U.S. patent application number 09/908093 was filed with the patent office on 2003-01-23 for secreted and transmembrane polypeptides and nucleic acids encoding the same.
This patent application is currently assigned to Genentech, Inc.. Invention is credited to Ashkenazi, Avi, Botstein, David, Desnoyers, Luc, Eaton, Dan L., Ferrara, Napoleone, Filvaroff, Ellen, Fong, Sherman, Gao, Wei-Qiang, Gerber, Hanspeter, Gerritsen, Mary E., Goddard, Audrey, Godowski, Paul J., Grimaldi, J. Christopher, Gurney, Austin L., Hillan, Kenneth J., Kljavin, Ivar J., Mather, Jennie P., Pan, James, Paoni, Nicholas F., Roy, Margaret Ann, Stewart, Timothy A., Tumas, Daniel, Williams, P. Mickey, Wood, William I..
Application Number | 20030017498 09/908093 |
Document ID | / |
Family ID | 43706158 |
Filed Date | 2003-01-23 |
United States Patent
Application |
20030017498 |
Kind Code |
A1 |
Ashkenazi, Avi ; et
al. |
January 23, 2003 |
Secreted and transmembrane polypeptides and nucleic acids encoding
the same
Abstract
The present invention is directed to novel polypeptides and to
nucleic acid molecules encoding those polypeptides. Also provided
herein are vectors and host cells comprising those nucleic acid
sequences, chimeric polypeptide molecules comprising the
polypeptides of the present invention fused to heterologous
polypeptide sequences, antibodies which bind to the polypeptides of
the present invention and to methods for producing the polypeptides
of the present invention.
Inventors: |
Ashkenazi, Avi; (San Mateo,
CA) ; Botstein, David; (Belmont, CA) ;
Desnoyers, Luc; (San Francisco, CA) ; Eaton, Dan
L.; (San Rafael, CA) ; Ferrara, Napoleone;
(San Francisco, CA) ; Filvaroff, Ellen; (San
Francisco, CA) ; Fong, Sherman; (Alameda, CA)
; Gao, Wei-Qiang; (Palo Alto, CA) ; Gerber,
Hanspeter; (San Francisco, CA) ; Gerritsen, Mary
E.; (San Mateo, CA) ; Goddard, Audrey; (San
Francisco, CA) ; Godowski, Paul J.; (Burlingame,
CA) ; Grimaldi, J. Christopher; (San Francisco,
CA) ; Gurney, Austin L.; (Belmont, CA) ;
Hillan, Kenneth J.; (San Francisco, CA) ; Kljavin,
Ivar J.; (Lafayette, CA) ; Mather, Jennie P.;
(Milllbrae, CA) ; Pan, James; (Belmont, CA)
; Paoni, Nicholas F.; (Belmont, CA) ; Roy,
Margaret Ann; (San Francisco, CA) ; Stewart, Timothy
A.; (San Francisco, CA) ; Tumas, Daniel;
(Orinda, CA) ; Williams, P. Mickey; (Half Moon
Bay, CA) ; Wood, William I.; (Hillsborough,
CA) |
Correspondence
Address: |
KNOBBE, MARTENS, OLSON & BEAR, LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Assignee: |
Genentech, Inc.
|
Family ID: |
43706158 |
Appl. No.: |
09/908093 |
Filed: |
July 17, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09908093 |
Jul 17, 2001 |
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09665350 |
Sep 18, 2000 |
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60059115 |
Sep 17, 1997 |
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60059184 |
Sep 17, 1997 |
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60059122 |
Sep 17, 1997 |
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60059117 |
Sep 17, 1997 |
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60059113 |
Sep 17, 1997 |
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60059121 |
Sep 17, 1997 |
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60059119 |
Sep 17, 1997 |
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60059263 |
Sep 18, 1997 |
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60059266 |
Sep 18, 1997 |
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60062125 |
Oct 15, 1997 |
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60062287 |
Oct 17, 1997 |
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60062285 |
Oct 17, 1997 |
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60063486 |
Oct 21, 1997 |
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60062816 |
Oct 24, 1997 |
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60062814 |
Oct 24, 1997 |
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60063127 |
Oct 24, 1997 |
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60063120 |
Oct 24, 1997 |
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60063121 |
Oct 24, 1997 |
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60063045 |
Oct 24, 1997 |
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60063128 |
Oct 24, 1997 |
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60063329 |
Oct 27, 1997 |
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60063327 |
Oct 27, 1997 |
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60063549 |
Oct 28, 1997 |
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60063541 |
Oct 28, 1997 |
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60063550 |
Oct 28, 1997 |
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60063542 |
Oct 28, 1997 |
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60063544 |
Oct 28, 1997 |
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60063564 |
Oct 28, 1997 |
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60063734 |
Oct 29, 1997 |
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60063738 |
Oct 29, 1997 |
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Oct 29, 1997 |
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60064215 |
Oct 29, 1997 |
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Oct 29, 1997 |
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60063732 |
Oct 29, 1997 |
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60064103 |
Oct 31, 1997 |
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60063870 |
Oct 31, 1997 |
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60064248 |
Nov 3, 1997 |
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Nov 7, 1997 |
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Nov 12, 1997 |
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60065846 |
Nov 17, 1997 |
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60065693 |
Nov 18, 1997 |
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60066120 |
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60069425 |
Dec 12, 1997 |
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Jun 4, 1998 |
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60099803 |
Sep 10, 1998 |
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60100262 |
Sep 14, 1998 |
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60100858 |
Sep 17, 1998 |
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60104080 |
Oct 13, 1998 |
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60109304 |
Nov 20, 1998 |
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60113296 |
Dec 22, 1998 |
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60143048 |
Jul 7, 1999 |
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60145698 |
Jul 26, 1999 |
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Current U.S.
Class: |
435/7.1 ;
435/183; 435/320.1; 435/325; 435/69.1; 514/19.1; 514/19.6;
514/19.8; 514/8.9; 530/350; 530/388.1; 536/23.2 |
Current CPC
Class: |
G01N 33/68 20130101;
G01N 2333/485 20130101; C07K 14/47 20130101 |
Class at
Publication: |
435/7.1 ;
435/69.1; 435/183; 435/325; 435/320.1; 514/12; 530/350; 530/388.1;
536/23.2 |
International
Class: |
G01N 033/53; C07H
021/04; C12N 009/00; C12P 021/02; C12N 005/06; C07K 014/435; A61K
038/17 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 10, 1998 |
WO |
PCT/US98/18824 |
Sep 14, 1998 |
WO |
PCT/US98/19177 |
Sep 16, 1998 |
WO |
PCT/US98/19330 |
Sep 17, 1998 |
WO |
PCT/US98/19437 |
Dec 1, 1998 |
WO |
PCT/US98/25108 |
Sep 8, 1999 |
WO |
PCT/US99/20594 |
Sep 13, 1999 |
WO |
PCT/US99/20944 |
Sep 15, 1999 |
WO |
PCT/US99/21090 |
Sep 15, 1999 |
WO |
PCT/US99/21547 |
Oct 5, 1999 |
WO |
PCT/US99/23089 |
Nov 29, 1999 |
WO |
PCT/US99/28214 |
Nov 30, 1999 |
WO |
PCT/US99/28313 |
Dec 1, 1999 |
WO |
PCT/US99/28301 |
Dec 2, 1999 |
WO |
PCT/US99/28564 |
Dec 2, 1999 |
WO |
PCT/US99/28565 |
Dec 16, 1999 |
WO |
PCT/US99/30095 |
Dec 20, 1999 |
WO |
PCT/US99/30999 |
Dec 20, 1999 |
WO |
PCT/US99/30911 |
Jan 5, 2000 |
WO |
PCT/US00/00219 |
Feb 11, 2000 |
WO |
PCT/US00/03565 |
Feb 22, 2000 |
WO |
PCT/US00/04414 |
Feb 24, 2000 |
WO |
PCT/US00/05004 |
Mar 2, 2000 |
WO |
PCT/US00/05841 |
Mar 20, 2000 |
WO |
PCT/US00/07377 |
Mar 30, 2000 |
WO |
PCT/US00/08439 |
May 22, 2000 |
WO |
PCT/US00/14042 |
Jun 2, 2000 |
WO |
PCT/US00/15264 |
Jul 28, 2000 |
WO |
PCT/US00/20710 |
Aug 24, 2000 |
WO |
PCT/US00/23328 |
Claims
What is claimed is:
1. Isolated nucleic acid having at least 80% sequence identity to a
nucleotide sequence that encodes a polypeptide comprising an amino
acid sequence selected from the group consisting of the amino acid
sequence shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4), FIG.
6 (SEQ ID NO:12), FIG. 9 (SEQ ID NO:18), FIG. 11 (SEQ ID NO:23),
FIG. 13 (SEQ ID NO:28), FIG. 15 (SEQ ID NO:34), FIG. 17 (SEQ ID
NO:39), FIG. 19 (SEQ ID NO:49), FIG. 22 (SEQ ID NO:59), FIG. 24
(SEQ ID NO:64), FIG. 26 (SEQ ID NO:69), FIG. 28 (SEQ ID NO:71),
FIG. 30 (SEQ ID NO:73), FIG. 32 (SEQ ID NO:84), FIG. 34 (SEQ ID
NO:91), FIG. 36 (SEQ ID NO:96), FIG. 38 (SEQ ID NO:104), FIG. 40
(SEQ ID NO:109), FIG. 42 (SEQ ID NO:114), FIG. 44 (SEQ ID NO:119),
FIG. 46 (SEQ ID NO:127), FIG. 48 (SEQ ID NO:132), FIG. 50 (SEQ ID
NO:137), FIG. 52 (SEQ ID NO:142), FIG. 54 (SEQ ID NO:148), FIG. 56
(SEQ ID NO:153), FIG. 58 (SEQ ID NO:159), FIG. 60 (SEQ ID NO:164),
FIG. 62 (SEQ ID NO:170), FIG. 64 (SEQ ID NO:175), FIG. 66 (SEQ ID
NO:177), FIG. 68 (SEQ ID NO:185), FIG. 70 (SEQ ID NO:190), FIG. 72
(SEQ ID NO:195), FIG. 74 (SEQ ID NO:201), FIG. 76 (SEQ ID NO:207),
FIG. 78 (SEQ ID NO:213), FIG. 80 (SEQ ID NO:221), FIG. 82 (SEQ ID
NO:227), FIG. 84 (SEQ ID NO:236), FIG. 86 (SEQ ID NO:245), FIG. 88
(SEQ ID NO:250), FIG. 90 (SEQ ID NO:255), FIG. 92 (SEQ ID NO:257),
FIG. 94 (SEQ ID NO:259), FIG. 96 (SEQ ID NO:261), FIG. 98 (SEQ ID
NO:263), FIG. 100 (SEQ ID NO:285), FIG. 102 (SEQ ID NO:290), FIG.
104 (SEQ ID NO:292), FIG. 106 (SEQ ID NO:294), FIG. 108 (SEQ ID
NO:310), FIG. 110 (SEQ ID NO:315), FIG. 112 (SEQ ID NO:320), FIG.
114 (SEQ ID NO:325), FIG. 116 (SEQ ID NO:332), FIG. 118 (SEQ ID
NO:339), FIG. 120 (SEQ ID NO:341), FIG. 122 (SEQ ID NO:377) and
FIG. 124 (SEQ ID NO:423).
2. The nucleic acid of claim 1, wherein said nucleotide sequence
comprises a nucleotide sequence selected from the group consisting
of the sequence shown in FIG. 1 (SEQ ID NO:1), FIG. 3 (SEQ ID
NO:3), FIG. 5 (SEQ ID NO:11), FIG. 8 (SEQ ID NO:17), FIG. 10 (SEQ
ID NO:22), FIG. 12 (SEQ ID NO:27), FIG. 14 (SEQ ID NO:33), FIG. 16
(SEQ ID NO:38), FIG. 18 (SEQ ID NO:48), FIG. 21 (SEQ ID NO:58),
FIG. 23 (SEQ ID NO:63), FIG. 25 (SEQ ID NO:68), FIG. 27 (SEQ ID
NO:70), FIG. 29 (SEQ ID NO:72), FIG. 31 (SEQ ID NO:83), FIG. 33
(SEQ ID NO:90), FIG. 35 (SEQ ID NO:95), FIG. 37 (SEQ ID NO:103),
FIG. 39 (SEQ ID NO:108), FIG. 41 (SEQ ID NO:113), FIG. 43 (SEQ ID
NO:118), FIG. 45 (SEQ ID NO:126), FIG. 47 (SEQ ID NO:131), FIG. 49
(SEQ ID NO:136), FIG. 51 (SEQ ID NO:141), FIG. 53 (SEQ ID NO:147),
FIG. 55 (SEQ ID NO:152), FIG. 57 (SEQ ID NO:158), FIG. 59 (SEQ ID
NO:163), FIG. 61 (SEQ ID NO:169), FIG. 63 (SEQ ID NO:174), FIG. 65
(SEQ ID NO:176), FIG. 67 (SEQ ID NO:184), FIG. 69 (SEQ ID NO:189),
FIG. 71 (SEQ ID NO:194), FIG. 73 (SEQ ID NO:200), FIG. 75 (SEQ ID
NO:206), FIG. 77 (SEQ ID NO:212), FIG. 79 (SEQ ID NO:220), FIG. 81
(SEQ ID NO:226), FIG. 83 (SEQ ID NO:235), FIG. 85 (SEQ ID NO:244),
FIG. 87 (SEQ ID NO:249), FIG. 89 (SEQ ID NO:254), FIG. 91 (SEQ ID
NO:256), FIG. 93 (SEQ ID NO:258), FIG. 95 (SEQ ID NO:260), FIG. 97
(SEQ ID NO:262), FIG. 99 (SEQ ID NO:284), FIG. 101 (SEQ ID NO:289),
FIG. 103 (SEQ ID NO:291), FIG. 105 (SEQ ID NO:293), FIG. 107 (SEQ
ID NO:309), FIG. 109 (SEQ ID NO:314), FIG. 111 (SEQ ID NO:319),
FIG. 113 (SEQ ID NO:324), FIG. 115 (SEQ ID NO:331), FIG. 117 (SEQ
ID NO:338), FIG. 119 (SEQ ID NO:340), FIG. 121 (SEQ ID NO:376) and
FIG. 123 (SEQ ID NO:422), or the complement thereof.
3. The nucleic acid of claim 1, wherein said nucleotide sequence
comprises a nucleotide sequence selected from the group consisting
of the full-length coding sequence of the sequence shown in FIG. 1
(SEQ ID NO:1), FIG. 3 (SEQ ID NO:3), FIG. 5 (SEQ ID NO:1), FIG. 8
(SEQ ID NO:17), FIG. 10 (SEQ ID NO:22), FIG. 12 (SEQ ID NO:27),
FIG. 14 (SEQ ID NO:33), FIG. 16 (SEQ ID NO:38), FIG. 18 (SEQ ID
NO:48), FIG. 21 (SEQ ID NO:58), FIG. 23 (SEQ ID NO:63), FIG. 25
(SEQ ID NO:68), FIG. 27 (SEQ ID NO:70), FIG. 29 (SEQ ID NO:72),
FIG. 31 (SEQ ID NO:83), FIG. 33 (SEQ ID NO:90), FIG. 35 (SEQ ID
NO:95), FIG. 37 (SEQ ID NO:103), FIG. 39 (SEQ ID NO:108), FIG. 41
(SEQ ID NO:113), FIG. 43 (SEQ ID NO:118), FIG. 45 (SEQ ID NO:126),
FIG. 47 (SEQ ID NO:131), FIG. 49 (SEQ ID NO:136), FIG. 51 (SEQ ID
NO:141), FIG. 53 (SEQ ID NO:147), FIG. 55 (SEQ ID NO:152), FIG. 57
(SEQ ID NO:158), FIG. 59 (SEQ ID NO:163), FIG. 61 (SEQ ID NO:169),
FIG. 63 (SEQ ID NO:174), FIG. 65 (SEQ ID NO:176), FIG. 67 (SEQ ID
NO:184), FIG. 69 (SEQ ID NO:189), FIG. 71 (SEQ ID NO:194), FIG. 73
(SEQ ID NO:200), FIG. 75 (SEQ ID NO:206), FIG. 77 (SEQ ID NO:212),
FIG. 79 (SEQ ID NO:220), FIG. 81 (SEQ ID NO:226), FIG. 83 (SEQ ID
NO:235), FIG. 85 (SEQ ID NO:244), FIG. 87 (SEQ ID NO:249), FIG. 89
(SEQ ID NO:254), FIG. 91 (SEQ ID NO:256), FIG. 93 (SEQ ID NO:258),
FIG. 95 (SEQ ID NO:260), FIG. 97 (SEQ ID NO:262), FIG. 99 (SEQ ID
NO:284), FIG. 101 (SEQ ID NO:289), FIG. 103 (SEQ ID NO:291), FIG.
105 (SEQ ID NO:293), FIG. 107 (SEQ ID NO:309), FIG. 109 (SEQ ID
NO:314), FIG. 111 (SEQ ID NO:319), FIG. 113 (SEQ ID NO:324), FIG.
115 (SEQ ID NO:331), FIG. 117 (SEQ ID NO:338), FIG. 119 (SEQ ID
NO:340), FIG. 121 (SEQ ID NO:376) and FIG. 123 (SEQ ID NO:422), or
the complement thereof.
4. Isolated nucleic acid which comprises the full-length coding
sequence of the DNA deposited under accession number ATCC 209258,
ATCC 209256, ATCC 209264, ATCC 209250, ATCC 209375, ATCC 209378,
ATCC 209384, ATCC 209396, ATCC 209420, ATCC 209480, ATCC 209265,
ATCC 209257, ATCC 209262, ATCC 209253, ATCC 209402, ATCC 209401,
ATCC 209397, ATCC 209400, ATCC 209385, ATCC 209367, ATCC 209432,
ATCC 209263, ATCC 209251, ATCC 209255, ATCC 209252, ATCC 209373,
ATCC 209370, ATCC 209523, ATCC 209372, ATCC 209374, ATCC 209373,
ATCC 209382, ATCC 209383, ATCC 209403, ATCC 209398, ATCC 209399,
ATCC 209392, ATCC 209387, ATCC 209388, ATCC 209394, ATCC 209421,
ATCC 209393, ATCC 209418, ATCC 209485, ATCC 209483, ATCC 209482,
ATCC 209491, ATCC 209481, ATCC 209438, ATCC 209927, ATCC 209439,
ATCC 209489, ATCC 209433, ATCC 209488, ATCC 209434, ATCC 209395,
ATCC 209486, ATCC 209490, ATCC 209484, ATCC 209371 or ATCC
203553.
5. A vector comprising the nucleic acid of claim 1.
6. The vector of claim 5 operably linked to control sequences
recognized by a host cell transformed with the vector.
7. A host cell comprising the vector of claim 5.
8. The host cell of claim 7 wherein said cell is a CHO cell.
9. The host cell of claim 7 wherein said cell is an E. coli.
10. The host cell of claim 7 wherein said cell is a yeast cell.
11. A process for producing a PRO polypeptides comprising culturing
the host cell of claim 7 under conditions suitable for expression
of said PRO polypeptide and recovering said PRO polypeptide from
the cell culture.
12. Isolated native sequence PRO polypeptide having at least 80%
sequence identity to an amino acid sequence selected from the group
consisting of the amino acid sequence shown in FIG. 2 (SEQ ID
NO:2), FIG. 4 (SEQ ID NO:4), FIG. 6 (SEQ ID NO:12), FIG. 9 (SEQ ID
NO:18), FIG. 11 (SEQ ID NO:23), FIG. 13 (SEQ ID NO:28), FIG. 15
(SEQ ID NO:34), FIG. 17 (SEQ ID NO:39), FIG. 19 (SEQ ID NO:49),
FIG. 22 (SEQ ID NO:59), FIG. 24 (SEQ ID NO:64), FIG. 26 (SEQ ID
NO:69), FIG. 28 (SEQ ID NO:71), FIG. 30 (SEQ ID NO:73), FIG. 32
(SEQ ID NO:84), FIG. 34 (SEQ ID NO:91), FIG. 36 (SEQ ID NO:96),
FIG. 38 (SEQ ID NO:104), FIG. 40 (SEQ ID NO:109), FIG. 42 (SEQ ID
NO:114), FIG. 44 (SEQ ID NO:119), FIG. 46 (SEQ ID NO:127), FIG. 48
(SEQ ID NO:132), FIG. 50 (SEQ ID NO:137), FIG. 52 (SEQ ID NO:142),
FIG. 54 (SEQ ID NO:148), FIG. 56 (SEQ ID NO:153), FIG. 58 (SEQ ID
NO:159), FIG. 60 (SEQ ID NO:164), FIG. 62 (SEQ ID NO:170), FIG. 64
(SEQ ID NO:175), FIG. 66 (SEQ ID NO:177), FIG. 68 (SEQ ID NO:185),
FIG. 70 (SEQ ID NO:190), FIG. 72 (SEQ ID NO:195), FIG. 74 (SEQ ID
NO:201), FIG. 76 (SEQ ID NO:207), FIG. 78 (SEQ ID NO:213), FIG. 80
(SEQ ID NO:221), FIG. 82 (SEQ ID NO:227), FIG. 84 (SEQ ID NO:236),
FIG. 86 (SEQ ID NO:245), FIG. 88 (SEQ ID NO:250), FIG. 90 (SEQ ID
NO:255), FIG. 92 (SEQ ID NO:257), FIG. 94 (SEQ ID NO:259), FIG. 96
(SEQ ID NO:261), FIG. 98 (SEQ ID NO:263), FIG. 100 (SEQ ID NO:285),
FIG. 102 (SEQ ID NO:290), FIG. 104 (SEQ ID NO:292), FIG. 106 (SEQ
ID NO:294), FIG. 108 (SEQ ID NO:310), FIG. 110 (SEQ ID NO:315),
FIG. 112 (SEQ ID NO:320), FIG. 114 (SEQ ID NO:325), FIG. 116 (SEQ
ID NO:332), FIG. 118 (SEQ ID NO:339), FIG. 120 (SEQ ID NO:341),
FIG. 122 (SEQ ID NO:377) and FIG. 124 (SEQ ID NO:423).
13. Isolated PRO polypeptide having at least 80% sequence identity
to the amino acid sequence encoded by the nucleotide deposited
under accession number ATCC 209258, ATCC 209256, ATCC 209264, ATCC
209250, ATCC 209375, ATCC 209378, ATCC 209384, ATCC 209396, ATCC
209420, ATCC 209480, ATCC 209265, ATCC 209257, ATCC 209262, ATCC
209253, ATCC 209402, ATCC 209401, ATCC 209397, ATCC 209400, ATCC
209385, ATCC 209367, ATCC 209432, ATCC 209263, ATCC 209251, ATCC
209255, ATCC 209252, ATCC 209373, ATCC 209370, ATCC 209523, ATCC
209372, ATCC 209374, ATCC 209373, ATCC 209382, ATCC 209383, ATCC
209403, ATCC 209398, ATCC 209399, ATCC 209392, ATCC 209387, ATCC
209388, ATCC 209394, ATCC 209421, ATCC 209393, ATCC 209418, ATCC
209485, ATCC 209483, ATCC 209482, ATCC 209491, ATCC 209481, ATCC
209438, ATCC 209927, ATCC 209439, ATCC 209489, ATCC 209433, ATCC
209488, ATCC 209434, ATCC 209395, ATCC 209486, ATCC 209490, ATCC
209484, ATCC 209371 or ATCC 203553.
14. A chimeric molecule comprising a polypeptide according to claim
12 fused to a heterologous amino acid sequence.
15. The chimeric molecule of claim 14 wherein said heterologous
amino acid sequence is an epitope tag sequence.
16. The chimeric molecule of claim 14 wherein said heterologous
amino acid sequence is a Fc region of an immunoglobulin.
17. An antibody which specifically binds to a PRO polypeptide
according to claim 12.
18. The antibody of claim 17 wherein said antibody is a monoclonal
antibody.
19. Isolated nucleic acid having at least 80% nucleic acid sequence
identity to: (a) a nucleotide sequence encoding the polypeptide
shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4), FIG. 6 (SEQ ID
NO:12), FIG. 9 (SEQ ID NO:18), FIG. 11 (SEQ ID NO:23), FIG. 13 (SEQ
ID NO:28), FIG. 15 (SEQ ID NO:34), FIG. 17 (SEQ ID NO:39), FIG. 19
(SEQ ID NO:49), FIG. 22 (SEQ ID NO:59), FIG. 24 (SEQ ID NO:64),
FIG. 26 (SEQ ID NO:69), FIG. 28 (SEQ ID NO:71), FIG. 30 (SEQ ID
NO:73), FIG. 32 (SEQ ID NO:84), FIG. 34 (SEQ ID NO:91), FIG. 36
(SEQ ID NO:96), FIG. 38 (SEQ ID NO:104), FIG. 40 (SEQ ID NO:109),
FIG. 42 (SEQ ID NO:114), FIG. 44 (SEQ ID NO:119), FIG. 46 (SEQ ID
NO:127), FIG. 48 (SEQ ID NO:132), FIG. 50 (SEQ ID NO:137), FIG. 52
(SEQ ID NO:142), FIG. 54 (SEQ ID NO:148), FIG. 56 (SEQ ID NO:153),
FIG. 58 (SEQ ID NO:159), FIG. 60 (SEQ ID NO:164), FIG. 62 (SEQ ID
NO:170), FIG. 64 (SEQ ID NO:175), FIG. 66 (SEQ ID NO:177), FIG. 68
(SEQ ID NO:185), FIG. 70 (SEQ ID NO:190), FIG. 72 (SEQ ID NO:195),
FIG. 74 (SEQ ID NO:201), FIG. 76 (SEQ ID NO:207), FIG. 78 (SEQ ID
NO:213), FIG. 80 (SEQ ID NO:221), FIG. 82 (SEQ ID NO:227), FIG. 84
(SEQ ID NO:236), FIG. 86 (SEQ ID NO:245), FIG. 88 (SEQ ID NO:250),
FIG. 90 (SEQ ID NO:255), FIG. 92 (SEQ ID NO:257), FIG. 94 (SEQ ID
NO:259), FIG. 96 (SEQ ID NO:261), FIG. 98 (SEQ ID NO:263), FIG. 100
(SEQ ID NO:285), FIG. 102 (SEQ ID NO:290), FIG. 104 (SEQ ID
NO:292), FIG. 106 (SEQ ID NO:294), FIG. 108 (SEQ ID NO:310), FIG.
110 (SEQ ID NO:315), FIG. 112 (SEQ ID NO:320), FIG. 114 (SEQ ID
NO:325), FIG. 116 (SEQ ID NO:332), FIG. 118 (SEQ ID NO:339), FIG.
120 (SEQ ID NO:341), FIG. 122 (SEQ ID NO:377) or FIG. 124 (SEQ ID
NO:423), lacking its associated signal peptide; (b) a nucleotide
sequence encoding an extracellular domain of the polypeptide shown
in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4), FIG. 6 (SEQ ID
NO:12), FIG. 9 (SEQ ID NO:18), FIG. 11 (SEQ ID NO:23), FIG. 13 (SEQ
ID NO:28), FIG. 15 (SEQ ID NO:34), FIG. 17 (SEQ ID NO:39), FIG. 19
(SEQ ID NO:49), FIG. 22 (SEQ ID NO:59), FIG. 24 (SEQ ID NO:64),
FIG. 26 (SEQ ID NO:69), FIG. 28 (SEQ ID NO:71), FIG. 30 (SEQ ID
NO:73), FIG. 32 (SEQ ID NO:84), FIG. 34 (SEQ ID NO:91), FIG. 36
(SEQ ID NO:96), FIG. 38 (SEQ ID NO:104), FIG. 40 (SEQ ID NO:109),
FIG. 42 (SEQ ID NO:114), FIG. 44 (SEQ ID NO:119), FIG. 46 (SEQ ID
NO:127), FIG. 48 (SEQ ID NO:132), FIG. 50 (SEQ ID NO:137), FIG. 52
(SEQ ID NO:142), FIG. 54 (SEQ ID NO:148), FIG. 56 (SEQ ID NO:153),
FIG. 58 (SEQ ID NO:159), FIG. 60 (SEQ ID NO:164), FIG. 62 (SEQ ID
NO:170), FIG. 64 (SEQ ID NO:175), FIG. 66 (SEQ ID NO:177), FIG. 68
(SEQ ID NO:185), FIG. 70 (SEQ ID NO:190), FIG. 72 (SEQ ID NO:195),
FIG. 74 (SEQ ID NO:201), FIG. 76 (SEQ ID NO:207), FIG. 78 (SEQ ID
NO:213), FIG. 80 (SEQ ID NO:221), FIG. 82 (SEQ ID NO:227), FIG. 84
(SEQ ID NO:236), FIG. 86 (SEQ ID NO:245), FIG. 88 (SEQ ID NO:250),
FIG. 90 (SEQ ID NO:255), FIG. 92 (SEQ ID NO:257), FIG. 94 (SEQ ID
NO:259), FIG. 96 (SEQ ID NO:261), FIG. 98 (SEQ ID NO:263), FIG. 100
(SEQ ID NO:285), FIG. 102 (SEQ ID NO:290), FIG. 104 (SEQ ID
NO:292), FIG. 106 (SEQ ID NO:294), FIG. 108 (SEQ ID NO:310), FIG.
110 (SEQ ID NO:315), FIG. 112 (SEQ ID NO:320), FIG. 114 (SEQ ID
NO:325), FIG. 116 (SEQ ID NO:332), FIG. 118 (SEQ ID NO:339), FIG.
120 (SEQ ID NO:341), FIG. 122 (SEQ ID NO:377) or FIG. 124 (SEQ ID
NO:423), with its associated signal peptide; or (c) a nucleotide
sequence encoding an extracellular domain of the polypeptide shown
in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4), FIG. 6 (SEQ ID
NO:12), FIG. 9 (SEQ ID NO:18), FIG. 11 (SEQ ID NO:23), FIG. 13 (SEQ
ID NO:28), FIG. 15 (SEQ ID NO:34), FIG. 17 (SEQ ID NO:39), FIG. 19
(SEQ ID NO:49), FIG. 22 (SEQ ID NO:59), FIG. 24 (SEQ ID NO:64),
FIG. 26 (SEQ ID NO:69), FIG. 28 (SEQ ID NO:71), FIG. 30 (SEQ ID
NO:73), FIG. 32 (SEQ ID NO:84), FIG. 34 (SEQ ID NO:91), FIG. 36
(SEQ ID NO:96), FIG. 38 (SEQ ID NO:104), FIG. 40 (SEQ ID NO:109),
FIG. 42 (SEQ ID NO:114), FIG. 44 (SEQ ID NO:119), FIG. 46 (SEQ ID
NO:127), FIG. 48 (SEQ ID NO:132), FIG. 50 (SEQ ID NO:137), FIG. 52
(SEQ ID NO:142), FIG. 54 (SEQ ID NO:148), FIG. 56 (SEQ ID NO:153),
FIG. 58 (SEQ ID NO:159), FIG. 60 (SEQ ID NO:164), FIG. 62 (SEQ ID
NO:170), FIG. 64 (SEQ ID NO:175), FIG. 66 (SEQ ID NO:177), FIG. 68
(SEQ ID NO:185), FIG. 70 (SEQ ID NO:190), FIG. 72 (SEQ ID NO:195),
FIG. 74 (SEQ ID NO:201), FIG. 76 (SEQ ID NO:207), FIG. 78 (SEQ ID
NO:213), FIG. 80 (SEQ ID NO:221), FIG. 82 (SEQ ID NO:227), FIG. 84
(SEQ ID NO:236), FIG. 86 (SEQ ID NO:245), FIG. 88 (SEQ ID NO:250),
FIG. 90 (SEQ ID NO:255), FIG. 92 (SEQ ID NO:257), FIG. 94 (SEQ ID
NO:259), FIG. 96 (SEQ ID NO:261), FIG. 98 (SEQ ID NO:263), FIG. 100
(SEQ ID NO:285), FIG. 102 (SEQ ID NO:290), FIG. 104 (SEQ ID
NO:292), FIG. 106 (SEQ ID NO:294), FIG. 108 (SEQ ID NO:310), FIG.
110 (SEQ ID NO:315), FIG. 112 (SEQ ID NO:320), FIG. 114 (SEQ ID
NO:325), FIG. 116 (SEQ ID NO:332), FIG. 118 (SEQ ID NO:339), FIG.
120 (SEQ ID NO:341), FIG. 122 (SEQ ID NO:377) or FIG. 124 (SEQ ID
NO:423), lacking its associated signal peptide.
20. An isolated polypeptide having at least 80% amino acid sequence
identity to: (a) the polypeptide shown in FIG. 2 (SEQ ID NO:2),
FIG. 4 (SEQ ID NO:4), FIG. 6 (SEQ ID NO:12), FIG. 9 (SEQ ID NO:18),
FIG. 11 (SEQ ID NO:23), FIG. 13 (SEQ ID NO:28), FIG. 15 (SEQ ID
NO:34), FIG. 17 (SEQ ID NO:39), FIG. 19 (SEQ ID NO:49), FIG. 22
(SEQ ID NO:59), FIG. 24 (SEQ ID NO:64), FIG. 26 (SEQ ID NO:69),
FIG. 28 (SEQ ID NO:71), FIG. 30 (SEQ ID NO:73), FIG. 32 (SEQ ID
NO:84), FIG. 34 (SEQ ID NO:91), FIG. 36 (SEQ ID NO:96), FIG. 38
(SEQ ID NO:104), FIG. 40 (SEQ ID NO:109), FIG. 42 (SEQ ID NO:114),
FIG. 44 (SEQ ID NO:119), FIG. 46 (SEQ ID NO:127), FIG. 48 (SEQ ID
NO:132), FIG. 50 (SEQ ID NO:137), FIG. 52 (SEQ ID NO:142), FIG. 54
(SEQ ID NO:148), FIG. 56 (SEQ ID NO:153), FIG. 58 (SEQ ID NO:159),
FIG. 60 (SEQ ID NO:164), FIG. 62 (SEQ ID NO:170), FIG. 64 (SEQ ID
NO:175), FIG. 66 (SEQ ID NO:177), FIG. 68 (SEQ ID NO:185), FIG. 70
(SEQ ID NO:190), FIG. 72 (SEQ ID NO:195), FIG. 74 (SEQ ID NO:201),
FIG. 76 (SEQ ID NO:207), FIG. 78 (SEQ ID NO:213), FIG. 80 (SEQ ID
NO:221), FIG. 82 (SEQ ID NO:227), FIG. 84 (SEQ ID NO:236), FIG. 86
(SEQ ID NO:245), FIG. 88 (SEQ ID NO:250), FIG. 90 (SEQ ID NO:255),
FIG. 92 (SEQ ID NO:257), FIG. 94 (SEQ ID NO:259), FIG. 96 (SEQ ID
NO:261), FIG. 98 (SEQ ID NO:263), FIG. 100 (SEQ ID NO:285), FIG.
102 (SEQ ID NO:290), FIG. 104 (SEQ ID NO:292), FIG. 106 (SEQ ID
NO:294), FIG. 108 (SEQ ID NO:310), FIG. 110 (SEQ ID NO:315), FIG.
112 (SEQ ID NO:320), FIG. 114 (SEQ ID NO:325), FIG. 116 (SEQ ID
NO:332), FIG. 118 (SEQ ID NO:339), FIG. 120 (SEQ ID NO:341), FIG.
122 (SEQ ID NO:377) or FIG. 124 (SEQ ID NO:423), lacking its
associated signal peptide; (b) an extracellular domain of the
polypeptide shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4),
FIG. 6 (SEQ ID NO:12), FIG. 9 (SEQ ID NO:18), FIG. 11 (SEQ ID
NO:23), FIG. 13 (SEQ ID NO:28), FIG. 15 (SEQ ID NO:34), FIG. 17
(SEQ ID NO:39), FIG. 19 (SEQ ID NO:49), FIG. 22 (SEQ ID NO:59),
FIG. 24 (SEQ ID NO:64), FIG. 26 (SEQ ID NO:69), FIG. 28 (SEQ ID
NO:71), FIG. 30 (SEQ ID NO:73), FIG. 32 (SEQ ID NO:84), FIG. 34
(SEQ ID NO:91), FIG. 36 (SEQ ID NO:96), FIG. 38 (SEQ ID NO:104),
FIG. 40 (SEQ ID NO:109), FIG. 42 (SEQ ID NO:114), FIG. 44 (SEQ ID
NO:119), FIG. 46 (SEQ ID NO:127), FIG. 48 (SEQ ID NO:132), FIG. 50
(SEQ ID NO:137), FIG. 52 (SEQ ID NO:142), FIG. 54 (SEQ ID NO:148),
FIG. 56 (SEQ ID NO:153), FIG. 58 (SEQ ID NO:159), FIG. 60 (SEQ ID
NO:164), FIG. 62 (SEQ ID NO:170), FIG. 64 (SEQ ID NO:175), FIG. 66
(SEQ ID NO:177), FIG. 68 (SEQ ID NO:185), FIG. 70 (SEQ ID NO:190),
FIG. 72 (SEQ ID NO:195), FIG. 74 (SEQ ID NO:201), FIG. 76 (SEQ ID
NO:207), FIG. 78 (SEQ ID NO:213), FIG. 80 (SEQ ID NO:221), FIG. 82
(SEQ ID NO:227), FIG. 84 (SEQ ID NO:236), FIG. 86 (SEQ ID NO:245),
FIG. 88 (SEQ ID NO:250), FIG. 90 (SEQ ID NO:255), FIG. 92 (SEQ ID
NO:257), FIG. 94 (SEQ ID NO:259), FIG. 96 (SEQ ID NO:261), FIG. 98
(SEQ ID NO:263), FIG. 100 (SEQ ID NO:285), FIG. 102 (SEQ ID
NO:290), FIG. 104 (SEQ ID NO:292), FIG. 106 (SEQ ID NO:294), FIG.
108 (SEQ ID NO:310), FIG. 110 (SEQ ID NO:315), FIG. 112 (SEQ ID
NO:320), FIG. 114 (SEQ ID NO:325), FIG. 116 (SEQ ID NO:332), FIG.
