR film with an
intensifying screen in -70.degree. C. freezer one hour to
overnight.
.sup.33P-Hybridization
[2219] A. Pretreatment of Frozen Sections
[2220] The slides were removed from the freezer, placed on
aluminium trays and thawed at room temperature for 5 minutes. The
trays were placed in 55.degree. C. incubator for five minutes to
reduce condensation. The slides were fixed for 10 minutes in 4%
paraformaldehyde on ice in the fume hood, and washed in
0.5.times.SSC for 5 minutes, at room temperature (25 ml
20.times.SSC+975 ml SQ H.sub.2O). After deproteination in 0.5
.mu.g/ml proteinase K for 10 minutes at 37.degree. C. (12.5 .mu.l
of 10 mg/ml stock in 250 ml prewarmed RNase-free RNAse buffer), the
sections were washed in 0.5.times.SSC for 10 minutes at room
temperature. The sections were dehydrated in 70%, 95%, 100%
ethanol, 2 minutes each.
[2221] B. Pretreatment of Paraffin-Embedded Sections
[2222] The slides were deparaffinized, placed in SQ H.sub.2O, and
rinsed twice in 2.times.SSC at room temperature, for 5 minutes each
time. The sections were deproteinated in 20 .mu.g/ml proteinase K
(500 .mu.l of 10 mg/ml in 250 ml RNase-free RNase buffer;
37.degree. C., 15 minutes)-human embryo, or 8.times. proteinase K
(100 .mu.l in 250 ml Rnase buffer, 37.degree. C., 30
minutes)--formalin tissues. Subsequent rinsing in 0.5.times.SSC and
dehydration were performed as described above.
[2223] C. Prehybridization
[2224] The slides were laid out in a plastic box lined with Box
buffer (4.times.SSC, 50% formamide)-saturated filter paper.
[2225] D. Hybridization
[2226] 1.0.times.10.sup.6 cpm probe and 1.0 .mu.l tRNA (50 mg/ml
stock) per slide were heated at 95.degree. C. for 3 minutes. The
slides were cooled on ice, and 48 .mu.l hybridization buffer were
added per slide. After vortexing, 50 .mu.l .sup.33P mix were added
to 50 .mu.l prehybridization on slide. The slides were incubated
overnight at 55.degree. C.
[2227] E. Washes
[2228] Washing was done 2.times.10 minutes with 2.times.SSC, EDTA
at room temperature (400 ml 20.times.SSC+16 ml 0.25M EDTA, Vf=4 L),
followed by RNaseA treatment at 37.degree. C. for 30 minutes (500
.mu.l of 10 mg/ml in 250 ml Rnase buffer=20 .mu.g/ml), The slides
were washed 2.times.10 minutes with 2.times.SSC, EDTA at room
temperature. The stringency wash conditions were as follows: 2
hours at 55.degree. C., 0.1.times.SSC, EDTA (20 ml 20.times.SSC+16
ml EDTA, Vf=4 L).
[2229] F. Oligonucleotides
[2230] In situ analysis was performed on a variety of DNA sequences
disclosed herein. The oligonucleotides employed for these analyses
were obtained so as to be complementary to the nucleic acids (or
the complements thereof) as shown in the accompanying figures.
[2231] G. Results
[2232] In situ analysis was performed on a variety of DNA sequences
disclosed herein the results of which are disclosed in Example
35.
Example 45
Preparation of Antibodies that Bind PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779
[2233] This example illustrates preparation of monoclonal
antibodies which can specifically bind PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779.
[2234] Techniques for producing the monoclonal antibodies are known
in the art and are described, for instance, in Goding, supra.
Immunogens that may be employed include purified PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 polypeptides, fusion proteins containing PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 polypeptides, and cells expressing recombinant PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptides on the cell surface. Selection of
the immunogen can be made by the skilled artisan without undue
experimentation.
[2235] Mice, such as Balb/c, are immunized with the PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 immunogen emulsified in complete Freund's adjuvant and
injected subcutaneously or intraperitoneally in an amount from
1-100 micrograms. Alternatively, the immunogen is emulsified in
MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, Mont.)
and injected into the animal's hind foot pads. The immunized mice
are then boosted 10 to 12 days later with additional immunogen
emulsified in the selected adjuvant. Thereafter, for several weeks,
the mice may also be boosted with additional immunization
injections. Serum samples may be periodically obtained from the
mice by retro-orbital bleeding for testing in ELISA assays to
detect anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236,
anti-PRO245, anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328,
anti-PRO344, anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731,
anti-PRO732, anti-PRO1003, anti-PRO1104, anti-PRO1151,
anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570,
anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725,
anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102,
anti-PRO10282, anti-PRO61709 or anti-PRO779 antibodies.
[2236] After a suitable antibody titer has been detected, the
animals "positive" for antibodies can be injected with a final
intravenous injection of PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779. Three to
four days later, the mice are sacrificed and the spleen cells are
harvested. The spleen cells are then fused (using 35% polyethylene
glycol) to a selected murine myeloma cell line such as P3X63AgU.1,
available from ATCC, No. CRL 1597. The fusions generate hybridoma
cells which can then be plated in 96 well tissue culture plates
containing HAT (hypoxanthine, aminopterin, and thymidine) medium to
inhibit proliferation of non-fused cells, myeloma hybrids, and
spleen cell hybrids.
[2237] The hybridoma cells will be screened in an ELISA for
reactivity against PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779. Determination of
"positive" hybridoma cells secreting the desired monoclonal
antibodies against PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 is within the skill
in the art.
[2238] The positive hybridoma cells can be injected
intraperitoneally into syngeneic Balb/c mice to produce ascites
containing the anti-PRO196, anti-PRO217, anti-PRO231, anti-PRO236,
anti-PRO245, anti-PRO246, anti-PRO258, anti-PRO287, anti-PRO328,
anti-PRO344, anti-PRO357, anti-PRO526, anti-PRO724, anti-PRO731,
anti-PRO732, anti-PRO1003, anti-PRO1104, anti-PRO1151,
anti-PRO1244, anti-PRO1298, anti-PRO1313, anti-PRO1570,
anti-PRO1886, anti-PRO1891, anti-PRO4409, anti-PRO5725,
anti-PRO5994, anti-PRO6097, anti-PRO7425, anti-PRO10102,
anti-PRO10282, anti-PRO61709 or anti-PRO779 monoclonal antibodies.
Alternatively, the hybridoma cells can be grown in tissue culture
flasks or roller bottles. Purification of the monoclonal antibodies
produced in the ascites can be accomplished using ammonium sulfate
precipitation, followed by gel exclusion chromatography.
Alternatively, affinity chromatography based upon binding of
antibody to protein A or protein G can be employed.
Example 46
Purification of PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 Polypeptides Using
Specific Antibodies
[2239] Native or recombinant PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptides may be purified by a variety of standard techniques in
the art of protein purification. For example, pro-PRO196,
pro-PRO217, pro-PRO231, pro-PRO236, pro-PRO245, pro-PRO246,
pro-PRO258, pro-PRO287, pro-PRO328, pro-PRO344, pro-PRO357,
pro-PRO526, pro-PRO724, pro-PRO731, pro-PRO732, pro-PRO1003,
pro-PRO1104, pro-PRO1151, pro-PRO1244, pro-PRO1298, pro-PRO1313,
pro-PRO1570, pro-PRO1886, pro-PRO1891, pro-PRO4409, pro-PRO5725,
pro-PRO5994, pro-PRO6097, pro-PRO7425, pro-PRO10102, pro-PRO10282,
pro-PRO61709 or pro-PRO779 polypeptide, mature PRO196, mature
PRO217, mature PRO231, mature PRO236, mature PRO245, mature PRO246,
mature PRO258, mature PRO287, mature PRO328, mature PRO344, mature
PRO357, mature PRO526, mature PRO724, mature PRO731, mature PRO732,
mature PRO1003, mature PRO1104, mature PRO1151, mature PRO1244,
mature PRO1298, mature PRO1313, mature PRO1570, mature PRO1886,
mature PRO1891, mature PRO4409, mature PRO5725, mature PRO5994,
mature PRO6097, mature PRO7425, mature PRO10102, mature PRO10282,
mature PRO61709 or mature PRO779 polypeptide, or pre-PRO196,
pre-PRO217, pre-PRO231, pre-PRO236, pre-PRO245, pre-PRO246,
pre-PRO258, pre-PRO287, pre-PRO328, pre-PRO344, pre-PRO357,
pre-PRO526, pre-PRO724, pre-PRO731, pre-PRO732, pre-PRO1003,
pre-PRO1104, pre-PRO1151, pre-PRO1244, pre-PRO1298, pre-PRO1313,
pre-PRO1570, pre-PRO1886, pre-PRO1891, pre-PRO4409, pre-PRO5725,
pre-PRO5994, pre-PRO6097, pre-PRO7425, pre-PRO10102, pre-PRO10282,
pre-PRO61709 or pre-PRO779 polypeptide is purified by
immunoaffinity chromatography using antibodies specific for the
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide of interest. In general,
an immunoaffinity column is constructed by covalently coupling the
anti-PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 polypeptide antibody to an
activated chromatographic resin.
[2240] Polyclonal immunoglobulins are prepared from immune sera
either by precipitation with ammonium sulfate or by purification on
immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway,
N.J.). Likewise, monoclonal antibodies are prepared from mouse
ascites fluid by ammonium sulfate precipitation or chromatography
on immobilized Protein A. Partially purified immunoglobulin is
covalently attached to a chromatographic resin such as
CnBr-activated SEPHAROSE.TM. (Pharmacia LKB Biotechnology). The
antibody is coupled to the resin, the resin is blocked, and the
derivative resin is washed according to the manufacturer's
instructions.
[2241] Such an immunoaffinity column is utilized in the
purification of PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide by
preparing a fraction from cells containing PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide in a soluble form. This preparation is derived by
solubilization of the whole cell or of a subcellular fraction
obtained via differential centrifugation by the addition of
detergent or by other methods well known in the art. Alternatively,
soluble polypeptide containing a signal sequence may be secreted in
useful quantity into the medium in which the cells are grown.
[2242] A soluble PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide-containing preparation is passed over the
immunoaffinity column, and the column is washed under conditions
that allow the preferential absorbance of PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide (e.g., high ionic strength buffers in the presence of
detergent). Then, the column is eluted under conditions that
disrupt antibody/PRO196, antibody/PRO217, antibody/PRO231,
antibody/PRO236, antibody/PRO245, antibody/PRO246, antibody/PRO258,
antibody/PRO287, antibody/PRO328, antibody/PRO344, antibody/PRO357,
antibody/PRO526, antibody/PRO724, antibody/PRO731, antibody/PRO732,
antibody/PRO1003, antibody/PRO1104, antibody/PRO1151,
antibody/PRO1244, antibody/PRO1298, antibody/PRO1313,
antibody/PRO1570, antibody/PRO1886, antibody/PRO1891,
antibody/PRO4409, antibody/PRO5725, antibody/PRO5994,
antibody/PRO6097, antibody/PRO7425, antibody/PRO10102,
antibody/PRO10282, antibody/PRO61709 or antibody/PRO779 polypeptide
binding (e.g., a low pH buffer such as approximately pH 2-3, or a
high concentration of a chaotrope such as urea or thiocyanate ion),
and PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide is collected.
Example 47
Drug Screening
[2243] This invention is particularly useful for screening
compounds by using PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptides or
binding fragment thereof in any of a variety of drug screening
techniques. The PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide or
fragment employed in such a test may either be free in solution,
affixed to a solid support, borne on a cell surface, or located
intracellularly. One method of drug screening utilizes eukaryotic
or prokaryotic host cells which are stably transformed with
recombinant nucleic acids expressing the PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide or fragment. Drugs are screened against such
transformed cells in competitive binding assays. Such cells, either
in viable or fixed form, can be used for standard binding assays.
One may measure, for example, the formation of complexes between
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide or a fragment and the
agent being tested. Alternatively, one can examine the diminution
in complex formation between the PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide and its target cell or target receptors caused by the
agent being tested.
[2244] Thus, the present invention provides methods of screening
for drugs or any other agents which can affect a PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 polypeptide-associated disease or disorder. These methods
comprise contacting such an agent with an PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide or fragment thereof and assaying (I) for the presence
of a complex between the agent and the PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide or fragment, or (ii) for the presence of a complex
between the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 polypeptide or fragment and
the cell, by methods well known in the art. In such competitive
binding assays, the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide or
fragment is typically labeled. After suitable incubation, free
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide or fragment is separated
from that present in bound form, and the amount of free or
uncomplexed label is a measure of the ability of the particular
agent to bind to PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide or to
interfere with the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246,
PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731,
PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313,
PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097,
PRO7425, PRO10102, PRO10282, PRO61709 or PRO779 polypeptide/cell
complex.
[2245] Another technique for drug screening provides high
throughput screening for compounds having suitable binding affinity
to a polypeptide and is described in detail in WO 84/03564,
published on Sep. 13, 1984. Briefly stated, large numbers of
different small peptide test compounds are synthesized on a solid
substrate, such as plastic pins or some other surface. As applied
to a PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 polypeptide, the peptide
test compounds are reacted with PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide and washed. Bound PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide is detected by methods well known in the art. Purified
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide can also be coated
directly onto plates for use in the aforementioned drug screening
techniques. In addition, non-neutralizing antibodies can be used to
capture the peptide and immobilize it on the solid support.
[2246] This invention also contemplates the use of competitive drug
screening assays in which neutralizing antibodies capable of
binding PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 polypeptide specifically
compete with a test compound for binding to PRO196, PRO217, PRO231,
PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357,
PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide or fragments thereof. In this manner, the antibodies
can be used to detect the presence of any peptide which shares one
or more antigenic determinants with PRO196, PRO217, PRO231, PRO236,
PRO245, PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526,
PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244,
PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725,
PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide.
Example 48
Rational Drug Design
[2247] The goal of rational drug design is to produce structural
analogs of biologically active polypeptide of interest (i.e., a
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide) or of small molecules
with which they interact, e.g., agonists, antagonists, or
inhibitors. Any of these examples can be used to fashion drugs
which are more active or stable forms of the PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 polypeptide or which enhance or interfere with the function
of the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258,
PRO287, PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732,
PRO1003, PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570,
PRO1886, PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425,
PRO10102, PRO10282, PRO61709 or PRO779 polypeptide in vivo (cf.,
Hodgson, Bio/Technology, 9: 19-21 (1991)).
[2248] In one approach, the three-dimensional structure of the
PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide, or of a PRO196, PRO217,
PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328, PRO344,
PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104, PRO1151,
PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891, PRO4409,
PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or
PRO779 polypeptide-inhibitor complex, is determined by x-ray
crystallography, by computer modeling or, most typically, by a
combination of the two approaches. Both the shape and charges of
the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide must be ascertained to
elucidate the structure and to determine active site(s) of the
molecule. Less often, useful information regarding the structure of
the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide may be gained by modeling
based on the structure of homologous proteins. In both cases,
relevant structural information is used to design analogous PRO196,
PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287, PRO328,
PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003, PRO1104,
PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886, PRO1891,
PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102, PRO10282,
PRO61709 or PRO779 polypeptide-like molecules or to identify
efficient inhibitors. Useful examples of rational drug design may
include molecules which have improved activity or stability as
shown by Braxton and Wells, Biochemistry, 31:7796-7801 (1992) or
which act as inhibitors, agonists, or antagonists of native
peptides as shown by Athauda et al., J. Biochem., 113:742-746
(1993).
[2249] It is also possible to isolate a target-specific antibody,
selected by functional assay, as described above, and then to solve
its crystal structure. This approach, in principle, yields a
pharmacore upon which subsequent drug design can be based. It is
possible to bypass protein crystallography altogether by generating
anti-idiotypic antibodies (anti-ids) to a functional,
pharmacologically active antibody. As a mirror image of a mirror
image, the binding site of the anti-ids would be expected to be an
analog of the original receptor. The anti-id could then be used to
identify and isolate peptides from banks of chemically or
biologically produced peptides. The isolated peptides would then
act as the pharmacore.
[2250] By virtue of the present invention, sufficient amounts of
the PRO196, PRO217, PRO231, PRO236, PRO245, PRO246, PRO258, PRO287,
PRO328, PRO344, PRO357, PRO526, PRO724, PRO731, PRO732, PRO1003,
PRO1104, PRO1151, PRO1244, PRO1298, PRO1313, PRO1570, PRO1886,
PRO1891, PRO4409, PRO5725, PRO5994, PRO6097, PRO7425, PRO10102,
PRO10282, PRO61709 or PRO779 polypeptide may be made available to
perform such analytical studies as X-ray crystallography. In
addition, knowledge of the PRO196, PRO217, PRO231, PRO236, PRO245,
PRO246, PRO258, PRO287, PRO328, PRO344, PRO357, PRO526, PRO724,
PRO731, PRO732, PRO1003, PRO1104, PRO1151, PRO1244, PRO1298,
PRO1313, PRO1570, PRO1886, PRO1891, PRO4409, PRO5725, PRO5994,
PRO6097, PRO7425, PRO10102, PRO10282, PRO61709 or PRO779
polypeptide amino acid sequence provided herein will provide
guidance to those employing computer modeling techniques in place
of or in addition to x-ray crystallography.
Sequence CWU 1
1
14212290DNAHomo sapiens 1ggctgagggg aggcccggag cctttctggg
gcctggggga tcctcttgca 50ctggtgggtg gagagaagcg cctgcagcca accagggtca
ggctgtgctc 100acagtttcct ctggcggcat gtaaaggctc cacaaaggag
ttgggagttc 150aaatgaggct gctgcggacg gcctgaggat ggaccccaag
ccctggacct 200gccgagcgtg gcactgaggc agcggctgac gctactgtga
gggaaagaag 250gttgtgagca gccccgcagg acccctggcc agccctggcc
ccagcctctg 300ccggagccct ctgtggaggc agagccagtg gagcccagtg
aggcagggct 350gcttggcagc caccggcctg caactcagga acccctccag
aggccatgga 400caggctgccc cgctgacggc cagggtgaag catgtgagga
gccgccccgg 450agccaagcag gagggaagag gctttcatag attctattca
caaagaataa 500ccaccatttt gcaaggacca tgaggccact gtgcgtgaca
tgctggtggc 550tcggactgct ggctgccatg ggagctgttg caggccagga
ggacggtttt 600gagggcactg aggagggctc gccaagagag ttcatttacc
taaacaggta 650caagcgggcg ggcgagtccc aggacaagtg cacctacacc
ttcattgtgc 700cccagcagcg ggtcacgggt gccatctgcg tcaactccaa
ggagcctgag 750gtgcttctgg agaaccgagt gcataagcag gagctagagc
tgctcaacaa 800tgagctgctc aagcagaagc ggcagatcga gacgctgcag
cagctggtgg 850aggtggacgg cggcattgtg agcgaggtga agctgctgcg
caaggagagc 900cgcaacatga actcgcgggt cacgcagctc tacatgcagc
tcctgcacga 950gatcatccgc aagcgggaca acgcgttgga gctctcccag
ctggagaaca 1000ggatcctgaa ccagacagcc gacatgctgc agctggccag
caagtacaag 1050gacctggagc acaagtacca gcacctggcc acactggccc
acaaccaatc 1100agagatcatc gcgcagcttg aggagcactg ccagagggtg
ccctcggcca 1150ggcccgtccc ccagccaccc cccgctgccc cgccccgggt
ctaccaacca 1200cccacctaca accgcatcat caaccagatc tctaccaacg
agatccagag 1250tgaccagaac ctgaaggtgc tgccaccccc tctgcccact
atgcccactc 1300tcaccagcct cccatcttcc accgacaagc cgtcgggccc
atggagagac 1350tgcctgcagg ccctggagga tggccacgac accagctcca
tctacctggt 1400gaagccggag aacaccaacc gcctcatgca ggtgtggtgc
gaccagagac 1450acgaccccgg gggctggacc gtcatccaga gacgcctgga
tggctctgtt 1500aacttcttca ggaactggga gacgtacaag caagggtttg
ggaacattga 1550cggcgaatac tggctgggcc tggagaacat ttactggctg
acgaaccaag 1600gcaactacaa actcctggtg accatggagg actggtccgg
ccgcaaagtc 1650tttgcagaat acgccagttt ccgcctggaa cctgagagcg
agtattataa 1700gctgcggctg gggcgctacc atggcaatgc gggtgactcc
tttacatggc 1750acaacggcaa gcagttcacc accctggaca gagatcatga
tgtctacaca 1800ggaaactgtg cccactacca gaagggaggc tggtggtata
acgcctgtgc 1850ccactccaac ctcaacgggg tctggtaccg cgggggccat