118 (SEQ ID NO:339), FIG. 120 (SEQ ID NO:341), FIG. 122 (SEQ ID
NO:377) or FIG. 124 (SEQ ID NO:423), with its associated signal
peptide; or (c) an extracellular domain of the polypeptide shown in
FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4), FIG. 6 (SEQ ID NO:212),
FIG. 9 (SEQ ID NO:218), FIG. 91 (SEQ ID NO:23), FIG. 13 (SEQ ID
NO:28), FIG. 15 (SEQ ID NO:34), FIG. 17 (SEQ ID NO:39), FIG. 19
(SEQ ID NO:49), FIG. 22 (SEQ ID NO:59), FIG. 24 (SEQ ID NO:64),
FIG. 26 (SEQ ID NO:69), FIG. 28 (SEQ ID NO:71), FIG. (SEQ ID
NO:73), FIG. 32 (SEQ ID NO:84), FIG. 34 (SEQ ID NO:91), FIG. 36
(SEQ ID NO:96), FIG. 38 (SEQ ID NO:104), FIG. 40 (SEQ ID NO:109),
FIG. 42 (SEQ ID NO:114), FIG. 44 (SEQ ID NO:119), FIG. 46 (SEQ ID
NO:127), FIG. 48 (SEQ ID NO:132), FIG. 50 (SEQ ID NO:137), FIG. 52
(SEQ ID NO:142), FIG. 54 (SEQ ID NO:148), FIG. 56 (SEQ ID NO:153),
FIG. 58 (SEQ ID NO:159), FIG. 60 (SEQ ID NO:164), FIG. 62 (SEQ ID
NO:170), FIG. 64 (SEQ ID NO:175), FIG. 66 (SEQ ID NO:177), FIG. 68
(SEQ ID NO:185), FIG. 70 (SEQ ID NO:190), FIG. 72 (SEQ ID NO:195),
FIG. 74 (SEQ ID NO:201), FIG. 76 (SEQ ID NO:207), FIG. 78 (SEQ ID
NO:213), FIG. 80 (SEQ ID NO:221), FIG. 82 (SEQ ID NO:227), FIG. 84
(SEQ ID NO:236), FIG. 86 (SEQ ID NO:245), FIG. 88 (SEQ ID NO:250),
FIG. 90 (SEQ ID NO:255), FIG. 92 (SEQ ID NO:257), FIG. 94 (SEQ ID
NO:259), FIG. 96 (SEQ ID NO:261), FIG. 98 (SEQ ID NO:263), FIG. 100
(SEQ ID NO:285), FIG. 102 (SEQ ID NO:290), FIG. 104 (SEQ ID
NO:292), FIG. 106 (SEQ ID NO:294), FIG. 108 (SEQ ID NO:310), FIG.
110 (SEQ ID NO:315), FIG. 112 (SEQ ID NO:320), FIG. 114 (SEQ ID
NO:325), FIG. 116 (SEQ ID NO:332), FIG. 118 (SEQ ID NO:339), FIG.
120 (SEQ ID NO:341), FIG. 122 (SEQ ID NO:377) or FIG. 124 (SEQ ID
NO:423), lacking its associated signal peptide.
21. A method of detecting a PRO245 polypeptide in a sample
suspected of containing a PRO245 polypeptide, said method
comprising contacting said sample with a PRO1868 polypeptide and
determining the formation of a PRO245/PRO1868 polypeptide conjugate
in said sample, wherein the formation of said conjugate is
indicative of the presence of a PRO245 polypeptide in said
sample.
22. The method according to claim 21, wherein said sample comprises
cells suspected of expressing said PRO245 polypeptide.
23. The method according to claim 21, wherein said PRO1868
polypeptide is labeled with a detectable label.
24. The method according to claim 21, wherein said PRO1868
polypeptide is attached to a solid support.
25. A method of detecting a PRO1868 polypeptide in a sample
suspected of containing a PRO1868 polypeptide, said method
comprising contacting said sample with a PRO245 polypeptide and
determining the formation of a PRO245/PRO1868 polypeptide conjugate
in said sample, wherein the formation of said conjugate is
indicative of the presence of a PRO1868 polypeptide in said
sample.
26. The method according to claim 25, wherein said sample comprises
cells suspected of expressing said PRO1868 polypeptide.
27. The method according to claim 25, wherein said PRO245
polypeptide is labeled with a detectable label.
28. The method according to claim 25, wherein said PRO245
polypeptide is attached to a solid support.
29. A method of linking a bioactive molecule to a cell expressing a
PRO245 polypeptide, said method comprising contacting said cell
with a PRO1868 polypeptide that is bound to said bioactive molecule
and allowing said PRO245 and PRO1868 polypeptides to bind to one
another, thereby linking said bioactive molecules to said cell.
30. The method according to claim 29, wherein said bioactive
molecule is a toxin, a radiolabel or an antibody.
31. The method according to claim 29, wherein said bioactive
molecule causes the death of said cell.
32. A method of linking a bioactive molecule to a cell expressing a
PRO1868 polypeptide, said method comprising contacting said cell
with a PRO245 polypeptide that is bound to said bioactive molecule
and allowing said PRO245 and PRO1868 polypeptides to bind to one
another, thereby linking said bioactive molecules to said cell.
33. The method according to claim 32, wherein said bioactive
molecule is a toxin, a radiolabel or an antibody.
34. The method according to claim 32, wherein said bioactive
molecule causes the death of said cell.
35. A method of modulating at least one biological activity of a
cell expressing a PRO245 polypeptide, said method comprising
contacting said cell with a PRO1868 polypeptide or an anti-PRO245
antibody, whereby said PRO1868 polypeptide or said anti-PRO245
antibody binds to said PRO245 polypeptide, thereby modulating at
least one biological activity of said cell.
36. The method according to claim 35, wherein said cell is
killed.
37. A method of modulating at least one biological activity of a
cell expressing a PRO1868 polypeptide, said method comprising
contacting said cell with a PRO245 polypeptide or an anti-PRO1868
antibody, whereby said PRO245 polypeptide or said anti-PRO1868
antibody binds to said PRO1868 polypeptide, thereby modulating at
least one biological activity of said cell.
38. The method according to claim 37, wherein said cell is killed.
Description
FIELD OF THE INVENTION
[0001] The present invention relates generally to the
identification and isolation of novel DNA and to the recombinant
production of novel polypeptides.
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 adhesin 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 immunoadhesins, 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] 1. PRO211 and PRO217
[0008] Epidermal growth factor (EGF) is a conventional mitogenic
factor that stimulates the proliferation of various types of cells
including epithelial cells and fibroblasts. EGF binds to and
activates the EGF receptor (EGFR), which initiates intracellular
signaling and subsequent effects. The EGFR is expressed in neurons
of the cerebral cortex, cerebellum, and hippocampus in addition to
other regions of the central nervous system (CNS). In addition, EGF
is also expressed in various regions of the CNS. Therefore, EGF
acts not only on mitotic cells, but also on postmitotic neurons. In
fact, many studies have indicated that EGF has neurotrophic or
neuromodulatory effects on various types of neurons in the CNS. For
example, EGF acts directly on cultured cerebral cortical and
cerebellar neurons, enhancing neurite outgrowth and survival. On
the other hand, EGF also acts on other cell types, including septal
cholinergic and mesencephalic dopaminergic neurons, indirectly
through glial cells. Evidence of the effects of EGF on neurons in
the CNS is accumulating, but the mechanisms of action remain
essentially unknown. EGF-induced signaling in mitotic cells is
better understood than in postmitotic neurons. Studies of cloned
pheochromocytoma PC12 cells and cultured cerebral cortical neurons
have suggested that the EGF-induced neurotrophic actions are
mediated by sustained activation of the EGFR and mitogen-activated
protein kinase (MAPK) in response to EGF. The sustained
intracellular signaling correlates with the decreased rate of EGFR
down-regulation, which might determine the response of neuronal
cells to EGF. It is likely that EGF is a multi-potent growth factor
that acts upon various types of cells including mitotic cells and
postmitotic neurons.
[0009] EGF is produced by the salivary and Brunner's glands of the
gastrointestinal system, kidney, pancreas, thyroid gland, pituitary
gland, and the nervous system, and is found in body fluids such as
saliva, blood, cerebrospinal fluid (CSF), urine, amniotic fluid,
prostatic fluid, pancreatic juice, and breast milk, Plata-Salaman,
Peptides 12: 653-663 (1991).
[0010] EGF is mediated by its membrane specific receptor, which
contains an intrinsic tyrosine kinase. Stoscheck et al., J. Cell
Biochem. 31: 135-152 (1986). EGF is believed to function by binding
to the extracellular portion of its receptor which induces a
transmembrane signal that activates the intrinsic tyrosine
kinase.
[0011] Purification and sequence analysis of the EGF-like domain
has revealed the presence of six conserved cysteine residues which
cross-bind to create three peptide loops, Savage et al., J. Biol.
Chem. 248: 7669-7672 (1979). It is now generally known that several
other peptides can react with the EGF receptor which share the same
generalized motif
X.sub.nCX.sub.7CX.sub.4/5CX.sub.10CXCX.sub.5GX.sub.2CX.sub.n, where
X represents any non-cysteine amino acid, and n is a variable
repeat number. Non isolated peptides having this motif include
TGF-.alpha., amphiregulin, schwannoma-derived growth factor (SDGF),
heparin-binding EGF-like growth factors and certain virally encoded
peptides (e.g., Vaccinia virus, Reisner, Nature 313: 801-803
(1985), Shope fibroma virus, Chang et al., Mol Cell Biol. 7:
535-540 (1987), Molluscum contagiosum, Porter and Archard, J. Gen.
Virol. 68: 673-682 (1987), and Myxoma virus, Upton et al., J.
Virol. 61: 1271-1275 (1987), Prigent and Lemoine, Prog. Growth
Factor Res. 4: 1-24 (1992).
[0012] EGF-like domains are not confined to growth factors but have
been observed in a variety of cell-surface and extracellular
proteins which have interesting properties in cell adhesion,
protein-protein interaction and development, Laurence and
Gusterson, Tumor Biol. 11: 229-261 (1990). These proteins include
blood coagulation factors (factors VI, IX, X, XII, protein C,
protein S, protein Z, tissue plasminogen activator, urokinase),
extracellular matrix components (laminin, cytotactin, entactin),
cell surface receptors (LDL receptor, thrombomodulin receptor) and
immunity-related proteins (complement C1r, uromodulin).
[0013] Even more interesting, the general structure pattern of
EGF-like precursors is preserved through lower organisms as well as
in mammalian cells. A number of genes with developmental
significance have been identified in invertebrates with EGF-like
repeats. For example, the notch gene of Drosophila encodes 36
tandemly arranged 40 amino acid repeats which show homology to EGF,
Wharton et al., Cell 43: 557-581 (1985). Hydropathy plots indicate
aputative membrane spanning domain, with the EGF-related sequences
being located on the extracellular side of the membrane. Other
homeotic genes with EGF-like repeats include Delta, 95F and 5ZD
which were identified using probes based on Notch, and the nematode
gene Lin-12 which encodes a putative receptor for a developmental
signal transmitted between two specified cells.
[0014] Specifically, EGF has been shown to have potential in the
preservation and maintenance of gastrointestinal mucosa and the
repair of acute and chronic mucosal lesions, Konturek et al., Eur.
J. Gastroenterol Hepatol. 7 (10), 933-37 (1995), including the
treatment of necrotizing enterocolitis, Zollinger-Ellison syndrome,
gastrointestinal ulceration gastrointestinal ulcerations and
congenital microvillus atrophy, Guglietta and Sullivan, Eur. J.
Gastroenterol Hepatol, 7(10), 945-50 (1995). Additionally, EGF has
been implicated in hair follicle differentiation; du Cros, J.
Invest. Dermatol. 101 (1 Suppl.), 106S-113S (1993), Hillier, Clin.
Endocrinol. 33(4), 427-28 (1990); kidney function, Hamm et al.,
Semin. Nephrol. 13 (1): 109-15 (1993), Harris, Am. J. Kidney Dis.
17(6): 627-30 (1991); tear fluid, van Setten et al., Int.
Ophthalmol L5(6); 359-62 (1991); vitamin K mediated blood
coagulation, Stenflo et al., Blood 78(7): 1637-51 (1991). EGF is
also implicated various skin disease characterized by abnormal
keratinocyte differentiation, e.g., psoriasis, epithelial cancers
such as squamous cell carcinomas of the lung, epidermoid carcinoma
of the vulva and gliomas. King et al., Am. J. Med. Sci. 296:
154-158 (1988).
[0015] Of great interest is mounting evidence that genetic
alterations in growth factors signaling pathways are closely linked
to developmental abnormalities and to chronic diseases including
cancer. Aaronson, Science 254: 1146-1153 (1991). For example,
c-erb-2 (also known as HER-2), a proto-oncogene with close
structural similarity to EGF receptor protein, is overexpressed in
human breast cancer. King et al., Science 229: 974-976 (1985);
Gullick, Hormones and their actions, Cooke et al., eds, Amsterdam,
Elsevier, pp 349-360 (1986).
[0016] We herein describe the identification and characterization
of novel polypeptides having homology to EGF, wherein those
polypeptides are herein designated PRO211 and PRO217.
[0017] 2. PRO230
[0018] Nepbritis is a condition characterized by inflammation of
the kidney affecting the structure and normal function of the
kidney. This condition can be chronic or acute and is generally
caused by infection, degenerative process or vascular disease. In
all cases, early detection is desirable so that the patient with
nephritis can begin treatment of the condition.
[0019] An approach to detecting nephritis is to determine the
antigens associated with nephritis and antibodies thereto. In
rabbit, a tubulointerstitial nephritis antigen (TIN-ag) has been
reported in Nelson, T. R., et al., J. Biol. Chem., 270(27):16265-70
(July 1995) (GENBANK/U24270). This study reports that the rabbit
TIN-ag is a basement membrane glycoprotein having a predicted amino
acid sequence which has a carboxyl-terminal region exhibiting 30%
homology with human preprocathepsin B, a member of the cystein
proteinase family of proteins. It is also reported that the rabbit
TIN-ag has a domain in the amino-terminal region containing an
epidermal growth factor-ike motif that shares homology with laminin
A and S chains, alpha 1 chain of type I collagen, von Willebrand's
factor and mucin, indicating structural and functional
similarities. Studies have also been conducted in mice. However, it
is desirable to identify tubulointerstitial nephritis antigens in
humans to aid in the development of early detection methods and
treatment of nephritis.
[0020] Proteins which have homology to tubulointerstitial nephritis
antigens are of particular interest to the medical and industrial
communities. Often, proteins having homology to each other have
similar function. It is also of interest when proteins having
homology do not have similar functions, indicating that certain
structural motifs identify information other than function, such as
locality of function. We herein describe the identification and
characterization of a novel polypeptide, designated hgerein as
PRO230, which has homology to tubulointerstitial nephritis
antigens.
[0021] 3. PRO232
[0022] Stem cells are undifferentiated cells capable of (a)
proliferation, (b) self maintenance, (c) the production of a large
number of differentiated functional progeny, (d) regeneration of
tissue after injury and/or (e) a flexibility in the use of these
options. Stem cells often express cell surface antigens which are
capable of serving as cell specific markers that can be exploited
to identify stem cells, thereby providing a means for identifying
and isolating specific stem cell populations.
[0023] Having possession of different stem cell populations will
allow for a number of important applications. For example,
possessing a specific stem cell population will allow for the
identification of growth factors and other proteins which are
involved in their proliferation and differentiation. In addition,
there may be as yet undiscovered proteins which are associated with
(1) the early steps of dedication of the stem cell to a particular
lineage, (2) prevention of such dedication, and (3) negative
control of stem cell proliferation, all of which may be identified
if one has possession of the stem cell population. Moreover, stem
cells are important and ideal targets for gene therapy where the
inserted genes promote the health of the individual into whom the
stem cells are transplanted. Finally, stem cells may play important
roles in transplantation of organs or tissues, for example liver
regeneration and skin grafting.
[0024] Given the importance of stem cells in various different
applications, efforts are currently being undertaken by both
industry and academia to identify new, native stem cell antigen
proteins so as to provide specific cell surface markers for
identifying stem cell populations as well as for providing insight
into the functional roles played by stem cell antigens in cell
proliferation and differentiation. We herein describe the
identification and characterization of novel polypeptides having
homology to a stem cell antigen, wherein those polypeptides are
herein designated as PRO232 polypeptides.
[0025] 4. PRO187
[0026] Growth factors are molecular signals or mediators that
enhance cell growth or proliferation, alone or in concert, by
binding to specific cell surface receptors. However, there are
other cellular reactions than only growth upon expression to growth
factors. As a result, growth factors are better characterized as
multifunctional and potent cellular regulators. Their biological
effects include proliferation, chemotaxis and stimulation of
extracellular matrix production. Growth factors can have both
stimulatory and inhibitory effects. For example, transforming
growth factor (TGF-.beta.) is highly pleiotropic and can stimulate
proliferation in some cells, especially connective tissue, while
being a potent inhibitor of proliferation in others, such as
lymphocytes and epithelial cells.
[0027] The physiological effect of growth stimulation or inhibition
by growth factors depends upon the state of development and
differentiation of the target tissue. The mechanism of local
cellular regulation by classical endocrine molecules involves
comprehends autocrine (same cell), juxtacrine (neighbor cell), and
paracrine (adjacent cells) pathways. Peptide growth factors are
elements of a complex biological language, providing the basis for
intercellular communication. They permit cells to convey
information between each other, mediate interaction between cells
and change gene expression. The effect of these multifunctional and
pluripotent factors is dependent on the presence or absence of
other peptides.
[0028] FGF-8 is a member of the fibroblast growth factors (FGFs)
which are a family of heparin-binding, potent mitogens for both
normal diploid fibroblasts and established cell lines,
Gospodarowicz et al. (1984), Proc. Natl. Acad. Sci. USA 81:6963.
The FGF family comprises acidic FGF (FGF-1), basic FGF (FGF-2),
INT-2 (FGF-3), K-FGF/HST (FGF-4), FGF-5, FGF-6, KGF (FGF-7), AIGF
(FGF-8) among others. All FGFs have two conserved cysteine residues
and share 30-50% sequence homology at the amino acid level. These
factors are mitogenic for a wide variety of normal diploid
mesoderm-derived and neural crest-derived cells, including
granulosa cells, adrenal cortical cells, chondrocytes, myoblasts,
corneal and vascular endothelial cells (bovine or human), vascular
smooth muscle cells, lens, retina and prostatic epithelial cells,
oligodendrocytes, astrocytes, chrondocytes, myoblasts and
osteoblasts.
[0029] Fibroblast growth factors can also stimulate a large number
of cell types in a non-mitogenic manner. These activities include
promotion of cell migration into wound area (chemotaxis),
initiation of new blood vessel formulation (angiogenesis),
modulation of nerve regeneration and survival (neurotrophism),
modulation of endocrine functions, and stimulation or suppression
of specific cellular protein expression, extracellular matrix
production and cell survival. Baird & Bohlen, Handbook of Exp.
Pharmacol. 95(1): 369418, Springer, (1990). These properties
provide a basis for using fibroblast growth factors in therapeutic
approaches to accelerate wound healing, nerve repair, collateral
blood vessel formation, and the like. For example, fibroblast
growth factors have been suggested to minimize myocardium damage in
heart disease and surgery (U.S. Pat. No. 4,378,347).
[0030] FGF-8, also known as androgen-induced growth factor (AIGF),
is a 215 amino acid protein which shares 3040% sequence homology
with the other members of the FGF family. FGF-8 has been proposed
to be under androgenic regulation and induction in the mouse
mammary carcinoma cell line SC3. Tanaka et al., Proc. Natl. Acad.
Sci. USA 89: 8928-8932 (1992); Sato et al., J. Steroid Biochem.
Molec. Biol. 47: 91-98 (1993). As a result, FGF-8 may have a local
role in the prostate, which is known to be an androgen-responsive
organ. FGF-8 can also be oncogenic, as it displays transforming
activity when transfected into NIH-3T3 fibroblasts. Kouhara et al.,
Oncogene 9 455462 (1994). While FGF-8 has been detected in heart,
brain, lung, kidney, testis, prostate and ovary, expression was
also detected in the absence of exogenous androgens. Schmitt et
al., J. Steroid Biochem. Mol. Biol. 57 (34): 173-78 (1996).
[0031] FGF-8 shares the property with several other FGFs of being
expressed at a variety of stages of murine embryogenesis, which
supports the theory that the various FGFs have multiple and perhaps
coordinated roles in differentiation and embryogenesis. Moreover,
FGF-8 has also been identified as a protooncogene that cooperates
with Wnt-1 in the process of mammary tumorigenesis (Shackleford et
al., Proc. Natl. Acad. Sci. USA 90, 740-744 (1993); Heikinheimo et
al., Mech. Dev. 48: 129-138 (1994)).
[0032] In contrast to the other FGFs, FGF-8 exists as three protein
isoforms, as a result of alternative splicing of the primary
transcript. Tanaka et al., supra. Normal adult expression of FGF-8
is weak and confined to gonadal tissue, however northern blot
analysis has indicated that FGF-8 .mu.mRNA is present from day 10
through day 12 or murine gestation, which suggests that FGF-8 is
important to normal development. Heikinheimo et al., Mech Dev.
48(2): 129-38 (1994). Further in situ hybridization assays between
day 8 and 16 of gestation indicated initial expression in the
surface ectoderm of the first bronchial arches, the frontonasal
process, the forebrain and the midbrain-hindbrain junction. At days
10-12, FGF-8 was expressed in the surface ectoderm of the forelimb
and hindlimb buds, the nasal its and nasopharynx, the infundibulum
and in the telencephalon, diencephalon and metencephalon.
Expression continues in the developing hindlimbs through day 13 of
gestation, but is undetectable thereafter. The results suggest that
FGF-8 has a unique temporal and spatial pattern in embryogenesis
and suggests a role for this growth factor in multiple regions of
ectodermal differentiation in the post-gastrulation embryo. We
herein describe the identification of novel poypeptides having
homology to FGF-8, wherein those polypeptides are heein designated
PRO187 polypeptides.
[0033] 5. PRO265
[0034] Protein-protein interactions include receptor and antigen
complexes and signaling mechanisms. As more is known about the
structural and functional mechanisms underlying protein-protein
interactions, protein-protein interactions can be more easily
manipulated to regulate the particular result of the
protein-protein interaction. Thus, the underlying mechanisms of
protein-protein interactions are of interest to the scientific and
medical community.
[0035] All proteins containing leucine-rich repeats are thought to
be involved in protein-protein interactions. 35 Leucine-rich
repeats are short sequence motifs present in a number of proteins
with diverse functions and cellular locations. The crystal
structure of ribonuclease inhibitor protein has revealed that
leucine-rich repeats correspond to beta-alpha structural units.
These units are arranged so that they form a parallel beta-sheet
with one surface exposed to solvent, so that the protein acquires
an unusual, nonglubular shape. These two features have been
indicated as responsible for the protein-binding functions of
proteins containing leucine-rich repeats. See, Kobe and
Deisenhofer, Trends Biochem. Sci., 19(10):415-421 (October
1994).
[0036] A study has been reported on leucine-rich proteoglycans
which serve as tissue organizers, orienting and ordering collagen
fibrils during ontogeny and are involved in pathological processes
such as wound healing, tissue repair, and tumor stroma formation.
Iozzo, R. V., Crit. Rev. Biochem. Mol. Biol., 32(2):141-174 (1997).
Others studies implicating leucine rich proteins in wound healing
and tissue repair are De La Salle, C., et al., Vouv. Rev. Fr.
Hematol. (Germany), 37(4):215-222 (1995), reporting mutations in
the leucine rich motif in a complex associated with the bleeding
disorder Bernard-Soulier syndrome and Chlemetson, K. J., Thromb.
Haemost. (Germany), 74(1): 111-116 (July 1995), reporting that
platelets have leucine rich repeats. Another protein of particular
interest which has been reported to have leucine-rich repeats is
the SLIT protein which has been reported to be useful in treating
neuro-degenerative diseases such as Alzheimer's disease, nerve
damage such as in Parkinson's disease, and for diagnosis of cancer,
see, Artavanistsakonas, S. and Rothberg, J. M., WO9210518-Al by
Yale University. Other studies reporting on the biological
functions of proteins having leucine-rich repeats include: Tayar,
N., et al., Mol. Cell Endocrinol., (Ireland), 125(1-2):65-70
(December 1996) (gonadotropin receptor involvement); Miura, Y., et
al., Nippon Rinsho (Japan), 54(7):1784-1789 (July 1996) (apoptosis
involvement); Harris, P. C., et al., J. Am. Soc. Nephrol.,
6(4):1125-1133 (October 1995) (kidney disease involvement); and
Ruoslahti, E. I., et al., WO9110727-A by La Jolla Cancer Research
Foundation (decorin binding to transforming growth factor-.beta.
involvement for treatment for cancer, wound healing and scarring).
Also of particular interest is fibromodulin and its use to prevent
or reduce dermal scarring. A study of fibromodulin is found in U.S.
Pat. No. 5,654,270 to Ruoslahti, et al.
[0037] Efforts are therefore being undertaken by both industry and
academia to identify new proteins having leucine rich repeats to
better understand protein-protein interactions. Of particular
interest are those proteins having leucine rich repeats and
homology to known proteins having leucine rich repeats such as
fibromodulin, the SLIT protein and platelet glycoprotein V. Many
efforts are focused on the screening of mammalian recombinant DNA
libraries to identify the coding sequences for novel secreted and
membrane-bound proteins having leucine rich repeats. We herein
describe the identification and characterization of novel
polypeptides having homology to fibromodulin, herein designated as
PRO265 polypeptides.
[0038] 6. PRO219
[0039] Human matrilin-2 polypeptide is a member of the von
Willebrand factor type A-like module superfamily. von Willebrand
factor is a protein which plays an important role in the
maintenence of hemostasis. More specifically, von Willebrand factor
is a protein which is known to participate in platelet-vessel wall
interactions at the site of vascular injury via its ability to
interact and form a complex with Factor VIII. The absence of von
Willebrand factor in the blood causes an abnormality with the blood
platelets that prevents platelet adhesion to the vascular wall at
the site of the vascular injury. The result is the propensity for
brusing, nose bleeds, intestinal bleeding, and the like comprising
von Willebrand's disease.
[0040] Given the physiological importance of the blood clotting
factors, efforts are currently being undertaken by both industry
and academia to identify new, native proteins which may be involved
in the coagulation process. We herein describe the identification
of a novel full-length polypeptide which possesses homology to the
human matrilin-2 precursor polypeptide.
[0041] 7. PRO246
[0042] The cell surface protein HCAR is a membrane-bound protein
that acts as a receptor for subgroup C of the adenoviruses and
subgroup B of the coxsackieviruses. Thus, HCAR may provide a means
for mediating viral infection of cells in that the presence of the
HCAR receptor on the cellular surface provides a binding site for
viral particles, thereby facilitating viral infection.
[0043] In light of the physiological importance of membrane-bound
proteins and specficially those which serve a cell surface receptor
for viruses, efforts are currently being undertaken by both
industry and academia to identify new, native membrane-bound
receptor proteins. Many of these efforts are focused on the
screening of mammalian recombinant DNA libraries to identify the
coding sequences for novel receptor proteins. We herein describe a
novel membrane-bound polypeptide (designated herein as PRO246)
having homology to the cell surface protein HCAR and to various
tumor antigens including A33 and carcinoembryonic antigen, wherein
this polypeptide may be a novel cell surface virus receptor or
tumor antigen.
[0044] 8. PRO228
[0045] There are a number of known seven transmembrane proteins and
within this family is a group which includes CD97 and EMR1. CD97 is
a seven-span transmembrane receptor which has a cellular ligand,
CD55, DAF. Hamann, et al., J. Exp. Med. (U.S.), 184(3):1189 (1996).
Additionally, CD97 has been reported as being a dedifferentiation
marker in human thyroid carcinomas and as associated with
inflammation. Aust, et al., Cancer Res. (U.S.), 57(9):1798 (1997);
Gray, et al., J. Immunol. (U.S.), 157(12):5438 (1996). CD97 has
also been reported as being related to the secretin receptor
superfamily, but unlike known members of that family, CD97 and EMR1
have extended extracellular regions that possess several EGF
domains at the N-terminus. Hamann, et al., Genomics, 32(1):144
(1996); Harmann, et al., J. Immunol., 155(4):1942 (1995). EMR1 is
further described in Lin, et al., Genomics, 41(3):301 (1997) and
Baud, et al., Genomics, 26(2):334 (1995). While CD97 and EMR1
appear to be related to the secretin receptors, a known member of
the secretin family of G protein-coupled receptors includes the
alpha-latroxin receptor, latrophilin, which has been described as
calcium independent and abundant among neuronal tissues. Lelianova,
et al., J. Biol. Chem., 272(34), 21504 (1997); Davletov, et al., J.
Biol. Chem. (U.S.), 271(38):23239 (1996). Both members of the
secretin receptor superfamily and non-members which are related to
the secretin receptor superfamily, or CRF and calcitonin receptors
are of interest. In particular, new members of these families,
identified by their homology to known proteins, are of
interest.
[0046] Efforts are being undertaken by both industry and academia
to identify new membrane-bound receptor proteins, particularly
transmembrane proteins with EGF repeats and large N-terminuses
which may belong to the family of seven-transmembrane proteins of
which CD97 and EMR1 are members. We herein describe the
identification and charactization of novel polypeptides having
homology to CD97 and EMR1, designated herein as PRO228
polypeptides.
[0047] 9. PRO533
[0048] Growth factors are molecular signals or mediators that
enhance cell growth or proliferation, alone or in concert, by
binding to specific cell surface receptors. However, there are
other cellular reactions than only growth upon expression to growth
factors. As a result, growth factors are better characterized as
multifunctional and potent cellular regulators. Their biological
effects include proliferation, chemotaxis and stimulation of
extracellular matrix production. Growth factors can have both
stimulatory and inhibitory effects. For example, transforming
growth factors (TGF-.beta.) is highly pleiotropic and can stimulate
proliferation in some cells, especially connective tissues, while
being a potent inhibitor of proliferation in others, such as
lymphocytes and epithelial cells.
[0049] The physiological effect of growth stimulation or inhibition
by growth factors depends upon the state of development and
differentiation of the target tissue. The mechanism of local
cellular regulation by classical endocrine molecules comprehends
autocrine (same cell), juxtacrine (neighbor cell), and paracrine
(adjacent cell) pathways. Peptide growth factors are elements of a
complex biological language, providing the basis for intercellular
communication. They permit cells to convey information between each
other, mediate interaction between cells and change gene
expression. The effect of these multifunctional and pluripotent
factors is dependent on the presence or absence of other
peptides.
[0050] Fibroblast growth factors (FGFs) are a family of
heparin-binding, potent mitogens for both normal diploid
fibroblasts and established cell lines, Godpodarowicz, D. et al.
(1984), Proc. Natl. Acad. Sci. USA 81: 6983, the FGF family
comprises acidic FGF (FGF-1), basic FGF (FGF-2), INT-2 (FGF-3),
K-FGF/HST (FGF-4), FGF-5, FGF-6, KGF (FGF-7), AIGF (FGF-8) among
others. All FGFs have two conserved cysteine residues and share
30-50% sequence homology at the amino acid level. These factors are
mitogenic for a wide variety of normal diploid mesoderm-derived and
neural crest-derived cells, inducing granulosa cells, adrenal
cortical cells, chrondocytes, myoblasts, corneal and vascular
endothelial cells (bovine or human), vascular smooth muscle cells,
lens, retina and prostatic epithelial cells, oligodendrocytes,
astrocytes, chrondocytes, myoblasis and osteoblasts.
[0051] Fibroblast growth factors can also stimulate a large number
of cell types in a non-mitogenic manner. These activities include
promotion of cell migration into a wound area (chemotaxis),
initiation of new blood vessel formulation (angiogenesis),
modulation of nerve regeneration and survival (neurotrophism),
modulation of endocrine functions, and stimulation or suppression
of specific cellular protein expression, extracellular matrix
production and cell survival. Baird, A. & Bohlen, P., Handbook
of Exp. Phrmacol. 95(1): 369-418 (1990). These properties provide a
basis for using fibroblast growth factors in therapeutic approaches
to accelerate wound healing, nerve repair, collateral blood vessel
formation, and the like. For example, fibroblast growth factors,
have been suggested to mininiize myocardium damage in heart disease
and surgery (U.S. Pat. No. 4,378,437).
[0052] We herein describe the identification and characterization
of novel polypeptides having homology to FGF, herein designated
PRO533 polypeptides.
[0053] 10. PRO245
[0054] Some of the most important proteins involved in the above
described regulation and modulation of cellular processes are the
enzymes which regulate levels of protein phosphorylation in the
cell. For example, it is known that the transduction of signals
that regulate cell growth and differentiation is regulated at least
in part by phosphorylation and dephosphorylation of various
cellular proteins. The enzymes that catalyze these processes
include the protein kinases, which function to phosphorylate
various cellular proteins, and the protein phosphatases, which
function to remove phosphate residues from various cellular
proteins. The balance of the level of protein phosphorylation in
the cell is thus mediated by the relative activities of these two
types of enzymes.
[0055] Although many protein kinase enzymes have been identified,
the physiological role played by many of these catalytic proteins
has yet to be elucidated. It is well known, however, that a number
of the known protein kinases function to phosphorylate tyrosine
residues in proteins, thereby leading to a variety of different
effects. Perhaps most importantly, there has been a great deal of
interest in the protein tyrosine kinases since the discovery that
many oncogene products and growth factors possess intrinsic protein
tyrosine kinase activity. There is, therefore, a desire to identify
new members of the protein tyrosine kinase family.
[0056] Given the physiological importance of the protein kinases,
efforts are being undertaken by both industry and academia to
identify new, native kinase proteins. Many of these efforts are
focused on the screening of mammalian recombinant DNA libraries to
identify the coding sequences for novel kinase proteins. We herein
describe the identification and characterization of novel
polypeptides having homology to tyrosine kinase proteins,
designated herein as PRO245 polypeptides.
[0057] 11. PRO220, PRO221 and PRO227
[0058] Protein-protein interactions include receptor and antigen
complexes and signaling mechanisms. As more is known about the
structural and functional mechanisms underlying protein-protein
interactions, protein-protein interactions can be more easily
manipulated to regulate the particular result of the
protein-protein interaction. Thus, the underlying mechanisms of
protein-protein interactions are of interest to the scientific and
medical community.
[0059] All proteins containing leucine-rich repeats are thought to
be involved in protein-protein interactions. Leucine-rich repeats
are short sequence motifs present in a number of proteins with
diverse functions and cellular locations. The crystal structure of
ribonuclease inhibitor protein has revealed that leucine-rich
repeats correspond to beta-alpha structural units. These units are
arranged so that they form a parallel beta-sheet with one surface
exposed to solvent, so that the protein acquires an unusual,
nonglubular shape. These two features have been indicated as
responsible for the protein-binding functions of proteins
containing leucine-rich repeats. See, Kobe and Deisenhofer, Trends
Biochem. Sci., 19(10):415-421 (October 1994).
[0060] A study has been reported on leucine-rich proteoglycans
which serve as tissue organizers, orienting and ordering collagen
fibrils during ontogeny and are involved in pathological processes
such as wound healing, tissue repair, and tumor stroma formation.
Iozzo, R. V., Crit. Rev. Biochem. Mol. Biol., 32(2):141-174 (1997).
Others studies implicating leucine rich proteins in wound healing
and tissue repair are De La Salle, C., et al., Vouv. Rev. Fr.
Hematol. (Germany), 37(4):215-222 (1995), reporting mutations in
the leucine rich motif in a complex associated with the bleeding
disorder Bernard-Soulier syndrome and Chlemetson, K. J., Thromb.
Haemost. (Germany), 74(1):111-116 (July 1995), reporting that
platelets have leucine rich repeats. Another protein of particular
interest which has been reported to have leucine-rich repeats is
the SLIT protein which has been reported to be useful in treating
neuro-degenerative diseases such as Alzheimer's disease, nerve
damage such as in Parkinson's disease, and for diagnosis of cancer,
see, Artavanistsakonas, S. and Rothberg, J. M., WO9210518-Al by
Yale University. Other studies reporting on the biological
functions of proteins having leucine-rich repeats include: Tayar,
N., et al., Mol. Cell Endocrinol., (Ireland), 125(1-2):65-70
(December 1996) (gonadotropin receptor involvement); Miura, Y., et
al., Nippon Rinsho (Japan), 54(7): 1784-1789 (July 1996) (apoptosis
involvement); Harris, P. C., et al., J. Am. Soc. Nephrol.,
6(4):1125-1133 (October 1995) (kidney disease involvement); and
Ruoslahti, E. I., et al., WO9110727-A by La Jolla Cancer Research
Foundation (decorin binding to transforming growth factors
involvement for treatment for cancer, wound healing and
scarring).
[0061] Efforts are therefore being undertaken by both industry and
academia to identify new proteins having leucine rich repeats to
better understand protein-protein interactions. Of particular
interest are those proteins having leucine rich repeats and
homology to known proteins having leucine rich repeats such as the
SLIT protein and platelet glycoprotein V.
[0062] 12. PRO258
[0063] Immunoglobulins are antibody molecules, the proteins that
function both as receptors for antigen on the B-cell membrane and
as the secreted products of the plasma cell. Like all antibody
molecules, immunoglobulins perform two major functions: they bind
specifically to an antigen and they participate in a limited number
of biological effector functions. Therefore, new members of the Ig
superfamily are always of interest. Molecules which act as
receptors by various viruses and those which act to regulate immune
function are of particular interest. Also of particular interest
are those molecules which have homology to known Ig family members
which act as virus receptors or regulate immune function. Thus,
molecules having homology to poliovirus receptors, CRTAM and CD166
(a ligand for lymphocyte antigen CD6) are of particular
interest.
[0064] Extracellular and membrane-bound proteins play important
roles in 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, usually at a
membrane-bound receptor protein.
[0065] We herein describe the identification and characterization
of novel polypeptides having homology to CRTAM, designated herein
as PRO258 polypeptides.
[0066] 13. PRO266
[0067] Protein-protein interactions include receptor and antigen
complexes and signaling mechanisms. As more is known about the
structural and functional mechanisms underlying protein-protein
interactions, protein-protein interactions can be more easily
manipulated to regulate the particular result of the
protein-protein interaction. Thus, the underlying mechanisms of
protein-protein interactions are of interest to the scientific and
medical community.