taccggagcc 1900gctaccagga cggagtctac tgggctgagt tccgaggagg
ctcttactca 1950ctcaagaaag tggtgatgat gatccgaccg aaccccaaca
ccttccacta 2000agccagctcc ccctcctgac ctctcgtggc cattgccagg
agcccaccct 2050ggtcacgctg gccacagcac aaagaacaac tcctcaccag
ttcatcctga 2100ggctgggagg accgggatgc tggattctgt tttccgaagt
cactgcagcg 2150gatgatggaa ctgaatcgat acggtgtttt ctgtccctcc
tactttcctt 2200cacaccagac agcccctcat gtctccagga caggacagga
ctacagacaa 2250ctctttcttt aaataaatta agtctctaca ataaaaaaaa
22902493PRTHomo sapiens 2Met Arg Pro Leu Cys Val Thr Cys Trp Trp
Leu Gly Leu Leu Ala1 5 10 15Ala Met Gly Ala Val Ala Gly Gln Glu Asp
Gly Phe Glu Gly Thr 20 25 30Glu Glu Gly Ser Pro Arg Glu Phe Ile Tyr
Leu Asn Arg Tyr Lys 35 40 45Arg Ala Gly Glu Ser Gln Asp Lys Cys Thr
Tyr Thr Phe Ile Val 50 55 60Pro Gln Gln Arg Val Thr Gly Ala Ile Cys
Val Asn Ser Lys Glu 65 70 75Pro Glu Val Leu Leu Glu Asn Arg Val His
Lys Gln Glu Leu Glu 80 85 90Leu Leu Asn Asn Glu Leu Leu Lys Gln Lys
Arg Gln Ile Glu Thr 95 100 105Leu Gln Gln Leu Val Glu Val Asp Gly
Gly Ile Val Ser Glu Val 110 115 120Lys Leu Leu Arg Lys Glu Ser Arg
Asn Met Asn Ser Arg Val Thr 125 130 135Gln Leu Tyr Met Gln Leu Leu
His Glu Ile Ile Arg Lys Arg Asp 140 145 150Asn Ala Leu Glu Leu Ser
Gln Leu Glu Asn Arg Ile Leu Asn Gln 155 160 165Thr Ala Asp Met Leu
Gln Leu Ala Ser Lys Tyr Lys Asp Leu Glu 170 175 180His Lys Tyr Gln
His Leu Ala Thr Leu Ala His Asn Gln Ser Glu 185 190 195Ile Ile Ala
Gln Leu Glu Glu His Cys Gln Arg Val Pro Ser Ala 200 205 210Arg Pro
Val Pro Gln Pro Pro Pro Ala Ala Pro Pro Arg Val Tyr 215 220 225Gln
Pro Pro Thr Tyr Asn Arg Ile Ile Asn Gln Ile Ser Thr Asn 230 235
240Glu Ile Gln Ser Asp Gln Asn Leu Lys Val Leu Pro Pro Pro Leu 245
250 255Pro Thr Met Pro Thr Leu Thr Ser Leu Pro Ser Ser Thr Asp Lys
260 265 270Pro Ser Gly Pro Trp Arg Asp Cys Leu Gln Ala Leu Glu Asp
Gly 275 280 285His Asp Thr Ser Ser Ile Tyr Leu Val Lys Pro Glu Asn
Thr Asn 290 295 300Arg Leu Met Gln Val Trp Cys Asp Gln Arg His Asp
Pro Gly Gly 305 310 315Trp Thr Val Ile Gln Arg Arg Leu Asp Gly Ser
Val Asn Phe Phe 320 325 330Arg Asn Trp Glu Thr Tyr Lys Gln Gly Phe
Gly Asn Ile Asp Gly 335 340 345Glu Tyr Trp Leu Gly Leu Glu Asn Ile
Tyr Trp Leu Thr Asn Gln 350 355 360Gly Asn Tyr Lys Leu Leu Val Thr
Met Glu Asp Trp Ser Gly Arg 365 370 375Lys Val Phe Ala Glu Tyr Ala
Ser Phe Arg Leu Glu Pro Glu Ser 380 385 390Glu Tyr Tyr Lys Leu Arg
Leu Gly Arg Tyr His Gly Asn Ala Gly 395 400 405Asp Ser Phe Thr Trp
His Asn Gly Lys Gln Phe Thr Thr Leu Asp 410 415 420Arg Asp His Asp
Val Tyr Thr Gly Asn Cys Ala His Tyr Gln Lys 425 430 435Gly Gly Trp
Trp Tyr Asn Ala Cys Ala His Ser Asn Leu Asn Gly 440 445 450Val Trp
Tyr Arg Gly Gly His Tyr Arg Ser Arg Tyr Gln Asp Gly 455 460 465Val
Tyr Trp Ala Glu Phe Arg Gly Gly Ser Tyr Ser Leu Lys Lys 470 475
480Val Val Met Met Ile Arg Pro Asn Pro Asn Thr Phe His 485
49032033DNAHomo sapiens 3ccaggccggg aggcgacgcg cccagccgtc
taaacgggaa cagccctggc 50tgagggagct gcagcgcagc agagtatctg acggcgccag
gttgcgtagg 100tgcggcacga ggagttttcc cggcagcgag gaggtcctga
gcagcatggc 150ccggaggagc gccttccctg ccgccgcgct ctggctctgg
agcatcctcc 200tgtgcctgct ggcactgcgg gcggaggccg ggccgccgca
ggaggagagc 250ctgtacctat ggatcgatgc tcaccaggca agagtactca
taggatttga 300agaagatatc ctgattgttt cagaggggaa aatggcacct
tttacacatg 350atttcagaaa agcgcaacag agaatgccag ctattcctgt
caatatccat 400tccatgaatt ttacctggca agctgcaggg caggcagaat
acttctatga 450attcctgtcc ttgcgctccc tggataaagg catcatggca
gatccaaccg 500tcaatgtccc tctgctggga acagtgcctc acaaggcatc
agttgttcaa 550gttggtttcc catgtcttgg aaaacaggat ggggtggcag
catttgaagt 600ggatgtgatt gttatgaatt ctgaaggcaa caccattctc
caaacacctc 650aaaatgctat cttctttaaa acatgtcaac aagctgagtg
cccaggcggg 700tgccgaaatg gaggcttttg taatgaaaga cgcatctgcg
agtgtcctga 750tgggttccac ggacctcact gtgagaaagc cctttgtacc
ccacgatgta 800tgaatggtgg actttgtgtg actcctggtt tctgcatctg
cccacctgga 850ttctatggag tgaactgtga caaagcaaac tgctcaacca
cctgctttaa 900tggagggacc tgtttctacc ctggaaaatg tatttgccct
ccaggactag 950agggagagca gtgtgaaatc agcaaatgcc cacaaccctg
tcgaaatgga 1000ggtaaatgca ttggtaaaag caaatgtaag tgttccaaag
gttaccaggg 1050agacctctgt tcaaagcctg tctgcgagcc tggctgtggt
gcacatggaa 1100cctgccatga acccaacaaa tgccaatgtc aagaaggttg
gcatggaaga 1150cactgcaata aaaggtacga agccagcctc atacatgccc
tgaggccagc 1200aggcgcccag ctcaggcagc acacgccttc acttaaaaag
gccgaggagc 1250ggcgggatcc acctgaatcc aattacatct ggtgaactcc
gacatctgaa 1300acgttttaag ttacaccaag ttcatagcct ttgttaacct
ttcatgtgtt 1350gaatgttcaa ataatgttca ttacacttaa gaatactggc
ctgaatttta 1400ttagcttcat tataaatcac tgagctgata tttactcttc
cttttaagtt 1450ttctaagtac gtctgtagca tgatggtata gattttcttg
tttcagtgct 1500ttgggacaga ttttatatta tgtcaattga tcaggttaaa
attttcagtg 1550tgtagttggc agatattttc aaaattacaa tgcatttatg
gtgtctgggg 1600gcaggggaac atcagaaagg ttaaattggg caaaaatgcg
taagtcacaa 1650gaatttggat ggtgcagtta atgttgaagt tacagcattt
cagattttat 1700tgtcagatat ttagatgttt gttacatttt taaaaattgc
tcttaatttt 1750taaactctca atacaatata ttttgacctt accattattc
cagagattca 1800gtattaaaaa aaaaaaaatt acactgtggt agtggcattt
aaacaatata 1850atatattcta aacacaatga aatagggaat ataatgtatg
aactttttgc 1900attggcttga agcaatataa tatattgtaa acaaaacaca
gctcttacct 1950aataaacatt ttatactgtt tgtatgtata aaataaaggt
gctgctttag 2000ttttttggaa aaaaaaaaaa aaaaaaaaaa aaa 20334379PRTHomo
sapiens 4Met Ala Arg Arg Ser Ala Phe Pro Ala Ala Ala Leu Trp Leu
Trp1 5 10 15Ser Ile Leu Leu Cys Leu Leu Ala Leu Arg Ala Glu Ala Gly
Pro 20 25 30Pro Gln Glu Glu Ser Leu Tyr Leu Trp Ile Asp Ala His Gln
Ala 35 40 45Arg Val Leu Ile Gly Phe Glu Glu Asp Ile Leu Ile Val Ser
Glu 50 55 60Gly Lys Met Ala Pro Phe Thr His Asp Phe Arg Lys Ala Gln
Gln 65 70 75Arg Met Pro Ala Ile Pro Val Asn Ile His Ser Met Asn Phe
Thr 80 85 90Trp Gln Ala Ala Gly Gln Ala Glu Tyr Phe Tyr Glu Phe Leu
Ser 95 100 105Leu Arg Ser Leu Asp Lys Gly Ile Met Ala Asp Pro Thr
Val Asn 110 115 120Val Pro Leu Leu Gly Thr Val Pro His Lys Ala Ser
Val Val Gln 125 130 135Val Gly Phe Pro Cys Leu Gly Lys Gln Asp Gly
Val Ala Ala Phe 140 145 150Glu Val Asp Val Ile Val Met Asn Ser Glu
Gly Asn Thr Ile Leu 155 160 165Gln Thr Pro Gln Asn Ala Ile Phe Phe
Lys Thr Cys Gln Gln Ala 170 175 180Glu Cys Pro Gly Gly Cys Arg Asn
Gly Gly Phe Cys Asn Glu Arg 185 190 195Arg Ile Cys Glu Cys Pro Asp
Gly Phe His Gly Pro His Cys Glu 200 205 210Lys Ala Leu Cys Thr Pro
Arg Cys Met Asn Gly Gly Leu Cys Val 215 220 225Thr Pro Gly Phe Cys
Ile Cys Pro Pro Gly Phe Tyr Gly Val Asn 230 235 240Cys Asp Lys Ala
Asn Cys Ser Thr Thr Cys Phe Asn Gly Gly Thr 245 250 255Cys Phe Tyr
Pro Gly Lys Cys Ile Cys Pro Pro Gly Leu Glu Gly 260 265 270Glu Gln
Cys Glu Ile Ser Lys Cys Pro Gln Pro Cys Arg Asn Gly 275 280 285Gly
Lys Cys Ile Gly Lys Ser Lys Cys Lys Cys Ser Lys Gly Tyr 290 295
300Gln Gly Asp Leu Cys Ser Lys Pro Val Cys Glu Pro Gly Cys Gly 305
310 315Ala His Gly Thr Cys His Glu Pro Asn Lys Cys Gln Cys Gln Glu
320 325 330Gly Trp His Gly Arg His Cys Asn Lys Arg Tyr Glu Ala Ser
Leu 335 340 345Ile His Ala Leu Arg Pro Ala Gly Ala Gln Leu Arg Gln
His Thr 350 355 360Pro Ser Leu Lys Lys Ala Glu Glu Arg Arg Asp Pro
Pro Glu Ser 365 370 375Asn Tyr Ile Trp51514DNAHomo sapiens
5ggggtctccc tcagggccgg gaggcacagc ggtccctgct tgctgaaggg
50ctggatgtac gcatccgcag gttcccgcgg acttgggggc gcccgctgag
100ccccggcgcc cgcagaagac ttgtgtttgc ctcctgcagc ctcaacccgg
150agggcagcga gggcctacca ccatgatcac tggtgtgttc agcatgcgct
200tgtggacccc agtgggcgtc ctgacctcgc tggcgtactg cctgcaccag
250cggcgggtgg ccctggccga gctgcaggag gccgatggcc agtgtccggt
300cgaccgcagc ctgctgaagt tgaaaatggt gcaggtcgtg tttcgacacg
350gggctcggag tcctctcaag ccgctcccgc tggaggagca ggtagagtgg
400aacccccagc tattagaggt cccaccccaa actcagtttg attacacagt
450caccaatcta gctggtggtc cgaaaccata ttctccttac gactctcaat
500accatgagac caccctgaag gggggcatgt ttgctgggca gctgaccaag
550gtgggcatgc agcaaatgtt tgccttggga gagagactga ggaagaacta
600tgtggaagac attccctttc tttcaccaac cttcaaccca caggaggtct
650ttattcgttc cactaacatt tttcggaatc tggagtccac ccgttgtttg
700ctggctgggc ttttccagtg tcagaaagaa ggacccatca tcatccacac
750tgatgaagca gattcagaag tcttgtatcc caactaccaa agctgctgga
800gcctgaggca gagaaccaga ggccggaggc agactgcctc tttacagcca
850ggaatctcag aggatttgaa aaaggtgaag gacaggatgg gcattgacag
900tagtgataaa gtggacttct tcatcctcct ggacaacgtg gctgccgagc
950aggcacacaa cctcccaagc tgccccatgc tgaagagatt tgcacggatg
1000atcgaacaga gagctgtgga cacatccttg tacatactgc ccaaggaaga
1050cagggaaagt cttcagatgg cagtaggccc attcctccac atcctagaga
1100gcaacctgct gaaagccatg gactctgcca ctgcccccga caagatcaga
1150aagctgtatc tctatgcggc tcatgatgtg accttcatac cgctcttaat
1200gaccctgggg atttttgacc acaaatggcc accgtttgct gttgacctga
1250ccatggaact ttaccagcac ctggaatcta aggagtggtt tgtgcagctc
1300tattaccacg ggaaggagca ggtgccgaga ggttgccctg atgggctctg
1350cccgctggac atgttcttga atgccatgtc agtttatacc ttaagcccag
1400aaaaatacca tgcactctgc tctcaaactc aggtgatgga agttggaaat
1450gaagagtaac tgatttataa aagcaggatg tgttgatttt aaaataaagt
1500gcctttatac aatg 15146428PRTHomo sapiens 6Met Ile Thr Gly Val
Phe Ser Met Arg Leu Trp Thr Pro Val Gly1 5 10 15Val Leu Thr Ser Leu
Ala Tyr Cys Leu His Gln Arg Arg Val Ala 20 25 30Leu Ala Glu Leu Gln
Glu Ala Asp Gly Gln Cys Pro Val Asp Arg 35 40 45Ser Leu Leu Lys Leu
Lys Met Val Gln Val Val Phe Arg His Gly 50 55 60Ala Arg Ser Pro Leu
Lys Pro Leu Pro Leu Glu Glu Gln Val Glu 65 70 75Trp Asn Pro Gln Leu
Leu Glu Val Pro Pro Gln Thr Gln Phe Asp 80 85 90Tyr Thr Val Thr Asn
Leu Ala Gly Gly Pro Lys Pro Tyr Ser Pro 95 100 105Tyr Asp Ser Gln
Tyr His Glu Thr Thr Leu Lys Gly Gly Met Phe 110 115 120Ala Gly Gln
Leu Thr Lys Val Gly Met Gln Gln Met Phe Ala Leu 125 130 135Gly Glu
Arg Leu Arg Lys Asn Tyr Val Glu Asp Ile Pro Phe Leu 140 145 150Ser
Pro Thr Phe Asn Pro Gln Glu Val Phe Ile Arg Ser Thr Asn 155 160
165Ile Phe Arg Asn Leu Glu Ser Thr Arg Cys Leu Leu Ala Gly Leu 170
175 180Phe Gln Cys Gln Lys Glu Gly Pro Ile Ile Ile His Thr Asp Glu
185 190 195Ala Asp Ser Glu Val Leu Tyr Pro Asn Tyr Gln Ser Cys Trp
Ser 200 205 210Leu Arg Gln Arg Thr Arg Gly Arg Arg Gln Thr Ala Ser
Leu Gln 215 220 225Pro Gly Ile Ser Glu Asp Leu Lys Lys Val Lys Asp
Arg Met Gly 230 235 240Ile Asp Ser Ser Asp Lys Val Asp Phe Phe Ile
Leu Leu Asp Asn 245 250 255Val Ala Ala Glu Gln Ala His Asn Leu Pro
Ser Cys Pro Met Leu 260 265 270Lys Arg Phe Ala Arg Met Ile Glu Gln
Arg Ala Val Asp Thr Ser 275 280 285Leu Tyr Ile Leu Pro Lys Glu Asp
Arg Glu Ser Leu Gln Met Ala 290 295 300Val Gly Pro Phe Leu His Ile
Leu Glu Ser Asn Leu Leu Lys Ala 305 310 315Met Asp Ser Ala Thr Ala
Pro Asp Lys Ile Arg Lys Leu Tyr Leu 320 325 330Tyr Ala Ala His Asp
Val Thr Phe Ile Pro Leu Leu Met Thr Leu 335 340 345Gly Ile Phe
Asp His Lys Trp Pro Pro Phe Ala Val Asp Leu Thr 350 355 360Met Glu
Leu Tyr Gln His Leu Glu Ser Lys Glu Trp Phe Val Gln 365 370 375Leu
Tyr Tyr His Gly Lys Glu Gln Val Pro Arg Gly Cys Pro Asp 380 385
390Gly Leu Cys Pro Leu Asp Met Phe Leu Asn Ala Met Ser Val Tyr 395
400 405Thr Leu Ser Pro Glu Lys Tyr His Ala Leu Cys Ser Gln Thr Gln
410 415 420Val Met Glu Val Gly Asn Glu Glu 42573106DNAHomo
sapiensUnsure1683Unknown base 7aggctcccgc gcgcggctga gtgcggactg
gagtgggaac ccgggtcccc 50gcgcttagag aacacgcgat gaccacgtgg agcctccggc
ggaggccggc 100ccgcacgctg ggactcctgc tgctggtcgt cttgggcttc
ctggtgctcc 150gcaggctgga ctggagcacc ctggtccctc tgcggctccg
ccatcgacag 200ctggggctgc aggccaaggg ctggaacttc atgctggagg
attccacctt 250ctggatcttc gggggctcca tccactattt ccgtgtgccc
agggagtact 300ggagggaccg cctgctgaag atgaaggcct gtggcttgaa
caccctcacc 350acctatgttc cgtggaacct gcatgagcca gaaagaggca
aatttgactt 400ctctgggaac ctggacctgg aggccttcgt cctgatggcc
gcagagatcg 450ggctgtgggt gattctgcgt ccaggcccct acatctgcag
tgagatggac 500ctcgggggct tgcccagctg gctactccaa gaccctggca
tgaggctgag 550gacaacttac aagggcttca ccgaagcagt ggacctttat
tttgaccacc 600tgatgtccag ggtggtgcca ctccagtaca agcgtggggg
acctatcatt 650gccgtgcagg tggagaatga atatggttcc tataataaag
accccgcata 700catgccctac gtcaagaagg cactggagga ccgtggcatt
gtggaactgc 750tcctgacttc agacaacaag gatgggctga gcaaggggat
tgtccaggga 800gtcttggcca ccatcaactt gcagtcaaca cacgagctgc
agctactgac 850cacctttctc ttcaacgtcc aggggactca gcccaagatg
gtgatggagt 900actggacggg gtggtttgac tcgtggggag gccctcacaa
tatcttggat 950tcttctgagg ttttgaaaac cgtgtctgcc attgtggacg
ccggctcctc 1000catcaacctc tacatgttcc acggaggcac caactttggc
ttcatgaatg 1050gagccatgca cttccatgac tacaagtcag atgtcaccag
ctatgactat 1100gatgctgtgc tgacagaagc cggcgattac acggccaagt
acatgaagct 1150tcgagacttc ttcggctcca tctcaggcat ccctctccct
cccccacctg 1200accttcttcc caagatgccg tatgagccct taacgccagt
cttgtacctg 1250tctctgtggg acgccctcaa gtacctgggg gagccaatca
agtctgaaaa 1300gcccatcaac atggagaacc tgccagtcaa tgggggaaat
ggacagtcct 1350tcgggtacat tctctatgag accagcatca cctcgtctgg
catcctcagt 1400ggccacgtgc atgatcgggg gcaggtgttt gtgaacacag
tatccatagg 1450attcttggac tacaagacaa cgaagattgc tgtccccctg
atccagggtt 1500acaccgtgct gaggatcttg gtggagaatc gtgggcgagt
caactatggg 1550gagaatattg atgaccagcg caaaggctta attggaaatc
tctatctgaa 1600tgattcaccc ctgaaaaact tcagaatcta tagcctggat
atgaagaaga 1650gcttctttca gaggttcggc ctggacaaat ggngttccct
cccagaaaca 1700cccacattac ctgctttctt cttgggtagc ttgtccatca
gctccacgcc 1750ttgtgacacc tttctgaagc tggagggctg ggagaagggg
gttgtattca 1800tcaatggcca gaaccttgga cgttactgga acattggacc
ccagaagacg 1850ctttacctcc caggtccctg gttgagcagc ggaatcaacc
aggtcatcgt 1900ttttgaggag acgatggcgg gccctgcatt acagttcacg
gaaacccccc 1950acctgggcag gaaccagtac attaagtgag cggtggcacc
ccctcctgct 2000ggtgccagtg ggagactgcc gcctcctctt gacctgaagc
ctggtggctg 2050ctgccccacc cctcactgca aaagcatctc cttaagtagc
aacctcaggg 2100actgggggct acagtctgcc cctgtctcag ctcaaaaccc
taagcctgca 2150gggaaaggtg ggatggctct gggcctggct ttgttgatga
tggctttcct 2200acagccctgc tcttgtgccg aggctgtcgg gctgtctcta
gggtgggagc 2250agctaatcag atcgcccagc ctttggccct cagaaaaagt
gctgaaacgt 2300gcccttgcac cggacgtcac agccctgcga gcatctgctg
gactcaggcg 2350tgctctttgc tggttcctgg gaggcttggc cacatccctc
atggccccat 2400tttatccccg aaatcctggg tgtgtcacca gtgtagaggg
tggggaaggg 2450gtgtctcacc tgagctgact ttgttcttcc ttcacaacct
tctgagcctt 2500ctttgggatt ctggaaggaa ctcggcgtga gaaacatgtg
acttcccctt 2550tcccttccca ctcgctgctt cccacagggt gacaggctgg
gctggagaaa 2600cagaaatcct caccctgcgt cttcccaagt tagcaggtgt
ctctggtgtt 2650cagtgaggag gacatgtgag tcctggcaga agccatggcc
catgtctgca 2700catccaggga ggaggacaga aggcccagct cacatgtgag
tcctggcaga 2750agccatggcc catgtctgca catccaggga ggaggacaga
aggcccagct 2800cacatgtgag tcctggcaga agccatggcc catgtctgca
catccaggga 2850ggaggacaga aggcccagct cacatgtgag tcctggcaga
agccatggcc 2900catgtctgca catccaggga ggaggacaga aggcccagct
cagtggcccc 2950cgctccccac cccccacgcc cgaacagcag gggcagagca
gccctccttc 3000gaagtgtgtc caagtccgca tttgagcctt gttctggggc
ccagcccaac 3050acctggcttg ggctcactgt cctgagttgc agtaaagcta
taaccttgaa 3100tcacaa 31068636PRTHomo sapiensUnsure539Unknown amino
acid 8Met Thr Thr Trp Ser Leu Arg Arg Arg Pro Ala Arg Thr Leu Gly1
5 10 15Leu Leu Leu Leu Val Val Leu Gly Phe Leu Val Leu Arg Arg Leu
20 25 30Asp Trp Ser Thr Leu Val Pro Leu Arg Leu Arg His Arg Gln Leu
35 40 45Gly Leu Gln Ala Lys Gly Trp Asn Phe Met Leu Glu Asp Ser Thr
50 55 60Phe Trp Ile Phe Gly Gly Ser Ile His Tyr Phe Arg Val Pro Arg
65 70 75Glu Tyr Trp Arg Asp Arg Leu Leu Lys Met Lys Ala Cys Gly Leu
80 85 90Asn Thr Leu Thr Thr Tyr Val Pro Trp Asn Leu His Glu Pro Glu
95 100 105Arg Gly Lys Phe Asp Phe Ser Gly Asn Leu Asp Leu Glu Ala
Phe 110 115 120Val Leu Met Ala Ala Glu Ile Gly Leu Trp Val Ile Leu
Arg Pro 125 130 135Gly Pro Tyr Ile Cys Ser Glu Met Asp Leu Gly Gly
Leu Pro Ser 140 145 150Trp Leu Leu Gln Asp Pro Gly Met Arg Leu Arg
Thr Thr Tyr Lys 155 160 165Gly Phe Thr Glu Ala Val Asp Leu Tyr Phe
Asp His Leu Met Ser 170 175 180Arg Val Val Pro Leu Gln Tyr Lys Arg
Gly Gly Pro Ile Ile Ala 185 190 195Val Gln Val Glu Asn Glu Tyr Gly
Ser Tyr Asn Lys Asp Pro Ala 200 205 210Tyr Met Pro Tyr Val Lys Lys
Ala Leu Glu Asp Arg Gly Ile Val 215 220 225Glu Leu Leu Leu Thr Ser
Asp Asn Lys Asp Gly Leu Ser Lys Gly 230 235 240Ile Val Gln Gly Val
Leu Ala Thr Ile Asn Leu Gln Ser Thr His 245 250 255Glu Leu Gln Leu
Leu Thr Thr Phe Leu Phe Asn Val Gln Gly Thr 260 265 270Gln Pro Lys
Met Val Met Glu Tyr Trp Thr Gly Trp Phe Asp Ser 275 280 285Trp Gly
Gly Pro His Asn Ile Leu Asp Ser Ser Glu Val Leu Lys 290 295 300Thr
Val Ser Ala Ile Val Asp Ala Gly Ser Ser Ile Asn Leu Tyr 305 310
315Met Phe His Gly Gly Thr Asn Phe Gly Phe Met Asn Gly Ala Met 320
325 330His Phe His Asp Tyr Lys Ser Asp Val Thr Ser Tyr Asp Tyr Asp
335 340 345Ala Val Leu Thr Glu Ala Gly Asp Tyr Thr Ala Lys Tyr Met
Lys 350 355 360Leu Arg Asp Phe Phe Gly Ser Ile Ser Gly Ile Pro Leu
Pro Pro 365 370 375Pro Pro Asp Leu Leu Pro Lys Met Pro Tyr Glu Pro
Leu Thr Pro 380 385 390Val Leu Tyr Leu Ser Leu Trp Asp Ala Leu Lys
Tyr Leu Gly Glu 395 400 405Pro Ile Lys Ser Glu Lys Pro Ile Asn Met
Glu Asn Leu Pro Val 410 415 420Asn Gly Gly Asn Gly Gln Ser Phe Gly
Tyr Ile Leu Tyr Glu Thr 425 430 435Ser Ile Thr Ser Ser Gly Ile Leu
Ser Gly His Val His Asp Arg 440 445 450Gly Gln Val Phe Val Asn Thr
Val Ser Ile Gly Phe Leu Asp Tyr 455 460 465Lys Thr Thr Lys Ile Ala
Val Pro Leu Ile Gln Gly Tyr Thr Val 470 475 480Leu Arg Ile Leu Val
Glu Asn Arg Gly Arg Val Asn Tyr Gly Glu 485 490 495Asn Ile Asp Asp
Gln Arg Lys Gly Leu Ile Gly Asn Leu Tyr Leu 500 505 510Asn Asp Ser
Pro Leu Lys Asn Phe Arg Ile Tyr Ser Leu Asp Met 515 520 525Lys Lys
Ser Phe Phe Gln Arg Phe Gly Leu Asp Lys Trp Xaa Ser 530 535 540Leu
Pro Glu Thr Pro Thr Leu Pro Ala Phe Phe Leu Gly Ser Leu 545 550
555Ser Ile Ser Ser Thr Pro Cys Asp Thr Phe Leu Lys Leu Glu Gly 560
565 570Trp Glu Lys Gly Val Val Phe Ile Asn Gly Gln Asn Leu Gly Arg
575 580 585Tyr Trp Asn Ile Gly Pro Gln Lys Thr Leu Tyr Leu Pro Gly
Pro 590 595 600Trp Leu Ser Ser Gly Ile Asn Gln Val Ile Val Phe Glu
Glu Thr 605 610 615Met Ala Gly Pro Ala Leu Gln Phe Thr Glu Thr Pro
His Leu Gly 620 625 630Arg Asn Gln Tyr Ile Lys 63591295DNAHomo
sapiens 9cccagaagtt caagggcccc cggcctcctg cgctcctgcc gccgggaccc
50tcgacctcct cagagcagcc ggctgccgcc ccgggaagat ggcgaggagg
100agccgccacc gcctcctcct gctgctgctg cgctacctgg tggtcgccct
150gggctatcat aaggcctatg ggttttctgc cccaaaagac caacaagtag
200tcacagcagt agagtaccaa gaggctattt tagcctgcaa aaccccaaag
250aagactgttt cctccagatt agagtggaag aaactgggtc ggagtgtctc
300ctttgtctac tatcaacaga ctcttcaagg tgattttaaa aatcgagctg
350agatgataga tttcaatatc cggatcaaaa atgtgacaag aagtgatgcg
400gggaaatatc gttgtgaagt tagtgcccca tctgagcaag gccaaaacct
450ggaagaggat acagtcactc tggaagtatt agtggctcca gcagttccat
500catgtgaagt accctcttct gctctgagtg gaactgtggt agagctacga
550tgtcaagaca aagaagggaa tccagctcct gaatacacat ggtttaagga
600tggcatccgt ttgctagaaa atcccagact tggctcccaa agcaccaaca
650gctcatacac aatgaataca aaaactggaa ctctgcaatt taatactgtt
700tccaaactgg acactggaga atattcctgt gaagcccgca attctgttgg
750atatcgcagg tgtcctggga aacgaatgca agtagatgat ctcaacataa
800gtggcatcat agcagccgta gtagttgtgg ccttagtgat ttccgtttgt
850ggccttggtg tatgctatgc tcagaggaaa ggctactttt caaaagaaac
900ctccttccag aagagtaatt cttcatctaa agccacgaca atgagtgaaa
950atgtgcagtg gctcacgcct gtaatcccag cactttggaa ggccgcggcg
1000ggcggatcac gaggtcagga gttctagacc agtctggcca atatggtgaa
1050accccatctc tactaaaata caaaaattag ctgggcatgg tggcatgtgc
1100ctgcagttcc agctgcttgg gagacaggag aatcacttga acccgggagg
1150cggaggttgc agtgagctga gatcacgcca ctgcagtcca gcctgggtaa
1200cagagcaaga ttccatctca aaaaataaaa taaataaata aataaatact
1250ggtttttacc tgtagaattc ttacaataaa tatagcttga tattc
129510312PRTHomo sapiens 10Met Ala Arg Arg Ser Arg His Arg Leu Leu
Leu Leu Leu Leu Arg1 5 10 15Tyr Leu Val Val Ala Leu Gly Tyr His Lys
Ala Tyr Gly Phe Ser 20 25 30Ala Pro Lys Asp Gln Gln Val Val Thr Ala
Val Glu Tyr Gln Glu 35 40 45Ala Ile Leu Ala Cys Lys Thr Pro Lys Lys
Thr Val Ser Ser Arg 50 55 60Leu Glu Trp Lys Lys Leu Gly Arg Ser Val
Ser Phe Val Tyr Tyr 65 70 75Gln Gln Thr Leu Gln Gly Asp Phe Lys Asn
Arg Ala Glu Met Ile 80 85 90Asp Phe Asn Ile Arg Ile Lys Asn Val Thr
Arg Ser Asp Ala Gly 95 100 105Lys Tyr Arg Cys Glu Val Ser Ala Pro
Ser Glu Gln Gly Gln Asn 110 115 120Leu Glu Glu Asp Thr Val Thr Leu
Glu Val Leu Val Ala Pro Ala 125 130 135Val Pro Ser Cys Glu Val Pro
Ser Ser Ala Leu Ser Gly Thr Val 140 145 150Val Glu Leu Arg Cys Gln
Asp Lys Glu Gly Asn Pro Ala Pro Glu 155 160 165Tyr Thr Trp Phe Lys
Asp Gly Ile Arg Leu Leu Glu Asn Pro Arg 170 175 180Leu Gly Ser Gln
Ser Thr Asn Ser Ser Tyr Thr Met Asn Thr Lys 185 190 195Thr Gly Thr
Leu Gln Phe Asn Thr Val Ser Lys Leu Asp Thr Gly 200 205 210Glu Tyr
Ser Cys Glu Ala Arg Asn Ser Val Gly Tyr Arg Arg Cys 215 220 225Pro
Gly Lys Arg Met Gln Val Asp Asp Leu Asn Ile Ser Gly Ile 230 235
240Ile Ala Ala Val Val Val Val Ala Leu Val Ile Ser Val Cys Gly 245
250 255Leu Gly Val Cys Tyr Ala Gln Arg Lys Gly Tyr Phe Ser Lys Glu
260 265 270Thr Ser Phe Gln Lys Ser Asn Ser Ser Ser Lys Ala Thr Thr
Met 275 280 285Ser Glu Asn Val Gln Trp Leu Thr Pro Val Ile Pro Ala
Leu Trp 290 295 300Lys Ala Ala Ala Gly Gly Ser Arg Gly Gln Glu Phe
305 310111813DNAHomo sapiens 11ggagccgccc tgggtgtcag cggctcggct
cccgcgcacg ctccggccgt 50cgcgcagcct cggcacctgc aggtccgtgc gtcccgcggc
tggcgcccct 100gactccgtcc cggccaggga gggccatgat ttccctcccg
gggcccctgg 150tgaccaactt gctgcggttt ttgttcctgg ggctgagtgc
cctcgcgccc 200ccctcgcggg cccagctgca actgcacttg cccgccaacc
ggttgcaggc 250ggtggaggga ggggaagtgg tgcttccagc gtggtacacc
ttgcacgggg 300aggtgtcttc atcccagcca tgggaggtgc cctttgtgat
gtggttcttc 350aaacagaaag aaaaggagga tcaggtgttg tcctacatca
atggggtcac 400aacaagcaaa cctggagtat ccttggtcta ctccatgccc
tcccggaacc 450tgtccctgcg gctggagggt ctccaggaga aagactctgg
cccctacagc 500tgctccgtga atgtgcaaga caaacaaggc aaatctaggg
gccacagcat 550caaaacctta gaactcaatg tactggttcc tccagctcct
ccatcctgcc 600gtctccaggg tgtgccccat gtgggggcaa acgtgaccct
gagctgccag 650tctccaagga gtaagcccgc tgtccaatac cagtgggatc
ggcagcttcc 700atccttccag actttctttg caccagcatt agatgtcatc
cgtgggtctt 750taagcctcac caacctttcg tcttccatgg ctggagtcta
tgtctgcaag 800gcccacaatg aggtgggcac tgcccaatgt aatgtgacgc
tggaagtgag 850cacagggcct ggagctgcag tggttgctgg agctgttgtg
ggtaccctgg 900ttggactggg gttgctggct gggctggtcc tcttgtacca
ccgccggggc 950aaggccctgg aggagccagc caatgatatc aaggaggatg
ccattgctcc 1000ccggaccctg ccctggccca agagctcaga cacaatctcc
aagaatggga 1050ccctttcctc tgtcacctcc gcacgagccc tccggccacc
ccatggccct 1100cccaggcctg gtgcattgac ccccacgccc agtctctcca
gccaggccct 1150gccctcacca agactgccca cgacagatgg ggcccaccct
caaccaatat 1200cccccatccc tggtggggtt tcttcctctg gcttgagccg
catgggtgct 1250gtgcctgtga tggtgcctgc ccagagtcaa gctggctctc
tggtatgatg 1300accccaccac tcattggcta aaggatttgg ggtctctcct
tcctataagg 1350gtcacctcta gcacagaggc ctgagtcatg ggaaagagtc
acactcctga 1400cccttagtac tctgccccca cctctcttta ctgtgggaaa
accatctcag 1450taagacctaa gtgtccagga gacagaagga gaagaggaag
tggatctgga 1500attgggagga gcctccaccc acccctgact cctccttatg
aagccagctg 1550ctgaaattag ctactcacca agagtgaggg gcagagactt
ccagtcactg 1600agtctcccag gcccccttga tctgtacccc acccctatct
aacaccaccc 1650ttggctccca ctccagctcc ctgtattgat ataacctgtc
aggctggctt 1700ggttaggttt tactggggca gaggataggg aatctcttat
taaaactaac 1750atgaaatatg tgttgttttc atttgcaaat ttaaataaag
atacataatg 1800tttgtatgaa aaa 181312390PRTHomo sapiens 12Met Ile
Ser Leu Pro Gly Pro Leu Val Thr Asn Leu Leu Arg Phe1 5 10 15Leu Phe
Leu Gly Leu Ser Ala Leu Ala Pro Pro Ser Arg Ala Gln 20 25 30Leu Gln
Leu His Leu Pro Ala Asn Arg Leu Gln Ala Val Glu Gly 35 40 45Gly Glu
Val Val Leu Pro Ala Trp Tyr Thr Leu His Gly Glu Val 50 55 60Ser Ser
Ser Gln Pro Trp Glu Val Pro Phe Val Met Trp Phe Phe 65 70 75Lys Gln
Lys Glu Lys Glu Asp Gln Val Leu Ser Tyr Ile Asn Gly 80 85 90Val Thr
Thr Ser Lys Pro Gly Val Ser Leu Val Tyr Ser Met Pro 95 100
105Ser Arg Asn Leu Ser Leu Arg Leu Glu Gly Leu Gln Glu Lys Asp 110
115 120Ser Gly Pro Tyr Ser Cys Ser Val Asn Val Gln Asp Lys Gln Gly
125 130 135Lys Ser Arg Gly His Ser Ile Lys Thr Leu Glu Leu Asn Val
Leu 140 145 150Val Pro Pro Ala Pro Pro Ser Cys Arg Leu Gln Gly Val
Pro His 155 160 165Val Gly Ala Asn Val Thr Leu Ser Cys Gln Ser Pro
Arg Ser Lys 170 175 180Pro Ala Val Gln Tyr Gln Trp Asp Arg Gln Leu
Pro Ser Phe Gln 185 190 195Thr Phe Phe Ala Pro Ala Leu Asp Val Ile
Arg Gly Ser Leu Ser 200 205 210Leu Thr Asn Leu Ser Ser Ser Met Ala
Gly Val Tyr Val Cys Lys 215 220 225Ala His Asn Glu Val Gly Thr Ala
Gln Cys Asn Val Thr Leu Glu 230 235 240Val Ser Thr Gly Pro Gly Ala
Ala Val Val Ala Gly Ala Val Val 245 250 255Gly Thr Leu Val Gly Leu
Gly Leu Leu Ala Gly Leu Val Leu Leu 260 265 270Tyr His Arg Arg Gly
Lys Ala Leu Glu Glu Pro Ala Asn Asp Ile 275 280 285Lys Glu Asp Ala
Ile Ala Pro Arg Thr Leu Pro Trp Pro Lys Ser 290 295 300Ser Asp Thr
Ile Ser Lys Asn Gly Thr Leu Ser Ser Val Thr Ser 305 310 315Ala Arg
Ala Leu Arg Pro Pro His Gly Pro Pro Arg Pro Gly Ala 320 325 330Leu
Thr Pro Thr Pro Ser Leu Ser Ser Gln Ala Leu Pro Ser Pro 335 340
345Arg Leu Pro Thr Thr Asp Gly Ala His Pro Gln Pro Ile Ser Pro 350
355 360Ile Pro Gly Gly Val Ser Ser Ser Gly Leu Ser Arg Met Gly Ala
365 370 375Val Pro Val Met Val Pro Ala Gln Ser Gln Ala Gly Ser Leu
Val 380 385 390131685DNAHomo sapiens 13cccacgcgtc cgcacctcgg
ccccgggctc cgaagcggct cgggggcgcc 50ctttcggtca acatcgtagt ccaccccctc
cccatcccca gcccccgggg 100attcaggctc gccagcgccc agccagggag
ccggccggga agcgcgatgg 150gggccccagc cgcctcgctc ctgctcctgc
tcctgctgtt cgcctgctgc 200tgggcgcccg gcggggccaa cctctcccag
gacgacagcc agccctggac 250atctgatgaa acagtggtgg ctggtggcac
cgtggtgctc aagtgccaag 300tgaaagatca cgaggactca tccctgcaat
ggtctaaccc tgctcagcag 350actctctact ttggggagaa gagagccctt
cgagataatc gaattcagct 400ggttacctct acgccccacg agctcagcat
cagcatcagc aatgtggccc 450tggcagacga gggcgagtac acctgctcaa
tcttcactat gcctgtgcga 500actgccaagt ccctcgtcac tgtgctagga
attccacaga agcccatcat 550cactggttat aaatcttcat tacgggaaaa
agacacagcc accctaaact 600gtcagtcttc tgggagcaag cctgcagccc
ggctcacctg gagaaagggt 650gaccaagaac tccacggaga accaacccgc
atacaggaag atcccaatgg 700taaaaccttc actgtcagca gctcggtgac
attccaggtt acccgggagg 750atgatggggc gagcatcgtg tgctctgtga
accatgaatc tctaaaggga 800gctgacagat ccacctctca acgcattgaa
gttttataca caccaactgc 850gatgattagg ccagaccctc cccatcctcg
tgagggccag aagctgttgc 900tacactgtga gggtcgcggc aatccagtcc
cccagcagta cctatgggag 950aaggagggca gtgtgccacc cctgaagatg
acccaggaga gtgccctgat 1000cttccctttc ctcaacaaga gtgacagtgg
cacctacggc tgcacagcca 1050ccagcaacat gggcagctac aaggcctact
acaccctcaa tgttaatgac 1100cccagtccgg tgccctcctc ctccagcacc
taccacgcca tcatcggtgg 1150gatcgtggct ttcattgtct tcctgctgct
catcatgctc atcttccttg 1200gccactactt gatccggcac aaaggaacct
acctgacaca tgaggcaaaa 1250ggctccgacg atgctccaga cgcggacacg
gccatcatca atgcagaagg 1300cgggcagtca ggaggggacg acaagaagga
atatttcatc tagaggcgcc 1350tgcccacttc ctgcgccccc caggggccct
gtggggactg ctggggccgt 1400caccaacccg gacttgtaca gagcaaccgc
agggccgccc ctcccgcttg 1450ctccccagcc cacccacccc cctgtacaga
atgtctgctt tgggtgcggt 1500tttgtactcg gtttggaatg gggagggagg
agggcggggg gaggggaggg 1550ttgccctcag ccctttccgt ggcttctctg
catttgggtt attattattt 1600ttgtaacaat cccaaatcaa atctgtctcc
aggctggaga ggcaggagcc 1650ctggggtgag aaaagcaaaa aacaaacaaa aaaca
168514398PRTHomo sapiens 14Met Gly Ala Pro Ala Ala Ser Leu Leu Leu
Leu Leu Leu Leu Phe1 5 10 15Ala Cys Cys Trp Ala Pro Gly Gly Ala Asn
Leu Ser Gln Asp Asp 20 25 30Ser Gln Pro Trp Thr Ser Asp Glu Thr Val
Val Ala Gly Gly Thr 35 40 45Val Val Leu Lys Cys Gln Val Lys Asp His
Glu Asp Ser Ser Leu 50 55 60Gln Trp Ser Asn Pro Ala Gln Gln Thr Leu
Tyr Phe Gly Glu Lys 65 70 75Arg Ala Leu Arg Asp Asn Arg Ile Gln Leu
Val Thr Ser Thr Pro 80 85 90His Glu Leu Ser Ile Ser Ile Ser Asn Val
Ala Leu Ala Asp Glu 95 100 105Gly Glu Tyr Thr Cys Ser Ile Phe Thr
Met Pro Val Arg Thr Ala 110 115 120Lys Ser Leu Val Thr Val Leu Gly
Ile Pro Gln Lys Pro Ile Ile 125 130 135Thr Gly Tyr Lys Ser Ser Leu
Arg Glu Lys Asp Thr Ala Thr Leu 140 145 150Asn Cys Gln Ser Ser Gly
Ser Lys Pro Ala Ala Arg Leu Thr Trp 155 160 165Arg Lys Gly Asp Gln
Glu Leu His Gly Glu Pro Thr Arg Ile Gln 170 175 180Glu Asp Pro Asn
Gly Lys Thr Phe Thr Val Ser Ser Ser Val Thr 185 190 195Phe Gln Val
Thr Arg Glu Asp Asp Gly Ala Ser Ile Val Cys Ser 200 205 210Val Asn
His Glu Ser Leu Lys Gly Ala Asp Arg Ser Thr Ser Gln 215 220 225Arg
Ile Glu Val Leu Tyr Thr Pro Thr Ala Met Ile Arg Pro Asp 230 235
240Pro Pro His Pro Arg Glu Gly Gln Lys Leu Leu Leu His Cys Glu 245
250 255Gly Arg Gly Asn Pro Val Pro Gln Gln Tyr Leu Trp Glu Lys Glu
260 265 270Gly Ser Val Pro Pro Leu Lys Met Thr Gln Glu Ser Ala Leu
Ile 275 280 285Phe Pro Phe Leu Asn Lys Ser Asp Ser Gly Thr Tyr Gly
Cys Thr 290 295 300Ala Thr Ser Asn Met Gly Ser Tyr Lys Ala Tyr Tyr
Thr Leu Asn 305 310 315Val Asn Asp Pro Ser Pro Val Pro Ser Ser Ser
Ser Thr Tyr His 320 325 330Ala Ile Ile Gly Gly Ile Val Ala Phe Ile
Val Phe Leu Leu Leu 335 340 345Ile Met Leu Ile Phe Leu Gly His Tyr
Leu Ile Arg His Lys Gly 350 355 360Thr Tyr Leu Thr His Glu Ala Lys
Gly Ser Asp Asp Ala Pro Asp 365 370 375Ala Asp Thr Ala Ile Ile Asn
Ala Glu Gly Gly Gln Ser Gly Gly 380 385 390Asp Asp Lys Lys Glu Tyr
Phe Ile 395152026DNAHomo sapiens 15cggacgcgtg ggattcagca gtggcctgtg
gctgccagag cagctcctca 50ggggaaacta agcgtcgagt cagacggcac cataatcgcc
tttaaaagtg 100cctccgccct gccggccgcg tatcccccgg ctacctgggc
cgccccgcgg 150cggtgcgcgc gtgagaggga gcgcgcgggc agccgagcgc
cggtgtgagc 200cagcgctgct gccagtgtga gcggcggtgt gagcgcggtg
ggtgcggagg 250ggcgtgtgtg ccggcgcgcg cgccgtgggg tgcaaacccc
gagcgtctac 300gctgccatga ggggcgcgaa cgcctgggcg ccactctgcc
tgctgctggc 350tgccgccacc cagctctcgc ggcagcagtc cccagagaga
cctgttttca 400catgtggtgg cattcttact ggagagtctg gatttattgg
cagtgaaggt 450tttcctggag tgtaccctcc aaatagcaaa tgtacttgga
aaatcacagt 500tcccgaagga aaagtagtcg ttctcaattt ccgattcata
gacctcgaga 550gtgacaacct gtgccgctat gactttgtgg atgtgtacaa
tggccatgcc 600aatggccagc gcattggccg cttctgtggc actttccggc
ctggagccct 650tgtgtccagt ggcaacaaga tgatggtgca gatgatttct
gatgccaaca 700cagctggcaa tggcttcatg gccatgttct ccgctgctga
accaaacgaa 750agaggggatc agtattgtgg aggactcctt gacagacctt
ccggctcttt 800taaaaccccc aactggccag accgggatta ccctgcagga
gtcacttgtg 850tgtggcacat tgtagcccca aagaatcagc ttatagaatt
aaagtttgag 900aagtttgatg tggagcgaga taactactgc cgatatgatt
atgtggctgt 950gtttaatggc ggggaagtca acgatgctag aagaattgga
aagtattgtg 1000gtgatagtcc acctgcgcca attgtgtctg agagaaatga
acttcttatt 1050cagtttttat cagacttaag tttaactgca gatgggttta
ttggtcacta 1100catattcagg ccaaaaaaac tgcctacaac tacagaacag
cctgtcacca 1150ccacattccc tgtaaccacg ggtttaaaac ccaccgtggc
cttgtgtcaa 1200caaaagtgta gacggacggg gactctggag ggcaattatt
gttcaagtga 1250ctttgtatta gccggcactg ttatcacaac catcactcgc
gatgggagtt 1300tgcacgccac agtctcgatc atcaacatct acaaagaggg
aaatttggcg 1350attcagcagg cgggcaagaa catgagtgcc aggctgactg
tcgtctgcaa 1400gcagtgccct ctcctcagaa gaggtctaaa ttacattatt
atgggccaag 1450taggtgaaga tgggcgaggc aaaatcatgc caaacagctt
tatcatgatg 1500ttcaagacca agaatcagaa gctcctggat gccttaaaaa
ataagcaatg 1550ttaacagtga actgtgtcca tttaagctgt attctgccat
tgcctttgaa 1600agatctatgt tctctcagta gaaaaaaaaa tacttataaa
attacatatt 1650ctgaaagagg attccgaaag atgggactgg ttgactcttc
acatgatgga 1700ggtatgaggc ctccgagata gctgagggaa gttctttgcc
tgctgtcaga 1750ggagcagcta tctgattgga aacctgccga cttagtgcgg
tgataggaag 1800ctaaaagtgt caagcgttga cagcttggaa gcgtttattt
atacatctct 1850gtaaaaggat attttagaat tgagttgtgt gaagatgtca
aaaaaagatt 1900ttagaagtgc aatatttata gtgttatttg tttcaccttc
aagcctttgc 1950cctgaggtgt tacaatcttg tcttgcgttt tctaaatcaa
tgcttaataa 2000aatattttta aaggaaaaaa aaaaaa 202616415PRTHomo
sapiens 16Met Arg Gly Ala Asn Ala Trp Ala Pro Leu Cys Leu Leu Leu
Ala1 5 10 15Ala Ala Thr Gln Leu Ser Arg Gln Gln Ser Pro Glu Arg Pro
Val 20 25 30Phe Thr Cys Gly Gly Ile Leu Thr Gly Glu Ser Gly Phe Ile
Gly 35 40 45Ser Glu Gly Phe Pro Gly Val Tyr Pro Pro Asn Ser Lys Cys
Thr 50 55 60Trp Lys Ile Thr Val Pro Glu Gly Lys Val Val Val Leu Asn
Phe 65 70 75Arg Phe Ile Asp Leu Glu Ser Asp Asn Leu Cys Arg Tyr Asp
Phe 80 85 90Val Asp Val Tyr Asn Gly His Ala Asn Gly Gln Arg Ile Gly
Arg 95 100 105Phe Cys Gly Thr Phe Arg Pro Gly Ala Leu Val Ser Ser
Gly Asn 110 115 120Lys Met Met Val Gln Met Ile Ser Asp Ala Asn Thr
Ala Gly Asn 125 130 135Gly Phe Met Ala Met Phe Ser Ala Ala Glu Pro
Asn Glu Arg Gly 140 145 150Asp Gln Tyr Cys Gly Gly Leu Leu Asp Arg
Pro Ser Gly Ser Phe 155 160 165Lys Thr Pro Asn Trp Pro Asp Arg Asp
Tyr Pro Ala Gly Val Thr 170 175 180Cys Val Trp His Ile Val Ala Pro
Lys Asn Gln Leu Ile Glu Leu 185 190 195Lys Phe Glu Lys Phe Asp Val
Glu Arg Asp Asn Tyr Cys Arg Tyr 200 205 210Asp Tyr Val Ala Val Phe
Asn Gly Gly Glu Val Asn Asp Ala Arg 215 220 225Arg Ile Gly Lys Tyr
Cys Gly Asp Ser Pro Pro Ala Pro Ile Val 230 235 240Ser Glu Arg Asn
Glu Leu Leu Ile Gln Phe Leu Ser Asp Leu Ser 245 250 255Leu Thr Ala
Asp Gly Phe Ile Gly His Tyr Ile Phe Arg Pro Lys 260 265 270Lys Leu
Pro Thr Thr Thr Glu Gln Pro Val Thr Thr Thr Phe Pro 275 280 285Val
Thr Thr Gly Leu Lys Pro Thr Val Ala Leu Cys Gln Gln Lys 290 295
300Cys Arg Arg Thr Gly Thr Leu Glu Gly Asn Tyr Cys Ser Ser Asp 305
310 315Phe Val Leu Ala Gly Thr Val Ile Thr Thr Ile Thr Arg Asp Gly
320 325 330Ser Leu His Ala Thr Val Ser Ile Ile Asn Ile Tyr Lys Glu
Gly 335 340 345Asn Leu Ala Ile Gln Gln Ala Gly Lys Asn Met Ser Ala
Arg Leu 350 355 360Thr Val Val Cys Lys Gln Cys Pro Leu Leu Arg Arg
Gly Leu Asn 365 370 375Tyr Ile Ile Met Gly Gln Val Gly Glu Asp Gly
Arg Gly Lys Ile 380 385 390Met Pro Asn Ser Phe Ile Met Met Phe Lys
Thr Lys Asn Gln Lys 395 400 405Leu Leu Asp Ala Leu Lys Asn Lys Gln
Cys 410 415171875DNAHomo sapiens 17gacggctggc caccatgcac ggctcctgca
gtttcctgat gcttctgctg 50ccgctactgc tactgctggt ggccaccaca ggccccgttg
gagccctcac 100agatgaggag aaacgtttga tggtggagct gcacaacctc
taccgggccc 150aggtatcccc gacggcctca gacatgctgc acatgagatg
ggacgaggag 200ctggccgcct tcgccaaggc ctacgcacgg cagtgcgtgt
ggggccacaa 250caaggagcgc gggcgccgcg gcgagaatct gttcgccatc
acagacgagg 300gcatggacgt gccgctggcc atggaggagt ggcaccacga
gcgtgagcac 350tacaacctca gcgccgccac ctgcagccca ggccagatgt
gcggccacta 400cacgcaggtg gtatgggcca agacagagag gatcggctgt
ggttcccact 450tctgtgagaa gctccagggt gttgaggaga ccaacatcga
attactggtg 500tgcaactatg agcctccggg gaacgtgaag gggaaacggc
cctaccagga 550ggggactccg tgctcccaat gtccctctgg ctaccactgc
aagaactccc 600tctgtgaacc catcggaagc ccggaagatg ctcaggattt
gccttacctg 650gtaactgagg ccccatcctt ccgggcgact gaagcatcag
actctaggaa 700aatgggtact ccttcttccc tagcaacggg gattccggct
ttcttggtaa 750cagaggtctc aggctccctg gcaaccaagg ctctgcctgc
tgtggaaacc 800caggccccaa cttccttagc aacgaaagac ccgccctcca
tggcaacaga 850ggctccacct tgcgtaacaa ctgaggtccc ttccattttg
gcagctcaca 900gcctgccctc cttggatgag gagccagtta ccttccccaa
atcgacccat 950gttcctatcc caaaatcagc agacaaagtg acagacaaaa
caaaagtgcc 1000ctctaggagc ccagagaact ctctggaccc caagatgtcc
ctgacagggg 1050caagggaact cctaccccat gcccaggagg aggctgaggc
tgaggctgag 1100ttgcctcctt ccagtgaggt cttggcctca gtttttccag
cccaggacaa 1150gccaggtgag ctgcaggcca cactggacca cacggggcac
acctcctcca 1200agtccctgcc caatttcccc aatacctctg ccaccgctaa
tgccacgggt 1250gggcgtgccc tggctctgca gtcgtccttg ccaggtgcag
agggccctga 1300caagcctagc gttgtgtcag ggctgaactc gggccctggt
catgtgtggg 1350gccctctcct gggactactg ctcctgcctc ctctggtgtt
ggctggaatc 1400ttctgaatgg gataccactc aaagggtgaa gaggtcagct
gtcctcctgt 1450catcttcccc accctgtccc cagcccctaa acaagatact
tcttggttaa 1500ggccctccgg aagggaaagg ctacggggca tgtgcctcat
cacaccatcc 1550atcctggagg cacaaggcct ggctggctgc gagctcagga
ggccgcctga 1600ggactgcaca ccgggcccac acctctcctg cccctccctc
ctgagtcctg 1650ggggtgggag gatttgaggg agctcactgc ctacctggcc
tggggctgtc 1700tgcccacaca gcatgtgcgc tctccctgag tgcctgtgta
gctggggatg 1750gggattccta ggggcagatg aaggacaagc cccactggag
tggggttctt 1800tgagtggggg aggcagggac gagggaagga aagtaactcc
tgactctcca 1850ataaaaacct gtccaacctg tgaaa 187518463PRTHomo sapiens
18Met His Gly Ser Cys Ser Phe Leu Met Leu Leu Leu Pro Leu Leu1 5 10
15Leu Leu Leu Val Ala Thr Thr Gly Pro Val Gly Ala Leu Thr Asp 20 25
30Glu Glu Lys Arg Leu Met Val Glu Leu His Asn Leu Tyr Arg Ala 35 40
45Gln Val Ser Pro Thr Ala Ser Asp Met Leu His Met Arg Trp Asp 50 55
60Glu Glu Leu Ala Ala Phe Ala Lys Ala Tyr Ala Arg Gln Cys Val 65 70
75Trp Gly His Asn Lys Glu Arg Gly Arg Arg Gly Glu Asn Leu Phe 80 85
90Ala Ile Thr Asp Glu Gly Met Asp Val Pro Leu Ala Met Glu Glu 95
100 105Trp His His Glu Arg Glu His Tyr Asn Leu Ser Ala Ala Thr Cys
110 115 120Ser Pro Gly Gln Met Cys Gly His Tyr Thr Gln Val Val Trp
Ala 125 130 135Lys Thr Glu Arg Ile Gly Cys Gly Ser His Phe Cys Glu
Lys Leu 140 145 150Gln Gly Val Glu Glu Thr Asn Ile Glu Leu Leu Val
Cys Asn Tyr 155 160
165Glu Pro Pro Gly Asn Val Lys Gly Lys Arg Pro Tyr Gln Glu Gly 170
175 180Thr Pro Cys Ser Gln Cys Pro Ser Gly Tyr His Cys Lys Asn Ser
185 190 195Leu Cys Glu Pro Ile Gly Ser Pro Glu Asp Ala Gln Asp Leu
Pro 200 205 210Tyr Leu Val Thr Glu Ala Pro Ser Phe Arg Ala Thr Glu
Ala Ser 215 220 225Asp Ser Arg Lys Met Gly Thr Pro Ser Ser Leu Ala
Thr Gly Ile 230 235 240Pro Ala Phe Leu Val Thr Glu Val Ser Gly Ser
Leu Ala Thr Lys 245 250 255Ala Leu Pro Ala Val Glu Thr Gln Ala Pro
Thr Ser Leu Ala Thr 260 265 270Lys Asp Pro Pro Ser Met Ala Thr Glu
Ala Pro Pro Cys Val Thr 275 280 285Thr Glu Val Pro Ser Ile Leu Ala
Ala His Ser Leu Pro Ser Leu 290 295 300Asp Glu Glu Pro Val Thr Phe
Pro Lys Ser Thr His Val Pro Ile 305 310 315Pro Lys Ser Ala Asp Lys
Val Thr Asp Lys Thr Lys Val Pro Ser 320 325 330Arg Ser Pro Glu Asn
Ser Leu Asp Pro Lys Met Ser Leu Thr Gly 335 340 345Ala Arg Glu Leu
Leu Pro His Ala Gln Glu Glu Ala Glu Ala Glu 350 355 360Ala Glu Leu
Pro Pro Ser Ser Glu Val Leu Ala Ser Val Phe Pro 365 370 375Ala Gln
Asp Lys Pro Gly Glu Leu Gln Ala Thr Leu Asp His Thr 380 385 390Gly
His Thr Ser Ser Lys Ser Leu Pro Asn Phe Pro Asn Thr Ser 395 400
405Ala Thr Ala Asn Ala Thr Gly Gly Arg Ala Leu Ala Leu Gln Ser 410
415 420Ser Leu Pro Gly Ala Glu Gly Pro Asp Lys Pro Ser Val Val Ser
425 430 435Gly Leu Asn Ser Gly Pro Gly His Val Trp Gly Pro Leu Leu
Gly 440 445 450Leu Leu Leu Leu Pro Pro Leu Val Leu Ala Gly Ile Phe
455 460191377DNAHomo sapiens 19gactagttct cttggagtct gggaggagga
aagcggagcc ggcagggagc 50gaaccaggac tggggtgacg gcagggcagg gggcgcctgg
ccggggagaa 100gcgcgggggc tggagcacca ccaactggag ggtccggagt
agcgagcgcc 150ccgaaggagg ccatcgggga gccgggaggg gggactgcga
gaggaccccg 200gcgtccgggc tcccggtgcc agcgctatga ggccactcct
cgtcctgctg 250ctcctgggcc tggcggccgg ctcgccccca ctggacgaca
acaagatccc 300cagcctctgc ccggggcacc ccggccttcc aggcacgccg
ggccaccatg 350gcagccaggg cttgccgggc cgcgatggcc gcgacggccg
cgacggcgcg 400cccggggctc cgggagagaa aggcgagggc gggaggccgg
gactgccggg 450acctcgaggg gaccccgggc cgcgaggaga ggcgggaccc
gcggggccca 500ccgggcctgc cggggagtgc tcggtgcctc cgcgatccgc
cttcagcgcc 550aagcgctccg agagccgggt gcctccgccg tctgacgcac
ccttgccctt 600cgaccgcgtg ctggtgaacg agcagggaca ttacgacgcc
gtcaccggca 650agttcacctg ccaggtgcct ggggtctact acttcgccgt
ccatgccacc 700gtctaccggg ccagcctgca gtttgatctg gtgaagaatg
gcgaatccat 750tgcctctttc ttccagtttt tcggggggtg gcccaagcca
gcctcgctct 800cggggggggc catggtgagg ctggagcctg aggaccaagt
gtgggtgcag 850gtgggtgtgg gtgactacat tggcatctat gccagcatca
agacagacag 900caccttctcc ggatttctgg tgtactccga ctggcacagc
tccccagtct 950ttgcttagtg cccactgcaa agtgagctca tgctctcact
cctagaagga 1000gggtgtgagg ctgacaacca ggtcatccag gagggctggc
ccccctggaa 1050tattgtgaat gactagggag gtggggtaga gcactctccg
tcctgctgct 1100ggcaaggaat gggaacagtg gctgtctgcg atcaggtctg
gcagcatggg 1150gcagtggctg gatttctgcc caagaccaga ggagtgtgct
gtgctggcaa 1200gtgtaagtcc cccagttgct ctggtccagg agcccacggt
ggggtgctct 1250cttcctggtc ctctgcttct ctggatcctc cccaccccct
cctgctcctg 1300gggccggccc ttttctcaga gatcactcaa taaacctaag
aaccctcata 1350aaaaaaaaaa aaaaaaaaaa aaaaaaa 137720243PRTHomo
sapiens 20Met Arg Pro Leu Leu Val Leu Leu Leu Leu Gly Leu Ala Ala
Gly1 5 10 15Ser Pro Pro Leu Asp Asp Asn Lys Ile Pro Ser Leu Cys Pro
Gly 20 25 30His Pro Gly Leu Pro Gly Thr Pro Gly His His Gly Ser Gln
Gly 35 40 45Leu Pro Gly Arg Asp Gly Arg Asp Gly Arg Asp Gly Ala Pro
Gly 50 55 60Ala Pro Gly Glu Lys Gly Glu Gly Gly Arg Pro Gly Leu Pro
Gly 65 70 75Pro Arg Gly Asp Pro Gly Pro Arg Gly Glu Ala Gly Pro Ala
Gly 80 85 90Pro Thr Gly Pro Ala Gly Glu Cys Ser Val Pro Pro Arg Ser
Ala 95 100 105Phe Ser Ala Lys Arg Ser Glu Ser Arg Val Pro Pro Pro
Ser Asp 110 115 120Ala Pro Leu Pro Phe Asp Arg Val Leu Val Asn Glu
Gln Gly His 125 130 135Tyr Asp Ala Val Thr Gly Lys Phe Thr Cys Gln
Val Pro Gly Val 140 145 150Tyr Tyr Phe Ala Val His Ala Thr Val Tyr
Arg Ala Ser Leu Gln 155 160 165Phe Asp Leu Val Lys Asn Gly Glu Ser
Ile Ala Ser Phe Phe Gln 170 175 180Phe Phe Gly Gly Trp Pro Lys Pro
Ala Ser Leu Ser Gly Gly Ala 185 190 195Met Val Arg Leu Glu Pro Glu
Asp Gln Val Trp Val Gln Val Gly 200 205 210Val Gly Asp Tyr Ile Gly
Ile Tyr Ala Ser Ile Lys Thr Asp Ser 215 220 225Thr Phe Ser Gly Phe
Leu Val Tyr Ser Asp Trp His Ser Ser Pro 230 235 240Val Phe
Ala212555DNAHomo sapiens 21ggggcgggtg gacgcggact cgaacgcagt
tgcttcggga cccaggaccc 50cctcgggccc gacccgccag gaaagactga ggccgcggcc
tgccccgccc 100ggctccctgc gccgccgccg cctcccggga cagaagatgt
gctccagggt 150ccctctgctg ctgccgctgc tcctgctact ggccctgggg
cctggggtgc 200agggctgccc atccggctgc cagtgcagcc agccacagac
agtcttctgc 250actgcccgcc aggggaccac ggtgccccga gacgtgccac
ccgacacggt 300ggggctgtac gtctttgaga acggcatcac catgctcgac
gcaagcagct 350ttgccggcct gccgggcctg cagctcctgg acctgtcaca
gaaccagatc 400gccagcctgc gcctgccccg cctgctgctg ctggacctca
gccacaacag 450cctcctggcc ctggagcccg gcatcctgga cactgccaac
gtggaggcgc 500tgcggctggc tggtctgggg ctgcagcagc tggacgaggg
gctcttcagc 550cgcttgcgca acctccacga cctggatgtg tccgacaacc
agctggagcg 600agtgccacct gtgatccgag gcctccgggg cctgacgcgc
ctgcggctgg 650ccggcaacac ccgcattgcc cagctgcggc ccgaggacct
ggccggcctg 700gctgccctgc aggagctgga tgtgagcaac ctaagcctgc
aggccctgcc 750tggcgacctc tcgggcctct tcccccgcct gcggctgctg
gcagctgccc 800gcaacccctt caactgcgtg tgccccctga gctggtttgg
cccctgggtg 850cgcgagagcc acgtcacact ggccagccct gaggagacgc
gctgccactt 900cccgcccaag aacgctggcc ggctgctcct ggagcttgac
tacgccgact 950ttggctgccc agccaccacc accacagcca cagtgcccac
cacgaggccc 1000gtggtgcggg agcccacagc cttgtcttct agcttggctc
ctacctggct 1050tagccccaca gcgccggcca ctgaggcccc cagcccgccc
tccactgccc 1100caccgactgt agggcctgtc ccccagcccc aggactgccc
accgtccacc 1150tgcctcaatg ggggcacatg ccacctgggg acacggcacc
acctggcgtg 1200cttgtgcccc gaaggcttca cgggcctgta ctgtgagagc
cagatggggc 1250aggggacacg gcccagccct acaccagtca cgccgaggcc
accacggtcc 1300ctgaccctgg gcatcgagcc ggtgagcccc acctccctgc
gcgtggggct 1350gcagcgctac ctccagggga gctccgtgca gctcaggagc
ctccgtctca 1400cctatcgcaa cctatcgggc cctgataagc ggctggtgac
gctgcgactg 1450cctgcctcgc tcgctgagta cacggtcacc cagctgcggc
ccaacgccac 1500ttactccgtc tgtgtcatgc ctttggggcc cgggcgggtg
ccggagggcg 1550aggaggcctg cggggaggcc catacacccc cagccgtcca
ctccaaccac 1600gccccagtca cccaggcccg cgagggcaac ctgccgctcc
tcattgcgcc 1650cgccctggcc gcggtgctcc tggccgcgct ggctgcggtg
ggggcagcct 1700actgtgtgcg gcgggggcgg gccatggcag cagcggctca
ggacaaaggg 1750caggtggggc caggggctgg gcccctggaa ctggagggag
tgaaggtccc 1800cttggagcca ggcccgaagg caacagaggg cggtggagag
gccctgccca 1850gcgggtctga gtgtgaggtg ccactcatgg gcttcccagg
gcctggcctc 1900cagtcacccc tccacgcaaa gccctacatc taagccagag
agagacaggg 1950cagctggggc cgggctctca gccagtgaga tggccagccc
cctcctgctg 2000ccacaccacg taagttctca gtcccaacct cggggatgtg
tgcagacagg 2050gctgtgtgac cacagctggg ccctgttccc tctggacctc
ggtctcctca 2100tctgtgagat gctgtggccc agctgacgag ccctaacgtc
cccagaaccg 2150agtgcctatg aggacagtgt ccgccctgcc ctccgcaacg
tgcagtccct 2200gggcacggcg ggccctgcca tgtgctggta acgcatgcct
gggccctgct 2250gggctctccc actccaggcg gaccctgggg gccagtgaag
gaagctcccg 2300gaaagagcag agggagagcg ggtaggcggc tgtgtgactc
tagtcttggc 2350cccaggaagc gaaggaacaa aagaaactgg aaaggaagat
gctttaggaa 2400catgttttgc ttttttaaaa tatatatata tttataagag
atcctttccc 2450atttattctg ggaagatgtt tttcaaactc agagacaagg
actttggttt 2500ttgtaagaca aacgatgata tgaaggcctt ttgtaagaaa
aaataaaaaa 2550aaaaa 255522598PRTHomo sapiens 22Met Cys Ser Arg Val
Pro Leu Leu Leu Pro Leu Leu Leu Leu Leu1 5 10 15Ala Leu Gly Pro Gly
Val Gln Gly Cys Pro Ser Gly Cys Gln Cys 20 25 30Ser Gln Pro Gln Thr
Val Phe Cys Thr Ala Arg Gln Gly Thr Thr 35 40 45Val Pro Arg Asp Val
Pro Pro Asp Thr Val Gly Leu Tyr Val Phe 50 55 60Glu Asn Gly Ile Thr
Met Leu Asp Ala Ser Ser Phe Ala Gly Leu 65 70 75Pro Gly Leu Gln Leu
Leu Asp Leu Ser Gln Asn Gln Ile Ala Ser 80 85 90Leu Arg Leu Pro Arg
Leu Leu Leu Leu Asp Leu Ser His Asn Ser 95 100 105Leu Leu Ala Leu
Glu Pro Gly Ile Leu Asp Thr Ala Asn Val Glu 110 115 120Ala Leu Arg
Leu Ala Gly Leu Gly Leu Gln Gln Leu Asp Glu Gly 125 130 135Leu Phe
Ser Arg Leu Arg Asn Leu His Asp Leu Asp Val Ser Asp 140 145 150Asn
Gln Leu Glu Arg Val Pro Pro Val Ile Arg Gly Leu Arg Gly 155 160
165Leu Thr Arg Leu Arg Leu Ala Gly Asn Thr Arg Ile Ala Gln Leu 170
175 180Arg Pro Glu Asp Leu Ala Gly Leu Ala Ala Leu Gln Glu Leu Asp
185 190 195Val Ser Asn Leu Ser Leu Gln Ala Leu Pro Gly Asp Leu Ser
Gly 200 205 210Leu Phe Pro Arg Leu Arg Leu Leu Ala Ala Ala Arg Asn
Pro Phe 215 220 225Asn Cys Val Cys Pro Leu Ser Trp Phe Gly Pro Trp
Val Arg Glu 230 235 240Ser His Val Thr Leu Ala Ser Pro Glu Glu Thr
Arg Cys His Phe 245 250 255Pro Pro Lys Asn Ala Gly Arg Leu Leu Leu
Glu Leu Asp Tyr Ala 260 265 270Asp Phe Gly Cys Pro Ala Thr Thr Thr
Thr Ala Thr Val Pro Thr 275 280 285Thr Arg Pro Val Val Arg Glu Pro
Thr Ala Leu Ser Ser Ser Leu 290 295 300Ala Pro Thr Trp Leu Ser Pro
Thr Ala Pro Ala Thr Glu Ala Pro 305 310 315Ser Pro Pro Ser Thr Ala
Pro Pro Thr Val Gly Pro Val Pro Gln 320 325 330Pro Gln Asp Cys Pro
Pro Ser Thr Cys Leu Asn Gly Gly Thr Cys 335 340 345His Leu Gly Thr
Arg His His Leu Ala Cys Leu Cys Pro Glu Gly 350 355 360Phe Thr Gly