[0068] All proteins containing leucine-rich repeats are thought to
be involved in protein-protein interactions. Leucine-rich repeats
are short sequence motifs present in a number of proteins with
diverse functions and cellular locations. The crystal structure of
ribonuclease inhibitor protein has revealed that leucine-rich
repeats correspond to beta-alpha structural units. These units are
arranged so that they form a parallel beta-sheet with one surface
exposed to solvent, so that the protein acquires an unusual,
nonglobular shape. These two features have been indicated as
responsible for the protein-binding functions of proteins
containing leucine-rich repeats. See, Kobe and Deisenhofer, Trends
Biochem. Sci., 19(10):415421 (October 1994).
[0069] A study has been reported on leucine-rich proteoglycans
which serve as tissue organizers, orienting and ordering collagen
fibrils during ontogeny and are involved in pathological processes
such as wound healing, tissue repair, and tumor stroma formation.
Iozzo, R. V., Crit. Rev. Biochem. Mol. Biol., 32(2):141-174 (1997).
Others studies implicating leucine rich proteins in wound healing
and tissue repair are De La Salle, C., et al., Vouv. Rev. Fr.
Hematol. (Germany), 37(4):215-222 (1995), reporting mutations in
the leucine rich motif in a complex associated with the bleeding
disorder Bernard-Soulier syndrome and Chlemetson, K. J., Thromb.
Haemost. (Germany), 74(1):111-116 (July 1995), reporting that
platelets have leucine rich repeats. Another protein of particular
interest which has been reported to have leucine-rich repeats is
the SLIT protein which has been reported to be useful in treating
neuro-degenerative diseases such as Alzheimer's disease, nerve
damage such as in Parkinson's disease, and for diagnosis of cancer,
see, Artavanistsakonas, S. and Rothberg, J. M., WO9210518-A1 by
Yale University. Other studies reporting on the biological
functions of proteins having leucine-rich repeats include: Tayar,
N., et al., Mol. Cell Endocrinol., (Ireland), 125(1-2):65-70
(December 1996) (gonadotropin receptor involvement); Miura, Y., et
al., Nippon Rinsho (Japan), 54(7): 1784-1789 (July 1996) (apoptosis
involvement); Harris, P. C., et al., J. Am. Soc. Nephrol.,
6(4):1125-1133 (October 1995) (kidney disease involvement); and
Ruoslahti, E. I., et al., WO9110727-A by La Jolla Cancer Research
Foundation (decorin binding to transforming growth factors
involvement for treatment for cancer, wound healing and
scarring).
[0070] Efforts are therefore being undertaken by both industry and
academia to identify new proteins having leucine rich repeats to
better understand protein-protein interactions, neuronal
development and adhesin molecules. Of particular interest are those
proteins having leucine rich repeats and homology to known proteins
having leucine rich repeats such as the SLIT protein. We herein
describe novel polypeptides having homology to SLIT, designated
herein as PRO266 polypeptides.
[0071] 14. PRO269
[0072] Thrombomodulin binds to and regulates the activity of
thrombin. It is important in the control of blood coagulation.
Thrombomodulin functions as a natural anticoagulant by accelerating
the activation of protein C by thrombin. Soluble thrombomodulin may
have therapeutic use as an antithrombotic agent with reduced risk
for hemorrhage as compared with heparin. Thrombomodulin is a cell
surface trans-membrane glycoprotein, present on endothelial cells
and platelets. A smaller, functionally active form of
thrombomodulin circulates in the plasma and is also found in urine.
(In Haeberli, A., Human Protein Data, VCH Oub., N.Y., 1992).
Peptides having homology to thrombomodulin are particularly
desirable.
[0073] We herein describe the identification and characterization
of novel polypeptides having homology to thrombomodulin, designated
herein as PRO269 polypeptides.
[0074] 15. PRO287
[0075] Procollagen C-proteinase enhancer protein binds to and
enhances the activity of bone morphogenic protein "BMP1
"/procollagen C-proteinase (PCP). It plays a role in extracellular
matrix deposition. BMP1 proteins may be used to induce bone and/or
cartilage formation and in wound healing and tissue repair.
Therefore, procollagen C-proteinase enhancer protein, BMP1 and
proteins having homology thereto, are of interest to the scientific
and medical communities.
[0076] We herein describe the identification and characterization
of novel polypeptides having homology to procollagen C-proteinase
enhancer protein precursor and procollagen C-proteinase enhancer
protein, designated herein as PRO287 polypeptides.
[0077] 16. PRO214
[0078] Growth factors are molecular signals or mediators that
enhances cell growth or proliferation, alone or in concert, by
binding to specific cell surface receptors. However, there are
other cellular reactions than only growth upon expression to growth
factors. As a result, growth factors are better characterized as
multifunctional and potent cellular regulators. Their biological
effects include proliferation, chemotaxis and stimulation of
extracellular matrix production. Growth factors can have both
stimulatory and inhibitory effects. For example, transforming
growth factor .beta. (TGF-.beta.) is highly pleiotropic and can
stimulate proliferation in some cells, especially connective
tissue, while being a potent inhibitor of proliferation in others,
such as lymphocytes and epithelial cells.
[0079] The physiological effect of growth stimulation or inhibition
by growth factors depends upon the state of development and
differentiation of the target tissue. The mechanism of local
cellular regulation by classical endocrine molecules involves
comprehends autocrine (same cell), juxtacrine (neighbor cell), and
paracrine (adjacent cells) pathways. Peptide growth factors are
elements of a complex biological language, providing the basis for
intercellular communication. They permit cells to convey
information between each other, mediate interaction between cells
and change gene expression. The effect of these multifunctional and
pluripotent factors is dependent on the presence or absence of
other peptides.
[0080] Epidermal growth factor (EGF) is a conventional mitogenic
factor that stimulates the proliferation of various types of cells
including epithelial cells and fibroblasts. EGF binds to and
activates the EGF receptor (EGFR), which initiates intracellular
signaling and subsequent effects. The EGFR is expressed in neurons
of the cerebral cortex, cerebellum, and hippocampus in addition to
other regions of the central nervous system (CNS). In addition, EGF
is also expressed in various regions of the CNS. Therefore, EGF
acts not only on mitotic cells, but also on postnitotic neurons. In
fact, many studies have indicated that EGF has neurotrophic or
neuromodulatory effects on various types of neurons in the CNS. For
example, EGF acts directly on cultured cerebral cortical and
cerebellar neurons, enhancing neurite outgrowth and survival. On
the other hand, EGF also acts on other cell types, including septal
cholinergic and mesencephalic dopaminergic neurons, indirectly
through glial cells. Evidence of the effects of EGF on neurons in
the CNS is accumulating, but the mechanisms of action remain
essentially unknown. EGF-induced signaling in mitotic cells is
better understood than in postmilotic neurons. Studies of cloned
pheochromocytoma PC12 cells and cultured cerebral cortical neurons
have suggested that the EGF-induced neurotrophic actions are
mediated by sustained activation of the EGFR and mitogen-activated
protein kinase (MAPK) in response to EGF. The sustained
intracellular signaling correlates with the decreased rate of EGFR
down-regulation, which might determine the response of neuronal
cells to EGF. It is likely that EGF is a multi-potent growth factor
that acts upon various types of cells including mitotic cells and
postmitotic neurons.
[0081] EGF is produced by the salivary and Brunner's glands of the
gastrointestinal system, kidney, pancreas, thyroid gland, pituitary
gland, and the nervous system, and is found in body fluids such as
saliva, blood, cerebrospinal fluid (CSF), urine, amniotic fluid,
prostatic fluid, pancreaticjuice, and breast milk, Plata-Salaman, C
R Peptides 12: 653-663 (1991).
[0082] EGF is mediated by its membrane specific receptor, which
contains an intrinsic tyrosine kinase. Stoscheck C M et al., J.
Cell Biochem. 31: 135-152 (1986). EGF is believed to function by
binding to the extracellular portion of its receptor which induces
a transmembrane signal that activates the intrinsic tyrosine
kinase.
[0083] Purification and sequence analysis of the EGF-like domain
has revealed the presence of six conserved cysteine residues which
cross-bind to create three peptide loops, Savage C R et al., J.
Biol. Chem. 248: 7669-7672 (1979). It is now generally known that
several other peptides can react with the EGF receptor which share
the same generalized motif
X.sub.xCX.sub.7CX.sub.4/5CX.sub.10CXCX.sub.5GX.sub.2CX.sub.n, where
X represents any non-cysteine amino acid, and n is a variable
repeat number. Non isolated peptides having this motif include
TGF-.alpha., amphiregulin, schwannoma-derived growth factor (SDGF),
heparin-binding EGF-like growth factors and certain virally encoded
peptides (e.g., Vaccinia virus, Reisner A H, Nature 313: 801-803
(1985), Shope fibroma virus, Chang W., et al., Mol Cell Biol. 7:
535-540 (1987), Molluscum contagiosum, Porter C D & Archard L
C, J. Gen. Virol. 68: 673-682 (1987), and Myxoma virus, Upton C et
al., J. Virol. 61: 1271-1275 (1987). Prigent SA & Lemoine N.
R., Prog. Growth Factor Res. 4: 1-24 (1992).
[0084] EGF-like domains are not confined to growth factors but have
been observed in a variety of cell-surface and extracellular
proteins which have interesting properties in cell adhesion,
protein-protein interaction and development, Laurence D J R &
Gusterson B A, Tumor Biol. 11: 229-261 (1990). These proteins
include blood coagulation factors (factors VI, IX, X, XII, protein
C, protein S, protein Z, tissue plasminogen activator, urokinase),
extracellular matrix components (laminin, cytotactin, entactin),
cell surface receptors (LDL receptor, thrombomodulin receptor) and
immunity-related proteins (complement C1r, uromodulin).
[0085] Even more interesting, the general structure pattern of
EGF-like precursors is preserved through lower organisms as well as
in mammalian cells. A number of genes with developmental
significance have been identified in invertebrates with EGF-like
repeats. For example, the notch gene of Drosophila encodes 36
tandemly arranged 40 amino acid repeats which show homology to EGF,
Wharton W et al., Cell 43: 557-581 (1985). Hydropathy plots
indicate a putative membrane spanning domain, with the EGF-related
sequences being located on the extracellular side of the membrane.
Other homeotic genes with EGF-like repeats include Delta, 95F and
5ZD which were identified using probes based on Notch, and the
nematode gene Lin-12 which encodes a putative receptor for a
developmental signal transmitted between two specified cells.
[0086] Specifically, EGF has been shown to have potential in the
preservation and maintenance of gastrointestinal mucosa and the
repair of acute and chronic mucosal lesions, Konturek, P C et al.,
Eur. J. Gastroenterol Hepatol. 7 (10), 933-37 (1995), including the
treatment of necrotizing enterocolitis, Zollinger-Ellison syndrome,
gastrointestinal ulceration gastrointestinal ulcerations and
congenital microvillus atrophy, A. Guglietta & P B Sullivan,
Eur. J. Gastroenterol Hepatol, 7(10), 945-50 (1995). Additionally,
EGF has been implicated in hair follicle differentiation; C. L. du
Cros, J. Invest. Dermatol. 101 (1 Suppl.), 106S-113S (1993), SG
Hillier, Clin. Endocrinol. 33(4),427-28(1990); kidney function, L.
L. Hamm et al., Semin. Nephrol. 13 (1): 109-15 (1993), R C Harris,
Am. J. Kidney Dis. L7(6): 627-30 (1991); tear fluid, G B van Setten
et al., Int. Ophthalmol 15(6); 359-62 (1991); vitamin K mediated
blood coagulation, J. Stenflo et al., Blood 78(7): 1637-51 (1991).
EGF is also implicated various skin disease characterized by
abnormal keratinocyte differentiation, e.g., psoriasis, epithelial
cancers such as squamous cell carcinomas of the lung, epidermoid
carcinoma of the vulva and gliomas. King, L E et al., Am. J. Med.
Sci. 296: 154-158 (1988).
[0087] Of great interest is mounting evidence that genetic
alterations in growth factors signaling pathways are closely linked
to developmental abnormalities and to chronic diseases including
cancer. Aaronson S A, Science 254: 1146-1153 (1991). For example,
c-erb-2 (also known as HER-2), a proto-oncogene with close
structural similarity to EGF receptor protein, is overexpressed in
human breast cancer. King et al., Science 229: 974-976 (1985);
Gullick, W J, Hormones and their actions, Cooke B A et al., eds,
Amsterdam, Elsevier, pp 349-360 (1986).
[0088] 17. PRO317
[0089] The TGF-.beta. supergene family, or simply TGF-.beta.
superfamily, a group of secreted proteins, includes a large number
of related growth and differentiation factors expressed in
virtually all phyla. Superfamily members bind to specific cell
surface receptors that activate signal transduction mechanisms to
elicit their multifunctional cytokine effects. Kolodziejczyk and
Hall, Biochem. Cell. Biol., 74: 299-314 (1996); Attisano and Wrana,
Cytokine Growth Factor Rev., 7: 327-339 (1996); and Hill, Cellular
Signaling, 8: 533-544 (1996).
[0090] Members of this family include five distinct forms of
TGF-.beta. (Sporn and Roberts, in Peptide Growth Factors and Their
Receptors, Sporn and Roberts, eds. (Springer-Verlag: Berlin, 1990)
pp. 419-472), as well as the differentiation factors vgl (Weeks and
Melton, Cell, 51: 861-867 (1987)) and DPP-C polypeptide (Padgett et
al., Nature, 325: 81-84 (1987)), the hormones activin and inhibin
(Mason et al., Nature, 318: 659-663 (1985); Mason et al., Growth
Factors, 1: 77-88 (1987)), the Mullerian-inhibiting substance (MIS)
(Cate et al., Cell, 45: 685-698 (1986)), the bone morphogenetic
proteins (BMPs) (Wozney et al., Science, 242: 1528-1534 (1988); PCT
WO 88/00205 published Jan. 14, 1988; U.S. Pat. No. 4,877,864 issued
Oct. 31, 1989), the developmentally regulated proteins Vgr-1 (Lyons
et al., Proc. Natl. Acad. Sci. USA. 86: 45544558 (1989)) and Vgr-2
(Jones et al., Molec. Endocrinol., 6: 1961-1968 (1992)), the mouse
growth differentiation factor (GDF), such as GDF-3 and GDF-9
(Kingsley, Genes Dev., 8: 133-146 (1994); McPherron and Lee, J.
Biol. Chem., 268: 3444-3449 (1993)), the mouse lefty/Stral (Meno et
al., Nature, 381: 151-155 (1996); Bouillet et al., Dev. Biol., 170:
420-433 (1995)), glial cell line-derived neurotrophic factor (GDNF)
(Lin et al., Science, 260: 1130-1132 (1993), neurturin (Kotzbauer
et al., Nature, 384: 467470 (1996)), and endometrial
bleeding-associated factor (EBAF) (Kothapalli et al., J. Clin.
Invest., 99: 2342-2350 (1997)). The subset BMP-2A and BMP-2B is
approximately 75% homologous in sequence to DPP-C and may represent
the mammalian equivalent of that protein.
[0091] The proteins of the TGF-.beta. superfamily are
disulfide-linked homo- or heterodimers encoded by larger precursor
polypeptide chains containing a hydrophobic signal sequence, a long
and relatively poorly conserved N-terminal pro region of several
hundred amino acids, a cleavage site (usually polybasic), and a
shorter and more highly conserved C-terminal region. This
C-terminal region corresponds to the processed mature protein and
contains approximately 100 amino acids with a characteristic
cysteine motif, i.e., the conservation of seven of the nine
cysteine residues of TGF-.beta. among all known family members.
Although the position of the cleavage site between the mature and
pro regions varies among the family members, the C-terminus of all
of the proteins is in the identical position, ending in the
sequence Cys-X-Cys-X, but differing in every case from the
TGF-.beta.consensus C-terminus of Cys-Lys-Cys-Ser. Spom and
Roberts, 1990, supra.
[0092] There are at least five forms of TGF-.beta. currently
identified, TGF-.beta. 1, TGF-.beta.2, TGF-.beta.3, TGF-.beta.4,
and TGF-.beta.5. The activated form of TGF-.beta.1 is a homodimer
formed by dimerization of the carboxy-terminal 112 amino acids of a
390 amino acid precursor. Recombinant TGF-.beta.1 has been cloned
(Derynck et al., Nature, 316:701-705 (1985)) and expressed in
Chinese hamster ovary cells (Gentry et al., Mol. Cell. Biol., 7:
3418-3427 (1987)). Additionally, recombinant human TGF-.beta.2
(deMartin et al., EMBO J., 6: 3673 (1987)), as well as human and
porcine TGF-.beta.3 (Derynck et al., EMBO J., 7: 3737-3743 (1988);
ten Dijke et al., Proc. Natl. Acad. Sci. USA, 85: 4715 (1988)) have
been cloned. TGF-.beta.2 has a precursor form of 414 amino acids
and is also processed to a homodimer from the carboxy-terminal 112
amino acids that shares approximately 70% homology with the active
form of TGF-.beta.1 (Marquardt et al., J. Biol. Chem., 262: 12127
(1987)). See also EP 200,341; 169,016; 268,561; and 267,463; U.S.
Pat. No. 4,774,322; Cheifetz et al., Cell, 48: 409415 (1987);
Jakowlew et al., Molecular Endocrin., 2: 747-755 (1988); Derynck et
al., J. Biol. Chem., 261: 4377-4379 (1986); Sharples et al., DNA,
6: 239-244 (1987); Derynck et al., Nucl. Acids. Res., 15: 3188-3189
(1987); Derynck et al., Nucl. Acids. Res., 15: 3187 (1987); Seyedin
et al., J. Biol. Chem., 261: 5693-5695 (1986); Madisenet al., DNA,
7: 1-8 (1988); and Hanks et al., Proc. Natl. Acad. Sci. (U.S.A.),
85: 79-82 (1988).
[0093] TGF-.beta.4 and TGF-.beta.5 were cloned from a chicken
chondrocyte cDNA library (Jakowlew et al., Molec. Endocrinol., 2:
1186-1195 (1988)) and from a frog oocyte cDNA library,
respectively.
[0094] The pro region of TGF-.beta. associates non-covalently with
the mature TGF-.beta. dimer (Wakefield et al., J. Biol. Chem., 263:
7646-7654 (1988); Wakefield et al., Growth Factors, 1: 203-218
(1989)), and the pro regions are found to be necessary for proper
folding and secretion of the active mature dimers of both
TGF-.beta.and activin (Gray and Mason, Science, 247: 1328-1330
(1990)). The association between the mature and pro regions of
TGF-.beta. masks the biological activity of the mature dimer,
resulting in formation of an inactive latent form. Latency is not a
constant of the TGF-.beta., superfamily, since the presence of the
pro region has no effect on activin or inhibin biological
activity.
[0095] A unifying feature of the biology of the proteins from the
TGF-.beta. superfamily is their ability to regulate developmental
processes. TGF-.beta. has been shown to have numerous regulatory
actions on a wide variety of both normal and neoplastic cells.
TGF-.beta. is multifunctional, as it can either stimulate or
inhibit cell proliferation, differentiation, and other critical
processes in cell function (Sporn and Roberts, supra).
[0096] One member of the TGF-.beta. superfamily, EBAF, is expressed
in endometrium only in the late secretory phase and during abnormal
endometrial bleeding. Kothapalli et al., J. Clin. Invest., 99:
2342-2350 (1997). Human endometrium is unique in that it is the
only tissue in the body that bleeds at regular intervals. In
addition, abnormal endometrial bleeding is one of the most common
manifestations of gynecological diseases, and is a prime indication
for hysterectomy. In situ hybridization showed that the mRNA of
EBAF was expressed in the stroma without any significant mRNA
expression in the endometrial glands or endothelial cells.
[0097] The predicted protein sequence of EBAF showed a strong
homology to the protein encoded by mouse lefty/stra3 of the
TGF-.beta. superfamily. A motif search revealed that the predicted
EBAF protein contains most of the cysteine residues which are
conserved among the TGF-.beta.-related proteins and which are
necessary for the formation of the cysteine knot structure. The
EBAF sequence contains an additional cysteine residue, 12 amino
acids upstream from the first conserved cysteine residue. The only
other family members known to contain an additional cysteine
residue are TGF-.beta.s, inhibins, and GDF-3. EBAF, similar to
LEFTY, GDF-3/Vgr2, and GDF-9, lacks the cysteine residue that is
known to form the intermolecular disulfide bond. Therefore, EBAF
appears to be an additional member of the TGF-.beta. superfamily
with an unpaired cysteine residue that may not exist as a dimer.
However, hydrophobic contacts between the two monomer subunits may
promote dimer formation. Fluorescence in situ hybridization showed
that the ebaf gene is located on human chromosome 1 at band q42.
1.
[0098] Additional members of the TGF-.beta. superfamily, such as
those related to EBAF, are being searched for by industry and
academics. We herein describe the identification and
characterization of novel polypeptides having homology to EBAF,
designated herein as PRO317 polypeptides.
[0099] 18. PRO301
[0100] The widespread occurrence of cancer has prompted the
devotion of considerable resources and discovering new treatments
of treatment. One particular method involves the creation of tumor
or cancer specific monoclonal antibodies (mAbs) which are specific
to tumor antigens. Such mAbs, which can distinguish between normal
and cancerous cells are useful in the diagnosis, prognosis and
treatment of the disease. Particular antigens are known to be
associated with neoplastic diseases, such as colorectal cancer.
[0101] One particular antigen, the A33 antigen is expressed in more
than 90% of primary or metastatic colon cancers as well as normal
colon epithelium. Since colon cancer is a widespread disease, early
diagnosis and treatment is an important medical goal. Diagnosis and
treatment of colon cancer can be implemented using monoclonal
antibodies (mAbs) specific therefore having fluorescent, nuclear
magnetic or radioactive tags. Radioactive gene, toxins and/or drug
tagged mAbs can be used for treatment in situ with minimal patient
description. mAbs can also be used to diagnose during the diagnosis
and treatment of colon cancers. For example, when the serum levels
of the A33 antigen are elevated in a patient, a drop of the levels
after surgery would indicate the tumor resection was successful. On
the other hand, a subsequent rise in serum A33 antigen levels after
surgery would indicate that metastases of the original tumor may
have formed or that new primary tumors may have appeared. Such
monoclonal antibodies can be used in lieu of, or in conjunction
with surgery and/or other chemotherapies. For example, U.S. Pat.
No. 4,579,827 and U.S. Ser. No. 424,991 (E.P. 199,141) are directed
to therapeutic administration of monoclonal antibodies, the latter
of which relates to the application of anti-A33 mAb.
[0102] Many cancers of epithelial origin have adenovirus receptors.
In fact, adenovirus-derived vectors have been proposed as a means
of inserting antisense nucleic acids into tumors (U.S. Pat. No.
5,518,885). Thus, the association of viral receptors with
neoplastic tumors is not unexpected.
[0103] We herein describe the identification and characterization
of novel polypeptides having homology to certain cancer-associated
antigens, designated herein as PRO301 polypeptides.
[0104] 19. PRO224
[0105] Cholesterol uptake can have serious implications on one's
health. Cholesterol uptake provides cells with most of the
cholesterol they require for membrane synthesis. If this uptake is
blocked, cholesterol accumulates in the blood and can contribute to
the formation of atherosclerotic plaques in blood vessel walls.
Most cholesterol is transported in the blood bound to protein in
the form of complexes known as low-density lipoproteins (LDLs).
LDLs are endocytosed into cells via LDL receptor proteins.
Therefore, LDL receptor proteins, and proteins having homology
thereto, are of interest to the scientific and medical
communities.
[0106] Membrane-bound proteins and receptors can play an important
role in the formation, differentiation and maintenance of
multicellular organisms. The LDL receptors are an example of
membrane-bound proteins which are involved in the synthesis and
formation of cell membranes, wherein the health of an individual is
affected directly and indirectly by its function. Many
membrane-bound proteins act as receptors such as the LDL receptor.
These receptors can function to endocytose substrates or they can
function as a receptor for a channel. Other membrane-bound proteins
function as signals or antigens.
[0107] Membrane-bound proteins and receptor molecules have various
industrial applications, including as pharmaceutical and diagnostic
agents. The membrane-bound proteins can also be employed for
screening of potential peptide or small molecule regulators of the
relevant receptor/ligand interaction. In the case of the LDL
receptor, it is desirable to find molecules which enhance
endocytosis so as to lower blood cholesterol levels and plaque
formation. It is also desirable to identify molecules which inhibit
endocytosis so that these molecules can be avoided or regulated by
individuals having high blood cholesterol. Polypeptides which are
homologous to lipoprotein receptors but which do not function as
lipoprotein receptors are also of interest in the determination of
the function of the fragments which show homology.
[0108] The following studies report on previously known low density
lipoprotein receptors and related proteins including
apolipoproteins: Sawamura, et al., Nippon Chemiphar Co, Japan
patent application J09098787; Novak, S., et al., J. Biol. Chem.,
271:(20)11732-6 (1996); Blaas, D., J. Virol., 69(11)7244-7
(November 1995); Scott, J., J. Inherit. Metab. Dis. (UK), 9/Supp. 1
(3-16) (1986); Yamamoto, et al., Cell, 39:27-38 (1984); Rebece, et
al., Neurobiol. Aging, 15:5117(1994); Novak, S., et al., J. Biol.
Chemistry, 271:11732-11736(1996); and Sestavel and Fruchart, Cell
Mol. Biol., 40(4):461-81 (June 1994). These publications and others
published prior to the filing of this application provide further
background to peptides already known in the art.
[0109] Efforts are being undertaken by both industry and academia
to identify new, native membrane-bound receptor proteins,
particularly those having homology to lipoprotein receptors. We
herein describe the identification and characterization of novel
polypeptides having homology to lipoprotein receptors, designated
herein as PRO224 polypeptides.
[0110] 20. PRO222
[0111] Complement is a group of proteins found in the blood that
are important in humoral immunity and inflammation. Complement
proteins are sequentially activated by antigen-antibody complexes
or by proteolytic enzymes. When activated, complement proteins kill
bacteria and other microorganisms, affect vascular permeability,
release histamine and attract white blood cells. Complement also
enhances phagocytosis when bound to target cells. In order to
prevent harm to autologous cells, the complement activation pathway
is tightly regulated.
[0112] Deficiencies in the regulation of complement activation or
in the complement proteins themselves may lead to immune-complex
diseases, such as systemic lupus erythematosus, and may result in
increased susceptibility to bacterial infection. In all cases,
early detection of complement deficiency is desirable so that the
patient can begin treatment. Thus, research efforts are currently
directed toward identification of soluble and membrane proteins
that regulate complement activation.
[0113] Proteins known to be important in regulating complement
activation in humans include Factor H and Complement receptor type
1 (CR1). Factor H is a 150 kD soluble serum protein that interacts
with complement protein C3b to accelerate the decay of C3
convertase and acts as a cofactor for Factor I-mediated cleavage of
complement protein C4b. Complement receptor type 1 is a 190-280 kD
membrane bound protein found in mast cells and most blood cells.
CR1 interacts with complement proteins C3b, C4b, and C3b to
accelerate dissociation of C3 convertases, acts as a cofactor for
Factor I-mediated cleavage of C3b and C4b, and binds immune
complexes and promotes their dissolution and phagocytosis.
[0114] Proteins which have homology to complement proteins are of
particular interest to the medical and industrial communities.
Often, proteins having homology to each other have similar
function. It is also of interest when proteins having homology do
not have similar functions, indicating that certain structural
motifs identify information other than function, such as locality
of function.
[0115] Efforts are being undertaken by both industry and academia
to identify new, native secreted and membrane-bound proteins,
particularly those having homology to known proteins involved in
the complement pathway. Proteins involved in the complement pathway
were reviewed in Birmingham DJ (1995), Critical Reviews in
Immunology, 15(2):133-154 and in Abbas A K, et al. (1994) Cellular
and Molecular Immunology, 2nd Ed. W.B. Saunders Company,
Philadelphia, pp 295-315.
[0116] We herein describe the identification and characterization
of novel polypeptides having homology to complement receptors,
designated herein as PRO222 polypeptides.
[0117] 21. PRO234
[0118] The successful function of many systems within multicellular
organisms is dependent on cell-cell interactions. Such interactions
are affected by the alignment of particular ligands with particular
receptors in a manner which allows for ligand-receptor binding and
thus a cell-cell adhesion. While protein-protein interactions in
cell recognition have been recognized for some time, only recently
has the role of carbohydrates in physiologically relevant
recognition been widely considered (see B. K. Brandley et al., J.
Leuk. Biol. 40: 97 (1986) and N. Sharon et al., Science 246: 227
(1989). Oligosaccharides are well positioned to act as recognition
novel lectins due to their cell surface location and structural
diversity. Many oligosaccharide structures can be created through
the differential activities of a smaller number of
glycosyltransferases. The diverse structures of oligosaccharides
can be generated by transcription of relatively few gene products,
which suggests that the oligosaccharides are a plausible mechanism
by which is directed a wide range of cell-cell interactions.
Examples of differential expression of cell surface carbohydrates
and putative carbohydrate binding proteins (lectins) on interacting
cells have been described (J. Dodd & T. M. Jessel, J. Neurosci.
5: 3278 (1985); L. J. Regan et al., Proc. Natl. Acad. Sci. USA 83:
2248 (1986); M. Constantine-Paton et al., Nature 324: 459 (1986);
and M. Tiemeyer et al., J. Biol. Chem. 263: 1671 (1989). One
interesting member of the lectin family are selecting.
[0119] The migration of leukocytes to sites of acute or chronic
inflammation involves adhesive interactions between these cells and
the endothelium. This specific adhesion is the initial event in the
cascade that is initiated by inflammatory insults, and it is,
therefore, of paramount importance to the regulated defense of the
organism.
[0120] The types of cell adhesion molecules that are involved in
the interaction between leukocytes and the endothelium during an
inflammatory response currently stands at four: (1) selectins; (2)
(carbohydrate and glycoprotein) ligands for selecting; (3)
integrins; and (4) integrin ligands, which are members of the
immunoglobulin gene superfamily.
[0121] The selectins are cell adhesion molecules that are unified
both structurally and functionally. Structurally, selectins are
characterized by the inclusion of a domain with homology to a
calcium-dependent lectin (C-lectins), an epidermal growth factor
(egf)-like domain and several complement binding-like domains,
Bevilacqua, M. P. et al., Science 243: 1160-1165 (1989); Johnston
et al., Cell 56: 1033-1044 (1989); Lasky et al, Cell 56: 1045-1055
(1989); Siegalman, M. et al., Science 243: 1165-1172 (1989);
Stoolman, L. M., Cell 56: 907-910 (1989). Functionally, selectins
share the common property of their ability to mediate cell binding
through interactions between their lectin domains and cell surface
carbohydrate ligands (Brandley, B, et al., Cell 63, 861-863 (1990);
Springer, T. and Lasky, L. A., Nature 349 19-197 (1991);
Bevilacqua, M. P. and Nelson, R. M., J. Clin. Invest. 91 379-387
(1993) and Tedder et al., J. Exp. Med. 170: 123-133 (1989).
[0122] There are three members identified so far in the selectin
family of cell adhesion molecules: L-selectin (also called
peripheral lymph node homing receptor (pnHR), LEC-CAM-1, LAM-1,
gp90.sup.MEL, gp100.sup.MEL, gp10.sup.MEL, MEL-14 antigen, Leu-8
antigen, TQ-1 antigen, DREG antigen), E-selectin (LEC-CAM-2,
LECAM-2, ELAM-1) and P-selectin (LEC-CAM-3, LECAM-3, GMP-140,
PADGEM).
[0123] The identification of the C-lectin domain has led to an
intense effort to define carbohydrate binding ligands for proteins
containing such domains. E-selectin is believed to recognize the
carbohydrate sequence NeuNAc.alpha.2-3Gal.beta.14(Fuc.alpha.1-3)
GlcNAc (sialyl-Lewis x, or sLeX) and related oligosaccharides, Berg
et al., J. Biol. Chem. 265: 14869-14872 (1991); Lowe et al., Cell
63: 475-484 (1990); Phillips et al., Science 250: 1130-1132(1990);
Tiemeyer et al., Proc. Natl. Acad. Sci. USA 88:
1138-1142(1991).
[0124] L-selectin, which comprises a lectin domain, performs its
adhesive function by recognizing carbohydrate-containing ligands on
endothelial cells. L-selectin is expressed on the surface of
leukocytes, such as lymphocytes, neutrophils, monocytes and
eosinophils, and is involved with the trafficking of lymphocytes to
peripheral lymphoid tissues (Gallatin et al., Nature 303: 30-34
(1983)) and with acute neutrophil-medicated inflammatory responses
(Watson, S. R., Nature 349: 164-167 (1991)). The amino acid
sequence of L-selectin and the encoding nucleic acid sequence are,
for example, disclosed in U.S. Pat. No. 5,098,833 issued Mar. 24,
1992.
[0125] L-selectin (LECAM-1) is particularly interesting because of
its ability to block neutrophil influx (Watson et al., Nature 349:
164-167 (1991). It is expressed in chronic lymphocytic leukemia
cells which bind to HEV (Spertini et al., Nature 349: 691-694
(1991). It is also believed that HEV structures at sites of chronic
inflammation are associated with the symptoms of diseases such as
rheumatoid arthritis, psoriasis and multiple sclerosis.
[0126] E-selectin (ELAM-1), is particularly interesting because of
its transient expression on endothelial cells in response to IL-1
or TNF. Bevilacqua et al., Science 243: 1160 (1989). The time
course of this induced expression (2-8 h) suggests a role for this
receptor in initial neutrophil induced extravasation in response to
infection and injury. It has further been reported that anti-ELAM-1
antibody blocks the influx of neutrophils in a primate asthma model
and thus is beneficial for preventing airway obstruction resulting
from the inflammatory response. Gundel et al., J. Clin. Invest. 88:
1407 (1991).
[0127] The adhesion of circulating neutrophils to stimulated
vascular endothelium is a primary event of the inflammatory
response. P-selectin has been reported to recognize the Lewis x
structure (Gal.beta.1-4(Fuc.alpha.1-3) GlcNAc), Larsen et al., Cell
63: 467-474(1990). Others report that an additional terminal linked
sialic acid is required for high affinity binding, Moore et al., J.
Cell. Biol. 112: 491-499 (1991). P-selectin has been shown to be
significant in acute lung injury. Anti-P-selectin antibody has been
shown to have strong protective effects in a rodent lung injury
model. M. S. Mulligan et al., J. Clin. Invest. 90: 1600 (1991).
[0128] We herein describe the identification and characterization
of novel polypeptides having homology to lectin proteins, herein
designated as PRO234 polypeptides.
[0129] 22. PRO231
[0130] Some of the most important proteins involved in the above
described regulation and modulation of cellular processes are the
enzymes which regulate levels of protein phosphorylation in the
cell. For example, it is known that the transduction of signals
that regulate cell growth and differentiation is regulated at least
in part by phosphorylation and dephosphorylation of various
cellular proteins. The enzymes that catalyze these processes
include the protein kinases, which function to phosphorylate
various cellular proteins, and the protein phosphatases, which
function to remove phosphate residues from various cellular
proteins. The balance of the level of protein phosphorylation in
the cell is thus mediated by the relative activities of these two
types of enzymes.
[0131] Protein phosphatases represent a growing family of enzymes
that are found in many diverse forms, including both membrane-bound
and soluble forms. While many protein phosphatases have been
described, the functions of only a very few are beginning to be
understood (Tonks, Semin. Cell Biol. 4:373453 (1993) and Dixon,
Recent Prog. Horm. Res. 51:405414 (1996)). However, in general, it
appears that many of the protein phosphatases function to modulate
the positive or negative signals induced by various protein
kinases. Therefore, it is likely that protein phosphatases play
critical roles in numerous and diverse cellular processes.
[0132] Given the physiological importance of the protein
phosphatases, efforts are being undertaken by both industry and
academia to identify new, native phosphatase proteins. Many of
these efforts are focused on the screening of mammalian recombinant
DNA libraries to identify the coding sequences for novel
phosphatase 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)].
[0133] We herein describe the identification and characterization
of novel polypeptides having homology to acid phosphatases,
designated herein as PRO231 polypeptides.
[0134] 23. PRO229
[0135] Scavenger receptors are known to protect IgG molecules from
catabolic degradation. Riechmann and Hollinger, Nature
Biotechnology, 15:617 (1997). In particular, studies of the CH2 and
CH3 domains have shown that specific sequences of these domains are
important in determining the half-lives of antibodies. Ellerson, et
al., J. Immunol., 116: 510 (1976); Yasmeen, et al., J. Immunol.
116: 518 (1976; Pollock, et al., Eur. J. Immunol., 20: 2021 (1990).
Scavenger receptor proteins and antibodies thereto are further
reported in U.S. Pat. No. 5,510,466 to Krieger, et al. Due to the
ability of scavenger receptors to increase the half-life of
polypeptides and their involvement in immune function, molecules
having homology to scavenger receptors are of importance to the
scientific and medical community.
[0136] Efforts are being undertaken by both industry and academia
to identify new, native secreted and membrane-bound receptor
proteins, particularly those having homology to scavenger
receptors. Many efforts are focused on the screening of mammalian
recombinant DNA libraries to identify the coding sequences for
novel secreted and membrane-bound receptor 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)].
[0137] We herein describe the identification and characterization
of novel polypeptides having homology to scavenger receptors,
designated herein as PRO229 polypeptides.
[0138] 24. PRO238
[0139] Oxygen free radicals and antioxidants appear to play an
important role in the central nervous system after cerebral
ischemia and reperfusion. Moreover, cardiac injury, related to
ischaemia and reperfusion has been reported to be caused by the
action of free radicals. Additionally, studies have reported that
the redox state of the cell is a pivotal determinant of the fate of
the cells. Furthermore, reactive oxygen species have been reported
to be cytotoxic, causing inflammatory disease, including tissue
necrosis, organ failure, atherosclerosis, infertility, birth
defects, premature aging, mutations and malignancy. Thus, the
control of oxidation and reduction is important for a number of
reasons including for control and prevention of strokes, heart
attacks, oxidative stress and hypertension. In this regard,
reductases, and particularly, oxidoreductases, are of interest.
Publications further describing this subject matter include Kelsey,
et al., Br. J. Cancer, 76(7):8524 (1997); Friedrich and Weiss, J.
Theor. Biol., 187(4):52940 (1997) and Pieulle, et al., J.
Bacteriol., 179(18):5684-92 (1997).
[0140] Efforts are being undertaken by both industry and academia
to identify new, native secreted and membrane-bound receptor
proteins, particularly secreted proteins which have homology to
reductase. Many efforts are focused on the screening of mammalian
recombinant DNA libraries to identify the coding sequences for
novel secreted and membrane-bound receptor 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)].
[0141] We herein describe the identification and characterization
of novel polypeptides having homology to reductase, designated
herein as PRO238 polypeptides.
[0142] 25. PRO233
[0143] Studies have reported that the redox state of the cell is an
important determinant of the fate of the cell. Furthermore,
reactive oxygen species have been reported to be cytotoxic, causing
inflammatory disease, including tissue necrosis, organ failure,
atherosclerosis, infertility, birth defects, premature aging,
mutations and malignancy. Thus, the control of oxidation and
reduction is important for a number of reasons, including the
control and prevention of strokes, heart attacks, oxidative stress
and hypertension. Oxygen free radicals and antioxidants appear to
play an important role in the central nervous system after cerebral
ischemia and reperfusion. Moreover, cardiac injury, related to
ischaemia and reperfusion has been reported to be caused by the
action of free radicals. In this regard, reductases, and
particularly, oxidoreductases, are of interest. In addition, the
transcription factors, NF-kappa B and AP-1, are known to be
regulated by redox state and to affect the expression of a large
variety of genes thought to be involved in the pathogenesis of
AIDS, cancer, atherosclerosis and diabetic complications.
Publications further describing this subject matter include Kelsey,
et al., Br. J. Cancer, 76(7):8524 (1997); Friedrich and Weiss, J.
Theor. Biol., 187(4):529-40 (1997) and Pieulle, et al., J.
Bacteriol., 179(18):5684-92 (1997). Given the physiological
importance of redox reactions in vivo, efforts are currently being
under taken to identify new, native proteins which are involved in
redox reactions. We describe herein the identification of novel
polypeptides which have homology to reductase, designated herein as
PRO233 polypeptides.
[0144] 26. PRO223
[0145] The carboxypeptidase family of exopeptidases constitutes a
diverse group of enzymes that hydrolyze carboxyl-terminal amide
bonds in polypeptides, wherein a large number of mammalian tissues
produce these enzymes. Many of the carboxypeptidase enzymes that
have been identified to date exhibit rather strong cleavage
specificities for certain amino acids in polypeptides. For example,
carboxypeptidase enzymes have been identified which prefer lysine,
arginine, serine or amino acids with either aromatic or branched
aliphatic side chains as substrates at the carboxyl terminus of the
polypeptide.
[0146] With regard to the serine carboxypeptidases, such amino acid
specific enzymes have been identified from a variety of different
mammalian and non-mammalian organisms. The mammalian serine
carboxypeptidase enzymes play important roles in many different
biological processes including, for example, protein digestion,
activation, inactivation, or modulation of peptide hormone
activity, and alteration of the physical properties of proteins and
enzymes.
[0147] In light of the physiological importance of the serine
carboxypeptidases, efforts are being undertaken by both industry
and academia to identify new, native secreted and membrane-bound
receptor proteins and specifically novel carboxypeptidases. Many of
these efforts are focused on the screening of mammalian recombinant
DNA libraries to identify the coding sequences for novel secreted
and membrane-bound receptor proteins. We describe herein novel
polypeptides having homology to one or more serine carboxypeptidase
polypeptides, designated herein as PRO223 polypeptides.
[0148] 27. PRO235
[0149] Plexin was first identified in Xenopus tadpole nervous
system as a membrane glycoprotein which was shown to mediate cell
adhesion via a homophilic binding mechanism in the presence of
calcium ions. Strong evolutionary conservation between Xenopus,
mouse and human homologs of plexin has been observed. [Kaneyama et
al., Biochem. And Biophys. Res. Comm. 226: 52 4529 (1996)]. Given
the physiological importance of cell adhesion mechanisms in vivo,
efforts are currently being under taken to identify new, native
proteins which are involved in cell adhesion. We describe herein
the identification of a novel polypeptide which has homology to
plexin, designated herein as PRO235.
[0150] 28. PRO236 and PRO262
[0151] .beta.-galactosidase is a well known enzymatic protein which
functions to hydrolyze .beta.-galactoside molecules.
.beta.-galactosidase has been employed for a variety of different
applications, both in vitro and in vivo and has proven to be an
extremely useful research tool. As such, there is an interest in
obtaining novel polypeptides which exhibit homology to the
.beta.-galactosidase polypeptide.
[0152] Given the strong interest in obtaining novel polypeptides
having homology to .beta.-galactosidase, efforts are currently
being undertaken by both industry and academia to identify new,
native .beta.-galactosidase homolog proteins. Many of these efforts
are focused on the screening of mammalian recombinant DNA libraries
to identify the coding sequences for novel
.beta.-galactosidase-like 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)]. We herein describe novel poylpeptides having
siginificant homology to the .beta.-galactosidase enzyme,
designated herein as PRO236 and PRO262 polypeptides.
[0153] 29. PRO239
[0154] Densin is a glycoprotein which has been isolated from the
brain which has all the hallmarks of an adhesion molecule. It is
highly concentrated at synaptic sites in the brain and is expressed
prominently in dendritic processes in developing neurons. Densin
has been characterized as a member of the O-linked
sialoglycoproteins. Densin has relevance to medically important
processes such as regeneration. Given the physiological importance
of synaptic processes and cell adhesion mechanisms in vivo, efforts
are currently being under taken to identify new, native proteins
which are involved in synaptic machinery and cell adhesion. We
describe herein the identification of novel polypeptides which have
homology to densin, designated herein as PRO239 polypeptides.
[0155] 30. PRO257
[0156] Ebnerin is a cell surface protein associated with von Ebner
glands in mammals. Efforts are being undertaken by both industry
and academia to identify new, native cell surface receptor proteins
and specifically those which possess sequence homology to cell
surface proteins such as ebnerin. Many of these efforts are focused
on the screening of mammalian recombinant DNA libraries to identify
the coding sequences for novel receptor proteins. We herein
describe the identification of novel polypeptides having
significant homology to the von Ebner's gland-associated protein
ebnerin, designated herein as PRO257 polypeptides.
[0157] 31. PRO260
[0158] Fucosidases are enzymes that remove fucose residues from
fucose containing proteoglycans. In some pathological conditions,
such as cancer, rheumatoid arthritis, and diabetes, there is an
abnormal fucosylation of serum proteins. Therefore, fucosidases,
and proteins having homology to fucosidase, are of importance to
the study and abrogation of these conditions. In particular,
proteins having homology to the alpha-1-fucosidase precursor are of
interest. Fucosidases and fucosidase inhibitors are further
described in U.S. Pat. Nos. 5,637,490, 5,382,709, 5,240,707,
5,153,325, 5,100,797, 5,096,909 and 5,017,704. Studies are also
reported in Valk, et al., J. Virol., 71(9):6796 (1997), Aktogu, et
al., Monaldi. Arch. Chest Dis. (Italy), 52(2):118 (1997) and
Focarelli, et al., Biochem. Biophys. Res. Commun. (U.S.), 234(1):54
(1997).
[0159] Efforts are being undertaken by both industry and academia
to identify new, native secreted and membrane-bound receptor
proteins. Of particular interest are proteins having homology to
the alpha-1-fucosidase precursor. Many efforts are focused on the
screening of mammalian recombinant DNA libraries to identify the
coding sequences for novel secreted and membrane-bound receptor
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)].
We herein describe the identification and characterization of novel
polypeptides having homology to fucosidases, designated herein as
PRO260 polypeptides.
[0160] 32. PRO263
[0161] CD44 is a cell surface adhesion molecule involved in
cell-cell and cell-matrix interactions. Hyaluronic acid, a
component of the extracellular matrix is a major ligand. Other
ligands include collagen, fibronectin, laminin, chrondroitin
sulfate, mucosal addressin, serglycin and osteoponin. CD44 is also
important in regulating cell traffic, lymph node homing,
transmission of growth signals, and presentation of chemokines and
growth factors to traveling cells. CD44 surface proteins are
associated with metastatic tumors and CD44 has been used as a
marker for HIV infection. Certain splice variants are associated
with metastasis and poor prognosis of cancer patients. Therefore,
molecules having homology with CD44 are of particular interest, as
their homology indicates that they may have functions related to
those functions of CD44. CD44 is further described in U.S. Pat.
Nos. 5,506,119, 5,504,194 and 5,108,904; Gerberick, et al.,
Toxicol. Appl. Pharmacol., 146(1):1 (1997); Wittig, et al.,
Immunol. Letters (Netherlands), 57(1-3):217 (1997); and Oliveira
and Odell, Oral Oncol. (England), 33(4):260 (1997).
[0162] Efforts are being undertaken by both industry and academia
to identify new, native secreted and membrane-bound receptor
proteins, particularly transmembrane proteins with homology to CD44
antigen. Many efforts are focused on the screening of mammalian
recombinant DNA libraries to identify the coding sequences for
novel secreted and membrane-bound receptor 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)].
[0163] We herein describe the identification and characterization
of novel polypeptides having homology to CD44 antigen, designated
herein as PRO263 polypeptides.
[0164] 33. PRO270
[0165] Thioredoxins effect reduction-oxidation (redox) state. Many
diseases are potentially related to redox state and reactive oxygen
species may play a role in many important biological processes. The
transcription factors, NF-kappa B and AP-1, are regulated by redox
state and are known to affect the expression of a large variety of
genes thought to be involved in the pathogenesis of AIDS, cancer,
atherosclerosis and diabetic complications. Such proteins may also
play a role in cellular antioxidant defense, and in pathological
conditions involving oxidative stress such as stroke and
inflammation in addition to having a role in apoptosis. Therefore,
thioredoxins, and proteins having homology thereto, are of interest
to the scientific and medical communities.
[0166] We herein describe the identification and characterization
of novel polypeptides having homology to thioredoxin, designated
herein as PRO270 polypeptides.
[0167] 34. PRO271
[0168] The proteoglycan link protein is a protein which is
intimately associated with various extracellular matrix proteins
and more specifically with proteins such as collagen. For example,
one primary component of collagen is a large proteoglycan called
aggrecan. This molecule is retained by binding to the
glycosaminoglycan hyaluronan through the amino terminal G1 globular
domain of the core protein. This binding is stabilized by the
proteoglycan link protein which is a protein that is also
associated with other tissues containing hyaluronan binding
proteoglycans such as versican.
[0169] Link protein has been identified as a potential target for
autoimmune antibodies in individuals who suffer from juvenile
rheumatoid arthritis (see Guerassimov et al., J. Rheumatology
24(5):959-964 (1997)). As such, there is strong interest in
identifying novel proteins having homology to link protein. We
herein describe the identification and characterization of novel
polypeptides having such homology, designated herein as PRO271
polypeptides.
[0170] 35. PRO272
[0171] Reticulocalbin is an endoplasmic reticular protein which may
be involved in protein transport and luminal protein processing.
Reticulocalbin resides in the lumen of the endoplasmic rerticulum,
is known to bind calcium, and may be involved in a luminal
retention mechanism of the endoplasmic reticulum. It contains six
domains of the EF-hand motif associated with high affinity calcium
binding. We describe herein the identification and characterization
of a novel polypeptide which has homology to the reticulocalbin
protein, designated herein as PRO272.
[0172] 36. PRO294
[0173] Collagen, a naturally occurring protein, finds wide
application in industry. Chemically hydrolyzed natural collagen can
be denatured and renatured by heating and cooling to produce
gelatin, which is used in photographic and medical, among other
applications. Collagen has important properties such as the ability
to form interchain aggregates having a conformation designated as a
triple helix. We herein describe the identification and
characterization of a novel polypeptide which has homology to
portions of the collagen molecule, designated herein as PRO294.
[0174] 37. PRO295
[0175] The integrins comprise a supergene family of cell-surface
glycoprotein receptors that promote cellular adhesion. Each cell
has numerous receptors that define its cell adhesive capabilities.
Integrins are involved in a wide variety of interaction between
cells and other cells or matrix components. The integrins are of
particular importance in regulating movement and function of immune
system cells The platelet IIb/IIIA integrin complex is of
particular importance in regulating platelet aggregation. A member
of the integrin family, integrin .beta.-6, is expressed on
epithelial cells and modulates epithelial inflammation. Another
integrin, leucocyte-associated antigen-1 (LFA-1) is important in
the adhesion of lymphocytes during an immune response. The
integrins are expressed as heterodimers of non-covalently
associated alpha and beta subunits. Given the physiological
importance of cell adhesion mechanisms in vivo, efforts are
currently being under taken to identify new, native proteins which
are involved in cell adhesion. We describe herein the
identification and characterization of a novel polypeptide which
has homology to integrin, designated herein as PRO295.
[0176] 38. PRO293
[0177] Protein-protein interactions include receptor and antigen
complexes and signaling mechanisms. As more is known about the
structural and functional mechanisms underlying protein-protein
interactions, protein-protein interactions can be more easily
manipulated to regulate the particular result of the
protein-protein interaction. Thus, the underlying mechanisms of
protein-protein interactions are of interest to the scientific and
medical community.
[0178] All proteins containing leucine-rich repeats are thought to
be involved in protein-protein interactions. Leucine-rich repeats
are short sequence motifs present in a number of proteins with
diverse functions and cellular locations. The crystal structure of
ribonuclease inhibitor protein has revealed that leucine-rich
repeats correspond to beta-alpha structural units. These units are
arranged so that they form a parallel beta-sheet with one surface
exposed to solvent, so that the protein acquires an unusual,
nonglubular shape. These two features have been indicated as
responsible for the protein-binding functions of proteins
containing leucine-rich repeats. See, Kobe and Deisenhofer, Trends
Biochem. Sci., 19(10):415421 (October 1994).
[0179] A study has been reported on leucine-rich proteoglycans
which serve as tissue organizers, orienting and ordering collagen
fibrils during ontogeny and are involved in pathological processes
such as wound healing, tissue repair, and tumor stroma formation.
Iozzo, R. V., Crit. Rev. Biochem. Mol. Biol., 32(2):141-174 (1997).
Others studies implicating leucine rich proteins in wound healing
and tissue repair are De La Salle, C., et al., Vouv. Rev. Fr.
Hematol. (Germany), 37(4):215-222 (1995), reporting mutations in
the leucine rich motif in a complex associated with the bleeding
disorder Bernard-Soulier syndrome and Chlemetson, K. J., Thromb.
Haemost. (Germany), 74(1): 111-116 (July 1995), reporting that
platelets have leucine rich repeats. Another protein of particular
interest which has been reported to have leucine-rich repeats is
the SLIT protein which has been reported to be useful in treating
neuro-degenerative diseases such as Alzheimer's disease, nerve
damage such as in Parkinson's disease, and for diagnosis of cancer,
see, Artavanistsakonas, S. and Rothberg, J. M., WO9210518-Al by
Yale University. Other studies reporting on the biological
functions of proteins having leucine-rich repeats include: Tayar,
N., et al., Mol. Cell Endocrinol., (Ireland), 125(1-2):65-70
(December 1996) (gonadotropin receptor involvement); Miura, Y., et
al., Nippon Rinsho (Japan), 54(7):1784-1789 (July 1996) (apoptosis
involvement); Harris, P. C., et al., J. Am. Soc. Nephrol.,
6(4):1125-1133 (October 1995) (kidney disease involvement); and
Ruoslahti, E. I., et al., WO9110727-A by La Jolla Cancer Research
Foundation (decorin binding to transforming growth factor.beta.
involvement for treatment for cancer, wound healing and
scarring).
[0180] Efforts are therefore being undertaken by both industry and
academia to identify new proteins having leucine rich repeats to
better understand protein-protein interactions. Of particular
interest are those proteins having leucine rich repeats and
homology to known neuronal leucine rich repeat proteins. Many
efforts are focused on the screening of mammalian recombinant DNA
libraries to identify the coding sequences for novel secreted and
membrane-bound proteins having leucine rich repeats. 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)].
[0181] We describe herein the identification and characterization
of a novel polypeptide which has homology to leucine rich repeat
proteins, designated herein as PRO293.
[0182] 39. PRO247
[0183] Protein-protein interactions include receptor and antigen
complexes and signaling mechanisms. As more is known about the
structural and functional mechanisms underlying protein-protein
interactions, protein-protein interactions can be more easily
manipulated to regulate the particular result of the
protein-protein interaction. Thus, the underlying mechanisms of
protein-protein interactions are of interest to the scientific and
medical community.
[0184] All proteins containing leucine-rich repeats are thought to
be involved in protein-protein interactions. Leucine-rich repeats
are short sequence motifs present in a number of proteins with
diverse functions and cellular locations. The crystal structure of
ribonuclease inhibitor protein has revealed that leucine-rich
repeats correspond to beta-alpha structural units. These units are
arranged so that they form a parallel beta-sheet with one surface
exposed to solvent, so that the protein acquires an unusual,
nonglubular shape. These two features have been indicated as
responsible for the protein-binding functions of proteins
containing leucine-rich repeats. See, Kobe and Deisenhofer, Trends
Biochem. Sci., 19(10):415-421 (October 1994).
[0185] A study has been reported on leucine-rich proteoglycans
which serve as tissue organizers, orienting and ordering collagen
fibrils during ontogeny and are involved in pathological processes
such as wound healing, tissue repair, and tumor stroma formation.
Iozzo, R. V., Crit. Rev. Biochem. Mol. Biol., 32(2):141-174 (1997).
Others studies implicating leucine rich proteins in wound healing
and tissue repair are De La Salle, C., et al., Vouv. Rev. Fr.
Hematol. (Germany), 37(4):215-222 (1995), reporting mutations in
the leucine rich motif in a complex associated with the bleeding
disorder Bernard-Soulier syndrome and Chlemetson, K. J., Thromb.
Haemost. (Germany), 74(1):111-116 (July 1995), reporting that
platelets have leucine rich repeats. Another protein of particular
interest which has been reported to have leucine-rich repeats is
the SLIT protein which has been reported to be useful in treating
neuro-degenerative diseases such as Alzheimer's disease, nerve
damage such as in Parkinson's disease, and for diagnosis of cancer,
see, Artavanistsakonas, S. and Rothberg, J. M., WO9210518-Al by
Yale University. Other studies reporting on the biological
functions of proteins having leucine-rich repeats include: Tayar,
N., et al., Mol. Cell Endocrinol., (Ireland), 125(1-2):65-70
(December 1996) (gonadotropin receptor involvement); Miura, Y., et
al., Nippon Rinsho (Japan), 54(7): 1784-1789 (July 1996) (apoptosis
involvement); Harris, P. C., et al., J. Am. Soc. Nephrol.,
6(4):1125-1133 (October 1995) (kidney disease involvement); and
Ruoslahti, E. I., et al., WO9110727-A by La Jolla Cancer Research
Foundation (decorin binding to transforming growth factors
involvement for treatment for cancer, wound healing and
scarring).
[0186] Densin is a glycoprotein which has been isolated from the
brain which has all the hallmarks of an adhesion molecule. It is
highly concentrated at synaptic sites in the brain and is expressed
prominently in dendritic processes in developing neurons. Densin
has been characterized as a member of the 0-linked
sialoglycoproteins. Densin has relevance to medically important
processes such as regeneration. Given the physiological importance
of synaptic processes and cell adhesion mechanisms in vivo, efforts
are currently being under taken to identify new, native proteins
which are involved in synaptic machinery and cell adhesion. Densin
is further described in Kennedy, M. B, Trends Neurosci. (England),
20(6):264 (1997) and Apperson, et al., J. Neurosci., 16(21):6839
(1996).
[0187] Efforts are therefore being undertaken by both industry and
academia to identify new proteins having leucine rich repeats to
better understand protein-protein interactions. Of particular
interest are those proteins having leucine rich repeats and
homology to known proteins having leucine rich repeats such as
KIAA0231 and densin. Many efforts are focused on the screening of
mammalian recombinant DNA libraries to identify the coding
sequences for novel secreted and membrane-bound proteins having
leucine rich repeats. 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)].
[0188] We describe herein the identification and characterization
of a novel polypeptide which has homology to leucine rich repeat
proteins, designated herein as PRO247.
[0189] 40. PRO302, PRO303, PRO304, PRO307 and PRO343
[0190] Proteases are enzymatic proteins which are involved in a
large number of very important biological processes in mammalian
and non-mammalian organisms. Numerous different protease enzymes
from a variety of different mammalian and non-mammalian organisms
have been both identified and characterized. The mammalian protease
enzymes play important roles in many different biological processes
including, for example, protein digestion, activation,
inactivation, or modulation of peptide hormone activity, and
alteration of the physical properties of proteins and enzymes.
[0191] In light of the important physiological roles played by
protease enzymes, efforts are currently being undertaken by both
industry and academia to identify new, native protease homologs.
Many of these efforts are focused on the screening of mammalian
recombinant DNA libraries to identify the coding sequences for
novel secreted and membrane-bound receptor 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)]. We herein describe
the identification of novel polypeptides having homology to various
protease enzymes, designated herein as PRO302, PRO303, PRO304,
PRO307 and PRO343 polypeptides.
[0192] 41. PRO328
[0193] The GLIP protein family has been characterized as comprising
zinc-finger proteins which play important roles in embryogenesis.
These proteins may function as transcriptional regulatory proteins
and are known to be amplified in a subset of human tumors. Glioma
pathogenesis protein is structurally related to a group of plant
pathogenesis-related proteins. It is highly expressed in
glioblastoma. See US Pat. Nos. 5,582,981 (issued Dec. 10, 1996) and
5,322,801 (issued Jun. 21, 1996), Ellington, A. D. et al., Nature,
346:818 (1990), Grindley, J. C. et al., Dev. Biol., 188(2):337
(1997), Marine, J. C. et al., Mech. Dev., 63(2):211 (1997), The
CRISP or cysteine rich secretory protein family are a group of
proteins which are also structurally related to a group of plant
pathogenesis proteins. [Schwidetzky, U., Biochem. J., 321:325
(1997), Pfisterer, P., Mol. Cell Biol., 16(11):6160 (1996),
Kratzschmar, J. Eur. J. Biochem., 236(3):827 (1996)]. We describe
herein the identification of a novel polypeptide which has homology
to GLIP and CRISP, designated herein as PRO328 polypeptides.
[0194] 42. PRO335, PRO331 and PRO326
[0195] Protein-protein interactions include receptor and antigen
complexes and signaling mechanisms. As more is known about the
structural and functional mechanisms underlying protein-protein
interactions, protein-protein interactions can be more easily
manipulated to regulate the particular result of the
protein-protein interaction. Thus, the underlying mechanisms of
protein-protein interactions are of interest to the scientific and
medical community.
[0196] All proteins containing leucine-rich repeats are thought to
be involved in protein-protein interactions. Leucine-rich repeats
are short sequence motifs present in a number of proteins with
diverse functions and cellular locations. The crystal structure of
ribonuclease inhibitor protein has revealed that leucine-rich
repeats correspond to beta-alpha structural units. These units are
arranged so that they form a parallel beta-sheet with one surface
exposed to solvent, so that the protein acquires an unusual,
nonglubular shape. These two features have been indicated as
responsible for the protein-binding functions of proteins
containing leucine-rich repeats. See, Kobe and Deisenhofer, Trends
Biochem. Sci., 19(10):415421 (October 1994).
[0197] A study has been reported on leucine-rich proteoglycans
which serve as tissue organizers, orienting and ordering collagen
fibrils during ontogeny and are involved in pathological processes
such as wound healing, tissue repair, and tumor stroma formation.
Iozzo, R. V., Crit. Rev. Biochem. Mol. Biol., 32(2):141-174 (1997).
Others studies implicating leucine rich proteins in wound healing
and tissue repair are De La Salle, C., et al., Vouv. Rev. Fr.
Hematol. (Germany), 37(4):215-222 (1995), reporting mutations in
the leucine rich motif in a complex associated with the bleeding
disorder Bernard-Soulier syndrome, Chlemetson, K. J., Thromb.
Haemost. (Germany), 74(1): 111-116 (July 1995), reporting that
platelets have leucine rich repeats and Ruoslahti, E. I., et al.,
WO9110727-A by La Jolla Cancer Research Foundation reporting that
decorin binding to transforming growth factors has involvement in a
treatment for cancer, wound healing and scarring. Related by
function to this group of proteins is the insulin like growth
factor (IGF), in that it is useful in wound-healing and associated
therapies concerned with re-growth of tissue, such as connective
tissue, skin and bone; in promoting body growth in humans and
animals; and in stimulating other growth-related processes. The
acid labile subunit of IGF (ALS) is also of interest in that it
increases the half-life of IGF and is part of the IGF complex in
vivo.
[0198] Another protein which has been reported to have leucine-rich
repeats is the SLIT protein which has been reported to be useful in
treating neuro-degenerative diseases such as Alzheimer's disease,
nerve damage such as in Parkinson's disease, and for diagnosis of
cancer, see, Artavanistsakonas, S. and Rothberg, J. M.,
WO9210518-Al by Yale University. Of particular interest is LIG-1, a
membrane glycoprotein that is expressed specifically in glial cells
in the mouse brain, and has leucine rich repeats and
immunoglobulin-like domains. Suzuki, et al., J. Biol. Chem. (U.S.),
271(37):22522 (1996). Other studies reporting on the biological
functions of proteins having leucine rich repeats include: Tayar,
N., et al., Mol. Cell Endocrinol., (Ireland), 125(1-2):65-70
(December 1996) (gonadotropin receptor involvement); Miura, Y., et
al., Nippon Rinsho (Japan), 54(7):1784-1789 (July 1996) (apoptosis
involvement); Harris, P. C., et al., J. Am. Soc. Nephrol.,
6(4):1125-1133 (October 1995) (kidney disease involvement).
[0199] Efforts are therefore being undertaken by both industry and
academia to identify new proteins having leucine rich repeats to
better understand protein-protein interactions. Of particular
interest are those proteins having leucine rich repeats and
homology to known proteins having leucine rich repeats such as
LIG-1, ALS and decorin. Many efforts are focused on the screening
of mammalian recombinant DNA libraries to identify the coding
sequences for novel secreted and membrane-bound proteins having
leucine rich repeats. 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)].
[0200] We describe herein the identification and characterization
of novel polypeptides which have homology to proteins of the
leucine rich repeat superfamily, designated herein as PRO335,
PRO331 and PRO326 polypeptides.
[0201] 43. PRO332
[0202] Secreted proteins comprising a repeat characterized by an
arrangement of conserved leucine residues (leucine-rich repeat
motif) have diverse biological roles. Certain proteoglycans, such
as biglycan, fibromodulin and decorin, are, for example,
characterized by the presence of a leucine-rich repeat of about 24
amino acids [Ruoslahti, Ann. Rev. Cell. Biol. 4 229-255 (1988);
Oldberg et al., EMBO J. 8, 2601-2604 (1989)]. In general,
proteoglycans are believed to play a role in regulating
extracellular matrix, cartilage or bone function. The proteoglycan
decorin binds to collagen type I and II and affects the rate of
fibril formation. Fibromodulin also binds collagen and delays
fibril formation. Both fibromodulin and decorin inhibit the
activity of transforming growth factor beta (TGF-.beta.) (U.S. Pat.
No. 5,583,103 issued Dec. 10, 1996). TGF-.beta. is known to play a
key role in the induction of extracellular matrix and has been
implicated in the development of fibrotic diseases, such as cancer
and glomerulonephritis. Accordingly, proteoglycans have been
proposed for the treatment of fibrotic cancer, based upon their
ability to inhibit TGF-.beta.'s growth stimulating activity on the
cancer cell. Proteoglycans have also been described as potentially
useful in the treatment of other proliferative pathologies,
including rheumatoid arthritis, arteriosclerosis, adult respiratory
distress syndrome, cirrhosis of the liver, fibrosis of the lungs,
post-myocardial infarction, cardiac fibrosis, post-angioplasty
restenosis, renal interstitial fibrosis and certain dermal fibrotic
conditions, such as keloids and scarring, which might result from
burn injuries, other invasive skin injuries, or cosmetic or
reconstructive surgery (U.S. Pat. No. 5,654,270, issued Aug. 5,
1997).
[0203] We describe herein the identification and characterization
of novel polypeptides which have homology to proteins of the
leucine rich repeat superfamily, designated herein as PRO332
polypeptides.
[0204] 44. PRO334
[0205] Microfibril bundles and proteins found in association with
these bundles, particularly attachment molecules, are of interest
in the field of dermatology, particularly in the study of skin
which has been damaged from aging, injuries or the sun. Fibrillin
microfibrils define the continuous elastic network of skin, and are
present in dermis as microfibril bundles devoid of measurable
elastin extending from the dermal-epithelial junction and as
components of the thick elastic fibres present in the deep
reticular dermis. Moreover, Marfan syndrome has been linked to
mutations which interfere with multimerization of fibrillin
monomers or other connective tissue elements.
[0206] Fibulin-1 is a modular glycoprotein with amino-terninal
anaphlatoxin-like modules followed by nine epidermal growth factor
(EGF)-like modules and, depending on alternative splicing, four
possible carboxyl termini. Fibulin-2 is a novel extracellular
matrix protein frequently found in close association with
microfibrils containing either fibronectin or fibrillin. Thus,
fibrillin, fibulin, and molecules related thereto are of interest,
particularly for the use of preventing skin from being damaged from
aging, injuries or the sun, or for restoring skin damaged from
same. Moreover, these molecules are generally of interest in the
study of connective tissue and attachment molecules and related
mechanisms. Fibrillin, fibulin and related molecules are further
described in Adams, et al., J. Mol. Biol., 272(2):226-36 (1997);
Kielty and Shuttleworth, Microsc. Res. Tech., 38(4):413-27 (1997);
and Child, J. Card. Surg. 12(2Supp.):131-5 (1997).
[0207] Currently, efforts are being undertaken by both industry and
academia to identify new, native secreted and membrane-bound
receptor proteins, particularly secreted proteins which have
homology to fibulin and fibrillin. Many efforts are focused on the
screening of mammalian recombinant DNA libraries to identify the
coding sequences for novel secreted and membrane-bound receptor
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)].
[0208] We herein describe the identification and characterization
of novel polypeptides having homology to fibulin and fibrillin,
designated herein as PRO334 polypeptides.
[0209] 45. PRO346
[0210] The widespread occurrence of cancer has prompted the
devotion of considerable resources and discovering new treatments
of treatment. One particular method involves the creation of tumor
or cancer specific monoclonal antibodies (mAbs) which are specific
to tumor antigens. Such mAbs, which can distinguish between normal
and cancerous cells are useful in the diagnosis, prognosis and
treatment of the disease. Particular antigens are known to be
associated with neoplastic diseases, such as colorectal and breast
cancer. Since colon cancer is a widespread disease, early diagnosis
and treatment is an important medical goal. Diagnosis and treatment
of cancer can be implemented using monoclonal antibodies (mAbs)
specific therefore having fluorescent, nuclear magnetic or
radioactive tags. Radioactive genes, toxins and/or drug tagged mAbs
can be used for treatment in situ with minimal patient
description.
[0211] Carcinoembryonic antigen (CEA) is a glycoprotein found in
human colon cancer and the digestive organs of a 2-6 month human
embryos. CEA is a known human tumor marker and is widely used in
the diagnosis of neoplastic diseases, such as colon cancer. For
example, when the serum levels of CEA are elevated in a patient, a
drop of CEA levels after surgery would indicate the tumor resection
was successful. On the other hand, a subsequent rise in serum CEA
levels after surgery would indicate that metastases of the original
tumor may have formed or that new primary tumors may have appeared.
CEA may also be a target for mAb, antisense nucleotides
[0212] 46. PRO268
[0213] Protein disulfide isomerase is an enzymatic protein which is
involved in the promotion of correct refolding of proteins through
the establishment of correct disulfide bond formation. Protein
disulfide isomerase was initially identified based upon its ability
to catalyze the renaturation of reduced denatured RNAse (Goldberger
et al., J. Biol. Chem. 239:1406-1410 (1964) and Epstein et al.,
Cold Spring Harbor Symp. Quant. Biol. 28:439-449 (1963)). Protein
disulfide isomerase has been shown to be a resident enzyme of the
endoplasmic reticulum which is retained in the endoplasmic
reticulum via a -KDEL or -HDEL amino acid sequence at its
C-terminus.
[0214] Given the importance of disulfide bond-forming enzymes and
their potential uses in a number of different applications, for
example in increasing the yield of correct refolding of
recombinantly produced proteins, efforts are currently being
undertaken by both industry and academia to identify new, native
proteins having homology to protein disulfide isomerase. Many of
these efforts are focused on the screening of mammalian recombinant
DNA libraries to identify the coding sequences for novel protein
disulfide isomerase homologs. We herein describe a novel
polypeptide having homology to protein disulfide isomerase,
designated herein as PRO268.
[0215] 47. PRO330
[0216] Prolyl 4-hydroxylase is an enzyme which functions to
post-translationally hydroxylate proline residues at the Y position
of the amino acid sequence Gly-X-Y, which is a repeating three
amino acid sequence found in both collagen and procollagen.
Hydroxylation of proline residues at the Y position of the Gly-X-Y
amino acid triplet to form 4-hydroxyproline residues at those
positions is required before newly synthesized collagen polypeptide
chains may fold into their proper three-dimensional triple-helical
conformation. If hydroxylation does not occur, synthesized collagen
polypeptides remain non-helical, are poorly secreted by cells and
cannot assemble into stable functional collagen fibrils. Vuorio et
al., Proc. Natl. Acad. Sci. USA 89:7467-7470 (1992). Prolyl
4-hydroxylase is comprised of at least two different polypeptide
subunits, alpha and beta.
[0217] Efforts are being undertaken by both industry and academia
to identify new, native secreted and membrane-bound receptor
proteins. Many efforts are focused on the screening of mammalian
recombinant DNA libraries to identify the coding sequences for
novel secreted and membrane-bound receptor 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)]. Based upon these
efforts, Applicants have herein identified and describe a novel
polypeptide having homology to the alpha subunit of prolyl
4-hydroxylase, designated herein as PRO330.
[0218] 48. PRO339 and PRO310
[0219] Fringe is a protein which specifically blocks
serrate-mediated activation of notch in the dorsal compartment of
the Drosophila wing imaginal disc. Fleming, et al., Development,
124(15):2973-81 (1997). Therefore, fringe is of interest for both
its role in development as well as its ability to regulate serrate,
particularly serrate's signaling abilities. Also of interest are
novel polypeptides which may have a role in development and/or the
regulation of serrate-like molecules. Of particular interest are
novel polypeptides having homology to fringe as identified and
described herein, designated herein as PRO339 and PRO310
polypeptides.
[0220] 49. PRO244
[0221] Lectins are a class of proteins comprising a region that
binds carbohydrates specifically and non-covalently. Numerous
lectins have been identified in higher animals, both membrane-bound
and soluble, and have been implicated in a variety of
cell-recognition phenomena and tumor metastasis.
[0222] Most lectins can be classified as either C-type
(calcium-dependent) or S-type (thiol-dependent).
[0223] Lectins are thought to play a role in regulating cellular
events that are initiated at the level of the plasma membrane. For
example, plasma membrane associated molecules are involved in the
activation of various subsets of lymphoid cells, e.g.
T-lymphocytes, and it is known that cell surface molecules are
responsible for activation of these cells and consequently their
response during an immune reaction.
[0224] A particular group of cell adhesion molecules, selecting,
belong in the superfamily of C-type lectins. This group includes
L-selectin (peripheral lymph node homing receptor (pnHR),
LEC-CAM-1, LAM-1, gp90.sup.MEL, gp.sub.100.sup.MEL, gp110.sup.MEL,
MEL-14 antigen, Leu-8 antigen, TQ-1 antigen, DREG antigen),
E-selectin (LEC-CAM-2, LECAM-2, ELAM-1), and P-selectin (LEC-CAM-3,
LECAM-3, GMP-140, PADGEM). The structure of selectins consists of a
C-type lectin (carbohydrate binding) domain, an epidermal growth
factor-like (EGF-like) motif, and variable numbers of complement
regulatory (CR) motifs. Selectins are associated with leukocyte
adhesion, e.g. the attachment of neutrophils to venular endothelial
cells adjacent to inflammation (E-selectin), or with the
trafficking of lymphocytes from blood to secondary lymphoid organs,
e.g. lymph nodes and Peyer's patches (L-selectin).
[0225] Another exemplary lectin is the cell-associated macrophage
antigen, Mac-2 that is believed to be involved in cell adhesion and
immune responses. Macrophages also express a lectin that recognizes
Tn Ag, a human carcinoma-associated epitope.
[0226] Another C-type lectin is CD95 (Fas antigen/APO-1) that is an
important mediator of immunologically relevant regulated or
programmed cell death (apoptosis). "Apoptosis" is a non-necrotic
cell death that takes place in metazoan animal cells following
activation of an intrinsic cell suicide program. The cloning of Fas
antigen is described in PCT publication WO 91/10448, and European
patent application EP510691. The mature Fas molecule consists of
319 amino acids of which 157 are extracellular, 17 constitute the
transmembrane domain, and 145 are intracellular. Increased levels
of Fas expression at T cell surface have been associated with tumor
cells and HIV-infected cells. Ligation of CD95 triggers apoptosis
in the presence of interleukin-1 (IL-2).
[0227] C-type lectins also include receptors for oxidized
low-density lipoprotein (LDL). This suggests a possible role in the
pathogenesis of atherosclerosis.
[0228] We herein describe the identification and characterization
of novel polypeptides having homology to C-type lectins, designated
herein as PRO244 polypeptides.
SUMMARY OF THE INVENTION
[0229] 1. PRO211 and PRO217
[0230] Applicants have identified cDNA clones that encode novel
polypeptides having homology to EGF, designated in the present
application as "PRO211" and "PRO217" polypeptides.
[0231] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO211 or PRO217
polypeptide. In one aspect, the isolated nucleic acid comprises DNA
encoding EGF-like homologue PRO211 and PRO217 polypeptides of FIG.