Leu Tyr Cys Glu Ser Gln Met Gly Gln Gly Thr Arg 365 370 375Pro Ser
Pro Thr Pro Val Thr Pro Arg Pro Pro Arg Ser Leu Thr 380 385 390Leu
Gly Ile Glu Pro Val Ser Pro Thr Ser Leu Arg Val Gly Leu 395 400
405Gln Arg Tyr Leu Gln Gly Ser Ser Val Gln Leu Arg Ser Leu Arg 410
415 420Leu Thr Tyr Arg Asn Leu Ser Gly Pro Asp Lys Arg Leu Val Thr
425 430 435Leu Arg Leu Pro Ala Ser Leu Ala Glu Tyr Thr Val Thr Gln
Leu 440 445 450Arg Pro Asn Ala Thr Tyr Ser Val Cys Val Met Pro Leu
Gly Pro 455 460 465Gly Arg Val Pro Glu Gly Glu Glu Ala Cys Gly Glu
Ala His Thr 470 475 480Pro Pro Ala Val His Ser Asn His Ala Pro Val
Thr Gln Ala Arg 485 490 495Glu Gly Asn Leu Pro Leu Leu Ile Ala Pro
Ala Leu Ala Ala Val 500 505 510Leu Leu Ala Ala Leu Ala Ala Val Gly
Ala Ala Tyr Cys Val Arg 515 520 525Arg Gly Arg Ala Met Ala Ala Ala
Ala Gln Asp Lys Gly Gln Val 530 535 540Gly Pro Gly Ala Gly Pro Leu
Glu Leu Glu Gly Val Lys Val Pro 545 550 555Leu Glu Pro Gly Pro Lys
Ala Thr Glu Gly Gly Gly Glu Ala Leu 560 565 570Pro Ser Gly Ser Glu
Cys Glu Val Pro Leu Met Gly Phe Pro Gly 575 580 585Pro Gly Leu Gln
Ser Pro Leu His Ala Lys Pro Tyr Ile 590 595232236DNAHomo sapiens
23ggcgccggtg caccgggcgg gctgagcgcc tcctgcggcc cggcctgcgc
50gccccggccc gccgcgccgc ccacgcccca accccggccc gcgcccccta
100gcccccgccc gggcccgcgc ccgcgcccgc gcccaggtga gcgctccgcc
150cgccgcgagg ccccgccccg gcccgccccc gccccgcccc ggccggcggg
200ggaaccgggc ggattcctcg cgcgtcaaac cacctgatcc cataaaacat
250tcatcctccc ggcggcccgc gctgcgagcg ccccgccagt ccgcgccgcc
300gccgccctcg ccctgtgcgc cctgcgcgcc ctgcgcaccc gcggcccgag
350cccagccaga gccgggcgga gcggagcgcg ccgagcctcg tcccgcggcc
400gggccggggc cgggccgtag cggcggcgcc tggatgcgga cccggccgcg
450gggagacggg cgcccgcccc gaaacgactt tcagtccccg acgcgccccg
500cccaacccct acgatgaaga gggcgtccgc tggagggagc cggctgctgg
550catgggtgct gtggctgcag gcctggcagg tggcagcccc atgcccaggt
600gcctgcgtat gctacaatga gcccaaggtg acgacaagct gcccccagca
650gggcctgcag gctgtgcccg tgggcatccc tgctgccagc cagcgcatct
700tcctgcacgg caaccgcatc tcgcatgtgc cagctgccag cttccgtgcc
750tgccgcaacc tcaccatcct gtggctgcac tcgaatgtgc tggcccgaat
800tgatgcggct gccttcactg gcctggccct cctggagcag ctggacctca
850gcgataatgc acagctccgg tctgtggacc ctgccacatt ccacggcctg
900ggccgcctac acacgctgca cctggaccgc tgcggcctgc aggagctggg
950cccggggctg ttccgcggcc tggctgccct gcagtacctc tacctgcagg
1000acaacgcgct gcaggcactg cctgatgaca ccttccgcga cctgggcaac
1050ctcacacacc tcttcctgca cggcaaccgc atctccagcg tgcccgagcg
1100cgccttccgt gggctgcaca gcctcgaccg tctcctactg caccagaacc
1150gcgtggccca tgtgcacccg catgccttcc gtgaccttgg ccgcctcatg
1200acactctatc tgtttgccaa caatctatca gcgctgccca ctgaggccct
1250ggcccccctg cgtgccctgc agtacctgag gctcaacgac aacccctggg
1300tgtgtgactg ccgggcacgc ccactctggg cctggctgca gaagttccgc
1350ggctcctcct ccgaggtgcc ctgcagcctc ccgcaacgcc tggctggccg
1400tgacctcaaa cgcctagctg ccaatgacct gcagggctgc gctgtggcca
1450ccggccctta ccatcccatc tggaccggca gggccaccga tgaggagccg
1500ctggggcttc ccaagtgctg ccagccagat gccgctgaca aggcctcagt
1550actggagcct ggaagaccag cttcggcagg caatgcgctg aagggacgcg
1600tgccgcccgg tgacagcccg ccgggcaacg gctctggccc acggcacatc
1650aatgactcac cctttgggac tctgcctggc tctgctgagc ccccgctcac
1700tgcagtgcgg cccgagggct ccgagccacc agggttcccc acctcgggcc
1750ctcgccggag gccaggctgt tcacgcaaga accgcacccg cagccactgc
1800cgtctgggcc aggcaggcag cgggggtggc gggactggtg actcagaagg
1850ctcaggtgcc ctacccagcc tcacctgcag cctcaccccc ctgggcctgg
1900cgctggtgct gtggacagtg cttgggccct gctgaccccc agcggacaca
1950agagcgtgct cagcagccag gtgtgtgtac atacggggtc tctctccacg
2000ccgccaagcc agccgggcgg ccgacccgtg gggcaggcca ggccaggtcc
2050tccctgatgg acgcctgccg cccgccaccc ccatctccac cccatcatgt
2100ttacagggtt cggcggcagc gtttgttcca gaacgccgcc tcccacccag
2150atcgcggtat atagagatat gcattttatt ttacttgtgt aaaaatatcg
2200gacgacgtgg aataaagagc tcttttctta aaaaaa 223624473PRTHomo
sapiens 24Met Lys Arg Ala Ser Ala Gly Gly Ser Arg Leu Leu Ala Trp
Val1 5 10
15Leu Trp Leu Gln Ala Trp Gln Val Ala Ala Pro Cys Pro Gly Ala 20 25
30Cys Val Cys Tyr Asn Glu Pro Lys Val Thr Thr Ser Cys Pro Gln 35 40
45Gln Gly Leu Gln Ala Val Pro Val Gly Ile Pro Ala Ala Ser Gln 50 55
60Arg Ile Phe Leu His Gly Asn Arg Ile Ser His Val Pro Ala Ala 65 70
75Ser Phe Arg Ala Cys Arg Asn Leu Thr Ile Leu Trp Leu His Ser 80 85
90Asn Val Leu Ala Arg Ile Asp Ala Ala Ala Phe Thr Gly Leu Ala 95
100 105Leu Leu Glu Gln Leu Asp Leu Ser Asp Asn Ala Gln Leu Arg Ser
110 115 120Val Asp Pro Ala Thr Phe His Gly Leu Gly Arg Leu His Thr
Leu 125 130 135His Leu Asp Arg Cys Gly Leu Gln Glu Leu Gly Pro Gly
Leu Phe 140 145 150Arg Gly Leu Ala Ala Leu Gln Tyr Leu Tyr Leu Gln
Asp Asn Ala 155 160 165Leu Gln Ala Leu Pro Asp Asp Thr Phe Arg Asp
Leu Gly Asn Leu 170 175 180Thr His Leu Phe Leu His Gly Asn Arg Ile
Ser Ser Val Pro Glu 185 190 195Arg Ala Phe Arg Gly Leu His Ser Leu
Asp Arg Leu Leu Leu His 200 205 210Gln Asn Arg Val Ala His Val His
Pro His Ala Phe Arg Asp Leu 215 220 225Gly Arg Leu Met Thr Leu Tyr
Leu Phe Ala Asn Asn Leu Ser Ala 230 235 240Leu Pro Thr Glu Ala Leu
Ala Pro Leu Arg Ala Leu Gln Tyr Leu 245 250 255Arg Leu Asn Asp Asn
Pro Trp Val Cys Asp Cys Arg Ala Arg Pro 260 265 270Leu Trp Ala Trp
Leu Gln Lys Phe Arg Gly Ser Ser Ser Glu Val 275 280 285Pro Cys Ser
Leu Pro Gln Arg Leu Ala Gly Arg Asp Leu Lys Arg 290 295 300Leu Ala
Ala Asn Asp Leu Gln Gly Cys Ala Val Ala Thr Gly Pro 305 310 315Tyr
His Pro Ile Trp Thr Gly Arg Ala Thr Asp Glu Glu Pro Leu 320 325
330Gly Leu Pro Lys Cys Cys Gln Pro Asp Ala Ala Asp Lys Ala Ser 335
340 345Val Leu Glu Pro Gly Arg Pro Ala Ser Ala Gly Asn Ala Leu Lys
350 355 360Gly Arg Val Pro Pro Gly Asp Ser Pro Pro Gly Asn Gly Ser
Gly 365 370 375Pro Arg His Ile Asn Asp Ser Pro Phe Gly Thr Leu Pro
Gly Ser 380 385 390Ala Glu Pro Pro Leu Thr Ala Val Arg Pro Glu Gly
Ser Glu Pro 395 400 405Pro Gly Phe Pro Thr Ser Gly Pro Arg Arg Arg
Pro Gly Cys Ser 410 415 420Arg Lys Asn Arg Thr Arg Ser His Cys Arg
Leu Gly Gln Ala Gly 425 430 435Ser Gly Gly Gly Gly Thr Gly Asp Ser
Glu Gly Ser Gly Ala Leu 440 445 450Pro Ser Leu Thr Cys Ser Leu Thr
Pro Leu Gly Leu Ala Leu Val 455 460 465Leu Trp Thr Val Leu Gly Pro
Cys 470253240DNAHomo sapiens 25cggacgcgtg ggcggacgcg tgggcctggg
caagggccgg ggcgccgggc 50cgagccacct cttcccctcc cccgcttccc tgtcgcgctc
cgctggctgg 100acgcgctgga ggagtggagc agcacccggc cggccctggg
ggctgacagt 150cggcaaagtt tggcccgaag aggaagtggt ctcaaacccc
ggcaggtggc 200gaccaggcca gaccaggggc gctcgctgcc tgcgggcggg
ctgtaggcga 250gggcgcgccc cagtgccgag acccggggct tcaggagccg
gccccgggag 300agaagagtgc ggcggcggac ggagaaaaca actccaaagt
tggcgaaagg 350caccgcccct actcccgggc tgccgccgcc tccccgcccc
cagccctggc 400atccagagta cgggtcgagc ccgggccatg gagcccccct
ggggaggcgg 450caccagggag cctgggcgcc cggggctccg ccgcgacccc
atcgggtaga 500ccacagaagc tccgggaccc ttccggcacc tctggacagc
ccaggatgct 550gttggccacc ctcctcctcc tcctccttgg aggcgctctg
gcccatccag 600accggattat ttttccaaat catgcttgtg aggacccccc
agcagtgctc 650ttagaagtgc agggcacctt acagaggccc ctggtccggg
acagccgcac 700ctcccctgcc aactgcacct ggctcatcct gggcagcaag
gaacagactg 750tcaccatcag gttccagaag ctacacctgg cctgtggctc
agagcgctta 800accctacgct cccctctcca gccactgatc tccctgtgtg
aggcacctcc 850cagccctctg cagctgcccg ggggcaacgt caccatcact
tacagctatg 900ctggggccag agcacccatg ggccagggct tcctgctctc
ctacagccaa 950gattggctga tgtgcctgca ggaagagttt cagtgcctga
accaccgctg 1000tgtatctgct gtccagcgct gtgatggggt tgatgcctgt
ggcgatggct 1050ctgatgaagc aggttgcagc tcagacccct tccctggcct
gaccccaaga 1100cccgtcccct ccctgccttg caatgtcacc ttggaggact
tctatggggt 1150cttctcctct cctggatata cacacctagc ctcagtctcc
cacccccagt 1200cctgccattg gctgctggac ccccatgatg gccggcggct
ggccgtgcgc 1250ttcacagccc tggacttggg ctttggagat gcagtgcatg
tgtatgacgg 1300ccctgggccc cctgagagct cccgactact gcgtagtctc
acccacttca 1350gcaatggcaa ggctgtcact gtggagacac tgtctggcca
ggctgttgtg 1400tcctaccaca cagttgcttg gagcaatggt cgtggcttca
atgccaccta 1450ccatgtgcgg ggctattgct tgccttggga cagaccctgt
ggcttaggct 1500ctggcctggg agctggcgaa ggcctaggtg agcgctgcta
cagtgaggca 1550cagcgctgtg acggctcatg ggactgtgct gacggcacag
atgaggagga 1600ctgcccaggc tgcccacctg gacacttccc ctgtggggct
gctggcacct 1650ctggtgccac agcctgctac ctgcctgctg accgctgcaa
ctaccagact 1700ttctgtgctg atggagcaga tgagagacgc tgtcggcatt
gccagcctgg 1750caatttccga tgccgggacg agaagtgcgt gtatgagacg
tgggtgtgcg 1800atgggcagcc agactgtgcg gacggcagtg atgagtggga
ctgctcctat 1850gttctgcccc gcaaggtcat tacagctgca gtcattggca
gcctagtgtg 1900cggcctgctc ctggtcatcg ccctgggctg cacctgcaag
ctctatgcca 1950ttcgcaccca ggagtacagc atctttgccc ccctctcccg
gatggaggct 2000gagattgtgc agcagcaggc acccccttcc tacgggcagc
tcattgccca 2050gggtgccatc ccacctgtag aagactttcc tacagagaat
cctaatgata 2100actcagtgct gggcaacctg cgttctctgc tacagatctt
acgccaggat 2150atgactccag gaggtggccc aggtgcccgc cgtcgtcagc
ggggccgctt 2200gatgcgacgc ctggtacgcc gtctccgccg ctggggcttg
ctccctcgaa 2250ccaacacccc ggctcgggcc tctgaggcca gatcccaggt
cacaccttct 2300gctgctcccc ttgaggccct agatggtggc acaggtccag
cccgtgaggg 2350cggggcagtg ggtgggcaag atggggagca ggcaccccca
ctgcccatca 2400aggctcccct cccatctgct agcacgtctc cagcccccac
tactgtccct 2450gaagccccag ggccactgcc ctcactgccc ctagagccat
cactattgtc 2500tggagtggtg caggccctgc gaggccgcct gttgcccagc
ctggggcccc 2550caggaccaac ccggagcccc cctggacccc acacagcagt
cctggccctg 2600gaagatgagg acgatgtgct actggtgcca ctggctgagc
cgggggtgtg 2650ggtagctgag gcagaggatg agccactgct tacctgaggg
gacctggggg 2700ctctactgag gcctctcccc tgggggctct actcatagtg
gcacaacctt 2750ttagaggtgg gtcagcctcc cctccaccac ttccttccct
gtccctggat 2800ttcagggact tggtgggcct cccgttgacc ctatgtagct
gctataaagt 2850taagtgtccc tcaggcaggg agagggctca cagagtctcc
tctgtacgtg 2900gccatggcca gacaccccag tcccttcacc accacctgct
ccccacgcca 2950ccaccatttg ggtggctgtt tttaaaaagt aaagttctta
gaggatcata 3000ggtctggaca ctccatcctt gccaaacctc tacccaaaag
tggccttaag 3050caccggaatg ccaattaact agagaccctc cagcccccaa
ggggaggatt 3100tgggcagaac ctgaggtttt gccatccaca atccctccta
cagggcctgg 3150ctcacaaaaa gagtgcaaca aatgcttcta ttccatagct
acggcattgc 3200tcagtaagtt gaggtcaaaa ataaaggaat catacatctc
324026713PRTHomo sapiens 26Met Leu Leu Ala Thr Leu Leu Leu Leu Leu
Leu Gly Gly Ala Leu1 5 10 15Ala His Pro Asp Arg Ile Ile Phe Pro Asn
His Ala Cys Glu Asp 20 25 30Pro Pro Ala Val Leu Leu Glu Val Gln Gly
Thr Leu Gln Arg Pro 35 40 45Leu Val Arg Asp Ser Arg Thr Ser Pro Ala
Asn Cys Thr Trp Leu 50 55 60Ile Leu Gly Ser Lys Glu Gln Thr Val Thr
Ile Arg Phe Gln Lys 65 70 75Leu His Leu Ala Cys Gly Ser Glu Arg Leu
Thr Leu Arg Ser Pro 80 85 90Leu Gln Pro Leu Ile Ser Leu Cys Glu Ala
Pro Pro Ser Pro Leu 95 100 105Gln Leu Pro Gly Gly Asn Val Thr Ile
Thr Tyr Ser Tyr Ala Gly 110 115 120Ala Arg Ala Pro Met Gly Gln Gly
Phe Leu Leu Ser Tyr Ser Gln 125 130 135Asp Trp Leu Met Cys Leu Gln
Glu Glu Phe Gln Cys Leu Asn His 140 145 150Arg Cys Val Ser Ala Val
Gln Arg Cys Asp Gly Val Asp Ala Cys 155 160 165Gly Asp Gly Ser Asp
Glu Ala Gly Cys Ser Ser Asp Pro Phe Pro 170 175 180Gly Leu Thr Pro
Arg Pro Val Pro Ser Leu Pro Cys Asn Val Thr 185 190 195Leu Glu Asp
Phe Tyr Gly Val Phe Ser Ser Pro Gly Tyr Thr His 200 205 210Leu Ala
Ser Val Ser His Pro Gln Ser Cys His Trp Leu Leu Asp 215 220 225Pro
His Asp Gly Arg Arg Leu Ala Val Arg Phe Thr Ala Leu Asp 230 235
240Leu Gly Phe Gly Asp Ala Val His Val Tyr Asp Gly Pro Gly Pro 245
250 255Pro Glu Ser Ser Arg Leu Leu Arg Ser Leu Thr His Phe Ser Asn
260 265 270Gly Lys Ala Val Thr Val Glu Thr Leu Ser Gly Gln Ala Val
Val 275 280 285Ser Tyr His Thr Val Ala Trp Ser Asn Gly Arg Gly Phe
Asn Ala 290 295 300Thr Tyr His Val Arg Gly Tyr Cys Leu Pro Trp Asp
Arg Pro Cys 305 310 315Gly Leu Gly Ser Gly Leu Gly Ala Gly Glu Gly
Leu Gly Glu Arg 320 325 330Cys Tyr Ser Glu Ala Gln Arg Cys Asp Gly
Ser Trp Asp Cys Ala 335 340 345Asp Gly Thr Asp Glu Glu Asp Cys Pro
Gly Cys Pro Pro Gly His 350 355 360Phe Pro Cys Gly Ala Ala Gly Thr
Ser Gly Ala Thr Ala Cys Tyr 365 370 375Leu Pro Ala Asp Arg Cys Asn
Tyr Gln Thr Phe Cys Ala Asp Gly 380 385 390Ala Asp Glu Arg Arg Cys
Arg His Cys Gln Pro Gly Asn Phe Arg 395 400 405Cys Arg Asp Glu Lys
Cys Val Tyr Glu Thr Trp Val Cys Asp Gly 410 415 420Gln Pro Asp Cys
Ala Asp Gly Ser Asp Glu Trp Asp Cys Ser Tyr 425 430 435Val Leu Pro
Arg Lys Val Ile Thr Ala Ala Val Ile Gly Ser Leu 440 445 450Val Cys
Gly Leu Leu Leu Val Ile Ala Leu Gly Cys Thr Cys Lys 455 460 465Leu
Tyr Ala Ile Arg Thr Gln Glu Tyr Ser Ile Phe Ala Pro Leu 470 475
480Ser Arg Met Glu Ala Glu Ile Val Gln Gln Gln Ala Pro Pro Ser 485
490 495Tyr Gly Gln Leu Ile Ala Gln Gly Ala Ile Pro Pro Val Glu Asp
500 505 510Phe Pro Thr Glu Asn Pro Asn Asp Asn Ser Val Leu Gly Asn
Leu 515 520 525Arg Ser Leu Leu Gln Ile Leu Arg Gln Asp Met Thr Pro
Gly Gly 530 535 540Gly Pro Gly Ala Arg Arg Arg Gln Arg Gly Arg Leu
Met Arg Arg 545 550 555Leu Val Arg Arg Leu Arg Arg Trp Gly Leu Leu
Pro Arg Thr Asn 560 565 570Thr Pro Ala Arg Ala Ser Glu Ala Arg Ser
Gln Val Thr Pro Ser 575 580 585Ala Ala Pro Leu Glu Ala Leu Asp Gly
Gly Thr Gly Pro Ala Arg 590 595 600Glu Gly Gly Ala Val Gly Gly Gln
Asp Gly Glu Gln Ala Pro Pro 605 610 615Leu Pro Ile Lys Ala Pro Leu
Pro Ser Ala Ser Thr Ser Pro Ala 620 625 630Pro Thr Thr Val Pro Glu
Ala Pro Gly Pro Leu Pro Ser Leu Pro 635 640 645Leu Glu Pro Ser Leu
Leu Ser Gly Val Val Gln Ala Leu Arg Gly 650 655 660Arg Leu Leu Pro
Ser Leu Gly Pro Pro Gly Pro Thr Arg Ser Pro 665 670 675Pro Gly Pro
His Thr Ala Val Leu Ala Leu Glu Asp Glu Asp Asp 680 685 690Val Leu
Leu Val Pro Leu Ala Glu Pro Gly Val Trp Val Ala Glu 695 700 705Ala
Glu Asp Glu Pro Leu Leu Thr 710274313DNAHomo sapiens 27gtcccacatc
ctgctcaact gggtcaggtc cctcttagac cagctcttgt 50ccatcatttg ctgaagtgga
ccaactagtt ccccagtagg gggtctcccc 100tggcaattct tgatcggcgt
ttggacatct cagatcgctt ccaatgaaga 150tggccttgcc ttggggtcct
gcttgtttca taatcatcta actatgggac 200aaggttgtgc cggcagctct
gggggaagga gcacggggct gatcaagcca 250tccaggaaac actggaggac
ttgtccagcc ttgaaagaac tctagtggtt 300tctgaatcta gcccacttgg
cggtaagcat gatgcaactt ctgcaacttc 350tgctggggct tttggggcca
ggtggctact tatttctttt aggggattgt 400caggaggtga ccactctcac
ggtgaaatac caagtgtcag aggaagtgcc 450atctggtaca gtgatcggga
agctgtccca ggaactgggc cgggaggaga 500ggcggaggca agctggggcc
gccttccagg tgttgcagct gcctcaggcg 550ctccccattc aggtggactc
tgaggaaggc ttgctcagca caggcaggcg 600gctggatcga gagcagctgt
gccgacagtg ggatccctgc ctggtttcct 650ttgatgtgct tgccacaggg
gatttggctc tgatccatgt ggagatccaa 700gtgctggaca tcaatgacca
ccagccacgg tttcccaaag gcgagcagga 750gctggaaatc tctgagagcg
cctctctgcg aacccggatc cccctggaca 800gagctcttga cccagacaca
ggccctaaca ccctgcacac ctacactctg 850tctcccagtg agcactttgc
cttggatgtc attgtgggcc ctgatgagac 900caaacatgca gaactcatag
tggtgaagga gctggacagg gaaatccatt 950cattttttga tctggtgtta
actgcctatg acaatgggaa cccccccaag 1000tcaggtacca gcttggtcaa
ggtcaacgtc ttggactcca atgacaatag 1050ccctgcgttt gctgagagtt
cactggcact ggaaatccaa gaagatgctg 1100cacctggtac gcttctcata
aaactgaccg ccacagaccc tgaccaaggc 1150cccaatgggg aggtggagtt
cttcctcagt aagcacatgc ctccagaggt 1200gctggacacc ttcagtattg
atgccaagac aggccaggtc attctgcgtc 1250gacctctaga ctatgaaaag
aaccctgcct acgaggtgga tgttcaggca 1300agggacctgg gtcccaatcc
tatcccagcc cattgcaaag ttctcatcaa 1350ggttctggat gtcaatgaca
acatcccaag catccacgtc acatgggcct 1400cccagccatc actggtgtca
gaagctcttc ccaaggacag ttttattgct 1450cttgtcatgg cagatgactt
ggattcagga cacaatggtt tggtccactg 1500ctggctgagc caagagctgg
gccacttcag gctgaaaaga actaatggca 1550acacatacat gttgctaacc
aatgccacac tggacagaga gcagtggccc 1600aaatataccc tcactctgtt
agcccaagac caaggactcc agcccttatc 1650agccaagaaa cagctcagca
ttcagatcag tgacatcaac gacaatgcac 1700ctgtgtttga gaaaagcagg
tatgaagtct ccacgcggga aaacaactta 1750ccctctcttc acctcattac
catcaaggct catgatgcag acttgggcat 1800taatggaaaa gtctcatacc
gcatccagga ctccccagtt gctcacttag 1850tagctattga ctccaacaca
ggagaggtca ctgctcagag gtcactgaac 1900tatgaagaga tggccggctt
tgagttccag gtgatcgcag aggacagcgg 1950gcaacccatg cttgcatcca
gtgtctctgt gtgggtcagc ctcttggatg 2000ccaatgataa tgccccagag
gtggtccagc ctgtgctcag cgatggaaaa 2050gccagcctct ccgtgcttgt
gaatgcctcc acaggccacc tgctggtgcc 2100catcgagact cccaatggct
tgggcccagc gggcactgac acacctccac 2150tggccactca cagctcccgg
ccattccttt tgacaaccat tgtggcaaga 2200gatgcagact cgggggcaaa
tggagagccc ctctacagca tccgcaatgg 2250aaatgaagcc cacctcttca
tcctcaaccc tcatacgggg cagctgttcg 2300tcaatgtcac caatgccagc
agcctcattg ggagtgagtg ggagctggag 2350atagtagtag aggaccaggg
aagccccccc ttacagaccc gagccctgtt 2400gagggtcatg tttgtcacca
gtgtggacca cctgagggac tcagcccgca 2450agcctggggc cttgagcatg
tcgatgctga cggtgatctg cctggctgta 2500ctgttgggca tcttcgggtt
gatcctggct ttgttcatgt ccatctgccg 2550gacagaaaag aaggacaaca
gggcctacaa ctgtcgggag gccgagtcca 2600cctaccgcca gcagcccaag
aggccccaga aacacattca gaaggcagac 2650atccacctcg tgcctgtgct
caggggtcag gcaggtgagc cttgtgaagt 2700cgggcagtcc cacaaagatg
tggacaagga ggcgatgatg gaagcaggct 2750gggacccctg cctgcaggcc
cccttccacc tcaccccgac cctgtacagg 2800acgctgcgta atcaaggcaa
ccagggagca ccggcggaga gccgagaggt 2850gctgcaagac acggtcaacc
tccttttcaa ccatcccagg cagaggaatg 2900cctcccggga gaacctgaac
cttcccgagc cccagcctgc
cacaggccag 2950ccacgttcca ggcctctgaa ggttgcaggc agccccacag
ggaggctggc 3000tggagaccag ggcagtgagg aagccccaca gaggccacca
gcctcctctg 3050caaccctgag acggcagcga catctcaatg gcaaagtgtc
ccctgagaaa 3100gaatcagggc cccgtcagat cctgcggagc ctggtccggc
tgtctgtggc 3150tgccttcgcc gagcggaacc ccgtggagga gctcactgtg
gattctcctc 3200ctgttcagca aatctcccag ctgctgtcct tgctgcatca
gggccaattc 3250cagcccaaac caaaccaccg aggaaataag tacttggcca
agccaggagg 3300cagcaggagt gcaatcccag acacagatgg cccaagtgca
agggctggag 3350gccagacaga cccagaacag gaggaagggc ctttggatcc
tgaagaggac 3400ctctctgtga agcaactgct agaagaagag ctgtcaagtc
tgctggaccc 3450cagcacaggt ctggccctgg accggctgag cgcccctgac
ccggcctgga 3500tggcgagact ctctttgccc ctcaccacca actaccgtga
caatgtgatc 3550tccccggatg ctgcagccac ggaggagccg aggaccttcc
agacgttcgg 3600caaggcagag gcaccagagc tgagcccaac aggcacgagg
ctggccagca 3650cctttgtctc ggagatgagc tcactgctgg agatgctgct
ggaacagcgc 3700tccagcatgc ccgtggaggc cgcctccgag gcgctgcggc
ggctctcggt 3750ctgcgggagg accctcagtt tagacttggc caccagtgca
gcctcaggca 3800tgaaagtgca aggggaccca ggtggaaaga cggggactga
gggcaagagc 3850agaggcagca gcagcagcag caggtgcctg tgaacatacc
tcagacgcct 3900ctggatccaa gaaccagggg cctgaggatc tgtggacaag
agctggtttc 3950taaaatcttg taactcacta gctagcggcg gcctgagaac
tttagggtga 4000ctgatgctac ccccacagag gaggcaagag ccccaggact
aacagctgac 4050tgaccaaagc agccccttgt aagcagctct gagtcttttg
gaggacaggg 4100acggtttgtg gctgagataa gtgtttcctg gcaaaacata
tgtggagcac 4150aaagggtcag tcctctggca gaacagatgc cacggagtat
cacaggcagg 4200aaagggtggc cttcttgggt agcaggagtc agggggctgt
accctggggg 4250tgccaggaaa tgctctctga cctatcaata aaggaaaagc
agtaaaaaaa 4300aaaaaaaaaa aaa 4313281184PRTHomo sapiens 28Met Met
Gln Leu Leu Gln Leu Leu Leu Gly Leu Leu Gly Pro Gly1 5 10 15Gly Tyr
Leu Phe Leu Leu Gly Asp Cys Gln Glu Val Thr Thr Leu 20 25 30Thr Val
Lys Tyr Gln Val Ser Glu Glu Val Pro Ser Gly Thr Val 35 40 45Ile Gly
Lys Leu Ser Gln Glu Leu Gly Arg Glu Glu Arg Arg Arg 50 55 60Gln Ala
Gly Ala Ala Phe Gln Val Leu Gln Leu Pro Gln Ala Leu 65 70 75Pro Ile
Gln Val Asp Ser Glu Glu Gly Leu Leu Ser Thr Gly Arg 80 85 90Arg Leu
Asp Arg Glu Gln Leu Cys Arg Gln Trp Asp Pro Cys Leu 95 100 105Val
Ser Phe Asp Val Leu Ala Thr Gly Asp Leu Ala Leu Ile His 110 115
120Val Glu Ile Gln Val Leu Asp Ile Asn Asp His Gln Pro Arg Phe 125
130 135Pro Lys Gly Glu Gln Glu Leu Glu Ile Ser Glu Ser Ala Ser Leu
140 145 150Arg Thr Arg Ile Pro Leu Asp Arg Ala Leu Asp Pro Asp Thr
Gly 155 160 165Pro Asn Thr Leu His Thr Tyr Thr Leu Ser Pro Ser Glu
His Phe 170 175 180Ala Leu Asp Val Ile Val Gly Pro Asp Glu Thr Lys
His Ala Glu 185 190 195Leu Ile Val Val Lys Glu Leu Asp Arg Glu Ile
His Ser Phe Phe 200 205 210Asp Leu Val Leu Thr Ala Tyr Asp Asn Gly
Asn Pro Pro Lys Ser 215 220 225Gly Thr Ser Leu Val Lys Val Asn Val
Leu Asp Ser Asn Asp Asn 230 235 240Ser Pro Ala Phe Ala Glu Ser Ser
Leu Ala Leu Glu Ile Gln Glu 245 250 255Asp Ala Ala Pro Gly Thr Leu
Leu Ile Lys Leu Thr Ala Thr Asp 260 265 270Pro Asp Gln Gly Pro Asn
Gly Glu Val Glu Phe Phe Leu Ser Lys 275 280 285His Met Pro Pro Glu
Val Leu Asp Thr Phe Ser Ile Asp Ala Lys 290 295 300Thr Gly Gln Val
Ile Leu Arg Arg Pro Leu Asp Tyr Glu Lys Asn 305 310 315Pro Ala Tyr
Glu Val Asp Val Gln Ala Arg Asp Leu Gly Pro Asn 320 325 330Pro Ile
Pro Ala His Cys Lys Val Leu Ile Lys Val Leu Asp Val 335 340 345Asn
Asp Asn Ile Pro Ser Ile His Val Thr Trp Ala Ser Gln Pro 350 355
360Ser Leu Val Ser Glu Ala Leu Pro Lys Asp Ser Phe Ile Ala Leu 365
370 375Val Met Ala Asp Asp Leu Asp Ser Gly His Asn Gly Leu Val His
380 385 390Cys Trp Leu Ser Gln Glu Leu Gly His Phe Arg Leu Lys Arg
Thr 395 400 405Asn Gly Asn Thr Tyr Met Leu Leu Thr Asn Ala Thr Leu
Asp Arg 410 415 420Glu Gln Trp Pro Lys Tyr Thr Leu Thr Leu Leu Ala
Gln Asp Gln 425 430 435Gly Leu Gln Pro Leu Ser Ala Lys Lys Gln Leu
Ser Ile Gln Ile 440 445 450Ser Asp Ile Asn Asp Asn Ala Pro Val Phe
Glu Lys Ser Arg Tyr 455 460 465Glu Val Ser Thr Arg Glu Asn Asn Leu
Pro Ser Leu His Leu Ile 470 475 480Thr Ile Lys Ala His Asp Ala Asp
Leu Gly Ile Asn Gly Lys Val 485 490 495Ser Tyr Arg Ile Gln Asp Ser
Pro Val Ala His Leu Val Ala Ile 500 505 510Asp Ser Asn Thr Gly Glu
Val Thr Ala Gln Arg Ser Leu Asn Tyr 515 520 525Glu Glu Met Ala Gly
Phe Glu Phe Gln Val Ile Ala Glu Asp Ser 530 535 540Gly Gln Pro Met
Leu Ala Ser Ser Val Ser Val Trp Val Ser Leu 545 550 555Leu Asp Ala
Asn Asp Asn Ala Pro Glu Val Val Gln Pro Val Leu 560 565 570Ser Asp
Gly Lys Ala Ser Leu Ser Val Leu Val Asn Ala Ser Thr 575 580 585Gly
His Leu Leu Val Pro Ile Glu Thr Pro Asn Gly Leu Gly Pro 590 595
600Ala Gly Thr Asp Thr Pro Pro Leu Ala Thr His Ser Ser Arg Pro 605
610 615Phe Leu Leu Thr Thr Ile Val Ala Arg Asp Ala Asp Ser Gly Ala
620 625 630Asn Gly Glu Pro Leu Tyr Ser Ile Arg Asn Gly Asn Glu Ala
His 635 640 645Leu Phe Ile Leu Asn Pro His Thr Gly Gln Leu Phe Val
Asn Val 650 655 660Thr Asn Ala Ser Ser Leu Ile Gly Ser Glu Trp Glu
Leu Glu Ile 665 670 675Val Val Glu Asp Gln Gly Ser Pro Pro Leu Gln
Thr Arg Ala Leu 680 685 690Leu Arg Val Met Phe Val Thr Ser Val Asp
His Leu Arg Asp Ser 695 700 705Ala Arg Lys Pro Gly Ala Leu Ser Met
Ser Met Leu Thr Val Ile 710 715 720Cys Leu Ala Val Leu Leu Gly Ile
Phe Gly Leu Ile Leu Ala Leu 725 730 735Phe Met Ser Ile Cys Arg Thr
Glu Lys Lys Asp Asn Arg Ala Tyr 740 745 750Asn Cys Arg Glu Ala Glu
Ser Thr Tyr Arg Gln Gln Pro Lys Arg 755 760 765Pro Gln Lys His Ile
Gln Lys Ala Asp Ile His Leu Val Pro Val 770 775 780Leu Arg Gly Gln
Ala Gly Glu Pro Cys Glu Val Gly Gln Ser His 785 790 795Lys Asp Val
Asp Lys Glu Ala Met Met Glu Ala Gly Trp Asp Pro 800 805 810Cys Leu
Gln Ala Pro Phe His Leu Thr Pro Thr Leu Tyr Arg Thr 815 820 825Leu
Arg Asn Gln Gly Asn Gln Gly Ala Pro Ala Glu Ser Arg Glu 830 835
840Val Leu Gln Asp Thr Val Asn Leu Leu Phe Asn His Pro Arg Gln 845