2 (SEQ ID NO:2) and/or 4 (SEQ ID NO:4) indicated in FIG. 1 (SEQ ID
NO1) and/or FIG. 3 (SEQ ID NO:3), respectively, or is complementary
to such encoding nucleic acid sequence, and remains stably bound to
it under at least moderate, and optionally, under high stringency
conditions.
[0232] In another embodiment, the invention provides isolated
PRO211 and PRO217 EGF-like homologue PRO211 and PRO217
polypeptides. In particular, the invention provides isolated native
sequence PRO211 and PRO217 EGF-like homologue polypeptides, which
in one embodiment, includes an amino acid sequence comprising
residues: 1 to 353 of FIG. 2 (SEQ ID NO:2) or (2) 1 to 379 of FIG.
4 (SEQ ID NO:4).
[0233] 2. PRO230
[0234] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as "PRO230".
[0235] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO230 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO230 polypeptide having amino acid residues 1 through 467 of FIG.
6 (SEQ ID NO:12), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions. In another
embodiment, the invention provides isolated PRO230 polypeptide. In
particular, the invention provides isolated native sequence PRO230
polypeptide, which in one embodiment, includes an amino acid
sequence comprising residues 1 through 467 of FIG. 6 (SEQ ID
NO:12).
[0236] In another embodiment, the invention provides an expressed
sequence tag (EST) comprising the nucleotide sequence of SEQ ID
NO:13 (FIG. 7) which is herein designated as DNA20088.
[0237] 3. PRO232
[0238] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as "PRO232".
[0239] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO232 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO232 polypeptide having amino acid residues 1 to 114 of FIG. 9
(SEQ ID NO:18), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0240] In another embodiment, the invention provides isolated
PRO232 polypeptide. In particular, the invention provides isolated
native sequence PRO232 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 114 of
FIG. 9 (SEQ ID NO:18).
[0241] 4. PRO187
[0242] Applicants have identified a cDNA clone that encodes a novel
polypeptide, designated in the present application as "PRO187".
[0243] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO187 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO187 polypeptide of FIG. 11 (SEQ ID NO:23), or is complementary
to such encoding nucleic acid sequence, and remains stably bound to
it under at least moderate, and optionally, under high stringency
conditions. In another aspect, the invention provides a nucleic
acid comprising the coding sequence of FIG. 10 (SEQ ID NO:22) or
its complement. In another aspect, the invention provides a nucleic
acid of the full length protein of clone DNA27864-1155, deposited
with the ATCC under accession number ATCC 209375, alternatively the
coding sequence of clone DNA27864-1155, deposited under accession
number ATCC 209375.
[0244] In yet another embodiment, the invention provides isolated
PRO187 polypeptide. In particular, the invention provides isolated
native sequence PRO187 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 205 of
FIG. 11 (SEQ ID NO:23). Alternatively, the invention provides a
polypeptide encoded by the nucleic acid deposited under accession
number ATCC 209375.
[0245] 5. PRO265
[0246] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as "PRO265".
[0247] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO265 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO265 polypeptide having amino acid residues 1 to 660 of FIG. 13
(SEQ ID NO:28), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0248] In another embodiment, the invention provides isolated
PRO265 polypeptide. In particular, the invention provides isolated
native sequence PRO265 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 660 of
FIG. 13 (SEQ ID NO:28). An additional embodiment of the present
invention is directed to an isolated extracellular domain of a
PRO265 polypeptide.
[0249] 6. PRO219
[0250] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as "PRO219".
[0251] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO219 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO219 polypeptide having amino acid residues 1 to 915 of FIG. 15
(SEQ ID NO:34), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0252] In another embodiment, the invention provides isolated
PRO219 polypeptide. In particular, the invention provides isolated
native sequence PRO219 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 915 of
FIG. 15 (SEQ ID NO:34).
[0253] 7. PRO246
[0254] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as "PRO246".
[0255] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO246 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO246 polypeptide having amino acid residues 1 to 390 of FIG. 17
(SEQ ID NO:39), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0256] In another embodiment, the invention provides isolated
PRO246 polypeptide. In particular, the invention provides isolated
native sequence PRO246 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 390 of
FIG. 17 (SEQ ID NO:39). An additional embodiment of the present
invention is directed to an isolated extracellular domain of a
PRO246 polypeptide.
[0257] 8. PRO228
[0258] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to CD97, EMR1 and latrophilin, wherein
the polypeptide is designated in the present application as
"PRO228".
[0259] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO228 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO228 polypeptide having amino acid residues 1 to 690 of FIG. 19
(SEQ ID NO:49), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0260] In another embodiment, the invention provides isolated
PRO228 polypeptide. In particular, the invention provides isolated
native sequence PRO228 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 690 of
FIG. 19 (SEQ ID NO:49). An additional embodiment of the present
invention is directed to an isolated extracellular domain of a
PRO228 polypeptide.
[0261] In another embodiment, the invention provides an expressed
sequence tag (EST) comprising the nucleotide sequence of SEQ ID
NO:50, designated herein as DNA21951.
[0262] 9. PRO533
[0263] Applicants have identified a cDNA clone (DNA49435-1219) that
encodes a novel polypeptide, designated in the present application
as PRO533.
[0264] In one embodiment, the invention provides an isolated
nucleic acid molecule having at least about 80% sequence identity
to (a) a DNA molecule encoding a PRO533 polypeptide comprising the
sequence of amino acids 23 to 216 of FIG. 22 (SEQ ID NO:59), or (b)
the complement of the DNA molecule of (a). The sequence identity
preferably is about 85%, more preferably about 90%, most preferably
about 95%. In one aspect, the isolated nucleic acid has at least
about 80%, preferably at least about 85%, more preferably at least
about 90%, and most preferably at least about 95% sequence identity
with a polypeptide having amino acid residues 23 to 216 of FIG. 22
(SEQ ID NO:59). Preferably, the highest degree of sequence identity
occurs within the secreted portion (amino acids 23 to 216 of FIG.
22, SEQ ID NO:59). In a further embodiment, the isolated nucleic
acid molecule comprises DNA encoding a PRO533 polypeptide having
amino acid residues 1 to 216 of FIG. 22 (SEQ ID NO:59), or is
complementary to such encoding nucleic acid sequence, and remains
stably bound to it under at least moderate, and optionally, under
high stringency conditions. In another aspect, the invention
provides a nucleic acid of the full length protein of clone
DNA49435-1219, deposited with the ATCC under accession number ATCC
209480.
[0265] In yet another embodiment, the invention provides isolated
PRO533 polypeptide. In particular, the invention provides isolated
native sequence PRO533 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 23 to 216 of
FIG. 22 (SEQ ID NO:59). Native PRO533 polypeptides with or without
the native signal sequence (amino acids 1 to 22 in FIG. 22 (SEQ ID
NO:59)), and with or without the initiating methionine are
specifically included. Alternatively, the invention provides a
PRO533 polypeptide encoded by the nucleic acid deposited under
accession number ATCC 209480.
[0266] 10. PRO245
[0267] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as "PRO245".
[0268] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO245 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO245 polypeptide having amino acid residues 1 to 312 of FIG. 24
(SEQ ID NO:64), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0269] In another embodiment, the invention provides isolated
PRO245 polypeptide. In particular, the invention provides isolated
native sequence PRO245 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 312 of
FIG. 24 (SEQ ID NO:64).
[0270] 11. PRO220, PRO221 and PRO227
[0271] Applicants have identified cDNA clones that each encode
novel polypeptides, all having leucine rich repeats. These
polypeptides are designated in the present application as PRO220,
PRO221 and PRO227.
[0272] In one embodiment, the invention provides isolated nucleic
acid molecules comprising DNA respectively encoding PRO220, PRO221
and PRO227, respectively. In one aspect, provided herein is an
isolated nucleic acid comprises DNA encoding the PRO220 polypeptide
having amino acid residues 1 through 708 of FIG. 26 (SEQ ID NO:69),
or is complementary to such encoding nucleic acid sequence, and
remains stably bound to it under at least moderate, and optionally,
under high stringency conditions. Also provided herein is an
isolated nucleic acid comprises DNA encoding the PRO221 polypeptide
having amino acid residues 1 through 259 of FIG. 28 (SEQ ID NO:71),
or is complementary to such encoding nucleic acid sequence, and
remains stably bound to it under at least moderate, and optionally,
under high stringency conditions. Moreover, also provided herein is
an isolated nucleic acid comprises DNA encoding the PRO227
polypeptide having amino acid residues 1 through 620 of FIG. 30
(SEQ ID NO:73), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0273] In another embodiment, the invention provides isolated
PRO220, PRO221 and PRO227 polypeptides. in particular, provided
herein is the isolated native sequence for the PRO220 polypeptide,
which in one embodiment, includes an amino acid sequence comprising
residues 1 to 708 of FIG. 26 (SEQ ID NO:69). Additionally provided
herein is the isolated native sequence for the PRO221 polypeptide,
which in one embodiment, includes an amino acid sequence comprising
residues 1 to 259 of FIG. 28 (SEQ ID NO:71). Moreover, provided
herein is the isolated native sequence for the PRO227 polypeptide,
which in one embodiment, includes an amino acid sequence comprising
residues 1 to 620 of FIG. 30 (SEQ ID NO:73).
[0274] 12. PRO258
[0275] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to CRTAM and poliovirus receptor
precursors, wherein the polypeptide is designated in the present
application as "PRO258".
[0276] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO258 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO258 polypeptide having amino acid residues 1 to 398 of FIG. 32
(SEQ ID NO:84), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0277] In another embodiment, the invention provides isolated
PRO258 polypeptide. In particular, the invention provides isolated
native sequence PRO258 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 398 of
FIG. 32 (SEQ ID NO:84). An additional embodiment of the present
invention is directed to an isolated extracellular domain of a
PRO258 polypeptide.
[0278] 13. PRO266
[0279] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as "PRO266".
[0280] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO266 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO266 polypeptide having amino acid residues 1 to 696 of FIG. 34
(SEQ ID NO:91), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0281] In another embodiment, the invention provides isolated
PRO266 polypeptide. In particular, the invention provides isolated
native sequence PRO266 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 696 of
FIG. 34 (SEQ ID NO:91).
[0282] 14. PRO269
[0283] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as PRO269.
[0284] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO269 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO269 polypeptide having amino acid residues 1 to 490 of FIG. 36
(SEQ ID NO:96), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0285] In another embodiment, the invention provides isolated
PRO269 polypeptide. In particular, the invention provides isolated
native sequence PRO269 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 490 of
FIG. 36 (SEQ ID NO:96). An additional embodiment of the present
invention is directed to an isolated extracellular domain of a
PRO269 polypeptide.
[0286] 15. PRO287
[0287] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as "PRO287".
[0288] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO287 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO287 polypeptide having amino acid residues 1 to 415 of FIG. 38
(SEQ ID NO:104), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0289] In another embodiment, the invention provides isolated
PRO287 polypeptide. In particular, the invention provides isolated
native sequence PRO287 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 415 of
FIG. 38 (SEQ ID NO:104).
[0290] 16. PRO214
[0291] Applicants have identified a cDNA clone that encodes a novel
polypeptide, designated in the present application as "PRO214".
[0292] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO214 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO214 polypeptide of FIG. 40 (SEQ ID NO:109), or is complementary
to such encoding nucleic acid sequence, and remains stably bound to
it under at least moderate, and optionally, under high stringency
conditions. In another aspect, the invention provides a nucleic
acid comprising the coding sequence of FIG. 39 (SEQ ID NO:108) or
its complement. In another aspect, the invention provides a nucleic
acid of the full length protein of clone DNA32286-1191, deposited
with ATCC under accession number ATCC 209385.
[0293] In yet another embodiment, the invention provides isolated
PRO214 polypeptide. In particular, the invention provides isolated
native sequence PRO214 polypeptide, which in one embodiment,
includes an amino acid sequence comprising the residues of FIG. 40
(SEQ ID NO:109). Alternatively, the invention provides a
polypeptide encoded by the nucleic acid deposited under accession
number ATCC 209385.
[0294] 17. PRO317
[0295] Applicants have identified a cDNA clone that encodes a novel
polypeptide, designated in the present application as "PRO317".
[0296] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding PRO317 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA (SEQ ID
NO:113) encoding PRO317 polypeptide having amino acid residues 1 to
366 of FIG. 42, or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0297] In another embodiment, the invention provides isolated
PRO317 polypeptide. In particular, the invention provides isolated
native-sequence PRO317 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 366 of
FIG. 42 (SEQ ID NO:114).
[0298] In yet another embodiment, the invention supplies a method
of detecting the presence of PRO317 in a sample, the method
comprising:
[0299] a) contacting a detectable anti-PRO317 antibody with a
sample suspected of containing PRO317; and
[0300] b) detecting binding of the antibody to the sample; wherein
the sample is selected from the group consisting of a body fluid, a
tissue sample, a cell extract, and a cell culture medium.
[0301] In a still further embodiment a method is provided for
determining the presence of PRO317 mRNA in a sample, the method
comprising:
[0302] a) contacting a sample suspected of containing PRO317 mRNA
with a detectable nucleic acid probe that hybridizes under moderate
to stringent conditions to PRO317 mRNA; and
[0303] b) detecting hybridization of the probe to the sample.
[0304] Preferably, in this method the sample is a tissue sample and
the detecting step is by in situ hybridization, or the sample is a
cell extract and detection is by Northern analysis.
[0305] Further, the invention provides a method for treating a
PRO317-associated disorder comprising administering to a mammal an
effective amount of the PRO317 polypeptide or a composition thereof
containing a carrier, or with an effective amount of a PRO317
agonist or PRO317 antagonist, such as an antibody which binds
specifically to PRO317.
[0306] 18. PRO301
[0307] Applicants have identified a cDNA clone (DNA40628-1216) that
encodes a novel polypeptide, designated in the present application
as "PRO301 ".
[0308] In one embodiment, the invention provides an isolated
nucleic acid molecule having at least about 80% sequence identity
to (a) a DNA molecule encoding a PRO301 polypeptide comprising the
sequence of amino acids 28 to 258 of FIG. 44 (SEQ ID NO:119), or
(b) the complement of the DNA molecule of (a). The sequence
identity preferably is about 85%, more preferably about 90%, most
preferably about 95%. In one aspect, the isolated nucleic acid has
at least about 80%, preferably at least about 85%, more preferably
at least about 90%, and most preferably at least about 95% sequence
identity with a polypeptide having amino acid residues 28 to 258 of
FIG. 44 (SEQ ID NO:119). Preferably, the highest degree of sequence
identity occurs within the extracellular domains (amino acids 28 to
258 of FIG. 44, SEQ ID NO:119). In a further embodiment, the
isolated nucleic acid molecule comprises DNA encoding a PRO301
polypeptide having amino acid residues 28 to 299 of FIG. 44 (SEQ ID
NO:119), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions. In another
aspect, the invention provides a nucleic acid of the full length
protein of clone DNA40628-1216, deposited with the ATCC under
accession number ATCC 209432, alternatively the coding sequence of
clone DNA40628-1216, deposited under accession number ATCC
209432.
[0309] In yet another embodiment, the invention provides isolated
PRO301 polypeptide. In particular, the invention provides isolated
native sequence PRO301 polypeptide, which in one embodiment,
includes an amino acid sequence comprising the extracellular domain
residues 28 to 258 of FIG. 44 (SEQ ID NO:119). Native PRO301
polypeptides with or without the native signal sequence (amino
acids 1 to 27 in FIG. 44 (SEQ ID NO:119), and with or without the
initiating methionine are specifically included. Additionally, the
sequences of the invention may also comprise the transmembrane
domain (residues 236 to about 258 in FIG. 44; SEQ ID NO:119) and/or
the intracellular domain (about residue 259 to 299 in FIG. 44; SEQ
ID NO:119). Alternatively, the invention provides a PRO301
polypeptide encoded by the nucleic acid deposited under accession
number ATCC 209432.
[0310] 19. PRO224
[0311] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as "PRO224".
[0312] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO224 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO224 polypeptide having amino acid residues 1 to 282 of FIG. 46
(SEQ ID NO:127), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0313] In another embodiment, the invention provides isolated
PRO224 polypeptide. In particular, the invention provides isolated
native sequence PRO224 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 282 of
FIG. 46 (SEQ ID NO:127).
[0314] 20. PRO222
[0315] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as "PRO222".
[0316] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO222 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO222 polypeptide having amino acid residues 1 to 490 of FIG. 48
(SEQ ID NO:132), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0317] In another embodiment, the invention provides isolated
PRO222 polypeptide. In particular, the invention provides isolated
native sequence PRO222 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 490 of
FIG. 48 (SEQ ID NO:132).
[0318] 21. PRO234
[0319] Applicants have identified a cDNA clone that encodes a novel
lectin polypeptide molecule, designated in the present application
as "PRO234".
[0320] In one embodiment, the invention provides an isolated
nucleic acid encoding a novel lectin comprising DNA encoding a
PRO234 polypeptide. In one aspect, the isolated nucleic acid
comprises the DNA encoding PRO234 polypeptides having amino acid
residues 1 to 382 of FIG. 50 (SEQ ID NO:137), or is complementary
to such encoding nucleic acid sequence, and remains stably bound to
it under at least moderate, and optionally, under high stringency
conditions. In another aspect, the invention provides an isolated
nucleic acid molecule comprising the nucleotide sequence of FIG. 49
(SEQ ID NO:136).
[0321] In another embodiment, the invention provides isolated novel
PRO234 polypeptides. In particular, the invention provides isolated
native sequence PRO234 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 382 of
FIG. 50 (SEQ ID NO:137).
[0322] In yet another embodiment, the invention provides
oligonucleotide probes useful for isolating genomic and cDNA
nucleotide sequences.
[0323] 22. PRO231
[0324] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to a putative acid phosphatase, wherein
the polypeptide is designated in the present application as
"PRO231".
[0325] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO231 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO231 polypeptide having amino acid residues 1 to 428 of FIG. 52
(SEQ ID NO:142), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0326] In another embodiment, the invention provides isolated
PRO231 polypeptide. In particular, the invention provides isolated
native sequence PRO231 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 428 of
FIG. 52 (SEQ ID NO:142).
[0327] 23. PRO229
[0328] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to scavenger receptors wherein the
polypeptide is designated in the present application as
"PRO229".
[0329] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO229 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO229 polypeptide having amino acid residues 1 to 347 of FIG. 54
(SEQ ID NO:148), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0330] In another embodiment, the invention provides isolated
PRO229 polypeptide. In particular, the invention provides isolated
native sequence PRO229 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 347 of
FIG. 54 (SEQ ID NO:148).
[0331] 24. PRO238
[0332] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to reductase, wherein the polypeptide
is designated in the present application as "PRO238".
[0333] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO238 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO238 polypeptide having amino acid residues 1 to 310 of FIG. 56
(SEQ ID NO:153), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0334] In another embodiment, the invention provides isolated
PRO238 polypeptide. In particular, the invention provides isolated
native sequence PRO238 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 310 of
FIG. 56 (SEQ ID NO:153).
[0335] 25. PRO233
[0336] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as "PRO233".
[0337] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO233 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO233 polypeptide having amino acid residues 1 to 300 of FIG. 58
(SEQ ID NO:159), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0338] In another embodiment, the invention provides isolated
PRO233 polypeptide. In particular, the invention provides isolated
native sequence PRO233 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 300 of
FIG. 58 (SEQ ID NO:159).
[0339] 26. PRO223
[0340] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to serine carboxypeptidase
polypeptides, wherein the polypeptide is designated in the present
application as "PRO223 ".
[0341] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO223 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO223 polypeptide having amino acid residues 1 to 476 of FIG. 60
(SEQ ID NO:164), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0342] In another embodiment, the invention provides isolated
PRO223 polypeptide. In particular, the invention provides isolated
native sequence PRO223 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 476 of
FIG. 60 (SEQ ID NO:164).
[0343] 27. PRO235
[0344] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as "PRO235".
[0345] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO235 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO235 polypeptide having amino acid residues 1 to 552 of FIG. 62
(SEQ ID NO:170), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0346] In another embodiment, the invention provides isolated
PRO235 polypeptide. In particular, the invention provides isolated
native sequence PRO235 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 552 of
FIG. 62 (SEQ ID NO:170).
[0347] 28. PRO236 and PRO262
[0348] Applicants have identified cDNA clones that encode novel
polypeptides having homology to .beta.-galactosidase, wherein those
polypeptides are designated in the present application as "PRO236"
and "PRO262".
[0349] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO236 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO236 polypeptide having amino acid residues 1 to 636 of FIG. 64
(SEQ ID NO:175), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0350] In another embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO262 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO262 polypeptide having amino acid residues 1 to 654 of FIG. 66
(SEQ ID NO:177), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0351] In another embodiment, the invention provides isolated
PRO236 polypeptide. In particular, the invention provides isolated
native sequence PRO236 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 636 of
FIG. 64 (SEQ ID NO:175).
[0352] In another embodiment, the invention provides isolated
PRO262 polypeptide. In particular, the invention provides isolated
native sequence PRO262 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 654 of
FIG. 66 (SEQ ID NO:177).
[0353] 29. PRO239
[0354] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as "PRO239".
[0355] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO239 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO239 polypeptide having amino acid residues 1 to 501 of FIG. 68
(SEQ ID NO:185), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0356] In another embodiment, the invention provides isolated
PRO239 polypeptide. In particular, the invention provides isolated
native sequence PRO239 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 501 of
FIG. 68 (SEQ ID NO:185).
[0357] 30. PRO257
[0358] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as "PRO257".
[0359] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO257 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO257 polypeptide having amino acid residues 1 to 607 of FIG. 70
(SEQ ID NO:190), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0360] In another embodiment, the invention provides isolated
PRO257 polypeptide. In particular, the invention provides isolated
native sequence PRO257 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 607 of
FIG. 70 (SEQ ID NO:190). An additional embodiment of the present
invention is directed to an isolated extracellular domain of a
PRO257 polypeptide.
[0361] 31. PRO260
[0362] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as "PRO260".
[0363] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO260 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO260 polypeptide having amino acid residues 1 to 467 of FIG. 72
(SEQ ID NO:195), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0364] In another embodiment, the invention provides isolated
PRO260 polypeptide. In particular, the invention provides isolated
native sequence PRO260 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 467 of
FIG. 72 (SEQ ID NO:195).
[0365] 32. PRO263
[0366] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to CD44 antigen, wherein the
polypeptide is designated in the present application as
"PRO263".
[0367] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO263 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO263 polypeptide having amino acid residues 1 to 322 of FIG. 74
(SEQ ID NO:201), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0368] In another embodiment, the invention provides isolated
PRO263 polypeptide. In particular, the invention provides isolated
native sequence PRO263 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 322 of
FIG. 74 (SEQ ID NO:201). An additional embodiment of the present
invention is directed to an isolated extracellular domain of a
PRO263 polypeptide.
[0369] 33. PRO270
[0370] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as "PRO270".
[0371] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO270 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA which
includes the sequence encoding the PRO270 polypeptide having amino
acid residues 1 to 296 of FIG. 76 (SEQ ID NO:207), or is
complementary to such encoding nucleic acid sequence, and remains
stably bound to it under at least moderate, and optionally, under
high stringency conditions.
[0372] In another embodiment, the invention provides isolated
PRO270 polypeptide. In particular, the invention provides isolated
native sequence PRO270 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 296 of
FIG. 76 (SEQ ID NO:207).
[0373] 34. PRO271
[0374] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to the proteoglycan link protein,
wherein the polypeptide is designated in the present application as
"PRO271".
[0375] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO271 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO271 polypeptide having amino acid residues 1 to 360 of FIG. 78
(SEQ ID NO:213), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0376] In another embodiment, the invention provides isolated
PRO271 polypeptide. In particular, the invention provides isolated
native sequence PRO271 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 360 of
FIG. 78 (SEQ ID NO:213).
[0377] 35. PRO272
[0378] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as "PRO272".
[0379] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO272 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO272 polypeptide having amino acid residues 1 to 328 of FIG. 80
(SEQ ID NO:221), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0380] In another embodiment, the invention provides isolated
PRO272 polypeptide. In particular, the invention provides isolated
native sequence PRO272 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 328 of
FIG. 80 (SEQ ID NO:211).
[0381] 36. PRO294
[0382] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as "PRO294".
[0383] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO294 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO294 polypeptide having amino acid residues 1 to 550 of FIG. 82
(SEQ ID NO:227), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0384] In another embodiment, the invention provides isolated
PRO294 polypeptide. In particular, the invention provides isolated
native sequence PRO294 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 550 of
FIG. 82 (SEQ ID NO:227).
[0385] 37. PRO295
[0386] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as "PRO295".
[0387] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO295 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO295 polypeptide having amino acid residues 1 to 350 of FIG. 84
(SEQ ID NO:236), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0388] In another embodiment, the invention provides isolated
PRO295 polypeptide. In particular, the invention provides isolated
native sequence PRO295 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 350 of
FIG. 84 (SEQ ID NO:236).
[0389] 38. PRO293
[0390] Applicants have identified a cDNA clone that encodes a novel
human neuronal leucine rich repeat polypeptide, wherein the
polypeptide is designated in the present application as
"PRO293".
[0391] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO293 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO293 polypeptide having amino acid residues 1 to 713 of FIG. 86
(SEQ ID NO:245), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0392] In another embodiment, the invention provides isolated
PRO293 polypeptide. In particular, the invention provides isolated
native sequence PRO293 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 713 of
FIG. 86 (SEQ ID NO:245). An additional embodiment of the present
invention is directed to an isolated extracellular domain of a
PRO293 polypeptide.
[0393] 39. PRO247
[0394] Applicants have identified a cDNA clone that encodes a novel
polypeptide having leucine rich repeats wherein the polypeptide is
designated in the present application as "PRO247".
[0395] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO247 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO247 polypeptide having amino acid residues 1 to 546 of FIG. 88
(SEQ ID NO:250), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0396] In another embodiment, the invention provides isolated
PRO247 polypeptide. In particular, the invention provides isolated
native sequence PRO247 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 546 of
FIG. 88 (SEQ ID NO:250). An additional embodiment of the present
invention is directed to an isolated extracellular domain of a
PRO247 polypeptide.
[0397] 40. PRO302, PRO303, PRO304, PRO307 and PRO343
[0398] Applicants have identified cDNA clones that encode novel
polypeptides having homology to various proteases, wherein those
polypeptide are designated in the present application as "PRO302",
"PRO303", "PRO304", "PRO307" and "PRO343" polypeptides.
[0399] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO302 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO302 polypeptide having amino acid residues 1 to 452 of FIG. 90
(SEQ ID NO:255), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0400] In another embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO303 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO303 polypeptide having amino acid residues 1 to 314 of FIG. 92
(SEQ ID NO:257), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0401] In yet another embodiment, the invention provides an
isolated nucleic acid molecule comprising DNA encoding a PRO304
polypeptide. In one aspect, the isolated nucleic acid comprises DNA
encoding the PRO304 polypeptide having amino acid residues 1 to 556
of FIG. 94 (SEQ ID NO:259), or is complementary to such encoding
nucleic acid sequence, and remains stably bound to it under at
least moderate, and optionally, under high stringency
conditions.
[0402] In another embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO307 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO307 polypeptide having amino acid residues 1 to 383 of FIG. 96
(SEQ ID NO:261), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0403] In another embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO343 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO343 polypeptide having amino acid residues 1 to 317 of FIG. 98
(SEQ ID NO:263), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0404] In another embodiment, the invention provides isolated
PRO302 polypeptide. In particular, the invention provides isolated
native sequence PRO302 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 452 of
FIG. 90 (SEQ ID NO:255). In another embodiment, the invention
provides isolated PRO303 polypeptide. In particular, the invention
provides isolated native sequence PRO303 polypeptide, which in one
embodiment, includes an amino acid sequence comprising residues 1
to 314 of FIG. 92 (SEQ ID NO:257).
[0405] In another embodiment, the invention provides isolated
PRO304 polypeptide. In particular, the invention provides isolated
native sequence PRO304 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 556 of
FIG. 94 (SEQ ID NO:259).
[0406] In another embodiment, the invention provides isolated
PRO307 polypeptide. In particular, the invention provides isolated
native sequence PRO307 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 383 of
FIG. 96 (SEQ ID NO:261).
[0407] In another embodiment, the invention provides isolated
PRO343 polypeptide. In particular, the invention provides isolated
native sequence PRO343 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 317 of
FIG. 98 (SEQ ID NO:263).
[0408] 41. PRO328
[0409] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as "PRO328".
[0410] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO328 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO328 polypeptide having amino acid residues 1 to 463 of FIG. 100
(SEQ ID NO:285), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0411] In another embodiment, the invention provides isolated
PRO328 polypeptide. In particular, the invention provides isolated
native sequence PRO328 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 463 of
FIG. 100 (SEQ ID NO:285). An additional embodiment of the present
invention is directed to an isolated extracellular domain of a
PRO306 polypeptide.
[0412] 42. PRO335, PRO331 and PRO326
[0413] Applicants have identified three cDNA clones that
respectively encode three novel polypeptides, each having leucine
rich repeats and homology to LIG-1 and ALS. These polypeptides are
designated in the present application as PRO335, PRO331 and PRO326,
respectively.
[0414] In one embodiment, the invention provides three isolated
nucleic acid molecules comprising DNA respectively encoding PRO335,
PRO331 and PRO326, respectively. In one aspect, herein is provided
an isolated nucleic acid comprising DNA encoding the PRO335
polypeptide having amino acid residues 1 through 1059 of FIG. 102
(SEQ ID NO:290), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions. Also provided
herein is an isolated nucleic acid comprises DNA encoding the
PRO331 polypeptide having amino acid residues 1 through 640 of FIG.
104 (SEQ ID NO:292), or is complementary to such encoding nucleic
acid sequence, and remains stably bound to it under at least
moderate, and optionally, under high stringency conditions.
Additionally provided herein is an isolated nucleic acid comprises
DNA encoding the PRO326 polypeptide having amino acid residues 1
through 1119 of FIG. 106 (SEQ ID NO:294), or is complementary to
such encoding nucleic acid sequence, and remains stably bound to it
under at least moderate, and optionally, under high stringency
conditions.
[0415] In another embodiment, the invention provides isolated
PRO335, PRO331 and PRO326 polypeptides or extracellular domains
thereof. In particular, the invention provides isolated native
sequence for the PRO335 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 through 1059
of FIG. 102 (SEQ ID NO:290). Also provided herein is the isolated
native sequence for the PRO331 polypeptide, which in one
embodiment, includes an amino acid sequence comprising residues 1
through 640 of FIG. 104 (SEQ ID NO:292). Also provided herein is
the isolated native sequence for the PRO326 polypeptide, which in
one embodiment, includes an amino acid sequence comprising residues
1 through 1119 of FIG. 106 (SEQ ID NO:294).
[0416] 43. PRO332
[0417] Applicants have identified a cDNA clone (DNA40982-1235) that
encodes a novel polypeptide, designated in the present application
as "PRO332."
[0418] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA having at least about 80%
sequence identity to (a) a DNA molecule encoding a PRO358
polypeptide comprising the sequence of amino acids 49 to 642 of
FIG. 108 (SEQ ID NO:310), or (b) the complement of the DNA molecule
of (a). The sequence identity preferably is about 85%, more
preferably about 90%, most preferably about 95%. In one aspect, the
isolated nucleic acid has at least about 80%, preferably at least
about 85%, more preferably at least about 90%, and most preferably
at least about 95% sequence identity with a polypeptide having
amino acid residues 1 to 642 of FIG. 108 (SEQ ID NO:310).
Preferably, the highest degree of sequence identity occurs within
the leucine-rich repeat domains (amino acids 116 to 624 of FIG.
108, SEQ ID NO:310). In a further embodiment, the isolated nucleic
acid molecule comprises DNA encoding a PRO332 polypeptide having
amino acid residues 49 to 642 of FIG. 108 (SEQ ID NO:310), or is
complementary to such encoding nucleic acid sequence, and remains
stably bound to it under at least moderate, and optionally, under
high stringency conditions.
[0419] In another embodiment, the invention provides isolated
PRO332 polypeptides. In particular, the invention provides isolated
native sequence PRO332 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 49 to 624 of
FIG. 108 (SEQ ID NO:310). Native PRO332 polypeptides with or
without the native signal sequence (amino acids 1 to 48 in FIG.
108, SEQ ID NO:310), and with or without the initiating methionine
are specifically included.
[0420] 44. PRO334
[0421] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to fibulin and fibrillin, wherein the
polypeptide is designated in the present application as
"PRO334".
[0422] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO334 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO334 polypeptide having amino acid residues 1 to 509 of FIG. 110
(SEQ ID NO:315), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0423] In another embodiment, the invention provides isolated
PRO334 polypeptide. In particular, the invention provides isolated
native sequence PRO334 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 509 of
FIG. 110 (SEQ ID NO:315).
[0424] 45. PRO346
[0425] Applicants have identified a cDNA clone (DNA44167-1243) that
encodes a novel polypeptide, designated in the present application
as "PRO346." In one embodiment, the invention provides an isolated
nucleic acid molecule having at least about 80% sequence identity
to (a) a DNA molecule encoding a PRO346 polypeptide comprising the
sequence of amino acids 19 to 339 of FIG. 112 (SEQ ID NO:320), or
(b) the complement of the DNA molecule of (a). The sequence
identity preferably is about 85%, more preferably about 90%, most
preferably about 95%. In one aspect, the isolated nucleic acid has
at least about 80%, preferably at least about 85%, more preferably
at least about 90%, and most preferably at least about 95% sequence
identity with a polypeptide having amino acid residues 19 to 339 of
FIG. 112 (SEQ ID NO:320). Preferably, the highest degree of
sequence identity occurs within the extracellular domains (amino
acids 19 to 339 of FIG. 112, SEQ ID NO:320). In alternative
embodiments, the polypeptide by which the homology is measured
comprises the residues 1-339, 19-360 or 19-450 of FIG. 112, SEQ ID
NO:320). In a further embodiment, the isolated nucleic acid
molecule comprises DNA encoding a PRO346 polypeptide having amino
acid residues 19 to 339 of FIG. 112 (SEQ ID NO:320), alternatively
residues 1-339, 19-360 or 19-450 of FIG. 112 (SEQ ID NO:320) or is
complementary to such encoding nucleic acid sequence, and remains
stably bound to it under at least moderate, and optionally, under
high stringency conditions. In another aspect, the invention
provides a nucleic acid of the full length protein of clone
DNA44167-1243, deposited with the ATCC under accession number ATCC
209434, alternatively the coding sequence of clone DNA44167-1243,
deposited under accession number ATCC 209434.
[0426] In yet another embodiment, the invention provides isolated
PRO346 polypeptide. In particular, the invention provides isolated
native sequence PRO346 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 19 to 339 of
FIG. 112 (SEQ ID NO:320). Native PRO346 polypeptides with or
without the native signal sequence (residues 1 to 18 in FIG. 112
(SEQ ID NO:320), with or without the initiating methionine, with or
without the transmembrane domain (residues 340 to 360) and with or
without the intracellular domain (residues 361 to 450) are
specifically included. Alternatively, the invention provides a
PRO346 polypeptide encoded by the nucleic acid deposited under
accession number ATCC 209434.
[0427] 46. PRO268
[0428] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to protein disulfide isomerase, wherein
the polypeptide is designated in the present application as
"PRO268".
[0429] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO268 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO268 polypeptide having amino acid residues 1 to 280 of FIG. 114
(SEQ ID NO:325), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0430] In another embodiment, the invention provides isolated
PRO268 polypeptide. In particular, the invention provides isolated
native sequence PRO268 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 280 of
FIG. 114 (SEQ ID NO:325). An additional embodiment of the present
invention is directed to an isolated extracellular domain of a
PRO268 polypeptide.
[0431] 47. PRO330
[0432] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to the alpha subunit of prolyl
4-hydroxylase, wherein the polypeptide is designated in the present
application as "PRO330".
[0433] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO330 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO330 polypeptide having amino acid residues 1 to 533 of FIG. 116
(SEQ ID NO:332), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0434] In another embodiment, the invention provides isolated
PRO330 polypeptide. In particular, the invention provides isolated
native sequence PRO330 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 533 of
FIG. 116 (SEQ ID NO:332).
[0435] 48. PRO339 and PRO310
[0436] Applicants have identified two cDNA clones wherein each
clone encodes a novel polypeptide having homology to fringe,
wherein the polypeptides are designated in the present application
as "PRO339" and "PRO310".
[0437] In one embodiment, the invention provides isolated nucleic
acid molecules comprising DNA encoding a PRO339 and/or a PRO310
polypeptide. In one aspect, the isolated nucleic acid comprises DNA
encoding the PRO339 polypeptide having amino acid residues 1 to 772
of FIG. 118 (SEQ ID NO:339), or is complementary to such encoding
nucleic acid sequence, and remains stably bound to it under at
least moderate, and optionally, under high stringency conditions.
In another aspect, the isolated nucleic acid comprises DNA encoding
the PRO310 polypeptide having amino acid residues 1 to 318 of FIG.
120 (SEQ ID NO:341), or is complementary to such encoding nucleic
acid sequence, and remains stably bound to it under at least
moderate, and optionally, under high stringency conditions.
[0438] In another embodiment, the invention provides isolated
PRO339 as well as isolated PRO310 polypeptides. In particular, the
invention provides isolated native sequence PRO339 polypeptide,
which in one embodiment, includes an amino acid sequence comprising
residues 1 to 772 of FIG. 118 (SEQ ID NO:339). The invention
further provides isolated native sequence PRO310 polypeptide, which
in one embodiment, includes an amino acid sequence comprising
residues 1 to 318 of FIG. 120 (SEQ ID NO:341).
[0439] 49. PRO244
[0440] Applicants have identified a cDNA clone that encodes a novel
polypeptide, designated in the present application as "PRO244".
[0441] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding PRO244 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding
PRO244 polypeptide having amino acid residues 1 to 219 of FIG. 122
(SEQ ID NO:377), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0442] In another embodiment, the invention provides isolated
PRO244 polypeptide. In particular, the invention provides isolated
native sequence PRO244 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 219 of
FIG. 122 (SEQ ID NO:377).
[0443] 50. Additional Embodiments
[0444] In other embodiments of the present invention, 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.
[0445] In other embodiments, 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.
[0446] In another embodiment, the invention provides an antibody
which specifically binds to any of the above or below described
polypeptides. Optionally, the antibody is a monoclonal antibody,
humanized antibody, antibody fragment or single-chain antibody.