850 855Arg Asn Ala Ser Arg Glu Asn Leu Asn Leu Pro Glu Pro Gln Pro
860 865 870Ala Thr Gly Gln Pro Arg Ser Arg Pro Leu Lys Val Ala Gly
Ser 875 880 885Pro Thr Gly Arg Leu Ala Gly Asp Gln Gly Ser Glu Glu
Ala Pro 890 895 900Gln Arg Pro Pro Ala Ser Ser Ala Thr Leu Arg Arg
Gln Arg His 905 910 915Leu Asn Gly Lys Val Ser Pro Glu Lys Glu Ser
Gly Pro Arg Gln 920 925 930Ile Leu Arg Ser Leu Val Arg Leu Ser Val
Ala Ala Phe Ala Glu 935 940 945Arg Asn Pro Val Glu Glu Leu Thr Val
Asp Ser Pro Pro Val Gln 950 955 960Gln Ile Ser Gln Leu Leu Ser Leu
Leu His Gln Gly Gln Phe Gln 965 970 975Pro Lys Pro Asn His Arg Gly
Asn Lys Tyr Leu Ala Lys Pro Gly 980 985 990Gly Ser Arg Ser Ala Ile
Pro Asp Thr Asp Gly Pro Ser Ala Arg 995 1000 1005Ala Gly Gly Gln
Thr Asp Pro Glu Gln Glu Glu Gly Pro Leu Asp 1010 1015 1020Pro Glu
Glu Asp Leu Ser Val Lys Gln Leu Leu Glu Glu Glu Leu 1025 1030
1035Ser Ser Leu Leu Asp Pro Ser Thr Gly Leu Ala Leu Asp Arg Leu
1040 1045 1050Ser Ala Pro Asp Pro Ala Trp Met Ala Arg Leu Ser Leu
Pro Leu 1055 1060 1065Thr Thr Asn Tyr Arg Asp Asn Val Ile Ser Pro
Asp Ala Ala Ala 1070 1075 1080Thr Glu Glu Pro Arg Thr Phe Gln Thr
Phe Gly Lys Ala Glu Ala 1085 1090 1095Pro Glu Leu Ser Pro Thr Gly
Thr Arg Leu Ala Ser Thr Phe Val 1100 1105 1110Ser Glu Met Ser Ser
Leu Leu Glu Met Leu Leu Glu Gln Arg Ser 1115 1120 1125Ser Met Pro
Val Glu Ala Ala Ser Glu Ala Leu Arg Arg Leu Ser 1130 1135 1140Val
Cys Gly Arg Thr Leu Ser Leu Asp Leu Ala Thr Ser Ala Ala 1145 1150
1155Ser Gly Met Lys Val Gln Gly Asp Pro Gly Gly Lys Thr Gly Thr
1160 1165 1170Glu Gly Lys Ser Arg Gly Ser Ser Ser Ser Ser Arg Cys
Leu 1175 1180293127DNAHomo sapiens 29tctcgcagat agtaaataat
ctcggaaagg cgagaaagaa gctgtctcca 50tcttgtctgt atccgctgct cttgtgacgt
tgtggagatg gggagcgtcc 100tggggctgtg ctccatggcg agctggatac
catgtttgtg tggaagtgcc 150ccgtgtttgc tatgccgatg ctgtcctagt
ggaaacaact ccactgtaac 200tagattgatc tatgcacttt tcttgcttgt
tggagtatgt gtagcttgtg 250taatgttgat accaggaatg gaagaacaac
tgaataagat tcctggattt 300tgtgagaatg agaaaggtgt tgtcccttgt
aacattttgg ttggctataa 350agctgtatat cgtttgtgct ttggtttggc
tatgttctat cttcttctct 400ctttactaat gatcaaagtg aagagtagca
gtgatcctag agctgcagtg 450cacaatggat tttggttctt taaatttgct
gcagcaattg caattattat 500tggggcattc ttcattccag aaggaacttt
tacaactgtg tggttttatg 550taggcatggc aggtgccttt tgtttcatcc
tcatacaact agtcttactt 600attgattttg cacattcatg gaatgaatcg
tgggttgaaa aaatggaaga 650agggaactcg agatgttggt atgcagcctt
gttatcagct acagctctga 700attatctgct gtctttagtt gctatcgtcc
tgttctttgt ctactacact 750catccagcca gttgttcaga aaacaaggcg
ttcatcagtg tcaacatgct 800cctctgcgtt ggtgcttctg taatgtctat
actgccaaaa atccaagaat 850cacaaccaag atctggtttg ttacagtctt
cagtaattac agtctacaca 900atgtatttga catggtcagc tatgaccaat
gaaccagaaa caaattgcaa 950cccaagtcta ctaagcataa ttggctacaa
tacaacaagc actgtcccaa 1000aggaagggca gtcagtccag tggtggcatg
ctcaaggaat tataggacta 1050attctctttt tgttgtgtgt attttattcc
agcatccgta cttcaaacaa 1100tagtcaggtt aataaactga ctctaacaag
tgatgaatct acattaatag 1150aagatggtgg agctagaagt gatggatcac
tggaggatgg ggacgatgtt 1200caccgagctg tagataatga aagggatggt
gtcacttaca gttattcctt 1250ctttcacttc atgcttttcc tggcttcact
ttatatcatg atgaccctta 1300ccaactggtc caggtatgaa ccctctcgtg
agatgaaaag tcagtggaca 1350gctgtctggg tgaaaatctc ttccagttgg
attggcatcg tgctgtatgt 1400ttggacactc gtggcaccac ttgttcttac
aaatcgtgat tttgactgag 1450tgagacttct agcatgaaag tcccactttg
attattgctt atttgaaaac 1500agtattccca acttttgtaa agttgtgtat
gtttttgctt cccatgtaac 1550ttctccagtg ttctggcatg aattagattt
tactgcttgt cattttgtta 1600ttttcttacc aagtgcattg atatgtgaag
tagaatgaat tgcagaggaa 1650agttttatga atatggtgat gagttagtaa
aagtggccat tattgggctt 1700attctctgct ctatagttgt gaaatgaaga
gtaaaaacaa atttgtttga 1750ctattttaaa attatattag accttaagct
gttttagcaa gcattaaagc 1800aaatgtatgg ctgccttttg aaatatttga
tgtgttgcct ggcaggatac 1850tgcaaagaac atggtttatt ttaaaattta
taaacaagtc acttaaatgc 1900cagttgtctg aaaaatctta taaggtttta
cccttgatac ggaatttaca 1950caggtaggga gtgtttagtg gacaatagtg
taggttatgg atggaggtgt 2000cggtactaaa ttgaataacg agtaaataat
cttacttggg tagagatggc 2050ctttgccaac aaagtgaact gttttggttg
ttttaaactc atgaagtatg 2100ggttcagtgg aaatgtttgg aactctgaag
gatttagaca aggttttgaa 2150aaggataatc atgggttaga aggaagtgtt
ttgaaagtca ctttgaaagt 2200tagttttggg cccagcacgg tagctcaccc
ttggtaatcc cagcactttg 2250ggagcttaag tgggtagatt acttgagccc
aggaattcag accagcttgg 2300cacatggtga acctgttcta taaaaataat
ctggctttga gcatatgcct 2350gtggtccagc actgagaggc tagtgaagat
tgctgagccc agagccaaag 2400gttgcagtga gcaagtcacg tcactgcact
ctagctggca cagagtaagc 2450caaaaaaata tatatatatt gaaatcaagg
aggcaaaatt ttgacaggga 2500aggaagtaac tgcaaaacca ctaggcttta
gtaggtactt atataaaatc 2550tagtccagtt ctctcattta aaaaaatgaa
gacactgaaa tacagactta 2600aatagctcag atagctaatt aggaaatttc
aagttggcca ataatagcat 2650tctctctgac atttaaaaat aatttctatt
caaaatacat gcatattgat 2700ttacacctca tactgtgata attaatgtga
tgtggattgc tggtgtccag 2750catgacccat aaacaggtca gaagaatgat
ggaatgtttt agaataaact 2800cctgcttata gtatactaca cagttcaaaa
gatgtttaaa atgcttttgt 2850atttactgcc atgtaattga aatatataga
ttattgtaac ctttcaacct 2900gaaaatcaag cagtatgaga gtttagttat
ttgtatgtgt cactagtgtc 2950taatgaagct tttaaaatct acaatttctt
ctttaaaaat atttattaat 3000gtgaatggaa tataacaatt cagcttaatt
ccccaacctt attctgtgtg 3050tagacattgt attccacaat tttgaatggc
tgtgttttac ctctaaataa 3100atgaattcag agaaaaaaaa aaaaaaa
312730453PRTHomo sapiens 30Met Gly Ser Val Leu Gly Leu Cys Ser Met
Ala Ser Trp Ile Pro1 5 10 15Cys Leu Cys Gly Ser Ala Pro Cys Leu Leu
Cys Arg Cys Cys Pro 20 25 30Ser Gly Asn Asn Ser Thr Val Thr Arg Leu
Ile Tyr Ala Leu Phe 35 40 45Leu Leu Val Gly Val Cys Val Ala Cys Val
Met Leu Ile Pro Gly 50 55 60Met Glu Glu Gln Leu Asn Lys Ile Pro Gly
Phe Cys Glu Asn Glu 65 70 75Lys Gly Val Val Pro Cys Asn Ile Leu Val
Gly Tyr Lys Ala Val 80 85 90Tyr Arg Leu Cys Phe Gly Leu Ala Met Phe
Tyr Leu Leu Leu Ser 95 100 105Leu Leu Met Ile Lys Val Lys Ser Ser
Ser Asp Pro Arg Ala Ala 110 115 120Val His Asn Gly Phe Trp Phe Phe
Lys Phe Ala Ala Ala Ile Ala 125 130 135Ile Ile Ile Gly Ala Phe Phe
Ile Pro Glu Gly Thr Phe Thr Thr 140 145 150Val Trp Phe Tyr Val Gly
Met Ala Gly Ala Phe Cys Phe Ile Leu 155 160 165Ile Gln Leu Val Leu
Leu Ile Asp Phe Ala His Ser Trp Asn Glu 170 175 180Ser Trp Val Glu
Lys Met Glu Glu Gly Asn Ser Arg Cys Trp Tyr 185 190 195Ala Ala Leu
Leu Ser Ala Thr Ala Leu Asn Tyr Leu Leu Ser Leu 200 205 210Val Ala
Ile Val Leu Phe Phe Val Tyr Tyr Thr His Pro Ala Ser 215 220 225Cys
Ser Glu Asn Lys Ala Phe Ile Ser Val Asn Met Leu Leu Cys 230 235
240Val Gly Ala Ser Val Met Ser Ile Leu Pro Lys Ile Gln Glu Ser 245
250 255Gln Pro Arg Ser Gly Leu Leu Gln Ser Ser Val Ile Thr Val Tyr
260 265 270Thr Met Tyr Leu Thr Trp Ser Ala Met Thr Asn Glu Pro Glu
Thr 275
280 285Asn Cys Asn Pro Ser Leu Leu Ser Ile Ile Gly Tyr Asn Thr Thr
290 295 300Ser Thr Val Pro Lys Glu Gly Gln Ser Val Gln Trp Trp His
Ala 305 310 315Gln Gly Ile Ile Gly Leu Ile Leu Phe Leu Leu Cys Val
Phe Tyr 320 325 330Ser Ser Ile Arg Thr Ser Asn Asn Ser Gln Val Asn
Lys Leu Thr 335 340 345Leu Thr Ser Asp Glu Ser Thr Leu Ile Glu Asp
Gly Gly Ala Arg 350 355 360Ser Asp Gly Ser Leu Glu Asp Gly Asp Asp
Val His Arg Ala Val 365 370 375Asp Asn Glu Arg Asp Gly Val Thr Tyr
Ser Tyr Ser Phe Phe His 380 385 390Phe Met Leu Phe Leu Ala Ser Leu
Tyr Ile Met Met Thr Leu Thr 395 400 405Asn Trp Ser Arg Tyr Glu Pro
Ser Arg Glu Met Lys Ser Gln Trp 410 415 420Thr Ala Val Trp Val Lys
Ile Ser Ser Ser Trp Ile Gly Ile Val 425 430 435Leu Tyr Val Trp Thr
Leu Val Ala Pro Leu Val Leu Thr Asn Arg 440 445 450Asp Phe
Asp31485DNAHomo sapiens 31gctcaagacc cagcagtggg acagccagac
agacggcacg atggcactga 50gctcccagat ctgggccgct tgcctcctgc tcctcctcct
cctcgccagc 100ctgaccagtg gctctgtttt cccacaacag acgggacaac
ttgcagagct 150gcaaccccag gacagagctg gagccagggc cagctggatg
cccatgttcc 200agaggcgaag gaggcgagac acccacttcc ccatctgcat
tttctgctgc 250ggctgctgtc atcgatcaaa gtgtgggatg tgctgcaaga
cgtagaacct 300acctgccctg cccccgtccc ctcccttcct tatttattcc
tgctgcccca 350gaacataggt cttggaataa aatggctggt tcttttgttt
tccaaaaaaa 400aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 450aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaa 4853284PRTHomo
sapiens 32Met Ala Leu Ser Ser Gln Ile Trp Ala Ala Cys Leu Leu Leu
Leu1 5 10 15Leu Leu Leu Ala Ser Leu Thr Ser Gly Ser Val Phe Pro Gln
Gln 20 25 30Thr Gly Gln Leu Ala Glu Leu Gln Pro Gln Asp Arg Ala Gly
Ala 35 40 45Arg Ala Ser Trp Met Pro Met Phe Gln Arg Arg Arg Arg Arg
Asp 50 55 60Thr His Phe Pro Ile Cys Ile Phe Cys Cys Gly Cys Cys His
Arg 65 70 75Ser Lys Cys Gly Met Cys Cys Lys Thr 80331245DNAHomo
sapiens 33ggcctcggtt caaacgaccc ggtgggtcta cagcggaagg gagggagcga
50aggtaggagg cagggcttgc ctcactggcc accctcccaa ccccaagagc
100ccagccccat ggtccccgcc gccggcgcgc tgctgtgggt cctgctgctg
150aatctgggtc cccgggcggc gggggcccaa ggcctgaccc agactccgac
200cgaaatgcag cgggtcagtt tacgctttgg gggccccatg acccgcagct
250accggagcac cgcccggact ggtcttcccc ggaagacaag gataatccta
300gaggacgaga atgatgccat ggccgacgcc gaccgcctgg ctggaccagc
350ggctgccgag ctcttggccg ccacggtgtc caccggcttt agccggtcgt
400ccgccattaa cgaggaggat gggtcttcag aagagggggt tgtgattaat
450gccggaaagg atagcaccag cagagagctt cccagtgcga ctcccaatac
500agcggggagt tccagcacga ggtttatagc caatagtcag gagcctgaaa
550tcaggctgac ttcaagcctg ccgcgctccc ccgggaggtc tactgaggac
600ctgccaggct cgcaggccac cctgagccag tggtccacac ctgggtctac
650cccgagccgg tggccgtcac cctcacccac agccatgcca tctcctgagg
700atctgcggct ggtgctgatg ccctggggcc cgtggcactg ccactgcaag
750tcgggcacca tgagccggag ccggtctggg aagctgcacg gcctttccgg
800gcgccttcga gttggggcgc tgagccagct ccgcacggag cacaagcctt
850gcacctatca acaatgtccc tgcaaccgac ttcgggaaga gtgccccctg
900gacacaagtc tctgtactga caccaactgt gcctctcaga gcaccaccag
950taccaggacc accactaccc ccttccccac catccacctc agaagcagtc
1000ccagcctgcc acccgccagc ccctgcccag ccctggcttt ttggaaacgg
1050gtcaggattg gcctggagga tatttggaat agcctctctt cagtgttcac
1100agagatgcaa ccaatagaca gaaaccagag gtaatggcca cttcatccac
1150atgaggagat gtcagtatct caacctctct tgccctttca atcctagcac
1200ccactagata tttttagtac agaaaaacaa aactggaaaa cacaa
124534341PRTHomo sapiens 34Met Val Pro Ala Ala Gly Ala Leu Leu Trp
Val Leu Leu Leu Asn1 5 10 15Leu Gly Pro Arg Ala Ala Gly Ala Gln Gly
Leu Thr Gln Thr Pro 20 25 30Thr Glu Met Gln Arg Val Ser Leu Arg Phe
Gly Gly Pro Met Thr 35 40 45Arg Ser Tyr Arg Ser Thr Ala Arg Thr Gly
Leu Pro Arg Lys Thr 50 55 60Arg Ile Ile Leu Glu Asp Glu Asn Asp Ala
Met Ala Asp Ala Asp 65 70 75Arg Leu Ala Gly Pro Ala Ala Ala Glu Leu
Leu Ala Ala Thr Val 80 85 90Ser Thr Gly Phe Ser Arg Ser Ser Ala Ile
Asn Glu Glu Asp Gly 95 100 105Ser Ser Glu Glu Gly Val Val Ile Asn
Ala Gly Lys Asp Ser Thr 110 115 120Ser Arg Glu Leu Pro Ser Ala Thr
Pro Asn Thr Ala Gly Ser Ser 125 130 135Ser Thr Arg Phe Ile Ala Asn
Ser Gln Glu Pro Glu Ile Arg Leu 140 145 150Thr Ser Ser Leu Pro Arg
Ser Pro Gly Arg Ser Thr Glu Asp Leu 155 160 165Pro Gly Ser Gln Ala
Thr Leu Ser Gln Trp Ser Thr Pro Gly Ser 170 175 180Thr Pro Ser Arg
Trp Pro Ser Pro Ser Pro Thr Ala Met Pro Ser 185 190 195Pro Glu Asp
Leu Arg Leu Val Leu Met Pro Trp Gly Pro Trp His 200 205 210Cys His
Cys Lys Ser Gly Thr Met Ser Arg Ser Arg Ser Gly Lys 215 220 225Leu
His Gly Leu Ser Gly Arg Leu Arg Val Gly Ala Leu Ser Gln 230 235
240Leu Arg Thr Glu His Lys Pro Cys Thr Tyr Gln Gln Cys Pro Cys 245
250 255Asn Arg Leu Arg Glu Glu Cys Pro Leu Asp Thr Ser Leu Cys Thr
260 265 270Asp Thr Asn Cys Ala Ser Gln Ser Thr Thr Ser Thr Arg Thr
Thr 275 280 285Thr Thr Pro Phe Pro Thr Ile His Leu Arg Ser Ser Pro
Ser Leu 290 295 300Pro Pro Ala Ser Pro Cys Pro Ala Leu Ala Phe Trp
Lys Arg Val 305 310 315Arg Ile Gly Leu Glu Asp Ile Trp Asn Ser Leu
Ser Ser Val Phe 320 325 330Thr Glu Met Gln Pro Ile Asp Arg Asn Gln
Arg 335 340353089DNAHomo sapiens 35caggaaccct ctctttgggt ctggattggg
acccctttcc agtaccattt 50tttctagtga accacgaagg gacgatacca gaaaacaccc
tcaacccaaa 100ggaaatagac tacagcccca attggctgac tttggctata
gaaaaaagaa 150aggaacgaaa agagacagtt ttttttggaa agctaagtct
tccctttatc 200gagtcaagaa accccccctt cttgagctat ttacagcttt
taacaattga 250gtaaagtacg ctccggtcac catggtgaca gccgccctgg
gtcccgtctg 300ggcagcgctc ctgctctttc tcctgatgtg tgagatccgt
atggtggagc 350tcacctttga cagagctgtg gccagcggct gccaacggtg
ctgtgactct 400gaggaccccc tggatcctgc ccatgtatcc tcagcctctt
cctccggccg 450cccccacgcc ctgcctgaga tcagacccta cattaatatc
accatcctga 500agggtgacaa aggggaccca ggcccaatgg gcctgccagg
gtacatgggc 550agggagggtc cccaagggga gcctggccct cagggcagca
agggtgacaa 600gggggagatg ggcagccccg gcgccccgtg ccagaagcgc
ttcttcgcct 650tctcagtggg ccgcaagacg gccctgcaca gcggcgagga
cttccagacg 700ctgctcttcg aaagggtctt tgtgaacctt gatgggtgct
ttgacatggc 750gaccggccag tttgctgctc ccctgcgtgg catctacttc
ttcagcctca 800atgtgcacag ctggaattac aaggagacgt acgtgcacat
tatgcataac 850cagaaagagg ctgtcatcct gtacgcgcag cccagcgagc
gcagcatcat 900gcagagccag agtgtgatgc tggacctggc ctacggggac
cgcgtctggg 950tgcggctctt caagcgccag cgcgagaacg ccatctacag
caacgacttc 1000gacacctaca tcaccttcag cggccacctc atcaaggccg
aggacgactg 1050agggcctctg ggccaccctc ccggctggag agctcaggtg
ctggtcccgt 1100cccctgcagg gctcagtttg cactgctgtg aagcaggaag
gccagggagg 1150tccccgggga cctggcattc tggggagacc ctgcttctat
cttggctgcc 1200atcatccctc ccagcctatt tctgctcctc tcttctctct
tggacctatt 1250ttaagaagct tgctaaccta aatattctag aactttccca
gcctcgtagc 1300ccagcacttc tcaaacttgg aaatgcatgc gaatcacccg
gggttcgtgt 1350taaatgcaga ttctgactca gcaggtctga gtgggtccag
gattctgtgt 1400ttctcatatg ttcctgggtg atgctgatgg ggtcagtcta
tgaaccacac 1450tggagcaacc aggttctagg actttctcaa tattctagta
ctttctgaac 1500attctggaat cctccccaca ttctagaatt ctcccaacat
ttttttttct 1550tgagacagag tcttgctctg ttgcccaggc tagagtgcag
tggtgcaatc 1600tcagttcact gcaacctctg cctcccgggt tcaagcgatt
cttctgcctc 1650agcctcccta gtggctggga ttacaggcgc ctgctaccat
gcctggctaa 1700tttttgtatt tttagtagag atggggtttc accatattgg
ccaggctggt 1750cttgaactcc tgacttcagg tgacccaccc gcctcggcct
ctcaaaatgc 1800tgggattaca ggtgtgagcc accgtgcctg gccaattcca
acattcttaa 1850attctctcat ccctccaggg ctccccgtgc tatgttctct
ttaccccttc 1900cccctcttct cttgctcagg cctgcaccac tgcagccacc
gttcatttat 1950tcattcatta aacactgagc actcactctg tgctgggtcc
cgggaagggt 2000gagggggtca gacacaggcc ctgcccctgc cctcagtgac
tggccagtcc 2050agcccaggcg gggagagatg tgtacatagg ttttaaagca
gacccagagc 2100tcatgggggc ctgtgttctg ggtgttcagg tgctgctggt
cctccattac 2150ccactgctcc ccaaggctgg tgggacgggg tcccggtggc
aggggcaggt 2200atctccttcc cgttcctcat ccacctgccc agtgctcatc
gttacagcaa 2250accccagggg gccttggcca ggtcaagggt tctgtgagga
gaggacccag 2300gagtgtgggg gcatttgggg ggtgaagtgg cccccgaaga
atggaaccca 2350cacccatagc tctccccaca gctgatacgg catcctgcga
gaagacctgc 2400cctcctcact gggatcccct tcctgcctcc tcccagggct
ctgccagggc 2450cttgctcagt cccttccacc aaagtcatct gaacttccgt
ttccccaggg 2500cctccagctg ccctcagaca ctgatgtctg tccccaggtg
ctctctgccc 2550ctcatgcccc tctcaccggc ccagtgcccc gactctccag
gctttatcaa 2600ggtgctaagg cccgggtggg cagctcctcg tctcagagcc
ctcctccggc 2650ctggtgctgc ctttacaaac acctgcagga gaagggccac
ggaagcccca 2700ggctttagag ccctcagcag gtctggggag ctagagcaaa
ggagggacct 2750caggccttcc gtttcttctt ccagggtggg gtggcctggt
gttcccctag 2800ccttccaaac ccaggtggcc tgcccttctc cccagaggga
ggcggcctcc 2850gcccattggt gctcatgcag actctggggc tgaggtgccc
cggggggtga 2900tctctggtgc tcacagccga gggagccgtg gctccatggc
cagatgacgg 2950aaacagggtc tgaccaagtg ccaggaagac ctgtgctata
aaccaccctg 3000cctgatcctg cccctgcctg accccgccac gccctgccgt
ccagcatgat 3050taaagaatgc tgtctcctct tggaaaaaaa aaaaaaaaa
308936259PRTHomo sapiens 36Met Val Thr Ala Ala Leu Gly Pro Val Trp
Ala Ala Leu Leu Leu1 5 10 15Phe Leu Leu Met Cys Glu Ile Arg Met Val
Glu Leu Thr Phe Asp 20 25 30Arg Ala Val Ala Ser Gly Cys Gln Arg Cys
Cys Asp Ser Glu Asp 35 40 45Pro Leu Asp Pro Ala His Val Ser Ser Ala
Ser Ser Ser Gly Arg 50 55 60Pro His Ala Leu Pro Glu Ile Arg Pro Tyr
Ile Asn Ile Thr Ile 65 70 75Leu Lys Gly Asp Lys Gly Asp Pro Gly Pro
Met Gly Leu Pro Gly 80 85 90Tyr Met Gly Arg Glu Gly Pro Gln Gly Glu
Pro Gly Pro Gln Gly 95 100 105Ser Lys Gly Asp Lys Gly Glu Met Gly
Ser Pro Gly Ala Pro Cys 110 115 120Gln Lys Arg Phe Phe Ala Phe Ser
Val Gly Arg Lys Thr Ala Leu 125 130 135His Ser Gly Glu Asp Phe Gln
Thr Leu Leu Phe Glu Arg Val Phe 140 145 150Val Asn Leu Asp Gly Cys
Phe Asp Met Ala Thr Gly Gln Phe Ala 155 160 165Ala Pro Leu Arg Gly
Ile Tyr Phe Phe Ser Leu Asn Val His Ser 170 175 180Trp Asn Tyr Lys
Glu Thr Tyr Val His Ile Met His Asn Gln Lys 185 190 195Glu Ala Val
Ile Leu Tyr Ala Gln Pro Ser Glu Arg Ser Ile Met 200 205 210Gln Ser
Gln Ser Val Met Leu Asp Leu Ala Tyr Gly Asp Arg Val 215 220 225Trp
Val Arg Leu Phe Lys Arg Gln Arg Glu Asn Ala Ile Tyr Ser 230 235
240Asn Asp Phe Asp Thr Tyr Ile Thr Phe Ser Gly His Leu Ile Lys 245
250 255Ala Glu Asp Asp372213DNAHomo sapiens 37gagcgaacat ggcagcgcgt
tggcggtttt ggtgtgtctc tgtgaccatg 50gtggtggcgc tgctcatcgt ttgcgacgtt
ccctcagcct ctgcccaaag 100aaagaaggag atggtgttat ctgaaaaggt
tagtcagctg atggaatgga 150ctaacaaaag acctgtaata agaatgaatg
gagacaagtt ccgtcgcctt 200gtgaaagccc caccgagaaa ttactccgtt
atcgtcatgt tcactgctct 250ccaactgcat agacagtgtg tcgtttgcaa
gcaagctgat gaagaattcc 300agatcctggc aaactcctgg cgatactcca
gtgcattcac caacaggata 350ttttttgcca tggtggattt tgatgaaggc
tctgatgtat ttcagatgct 400aaacatgaat tcagctccaa ctttcatcaa
ctttcctgca aaagggaaac 450ccaaacgggg tgatacatat gagttacagg
tgcggggttt ttcagctgag 500cagattgccc ggtggatcgc cgacagaact
gatgtcaata ttagagtgat 550tagaccccca aattatgctg gtccccttat
gttgggattg cttttggctg 600ttattggtgg acttgtgtat cttcgaagaa
gtaatatgga atttctcttt 650aataaaactg gatgggcttt tgcagctttg
tgttttgtgc ttgctatgac 700atctggtcaa atgtggaacc atataagagg
accaccatat gcccataaga 750atccccacac gggacatgtg aattatatcc
atggaagcag tcaagcccag 800tttgtagctg aaacacacat tgttcttctg
tttaatggtg gagttacctt 850aggaatggtg cttttatgtg aagctgctac
ctctgacatg gatattggaa 900agcgaaagat aatgtgtgtg gctggtattg
gacttgttgt attattcttc 950agttggatgc tctctatttt tagatctaaa
tatcatggct acccatacag 1000ctttctgatg agttaaaaag gtcccagaga
tatatagaca ctggagtact 1050ggaaattgaa aaacgaaaat cgtgtgtgtt
tgaaaagaag aatgcaactt 1100gtatattttg tattacctct ttttttcaag
tgatttaaat agttaatcat 1150ttaaccaaag aagatgtgta gtgccttaac
aagcaatcct ctgtcaaaat 1200ctgaggtatt tgaaaataat tatcctctta
accttctctt cccagtgaac 1250tttatggaac atttaattta gtacaattaa
gtatattata aaaattgtaa 1300aactactact ttgttttagt tagaacaaag
ctcaaaacta ctttagttaa 1350cttggtcatc tgattttata ttgccttatc
caaagatggg gaaagtaagt 1400cctgaccagg tgttcccaca tatgcctgtt
acagataact acattaggaa 1450ttcattctta gcttcttcat ctttgtgtgg
atgtgtatac tttacgcatc 1500tttccttttg agtagagaaa ttatgtgtgt
catgtggtct tctgaaaatg 1550gaacaccatt cttcagagca cacgtctagc
cctcagcaag acagttgttt 1600ctcctcctcc ttgcatattt cctactgcgc
tccagcctga gtgatagagt 1650gagactctgt ctcaaaaaaa agtatctcta
aatacaggat tataatttct 1700gcttgagtat ggtgttaact accttgtatt
tagaaagatt tcagattcat 1750tccatctcct tagttttctt ttaaggtgac
ccatctgtga taaaaatata 1800gcttagtgct aaaatcagtg taacttatac
atggcctaaa atgtttctac 1850aaattagagt ttgtcactta ttccatttgt
acctaagaga aaaataggct 1900cagttagaaa aggactccct ggccaggcgc
agtgacttac gcctgtaatc 1950tcagcacttt gggaggccaa ggcaggcaga
tcacgaggtc aggagttcga 2000gaccatcctg gccaacatgg tgaaaccccg
tctctactaa aaatataaaa 2050attagctggg tgtggtggca ggagcctgta
atcccagcta cacaggaggc 2100tgaggcacga gaatcacttg aactcaggag
atggaggttt cagtgagccg 2150agatcacgcc actgcactcc agcctggcaa
cagagcgaga ctccatctca 2200aaaaaaaaaa aaa 221338335PRTHomo sapiens
38Met Ala Ala Arg Trp Arg Phe Trp Cys Val Ser Val Thr Met Val1 5 10
15Val Ala Leu Leu Ile Val Cys Asp Val Pro Ser Ala Ser Ala Gln 20 25
30Arg Lys Lys Glu Met Val Leu Ser Glu Lys Val Ser Gln Leu Met 35 40
45Glu Trp Thr Asn Lys Arg Pro Val Ile Arg Met Asn Gly Asp Lys 50 55
60Phe Arg Arg Leu Val Lys Ala Pro Pro Arg Asn Tyr Ser Val Ile 65 70
75Val Met Phe Thr Ala Leu Gln Leu His Arg Gln Cys Val Val Cys 80 85
90Lys Gln Ala Asp Glu Glu Phe Gln Ile Leu Ala Asn Ser Trp Arg 95
100 105Tyr Ser Ser Ala Phe Thr Asn Arg Ile Phe Phe Ala Met Val Asp
110 115 120Phe Asp Glu Gly Ser Asp Val Phe Gln Met Leu Asn Met Asn
Ser 125 130 135Ala Pro Thr Phe Ile Asn Phe
Pro Ala Lys Gly Lys Pro Lys Arg 140 145 150Gly Asp Thr Tyr Glu Leu
Gln Val Arg Gly Phe Ser Ala Glu Gln 155 160 165Ile Ala Arg Trp Ile
Ala Asp Arg Thr Asp Val Asn Ile Arg Val 170 175 180Ile Arg Pro Pro
Asn Tyr Ala Gly Pro Leu Met Leu Gly Leu Leu 185 190 195Leu Ala Val
Ile Gly Gly Leu Val Tyr Leu Arg Arg Ser Asn Met 200 205 210Glu Phe
Leu Phe Asn Lys Thr Gly Trp Ala Phe Ala Ala Leu Cys 215 220 225Phe
Val Leu Ala Met Thr Ser Gly Gln Met Trp Asn His Ile Arg 230 235
240Gly Pro Pro Tyr Ala His Lys Asn Pro His Thr Gly His Val Asn 245
250 255Tyr Ile His Gly Ser Ser Gln Ala Gln Phe Val Ala Glu Thr His
260 265 270Ile Val Leu Leu Phe Asn Gly Gly Val Thr Leu Gly Met Val
Leu 275 280 285Leu Cys Glu Ala Ala Thr Ser Asp Met Asp Ile Gly Lys
Arg Lys 290 295 300Ile Met Cys Val Ala Gly Ile Gly Leu Val Val Leu
Phe Phe Ser 305 310 315Trp Met Leu Ser Ile Phe Arg Ser Lys Tyr His
Gly Tyr Pro Tyr 320 325 330Ser Phe Leu Met Ser 335391648DNAHomo
sapiens 39caggccattt gcatcccact gtccttgtgt tcggagccag gccacaccgt
50cctcagcagt gtcatgtgtt aaaaacgcca agctgaatat atcatgcccc
100tattaaaact tgtacatggc tccccattgg tttttggaga aaagttcaag
150ctttttacct tggtgtctgc ctgtatccca gtgttcaggc tggctagacg
200gcggaagaag atcctatttt actgtcactt cccagatctg cttctcacca
250agagagattc ttttcttaaa cgactataca gggccccaat tgactggata
300gaggaataca ccacaggcat ggcagactgc atcttagtca acagccagtt
350cacagctgct gtttttaagg aaacattcaa gtccctgtct cacatagacc
400ctgatgtcct ctatccatct ctaaatgtca ccagctttga ctcagttgtt
450cctgaaaagc tggatgacct agtccccaag gggaaaaaat tcctgctgct
500ctccatcaac agatacgaaa ggaagaaaaa tctgactttg gcactggaag
550ccctagtaca gctgcgtgga agattgacat cccaagattg ggagagggtt
600catctgatcg tggcaggtgg ttatgacgag agagtcctgg agaatgtgga
650acattatcag gaattgaaga aaatggtcca acagtccgac cttggccagt
700atgtgacctt cttgaggtct ttctcagaca aacagaaaat ctccctcctc
750cacagctgca cgtgtgtgct ttacacacca agcaatgagc actttggcat
800tgtccctctg gaagccatgt acatgcagtg cccagtcatt gctgttaatt
850cgggtggacc cttggagtcc attgaccaca gtgtcacagg gtttctgtgt
900gagcctgacc cggtgcactt ctcagaagca atagaaaagt tcatccgtga
950accttcctta aaagccacca tgggcctggc tggaagagcc agagtgaagg
1000aaaaattttc ccctgaagca tttacagaac agctctaccg atatgttacc
1050aaactgctgg tataatcaga ttgtttttaa gatctccatt aatgtcattt
1100ttatggattg tagacccagt tttgaaacca aaaaagaaac ctagaatcta
1150atgcagaaga gatcttttaa aaaataaact tgagtcttga atgtgagcca
1200ctttcctata taccacacct ccctgtccac ttttcagaaa aaccatgtct
1250tttatgctat aatcattcca aattttgcca gtgttaagtt acaaatgtgg
1300tgtcattcca tgttcagcag agtattttaa ttatattttc tcgggattat
1350tgctcttctg tctataaatt ttgaatgata ctgtgcctta attggttttc
1400atagtttaag tgtgtatcat tatcaaagtt gattaatttg gcttcatagt
1450ataatgagag cagggctatt gtagttccca gattcaatcc accgaagtgt
1500tcactgtcat ctgttaggga atttttgttt gtcctgtctt tgcctggatc
1550catagcgaga gtgctctgta ttttttttaa gataatttgt atttttgcac
1600actgagatat aataaaaggt gtttatcata aaaaaaaaaa aaaaaaaa
164840323PRTHomo sapiens 40Met Pro Leu Leu Lys Leu Val His Gly Ser
Pro Leu Val Phe Gly1 5 10 15Glu Lys Phe Lys Leu Phe Thr Leu Val Ser
Ala Cys Ile Pro Val 20 25 30Phe Arg Leu Ala Arg Arg Arg Lys Lys Ile
Leu Phe Tyr Cys His 35 40 45Phe Pro Asp Leu Leu Leu Thr Lys Arg Asp
Ser Phe Leu Lys Arg 50 55 60Leu Tyr Arg Ala Pro Ile Asp Trp Ile Glu
Glu Tyr Thr Thr Gly 65 70 75Met Ala Asp Cys Ile Leu Val Asn Ser Gln
Phe Thr Ala Ala Val 80 85 90Phe Lys Glu Thr Phe Lys Ser Leu Ser His
Ile Asp Pro Asp Val 95 100 105Leu Tyr Pro Ser Leu Asn Val Thr Ser
Phe Asp Ser Val Val Pro 110 115 120Glu Lys Leu Asp Asp Leu Val Pro
Lys Gly Lys Lys Phe Leu Leu 125 130 135Leu Ser Ile Asn Arg Tyr Glu
Arg Lys Lys Asn Leu Thr Leu Ala 140 145 150Leu Glu Ala Leu Val Gln
Leu Arg Gly Arg Leu Thr Ser Gln Asp 155 160 165Trp Glu Arg Val His
Leu Ile Val Ala Gly Gly Tyr Asp Glu Arg 170 175 180Val Leu Glu Asn
Val Glu His Tyr Gln Glu Leu Lys Lys Met Val 185 190 195Gln Gln Ser
Asp Leu Gly Gln Tyr Val Thr Phe Leu Arg Ser Phe 200 205 210Ser Asp
Lys Gln Lys Ile Ser Leu Leu His Ser Cys Thr Cys Val 215 220 225Leu
Tyr Thr Pro Ser Asn Glu His Phe Gly Ile Val Pro Leu Glu 230 235
240Ala Met Tyr Met Gln Cys Pro Val Ile Ala Val Asn Ser Gly Gly 245
250 255Pro Leu Glu Ser Ile Asp His Ser Val Thr Gly Phe Leu Cys Glu
260 265 270Pro Asp Pro Val His Phe Ser Glu Ala Ile Glu Lys Phe Ile
Arg 275 280 285Glu Pro Ser Leu Lys Ala Thr Met Gly Leu Ala Gly Arg
Ala Arg 290 295 300Val Lys Glu Lys Phe Ser Pro Glu Ala Phe Thr Glu
Gln Leu Tyr 305 310 315Arg Tyr Val Thr Lys Leu Leu Val
320411181DNAHomo sapiens 41aagaccctct ctttcgctgt ttgagagtct
ctcggctcaa ggaccgggag 50gtaagaggtt tgggactgcc ccggcaactc cagggtgtct
ggtccacgac 100ctatcctagg cgccatgggt gtgataggta tacagctggt
tgttaccatg 150gtgatggcca gtgtcatgca gaagattata cctcactatt
ctcttgctcg 200atggctactc tgtaatggca gtttgaggtg gtatcaacat
cctacagaag 250aagaattaag aattcttgca gggaaacaac aaaaagggaa
aaccaaaaaa 300gataggaaat ataatggtca cattgaaagt aagccattaa
ccattccaaa 350ggatattgac cttcatctag aaacaaagtc agttacagaa
gtggatactt 400tagcattgca ttactttcca gaataccagt ggctggtgga
tttcacagtg 450gctgctacag ttgtgtatct agtaactgaa gtctactaca
attttatgaa 500gcctacacag gaaatgaata tcagcttagt ctggtgccta
cttgttttgt 550cttttgcaat caaagttcta ttttcattaa ctacacacta
ttttaaagta 600gaagatggtg gtgaaagatc tgtttgtgtc acctttggat
tttttttctt 650tgtcaaagca atggcagtgt tgattgtaac agaaaattat
ctggaatttg 700gacttgaaac agggtttaca aatttttcag acagtgcgat
gcagtttctt 750gaaaagcaag gtttagaatc tcagagtcct gtttcaaaac
ttactttcaa 800atttttcctg gctattttct gttcattcat tggggctttt
ttgacatttc 850ctggattacg actggctcaa atgcatctgg atgccctgaa
tttggcaaca 900gaaaaaatta cacaaacttt acttcatatc aacttcttgg
cacctttatt 950tatggttttg ctctgggtaa aaccaatcac caaagactac
attatgaacc 1000caccactggg caaagaaatt tccccatctg gaagatgaag
ataatagtat 1050ctaactcaca aggttatcat tggaataaat gaaagaacac
atgtaatgca 1100accagctgga attaagtgct taataaatgt tcttttcact
gctttgcctc 1150atcagaatta aaatagaaat acttgactag t 118142307PRTHomo
sapiens 42Met Gly Val Ile Gly Ile Gln Leu Val Val Thr Met Val Met
Ala1 5 10 15Ser Val Met Gln Lys Ile Ile Pro His Tyr Ser Leu Ala Arg
Trp 20 25 30Leu Leu Cys Asn Gly Ser Leu Arg Trp Tyr Gln His Pro Thr
Glu 35 40 45Glu Glu Leu Arg Ile Leu Ala Gly Lys Gln Gln Lys Gly Lys
Thr 50 55 60Lys Lys Asp Arg Lys Tyr Asn Gly His Ile Glu Ser Lys Pro
Leu 65 70 75Thr Ile Pro Lys Asp Ile Asp Leu His Leu Glu Thr Lys Ser
Val 80 85 90Thr Glu Val Asp Thr Leu Ala Leu His Tyr Phe Pro Glu Tyr
Gln 95 100 105Trp Leu Val Asp Phe Thr Val Ala Ala Thr Val Val Tyr
Leu Val 110 115 120Thr Glu Val Tyr Tyr Asn Phe Met Lys Pro Thr Gln
Glu Met Asn 125 130 135Ile Ser Leu Val Trp Cys Leu Leu Val Leu Ser
Phe Ala Ile Lys 140 145 150Val Leu Phe Ser Leu Thr Thr His Tyr Phe
Lys Val Glu Asp Gly 155 160 165Gly Glu Arg Ser Val Cys Val Thr Phe
Gly Phe Phe Phe Phe Val 170 175 180Lys Ala Met Ala Val Leu Ile Val
Thr Glu Asn Tyr Leu Glu Phe 185 190 195Gly Leu Glu Thr Gly Phe Thr
Asn Phe Ser Asp Ser Ala Met Gln 200 205 210Phe Leu Glu Lys Gln Gly
Leu Glu Ser Gln Ser Pro Val Ser Lys 215 220 225Leu Thr Phe Lys Phe
Phe Leu Ala Ile Phe Cys Ser Phe Ile Gly 230 235 240Ala Phe Leu Thr
Phe Pro Gly Leu Arg Leu Ala Gln Met His Leu 245 250 255Asp Ala Leu
Asn Leu Ala Thr Glu Lys Ile Thr Gln Thr Leu Leu 260 265 270His Ile
Asn Phe Leu Ala Pro Leu Phe Met Val Leu Leu Trp Val 275 280 285Lys
Pro Ile Thr Lys Asp Tyr Ile Met Asn Pro Pro Leu Gly Lys 290 295
300Glu Ile Ser Pro Ser Gly Arg 305432063DNAHomo sapiens
43gagagaggca gcagcttgct cagcggacaa ggatgctggg cgtgagggac
50caaggcctgc cctgcactcg ggcctcctcc agccagtgct gaccagggac
100ttctgacctg ctggccagcc aggacctgtg tggggaggcc ctcctgctgc
150cttggggtga caatctcagc tccaggctac agggagaccg ggaggatcac
200agagccagca tgttacagga tcctgacagt gatcaacctc tgaacagcct
250cgatgtcaaa cccctgcgca aaccccgtat ccccatggag accttcagaa
300aggtggggat ccccatcatc atagcactac tgagcctggc gagtatcatc
350attgtggttg tcctcatcaa ggtgattctg gataaatact acttcctctg
400cgggcagcct ctccacttca tcccgaggaa gcagctgtgt gacggagagc
450tggactgtcc cttgggggag gacgaggagc actgtgtcaa gagcttcccc
500gaagggcctg cagtggcagt ccgcctctcc aaggaccgat ccacactgca
550ggtgctggac tcggccacag ggaactggtt ctctgcctgt ttcgacaact
600tcacagaagc tctcgctgag acagcctgta ggcagatggg ctacagcaga
650gctgtggaga ttggcccaga ccaggatctg gatgttgttg aaatcacaga
700aaacagccag gagcttcgca tgcggaactc aagtgggccc tgtctctcag
750gctccctggt ctccctgcac tgtcttgcct gtgggaagag cctgaagacc
800ccccgtgtgg tgggtgggga ggaggcctct gtggattctt ggccttggca
850ggtcagcatc cagtacgaca aacagcacgt ctgtggaggg agcatcctgg
900acccccactg ggtcctcacg gcagcccact gcttcaggaa acataccgat
950gtgttcaact ggaaggtgcg ggcaggctca gacaaactgg gcagcttccc
1000atccctggct gtggccaaga tcatcatcat tgaattcaac cccatgtacc
1050ccaaagacaa tgacatcgcc ctcatgaagc tgcagttccc actcactttc
1100tcaggcacag tcaggcccat ctgtctgccc ttctttgatg aggagctcac
1150tccagccacc ccactctgga tcattggatg gggctttacg aagcagaatg
1200gagggaagat gtctgacata ctgctgcagg cgtcagtcca ggtcattgac
1250agcacacggt gcaatgcaga cgatgcgtac cagggggaag tcaccgagaa
1300gatgatgtgt gcaggcatcc cggaaggggg tgtggacacc tgccagggtg
1350acagtggtgg gcccctgatg taccaatctg accagtggca tgtggtgggc
1400atcgttagct ggggctatgg ctgcgggggc ccgagcaccc caggagtata
1450caccaaggtc tcagcctatc tcaactggat ctacaatgtc tggaaggctg
1500agctgtaatg ctgctgcccc tttgcagtgc tgggagccgc ttccttcctg
1550ccctgcccac ctggggatcc cccaaagtca gacacagagc aagagtcccc
1600ttgggtacac ccctctgccc acagcctcag catttcttgg agcagcaaag
1650ggcctcaatt cctgtaagag accctcgcag cccagaggcg cccagaggaa
1700gtcagcagcc ctagctcggc cacacttggt gctcccagca tcccagggag
1750agacacagcc cactgaacaa ggtctcaggg gtattgctaa gccaagaagg
1800aactttccca cactactgaa tggaagcagg ctgtcttgta aaagcccaga
1850tcactgtggg ctggagagga gaaggaaagg gtctgcgcca gccctgtccg
1900tcttcaccca tccccaagcc tactagagca agaaaccagt tgtaatataa
1950aatgcactgc cctactgttg gtatgactac cgttacctac tgttgtcatt
2000gttattacag ctatggccac tattattaaa gagctgtgta acatctctgg
2050caaaaaaaaa aaa 206344432PRTHomo sapiens 44Met Leu Gln Asp Pro
Asp Ser Asp Gln Pro Leu Asn Ser Leu Asp1 5 10 15Val Lys Pro Leu Arg
Lys Pro Arg Ile Pro Met Glu Thr Phe Arg 20 25 30Lys Val Gly Ile Pro
Ile Ile Ile Ala Leu Leu Ser Leu Ala Ser 35 40 45Ile Ile Ile Val Val
Val Leu Ile Lys Val Ile Leu Asp Lys Tyr 50 55 60Tyr Phe Leu Cys Gly
Gln Pro Leu His Phe Ile Pro Arg Lys Gln 65 70 75Leu Cys Asp Gly Glu
Leu Asp Cys Pro Leu Gly Glu Asp Glu Glu 80 85 90His Cys Val Lys Ser
Phe Pro Glu Gly Pro Ala Val Ala Val Arg 95 100 105Leu Ser Lys Asp
Arg Ser Thr Leu Gln Val Leu Asp Ser Ala Thr 110 115 120Gly Asn Trp
Phe Ser Ala Cys Phe Asp Asn Phe Thr Glu Ala Leu 125 130 135Ala Glu
Thr Ala Cys Arg Gln Met Gly Tyr Ser Arg Ala Val Glu 140 145 150Ile
Gly Pro Asp Gln Asp Leu Asp Val Val Glu Ile Thr Glu Asn 155 160
165Ser Gln Glu Leu Arg Met Arg Asn Ser Ser Gly Pro Cys Leu Ser 170
175 180Gly Ser Leu Val Ser Leu His Cys Leu Ala Cys Gly Lys Ser Leu
185 190 195Lys Thr Pro Arg Val Val Gly Gly Glu Glu Ala Ser Val Asp
Ser 200 205 210Trp Pro Trp Gln Val Ser Ile Gln Tyr Asp Lys Gln His
Val Cys 215 220 225Gly Gly Ser Ile Leu Asp Pro His Trp Val Leu Thr
Ala Ala His 230 235 240Cys Phe Arg Lys His Thr Asp Val Phe Asn Trp
Lys Val Arg Ala 245 250 255Gly Ser Asp Lys Leu Gly Ser Phe Pro Ser
Leu Ala Val Ala Lys 260 265 270Ile Ile Ile Ile Glu Phe Asn Pro Met
Tyr Pro Lys Asp Asn Asp 275 280 285Ile Ala Leu Met Lys Leu Gln Phe
Pro Leu Thr Phe Ser Gly Thr 290 295 300Val Arg Pro Ile Cys Leu Pro
Phe Phe Asp Glu Glu Leu Thr Pro 305 310 315Ala Thr Pro Leu Trp Ile
Ile Gly Trp Gly Phe Thr Lys Gln Asn 320 325 330Gly Gly Lys Met Ser
Asp Ile Leu Leu Gln Ala Ser Val Gln Val 335 340 345Ile Asp Ser Thr
Arg Cys Asn Ala Asp Asp Ala Tyr Gln Gly Glu 350 355 360Val Thr Glu
Lys Met Met Cys Ala Gly Ile Pro Glu Gly Gly Val 365 370 375Asp Thr
Cys Gln Gly Asp Ser Gly Gly Pro Leu Met Tyr Gln Ser 380 385 390Asp
Gln Trp His Val Val Gly Ile Val Ser Trp Gly Tyr Gly Cys 395 400
405Gly Gly Pro Ser Thr Pro Gly Val Tyr Thr Lys Val Ser Ala Tyr 410
415 420Leu Asn Trp Ile Tyr Asn Val Trp Lys Ala Glu Leu 425
430452714DNAHomo sapiens 45cggacgcgtg ggtggcaacc aggagaagcc
aaacttggtc ccccggctcg 50cggagtgcct gcgagcggtg ctcatggcgc tctatgaggt
cttctctcac 100ccggtcgagc gcagttaccg cgcggggctc tgctccaaag
ccgcgctgtt 150cctgctgctg gccgctgcgc tcacgtacat cccgccgctg
ctggtggcct 200tccggagcca cgggttttgg ctgaagcgga gcagctacga
ggagcagccg 250accgtgcgct tccaacacca ggtgctgctc gtggccctgc
tcggacccga 300aagcgacggg ttcctcgcct ggagcacgtt ccccgccttc
aaccggctgc 350aaggggatcg cctgcgcgtc ccgctcgttt cgactagaga
agaagacagg 400aaccaggatg ggaagacgga catgttacat tttaagctgg
agcttcccct 450gcagtccacg gagcacgttc tcggtgtgca gctcatcctg
actttctcct 500atcgattaca caggatggcg accctcgtga tgcagagcat
ggcgtttctc 550cagtcctcct ttcctgtccc gggatcccag ttatacgtga
acggagacct 600gaggctgcag cagaagcagc cgctgagctg tggtggccta
gatgcccgat 650acaacatatc cgtgatcaac gggaccagcc cctttgccta
tgactacgac 700ctcacccata ttgttgctgc ctaccaggag
aggaacgtta ccaccgtcct 750gaatgatccc aaccccatct ggctggtggg
cagggccgca gatgctccat 800ttgtgattaa tgctatcatc cgataccctg
tggaagtcat ttcttatcag 850ccaggattct gggagatggt aaagttcgcc
tgggtacagt atgtcagcat 900cctgcttatc ttcctctggg tgtttgaaag
aatcaagatc ttcgtgtttc 950agaatcaggt ggtgaccacc attcctgtga
cagtgacgcc ccggggagac 1000ttgtgtaagg agcacttatc ctagaaaggc
catttctgaa gactcagcag 1050gaccgtggct gcctcattgt catcttctgg
gaacatctta ggaccttttg 1100aaagagccca gcggacacct gcgggcttgt
gtgcttttcc ctcagagaca 1150acggttcttt ccggttttgc tctacacagt
tccgtatctt cagagctcct 1200gcagaattgt cagggactag tttgtggaaa
ggtctgagag ttcctggagg 1250ctataattag ctttttgggt tttccttctt
tgccttagcg ttgaatttca 1300ggagaaaatt gcagtcagtt cagacatctt
ggaaagagtc ccatctctgg 1350tcaagcagag acttttcctc tgttgaactg
aggaacacac tgtgcatttc 1400ttccttctgt tgtgagccac tcttactctt
ttcagggctc tcttgtgaca 1450aacatgccaa tcactagcac tttgcacccc
tgggcttctc catttcccat 1500tcacagcttt gatttccaga gctgaggcct
ttaactggag acctggaggg 1550gcagggccca agggcaaggg ccgcattagc
acaggcaatc agggagggcc 1600gctgaaggac acttggaccg tccacctgcc
ccagcccaac agtcagtcat 1650ctgtcatcag ctcagctgag cagccctgga
tctttgccgt actgtgactg 1700ggctctttgc cctatttttc cctctgtctg
tgcccctgga tggcaggctg 1750aagtcagagg ggctgtttca ttctcagccc
cctcagcagc actgggggaa 1800gaaagcattg tcacaacagg ttctttctgg
ccctcaccca acagcctggg 1850cacttggccc tcctcctcct tgacagccct
cccccttcct gcaaaggaca 1900ggggcgacag gggttggtgt tgggattggc
tcccgctgcc tgacaaccac 1950aagtttattt ggaaggctag cgggaagccc
agcggctggc gtttcccttg 2000actaaggaac agggtgccca tcagagtggg
gcgggcagct ttgggaagga 2050cacaagaagc agtaagagtg taaagaggat
gctggcctgg gcaggccagt 2100ccagcctggc cactagcaga ataccaagca
gtccagtgga ttaccctcgt 2150ggctaagcaa gtgtctgcag gagcagagat
ggctggaagg ggcctctgca 2200cacggaagat ggcttgttca gcccattcac
ctcctgagga tgtgggcagt 2250ctcctccaag aacacatgga gctgcttcct
gatcccaagc aggtcattgc 2300cactggaagg acatggcccc ggtgatccat
gcttcatgcc cacccagaaa 2350cacacccctc agtgtgtgcc tcagtttact
ttggagatca gttgtcgttt 2400ttagtgctcc tttaggctta ctaaaacagt
tttggaaaca aagctatttt 2450gaagtattca agcagaggaa ttccctaaca
ctgaccccct tgtctttttt 2500taatattcag gctgttttat atgcctaaat
ttttttctta agatctaaac 2550gaaaaatagt ttcttgttta aattcacata
aggcaatgag atatggaaag 2600atgacaagat acgtataaac attggtttgc
atcttattaa attattctaa 2650tgcaaatctt gtataaagaa cccatgatgt
tttgtaactt tctaattaaa 2700atgttcaaaa tgag 271446316PRTHomo sapiens
46Met Ala Leu Tyr Glu Val Phe Ser His Pro Val Glu Arg Ser Tyr1 5 10
15Arg Ala Gly Leu Cys Ser Lys Ala Ala Leu Phe Leu Leu Leu Ala 20 25
30Ala Ala Leu Thr Tyr Ile Pro Pro Leu Leu Val Ala Phe Arg Ser 35 40
45His Gly Phe Trp Leu Lys Arg Ser Ser Tyr Glu Glu Gln Pro Thr 50 55
60Val Arg Phe Gln His Gln Val Leu Leu Val Ala Leu Leu Gly Pro 65 70
75Glu Ser Asp Gly Phe Leu Ala Trp Ser Thr Phe Pro Ala Phe Asn 80 85
90Arg Leu Gln Gly Asp Arg Leu Arg Val Pro Leu Val Ser Thr Arg 95
100 105Glu Glu Asp Arg Asn Gln Asp Gly Lys Thr Asp Met Leu His Phe
110 115 120Lys Leu Glu Leu Pro Leu Gln Ser Thr Glu His Val Leu Gly
Val 125 130 135Gln Leu Ile Leu Thr Phe Ser Tyr Arg Leu His Arg Met
Ala Thr 140 145 150Leu Val Met Gln Ser Met Ala Phe Leu Gln Ser Ser
Phe Pro Val 155 160 165Pro Gly Ser Gln Leu Tyr Val Asn Gly Asp Leu
Arg Leu Gln Gln 170 175 180Lys Gln Pro Leu Ser Cys Gly Gly Leu Asp
Ala Arg Tyr Asn Ile 185 190 195Ser Val Ile Asn Gly Thr Ser Pro Phe
Ala Tyr Asp Tyr Asp Leu 200 205 210Thr His Ile Val Ala Ala Tyr Gln
Glu Arg Asn Val Thr Thr Val 215 220 225Leu Asn Asp Pro Asn Pro Ile
Trp Leu Val Gly Arg Ala Ala Asp 230 235 240Ala Pro Phe Val Ile Asn
Ala Ile Ile Arg Tyr Pro Val Glu Val 245 250 255Ile Ser Tyr Gln Pro
Gly Phe Trp Glu Met Val Lys Phe Ala Trp 260 265 270Val Gln Tyr Val
Ser Ile Leu Leu Ile Phe Leu Trp Val Phe Glu 275 280 285Arg Ile Lys
Ile Phe Val Phe Gln Asn Gln Val Val Thr Thr Ile 290 295 300Pro Val
Thr Val Thr Pro Arg Gly Asp Leu Cys Lys Glu His Leu 305 310
315Ser473582DNAHomo sapiens 47cggacgcgtg ggtccggcgg cctgaggctg
caccgggcac gggtcggccg 50caatccagcc tgggcggagc cggagttgcg agccgctgcc
tagaggccga 100ggagctcaca gctatgggct ggaggccccg gagagctcgg
gggaccccgt 150tgctgctgct gctactactg ctgctgctct ggccagtgcc
aggcgccggg 200gtgcttcaag gacatatccc tgggcagcca gtcaccccgc
actgggtcct 250ggatggacaa ccctggcgca ccgtcagcct ggaggagccg
gtctcgaagc 300cagacatggg gctggtggcc ctggaggctg aaggccagga
gctcctgctt 350gagctggaga agaaccacag gctgctggcc ccaggataca
tagaaaccca 400ctacggccca gatgggcagc cagtggtgct ggcccccaac
cacacggatc 450attgccacta ccaagggcga gtaaggggct tccccgactc
ctgggtagtc 500ctctgcacct gctctgggat gagtggcctg atcaccctca
gcaggaatgc 550cagctattat ctgcgtccct ggccaccccg gggctccaag
gacttctcaa 600cccacgagat ctttcggatg gagcagctgc tcacctggaa
aggaacctgt 650ggccacaggg atcctgggaa caaagcgggc atgaccagcc
ttcctggtgg 700tccccagagc aggggcaggc gagaagcgcg caggacccgg
aagtacctgg 750aactgtacat tgtggcagac cacaccctgt tcttgactcg
gcaccgaaac 800ttgaaccaca ccaaacagcg tctcctggaa gtcgccaact
acgtggacca 850gcttctcagg actctggaca ttcaggtggc gctgaccggc
ctggaggtgt 900ggaccgagcg ggaccgcagc cgcgtcacgc aggacgccaa
cgccacgctc 950tgggccttcc tgcagtggcg ccgggggctg tgggcgcagc
ggccccacga 1000ctccgcgcag ctgctcacgg gccgcgcctt ccagggcgcc
acagtgggcc 1050tggcgcccgt cgagggcatg tgccgcgccg agagctcggg
aggcgtgagc 1100acggaccact cggagctccc catcggcgcc gcagccacca
tggcccatga 1150gatcggccac agcctcggcc tcagccacga ccccgacggc
tgctgcgtgg 1200aggctgcggc cgagtccgga ggctgcgtca tggctgcggc
caccgggcac 1250ccgtttccgc gcgtgttcag cgcctgcagc cgccgccagc
tgcgcgcctt 1300cttccgcaag gggggcggcg cttgcctctc caatgccccg
gaccccggac 1350tcccggtgcc gccggcgctc tgcgggaacg gcttcgtgga
agcgggcgag 1400gagtgtgact gcggccctgg ccaggagtgc cgcgacctct
gctgctttgc 1450tcacaactgc tcgctgcgcc cgggggccca gtgcgcccac
ggggactgct 1500gcgtgcgctg cctgctgaag ccggctggag cgctgtgccg
ccaggccatg 1550ggtgactgtg acctccctga gttttgcacg ggcacctcct
cccactgtcc 1600cccagacgtt tacctactgg acggctcacc ctgtgccagg
ggcagtggct 1650actgctggga tggcgcatgt cccacgctgg agcagcagtg
ccagcagctc 1700tgggggcctg gctcccaccc agctcccgag gcctgtttcc
aggtggtgaa 1750ctctgcggga gatgctcatg gaaactgcgg ccaggacagc
gagggccact 1800tcctgccctg tgcagggagg gatgccctgt gtgggaagct
gcagtgccag 1850ggtggaaagc ccagcctgct cgcaccgcac atggtgccag
tggactctac 1900cgttcaccta gatggccagg aagtgacttg tcggggagcc
ttggcactcc 1950ccagtgccca gctggacctg cttggcctgg gcctggtaga
gccaggcacc 2000cagtgtggac ctagaatggt gtgccagagc aggcgctgca
ggaagaatgc 2050cttccaggag cttcagcgct gcctgactgc ctgccacagc
cacggggttt 2100gcaatagcaa ccataactgc cactgtgctc caggctgggc
tccacccttc 2150tgtgacaagc caggctttgg tggcagcatg gacagtggcc
ctgtgcaggc 2200tgaaaaccat gacaccttcc tgctggccat gctcctcagc
gtcctgctgc 2250ctctgctccc aggggccggc ctggcctggt gttgctaccg
actcccagga 2300gcccatctgc agcgatgcag ctggggctgc agaagggacc
ctgcgtgcag 2350tggccccaaa gatggcccac acagggacca ccccctgggc
ggcgttcacc 2400ccatggagtt gggccccaca gccactggac agccctggcc
cctggaccct 2450gagaactctc atgagcccag cagccaccct gagaagcctc
tgccagcagt 2500ctcgcctgac ccccaagcag atcaagtcca gatgccaaga
tcctgcctct 2550ggtgagaggt agctcctaaa atgaacagat ttaaagacag
gtggccactg 2600acagccactc caggaacttg aactgcaggg gcagagccag
tgaatcaccg 2650gacctccagc acctgcaggc agcttggaag tttcttcccc
gagtggagct 2700tcgacccacc cactccagga acccagagcc acattagaag
ttcctgaggg 2750ctggagaaca ctgcttgggc acactctcca gctcaataaa
ccatcagtcc 2800cagaagcaaa ggtcacacag cccctgacct ccctcaccag
tggaggctgg 2850gtagtgctgg ccatcccaaa agggctctgt cctgggagtc
tggtgtgtct 2900cctacatgca atttccacgg acccagctct gtggagggca
tgactgctgg 2950ccagaagcta gtggtcctgg ggccctatgg ttcgactgag
tccacactcc 3000cctgcagcct ggctggcctc tgcaaacaaa cataattttg
gggaccttcc 3050ttcctgtttc ttcccaccct gtcttctccc ctaggtggtt
cctgagcccc 3100cacccccaat cccagtgcta cacctgaggt tctggagctc
agaatctgac 3150agcctctccc ccattctgtg tgtgtccggg ggacagaggg
aaccatttaa 3200gaaaagatac caaagtagaa gtcaaaagaa agacatgttg
gctataggcg 3250tggtggctca tgcctataat cccagcactt tgggaagccg
gggtaggagg 3300atcaccagag gccagcaggt ccacaccagc ctgggcaaca
cagcaagaca 3350ccgcatctac agaaaaattt taaaattagc tgggcgtggt
ggtgtgtacc 3400tgtaggccta gctgctcagg aggctgaagc aggaggatca
cttgagcctg 3450agttcaacac tgcagtgagc tatggtggca ccactgcact
ccagcctggg 3500tgacagagca agaccctgtc tctaaaataa attttaaaag
gacttaaaaa 3550aaaaaaaaaa aaaaaaaaaa aaaaaaagaa aa 358248813PRTHomo
sapiens 48Met Gly Trp Arg Pro Arg Arg Ala Arg Gly Thr Pro Leu Leu
Leu1 5 10 15Leu Leu Leu Leu Leu Leu Leu Trp Pro Val Pro Gly Ala Gly
Val 20 25 30Leu Gln Gly His Ile Pro Gly Gln Pro Val Thr Pro His Trp
Val 35 40 45Leu Asp Gly Gln Pro Trp Arg Thr Val Ser Leu Glu Glu Pro
Val 50 55 60Ser Lys Pro Asp Met Gly Leu Val Ala Leu Glu Ala Glu Gly
Gln 65 70 75Glu Leu Leu Leu Glu Leu Glu Lys Asn His Arg Leu Leu Ala
Pro 80 85 90Gly Tyr Ile Glu Thr His Tyr Gly Pro Asp Gly Gln Pro Val
Val 95 100 105Leu Ala Pro Asn His Thr Asp His Cys His Tyr Gln Gly
Arg Val 110 115 120Arg Gly Phe Pro Asp Ser Trp Val Val Leu Cys Thr
Cys Ser Gly 125 130 135Met Ser Gly Leu Ile Thr Leu Ser Arg Asn Ala
Ser Tyr Tyr Leu 140 145 150Arg Pro Trp Pro Pro Arg Gly Ser Lys Asp
Phe Ser Thr His Glu 155 160 165Ile Phe Arg Met Glu Gln Leu Leu Thr
Trp Lys Gly Thr Cys Gly 170 175 180His Arg Asp Pro Gly Asn Lys Ala
Gly Met Thr Ser Leu Pro Gly 185 190 195Gly Pro Gln Ser Arg Gly Arg
Arg Glu Ala Arg Arg Thr Arg Lys 200 205 210Tyr Leu Glu Leu Tyr Ile
Val Ala Asp His Thr Leu Phe Leu Thr 215 220 225Arg His Arg Asn Leu
Asn His Thr Lys Gln Arg Leu Leu Glu Val 230 235 240Ala Asn Tyr Val
Asp Gln Leu Leu Arg Thr Leu Asp Ile Gln Val 245 250 255Ala Leu Thr
Gly Leu Glu Val Trp Thr Glu Arg Asp Arg Ser Arg 260 265 270Val Thr
Gln Asp Ala Asn Ala Thr Leu Trp Ala Phe Leu Gln Trp 275 280 285Arg
Arg Gly Leu Trp Ala Gln Arg Pro His Asp Ser Ala Gln Leu 290 295
300Leu Thr Gly Arg Ala Phe Gln Gly Ala Thr Val Gly Leu Ala Pro 305
310 315Val Glu Gly Met Cys Arg Ala Glu Ser Ser Gly Gly Val Ser Thr
320 325 330Asp His Ser Glu Leu Pro Ile Gly Ala Ala Ala Thr Met Ala
His 335 340 345Glu Ile Gly His Ser Leu Gly Leu Ser His Asp Pro Asp
Gly Cys 350 355 360Cys Val Glu Ala Ala Ala Glu Ser Gly Gly Cys Val
Met Ala Ala 365 370 375Ala Thr Gly His Pro Phe Pro Arg Val Phe Ser
Ala Cys Ser Arg 380 385 390Arg Gln Leu Arg Ala Phe Phe Arg Lys Gly
Gly Gly Ala Cys Leu 395 400 405Ser Asn Ala Pro Asp Pro Gly Leu Pro
Val Pro Pro Ala Leu Cys 410 415 420Gly Asn Gly Phe Val Glu Ala Gly
Glu Glu Cys Asp Cys Gly Pro 425 430 435Gly Gln Glu Cys Arg Asp Leu
Cys Cys Phe Ala His Asn Cys Ser 440 445 450Leu Arg Pro Gly Ala Gln
Cys Ala His Gly Asp Cys Cys Val Arg 455 460 465Cys Leu Leu Lys Pro
Ala Gly Ala Leu Cys Arg Gln Ala Met Gly 470 475 480Asp Cys Asp Leu
Pro Glu Phe Cys Thr Gly Thr Ser Ser His Cys 485 490 495Pro Pro Asp
Val Tyr Leu Leu Asp Gly Ser Pro Cys Ala Arg Gly 500 505 510Ser Gly
Tyr Cys Trp Asp Gly Ala Cys Pro Thr Leu Glu Gln Gln 515 520 525Cys
Gln Gln Leu Trp Gly Pro Gly Ser His Pro Ala Pro Glu Ala 530 535
540Cys Phe Gln Val Val Asn Ser Ala Gly Asp Ala His Gly Asn Cys 545
550 555Gly Gln Asp Ser Glu Gly His Phe Leu Pro Cys Ala Gly Arg Asp
560 565 570Ala Leu Cys Gly Lys Leu Gln Cys Gln Gly Gly Lys Pro Ser
Leu 575 580 585Leu Ala Pro His Met Val Pro Val Asp Ser Thr Val His
Leu Asp 590 595 600Gly Gln Glu Val Thr Cys Arg Gly Ala Leu Ala Leu
Pro Ser Ala 605 610 615Gln Leu Asp Leu Leu Gly Leu Gly Leu Val Glu
Pro Gly Thr Gln 620 625 630Cys Gly Pro Arg Met Val Cys Gln Ser Arg
Arg Cys Arg Lys Asn 635 640 645Ala Phe Gln Glu Leu Gln Arg Cys Leu
Thr Ala Cys His Ser His 650 655 660Gly Val Cys Asn Ser Asn His Asn
Cys His Cys Ala Pro Gly Trp 665 670 675Ala Pro Pro Phe Cys Asp Lys
Pro Gly Phe Gly Gly Ser Met Asp 680 685 690Ser Gly Pro Val Gln Ala
Glu Asn His Asp Thr Phe Leu Leu Ala 695 700 705Met Leu Leu Ser Val
Leu Leu Pro Leu Leu Pro Gly Ala Gly Leu 710 715 720Ala Trp Cys Cys
Tyr Arg Leu Pro Gly Ala His Leu Gln Arg Cys 725 730 735Ser Trp Gly
Cys Arg Arg Asp Pro Ala Cys Ser Gly Pro Lys Asp 740 745 750Gly Pro
His Arg Asp His Pro Leu Gly Gly Val His Pro Met Glu 755 760 765Leu
Gly Pro Thr Ala Thr Gly Gln Pro Trp Pro Leu Asp Pro Glu 770 775
780Asn Ser His Glu Pro Ser Ser His Pro Glu Lys Pro Leu Pro Ala 785
790 795Val Ser Pro Asp Pro Gln Ala Asp Gln Val Gln Met Pro Arg Ser
800 805 810Cys Leu Trp491675DNAHomo sapiens 49ggcacgaggg agcctccgtt
agggggtggg aaaggacttt gccataggtc 50gctgaggcca ccatctgctc tcttactggc
caagggcgta aaaagatagt 100cttcccatta gctagagagc aaaccccaga
aagcctattg gctgcgccgt 150ccgcgggcct tggtccgctt tgaaggcggg
ctgcggctgc gagaggaggg 200cgggcgggag gctagctgtt gtcgtggttg
ctcggaggca cgtgtgcagt 250cccggaagcg gcgaggggaa actgctccgc
gcgcgccgcg ggaggaggaa 300ccgcccggtc ctttagggtc cgggcccggc
cgggccatgg attcaatgcc 350tgagcccgcg tcccgctgtc ttctgcttct