[0447] In yet other embodiments, the invention provides
oligonucleotide probes useful for isolating genomic and cDNA
nucleotide sequences, wherein those probes may be derived from any
of the above or below described nucleotide sequences.
[0448] In other embodiments, the invention provides an isolated
nucleic acid molecule comprising a nucleotide sequence that encodes
a PRO polypeptide.
[0449] In one aspect, the isolated nucleic acid molecule comprises
a nucleotide sequence having at least about 80% sequence identity,
preferably at least about 81% sequence identity, more preferably at
least about 82% sequence identity, yet more preferably at least
about 83% sequence identity, yet more preferably at least about 84%
sequence identity, yet more preferably at least about 85% sequence
identity, yet more preferably at least about 86% sequence identity,
yet more preferably at least about 87% sequence identity, yet more
preferably at least about 88% sequence identity, yet more
preferably at least about 89% sequence identity, yet more
preferably at least about 90% sequence identity, yet more
preferably at least about 91% sequence identity, yet more
preferably at least about 92% sequence identity, yet more
preferably at least about 93% sequence identity, yet more
preferably at least about 94% sequence identity, yet more
preferably at least about 95% sequence identity, yet more
preferably at least about 96% sequence identity, yet more
preferably at least about 97% sequence identity, yet more
preferably at least about 98% sequence identity and yet more
preferably at least about 99% sequence identity to (a) a DNA
molecule encoding a PRO polypeptide having a full-length amino acid
sequence as disclosed herein, an amino acid sequence lacking the
signal peptide as disclosed herein or an extracellular domain of a
transmembrane protein, with or without the signal peptide, as
disclosed herein, or (b) the complement of the DNA molecule of
(a).
[0450] In other aspects, the isolated nucleic acid molecule
comprises a nucleotide sequence having at least about 80% sequence
identity, preferably at least about 81% sequence identity, more
preferably at least about 82% sequence identity, yet more
preferably at least about 83% sequence identity, yet more
preferably at least about 84% sequence identity, yet more
preferably at least about 85% sequence identity, yet more
preferably at least about 86% sequence identity, yet more
preferably at least about 87% sequence identity, yet more
preferably at least about 88% sequence identity, yet more
preferably at least about 89% sequence identity, yet more
preferably at least about 90% sequence identity, yet more
preferably at least about 91% sequence identity, yet more
preferably at least about 92% sequence identity, yet more
preferably at least about 93% sequence identity, yet more
preferably at least about 94% sequence identity, yet more
preferably at least about 95% sequence identity, yet more
preferably at least about 96% sequence identity, yet more
preferably at least about 97% sequence identity, yet more
preferably at least about 98% sequence identity and yet more
preferably at least about 99% sequence identity to (a) a DNA
molecule comprising the coding sequence of a full-length PRO
polypeptide cDNA as disclosed herein, the coding sequence of a PRO
polypeptide lacking the signal peptide as disclosed herein or the
coding sequence of an extracellular domain of a transmembrane PRO
polypeptide, with or without the signal peptide, as disclosed
herein, or (b) the complement of the DNA molecule of (a).
[0451] In a further aspect, the invention concerns an isolated
nucleic acid molecule comprising a nucleotide sequence having at
least about 80% sequence identity, preferably at least about 81%
sequence identity, more preferably at least about 82% sequence
identity, yet more preferably at least about 83% sequence identity,
yet more preferably at least about 84% sequence identity, yet more
preferably at least about 85% sequence identity, yet more
preferably at least about 86% sequence identity, yet more
preferably at least about 87% sequence identity, yet more
preferably at least about 88% sequence identity, yet more
preferably at least about 89% sequence identity, yet more
preferably at least about 90% sequence identity, yet more
preferably at least about 91% sequence identity, yet more
preferably at least about 92% sequence identity, yet more
preferably at least about 93% sequence identity, yet more
preferably at least about 94% sequence identity, yet more
preferably at least about 95% sequence identity, yet more
preferably at least about 96% sequence identity, yet more
preferably at least about 97% sequence identity, yet more
preferably at least about 98% sequence identity and yet more
preferably at least about 99% 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).
[0452] Another aspect the invention provides an isolated nucleic
acid molecule comprising a nucleotide sequence encoding a PRO
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 PRO polypeptides are
contemplated.
[0453] Another embodiment is directed to fragments of a PRO
polypeptide coding sequence, or the complement thereof, that may
find use as, for example, hybridization probes or for encoding
fragments of a PRO polypeptide that may optionally encode a
polypeptide comprising a binding site for an anti-PRO antibody.
Such nucleic acid fragments are usually at least about 20
nucleotides in length, preferably at least about 30 nucleotides in
length, mor e preferably at least about 40 nucleotides in length,
yet more preferably at least about 50 nucleotides in length, yet
more preferably at least about 60 nucleotides in length, yet more
preferably at least about 70 nucleotides in length, yet more
preferably at least about 80 nucleotides in length, yet more
preferably at least about 90 nucleotides in length, yet more
preferably at least about 100 nucleotides in length, yet more
preferably at least about 110 nucleotides in length, yet more
preferably at least about 120 nucleotides in length, yet more
preferably at least about 130 nucleotides in length, yet more
preferably at least about 140 nucleotides in length, yet more
preferably at least about 150 nucleotides in length, yet more
preferably at least about 160 nucleotides in length, yet more
preferably at least about 170 nucleotides in length, yet more
preferably at least about 180 nucleotides in length, yet more
preferably at least about 190 nucleotides in length, yet more
preferably at least about 200 nucleotides in length, yet more
preferably at least about 250 nucleotides in length, yet more
preferably at least about 300 nucleotides in length, yet more
preferably at least about 350 nucleotides in length, yet more
preferably at least about 400 nucleotides in length, yet more
preferably at least about 450 nucleotides in length, yet more
preferably at least about 500 nucleotides in length, yet more
preferably at least about 600 nucleotides in length, yet more
preferably at least about 700 nucleotides in length, yet more
preferably at least about 800 nucleotides in length, yet more
preferably at least about 900 nucleotides in length and yet more
preferably 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 PRO polypeptide-encoding nucleotide
sequence may be determined in a routine manner by aligning the PRO
polypeptide-encoding nucleotide sequence with other known
nucleotide sequences using any of a number of well known sequence
alignment programs and determining which PRO polypeptide-encoding
nucleotide sequence fragment(s) are novel. All of such PRO
polypeptide-encoding nucleotide sequences are contemplated herein.
Also contemplated are the PRO polypeptide fragments encoded by
these nucleotide molecule fragments, preferably those PRO
polypeptide fragments that comprise a binding site for an anti-PRO
antibody.
[0454] In another embodiment, the invention provides isolated PRO
polypeptide encoded by any of the isolated nucleic acid sequences
hereinabove identified.
[0455] In a certain aspect, the invention concerns an isolated PRO
polypeptide, comprising an amino acid sequence having at least
about 80% sequence identity, preferably at least about 81% sequence
identity, more preferably at least about 82% sequence identity, yet
more preferably at least about 83% sequence identity, yet more
preferably at least about 84% sequence identity, yet more
preferably at least about 85% sequence identity, yet more
preferably at least about 86% sequence identity, yet more
preferably at least about 87% sequence identity, yet more
preferably at least about 88% sequence identity, yet more
preferably at least about 89% sequence identity, yet more
preferably at least about 90% sequence identity, yet more
preferably at least about 91% sequence identity, yet more
preferably at least about 92% sequence identity, yet more
preferably at least about 93% sequence identity, yet more
preferably at least about 94% sequence identity, yet more
preferably at least about 95% sequence identity, yet more
preferably at least about 96% sequence identity, yet more
preferably at least about 97% sequence identity, yet more
preferably at least about 98% sequence identity and yet more
preferably at least about 99% sequence identity to a PRO
polypeptide having a full-length amino acid sequence as disclosed
herein, an amino acid sequence lacking the signal peptide as
disclosed herein or an extracellular domain of a transmembrane
protein, with or without the signal peptide, as disclosed
herein.
[0456] In a further aspect, the invention concerns an isolated PRO
polypeptide comprising an amino acid sequence having at least about
80% sequence identity, preferably at least about 81% sequence
identity, more preferably at least about 82% sequence identity, yet
more preferably at least about 83% sequence identity, yet more
preferably at least about 84% sequence identity, yet more
preferably at least about 85% sequence identity, yet more
preferably at least about 86% sequence identity, yet more
preferably at least about 87% sequence identity, yet more
preferably at least about 88% sequence identity, yet more
preferably at least about 89% sequence identity, yet more
preferably at least about 90% sequence identity, yet more
preferably at least about 91% sequence identity, yet more
preferably at least about 92% sequence identity, yet more
preferably at least about 93% sequence identity, yet more
preferably at least about 94% sequence identity, yet more
preferably at least about 95% sequence identity, yet more
preferably at least about 96% sequence identity, yet more
preferably at least about 97% sequence identity, yet more
preferably at least about 98% sequence identity and yet more
preferably at least about 99% sequence identity to an amino acid
sequence encoded by any of the human protein cDNAs deposited with
the ATCC as disclosed herein.
[0457] In a further aspect, the invention concerns an isolated PRO
polypeptide comprising an amino acid sequence scoring at least
about 80% positives, preferably at least about 81% positives, more
preferably at least about 82% positives, yet more preferably at
least about 83% positives, yet more preferably at least about 84%
positives, yet more preferably at least about 85% positives, yet
more preferably at least about 86% positives, yet more preferably
at least about 87% positives, yet more preferably at least about
88% positives, yet more preferably at least about 89% positives,
yet more preferably at least about 90% positives, yet more
preferably at least about 91% positives, yet more preferably at
least about 92% positives, yet more preferably at least about 93%
positives, yet more preferably at least about 94% positives, yet
more preferably at least about 95% positives, yet more preferably
at least about 96% positives, yet more preferably at least about
97% positives, yet more preferably at least about 98% positives and
yet more preferably at least about 99% positives when compared with
the amino acid sequence of a PRO polypeptide having a full-length
amino acid sequence as disclosed herein, an amino acid sequence
lacking the signal peptide as disclosed herein or an extracellular
domain of a transmembrane protein, with or without the signal
peptide, as disclosed herein.
[0458] In a specific aspect, the invention provides an isolated PRO
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 PRO polypeptide and
recovering the PRO polypeptide from the cell culture.
[0459] Another aspect the invention provides an isolated PRO
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 PRO polypeptide and recovering the
PRO polypeptide from the cell culture.
[0460] In yet another embodiment, the invention concerns agonists
and antagonists of a native PRO polypeptide as defined herein. In a
particular embodiment, the agonist or antagonist is an anti-PRO
antibody or a small molecule.
[0461] In a further embodiment, the invention concerns a method of
identifying agonists or antagonists to a PRO polypeptide which
comprise contacting the PRO polypeptide with a candidate molecule
and monitoring a biological activity mediated by said PRO
polypeptide. Preferably, the PRO polypeptide is a native PRO
polypeptide.
[0462] In a still further embodiment, the invention concerns a
composition of matter comprising a PRO polypeptide, or an agonist
or antagonist of a PRO polypeptide as herein described, or an
anti-PRO antibody, in combination with a carrier. Optionally, the
carrier is a pharmaceutically acceptable carrier.
[0463] Another embodiment of the present invention is directed to
the use of a PRO polypeptide, or an agonist or antagonist thereof
as hereinbefore described, or an anti-PRO antibody, for the
preparation of a medicament useful in the treatment of a condition
which is responsive to the PRO polypeptide, an agonist or
antagonist thereof or an anti-PRO antibody.
BRIEF DESCRIPTION OF THE DRAWINGS
[0464] FIG. 1 shows a nucleotide sequence (SEQ ID NO:1) of a native
sequence PRO211 cDNA, wherein SEQ ID NO:1 is a clone designated
herein as "DNA32292-1131 ".
[0465] 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.
[0466] 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".
[0467] 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.
[0468] FIG. 5 shows a nucleotide sequence (SEQ ID NO:11) of a
native sequence PRO230 cDNA, wherein SEQ ID NO:11 is a clone
designated herein as "DNA33223-1136".
[0469] FIG. 6 shows the amino acid sequence (SEQ ID NO:12) derived
from the coding sequence of SEQ ID NO:11 shown in FIG. 5.
[0470] FIG. 7 shows a nucleotide sequence designated herein as
DNA20088 (SEQ ID NO:13).
[0471] FIG. 8 shows a nucleotide sequence (SEQ ID NO:17) of a
native sequence PRO232 cDNA, wherein SEQ ID NO:17 is a clone
designated herein as "DNA34435-1140".
[0472] FIG. 9 shows the amino acid sequence (SEQ ID NO:18) derived
from the coding sequence of SEQ ID NO:17 shown in FIG. 8.
[0473] FIG. 10 shows a nucleotide sequence (SEQ ID NO:22) of a
native sequence PRO187 cDNA, wherein SEQ ID NO:22 is a clone
designated herein as "DNA27864-1155".
[0474] FIG. 11 shows the amino acid sequence (SEQ ID NO:23) derived
from the coding sequence of SEQ ID NO:22 shown in FIG. 10.
[0475] FIG. 12 shows a nucleotide sequence (SEQ ID NO:27) of a
native sequence PRO265 cDNA, wherein SEQ ID NO:27 is a clone
designated herein as "DNA36350-1158".
[0476] FIG. 13 shows the amino acid sequence (SEQ ID NO:28) derived
from the coding sequence of SEQ ID NO:27 shown in FIG. 12.
[0477] FIG. 14 shows a nucleotide sequence (SEQ ID NO:33) of a
native sequence PRO219 cDNA, wherein SEQ ID NO:33 is a clone
designated herein as "DNA32290-1164".
[0478] FIG. 15 shows the amino acid sequence (SEQ ID NO:34) derived
from the coding sequence of SEQ ID NO:33 shown in FIG. 14.
[0479] FIG. 16 shows a nucleotide sequence (SEQ ID NO:38) of a
native sequence PRO246 cDNA, wherein SEQ ID NO:38 is a clone
designated herein as "DNA35639-1172".
[0480] FIG. 17 shows the amino acid sequence (SEQ ID NO:39) derived
from the coding sequence of SEQ ID NO:38 shown in FIG. 16.
[0481] FIG. 18 shows a nucleotide sequence (SEQ ID NO:48) of a
native sequence PRO228 cDNA, wherein SEQ ID NO:48 is a clone
designated herein as "DNA33092-1202".
[0482] FIG. 19 shows the amino acid sequence (SEQ ID NO:49) derived
from the coding sequence of SEQ ID NO:48 shown in FIG. 18.
[0483] FIG. 20 shows a nucleotide sequence designated herein as
DNA21951 (SEQ ID NO:50).
[0484] FIG. 21 shows a nucleotide sequence (SEQ ID NO:58) of a
native sequence PRO533 cDNA, wherein SEQ ID NO:58 is a clone
designated herein as "DNA49435-1219".
[0485] FIG. 22 shows the amino acid sequence (SEQ ID NO:59) derived
from the coding sequence of SEQ ID NO:58 shown in FIG. 21.
[0486] FIG. 23 shows a nucleotide sequence (SEQ ID NO:63) of a
native sequence PRO245 cDNA, wherein SEQ ID NO:63 is a clone
designated herein as "DNA35638-1141 ".
[0487] FIG. 24 shows the amino acid sequence (SEQ ID NO:64) derived
from the coding sequence of SEQ ID NO:63 shown in FIG. 23.
[0488] FIG. 25 shows a nucleotide sequence (SEQ ID NO:68) of a
native sequence PRO220 cDNA, wherein SEQ ID NO:68 is a clone
designated herein as "DNA32298-1132".
[0489] FIG. 26 shows the amino acid sequence (SEQ ID NO:69) derived
from the coding sequence of SEQ ID NO:68 shown in FIG. 25.
[0490] FIG. 27 shows a nucleotide sequence (SEQ ID NO:70) of a
native sequence PRO221 cDNA, wherein SEQ ID NO:70 is a clone
designated herein as "DNA33089-1132".
[0491] FIG. 28 shows the amino acid sequence (SEQ ID NO:71) derived
from the coding sequence of SEQ ID NO:70 shown in FIG. 27.
[0492] FIG. 29 shows a nucleotide sequence (SEQ ID NO:72) of a
native sequence PRO227 cDNA, wherein SEQ ID NO:72 is a clone
designated herein as "DNA33786-1132".
[0493] FIG. 30 shows the amino acid sequence (SEQ ID NO:73) derived
from the coding sequence of SEQ ID NO:72 shown in FIG. 29.
[0494] FIG. 31 shows a nucleotide sequence (SEQ ID NO:83) of a
native sequence PRO258 cDNA, wherein SEQ ID NO:83 is a clone
designated herein as "DNA35918-1174".
[0495] FIG. 32 shows the amino acid sequence (SEQ ID NO:84) derived
from the coding sequence of SEQ ID NO:83 shown in FIG. 31.
[0496] FIG. 33 shows a nucleotide sequence (SEQ ID NO:90) of a
native sequence PRO266 cDNA, wherein SEQ ID NO:90 is a clone
designated herein as "DNA37150-1178".
[0497] FIG. 34 shows the amino acid sequence (SEQ ID NO:91) derived
from the coding sequence of SEQ ID NO:90 shown in FIG. 33.
[0498] FIG. 35 shows a nucleotide sequence (SEQ ID NO:95) of a
native sequence PRO269 cDNA, wherein SEQ ID NO:95 is a clone
designated herein as "DNA38260-1180".
[0499] FIG. 36 shows the amino acid sequence (SEQ ID NO:96) derived
from the coding sequence of SEQ ID NO:95 shown in FIG. 35.
[0500] FIG. 37 shows a nucleotide sequence (SEQ ID NO:103) of a
native sequence PRO287 cDNA, wherein SEQ ID NO:103 is a clone
designated herein as "DNA39969-1185".
[0501] FIG. 38 shows the amino acid sequence (SEQ ID NO:104)
derived from the coding sequence of SEQ ID NO:103 shown in FIG.
37.
[0502] FIG. 39 shows a nucleotide sequence (SEQ ID NO:108) of a
native sequence PRO214 cDNA, wherein SEQ ID NO:108 is a clone
designated herein as "DNA32286-1191".
[0503] FIG. 40 shows the amino acid sequence (SEQ ID NO:109)
derived from the coding sequence of SEQ ID NO:108 shown in FIG.
39.
[0504] FIG. 41 shows a nucleotide sequence (SEQ ID NO:113) of a
native sequence PRO317 cDNA, wherein SEQ ID NO:113 is a clone
designated herein as "DNA33461-1199".
[0505] FIG. 42 shows the amino acid sequence (SEQ ID NO:114)
derived from the coding sequence of SEQ ID NO:113 shown in FIG.
41.
[0506] FIG. 43 shows a nucleotide sequence (SEQ ID NO:118) of a
native sequence PRO301 cDNA, wherein SEQ ID NO:118 is a clone
designated herein as "DNA40628-1216".
[0507] FIG. 44 shows the amino acid sequence (SEQ ID NO:119)
derived from the coding sequence of SEQ ID NO:118 shown in FIG.
43.
[0508] FIG. 45 shows a nucleotide sequence (SEQ ID NO:126) of a
native sequence PRO224 cDNA, wherein SEQ ID NO:126 is a clone
designated herein as "DNA33221-1133".
[0509] FIG. 46 shows the amino acid sequence (SEQ ID NO:127)
derived from the coding sequence of SEQ ID NO:126 shown in FIG.
45.
[0510] FIG. 47 shows a nucleotide sequence (SEQ ID NO:131) of
anative sequence PRO222 cDNA, wherein SEQ ID NO:131 is a clone
designated herein as "DNA33107-1135".
[0511] FIG. 48 shows the amino acid sequence (SEQ ID NO:132)
derived from the coding sequence of SEQ ID NO:131 shown in FIG.
47.
[0512] FIG. 49 shows a nucleotide sequence (SEQ ID NO:136) of a
native sequence PRO234 cDNA, wherein SEQ ID NO:136 is a clone
designated herein as "DNA35557-1137".
[0513] FIG. 50 shows the amino acid sequence (SEQ ID NO:137)
derived from the coding sequence of SEQ ID NO:136 shown in FIG.
49.
[0514] FIG. 51 shows a nucleotide sequence (SEQ ID NO:141) of a
native sequence PRO231 cDNA, wherein SEQ ID NO:141 is a clone
designated herein as "DNA34434-1139".
[0515] FIG. 52 shows the amino acid sequence (SEQ ID NO:142)
derived from the coding sequence of SEQ ID NO:141 shown in FIG.
51.
[0516] FIG. 53 shows a nucleotide sequence (SEQ ID NO:147) of a
native sequence PRO229 cDNA, wherein SEQ ID NO:147 is a clone
designated herein as "DNA33100-1159".
[0517] FIG. 54 shows the amino acid sequence (SEQ ID NO:148)
derived from the coding sequence of SEQ ID NO:147 shown in FIG.
53.
[0518] FIG. 55 shows a nucleotide sequence (SEQ ID NO:152) of a
native sequence PRO238 cDNA, wherein SEQ ID NO:152 is a clone
designated herein as "DNA35600-1162".
[0519] FIG. 56 shows the amino acid sequence (SEQ ID NO:153)
derived from the coding sequence of SEQ ID NO:152 shown in FIG.
55.
[0520] FIG. 57 shows a nucleotide sequence (SEQ ID NO:158) of a
native sequence PRO233 cDNA, wherein SEQ ID NO:158 is a clone
designated herein as "DNA34436-1238".
[0521] FIG. 58 shows the amino acid sequence (SEQ ID NO:159)
derived from the coding sequence of SEQ ID NO:158 shown in FIG.
57.
[0522] FIG. 59 shows a nucleotide sequence (SEQ ID NO:163) of a
native sequence PRO223 cDNA, wherein SEQ ID NO:163 is a clone
designated herein as "DNA33206-1165".
[0523] FIG. 60 shows the amino acid sequence (SEQ ID NO:164)
derived from the coding sequence of SEQ ID NO:163 shown in FIG.
59.
[0524] FIG. 61 shows a nucleotide sequence (SEQ ID NO:169) of a
native sequence PRO235 cDNA, wherein SEQ ID NO:169 is a clone
designated herein as "DNA35558-1167".
[0525] FIG. 62 shows the amino acid sequence (SEQ ID NO:170)
derived from the coding sequence of SEQ ID NO:169 shown in FIG.
61.
[0526] FIG. 63 shows a nucleotide sequence (SEQ ID NO:174) of a
native sequence PRO236 cDNA, wherein SEQ ID NO:174 is a clone
designated herein as "DNA35599-1168".
[0527] FIG. 64 shows the amino acid sequence (SEQ ID NO:175)
derived from the coding sequence of SEQ ID NO:174 shown in FIG.
63.
[0528] FIG. 65 shows a nucleotide sequence (SEQ ID NO:176) of a
native sequence PRO262 cDNA, wherein SEQ ID NO:176 is a clone
designated herein as "DNA36992-1168".
[0529] FIG. 66 shows the amino acid sequence (SEQ ID NO:177)
derived from the coding sequence of SEQ ID NO:176 shown in FIG.
65.
[0530] FIG. 67 shows a nucleotide sequence (SEQ ID NO:184) of a
native sequence PRO239 cDNA, wherein SEQ ID NO:184 is a clone
designated herein as "DNA34407-1169".
[0531] FIG. 68 shows the amino acid sequence (SEQ ID NO:185)
derived from the coding sequence of SEQ ID NO:184 shown in FIG.
67.
[0532] FIG. 69 shows a nucleotide sequence (SEQ ID NO:189) of a
native sequence PRO257 cDNA, wherein SEQ ID NO:189 is a clone
designated herein as "DNA35841-1173".
[0533] FIG. 70 shows the amino acid sequence (SEQ ID NO:190)
derived from the coding sequence of SEQ ID NO:189 shown in FIG.
69.
[0534] FIG. 71 shows a nucleotide sequence (SEQ ID NO:194) of a
native sequence PRO260 cDNA, wherein SEQ ID NO:194 is a clone
designated herein as "DNA33470-1175".
[0535] FIG. 72 shows the amino acid sequence (SEQ ID NO:195)
derived from the coding sequence of SEQ ID NO:194 shown in FIG.
71.
[0536] FIG. 73 shows a nucleotide sequence (SEQ ID NO:200) of a
native sequence PRO263 cDNA, wherein SEQ ID NO:200 is a clone
designated herein as "DNA34431-1177".
[0537] FIG. 74 shows the amino acid sequence (SEQ ID NO:201)
derived from the coding sequence of SEQ ID NO:200 shown in FIG.
73.
[0538] FIG. 75 shows a nucleotide sequence (SEQ ID NO:206) of a
native sequence PRO270 cDNA, wherein SEQ ID NO:206 is a clone
designated herein as "DNA39510-1181".
[0539] FIG. 76 shows the amino acid sequence (SEQ ID NO:207)
derived from the coding sequence of SEQ ID NO:206 shown in FIG.
75.
[0540] FIG. 77 shows a nucleotide sequence (SEQ ID NO:212) of a
native sequence PRO271 cDNA, wherein SEQ ID NO:212 is a clone
designated herein as "DNA39423-1182".
[0541] FIG. 78 shows the amino acid sequence (SEQ ID NO:213)
derived from the coding sequence of SEQ ID NO:212 shown in FIG.
77.
[0542] FIG. 79 shows a nucleotide sequence (SEQ ID NO:220) of a
native sequence PRO272 cDNA, wherein SEQ ID NO:220 is a clone
designated herein as "DNA40620-1183".
[0543] FIG. 80 shows the amino acid sequence (SEQ ID NO:221)
derived from the coding sequence of SEQ ID NO:220 shown in FIG.
79.
[0544] FIG. 81 shows a nucleotide sequence (SEQ ID NO:226) of a
native sequence PRO294 cDNA, wherein SEQ ID NO:226 is a clone
designated herein as "DNA40604-1187".
[0545] FIG. 82 shows the amino acid sequence (SEQ ID NO:227)
derived from the coding sequence of SEQ ID NO:226 shown in FIG.
81.
[0546] FIG. 83 shows a nucleotide sequence (SEQ ID NO:235) of a
native sequence PRO295 cDNA, wherein SEQ ID NO:235 is a clone
designated herein as "DNA38268-1188".
[0547] FIG. 84 shows the amino acid sequence (SEQ ID NO:236)
derived from the coding sequence of SEQ ID NO:235 shown in FIG.
83.
[0548] FIG. 85 shows a nucleotide sequence (SEQ ID NO:244) of a
native sequence PRO293 cDNA, wherein SEQ ID NO:244 is a clone
designated herein as "DNA37151-1193".
[0549] FIG. 86 shows the amino acid sequence (SEQ ID NO:245)
derived from the coding sequence of SEQ ID NO:244 shown in FIG.
85.
[0550] FIG. 87 shows a nucleotide sequence (SEQ ID NO:249) of a
native sequence PRO247 cDNA, wherein SEQ ID NO:249 is a clone
designated herein as "DNA35673-1201".
[0551] FIG. 88 shows the amino acid sequence (SEQ ID NO:250)
derived from the coding sequence of SEQ ID NO:249 shown in FIG.
87.
[0552] FIG. 89 shows a nucleotide sequence (SEQ ID NO:254) of a
native sequence PRO302 cDNA, wherein SEQ ID NO:254 is a clone
designated herein as "DNA40370-1217".
[0553] FIG. 90 shows the amino acid sequence (SEQ ID NO:255)
derived from the coding sequence of SEQ ID NO:254 shown in FIG.
89.
[0554] FIG. 91 shows a nucleotide sequence (SEQ ID NO:256) of a
native sequence PRO303 cDNA, wherein SEQ ID NO:256 is a clone
designated herein as "DNA42551-1217".
[0555] FIG. 92 shows the amino acid sequence (SEQ ID NO:257)
derived from the coding sequence of SEQ ID NO:256 shown in FIG.
91.
[0556] FIG. 93 shows a nucleotide sequence (SEQ ID NO:258) of a
native sequence PRO304 cDNA, wherein SEQ ID NO:258 is a clone
designated herein as "DNA39520-1217".
[0557] FIG. 94 shows the amino acid sequence (SEQ ID NO:259)
derived from the coding sequence of SEQ ID NO:258 shown in FIG.
93.
[0558] FIG. 95 shows a nucleotide sequence (SEQ ID NO:260) of a
native sequence PRO307 cDNA, wherein SEQ ID NO:260 is a clone
designated herein as "DNA41225-1217".
[0559] FIG. 96 shows the amino acid sequence (SEQ ID NO:261)
derived from the coding sequence of SEQ ID NO:260 shown in FIG.
95.
[0560] FIG. 97 shows a nucleotide sequence (SEQ ID NO:262) of a
native sequence PRO343 cDNA, wherein SEQ ID NO:262 is a clone
designated herein as "DNA43318-1217".
[0561] FIG. 98 shows the amino acid sequence (SEQ ID NO:263)
derived from the coding sequence of SEQ ID NO:262 shown in FIG.
97.
[0562] FIG. 99 shows a nucleotide sequence (SEQ ID NO:284) of a
native sequence PRO328 cDNA, wherein SEQ ID NO:284 is a clone
designated herein as "DNA40587-1231".
[0563] FIG. 100 shows the amino acid sequence (SEQ ID NO:285)
derived from the coding sequence of SEQ ID NO:284 shown in FIG.
99.
[0564] FIG. 101 shows a nucleotide sequence (SEQ ID NO:289) of a
native sequence PRO335 cDNA, wherein SEQ ID NO:289 is a clone
designated herein as "DNA41388-1234".
[0565] FIG. 102 shows the amino acid sequence (SEQ ID NO:290)
derived from the coding sequence of SEQ ID NO:289 shown in FIG.
101.
[0566] FIG. 103 shows a nucleotide sequence (SEQ ID NO:291) of a
native sequence PRO331 cDNA, wherein SEQ ID NO:291 is a clone
designated herein as "DNA40981-1234".
[0567] FIG. 104 shows the amino acid sequence (SEQ ID NO:292)
derived from the coding sequence of SEQ ID NO:291 shown in FIG.
103.
[0568] FIG. 105 shows a nucleotide sequence (SEQ ID NO:293) of a
native sequence PRO326 cDNA, wherein SEQ ID NO:293 is a clone
designated herein as "DNA37140-1234".
[0569] FIG. 106 shows the amino acid sequence (SEQ ID NO:294)
derived from the coding sequence of SEQ ID NO:293 shown in FIG.
105.
[0570] FIG. 107 shows a nucleotide sequence (SEQ ID NO:309) of a
native sequence PRO332 cDNA, wherein SEQ ID NO:309 is a clone
designated herein as "DNA40982-1235".
[0571] FIG. 108 shows the amino acid sequence (SEQ ID NO:310)
derived from the coding sequence of SEQ ID NO:309 shown in FIG.
107.
[0572] FIG. 109 shows a nucleotide sequence (SEQ ID NO:314) of a
native sequence PRO334 cDNA, wherein SEQ ID NO:314 is a clone
designated herein as "DNA41379-1236".
[0573] FIG. 110 shows the amino acid sequence (SEQ ID NO:315)
derived from the coding sequence of SEQ ID NO:314 shown in FIG.
109.
[0574] FIG. 111 shows a nucleotide sequence (SEQ ID NO:319) of a
native sequence PRO346 cDNA, wherein SEQ ID NO:319 is a clone
designated herein as "DNA44167-1243".
[0575] FIG. 112 shows the amino acid sequence (SEQ ID NO:320)
derived from the coding sequence of SEQ ID NO:319 shown in FIG.
111.
[0576] FIG. 113 shows a nucleotide sequence (SEQ ID NO:324) of a
native sequence PRO268 cDNA, wherein SEQ ID NO:324 is a clone
designated herein as "DNA39427-1179".
[0577] FIG. 114 shows the amino acid sequence (SEQ ID NO:325)
derived from the coding sequence of SEQ ID NO:324 shown in FIG.
113.
[0578] FIG. 115 shows a nucleotide sequence (SEQ ID NO:331) of a
native sequence PRO330 cDNA, wherein SEQ ID NO:331 is a clone
designated herein as "DNA40603-1232".
[0579] FIG. 116 shows the amino acid sequence (SEQ ID NO:332)
derived from the coding sequence of SEQ ID NO:331 shown in FIG.
115.
[0580] FIG. 117 shows a nucleotide sequence (SEQ ID NO:338) of a
native sequence PRO339 cDNA, wherein SEQ ID NO:338 is a clone
designated herein as "DNA43466-1225".
[0581] FIG. 118 shows the amino acid sequence (SEQ ID NO:339)
derived from the coding sequence of SEQ ID NO:338 shown in FIG.
117.
[0582] FIG. 119 shows a nucleotide sequence (SEQ ID NO:340) of a
native sequence PRO310 cDNA, wherein SEQ ID NO:340 is a clone
designated herein as "DNA43046-1225".
[0583] FIG. 120 shows the amino acid sequence (SEQ ID NO:341)
derived from the coding sequence of SEQ ID NO:340 shown in FIG.
119.
[0584] FIG. 121 shows a nucleotide sequence (SEQ ID NO:376) of a
native sequence PRO244 cDNA, wherein SEQ ID NO:376 is a clone
designated herein as "DNA35668-1171".
[0585] FIG. 122 shows the amino acid sequence (SEQ ID NO:377)
derived from the coding sequence of SEQ ID NO:376 shown in FIG.
121.
[0586] FIG. 123 shows a nucleotide sequence (SEQ ID NO:422) of a
native sequence PRO1868 cDNA, wherein SEQ ID NO:422 is a clone
designated herein as "DNA77624-2515".
[0587] FIG. 124 shows the amino acid sequence (SEQ ID NO:423)
derived from the coding sequence of SEQ ID NO:422 shown in FIG.
123.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0588] I. Definitions
[0589] 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 PRO 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.
[0590] A "native sequence PRO polypeptide" comprises a polypeptide
having the same amino acid sequence as the corresponding PRO
polypeptide derived from nature. Such native sequence PRO
polypeptides can be isolated from nature or can be produced by
recombinant or synthetic means. The term "native sequence PRO
polypeptide" specifically encompasses naturally-occurring truncated
or secreted forms of the specific PRO polypeptide (e.g., an
extracellular domain sequence), naturally-occurring variant forms
(e.g., alternatively spliced forms) and naturally-occurring allelic
variants of the polypeptide. In various embodiments of the
invention, the native sequence PRO polypeptides disclosed herein
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 PRO 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 PRO polypeptides.
[0591] The PRO polypeptide "extracellular domain" or "ECD" refers
to a form of the PRO polypeptide which is essentially free of the
transmembrane and cytoplasmic domains. Ordinarily, a PRO
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 PRO 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 PRO 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 comtemplated by the present
invention.
[0592] The approximate location of the "signal peptides" of the
various PRO 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. 14:46834690 (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.
[0593] "PRO polypeptide variant" means an active PRO polypeptide as
defined above or below having at least about 80% amino acid
sequence identity with a full-length native sequence PRO
polypeptide sequence as disclosed herein, a PRO polypeptide
sequence lacking the signal peptide as disclosed herein, an
extracellular domain of a PRO polypeptide, with or without the
signal peptide, as disclosed herein or any other fragment of a
full-length PRO polypeptide sequence as disclosed herein. Such PRO
polypeptide variants include, for instance, PRO 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 PRO polypeptide variant will have at least about 80%
amino acid sequence identity, preferably at least about 81% amino
acid sequence identity, more preferably at least about 82% amino
acid sequence identity, more preferably at least about 83% amino
acid sequence identity, more preferably at least about 84% amino
acid sequence identity, more preferably at least about 85% amino
acid sequence identity, more preferably at least about 86% amino
acid sequence identity, more preferably at least about 87% amino
acid sequence identity, more preferably at least about 88% amino
acid sequence identity, more preferably at least about 89% amino
acid sequence identity, more preferably at least about 90% amino
acid sequence identity, more preferably at least about 91% amino
acid sequence identity, more preferably at least about 92% amino
acid sequence identity, more preferably at least about 93% amino
acid sequence identity, more preferably at least about 94% amino
acid sequence identity, more preferably at least about 95% amino
acid sequence identity, more preferably at least about 96% amino
acid sequence identity, more preferably at least about 97% amino
acid sequence identity, more preferably at least about 98% amino
acid sequence identity and most preferably at least about 99% amino
acid sequence identity with a full-length native sequence PRO
polypeptide sequence as disclosed herein, a PRO polypeptide
sequence lacking the signal peptide as disclosed herein, an
extracellular domain of a PRO polypeptide, with or without the
signal peptide, as disclosed herein or any other specifically
defined fragment of a full-length PRO polypeptide sequence as
disclosed herein. Ordinarily, PRO variant polypeptides are at least
about 10 amino acids in length, often at least about 20 amino acids
in length, more often at least about 30 amino acids in length, more
often at least about 40 amino acids in length, more often at least
about 50 amino acids in length, more often at least about 60 amino
acids in length, more often at least about 70 amino acids in
length, more often at least about 80 amino acids in length, more
often at least about 90 amino acids in length, more often at least
about 100 amino acids in length, more often at least about 150
amino acids in length, more often at least about 200 amino acids in
length, more often at least about 300 amino acids in length, or
more.
[0594] "Percent (%) amino acid sequence identity" with respect to
the PRO polypeptide sequence s 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 PRO
polypeptide sequence, after aligning the sequences and introducing
gaps, if necessary, t o achieve the maximum percent sequence id
entity, 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.
[0595] 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 {fraction (X/Y)}
[0596] 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.
[0597] 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. However, % amino acid sequence identity values may also be
obtained as described below by using the WU-BLAST-2 computer
program (Altschul et al., Methods in Enzymology 266:460-480
(1996)). Most of the WU-BLAST-2 search parameters are set to the
default values. Those not set to default values, i.e., the
adjustable parameters, are set with the following values: overlap
span=1, overlap fraction=0.125, word threshold (T)=11, and scoring
matrix=BLOSUM62. When WU-BLAST-2 is employed, a % amino acid
sequence identity value is determined by dividing (a) the number of
matching identical amino acid residues between the amino acid
sequence of the PRO polypeptide of interest having a sequence
derived from the native PRO polypeptide and the comparison amino
acid sequence of interest (i.e., the sequence against which the PRO
polypeptide of interest is being compared which may be a PRO
variant polypeptide) as determined by WU-BLAST-2 by (b) the total
number of amino acid residues of the PRO polypeptide of interest.