tcccttgctg ctgctgctgc 400tgctgctgct gccggccccg gagctgggcc
cgagccaggc cggagctgag 450gagaacgact gggttcgcct gcccagcaaa
tgcgaagtgt gtaaatatgt 500tgctgtggag ctgaagtcag cctttgagga
aaccggcaag accaaggagg 550tgattggcac gggctatggc atcctggacc
agaaggcctc tggagtcaaa 600tacaccaagt cggacttgcg gttaatcgaa
gtcactgaga ccatttgcaa 650gaggctcctg gattatagcc tgcacaagga
gaggaccggc agcaatcgat 700ttgccaaggg catgtcagag acctttgaga
cattacacaa cctggtacac 750aaaggggtca aggtggtgat
ggacatcccc tatgagctgt ggaacgagac 800ttctgcagag gtggctgacc
tcaagaagca gtgtgatgtg ctggtggaag 850agtttgagga ggtgatcgag
gactggtaca ggaaccacca ggaggaagac 900ctgactgaat tcctctgcgc
caaccacgtg ctgaagggaa aagacaccag 950ttgcctggca gagcagtggt
ccggcaagaa gggagacaca gctgccctgg 1000gagggaagaa gtccaagaag
aagagcagca gggccaaggc agcaggcggc 1050aggagtagca gcagcaaaca
aaggaaggag ctgggtggcc ttgagggaga 1100ccccagcccc gaggaggatg
agggcatcca gaaggcatcc cctctcacac 1150acagcccccc tgatgagctc
tgagcccacc cagcatcctc tgtcctgaga 1200cccctgattt tgaagctgag
gagtcagggg catggctctg gcaggccggg 1250atggccccgc agccttcagc
ccctccttgc cttggctgtg ccctcttctg 1300ccaaggaaag acacaagccc
caggaagaac tcagagccgt catgggtagc 1350ccacgccgtc ctttcccctc
cccaagtgtt tctctcctga cccagggttc 1400aggcaggcct tgtggtttca
ggactgcaag gactccagtg tgaactcagg 1450aggggcaggt gtcagaactg
ggcaccagga ctggagcccc ctccggagac 1500caaactcacc atccctcagt
cctccccaac agggtactag gactgcagcc 1550ccctgtagct cctctctgct
tacccctcct gtggacacct tgcactctgc 1600ctggcccttc ccagagccca
aagagtaaaa atgttctggt tctgatttct 1650gaaaaaaaaa aaaaaaaaaa ttcct
167550278PRTHomo sapiens 50Met Asp Ser Met Pro Glu Pro Ala Ser Arg
Cys Leu Leu Leu Leu1 5 10 15Pro Leu Leu Leu Leu Leu Leu Leu Leu Leu
Pro Ala Pro Glu Leu 20 25 30Gly Pro Ser Gln Ala Gly Ala Glu Glu Asn
Asp Trp Val Arg Leu 35 40 45Pro Ser Lys Cys Glu Val Cys Lys Tyr Val
Ala Val Glu Leu Lys 50 55 60Ser Ala Phe Glu Glu Thr Gly Lys Thr Lys
Glu Val Ile Gly Thr 65 70 75Gly Tyr Gly Ile Leu Asp Gln Lys Ala Ser
Gly Val Lys Tyr Thr 80 85 90Lys Ser Asp Leu Arg Leu Ile Glu Val Thr
Glu Thr Ile Cys Lys 95 100 105Arg Leu Leu Asp Tyr Ser Leu His Lys
Glu Arg Thr Gly Ser Asn 110 115 120Arg Phe Ala Lys Gly Met Ser Glu
Thr Phe Glu Thr Leu His Asn 125 130 135Leu Val His Lys Gly Val Lys
Val Val Met Asp Ile Pro Tyr Glu 140 145 150Leu Trp Asn Glu Thr Ser
Ala Glu Val Ala Asp Leu Lys Lys Gln 155 160 165Cys Asp Val Leu Val
Glu Glu Phe Glu Glu Val Ile Glu Asp Trp 170 175 180Tyr Arg Asn His
Gln Glu Glu Asp Leu Thr Glu Phe Leu Cys Ala 185 190 195Asn His Val
Leu Lys Gly Lys Asp Thr Ser Cys Leu Ala Glu Gln 200 205 210Trp Ser
Gly Lys Lys Gly Asp Thr Ala Ala Leu Gly Gly Lys Lys 215 220 225Ser
Lys Lys Lys Ser Ser Arg Ala Lys Ala Ala Gly Gly Arg Ser 230 235
240Ser Ser Ser Lys Gln Arg Lys Glu Leu Gly Gly Leu Glu Gly Asp 245
250 255Pro Ser Pro Glu Glu Asp Glu Gly Ile Gln Lys Ala Ser Pro Leu
260 265 270Thr His Ser Pro Pro Asp Glu Leu 27551633DNAHomo sapiens
51ctcctgcact aggctctcag ccagggatga tgcgctgctg ccgccgccgc
50tgctgctgcc ggcaaccacc ccatgccctg aggccgttgc tgttgctgcc
100cctcgtcctt ttacctcccc tggcagcagc tgcagcgggc ccaaaccgat
150gtgacaccat ataccagggc ttcgccgagt gtctcatccg cttgggggac
200agcatgggcc gcggaggcga gctggagacc atctgcaggt cttggaatga
250cttccatgcc tgtgcctctc aggtcctgtc aggctgtccg gaggaggcag
300ctgcagtgtg ggaatcacta cagcaagaag ctcgccaggc cccccgtccg
350aataacttgc acactctgtg cggtgccccg gtgcatgttc gggagcgcgg
400cacaggctcc gaaaccaacc aggagacgct gcgggctaca gcgcctgcac
450tccccatggc ccctgcgccc ccactgctgg cggctgctct ggctctggcc
500tacctcctga ggcctctggc ctagcttgtt gggttgggta gcagcgcccg
550tacctccagc cctgctctgg cggtggttgt ccaggctctg cagagcgcag
600cagggctttt cattaaaggt atttatattt gta 63352165PRTHomo sapiens
52Met Met Arg Cys Cys Arg Arg Arg Cys Cys Cys Arg Gln Pro Pro1 5 10
15His Ala Leu Arg Pro Leu Leu Leu Leu Pro Leu Val Leu Leu Pro 20 25
30Pro Leu Ala Ala Ala Ala Ala Gly Pro Asn Arg Cys Asp Thr Ile 35 40
45Tyr Gln Gly Phe Ala Glu Cys Leu Ile Arg Leu Gly Asp Ser Met 50 55
60Gly Arg Gly Gly Glu Leu Glu Thr Ile Cys Arg Ser Trp Asn Asp 65 70
75Phe His Ala Cys Ala Ser Gln Val Leu Ser Gly Cys Pro Glu Glu 80 85
90Ala Ala Ala Val Trp Glu Ser Leu Gln Gln Glu Ala Arg Gln Ala 95
100 105Pro Arg Pro Asn Asn Leu His Thr Leu Cys Gly Ala Pro Val His
110 115 120Val Arg Glu Arg Gly Thr Gly Ser Glu Thr Asn Gln Glu Thr
Leu 125 130 135Arg Ala Thr Ala Pro Ala Leu Pro Met Ala Pro Ala Pro
Pro Leu 140 145 150Leu Ala Ala Ala Leu Ala Leu Ala Tyr Leu Leu Arg
Pro Leu Ala 155 160 16553386DNAHomo sapiens 53ccaggatcag catggccgtc
cgccagtggg taatcgccct ggccttggct 50gccctccttg ttgtggacag ggaagtgcca
gtggcagcag gaaagctccc 100tttctcaaga atgcccatct gtgaacacat
ggtagagtct ccaacctgtt 150cccagatgtc caacctggtc tgcggcactg
atgggctcac atatacgaat 200gaatgccagc tctgcttggc ccggataaaa
accaaacagg acatccagat 250catgaaagat ggcaaatgct gatcccacag
gagcacctca agccatgaag 300tgtcagctgg agaacagtgg tgggcatgga
gaggatatga catgaaataa 350aagatccagc ccaactgaaa aaaaaaaaaa aaaaaa
3865486PRTHomo sapiens 54Met Ala Val Arg Gln Trp Val Ile Ala Leu
Ala Leu Ala Ala Leu1 5 10 15Leu Val Val Asp Arg Glu Val Pro Val Ala
Ala Gly Lys Leu Pro 20 25 30Phe Ser Arg Met Pro Ile Cys Glu His Met
Val Glu Ser Pro Thr 35 40 45Cys Ser Gln Met Ser Asn Leu Val Cys Gly
Thr Asp Gly Leu Thr 50 55 60Tyr Thr Asn Glu Cys Gln Leu Cys Leu Ala
Arg Ile Lys Thr Lys 65 70 75Gln Asp Ile Gln Ile Met Lys Asp Gly Lys
Cys 80 85552594DNAHomo sapiens 55gtggagttgg gtggtgtcgg gagcctctcc
ctgaggggca ccgcgtcttc 50aggagctggg cctccagtgc ggcgcgatgt caggcgcggt
gacagctctg 100tgagtccgag gccgcggccg tggcgctggg cggctgcggg
gcctgaccgg 150tccgctcatg gtgccgccac gacgccatcg cggggcagga
aggccagggg 200tgctgagttc ttcacctcct tttagactga gatctgccaa
gttttccggc 250attgctcttg aggatctcag aagggctctt aagacaagac
tgcaaatggt 300gtgtgtattt gtcatgaacc gaatgaattc ccagaacagt
ggtttcactc 350agcgcaggcg aatggctctt gggattgtta ttcttctgct
tgttgatgtg 400atatgggttg cttcctctga acttacttcg tatgttttta
cccagtacaa 450caaaccattc ttcagcacct ttgcaaaaac atctatgttt
gttttgtacc 500ttttgggctt tattatttgg aagccatgga gacaacagtg
tacaagagga 550cttcgcggaa agcatgctgc tttttttgca gatgctgaag
gttactttgc 600tgcttgcaca acagatacaa ctatgaatag ttctttgagt
gaacctctgt 650atgtgcctgt gaaattccat gatcttccaa gtgaaaaacc
tgagagcaca 700aacattgata ctgaaaaaac ccccaaaaag tctcgtgtga
ggttcagtaa 750tatcatggag attcgacagc ttccgtcaag tcatgcattg
gaagcaaagt 800tgtctcgcat gtcatatcct gtgaaagaac aagaatccat
actgaaaact 850gtggggaaac ttactgcaac tcaagtagcg aaaattagct
tttttttttg 900ctttgtgtgg tttttggcaa atttgtcata tcaagaagca
ctttcagaca 950cacaagttgc tatagttaat attttatctt caacttccgg
actttttacc 1000ttaatccttg ctgcagtatt tccaagtaac agtggagata
gatttaccct 1050ttctaaacta ttagctgtaa ttttaagcat tggaggcgtt
gtactggtaa 1100acctggcagg gtctgaaaaa cctgctggaa gagacacagt
aggttccatt 1150tggtctcttg ctggagccat gctctatgct gtctatattg
ttatgattaa 1200gagaaaagta gatagagaag acaagttgga tattccaatg
ttctttggtt 1250ttgtaggttt gtttaatctg ctgctcttat ggccaggttt
ctttttactt 1300cattatactg gatttgagga cttcgagttt cccaataaag
tagtattaat 1350gtgcattatc attaatggcc ttattggaac agtactctca
gagttcctgt 1400ggttgtgggg ctgctttctt acctcatcat tgataggcac
acttgcacta 1450agccttacaa tacctctgtc cataatagct gacatgtgta
tgcaaaaggt 1500gcagttttct tggttatttt ttgcaggagc tatccctgta
tttttttcat 1550tttttattgt aactctccta tgccattata ataattggga
tcctgtgatg 1600gtgggaatca gaagaatatt tgcttttata tgcagaaaac
atcgaattca 1650gagagttcca gaagacagcg aacagtgtga gagtctcatt
tctatgcaca 1700gtgtttctca ggaggatgga gctagttagc tgtctgttgt
ctgtagccca 1750gcttgataat ggaactatac agcgaagaga caatctctgg
caagtttttg 1800tagaaaaaat gtttcagtgc ctagtctgaa aaataacagt
ttgagttctt 1850tgaaactcta aaatatattt ttctcatacc tgttttcttc
attttcataa 1900tgaagcactt tgctatgtag ctgtgtacat atcactacag
ttataggaag 1950tttcagtcta cagtccatcc aaaggaccaa cctgccttac
acatctcaag 2000gaattcagct gttgaaatca tttgaactaa tcaaggaata
aatcctaatg 2050ttctgggact ttattttcac atgttaaatg ctggaatata
ttatgaaaat 2100gttttcaaga aatcacttaa gtgttcatag accagtattt
ctgacaggta 2150aaatgctaaa ataagctacc tgtaataagt gtggattata
tttttgggtt 2200ttgtagaata ttgcaaatta accacacaaa aaatgtttaa
tttatgcaac 2250aagcatgttt gtgcaaattt catgggactt taaaaagaat
aagtatttga 2300gaaaatatct ggttcactta cactacattt actgtattat
tcttttatag 2350cattaggtgc cttgtatttt aaatctgtga caaaccatgg
caaattttta 2400aaggggaagt attattataa aatgaagaaa tatgtatttc
taaaggctat 2450attgctgtaa acttaattga taaagctctg tttaatttag
agttttgaag 2500aaatagtctc ccttcaatta agaaattttc ataatggaat
gatttaaatt 2550gaagtgacaa agagtattat taaaatacaa tgtttataaa aaaa
259456523PRTHomo sapiens 56Met Val Pro Pro Arg Arg His Arg Gly Ala
Gly Arg Pro Gly Val1 5 10 15Leu Ser Ser Ser Pro Pro Phe Arg Leu Arg
Ser Ala Lys Phe Ser 20 25 30Gly Ile Ala Leu Glu Asp Leu Arg Arg Ala
Leu Lys Thr Arg Leu 35 40 45Gln Met Val Cys Val Phe Val Met Asn Arg
Met Asn Ser Gln Asn 50 55 60Ser Gly Phe Thr Gln Arg Arg Arg Met Ala
Leu Gly Ile Val Ile 65 70 75Leu Leu Leu Val Asp Val Ile Trp Val Ala
Ser Ser Glu Leu Thr 80 85 90Ser Tyr Val Phe Thr Gln Tyr Asn Lys Pro
Phe Phe Ser Thr Phe 95 100 105Ala Lys Thr Ser Met Phe Val Leu Tyr
Leu Leu Gly Phe Ile Ile 110 115 120Trp Lys Pro Trp Arg Gln Gln Cys
Thr Arg Gly Leu Arg Gly Lys 125 130 135His Ala Ala Phe Phe Ala Asp
Ala Glu Gly Tyr Phe Ala Ala Cys 140 145 150Thr Thr Asp Thr Thr Met
Asn Ser Ser Leu Ser Glu Pro Leu Tyr 155 160 165Val Pro Val Lys Phe
His Asp Leu Pro Ser Glu Lys Pro Glu Ser 170 175 180Thr Asn Ile Asp
Thr Glu Lys Thr Pro Lys Lys Ser Arg Val Arg 185 190 195Phe Ser Asn
Ile Met Glu Ile Arg Gln Leu Pro Ser Ser His Ala 200 205 210Leu Glu
Ala Lys Leu Ser Arg Met Ser Tyr Pro Val Lys Glu Gln 215 220 225Glu
Ser Ile Leu Lys Thr Val Gly Lys Leu Thr Ala Thr Gln Val 230 235
240Ala Lys Ile Ser Phe Phe Phe Cys Phe Val Trp Phe Leu Ala Asn 245
250 255Leu Ser Tyr Gln Glu Ala Leu Ser Asp Thr Gln Val Ala Ile Val
260 265 270Asn Ile Leu Ser Ser Thr Ser Gly Leu Phe Thr Leu Ile Leu
Ala 275 280 285Ala Val Phe Pro Ser Asn Ser Gly Asp Arg Phe Thr Leu
Ser Lys 290 295 300Leu Leu Ala Val Ile Leu Ser Ile Gly Gly Val Val
Leu Val Asn 305 310 315Leu Ala Gly Ser Glu Lys Pro Ala Gly Arg Asp
Thr Val Gly Ser 320 325 330Ile Trp Ser Leu Ala Gly Ala Met Leu Tyr
Ala Val Tyr Ile Val 335 340 345Met Ile Lys Arg Lys Val Asp Arg Glu
Asp Lys Leu Asp Ile Pro 350 355 360Met Phe Phe Gly Phe Val Gly Leu
Phe Asn Leu Leu Leu Leu Trp 365 370 375Pro Gly Phe Phe Leu Leu His
Tyr Thr Gly Phe Glu Asp Phe Glu 380 385 390Phe Pro Asn Lys Val Val
Leu Met Cys Ile Ile Ile Asn Gly Leu 395 400 405Ile Gly Thr Val Leu
Ser Glu Phe Leu Trp Leu Trp Gly Cys Phe 410 415 420Leu Thr Ser Ser
Leu Ile Gly Thr Leu Ala Leu Ser Leu Thr Ile 425 430 435Pro Leu Ser
Ile Ile Ala Asp Met Cys Met Gln Lys Val Gln Phe 440 445 450Ser Trp
Leu Phe Phe Ala Gly Ala Ile Pro Val Phe Phe Ser Phe 455 460 465Phe
Ile Val Thr Leu Leu Cys His Tyr Asn Asn Trp Asp Pro Val 470 475
480Met Val Gly Ile Arg Arg Ile Phe Ala Phe Ile Cys Arg Lys His 485
490 495Arg Ile Gln Arg Val Pro Glu Asp Ser Glu Gln Cys Glu Ser Leu
500 505 510Ile Ser Met His Ser Val Ser Gln Glu Asp Gly Ala Ser 515
52057983DNAHomo sapiens 57ggatgcagca gagaggagca gctggaagcc
gtggctgcgc tctcttccct 50ctgctgggcg tcctgttctt ccagggtgtt tatatcgtct
tttccttgga 100gattcgtgca gatgcccatg tccgaggtta tgttggagaa
aagatcaagt 150tgaaatgcac tttcaagtca acttcagatg tcactgacaa
gcttactata 200gactggacat atcgccctcc cagcagcagc cacacagtat
caatatttca 250ttatcagtct ttccagtacc caaccacagc aggcacattt
cgggatcgga 300tttcctgggt tggaaatgta tacaaagggg atgcatctat
aagtataagc 350aaccctacca taaaggacaa tgggacattc agctgtgctg
tgaagaatcc 400cccagatgtg caccataata ttcccatgac agagctaaca
gtcacagaaa 450ggggttttgg caccatgctt tcctctgtgg cccttctttc
catccttgtc 500tttgtgccct cagccgtggt ggttgctctg ctgctggtga
gaatggggag 550gaaggctgct gggctgaaga agaggagcag gtctggctat
aagaagtcat 600ctattgaggt ttccgatgac actgatcagg aggaggaaga
ggcgtgtatg 650gcgaggcttt gtgtccgttg cgctgagtgc ctggattcag
actatgaaga 700gacatattga tgaaagtctg tatgacacaa gaagagtcac
ctaaagacag 750gaaacatccc attccactgg cagctaaagc ctgtcagaga
aagtggagct 800ggcctggacc atagcgatgg acaatcctgg agatcatcag
taaagacttt 850aggaaccact tatttattga ataaatgttc ttgttgtatt
tataaactgt 900tcaggaagtc tcataagaga ctcatgactt cccctttcaa
tgaattatgc 950tgtaattgaa tgaagaaatt cttttcctga gca 98358235PRTHomo
sapiens 58Met Gln Gln Arg Gly Ala Ala Gly Ser Arg Gly Cys Ala Leu
Phe1 5 10 15Pro Leu Leu Gly Val Leu Phe Phe Gln Gly Val Tyr Ile Val
Phe 20 25 30Ser Leu Glu Ile Arg Ala Asp Ala His Val Arg Gly Tyr Val
Gly 35 40 45Glu Lys Ile Lys Leu Lys Cys Thr Phe Lys Ser Thr Ser Asp
Val 50 55 60Thr Asp Lys Leu Thr Ile Asp Trp Thr Tyr Arg Pro Pro Ser
Ser 65 70 75Ser His Thr Val Ser Ile Phe His Tyr Gln Ser Phe Gln Tyr
Pro 80 85 90Thr Thr Ala Gly Thr Phe Arg Asp Arg Ile Ser Trp Val Gly
Asn 95 100 105Val Tyr Lys Gly Asp Ala Ser Ile Ser Ile Ser Asn Pro
Thr Ile 110 115 120Lys Asp Asn Gly Thr Phe Ser Cys Ala Val Lys Asn
Pro Pro Asp 125 130 135Val His His Asn Ile Pro Met Thr Glu Leu Thr
Val Thr Glu Arg 140 145 150Gly Phe Gly Thr Met Leu Ser Ser Val Ala
Leu Leu Ser Ile Leu 155 160 165Val Phe Val Pro Ser Ala Val Val Val
Ala Leu Leu Leu Val Arg 170 175 180Met Gly Arg Lys Ala Ala Gly Leu
Lys Lys Arg Ser Arg Ser Gly 185 190 195Tyr Lys Lys Ser Ser Ile Glu
Val Ser Asp Asp Thr Asp Gln Glu 200 205 210Glu Glu Glu Ala Cys Met
Ala Arg Leu Cys Val Arg Cys Ala
Glu 215 220 225Cys Leu Asp Ser Asp Tyr Glu Glu Thr Tyr 230
235591794DNAHomo sapiens 59cccttggaag ctggaatcct gcaacaatgg
cccagggtgt cctctggatc 50ctactcggat tgctactgtg gtcagaccca gggacagcct
ccctgcccct 100gctcatggac tctgtcatcc aggccctggc tgagctggag
cagaaagtgc 150cagctgccaa gaccagacac acagcttctg cgtggctgat
gtcagctcca 200aactctggcc cccacaatcg cctctaccac ttcctgctgg
gggcatggag 250cctcaatgct acagagttgg atccctgccc actaagccca
gagctgttag 300gcctgaccaa ggaggtggcc cgacatgacg tacgagaagg
gaaggaatat 350ggggtggtgc tggcacctga tggctcgacc gtggctgtgg
agcctctgct 400ggcggggctg gaggcagggc tgcaagggcg cagggtcata
aatttgccct 450tggacagcat ggctgcccct tgggagactg gagatacctt
tccagatgtt 500gtggccattg ctccagatgt aagagccacc tcctccccag
gactcaggga 550tggctctcca gatgtcacca ctgcagatat tggagccaac
actccagatg 600ctacaaaagg ctgtccagat gtccaagctt ccttgccaga
tgccaaagcc 650aagtccccac cgaccatggt ggacagcctc ctggcagtca
ccctggctgg 700aaacctgggc ctgaccttcc tccgaggttc ccagacccag
agccatccag 750acctgggaac tgagggctgc tgggaccagc tctctgcccc
tcggaccttt 800acgcttttgg accccaaggc atctctgtta accatggcct
tcctcaatgg 850cgccctggat ggggtcatcc ttggagacta cctgagccgg
actcctgagc 900cccggccatc cctcagccac ttgctgagcc agtactatgg
ggctggggtg 950gccagagacc cagggttccg cagcaacttc cgacggcaga
acggtgctgc 1000tctgacttca gcctccatcc tggcccagca ggtgtgggga
acccttgtcc 1050ttctacagag gctggagcca gtacacctcc agcttcagtg
catgagccaa 1100gaacagctgg cccaggtggc tgccaatgct accaaggaat
tcactgaggc 1150cttcctggga tgcccggcca tccacccccg ctgccgctgg
ggagcggcgc 1200cttatcgggg ccgcccgaag ctgctgcagc tgccgctggg
attcttgtac 1250gtgcatcaca cctacgtgcc tgcaccaccc tgcacggact
tcacgcgctg 1300cgcagccaac atgcgctcca tgcagcgcta ccaccaggac
acgcaaggct 1350ggggagacat cggctacagt ttcgtggtgg gctcggacgg
ctacgtgtac 1400gagggacgcg gctggcactg ggtgggcgcc cacacgctcg
gccacaactc 1450ccggggcttc ggcgtggcca tagtgggcaa ctacaccgcg
gcgctgccca 1500ccgaggccgc tctgcgcacg gtgcgcgaca cgctcccgag
ttgtgcggtg 1550cgcgccggcc tcctgcggcc agactacgcg ctgctgggcc
accgccagct 1600ggtgcgcacc gactgccccg gcgacgcgct cttcgacctg
ctgcgcacct 1650ggccgcactt caccgcgact gttaagccaa gacctgccag
gagtgtctct 1700aagagatcca ggagggagcc acccccaagg accctgccag
ccacagacct 1750ccaataaaga cagcatggaa acaaaaaaaa aaaaaaaaaa aaaa
179460576PRTHomo sapiens 60Met Ala Gln Gly Val Leu Trp Ile Leu Leu
Gly Leu Leu Leu Trp1 5 10 15Ser Asp Pro Gly Thr Ala Ser Leu Pro Leu
Leu Met Asp Ser Val 20 25 30Ile Gln Ala Leu Ala Glu Leu Glu Gln Lys
Val Pro Ala Ala Lys 35 40 45Ala Arg His Thr Ala Ser Ala Trp Leu Met
Ser Ala Pro Asn Ser 50 55 60Gly Pro His Asn Arg Leu Tyr His Phe Leu
Leu Gly Ala Trp Ser 65 70 75Leu Asn Ala Thr Glu Leu Asp Pro Cys Pro
Leu Ser Pro Glu Leu 80 85 90Leu Gly Leu Thr Lys Glu Val Ala Gln His
Asp Val Arg Glu Gly 95 100 105Lys Glu Tyr Gly Val Val Leu Ala Pro
Asp Gly Ser Thr Val Ala 110 115 120Val Glu Pro Leu Leu Ala Gly Leu
Glu Ala Gly Leu Gln Gly Arg 125 130 135Arg Val Ile Asn Leu Pro Leu
Asp Ser Met Ala Ala Pro Trp Glu 140 145 150Thr Gly Asp Thr Phe Pro
Asp Val Val Ala Ile Ala Pro Asp Val 155 160 165Arg Ala Thr Ser Ser
Pro Gly Leu Arg Asp Gly Ser Pro Asp Val 170 175 180Thr Thr Ala Asp
Ile Gly Ala Asn Thr Pro Asp Ala Thr Lys Gly 185 190 195Cys Pro Asp
Val Gln Ala Ser Leu Pro Asp Ala Lys Ala Lys Ser 200 205 210Pro Pro
Thr Met Val Asp Ser Leu Leu Ala Val Thr Leu Ala Gly 215 220 225Asn
Leu Gly Leu Thr Phe Leu Arg Gly Ser Gln Thr Gln Ser His 230 235
240Pro Asp Leu Gly Thr Glu Gly Cys Trp Asp Gln Leu Ser Ala Pro 245
250 255Arg Thr Phe Thr Leu Leu Asp Pro Lys Ala Ser Leu Leu Thr Lys
260 265 270Ala Phe Leu Asn Gly Ala Leu Asp Gly Val Ile Leu Gly Asp
Tyr 275 280 285Leu Ser Arg Thr Pro Glu Pro Arg Pro Ser Leu Ser His
Leu Leu 290 295 300Ser Gln Tyr Tyr Gly Ala Gly Val Ala Arg Asp Pro
Gly Phe Arg 305 310 315Ser Asn Phe Arg Arg Gln Asn Gly Ala Ala Leu
Thr Ser Ala Ser 320 325 330Ile Leu Ala Gln Gln Val Trp Gly Thr Leu
Val Leu Leu Gln Arg 335 340 345Leu Glu Pro Val His Leu Gln Leu Gln
Cys Met Ser Gln Glu Gln 350 355 360Leu Ala Gln Val Ala Ala Asn Ala
Thr Lys Glu Phe Thr Glu Ala 365 370 375Phe Leu Gly Cys Pro Ala Ile
His Pro Arg Cys Arg Trp Gly Ala 380 385 390Ala Pro Tyr Gln Gly Arg
Pro Lys Leu Leu Gln Leu Pro Leu Gly 395 400 405Phe Leu Tyr Val His
His Thr Tyr Val Pro Ala Pro Pro Cys Thr 410 415 420Asp Phe Thr Arg
Cys Ala Ala Asn Met Arg Ser Met Gln Arg Tyr 425 430 435His Gln Asp
Thr Gln Gly Trp Gly Asp Ile Gly Tyr Ser Phe Val 440 445 450Val Gly
Ser Asp Gly Tyr Val Tyr Glu Gly Arg Gly Trp His Trp 455 460 465Val
Gly Ala His Thr Leu Gly His Asn Ser Arg Gly Phe Gly Val 470 475
480Ala Ile Val Gly Asn Tyr Thr Ala Ala Leu Pro Thr Glu Ala Ala 485
490 495Leu Arg Thr Val Arg Asp Thr Leu Pro Ser Cys Ala Val Arg Ala
500 505 510Gly Leu Leu Arg Pro Asp Tyr Ala Leu Leu Gly His Arg Gln
Leu 515 520 525Val Arg Thr Asp Cys Pro Gly Asp Ala Leu Phe Asp Leu
Leu Arg 530 535 540Thr Trp Pro His Phe Thr Ala Thr Val Lys Pro Arg
Pro Ala Arg 545 550 555Ser Val Ser Lys Arg Ser Arg Arg Glu Pro Pro
Pro Arg Thr Leu 560 565 570Pro Ala Thr Asp Leu Gln 575612732DNAHomo
sapiens 61agtcccagac gggcttttcc cagagagcta aaagagaagg gccagagaat
50gtcgtcccag ccagcaggga accagacctc ccccggggcc acagaggact
100actcctatgg cagctggtac atcgatgagc cccagggggg cgaggagctc
150cagccagagg gggaagtgcc ctcctgccac accagcatac cacccggcct
200gtaccacgcc tgcctggcct cgctgtcaat ccttgtgctg ctgctcctgg
250ccatgctggt gaggcgccgc cagctctggc ctgactgtgt gcgtggcagg
300cccggcctgc ccagccctgt ggatttcttg gctggggaca ggccccgggc
350agtgcctgct gctgttttca tggtcctcct gagctccctg tgtttgctgc
400tccccgacga ggacgcattg cccttcctga ctctcgcctc agcacccagc
450caagatggga aaactgaggc tccaagaggg gcctggaaga tactgggact
500gttctattat gctgccctct actaccctct ggctgcctgt gccacggctg
550gccacacagc tgcacacctg ctcggcagca cgctgtcctg ggcccacctt
600ggggtccagg tctggcagag ggcagagtgt ccccaggtgc ccaagatcta
650caagtactac tccctgctgg cctccctgcc tctcctgctg ggcctcggat
700tcctgagcct ttggtaccct gtgcagctgg tgagaagctt cagccgtagg
750acaggagcag gctccaaggg gctgcagagc agctactctg aggaatatct
800gaggaacctc ctttgcagga agaagctggg aagcagctac cacacctcca
850agcatggctt cctgtcctgg gcccgcgtct gcttgagaca ctgcatctac
900actccacagc caggattcca tctcccgctg aagctggtgc tttcagctac
950actgacaggg acggccattt accaggtggc cctgctgctg ctggtgggcg
1000tggtacccac tatccagaag gtgagggcag gggtcaccac ggatgtctcc
1050tacctgctgg ccggctttgg aatcgtgctc tccgaggaca agcaggaggt
1100ggtggagctg gtgaagcacc atctgtgggc tctggaagtg tgctacatct
1150cagccttggt cttgtcctgc ttactcacct tcctggtcct gatgcgctca
1200ctggtgacac acaggaccaa ccttcgagct ctgcaccgag gagctgccct
1250ggacttgagt cccttgcatc ggagtcccca tccctcccgc caagccatat
1300tctgttggat gagcttcagt gcctaccaga cagcctttat ctgccttggg
1350ctcctggtgc agcagatcat cttcttcctg ggaaccacgg ccctggcctt
1400cctggtgctc atgcctgtgc tccatggcag gaacctcctg ctcttccgtt
1450ccctggagtc ctcgtggccc ttctggctga ctttggccct ggctgtgatc
1500ctgcagaaca tggcagccca ttgggtcttc ctggagactc atgatggaca
1550cccacagctg accaaccggc gagtgctcta tgcagccacc tttcttctct
1600tccccctcaa tgtgctggtg ggtgccatgg tggccacctg gcgagtgctc
1650ctctctgccc tctacaacgc catccacctt ggccagatgg acctcagcct
1700gctgccaccg agagccgcca ctctcgaccc cggctactac acgtaccgaa
1750acttcttgaa gattgaagtc agccagtcgc atccagccat gacagccttc
1800tgctccctgc tcctgcaagc gcagagcctc ctacccagga ccatggcagc
1850cccccaggac agcctcagac caggggagga agacgaaggg atgcagctgc
1900tacagacaaa ggactccatg gccaagggag ctaggcccgg ggccagccgc
1950ggcagggctc gctggggtct ggcctacacg ctgctgcaca acccaaccct
2000gcaggtcttc cgcaagacgg ccctgttggg tgccaatggt gcccagccct
2050gagggcaggg aaggtcaacc cacctgccca tctgtgctga ggcatgttcc
2100tgcctaccat cctcctccct ccccggctct cctcccagca tcacaccagc
2150catgcagcca gcaggtcctc cggatcactg tggttgggtg gaggtctgtc
2200tgcactggga gcctcaggag ggctctgctc cacccacttg gctatgggag
2250agccagcagg ggttctggag aaaaaaactg gtgggttagg gccttggtcc
2300aggagccagt tgagccaggg cagccacatc caggcgtctc cctaccctgg
2350ctctgccatc agccttgaag ggcctcgatg aagccttctc tggaaccact
2400ccagcccagc tccacctcag ccttggcctt cacgctgtgg aagcagccaa
2450ggcacttcct caccccctca gcgccacgga cctctctggg gagtggccgg
2500aaagctcccg gtcctctggc ctgcagggca gcccaagtca tgactcagac
2550caggtcccac actgagctgc ccacactcga gagccagata tttttgtagt
2600ttttatgcct ttggctatta tgaaagaggt tagtgtgttc cctgcaataa
2650acttgttcct gagaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
2700aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa 273262667PRTHomo sapiens
62Met Ser Ser Gln Pro Ala Gly Asn Gln Thr Ser Pro Gly Ala Thr1 5 10
15Glu Asp Tyr Ser Tyr Gly Ser Trp Tyr Ile Asp Glu Pro Gln Gly 20 25
30Gly Glu Glu Leu Gln Pro Glu Gly Glu Val Pro Ser Cys His Thr 35 40
45Ser Ile Pro Pro Gly Leu Tyr His Ala Cys Leu Ala Ser Leu Ser 50 55
60Ile Leu Val Leu Leu Leu Leu Ala Met Leu Val Arg Arg Arg Gln 65 70
75Leu Trp Pro Asp Cys Val Arg Gly Arg Pro Gly Leu Pro Ser Pro 80 85
90Val Asp Phe Leu Ala Gly Asp Arg Pro Arg Ala Val Pro Ala Ala 95
100 105Val Phe Met Val Leu Leu Ser Ser Leu Cys Leu Leu Leu Pro Asp
110 115 120Glu Asp Ala Leu Pro Phe Leu Thr Leu Ala Ser Ala Pro Ser
Gln 125 130 135Asp Gly Lys Thr Glu Ala Pro Arg Gly Ala Trp Lys Ile
Leu Gly 140 145 150Leu Phe Tyr Tyr Ala Ala Leu Tyr Tyr Pro Leu Ala
Ala Cys Ala 155 160 165Thr Ala Gly His Thr Ala Ala His Leu Leu Gly
Ser Thr Leu Ser 170 175 180Trp Ala His Leu Gly Val Gln Val Trp Gln
Arg Ala Glu Cys Pro 185 190 195Gln Val Pro Lys Ile Tyr Lys Tyr Tyr
Ser Leu Leu Ala Ser Leu 200 205 210Pro Leu Leu Leu Gly Leu Gly Phe
Leu Ser Leu Trp Tyr Pro Val 215 220 225Gln Leu Val Arg Ser Phe Ser
Arg Arg Thr Gly Ala Gly Ser Lys 230 235 240Gly Leu Gln Ser Ser Tyr
Ser Glu Glu Tyr Leu Arg Asn Leu Leu 245 250 255Cys Arg Lys Lys Leu
Gly Ser Ser Tyr His Thr Ser Lys His Gly 260 265 270Phe Leu Ser Trp
Ala Arg Val Cys Leu Arg His Cys Ile Tyr Thr 275 280 285Pro Gln Pro
Gly Phe His Leu Pro Leu Lys Leu Val Leu Ser Ala 290 295 300Thr Leu
Thr Gly Thr Ala Ile Tyr Gln Val Ala Leu Leu Leu Leu 305 310 315Val
Gly Val Val Pro Thr Ile Gln Lys Val Arg Ala Gly Val Thr 320 325
330Thr Asp Val Ser Tyr Leu Leu Ala Gly Phe Gly Ile Val Leu Ser 335
340 345Glu Asp Lys Gln Glu Val Val Glu Leu Val Lys His His Leu Trp
350 355 360Ala Leu Glu Val Cys Tyr Ile Ser Ala Leu Val Leu Ser Cys
Leu 365 370 375Leu Thr Phe Leu Val Leu Met Arg Ser Leu Val Thr His
Arg Thr 380 385 390Asn Leu Arg Ala Leu His Arg Gly Ala Ala Leu Asp
Leu Ser Pro 395 400 405Leu His Arg Ser Pro His Pro Ser Arg Gln Ala
Ile Phe Cys Trp 410 415 420Met Ser Phe Ser Ala Tyr Gln Thr Ala Phe
Ile Cys Leu Gly Leu 425 430 435Leu Val Gln Gln Ile Ile Phe Phe Leu
Gly Thr Thr Ala Leu Ala 440 445 450Phe Leu Val Leu Met Pro Val Leu
His Gly Arg Asn Leu Leu Leu 455 460 465Phe Arg Ser Leu Glu Ser Ser
Trp Pro Phe Trp Leu Thr Leu Ala 470 475 480Leu Ala Val Ile Leu Gln
Asn Met Ala Ala His Trp Val Phe Leu 485 490 495Glu Thr His Asp Gly
His Pro Gln Leu Thr Asn Arg Arg Val Leu 500 505 510Tyr Ala Ala Thr
Phe Leu Leu Phe Pro Leu Asn Val Leu Val Gly 515 520 525Ala Met Val
Ala Thr Trp Arg Val Leu Leu Ser Ala Leu Tyr Asn 530 535 540Ala Ile
His Leu Gly Gln Met Asp Leu Ser Leu Leu Pro Pro Arg 545 550 555Ala
Ala Thr Leu Asp Pro Gly Tyr Tyr Thr Tyr Arg Asn Phe Leu 560 565
570Lys Ile Glu Val Ser Gln Ser His Pro Ala Met Thr Ala Phe Cys 575
580 585Ser Leu Leu Leu Gln Ala Gln Ser Leu Leu Pro Arg Thr Met Ala
590 595 600Ala Pro Gln Asp Ser Leu Arg Pro Gly Glu Glu Asp Glu Gly
Met 605 610 615Gln Leu Leu Gln Thr Lys Asp Ser Met Ala Lys Gly Ala
Arg Pro 620 625 630Gly Ala Ser Arg Gly Arg Ala Arg Trp Gly Leu Ala
Tyr Thr Leu 635 640 645Leu His Asn Pro Thr Leu Gln Val Phe Arg Lys
Thr Ala Leu Leu 650 655 660Gly Ala Asn Gly Ala Gln Pro
665634834DNAHomo sapiens 63gatgtggagc tggggtccct gcaagtcatg
aacaaaacga gaaagattat 50ggaacatggg ggggccacct tcatcaatgc ctttgtgact
acacccatgt 100gctgcccgtc acggtcctcc atgctcaccg ggaagtatgt
gcacaatcac 150aatgtctaca ccaacaacga gaactgctct tccccctcgt
ggcaggccat 200gcatgagcct cggacttttg ctgtatatct taacaacact
ggctacagaa 250cagccttttt tggaaaatac ctcaatgaat ataatggcag
ctacatcccc 300cctgggtggc gagaatggct tggattaatc aagaattctc
gcttctataa 350ttacactgtt tgtcgcaatg gcatcaaaga aaagcatgga
tttgattatg 400caaaggacta cttcacagac ttaatcacta acgagagcat
taattacttc 450aaaatgtcta agagaatgta tccccatagg cccgttatga
tggtgatcag 500ccacgctgcg ccccacggcc ccgaggactc agccccacag
ttttctaaac 550tgtaccccaa tgcttcccaa cacataactc ctagttataa
ctatgcacca 600aatatggata aacactggat tatgcagtac acaggaccaa
tgctgcccat 650ccacatggaa tttacaaaca ttctacagcg caaaaggctc
cagactttga 700tgtcagtgga tgattctgtg gagaggctgt ataacatgct
cgtggagacg 750ggggagctgg agaatactta catcatttac accgccgacc
atggttacca 800tattgggcag tttggactgg tcaaggggaa atccatgcca
tatgactttg 850atattcgtgt gccttttttt attcgtggtc caagtgtaga
accaggatca 900atagtcccac agatcgttct caacattgac ttggccccca
cgatcctgga 950tattgctggg ctcgacacac ctcctgatgt ggacggcaag
tctgtcctca 1000aacttctgga cccagaaaag ccaggtaaca ggtttcgaac
aaacaagaag 1050gccaaaattt ggcgtgatac attcctagtg gaaagaggca
aatttctacg
1100taagaaggaa gaatccagca agaatatcca acagtcaaat cacttgccca
1150aatatgaacg ggtcaaagaa ctatgccagc aggccaggta ccagacagcc
1200tgtgaacaac cggggcagaa gtggcaatgc attgaggata catctggcaa
1250gcttcgaatt cacaagtgta aaggacccag tgacctgctc acagtccggc
1300agagcacgcg gaacctctac gctcgcggct tccatgacaa agacaaagag
1350tgcagttgta gggagtctgg ttaccgtgcc agcagaagcc aaagaaagag
1400tcaacggcaa ttcttgagaa accaggggac tccaaagtac aagcccagat
1450ttgtccatac tcggcagaca cgttccttgt ccgtcgaatt tgaaggtgaa
1500atatatgaca taaatctgga agaagaagaa gaattgcaag tgttgcaacc
1550aagaaacatt gctaagcgtc atgatgaagg ccacaagggg ccaagagatc
1600tccaggcttc cagtggtggc aacaggggca ggatgctggc agatagcagc
1650aacgccgtgg gcccacctac cactgtccga gtgacacaca agtgttttat
1700tcttcccaat gactctatcc attgtgagag agaactgtac caatcggcca
1750gagcgtggaa ggaccataag gcatacattg acaaagagat tgaagctctg
1800caagataaaa ttaagaattt aagagaagtg agaggacatc tgaagagaag
1850gaagcctgag gaatgtagct gcagtaaaca aagctattac aataaagaga
1900aaggtgtaaa aaagcaagag aaattaaaga gccatcttca cccattcaag
1950gaggctgctc aggaagtaga tagcaaactg caacttttca aggagaacaa
2000ccgtaggagg aagaaggaga ggaaggagaa gagacggcag aggaaggggg
2050aagagtgcag cctgcctggc ctcacttgct tcacgcatga caacaaccac
2100tggcagacag ccccgttctg gaacctggga tctttctgtg cttgcacgag
2150ttctaacaat aacacctact ggtgtttgcg tacagttaat gagacgcata
2200attttctttt ctgtgagttt gctactggct ttttggagta ttttgatatg
2250aatacagatc cttatcagct cacaaataca gtgcacacgg tagaacgagg
2300cattttgaat cagctacacg tacaactaat ggagctcaga agctgtcaag
2350gatataagca gtgcaaccca agacctaaga atcttgatgt tggaaataaa
2400gatggaggaa gctatgacct acacagagga cagttatggg atggatggga
2450aggttaatca gccccgtctc actgcagaca tcaactggca aggcctagag
2500gagctacaca gtgtgaatga aaacatctat gagtacagac aaaactacag
2550acttagtctg gtggactgga ctaattactt gaaggattta gatagagtat
2600ttgcactgct gaagagtcac tatgagcaaa ataaaacaaa taagactcaa
2650actgctcaaa gtgacgggtt cttggttgtc tctgctgagc acgctgtgtc
2700aatggagatg gcctctgctg actcagatga agacccaagg cataaggttg
2750ggaaaacacc tcatttgacc ttgccagctg accttcaaac cctgcatttg
2800aaccgaccaa cattaagtcc agagagtaaa cttgaatgga ataacgacat
2850tccagaagtt aatcatttga attctgaaca ctggagaaaa accgaaaaat
2900ggacggggca tgaagagact aatcatctgg aaaccgattt cagtggcgat
2950ggcatgacag agctagagct cgggcccagc cccaggctgc agcccattcg
3000caggcacccg aaagaacttc cccagtatgg tggtcctgga aaggacattt
3050ttgaagatca actatatctt cctgtgcatt ccgatggaat ttcagttcat
3100cagatgttca ccatggccac cgcagaacac cgaagtaatt ccagcatagc
3150ggggaagatg ttgaccaagg tggagaagaa tcacgaaaag gagaagtcac
3200agcacctaga aggcagcgcc tcctcttcac tctcctctga ttagatgaaa
3250ctgttacctt accctaaaca cagtatttct ttttaacttt tttatttgta
3300aactaataaa ggtaatcaca gccaccaaca ttccaagcta ccctgggtac
3350ctttgtgcag tagaagctag tgagcatgtg agcaagcggt gtgcacacgg
3400agactcatcg ttataattta ctatctgcca agagtagaaa gaaaggctgg
3450ggatatttgg gttggcttgg ttttgatttt ttgcttgttt gtttgttttg
3500tactaaaaca gtattatctt ttgaatatcg tagggacata agtatataca
3550tgttatccaa tcaagatggc tagaatggtg cctttctgag tgtctaaaac
3600ttgacacccc tggtaaatct ttcaacacac ttccactgcc tgcgtaatga
3650agttttgatt catttttaac cactggaatt tttcaatgcc gtcattttca
3700gttagatgat tttgcacttt gagattaaaa tgccatgtct atttgattag
3750tcttattttt ttatttttac aggcttatca gtctcactgt tggctgtcat
3800tgtgacaaag tcaaataaac ccccaaggac gacacacagt atggatcaca
3850tattgtttga cattaagctt ttgccagaaa atgttgcatg tgttttacct
3900cgacttgcta aaatcgatta gcagaaaggc atggctaata atgttggtgg
3950tgaaaataaa taaataagta aacaaaatga agattgcctg ctctctctgt
4000gcctagcctc aaagcgttca tcatacatca tacctttaag attgctatat
4050tttgggttat tttcttgaca ggagaaaaag atctaaagat cttttatttt
4100catctttttt ggttttcttg gcatgactaa gaagcttaaa tgttgataaa
4150atatgactag ttttgaattt acaccaagaa cttctcaata aaagaaaatc
4200atgaatgctc cacaatttca acataccaca agagaagtta atttcttaac
4250attgtgttct atgattattt gtaagacctt caccaagttc tgatatcttt
4300taaagacata gttcaaaatt gcttttgaaa atctgtattc ttgaaaatat
4350ccttgttgtg tattaggttt ttaaatacca gctaaaggat tacctcactg
4400agtcatcagt accctcctat tcagctcccc aagatgatgt gtttttgctt
4450accctaagag aggttttctt cttattttta gataattcaa gtgcttagat
4500aaattatgtt ttctttaagt gtttatggta aactctttta aagaaaattt
4550aatatgttat agctgaatct ttttggtaac tttaaatctt tatcatagac
4600tctgtacata tgttcaaatt agctgcttgc ctgatgtgtg tatcatcggt
4650gggatgacag aacaaacata tttatgatca tgaataatgt gctttgtaaa
4700aagatttcaa gttattagga agcatactct gttttttaat catgtataat
4750attccatgat acttttatag aacaattctg gcttcaggaa agtctagaag
4800caatatttct tcaaataaaa ggtgtttaaa cttt 483464871PRTHomo sapiens
64Met Lys Tyr Ser Cys Cys Ala Leu Val Leu Ala Val Leu Gly Thr1 5 10
15Glu Leu Leu Gly Ser Leu Cys Ser Thr Val Arg Ser Pro Arg Phe 20 25
30Arg Gly Arg Ile Gln Gln Glu Arg Lys Asn Ile Arg Pro Asn Ile 35 40
45Ile Leu Val Leu Thr Asp Asp Gln Asp Val Glu Leu Gly Ser Leu 50 55
60Gln Val Met Asn Lys Thr Arg Lys Ile Met Glu His Gly Gly Ala 65 70
75Thr Phe Ile Asn Ala Phe Val Thr Thr Pro Met Cys Cys Pro Ser 80 85
90Arg Ser Ser Met Leu Thr Gly Lys Tyr Val His Asn His Asn Val 95
100 105Tyr Thr Asn Asn Glu Asn Cys Ser Ser Pro Ser Trp Gln Ala Met
110 115 120His Glu Pro Arg Thr Phe Ala Val Tyr Leu Asn Asn Thr Gly
Tyr 125 130 135Arg Thr Ala Phe Phe Gly Lys Tyr Leu Asn Glu Tyr Asn
Gly Ser 140 145 150Tyr Ile Pro Pro Gly Trp Arg Glu Trp Leu Gly Leu
Ile Lys Asn 155 160 165Ser Arg Phe Tyr Asn Tyr Thr Val Cys Arg Asn
Gly Ile Lys Glu 170 175 180Lys His Gly Phe Asp Tyr Ala Lys Asp Tyr
Phe Thr Asp Leu Ile 185 190 195Thr Asn Glu Ser Ile Asn Tyr Phe Lys
Met Ser Lys Arg Met Tyr 200 205 210Pro His Arg Pro Val Met Met Val
Ile Ser His Ala Ala Pro His 215 220 225Gly Pro Glu Asp Ser Ala Pro
Gln Phe Ser Lys Leu Tyr Pro Asn 230 235 240Ala Ser Gln His Ile Thr
Pro Ser Tyr Asn Tyr Ala Pro Asn Met 245 250 255Asp Lys His Trp Ile
Met Gln Tyr Thr Gly Pro Met Leu Pro Ile 260 265 270His Met Glu Phe
Thr Asn Ile Leu Gln Arg Lys Arg Leu Gln Thr 275 280 285Leu Met Ser
Val Asp Asp Ser Val Glu Arg Leu Tyr Asn Met Leu 290 295 300Val Glu
Thr Gly Glu Leu Glu Asn Thr Tyr Ile Ile Tyr Thr Ala 305 310 315Asp
His Gly Tyr His Ile Gly Gln Phe Gly Leu Val Lys Gly Lys 320 325
330Ser Met Pro Tyr Asp Phe Asp Ile Arg Val Pro Phe Phe Ile Arg 335
340 345Gly Pro Ser Val Glu Pro Gly Ser Ile Val Pro Gln Ile Val Leu
350 355 360Asn Ile Asp Leu Ala Pro Thr Ile Leu Asp Ile Ala Gly Leu
Asp 365 370 375Thr Pro Pro Asp Val Asp Gly Lys Ser Val Leu Lys Leu
Leu Asp 380 385 390Pro Glu Lys Pro Gly Asn Arg Phe Arg Thr Asn Lys
Lys Ala Lys 395 400 405Ile Trp Arg Asp Thr Phe Leu Val Glu Arg Gly
Lys Phe Leu Arg 410 415 420Lys Lys Glu Glu Ser Ser Lys Asn Ile Gln
Gln Ser Asn His Leu 425 430 435Pro Lys Tyr Glu Arg Val Lys Glu Leu
Cys Gln Gln Ala Arg Tyr 440 445 450Gln Thr Ala Cys Glu Gln Pro Gly
Gln Lys Trp Gln Cys Ile Glu 455 460 465Asp Thr Ser Gly Lys Leu Arg
Ile His Lys Cys Lys Gly Pro Ser 470 475 480Asp Leu Leu Thr Val Arg
Gln Ser Thr Arg Asn Leu Tyr Ala Arg 485 490 495Gly Phe His Asp Lys
Asp Lys Glu Cys Ser Cys Arg Glu Ser Gly 500 505 510Tyr Arg Ala Ser
Arg Ser Gln Arg Lys Ser Gln Arg Gln Phe Leu 515 520 525Arg Asn Gln
Gly Thr Pro Lys Tyr Lys Pro Arg Phe Val His Thr 530 535 540Arg Gln
Thr Arg Ser Leu Ser Val Glu Phe Glu Gly Glu Ile Tyr 545 550 555Asp
Ile Asn Leu Glu Glu Glu Glu Glu Leu Gln Val Leu Gln Pro 560 565
570Arg Asn Ile Ala Lys Arg His Asp Glu Gly His Lys Gly Pro Arg 575
580 585Asp Leu Gln Ala Ser Ser Gly Gly Asn Arg Gly Arg Met Leu Ala
590 595 600Asp Ser Ser Asn Ala Val Gly Pro Pro Thr Thr Val Arg Val
Thr 605 610 615His Lys Cys Phe Ile Leu Pro Asn Asp Ser Ile His Cys
Glu Arg 620 625 630Glu Leu Tyr Gln Ser Ala Arg Ala Trp Lys Asp His
Lys Ala Tyr 635 640 645Ile Asp Lys Glu Ile Glu Ala Leu Gln Asp Lys
Ile Lys Asn Leu 650 655 660Arg Glu Val Arg Gly His Leu Lys Arg Arg
Lys Pro Glu Glu Cys 665 670 675Ser Cys Ser Lys Gln Ser Tyr Tyr Asn
Lys Glu Lys Gly Val Lys 680 685 690Lys Gln Glu Lys Leu Lys Ser His
Leu His Pro Phe Lys Glu Ala 695 700 705Ala Gln Glu Val Asp Ser Lys
Leu Gln Leu Phe Lys Glu Asn Asn 710 715 720Arg Arg Arg Lys Lys Glu
Arg Lys Glu Lys Arg Arg Gln Arg Lys 725 730 735Gly Glu Glu Cys Ser
Leu Pro Gly Leu Thr Cys Phe Thr His Asp 740 745 750Asn Asn His Trp
Gln Thr Ala Pro Phe Trp Asn Leu Gly Ser Phe 755 760 765Cys Ala Cys
Thr Ser Ser Asn Asn Asn Thr Tyr Trp Cys Leu Arg 770 775 780Thr Val
Asn Glu Thr His Asn Phe Leu Phe Cys Glu Phe Ala Thr 785 790 795Gly
Phe Leu Glu Tyr Phe Asp Met Asn Thr Asp Pro Tyr Gln Leu 800 805
810Thr Asn Thr Val His Thr Val Glu Arg Gly Ile Leu Asn Gln Leu 815
820 825His Val Gln Leu Met Glu Leu Arg Ser Cys Gln Gly Tyr Lys Gln
830 835 840Cys Asn Pro Arg Pro Lys Asn Leu Asp Val Gly Asn Lys Asp
Gly 845 850 855Gly Ser Tyr Asp Leu His Arg Gly Gln Leu Trp Asp Gly
Trp Glu 860 865 870Gly651662DNAHomo sapiens 65gcggccgcgt cgaccgggcc
ctgcgggcgc ggggctgaag gcggaaccac 50gacgggcaga gagcacggag ccgggaagcc
cctgggcgcc cgtcggaggg 100ctatggagca gcggccgcgg ggctgcgcgg
cggtggcggc ggcgctcctc 150ctggtgctgc tgggggcccg ggcccagggc
ggcactcgta gccccaggtg 200tgactgtgcc ggtgacttcc acaagaagat
tggtctgttt tgttgcagag 250gctgcccagc ggggcactac ctgaaggccc
cttgcacgga gccctgcggc 300aactccacct gccttgtgtg tccccaagac
accttcttgg cctgggagaa 350ccaccataat tctgaatgtg cccgctgcca
ggcctgtgat gagcaggcct 400cccaggtggc gctggagaac tgttcagcag
tggccgacac ccgctgtggc 450tgtaagccag gctggtttgt ggagtgccag
gtcagccaat gtgtcagcag 500ttcacccttc tactgccaac catgcctaga
ctgcggggcc ctgcaccgcc 550acacacggct actctgttcc cgcagagata
ctgactgtgg gacctgcctg 600cctggcttct atgaacatgg cgatggctgc
gtgtcctgcc ccacgagcac 650cctggggagc tgtccagagc gctgtgccgc
tgtctgtggc tggaggcaga 700tgttctgggt ccaggtgctc ctggctggcc
ttgtggtccc cctcctgctt 750ggggccaccc tgacctacac ataccgccac
tgctggcctc acaagcccct 800ggttactgca gatgaagctg ggatggaggc
tctgacccca ccaccggcca 850cccatctgtc acccttggac agcgcccaca
cccttctagc acctcctgac 900agcagtgaga agatctgcac cgtccagttg
gtgggtaaca gctggacccc 950tggctacccc gagacccagg aggcgctctg
cccgcaggtg acatggtcct 1000gggaccagtt gcccagcaga gctcttggcc
ccgctgctgc gcccacactc 1050tcgccagagt ccccagccgg ctcgccagcc
atgatgctgc agccgggccc 1100gcagctctac gacgtgatgg acgcggtccc
agcgcggcgc tggaaggagt 1150tcgtgcgcac gctggggctg cgcgaggcag
agatcgaagc cgtggaggtg 1200gagatcggcc gcttccgaga ccagcagtac
gagatgctca agcgctggcg 1250ccagcagcag cccgcgggcc tcggagccgt
ttacgcggcc ctggagcgca 1300tggggctgga cggctgcgtg gaagacttgc
gcagccgcct gcagcgcggc 1350ccgtgacacg gcgcccactt gccacctagg
cgctctggtg gcccttgcag 1400aagccctaag tacggttact tatgcgtgta
gacattttat gtcacttatt 1450aagccgctgg cacggccctg cgtagcagca
ccagccggcc ccacccctgc 1500tcgcccctat cgctccagcc aaggcgaaga
agcacgaacg aatgtcgaga 1550gggggtgaag acatttctca acttctcggc
cggagtttgg ctgagatcgc 1600ggtattaaat ctgtgaaaga aaacaaaaaa
aaaaaaaaaa aaaaaaaagt 1650cgacgcggcc gc 166266417PRTHomo sapiens
66Met Glu Gln Arg Pro Arg Gly Cys Ala Ala Val Ala Ala Ala Leu1 5 10
15Leu Leu Val Leu Leu Gly Ala Arg Ala Gln Gly Gly Thr Arg Ser 20 25
30Pro Arg Cys Asp Cys Ala Gly Asp Phe His Lys Lys Ile Gly Leu 35 40
45Phe Cys Cys Arg Gly Cys Pro Ala Gly His Tyr Leu Lys Ala Pro 50 55
60Cys Thr Glu Pro Cys Gly Asn Ser Thr Cys Leu Val Cys Pro Gln 65 70
75Asp Thr Phe Leu Ala Trp Glu Asn His His Asn Ser Glu Cys Ala 80 85
90Arg Cys Gln Ala Cys Asp Glu Gln Ala Ser Gln Val Ala Leu Glu 95
100 105Asn Cys Ser Ala Val Ala Asp Thr Arg Cys Gly Cys Lys Pro Gly
110 115 120Trp Phe Val Glu Cys Gln Val Ser Gln Cys Val Ser Ser Ser
Pro 125 130 135Phe Tyr Cys Gln Pro Cys Leu Asp Cys Gly Ala Leu His
Arg His 140 145 150Thr Arg Leu Leu Cys Ser Arg Arg Asp Thr Asp Cys
Gly Thr Cys 155 160 165Leu Pro Gly Phe Tyr Glu His Gly Asp Gly Cys
Val Ser Cys Pro 170 175 180Thr Ser Thr Leu Gly Ser Cys Pro Glu Arg
Cys Ala Ala Val Cys 185 190 195Gly Trp Arg Gln Met Phe Trp Val Gln
Val Leu Leu Ala Gly Leu 200 205 210Val Val Pro Leu Leu Leu Gly Ala
Thr Leu Thr Tyr Thr Tyr Arg 215 220 225His Cys Trp Pro His Lys Pro
Leu Val Thr Ala Asp Glu Ala Gly 230 235 240Met Glu Ala Leu Thr Pro
Pro Pro Ala Thr His Leu Ser Pro Leu 245 250 255Asp Ser Ala His Thr
Leu Leu Ala Pro Pro Asp Ser Ser Glu Lys 260 265 270Ile Cys Thr Val
Gln Leu Val Gly Asn Ser Trp Thr Pro Gly Tyr 275 280 285Pro Glu Thr
Gln Glu Ala Leu Cys Pro Gln Val Thr Trp Ser Trp 290 295 300Asp Gln
Leu Pro Ser Arg Ala Leu Gly Pro Ala Ala Ala Pro Thr 305 310 315Leu
Ser Pro Glu Ser Pro Ala Gly Ser Pro Ala Met Met Leu Gln 320 325
330Pro Gly Pro Gln Leu Tyr Asp Val Met Asp Ala Val Pro Ala Arg 335
340 345Arg Trp Lys Glu Phe Val Arg Thr Leu Gly Leu Arg Glu Ala Glu
350 355 360Ile Glu Ala Val Glu Val Glu Ile Gly Arg Phe Arg Asp Gln
Gln 365 370 375Tyr Glu Met Leu Lys Arg Trp Arg Gln Gln Gln Pro Ala
Gly Leu 380 385 390Gly Ala Val Tyr Ala Ala Leu Glu Arg Met Gly Leu
Asp Gly Cys 395 400 405Val Glu Asp Leu Arg Ser Arg Leu Gln Arg Gly
Pro 410 4156743DNAArtificial sequenceoligonucleotide probe
67tgtaaaacga cggccagtta aatagacctg caattattaa tct
436841DNAArtificial sequenceoligonucleotide probe 68caggaaacag
ctatgaccac ctgcacacct gcaaatccat t 416933DNAArtificial
sequenceoligonucleotide probe 69gctgacgaac caaggcaact acaaactcct
ggt 337041DNAArtificial sequenceoligonucleotide probe 70tgcggccgga
ccagtcctcc atggtcacca ggagtttgta g 417133DNAArtificial
sequenceoligonucleotide probe 71ggtggtgaac tgcttgccgt tgtgccatgt
aaa 337222DNAArtificial sequenceoligonucleotide probe 72aaagacgcat
ctgcgagtgt cc 227323DNAArtificial sequenceoligonucleotide probe
73tgctgatttc acactgctct ccc 237449DNAArtificial
sequenceoligonucleotide probe 74cccacgatgt atgaatggtg gactttgtgt
gactcctggt ttctgcatc 497524DNAArtificial sequenceoligonucleotide
probe 75ccaactacca aagctgctgg agcc 247624DNAArtificial
sequenceoligonucleotide probe 76gcagctctat taccacggga agga
247724DNAArtificial sequenceoligonucleotide probe 77tccttcccgt
ggtaatagag ctgc 247845DNAArtificial sequenceoligonucleotide probe
78ggcagagaac cagaggccgg aggagactgc ctctttacag ccagg
457924DNAArtificial sequenceoligonucleotide probe 79tggctactcc
aagaccctgg catg 248024DNAArtificial sequenceoligonucleotide probe
80tggacaaatc cccttgctca gccc 248150DNAArtificial
sequenceoligonucleotide probe 81gggcttcacc gaagcagtgg acctttattt
tgaccacctg atgtccaggg 508222DNAArtificial sequenceoligonucleotide
probe 82atcgttgtga agttagtgcc cc 228323DNAArtificial
sequenceoligonucleotide probe 83acctgcgata tccaacagaa ttg
238448DNAArtificial sequenceoligonucleotide probe 84ggaagaggat
acagtcactc tggaagtatt agtggctcca gcagttcc 488522DNAArtificial
sequenceoligonucleotide probe 85agggtctcca ggagaaagac tc
228624DNAArtificial sequenceoligonucleotide probe 86attgtgggcc
ttgcagacat agac 248750DNAArtificial sequenceoligonucleotide probe
87ggccacagca tcaaaacctt agaactcaat gtactggttc ctccagctcc
508822DNAArtificial sequenceoligonucleotide probe 88gctaggaatt
ccacagaagc cc 228922DNAArtificial sequenceoligonucleotide probe
89aacctggaat gtcaccgagc tg 229026DNAArtificial
sequenceoligonucleotide probe 90cctagcacag tgacgaggga cttggc
269150DNAArtificial sequenceoligonucleotide probe 91aagacacagc
caccctaaac tgtcagtctt ctgggagcaa gcctgcagcc 509250DNAArtificial
sequenceoligonucleotide probe 92gccctggcag acgagggcga gtacacctgc
tcaatcttca ctatgcctgt 509322DNAArtificial sequenceoligonucleotide
probe 93ccgattcata gacctcgaga gt 229422DNAArtificial
sequenceoligonucleotide probe 94gtcaaggagt cctccacaat ac
229545DNAArtificial sequenceoligonucleotide probe 95gtgtacaatg
gccatgccaa tggccagcgc attggccgct tctgt 459619DNAArtificial
sequenceoligonucleotide probe 96tcctgcagtt tcctgatgc
199724DNAArtificial sequenceoligonucleotide probe 97ctcatattgc
acaccagtaa ttcg 249845DNAArtificial sequenceoligonucleotide probe
98atgaggagaa acgtttgatg gtggagctgc acaacctcta ccggg
459924DNAArtificial sequenceoligonucleotide probe 99tacaggccca
gtcaggacca gggg 2410018DNAArtificial sequenceoligonucleotide probe
100agccagcctc gctctcgg 1810118DNAArtificial sequenceoligonucleotide
probe 101gtctgcgatc aggtctgg 1810220DNAArtificial
sequenceoligonucleotide probe 102gaaagaggca atggattcgc
2010324DNAArtificial sequenceoligonucleotide probe 103gacttacact
tgccagcaca gcac 2410445DNAArtificial sequenceoligonucleotide probe
104ggagcaccac caactggagg gtccggagta gcgagcgccc cgaag
4510522DNAArtificial sequenceoligonucleotide probe 105ccctccactg
ccccaccgac tg 2210624DNAArtificial sequenceoligonucleotide probe
106cggttctggg gacgttaggg ctcg 2410725DNAArtificial
sequenceoligonucleotide probe 107ctgcccaccg tccacctgcc tcaat
2510845DNAArtificial sequenceoligonucleotide probe 108aggactgccc
accgtccacc tgcctcaatg ggggcacatg ccacc 4510945DNAArtificial
sequenceoligonucleotide probe 109acgcaaagcc ctacatctaa gccagagaga
gacagggcag ctggg 4511024DNAArtificial sequenceoligonucleotide probe
110tggctgccct gcagtacctc tacc 2411124DNAArtificial
sequenceoligonucleotide probe 111ccctgcaggt cattggcagc tagg
2411245DNAArtificial sequenceoligonucleotide probe 112aggcactgcc
tgatgacacc ttccgcgacc tgggcaacct cacac 4511320DNAArtificial
sequenceoligonucleotide probe 113ggctgtcact gtggagacac
2011418DNAArtificial sequenceoligonucleotide probe 114gcaaggtcat
tacagctg 1811523DNAArtificial sequenceoligonucleotide probe
115agaacatagg agcagtccca ctc 2311623DNAArtificial
sequenceoligonucleotide probe 116tgcctgctgc tgcacaatct cag
2311745DNAArtificial sequenceoligonucleotide probe 117ggctattgct
tgccttggga cagaccctgt ggcttaggct ctggc 4511824DNAArtificial
sequenceoligonucleotide probe 118gtaagcacat gcctccagag gtgc
2411924DNAArtificial sequenceoligonucleotide probe 119gtgacgtgga
tgcttgggat gttg 2412050DNAArtificial sequenceoligonucleotide probe
120tggacacctt cagtattgat gccaagacag gccaggtcat tctgcgtcga
5012122DNAArtificial sequenceoligonucleotide probe 121atgtttgtgt
ggaagtgccc cg 2212218DNAArtificial sequenceoligonucleotide probe
122gtcaacatgc tcctctgc 1812326DNAArtificial sequenceoligonucleotide
probe 123aatccattgt gcactgcagc tctagg 2612423DNAArtificial
sequenceoligonucleotide probe 124gagcatgcca ccactggact gac
2312554DNAArtificial sequenceoligonucleotide probe 125gccgatgctg
tcctagtgga aacaactcca ctgtaactag attgatctat 50gcac
5412625DNAArtificial sequenceoligonucleotide probe 126ccagacgctg
ctcttcgaaa gggtc 2512723DNAArtificial sequenceoligonucleotide probe
127ggtccccgta ggccaggtcc agc 2312850DNAArtificial
sequenceoligonucleotide probe 128ctacttcttc agcctcaatg tgcacagctg
gaattacaag gagacgtacg 5012924DNAArtificial sequenceoligonucleotide
probe 129gctgctttgc tcacaactgc tcgc 2413018DNAArtificial
sequenceoligonucleotide probe 130catgacacct tcctgctg
1813124DNAArtificial sequenceoligonucleotide probe 131cagccatggg
tgactgtgac ctcc 2413224DNAArtificial sequenceoligonucleotide probe
132ctcctgggag tcggtagcaa cacc 2413318DNAArtificial
sequenceoligonucleotide probe 133gggaggtcac agtcaccc
1813423DNAArtificial sequenceoligonucleotide probe 134ggctgggctt
tccaccctgg cac 2313540DNAArtificial sequenceoligonucleotide probe
135cagccatggg tgactgtgac ctccctgagt tttgcacggg 4013621DNAArtificial
sequenceoligonucleotide probe 136gagctgaagt cagcctttga g
2113720DNAArtificial sequenceoligonucleotide probe 137ctctgcagaa
gtctcgttcc 2013819DNAArtificial sequenceoligonucleotide probe
138ctgaccggtc cgctcatgg 1913921DNAArtificial
sequenceoligonucleotide probe 139cagcatgctt tccgcgaagt c
2114046DNAArtificial sequenceoligonucleotide probe 140ggcaggaagg
ccaggggtgc tgagttcttc acctcctttt agactg 4614127DNAArtificial
sequenceoligonucleotide probe 141ggcgctctgg tggcccttgc agaagcc
2714225DNAArtificial sequenceoligonucleotide probe 142ttcggccgag
aagttgagaa atgtc 25
* * * * *
References