For example, in the statement "a polypeptide comprising an the
amino acid sequence A which has or having at least 80% amino acid
sequence identity to the amino acid sequence B", the amino acid
sequence A is the comparison amino acid sequence of interest and
the amino acid sequence B is the amino acid sequence of the PRO
polypeptide of interest.
[0598] Percent amino acid sequence identity may also be determined
using the sequence comparison program NCBI-BLAST2 (Altschul et al.,
Nucleic Acids Res. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence
comparison program may be downloaded from
http://www.ncbi.nlm.nih.gov. NCBI-BLAST2 uses several search
parameters, wherein all of those search parameters are set to
default values including, for example, unmask=yes, strand all,
expected occurrences=10, minimum low complexity length=1515,
multi-pass e-value=0.01, constant for multi-pass=25, dropoff for
final gapped alignment=25 and scoring matrix=BLOSUM62.
[0599] In situations where NCBI-BLAST2 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 {fraction (X/Y)}
[0600] where X is the number of amino acid residues scored as
identical matches by the sequence alignment program NCBI-BLAST2 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.
[0601] "PRO variant polynucleotide" or "PRO variant nucleic acid
sequence" means a nucleic acid molecule which encodes an active PRO
polypeptide as defined below and which has at least about 80%
nucleic acid sequence identity with a nucleotide acid sequence
encoding a full-length native sequence PRO polypeptide sequence as
disclosed herein, a full-length native sequence PRO polypeptide
sequence lacking the signal peptide as disclosed herein, an
extracellular domain of a PRO polypeptide, with or without the
signal peptide, as disclosed herein or any other fragment of a
full-length PRO polypeptide sequence as disclosed herein.
Ordinarily, a PRO variant polynucleotide will have at least about
80% nucleic acid sequence identity, more preferably at least about
81% nucleic acid sequence identity, more preferably at least about
82% nucleic acid sequence identity, more preferably at least about
83% nucleic acid sequence identity, more preferably at least about
84% nucleic acid sequence identity, more preferably at least about
85% nucleic acid sequence identity, more preferably at least about
86% nucleic acid sequence identity, more preferably at least about
87% nucleic acid sequence identity, more preferably at least about
88% nucleic acid sequence identity, more preferably at least about
89% nucleic acid sequence identity, more preferably at least about
90% nucleic acid sequence identity, more preferably at least about
91% nucleic acid sequence identity, more preferably at least about
92% nucleic acid sequence identity, more preferably at least about
93% nucleic acid sequence identity, more preferably at least about
94% nucleic acid sequence identity, more preferably at least about
95% nucleic acid sequence identity, more preferably at least about
96% nucleic acid sequence identity, more preferably at least about
97% nucleic acid sequence identity, more preferably at least about
98% nucleic acid sequence identity and yet more preferably at least
about 99% nucleic acid sequence identity with a nucleic acid
sequence encoding a full-length native sequence PRO polypeptide
sequence as disclosed herein, a full-length native sequence PRO
polypeptide sequence lacking the signal peptide as disclosed
herein, an extracellular domain of a PRO polypeptide, with or
without the signal sequence, as disclosed herein or any other
fragment of a full-length PRO polypeptide sequence as disclosed
herein. Variants do not encompass the native nucleotide
sequence.
[0602] Ordinarily, PRO variant polynucleotides are at least about
30 nucleotides in length, often at least about 60 nucleotides in
length, more often at least about 90 nucleotides in length, more
often at least about 120 nucleotides in length, more often at least
about 150 nucleotides in length, more often at least about 180
nucleotides in length, more often at least about 210 nucleotides in
length, more often at least about 240 nucleotides in length, more
often at least about 270 nucleotides in length, more often at least
about 300 nucleotides in length, more often at least about 450
nucleotides in length, more often at least about 600 nucleotides in
length, more often at least about 900 nucleotides in length, or
more.
[0603] "Percent (%) nucleic acid sequence identity" with respect to
PRO-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 PRO 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.
[0604] 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 {fraction (W/Z)}
[0605] 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.
[0606] 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. However, % nucleic acid sequence identity values may also
be obtained as described below by using the WU-BLAST-2 computer
program (Altschul et al., Methods in Enzymology 266:460480 (1996)).
Most of the WU-BLAST-2 search parameters are set to the default
values. Those not set to default values, i.e., the adjustable
parameters, are set with the following values: overlap span=1,
overlap fraction=0.125, word threshold (T)=11, and scoring
matrix=BLOSUM62. When WU-BLAST-2 is employed, a % nucleic acid
sequence identity value is determined by dividing (a) the number of
matching identical nucleotides between the nucleic acid sequence of
the PRO polypeptide-encoding nucleic acid molecule of interest
having a sequence derived from the native sequence PRO
polypeptide-encoding nucleic acid and the comparison nucleic acid
molecule of interest (i.e., the sequence against which the PRO
polypeptide-encoding nucleic acid molecule of interest is being
compared which may be a variant PRO polynucleotide) as determined
by WU-BLAST-2 by (b) the total number of nucleotides of the PRO
polypeptide-encoding nucleic acid molecule of interest. For
example, in the statement "an isolated nucleic acid molecule
comprising a nucleic acid sequence A which has or having at least
80% nucleic acid sequence identity to the nucleic acid sequence B",
the nucleic acid sequence A is the comparison nucleic acid molecule
of interest and the nucleic acid sequence B is the nucleic acid
sequence of the PRO polypeptide-encoding nucleic acid molecule of
interest.
[0607] Percent nucleic acid sequence identity may also be
determined using the sequence comparison program NCBI-BLAST2
(Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)). The
NCBI-BLAST2 sequence comparison program may be downloaded from
http://www.ncbi.nlm.nih.gov. NCBI-BLAST2 uses several search
parameters, wherein all of those search parameters are set to
default values including, for example, unmask=yes, strand=all,
expected occurrences=10, minimum low complexity length=15/5,
multi-pass e-value=0.01, constant for multi-pass=25, dropoff for
final gapped alignment=25 and scoring matrix=BLOSUM62.
[0608] In situations where NCBI-BLAST2 is employed for 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 {fraction (W/Z)}
[0609] where W is the number of nucleotides scored as identical
matches by the sequence alignment program NCBI-BLAST2 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.
[0610] In other embodiments, PRO variant polynucleotides are
nucleic acid molecules that encode an active PRO polypeptide and
which are capable of hybridizing, preferably under stringent
hybridization and wash conditions, to nucleotide sequences encoding
a full-length PRO polypeptide as disclosed herein. PRO variant
polypeptides may be those that are encoded by a PRO variant
polynucleotide.
[0611] The term "positives", in the context of sequence comparison
performed as described above, includes residues in the sequences
compared that are not identical but have similar properties (e.g.
as a result of conservative substitutions, see Table 6 below). For
purposes herein, the % value of positives is determined by dividing
(a) the number of amino acid residues scoring a positive value
between the PRO polypeptide amino acid sequence of interest having
a sequence derived from the native PRO polypeptide sequence and the
comparison amino acid sequence of interest (i.e., the amino acid
sequence against which the PRO polypeptide sequence is being
compared) as determined in the BLOSUM62 matrix of WU-BLAST-2 by (b)
the total number of amino acid residues of the PRO polypeptide of
interest.
[0612] Unless specifically stated otherwise, the % value of
positives is calculated as described in the immediately preceding
paragraph. However, in the context of the amino acid sequence
identity comparisons performed as described for ALIGN-2 and
NCBI-BLAST-2 above, includes amino acid residues in the sequences
compared that are not only identical, but also those that have
similar properties. Amino acid residues that score a positive value
to an amino acid residue of interest are those that are either
identical to the amino acid residue of interest or are a preferred
substitution (as defined in Table 6 below) of the amino acid
residue of interest.
[0613] For amino acid sequence comparisons using ALIGN-2 or
NCBI-BLAST2, the % value of positives 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 % positives to, with, or against
a given amino acid sequence B) is calculated as follows:
100 times the fraction {fraction (X/Y)}
[0614] where X is the number of amino acid residues scoring a
positive value as defined above by the sequence alignment program
ALIGN-2 or NCBI-BLAST2 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 % positives of A
to B will not equal the % positives of B to A.
[0615] "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. In
preferred embodiments, 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 PRO
polypeptide natural environment will not be present. Ordinarily,
however, isolated polypeptide will be prepared by at least one
purification step.
[0616] An "isolated" PRO 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.
[0617] 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.
[0618] 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.
[0619] The term "antibody" is used in the broadest sense and
specifically covers, for example, single anti-PRO monoclonal
antibodies (including agonist, antagonist, and neutralizing
antibodies), anti-PRO antibody compositions with polyepitopic
specificity, single chain anti-PRO antibodies, and fragments of
anti-PRO antibodies (see below). 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.
[0620] "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).
[0621] "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 sodiumphosphate 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.
[0622] "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 trisodiumcitrate), 50
mM sodiumphosphate (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.
[0623] The term "epitope tagged" when used herein refers to a
chimeric polypeptide comprising a PRO 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).
[0624] As used herein, the term "immunoadhesin" designates
antibody-like molecules which combine the binding specificity of a
heterologous protein (an "adhesin") with the effector functions of
immunoglobulin constant domains. Structurally, the immunoadhesins
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 adhesin part of an
immunoadhesin 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
immunoadhesin 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.
[0625] "Active" or "activity" for the purposes herein refers to
form(s) of a PRO polypeptide which retain a biological and/or an
immunological activity of native or naturally-occurring PRO,
wherein "biological" activity refers to a biological function
(either inhibitory or stimulatory) caused by a native or
naturally-occurring PRO other than the ability to induce the
production of an antibody against an antigenic epitope possessed by
a native or naturally-occurring PRO 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 PRO.
[0626] The term "antagonist" is used in the broadest sense, and
includes any molecule that partially or fully blocks, inhibits, or
neutralizes a biological activity of a native PRO polypeptide
disclosed herein. In a similar manner, the term "agonist" is used
in the broadest sense and includes any molecule that mimics a
biological activity of a native PRO 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 PRO polypeptides,
peptides, antisense oligonucleotides, small organic molecules, etc.
Methods for identifying agonists or antagonists of a PRO
polypeptide may comprise contacting a PRO polypeptide with a
candidate agonist or antagonist molecule and measuring a detectable
change in one or more biological activities normally associated
with the PRO polypeptide.
[0627] "Treatment" 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. Those in need of treatment include those already with the
disorder as well as those prone to have the disorder or those in
whom the disorder is to be prevented.
[0628] "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.
[0629] "Intermittent" administration is treatment that is not
consecutively done without interruption, but rather is cyclic in
nature.
[0630] "Mammal" for purposes of treatment refers to any animal
classified as a mammal, including humans, 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.
[0631] Administration "in combination with" one or more further
therapeutic agents includes simultaneous (concurrent) and
consecutive administration in any order.
[0632] "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..
[0633] "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
(Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]); single-chain
antibody molecules; and multispecific antibodies formed from
antibody fragments.
[0634] Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, each with a
single antigen-binding site, and a residual "Fc" fragment, a
designation reflecting the ability to crystallize readily. Pepsin
treatment yields an F(ab').sub.2 fragment that has two
antigen-combining sites and is still capable of cross-linking
antigen.
[0635] "Fv" is the minimum antibody fragment which contains a
complete antigen-recognition and -binding site. This region
consists of a dimer of one heavy- and one light-chain variable
domain in tight, non-covalent association. It is in this
configuration that the three CDRs of each variable domain interact
to define an antigen-binding site on the surface of the
V.sub.H-V.sub.L dimer. Collectively, the six CDRs 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.
The Fab fragment also contains the constant domain of the light
chain and the first constant domain (CH1) of the heavy chain. Fab
fragments differ from Fab' fragments by the addition of a few
residues at the carboxy terminus of the heavy chain CHi 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.
[0636] The "light chains" of antibodies (immunoglobulins) 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.
[0637] Depending on the amino acid sequence of the constant domain
of their heavy chains, immunoglobulins can be assigned to different
classes. There are five major classes of immunoglobulins: IgA, IgD,
IgE, IgG, and IgM, and several of these may be further divided into
subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and
IgA2.
[0638] "Single-chain Fv" or "sFv" antibody fragments comprise the
V.sub.H and V.sub.L domains of antibody, wherein these domains are
present in a single polypeptide chain. Preferably, the Fv
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).
[0639] The term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a heavy-chain
variable domain (V.sub.H) connected to a light-chain variable
domain (V.sub.L) in the same polypeptide chain (V.sub.H-V.sub.L).
By using a linker that is too short to allow pairing between the
two domains on the same chain, the domains are forced to pair with
the complementary domains of another chain and create two
antigen-binding sites. Diabodies are described more fully in, for
example, EP 404,097; WO 93111161; and Hollinger et al., Proc. Natl.
Acad. Sci. USA, 90:6444-6448 (1993).
[0640] An "isolated" antibody is one which has been identified and
separated and/or recovered from a component of its natural
environment. Contaninant 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. In preferred
embodiments, 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.
[0641] 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.
[0642] 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. In certain embodiments, 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.
[0643] 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 PRO 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.
[0644] A "small molecule" is defined herein to have a molecular
weight below about 500 Daltons.
[0645] "PRO317-associated disorder" refers to a pathological
condition or disease wherein PRO317 is over- or underexpressed.
Such disorders include diseases of the female genital tract or of
the endometrium of a mammal, including hyperplasia, endometritis,
endometriosis, wherein the patient is at risk for infertility due
to endometrial factor, endometrioma, and endometrial cancer,
especially those diseases involving abnormal bleeding such as a
gynecological disease. They also include diseases involving
angiogenesis, wherein the angiogenesis results in a pathological
condition, such as cancer involving solid tumors (the therapy for
the disorder would result in decreased vascularization and a
decline in growth and metastasis of a variety of tumors).
Alternatively, the angiogenesis may be beneficial, such as for
ischemia, especially coronary ischemia. Hence, these disorders
include those found in patients whose hearts are functioning but
who have a blocked blood supply due to atherosclerotic coronary
artery disease, and those with a ftmctioning but underperfused
heart, including patients with coronary arterial disease who are
not optimal candidates for angioplasty and coronary artery by-pass
surgery. The disorders also include diseases involving the kidney
or originating from the kidney tissue, such as polycystic kidney
disease and chronic and acute renal failure.
1TABLE 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, 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 */ #deflne PINS0 8 /* penalty for a gap */ #define PINS1 4
/* penalty per residue */ struct jmp { short n[MAXJMP]; /* size
ofjmp (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 ofjmps */ }; 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 {fraction (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.vertline.(1
<<(`D`-`A`)).vertline.(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.vertline.(1<<(`E`-`A`)).vertline.(1<<(`Q`-`A`))
}; main(ac, av) main int ac; char *av[]; { prog = av[0]; if(ac !=
3) { fprintf(stderr,"usage: %s file1 file2.backslash.n", prog);
fprintf(stderr,"where file1 and file2 are two dna or two protein
sequences..backslash.n"); fprintf(stderr,"The sequences can be in
upper- or lower-case.backslash.n"); fprintf(stderr,"Any lines
beginning with `;` or `<` are ignored.backslash.n");
fprintf(stderr,"Output is in the file
.backslash."align.out.backslash.".b- ackslash.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 aligmnent, 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 row0, 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 .vertline. .vertline. 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 .vertline. .vertline. 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 .vertline. .vertline. (ndelx >= MAXJMP && xx >
dx[id].jp.x[ij]+MX) .vertline. .vertline. 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
.vertline. .vertline. (ndely[yy]>= MAXJMP && xx >
dx[id].jp.x[ij]+MX) .vertline. .vertline. 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] =
-ndeiy[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); } Page 4 of nw.c /*
* * 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, [mum], 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.backslash.n", prog, ofile); cleanup(1); } fprintf(fx, "<first
sequence: %s (length = %d).backslash.n", namex[0], len0);
fprintf(fx, "<second sequence: %s (length = %d).backslash.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(); } Page 1 of nwprint.c /* * 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, ".backslash.n");
fprintf(fx, "<%d match%s in an overlap of %d: %.2f percent
similarity.backslash.n", nm, (nm == 1)? "" : "es", lx, pct); Page 2
of nwprint.c 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, ".backslash.n<score: %d (match = %d,
mismatch = %d, gap penalty = %d + %d per base).backslash.n", smax,
DMAT, DMIS, DINS0, DINS1); else fprintf(fx, ".backslash.n<score:
%d (Dayhoff PAM 250 matrix, gap penalty = %d + %d per
residue).backslash.n", smax, PINS0, PINS1); if (endgaps)
fprintf(fx, "<endgaps penalized. left endgap: %d %s%s, right
endgap: %d %s%s.backslash.n", firstgap, (dna)? "base" : "residue",
(firstgap == 1)? "" :"s", lastgap, (dna)? "base" : "residue",
(lastgap == 1)? "" : "s"); else fprintf(fx, "<endgaps not
penalized.backslash.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]; } Page 3
of nwprint.c 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 .vertline. .vertline. !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]- = `.backslash.0`; Page 4 of nwprint.c ...dumpblock
(void) putc(`.backslash.n`, fx); for (i = 0; i < 2; i++) { if
(*out[i] && (*out[i] != ` ` .vertline. .vertline.
*(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 == ` ` .vertline. .vertline. *py == `-`) *pn = ` `; else {
if (i%10 == 0 .vertline. .vertline. (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
= `.backslash.0`; nc[ix] = i; for (pn = nline; *pn; pn++) (void)
putc(*pn, fx); (void) putc(`.backslash.n`, fx); } /* * put out a
line (name, [num], seq. [num]): dumpblock() */ static putline(ix)
putilne int ix; { Page 5 of nwprint.c ...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(`.backslash.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] .vertline. .vertline.
(*out[0] == ` ` && *(po[0]) == ` `) .vertline. .vertline.
!*out[1] .vertline. .vertline. (*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++ = `.backslash.n`; *px =
`.backslash.0`; } Page 6 of nwprint.c /* * 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)); } Page 7 of
nwprint.c /* * 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/flle.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.backslash.n", prog, file); exit(1); } tlen = natgc = 0; while
(fgets(line, 1024, fb)) { if (*line == `;` .vertline. .vertline.
*line == `<`.vertline. .vertline. *line == `>`) continue; for
(px = line; *px != `.backslash.n`;px++) if (isupper(*px) .vertline.
.vertline. islower(*px)) tlen++; } if ((pseq =
malloc((unsigned)(tlen+6))) == 0) { fprintf(stderr,"%s: malloc()
failed to get %d bytes for %s.backslash.n", prog, tlen+6, file);
exit(1); } pseq[0] = pseq[1] = pseq[2] = pseq[3] = `.backslash.0`;
Page 1 of nwsubr.c ...getseq py = pseq + 4; *len = tlen;
rewind(fp); while (fgets(line, 1024, fp)) { if (*line == `;`
.vertline. .vertline. *line == `<` .vertline. .vertline. *line
== `>`) continue; for (px = line; *px != `.backslash.n`; px++) {
if (isupper(*px)) *py++ = *px; else if (islower(*px)) *py++=
toupper(*px); if (index("ATGCU",*(py-1))) natgc++; } } *py++ =
`.backslash.0`; *py = `.backslash.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).backslash.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.backslash.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-)
Page 2 of nwsubr.c ...readjmps if (j < 0 &&
dx[dmax].offset && fj) { (void) lseek(fd, dx[chnax].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.backslash.n", prog); cleanup(I); } 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 .vertline. .vertline. eadgaps)? -siz : MAXCAP; 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 .vertline. .vertline. endgaps)?
siz : MAXGAP; i0++; } } else break; } /* reverse the order ofjmps
*/ 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; } } Page 3 of nwsubr.c /* * 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.backslash.n", prog, jname);
cleanup(1); } if ((fj = fopen(jname, "w")) == 0) { fprintf(stderr,
"%s: can't write %s.backslash.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); }
Page 4 of nwsubr.c
[0646]
2TABLE 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%
[0647]
3TABLE 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%
[0648]
4TABLE 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%
[0649]
5TABLE 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%
[0650] II. Compositions and Methods of the Invention
[0651] A. Full-length PRO Polypeptides
[0652] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO polypeptides. In particular, cDNAs
encoding various PRO 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.
[0653] 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 PRO 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.
[0654] 1. Full-length PRO211 and PRO217 Polypeptides
[0655] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO211 and PRO217. In particular, Applicants
have identified and isolated cDNA encoding PRO211 and PRO217
polypeptides, as disclosed in further detail in the Examples below.
Using BLAST (FastA format) sequence alignment computer programs,
Applicants found that cDNA sequences encoding full-length native
sequence PRO211 and PRO217 have homologies to known proteins having
EGF-like domains. Specifically, the cDNA sequence DNA32292-1131
(FIG. 1, SEQ ID NO:1) has certain identify and a Blast score of 209
with PAC6_RAT and certain identify and a Blast score of 206 with
Fibulin-1, isoform c precursor. The cDNA sequence DNA33094-1131
(FIG. 3, SEQ ID NO:3) has 36% identity and a Blast score of 336
with eastern newt tenascin, and 37% identity and a Blast score of
331 with human tenascin-X precursor. Accordingly, it is presently
believed that PRO211 and PRO217 polypeptides disclosed in the
present application are newly identified members of the EGF-like
family and possesses properties typical of the EGF-like protein
family.
[0656] 2. Full-length PRO230 Polypeptides
[0657] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO230. In particular, Applicants have
identified and isolated cDNA encoding a PRO230 polypeptide, as
disclosed in further detail in the Examples below. Using known
programs such as BLAST and FastA sequence alignment computer
programs, Applicants found that a cDNA sequence encoding
full-length native sequence PRO230 has 48% amino acid identity with
the rabbit tubulointerstitial nephritis antigen precursor.
Accordingly, it is presently believed that PRO230 polypeptide
disclosed in the present application is a newly identified member
of the tubulointerstitial nephritis antigen family and possesses
the ability to be recognized by human autoantibodies in certain
forms of tubulointerstitial nephritis.
[0658] 3. Full-length PRO232 Polypeptides
[0659] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO232. In particular, Applicants have
identified and isolated cDNA encoding a PRO232 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that a
portion of the full-length native sequence PRO232 (shown in FIG. 9
and SEQ ID NO:18) has 35% sequence identity with a stem cell
surface antigen from Gallus gallus. Accordingly, it is presently
believed that the PRO232 polypeptide disclosed in the present
application may be a newly identified stem cell antigen.
[0660] 4. Full-length PRO187 Polypeptides
[0661] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO187. In particular, Applicants have
identified and isolated cDNA encoding a PRO187 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that a
full-length native sequence PRO187 (shown in FIG. 15) has 74% amino
acid sequence identity and BLAST score of 310 with various
androgen-induced growth factors and FGF-8. Accordingly, it is
presently believed that PRO187 polypeptide disclosed in the present
application is a newly identified member of the FGF-8 protein
family and may possess identify activity or property typical of the
FGF-8-like protein family.
[0662] 5. Full-length PRO265 Polypeptides
[0663] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO265. In particular, Applicants have
identified and isolated cDNA encoding a PRO265 polypeptide, as
disclosed in further detail in the Examples below. Using programs
such as BLAST and FastA sequence alignment computer programs,
Applicants found that various portions of the PRO265 polypeptide
have significant homology with the fibromodulin protein and
fibromodulin precursor protein. Applicants have also found that the
DNA encoding the PRO265 polypeptide has significant homology with
platelet glycoprotein V, a member of the leucine rich related
protein family involved in skin and wound repair. Accordingly, it
is presently believed that PRO265 polypeptide disclosed in the
present application is a newly identified member of the leucine
rich repeat family and possesses protein protein binding
capabilities, as well as be involved in skin and wound repair as
typical of this family.
[0664] 6. Full-length PRO219 Polypeptides
[0665] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO219. In particular, Applicants have
identified and isolated cDNA encoding a PRO219 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
various portions of the PRO219 polypeptide have significant
homology with the mouse and human matrilin-2 precursor
polypeptides. Accordingly, it is presently believed that PRO219
polypeptide disclosed in the present application is related to the
matrilin-2 precursor polypeptide.
[0666] 7. Full-length PRO246 Polypeptides
[0667] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO246. In particular, Applicants have
identified and isolated cDNA encoding a PRO246 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that a
portion of the PRO246 polypeptide has significant homology with the
human cell surface protein HCAR. Accordingly, it is presently
believed that PRO246 polypeptide disclosed in the present
application may be a newly identified membrane-bound virus receptor
or tumor cell-specific antigen.
[0668] 8. Full-length PRO228 Polypeptides
[0669] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO228. In particular, Applicants have
identified and isolated cDNA encoding a PRO228 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
various portions of the PRO228 polypeptide have significant
homology with the EMR1 protein. Applicants have also found that the
DNA encoding the PRO228 polypeptide has significant homology with
latrophilin, macrophage-restricted cell surface glycoprotein,
B0457.1 and leucocyte antigen CD97 precursor. Accordingly, it is
presently believed that PRO228 polypeptide disclosed in the present
application is a newly identified member of the seven transmembrane
superfamily and possesses characteristics and functional properties
typical of this family. In particular, it is believed that PRO228
is a new member of the subgroup within this family to which CD97
and EMR1 belong.
[0670] 9. Full-length PRO533 Polypeptides
[0671] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO533. In particular, Applicants have
identified and isolated cDNA encoding a PRO533 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST-2
and FastA sequence alignment computer programs, Applicants found
that a full-length native sequence PRO533 (shown in FIG. 22 and SEQ
ID NO:59) has a Blast score of 509 and 53% amino acid sequence
identity with fibroblast growth factor (FGF). Accordingly, it is
presently believed that PRO533 disclosed in the present application
is a newly identified member of the fibroblast growth factor family
and may possess activity typical of such polypeptides.
[0672] 10. Full-length PRO245 Polypeptides
[0673] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO245. In particular, Applicants have
identified and isolated cDNA encoding a PRO245 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that a
portion of the amino acid sequence of the PRO245 polypeptide has
60% amino acid identity with the human c-myb protein. Accordingly,
it is presently believed that the PRO245 polypeptide disclosed in
the present application may be a newly identified member of the
transmembrane protein tyrosine kinase family.
[0674] 11. Full-length PRO220, PRO221 and PRO227 Polypeptides
[0675] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO220, PRO221 and PRO227. In particular,
Applicants have identified and isolated cDNAs encoding a PRO220,
PRO221 and PRO227 polypeptide, respectively, as disclosed in
further detail in the Examples below. Using BLAST and FastA
sequence alignment computer programs, PRO220 has amino acid
identity with the amino acid sequence of a leucine rich protein
wherein the identity is 87%. PRO220 additionally has amino acid
identity with the neuronal leucine rich protein wherein the
identity is 55%. The neuronal leucine rich protein is further
described in Taguchi, et al., Mol. Brain Res., 35:31-40 (1996).
[0676] PRO221 has amino acid identity with the SLIT protein
precursor, wherein different portions of these two proteins have
the respective percent identities of 39%, 38%, 34%, 31%, and 30%.
PRO227 has amino acid identity with the amino acid sequence of
platelet glycoprotein V precursor. The same results were obtained
for human glycoprotein V. Different portions of these two proteins
show the following percent identities of 30%, 28%, 28%, 31%, 35%,
39% and 27%.
[0677] Accordingly, it is presently believed that PRO220, PRO221
and PRO227 polypeptides disclosed in the present application are
newly identified members of the leucine rich repeat protein
superfamily and that each possesses protein-protein binding
capabilities typical of the leucine rich repeat protein
superfamily. It is also believed that they have capabilities
similar to those of SLIT, the leucine rich repeat protein and human
glycoprotein V.
[0678] 12. Full-length PRO258 Polypeptides
[0679] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO258. In particular, Applicants have
identified and isolated cDNA encoding a PRO258 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
various portions of the PRO258 polypeptide have significant
homology with the CRTAM and poliovirus receptors. Accordingly, it
is presently believed that PRO258 polypeptide disclosed in the
present application is a newly identified member of the Ig
superfamily and possesses virus receptor capabilities or regulates
immune function as typical of this family.
[0680] 13. Full-length PRO266 Polypeptides
[0681] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO266. In particular, Applicants have
identified and isolated cDNA encoding a PRO266 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
various portions of the PRO266 polypeptide have significant
homology with the SLIT protein from Drosophilia. Accordingly, it is
presently believed that PRO266 polypeptide disclosed in the present
application is a newly identified member of the leucine rich repeat
family and possesses ligand-ligand binding activity and neuronal
development typical of this family. SLIT has been shown to be
useful in the study and treatment of Alzheimer's disease, supra,
and thus, PRO266 may have involvement in the study and cure of this
disease.
[0682] 14. Full-length PRO269 Polypeptides
[0683] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO269. In particular, Applicants have
identified and isolated cDNA encoding a PRO269 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST,
FastA and sequence alignment computer programs, Applicants found
that the amino acid sequence encoded by nucleotides 314 to 1783 of
the full-length native sequence PRO269 (shown in FIG. 35 and SEQ ID
NO:95) has significant homology to human urinary thrombomodulin and
various thrombomodulin analogues respectively, to which it was
aligned. Accordingly, it is presently believed that PRO269
polypeptide disclosed in the present application is a newly
identified member of the thrombomodulin family.
[0684] 15. Full-length PRO287 Polypeptides
[0685] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO287. In particular, Applicants have
identified and isolated cDNA encoding a PRO287 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
various portions of the PRO287 polypeptide have significant
homology with the type 1 procollagen C-proteinase enhancer protein
precursor and type 1 procollagen C-proteinase enhancer protein.
Accordingly, it is presently believed that PRO287 polypeptide
disclosed in the present application is a newly identified member
of the C-proteinase enhancer protein family.
[0686] 16. Full-length PRO214 Polypeptides
[0687] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO214. In particular, Applicants have
identified and isolated cDNA encoding a PRO214 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that a
full-length native sequence PRO214 polypeptide (shown in FIG. 40
and SEQ ID NO:109) has 49% amino acid sequence identity with HT
protein, a known member of the EGF-family. The comparison resulted
in a BLAST score of 920, with 150 matching nucleotides.
Accordingly, it is presently believed that the PRO214 polypeptide
disclosed in the present application is a newly identified member
of the family comprising EGF domains and may possess activities or
properties typical of the EGF-domain containing family.
[0688] 17. Full-length PRO317 Polypeptides
[0689] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO317. In particular, cDNA encoding a
PRO317 polypeptide has been identified and isolated, as disclosed
in further detail in the Examples below. Using BLAST.TM. and
FastA.TM. sequence alignment computer programs, it was found that a
full-length native-sequence PRO317 (shown in FIG. 42 and SEQ ID
NO:114) has 92% amino acid sequence identity with EBAF-1. Further,
it is closely aligned with many other members of the
TGF-superfamily.
[0690] Accordingly, it is presently believed that PRO317 disclosed
in the present application is a newly identified member of the
TGF-superfamily and may possess properties that are therapeutically
useful in conditions of uterine bleeding, etc. Hence, PRO317 may be
useful in diagnosing or treating abnormal bleeding involved in
gynecological diseases, for example, to avoid or lessen the need
for a hysterectomy. PRO317 may also be useful as an agent that
affects angiogenesis in general, so PRO317 may be useful in
anti-tumor indications, or conversely, in treating coronary
ischemic conditions.
[0691] Library sources reveal that ESTs used to obtain the
consensus DNA for generating PRO317 primers and probes were found
in normal tissues (uterus, prostate, colon, and pancreas), in
several tumors (colon, brain (twice), pancreas, and mullerian
cell), and in a heart with ischemia. PRO317 has shown up in several
tissues as well, but it does look to have a greater concentration
in uterus. Hence, PRO317 may have a broader use by the body than
EBAF-1. It is contemplated that, at least for some indications,
PRO317 may have opposite effects from EBAF-1.
[0692] 18. Full-length PRO301 Polypeptides
[0693] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO301. In particular, Applicants have
identified and isolated cDNA encoding a PRO301 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that a
full-length native sequence PRO301 (shown in FIG. 44 and SEQ ID
NO:119) has a Blast score of 246 corresponding to 30% amino acid
sequence identity with human A33 antigen precursor. Accordingly, it
is presently believed that PRO301 disclosed in the present
application is a newly identified member of the A33 antigen protein
family and may be expressed in human neoplastic diseases such as
colorectal cancer.
[0694] 19. Full-length PRO224 Polypeptides
[0695] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO224. In particular, Applicants have
identified and isolated cDNA encoding a PRO224 polypeptide, as
disclosed in further detail in the Examples below. Using known
programs such as BLAST and FastA sequence alignment computer
programs, Applicants found that full-length native PRO224 (FIG. 46,
SEQ ID NO:127) has amino acid identity with apolipoprotein E
receptor 2906 from homo sapiens. The alignments of different
portions of these two polypeptides show amino acid identities of
37%, 36%, 30%, 44%, 44% and 28% respectively. Full-length native
PRO224 (FIG. 46, SEQ ID NO:127) also has amino acid identity with
very low-density lipoprotein receptor precursor from gall. The
alignments of different portions of these two polypeptides show
amino acid identities of 38%, 37%, 42%, 33%, and 37% respectively.
Additionally, full-length native PRO224 (FIG. 46, SEQ ID NO:127)
has amino acid identity with the chicken oocyte receptor P95 from
Gallus gallus. The alignments of different portions of these two
polypeptides show amino acid identities of 38%, 37%, 42%, 33%, and
37% respectively. Moreover, full-length native PRO224 (FIG. 46, SEQ
ID NO:127) has amino acid identity with very low density
lipoprotein receptor short form precursor from humans. The
alignments of different portions of these two polypeptides show
amino acid identities of 32%, 38%, 34%, 45%, and 31%, respectively.
Accordingly, it is presently believed that PRO224 polypeptide
disclosed in the present application is a newly identified member
of the low density lipoprotein receptor family and possesses the
structural characteristics required to have the functional ability
to recognize and endocytose low density lipoproteins typical of the
low density lipoprotein receptor family. (The alignments described
above used the following scoring parameters: T=7, S+65, S2=36,
Matrix: BLOSUM62.)
[0696] 20. Full-length PRO222 Polypeptides
[0697] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO222. In particular, Applicants have
identified and isolated cDNA encoding a PRO222 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that a
sequence encoding full-length native sequence PRO222 (shown in FIG.
48 and SEQ ID NO:132) has 25-26% amino acid identity with mouse
complement factor h precursor, has 27-29% amino acid identity with
complement receptor, has 2547% amino acid identity with mouse
complement C3b receptor type 2 long form precursor, has 40% amino
acid identity with human hypothetical protein kiaa0247.
Accordingly, it is presently believed that PRO222 polypeptide
disclosed in the present application is a newly identified member
of the complement receptor family and possesses activity typical of
the complement receptor family.
[0698] 21. Full-length PRO234 Polypeptides
[0699] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO234. In particular, Applicants have
identified and isolated cDNA encoding a PRO234 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST
(FastA-format) sequence alignment computer programs, Applicants
found that a cDNA sequence encoding full-length native sequence
PRO234 has 31% identity and Blast score of 134 with E-selectin
precursor. Accordingly, it is presently believed that the PRO234
polypeptides disclosed in the present application are newly
identified members of the lectin/selectin family and possess
activity typical of the lectin/selectin family.
[0700] 22. Full-length PRO231 Polypeptides
[0701] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO231. In particular, Applicants have
identified and isolated cDNA encoding a PRO231 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
the full-length native sequence PRO231 polypeptide (shown in FIG.
52 and SEQ ID NO:142) has 30% and 31% amino acid identity with
human and rat prostatic acid phosphatase precursor proteins,
respectively. Accordingly, it is presently believed that the PRO231
polypeptide disclosed in the present application may be a newly
identified member of the acid phosphatase protein family.
[0702] 23. Full-length PRO229 Polypeptides
[0703] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO229. In particular, Applicants have
identified and isolated cDNA encoding a PRO229 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
various portions of the PRO229 polypeptide have significant
homology with antigen wc1.1, M130 antigen, T cell surface
glycoprotein CD6 and CD6. It also is related to Sp-alpha.
Accordingly, it is presently believed that PRO229 polypeptide
disclosed in the present application is a newly identified member
of the family containing scavenger receptor homology, a sequence
motif found in a number of proteins involved in immune function and
thus possesses immune function and /or segments which resist
degradation, typical of this family.
[0704] 24. Full-length PRO238 Polypeptides
[0705] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO238. In particular, Applicants have
identified and isolated cDNA encoding a PRO238 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
various portions of the PRO238 polypeptide have significant
homology with reductases, including oxidoreductase and fatty
acyl-CoA reductase. Accordingly, it is presently believed that
PRO238 polypeptide disclosed in the present application is a newly
identified member of the reductase family and possesses reducing
activity typical of the reductase family.
[0706] 25. Full-length PRO233 Polypeptides
[0707] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO233. In particular, Applicants have
identified and isolated cDNA encoding a PRO233 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
various portions of the PRO233 polypeptide have significant
homology with the reductase protein. Applicants have also found
that the DNA encoding the PRO233 polypeptide has significant
homology with proteins from Caenorhabditis elegans. Accordingly, it
is presently believed that PRO233 polypeptide disclosed in the
present application is a newly identified member of the reductase
family and possesses the ability to effect the redox state of the
cell typical of the reductase family.
[0708] 26. Full-length PRO223 Polypeptides
[0709] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO223. In particular, Applicants have
identified and isolated cDNA encoding a PRO223 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
the PRO223 polypeptide has significant homology with various serine
carboxypeptidase polypeptides. Accordingly, it is presently
believed that PRO223 polypeptide disclosed in the present
application is a newly identified serine carboxypeptidase.
[0710] 27. Full-length PRO235 Polypeptides
[0711] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO235. In particular, Applicants have
identified and isolated cDNA encoding a PRO235 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
various portions of the PRO235 polypeptide have significant
homology with the various plexin proteins. Accordingly, it is
presently believed that PRO235 polypeptide disclosed in the present
application is a newly identified member of the plexin family and
possesses cell adhesion properties typical of the plexin
family.
[0712] 28. Full-length PRO236 and PRO262 Polypeptides
[0713] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO236 and PRO262. In particular, Applicants
have identified and isolated cDNA encoding PRO236 and PRO262
polypeptides, as disclosed in further detail in the Examples below.
Using BLAST and FastA sequence alignment computer programs,
Applicants found that various portions of the PRO236 and PRO262
polypeptides have significant homology with various
.beta.-galactosidase and .beta.-galactosidase precursor
polypeptides. Accordingly, it is presently believed that the PRO236
and PRO262 polypeptides disclosed in the present application are
newly identified .beta.-galactosidase homologs.
[0714] 29. Full-length PRO239 Polypeptides
[0715] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO239. In particular, Applicants have
identified and isolated cDNA encoding a PRO239 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
various portions of the PRO239 polypeptide have significant
homology with densin proteins. Accordingly, it is presently
believed that PRO239 polypeptide disclosed in the present
application is a newly identified member of the densin family and
possesses cell adhesion and the ability to effect synaptic
processes as is typical of the densin family.
[0716] 30. Full-length PRO257 Polypeptides
[0717] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO257. In particular, Applicants have
identified and isolated cDNA encoding a PRO257 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
various portions of the PRO257 polypeptide have significant
homology with the ebnerin precursor and ebnerin protein.
Accordingly, it is presently believed that PRO257 polypeptide
disclosed in the present application is a newly identified protein
member which is related to the ebnerin protein.
[0718] 31. Full-length PRO260 Polypeptides
[0719] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO260. In particular, Applicants have
identified and isolated cDNA encoding a PRO260 polypeptide, as
disclosed in further detail in the Examples below. Using programs
such as BLAST and FastA sequence alignment computer programs,
Applicants found that various portions of the PRO260 polypeptide
have significant homology with the alpha-1-fucosidase precursor.
Accordingly, it is presently believed that PRO260 polypeptide
disclosed in the present application is a newly identified member
of the fucosidase family and possesses enzymatic activity related
to fucose residues typical of the fucosidase family.
[0720] 32. Full-length PRO263 Polypeptides
[0721] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO263. In particular, Applicants have
identified and isolated cDNA encoding a PRO263 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
various portions of the PRO263 polypeptide have significant
homology with the CD44 antigen and related proteins. Accordingly,
it is presently believed that PRO263 polypeptide disclosed in the
present application is a newly identified member of the CD44
antigen family and possesses at least one of the properties
associated with these antigens, i.e., cancer and HIV marker,
cell-cell or cell-matrix interactions, regulating cell traffic,
lymph node homing, transmission of growth signals, and presentation
of chemokines and growth facors to traveling cells.
[0722] 33. Full-length PRO270 Polypeptides
[0723] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO270. In particular, Applicants have
identified and isolated cDNA encoding a PRO270 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST,
FastA and sequence alignment computer programs, Applicants found
that that various portions of the PRO270 polypeptide have
significant homology with various thioredoxin proteins.
Accordingly, it is presently believed that PRO270 polypeptide
disclosed in the present application is a newly identified member
of the thioredoxin family and possesses the ability to effect
reduction-oxidation (redox) state typical of the thioredoxin
family.
[0724] 34. Full-length PRO271 Polypeptides
[0725] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO271. In particular, Applicants have
identified and isolated cDNA encoding a PRO271 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
the PRO271 polypeptide has significant homology with various link
proteins and precursors thereof. Accordingly, it is presently
believed that PRO271 polypeptide disclosed in the present
application is a newly identified link protein homolog.
[0726] 35. Full-length PRO272 Polypeptides
[0727] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO272. In particular, Applicants have
identified and isolated cDNA encoding a PRO272 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
various portions of the PRO272 polypeptide have significant
homology with the human reticulocalbin protein and its precursors.
Applicants have also found that the DNA encoding the PRO272
polypeptide has significant homology with the mouse reticulocalbin
precursor protein. Accordingly, it is presently believed that
PRO272 polypeptide disclosed in the present application is a newly
identified member of the reticulocalbin family and possesses the
ability to bind calcium typical of the reticulocalbin family.
[0728] 36. Full-length PRO294 Polypeptides
[0729] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO294. In particular, Applicants have
identified and isolated cDNA encoding a PRO294 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
various portions of the PRO294 polypeptide have significant
homology with the various portions of a number of collagen
proteins. Accordingly, it is presently believed that PRO294
polypeptide disclosed in the present application is a newly
identified member of the collagen family.
[0730] 37. Full-length PRO295 Polypeptides
[0731] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO295. In particular, Applicants have
identified and isolated cDNA encoding a PRO295 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
various portions of the PRO295 polypeptide have significant
homology with integrin proteins. Accordingly, it is presently
believed that PRO295 polypeptide disclosed in the present
application is a newly identified member of the integrin family and
possesses cell adhesion typical of the integrin family.
[0732] 38. Full-length PRO293 Polypeptides
[0733] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO293. In particular, Applicants have
identified and isolated cDNA encoding a PRO293 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
portions of the PRO293 polypeptide have significant homology with
the neuronal leucine rich repeat proteins 1 and 2, (NLRR-1 and
NLRR-2), particularly NLRR-2. Accordingly, it is presently believed
that PRO293 polypeptide disclosed in the present application is a
newly identified member of the neuronal leucine rich repeat protein
family and possesses ligand-ligand binding activity typical of the
NRLL protein family.
[0734] 39. Full-length PRO247 Polypeptides
[0735] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO247. In particular, Applicants have
identified and isolated cDNA encoding a PRO247 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
various portions of the PRO247 polypeptide have significant
homology with densin. Applicants have also found that the DNA
encoding the PRO247 polypeptide has significant homology with a
number of other proteins, including KIAA0231. Accordingly, it is
presently believed that PRO247 polypeptide disclosed in the present
application is a newly identified member of the leucine rich repeat
family and possesses ligand binding abilities typical of this
family.
[0736] 40. Full-length PRO302, PRO303, PRO304, PRO307 and PRO343
Polypeptides
[0737] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO302, PRO303, PRO304, PRO307 and PRO343.
In particular, Applicants have identified and isolated cDNA
encoding PRO302, PRO303, PRO304, PRO307 and PRO343 polypeptides, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
various portions of the PRO302, PRO303, PRO304, PRO307 and PRO343
polypeptides have significant homology with various protease
proteins. Accordingly, it is presently believed that the PRO302,
PRO303, PRO304, PRO307 and PRO343 polypeptides disclosed in the
present application are newly identified protease proteins.
[0738] 41. Full-length PRO328 Polypeptides
[0739] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO328. In particular, Applicants have
identified and isolated cDNA encoding a PRO328 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
various portions of the PRO328 polypeptide have significant
homology with the human glioblastoma protein ("GLIP"). Further,
Applicants found that various portions of the PRO328 polypeptide
have significant homology with the cysteine rich secretory protein
("CRISP") as identified by BLAST homology [ECCRISP3.sub.--1,
S68683, and CRS3_HUMAN]. Accordingly, it is presently believed that
PRO328 polypeptide disclosed in the present application is a newly
identified member of the GLIP or CRISP families and possesses
transcriptional regulatory activity typical of the GLIP or CRISP
families.
[0740] 42. Full-length PRO335, PRO331 and PRO326 Polypeptides
[0741] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO335, PRO331 or PRO326. In particular,
Applicants have identified and isolated cDNA encoding a PRO335,
PRO331 or PRO326 polypeptide, as disclosed in further detail in the
Examples below. Using BLAST and FastA sequence alignment computer
programs, Applicants found that various portions of the PRO335,
PRO331 or PRO326 polypeptide have significant homology with LIG-1,
ALS and in the case of PRO331, additionally, decorin. Accordingly,
it is presently believed that the PRO335, PRO331 and PRO326
polypeptides disclosed in the present application are newly
identified members of the leucine rich repeat superfamily, and
particularly, are related to LIG-1 and possess the biological
functions of this family as discussed and referenced herein.
[0742] 43. Full-length PRO332 Polypeptides
[0743] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO332. In particular, Applicants have
identified and isolated cDNA encoding PRO332 polypeptides, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that a
full-length native sequence PRO332 (shown in FIG. 108 and SEQ ID
NO:310) has about 30-40% amino acid sequence identity with a series
of known proteoglycan sequences, including, for example,
fibromodulin and fibromodulin precursor sequences of various
species (FMOD_BOVIN, FMOD_CHICK, FMOD_RAT, FMOD_MOUSE, FMOD_HUMAN,
P_R36773), osteomodulin sequences (AB000114.sub.--1,
AB007848.sub.--1), decorin sequences (CFU83141.sub.--1,
OCU03394.sub.--1, P R42266, P_R42267, P_R42260, P R89439), keratan
sulfate proteoglycans (BTU48360.sub.--1, AF022890.sub.--1), corneal
proteoglycan (AF022256.sub.--1), and bonelcartilage proteoglycans
and proteoglycane precursors (PGS1_BOVIN, PGS2_MOUSE, PGS2_HUMAN).
Accordingly, it is presently believed that PRO332 disclosed in the
present application is a new proteoglycan-type molecule, and may
play a role in regulating extracellular matrix, cartilage, and/or
bone function.
[0744] 44. Full-length PRO334 Polypeptides
[0745] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO334. In particular, Applicants have
identified and isolated cDNA encoding a PRO334 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
various portions of the PRO334 polypeptide have significant
homology with fibulin and fibrillin. Accordingly, it is presently
believed that PRO334 polypeptide disclosed in the present
application is a newly identified member of the epidermal growth
factor family and possesses properties and activities typical of
this family.
[0746] 45. Full-length PRO346 Polypeptides
[0747] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO346. In particular, Applicants have
identified and isolated cDNA encoding a PRO346 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that a
full-length native sequence PRO346 (shown in FIG. 112 and SEQ ID
NO:320) has 28% amino acid sequence identity with carcinoembryonic
antigen. Accordingly, it is presently believed that PRO346
disclosed in the present application is a newly identified member
of the carcinoembryonic protein family and may be expressed in
association with neoplastic tissue disorders.
[0748] 46. Full-length PRO268 Polypeptides
[0749] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO268. In particular, Applicants have
identified and isolated cDNA encoding a PRO268 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
portions of the PRO268 polypeptide have significant homology with
the various protein disulfide isomerase proteins. Accordingly, it
is presently believed that PRO268 polypeptide disclosed in the
present application is a homolog of the protein disulfide isomerase
p5 protein.
[0750] 47. Full-length PRO330 Polypeptides
[0751] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO330. In particular, Applicants have
identified and isolated cDNA encoding a PRO330 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
various portions of the PRO330 polypeptide have significant
homology with the murine prolyl 4-hydroxylase alpha-II subunit
protein. Accordingly, it is presently believed that PRO330
polypeptide disclosed in the present application is a novel prolyl
4-hydroxylase subunit polypeptide.
[0752] 48. Full-length PRO339 and PRO310 Polypeptides
[0753] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO339 and PRO310. In particular, Applicants
have identified and isolated cDNA encoding a PRO339 polypeptide, as
disclosed in further detail in the Examples below. Applicants have
also identified and isolated cDNA encoding a PRO310 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
various portions of the PRO339 and PRO310 polypeptides have
significant homology with small secreted proteins from C. elegans
and are distantly related to fringe. PRO339 also shows homology to
collagen-like polymers. Sequences which were used to identify
PRO310, designated herein as DNA40533 and DNA42267, also show
homology to proteins from C. elegans. Accordingly, it is presently
believed that the PRO339 and PRO310 polypeptides disclosed in the
present application are newly identified member of the family of
proteins involved in development, and which may have regulatory
abilities similar to the capability of fringe to regulate
serrate.
[0754] 49. Full-length PRO244 Polypeptides
[0755] The present invention provides newly identified and isolated
nucleotide sequences encoding C-type lectins referred to in the
present application as PRO244. In particular, applicants have
identified and isolated cDNA encoding PRO244 polypeptides, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that a
full-length native sequence PRO244 (shown in FIG. 122 and SEQ ID
NO:377) has 43% amino acid sequence identity with the hepatic
lectin gallus gallus (LECH-CHICK), and 42% amino acid sequence
identity with an HIV gp120 binding C-type lectin (A46274).
Accordingly, it is presently believed that PRO244 disclosed in the
present application is a newly identified member of the C-lectin
superfamily and may play a role in immune function, apoptosis, or
in the pathogenesis of atherosclerosis. In addition, PRO244 may be
useful in identifying tumor-associated epitopes.
[0756] B. PRO Polypeptide Variants
[0757] In addition to the full-length native sequence PRO
polypeptides described herein, it is contemplated that PRO variants
can be prepared. PRO variants can be prepared by introducing
appropriate nucleotide changes into the PRO DNA, and/or by
synthesis of the desired PRO polypeptide. Those skilled in the art
will appreciate that amino acid changes may alter
post-translational processes of the PRO, such as changing the
number or position of glycosylation sites or altering the membrane
anchoring characteristics.
[0758] Variations in the native full-length sequence PRO or in
various domains of the PRO 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 PRO that results in a change in the amino acid sequence of the
PRO as compared with the native sequence PRO. 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 PRO. 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 PRO 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.
[0759] PRO 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 PRO
polypeptide.
[0760] PRO fragments may be prepared by any of a number of
conventional techniques. Desired peptide fragments may be
chemically synthesized. An alternative approach involves generating
PRO 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, PRO polypeptide fragments share at least
one biological and/or immunological activity with the native PRO
polypeptide disclosed herein.
[0761] In particular embodiments, 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 introduced and the products
screened.
6TABLE 6 Original Exemplary Preferred Residue Substitutions
Substitutions Ala (A) val; leu; ile val Arg (R) lys; gln; asn lys
Asn (N) gln; his; lys; arg gln Asp (D) glu glu Cys (C) ser ser Gln
(Q) asn asn Glu (E) asp asp Gly (G) pro; ala ala His (H) asn; gln;
lys; arg arg Ile (I) leu; val; met; ala; phe; leu norleucine Leu
(L) norleucine; ile; val; ile met; ala; phe Lys (K) arg; gln; asn
arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyr leu
Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser Trp (W) tyr; phe
tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met; phe; leu
ala; norleucine
[0762] Substantial modifications in function or immunological
identity of the PRO 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:
[0763] (1) hydrophobic: norleucine, met, ala, val, leu, ile;
[0764] (2) neutral hydrophilic: cys, ser, thr;
[0765] (3) acidic: asp, glu;
[0766] (4) basic: asn, gln, his, lys, arg;
[0767] (5) residues that influence chain orientation: gly, pro;
and
[0768] (6) aromatic: trp, tyr, phe.
[0769] 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.
[0770] 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 PRO variant DNA.
[0771] 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.
[0772] C. Modifications of PRO
[0773] Covalent modifications of PRO are included within the scope
of this invention. One type of covalent modification includes
reacting targeted amino acid residues of a PRO polypeptide with an
organic derivatizing agent that is capable of reacting with
selected side chains or the N- or C-terminal residues of the PRO.
Derivatization with bifunctional agents is useful, for instance,
for crosslinking PRO to a water-insoluble support matrix or surface
for use in the method for purifying anti-PRO 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.
[0774] 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.
[0775] Another type of covalent modification of the PRO 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 PRO (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 PRO. 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.
[0776] Addition of glycosylation sites to the PRO 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 PRO
(for 0-linked glycosylation sites). The PRO amino acid sequence may
optionally be altered through changes at the DNA level,
particularly by mutating the DNA encoding the PRO polypeptide at
preselected bases such that codons are generated that will
translate into the desired amino acids.
[0777] Another means of increasing the number of carbohydrate
moieties on the PRO 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 Sep. 11, 1987,
and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306
(1981).
[0778] Removal of carbohydrate moieties present on the PRO
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).
[0779] Another type of covalent modification of PRO comprises
linking the PRO 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. Nos.
4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or
4,179,337.
[0780] The PRO of the present invention may also be modified in a
way to form a chimeric molecule comprising PRO fused to another,
heterologous polypeptide or amino acid sequence.
[0781] In one embodiment, such a chimeric molecule comprises a
fusion of the PRO 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 PRO.
The presence of such epitope-tagged forms of the PRO can be
detected using an antibody against the tag polypeptide. Also,
provision of the epitope tag enables the PRO 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)].
[0782] In an alternative embodiment, the chimeric molecule may
comprise a fusion of the PRO 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 PRO polypeptide in place
of at least one variable region within an Ig molecule. In a
particularly preferred embodiment, 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.
[0783] D. Preparation of PRO
[0784] The description below relates primarily to production of PRO
by culturing cells transformed or transfected with a vector
containing PRO nucleic acid. It is, of course, contemplated that
alternative methods, which are well known in the art, may be
employed to prepare PRO. For instance, the PRO 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
PRO may be chemically synthesized separately and combined using
chemical or enzymatic methods to produce the full-length PRO.
[0785] 1. Isolation of DNA Encoding PRO
[0786] DNA encoding PRO may be obtained from a cDNA library
prepared from tissue believed to possess the PRO mRNA and to
express it at a detectable level. Accordingly, human PRO DNA can be
conveniently obtained from a cDNA library prepared from human
tissue, such as described in the Examples. The PRO-encoding gene
may also be obtained from a genomic library or by known synthetic
procedures (e.g., automated nucleic acid synthesis).
[0787] Libraries can be screened with probes (such as antibodies to
the PRO 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 Laboratorv Manual (New York:
Cold Spring Harbor Laboratory Press, 1989). An alternative means to
isolate the gene encoding PRO is to use PCR methodology [Sambrook
et al., supra; Dieffenbach et al., PCR Primer: A Laboratory Manual
(Cold Spring Harbor Laboratory Press, 1995)].
[0788] 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.
[0789] 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.
[0790] 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.
[0791] 2. Selection and Transformation of Host Cells
[0792] Host cells are transfected or transformed with expression or
cloning vectors described herein for PRO 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.
[0793] 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.
Formammalian cells without such cell walls, the calcium phosphate
precipitation method of Graham and van der Eb, Virology, 52:456457
(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).
[0794] 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 K5772 (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 Apr. 12, 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 miinmal 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 ptr3phoA E15 (argF-lac)169 degP ompTkan.sup.r; 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 disclosedin U.S. Pat. No. 4,946,783 issued
Aug. 7, 1990. Alternatively, in vitro methods of cloning, e.g., PCR
or other nucleic acid polymerase reactions, are suitable.
[0795] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for PRO-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 May 2, 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., 737 [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; Trichoderna reesia (EP 244,234); Neurospora crassa (Case
et al., Proc. Natl. Acad. Sci. USA, 76:5259-5263 [1979]);
Schwanniomyces such as Schwannionyces occidentalis (EP 394,538
published Oct. 31, 1990); and filamentous fungi such as, e.g.,
Neurospora, Penicillium, Tolypocladium (WO 91/00357 published Jan.
10, 1991), and Aspergillus hosts such as A. nidulans (3allance 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).
[0796] Suitable host cells for the expression of glycosylated PRO
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.
[0797] 3. Selection and Use of a Replicable Vector
[0798] The nucleic acid (e.g., cDNA or genomic DNA) encoding PRO
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.
[0799] The PRO 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 PRO-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 Apr. 4, 1990), or the signal described in WO 90/13646
published Nov. 15, 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.
[0800] 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 Grain-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.
[0801] 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.
[0802] An example of suitable selectable markers for mammalian
cells are those that enable the identification of cells competent
to take up the PRO-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)].
[0803] Expression and cloning vectors usually contain a promoter
operably linked to the PRO-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 PRO.
[0804] 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, hexolinase, pyruvate
decarboxylase, phosphofructokinase, glucose-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate
isomerase, phosphoglucose isomerase, and glucokinase.
[0805] 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.
[0806] PRO 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 Jul. 5, 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.
[0807] Transcription of a DNA encoding the PRO 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 PRO coding sequence, but
is preferably located at a site 5' from the promoter.
[0808] 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 PRO.
[0809] Still other methods, vectors, and host cells suitable for
adaptation to the synthesis of PRO 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.
[0810] 4. Detecting Gene Amplification/Expression
[0811] 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.
[0812] Gene expression, alternatively, may be measured by
iununological 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 PRO polypeptide or against a synthetic peptide
based on the DNA sequences provided herein or against exogenous
sequence fused to PRO DNA and encoding a specific antibody
epitope.
[0813] 5. Purification of Polypeptide
[0814] Forms of PRO 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 PRO can be
disrupted by various physical or chemical means, such as
freeze-thaw cycling, sonication, mechanical disruption, or cell
lysing agents.
[0815] It may be desired to purify PRO 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 PRO. 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 PRO
produced.
[0816] E. Uses for PRO
[0817] Nucleotide sequences (or their complement) encoding PRO 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. PRO nucleic acid will
also be useful for the preparation of PRO polypeptides by the
recombinant techniques described herein.
[0818] The full-length native sequence PRO gene, or portions
thereof, may be used as hybridization probes for a cDNA library to
isolate the full-length PRO cDNA or to isolate still other cDNAs
(for instance, those encoding naturally-occurring variants of PRO
or PRO from other species) which have a desired sequence identity
to the native PRO 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 PRO. By way of example, a screening method will comprise
isolating the coding region of the PRO 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 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 PRO 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.
[0819] Any EST sequences disclosed in the present application may
similarly be employed as probes, using the methods disclosed
herein.
[0820] Other useful fragments of the PRO nucleic acids include
antisense or sense oligonucleotides comprising a singe-stranded
nucleic acid sequence (either RNA or DNA) capable of binding to
target PRO mRNA (sense) or PRO DNA (antisense) sequences. Antisense
or sense oligonucleotides, according to the present invention,
comprise a fragment of the coding region of PRO 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).
[0821] 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 PRO proteins. 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.
[0822] 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.
[0823] 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).
[0824] 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.
[0825] 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.
[0826] Antisense 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.
[0827] The probes may also be employed in PCR techniques to
generate a pool of sequences for identification of closely related
PRO coding sequences.
[0828] Nucleotide sequences encoding a PRO can also be used to
construct hybridization probes for mapping the gene which encodes
that PRO 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.
[0829] When the coding sequences for PRO encode a protein which
binds to another protein (example, where the PRO is a receptor),
the PRO 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 PRO 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 PRO or a receptor for PRO. 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.
[0830] Nucleic acids which encode PRO or its modified forms can
also be used to generate either transgenic animals or "knock outs
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. In one embodiment, cDNA encoding PRO
can be used to clone genomic DNA encoding PRO in accordance with
established techniques and the genomic sequences used to generate
transgenic animals that contain cells which express DNA encoding
PRO. Methods for generating transgenic animals, particularly
animals such as mice or rats, have become conventional in the art
and are described, for example, in U.S. Pat. Nos. 4,736,866 and
4,870,009. Typically, particular cells would be targeted for PRO
transgene incorporation with tissue-specific enhancers. Transgenic
animals that include a copy of a transgene encoding PRO 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 PRO.
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.
[0831] Alternatively, non-human homologues of PRO can be used to
construct a PRO "knock out" animal which has a defective or altered
gene encoding PRO as a result of homologous recombination between
the endogenous gene encoding PRO and altered genomic DNA encoding
PRO introduced into an embryonic stem cell of the animal. For
example, cDNA encoding PRO can be used to clone genomic DNA
encoding PRO in accordance with established techniques. A portion
of the genomic DNA encoding PRO 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 PRO polypeptide.
[0832] Nucleic acid encoding the PRO 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 83:41434146 [1986]). The
oligonucleotides can be modified to enhance their uptake, e.g. by
substituting their negatively charged phosphodiester groups by
uncharged groups.
[0833] 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, 44294432
(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).
[0834] The PRO 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 The nucleic acid molecules
encoding the PRO 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 PRO nucleic acid
molecule of the present invention can be used as a chromosome
marker.
[0835] The PRO polypeptides and nucleic acid molecules of the
present invention may also be used for tissue typing, wherein the
PRO polypeptides of the present invention may be differentially
expressed in one tissue as compared to another. PRO nucleic acid
molecules will find use for generating probes for PCR, Northern
analysis, Southern analysis and Western analysis.
[0836] The PRO polypeptides described herein may also be employed
as therapeutic agents. The PRO polypeptides of the present
invention can be formulated according to known methods to prepare
pharmaceutically useful compositions, whereby the PRO 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.
[0837] 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.
[0838] 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.
[0839] 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.
[0840] 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.
[0841] When in vivo administration of a PRO 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. Nos. 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.
[0842] Where sustained-release administration of a PRO polypeptide
is desired in a formulation with release characteristics suitable
for the treatment of any disease or disorder requiring
administration of the PRO polypeptide, microencapsulation of the
PRO 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/Technologs
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.
[0843] 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. 141.
[0844] This invention encompasses methods of screening compounds to
identify those that mimic the PRO polypeptide (agonists) or prevent
the effect of the PRO polypeptide (antagonists). Screening assays
for antagonist drug candidates are designed to identify compounds
that bind or complex with the PRO polypeptides encoded by the genes
identified herein, or otherwise interfere with the interaction of
the encoded polypeptides 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.
[0845] 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.
[0846] All assays for antagonists are common in that they call for
contacting the drug candidate with a PRO polypeptide encoded by a
nucleic acid identified herein under conditions and for a time
sufficient to allow these two components to interact.
[0847] In binding assays, the interaction is binding and the
complex formed can be isolated or detected in the reaction mixture.
In a particular embodiment, the PRO 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 PRO polypeptide
and drying. Alternatively, an immobilized antibody, e.g., a
monoclonal antibody, specific for the PRO 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.
[0848] If the candidate compound interacts with but does not bind
to a particular PRO 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 GALI-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.
[0849] Compounds that interfere with the interaction of a gene
encoding a PRO 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.
[0850] To assay for antagonists, the PRO 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 PRO polypeptide indicates that the
compound is an antagonist to the PRO polypeptide. Alternatively,
antagonists may be detected by combining the PRO polypeptide and a
potential antagonist with membrane-bound PRO polypeptide receptors
or recombinant receptors under appropriate conditions for a
competitive inhibition assay. The PRO polypeptide can be labeled,
such as by radioactivity, such that the number of PRO 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 PRO
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 PRO polypeptide. Transfected cells that are
grown on glass slides are exposed to labeled PRO polypeptide. The
PRO 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.
[0851] As an alternative approach for receptor identification,
labeled PRO 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.
[0852] In another assay for antagonists, mammalian cells or a
membrane preparation expressing the receptor would be incubated
with labeled PRO polypeptide in the presence of the candidate
compound. The ability of the compound to enhance or block this
interaction could then be measured.
[0853] More specific examples of potential antagonists include an
oligonucleotide that binds to the fusions of immunoglobulin with
PRO 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 PRO polypeptide that recognizes the receptor but
imparts no effect, thereby competitively inhibiting the action of
the PRO polypeptide.
[0854] Another potential PRO 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 PRO 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 PRO polypeptide. The antisense RNA
oligonucleotide hybridizes to the mRNA in vivo and blocks
translation of the mRNA molecule into the PRO 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 PRO 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.
[0855] 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 PRO polypeptide, thereby
blocking the normal biological activity of the PRO 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.
[0856] 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, 4:469471 (1994),
and PCT publication No. WO 97/33551 (published Sep. 18, 1997).
[0857] 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.
[0858] 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.
[0859] With regard to the PRO211 and PRO217 polypeptide,
therapeutic indications include disorders associated with the
preservation and maintenance of gastrointestinal mucosa and the
repair of acute and chronic mucosal lesions (e.g., enterocolitis,
Zollinger-Ellison syndrome, gastrointestinal ulceration and
congenital microvillus atrophy), skin diseases associated with
abnormal keratinocyte differentiation (e.g., psoriasis, epithelial
cancers such as lung squamous cell carcinoma, epidermoid carcinoma
of the vulva and gliomas.
[0860] Since the PRO232 polypeptide and nucleic acid encoding it
possess sequence homology to a cell surface stem cell antigen and
its encoding nucleic acid, probes based upon the PRO232 nucleotide
sequence may be employed to identify other novel stem cell surface
antigen proteins. Soluble forms of the PRO232 polypeptide may be
employed as antagonists of membrane bound PRO232 activity both in
vitro and in vivo. PRO232 polypeptides may be employed in screening
assays designed to identify agonists or antagonists of the native
PRO232 polypeptide, wherein such assays may take the form of any
conventional cell-type or biochemical binding assay. Moreover, the
PRO232 polypeptide may serve as a molecular marker for the tissues
in which the polypeptide is specifically expressed.
[0861] With regard to the PRO187 polypeptides disclosed herein,
FGF-8 has been implicated in cellular differentiation and
embryogenesis, including the patterning which appears during limb
formation. FGF-8 and the PRO187 molecules of the invention
therefore are likely to have potent effects on cell growth and
development. Diseases which relate to cellular growth and
differentiation are therefore suitable targets for therapeutics
based on functionality similar to FGF-8. For example, diseases
related to growth or survival of nerve cells including Parkinson's
disease, Alzheimer's disease, ALS, neuropathies. Additionally,
disease related to uncontrolled cell growth, e.g., cancer, would
also be expected therapeutic targets.
[0862] With regard to the PRO265 polypeptides disclosed herein,
other methods for use with PRO265 are described in U.S. Pat. No.
5,654,270 to Ruoslahti et al. In particular, PRO265 can be used in
comparison with the fibromodulin disclosed therein to compare its
effects on reducing dermal scarring and other properties of the
fibromodulin described therein including where it is located and
with what it binds and does not.
[0863] The PRO219 polypeptides of the present invention which play
a regulatory role in the blood coagulation cascade may be employed
in vivo for therapeutic purposes as well as for in vitro purposes.
Those of ordinary skill in the art will well know how to employ
PRO219 polypeptides for such uses.
[0864] The PRO246 polypeptides of the present invention which serve
as cell surface receptors for one or more viruses will find other
uses. For example, extracellular domains derived from these PRO246
polypeptides may be employed therapeutically in vivo for lessening
the effects of viral infection. Those PRO246 polypeptides which
serves as tumor specific antigens may be exploited as therapeutic
targets for anti-tumor drugs, and the like. Those of ordinary skill
in the art will well know how to employ PRO246 polypeptides for
such uses.
[0865] Assays in which connective growth factor and other growth
factors are usually used should be performed with PRO261. An assay
to determine whether TGF beta induces PRO261, indicating a role in
cancer is performed as known in the art. Wound repair and tissue
growth assays are also performed with PRO261. The results are
applied accordingly.
[0866] PRO228 polypeptides should be used in assays in which EMR1,
CD97 and latrophilin would be used in to determine their relative
activities. The results can be applied accordingly. For example, a
competitive binding assay with PRO228 and CD97 can be performed
with the ligand for CD97, CD55.
[0867] Native PRO533 is a 216 amino acid polypeptide of which
residues 1-22 are the signal sequence. Residues 3 to 216 have a
Blast score of 509, corresponding to 53% homology to fibroblast
growth factor. At the nucleotide level, DNA47412, the EST from
which PCR oligos were generated to isolate the full length
DNA49435-1219, has been observed to map to 11p15. Sequence homology
to the 11p15 locus would indicate that PRO533 may have utility in
the treatment of Usher Syndrome or Atrophia areata.
[0868] As mentioned previously, fibroblast growth factors can act
upon cells in both a mitogenic and non-mitogenic manner. These
factors are mitogenic for a wide variety of normal diploid
mesoderm-derived and neural crest-derived cells, inducing granulosa
cells, adrenal cortical cells, chrondrocytes, myoblasts, corneal
and vascular endothelial cells (bovine or human), vascular smooth
muscle cells, lens, retina and prostatic epithelial cells,
oligodendrocytes, astrocytes, chrondocytes, myoblasts and
osteoblasts.
[0869] Non-mitogenic actions of fibroblast growth factors include
promotion of cell migration into a wound area (chemotaxis),
initiation of new blood vessel formulation (angiogenesis),
modulation of nerve regeneration and survival (neurotrophism),
modulation of endocrine functions, and stimulation or suppression
of specific cellular protein expression, extracellular matrix
production and cell survival. Baird, A. & Bohlen, P., Handbook
of Exp. Phrmacol. 95(1): 369-418 (1990). These properties provide a
basis for using fibroblast growth factors in therapeutic approaches
to accelerate wound healing, nerve repair, collateral blood vessel
formation, and the like. For example, fibroblast growth factors,
have been suggested to minimize myocardium damage in heart disease
and surgery (U.S. Pat. No. 4,378,437).
[0870] Since the PRO245 polypeptide and nucleic acid encoding it
possess sequence homology to a transmembrane protein tyrosine
kinase protein and its encoding nucleic acid, probes based upon the
PRO245 nucleotide sequence may be employed to identify other novel
transmembrane tyrosine kinase proteins. Soluble forms of the PRO245
polypeptide may be employed as antagonists of membrane bound PRO245
activity both in vitro and in vivo. PRO245 polypeptides may be
employed in screening assays designed to identify agonists or
antagonists of the native PRO245 polypeptide, wherein such assays
may take the form of any conventional cell-type or biochemical
binding assay. Moreover, the PRO245 polypeptide may serve as a
molecular marker for the tissues in which the polypeptide is
specifically expressed.
[0871] PRO220, PRO221 and PRO227 all have leucine rich repeats.
Additionally, PRO220 and PRO221 have homology to SLIT and leucine
rich repeat protein. Therefore, these proteins are useful in assays
described in the literature, supra, wherein the SLIT and leucine
rich repeat protein are used. Regarding the SLIT protein, PRO227
can be used in an assay to determine the affect of PRO227 on
neurodegenerative disease. Additionally, PRO227 has homology to
human glycoprotein V. In the case of PRO227, this polypeptide is
used in an assay to determine its affect on bleeding, clotting,
tissue repair and scarring.
[0872] The PRO266 polypeptide can be used in assays to determine if
it has a role in neurodegenerative diseases or their reversal.
[0873] PRO269 polypeptides and portions thereof which effect the
activity of thrombin may also be useful for in vivo therapeutic
purposes, as well as for various in vitro applications. In
addition, PRO269 polypeptides and portions thereof may have
therapeutic use as an antithrombotic agent with reduced risk for
hemorrhage as compared with heparin. Peptides having homology to
thrombomodulin are particularly desirable.
[0874] PRO287 polypeptides and portions thereof which effect the
activity of bone morphogenic protein "BMP1"/procollagen
C-proteinase (PCP) may also be useful for in vivo therapeutic
purposes, as well as for various in vitro applications. In
addition, PRO287 polypeptides and portions thereof may have
therapeutic applications in wound healing and tissue repair.
Peptides having homology to procollagen C-proteinase enhancer
protein and its precursor may also be used to induce bone and/or
cartilage formation and are therefore of particular interest to the
scientific and medical communities.
[0875] Therapeutic indications for PRO214 polypeptides include
disorders associated with the preservation and maintenance of
gastrointestinal mucosa and the repair of acute and chronic mucosal
lesions (e.g., enterocolitis, Zollinger-Ellison syndrome,
gastrointestinal ulceration and congenital microvillus atrophy),
skin diseases associated with abnormal keratinocyte differentiation
(e.g., psoriasis, epithelial cancers such as lung squamous cell
carcinoma, epidermoid carcinoma of the vulva and gliomas.
[0876] Studies on the generation and analysis of mice deficient in
members of the TGF-superfamily are reported in Matzuk, Trends in
Endocrinol. and Metabol., 6: 120-127 (1995).
[0877] The PRO317 polypeptide, as well as PRO317-specific
antibodies, inhibitors, agonists, receptors, or their analogs,
herein are useful in treating PRO317-associated disorders. Hence,
for example, they may be employed in modulating endometrial
bleeding angiogenesis, and may also have an effect on kidney
tissue. Endometrial bleeding can occur in gynecological diseases
such as endometrial cancer as abnormal bleeding. Thus, the
compositions herein may find use in diagnosing and treating
abnormal bleeding conditions in the endometrium, as by reducing or
eliminating the need for a hysterectomy. The molecules herein may
also find use in angiogenesis applications such as anti-tumor
indications for which the antibody against vascular endothelial
growth factor is used, or, conversely, ischemic indications for
which vascular endothelial growth factor is employed.
[0878] Bioactive compositions comprising PRO317 or agonists or
antagonists thereof may be administered in a suitable therapeutic
dose determined by any of several methodologies including clinical
studies on mammalian species to determine maximal tolerable dose
and on normal human subjects to determine safe dose. Additionally,
the bioactive agent may be complexed with a variety of well
established compounds or compositions which enhance stability or
pharmacological properties such as half-life. It is contemplated
that the therapeutic, bioactive composition may be delivered by
intravenous infusion into the bloodstream or any other effective
means which could be used for treating problems of the kidney,
uterus, endometrium, blood vessels, or related tissue, e.g., in the
heart or genital tract.
[0879] Dosages and administration of PRO317, PRO317 agonist, or
PRO317 antagonist in a pharmaceutical composition may be determined
by one of ordinary skill in the art of clinical pharmacology or
pharmacokinetics. See, for example, Mordenti and Rescigno,
Pharmaceutical Research, 9:17-25 (1992); Morenti et al.,
Pharmaceutical Research, 8:1351-1359 (1991); and Mordenti and
Chappell, "The use of interspecies scaling in toxicokinetics" in
Toxicokinetics and New Drug Development, Yacobi et al. (eds)
(Pergamon Press: NY, 1989), pp. 42-96. An effective amount of
PRO317, PRO317 agonist, or PRO317 antagonist to be employed
therapeutically will depend, for example, upon the therapeutic
objectives, the route of administration, and the condition of the
mammal. Accordingly, it will be necessary for the therapist to
titer the dosage and modify the route of administration as required
to obtain the optimal therapeutic effect. A typical daily dosage
might range from about 10 ng/kg to up to 100 mg/kg of the mammal's
body weight or more per day, preferably about 1 .mu.g/kg/day to 10
mg/kg/day. Typically, the clinician will administer PRO317, PRO317
agonist, or PRO317 antagonist, until a dosage is reached that
achieves the desired effect for treatment of the above mentioned
disorders.
[0880] PRO317 or an PRO317 agonist or PRO317 antagonist may be
administered alone or in combination with another to achieve the
desired pharmacological effect. PRO317 itself, or agonists or
antagonists of PRO317 can provide different effects when
administered therapeutically. Such compounds for treatment will be
formulated in a nontoxic, inert, pharmaceutically acceptable
aqueous carrier medium preferably at a pH of about 5 to 8, more
preferably 6 to 8, although the pH may vary according to the
characteristics of the PRO317, agonist, or antagonist being
formulated and the condition to be treated. Characteristics of the
treatment compounds include solubility of the molecule, half-life,
and antigenicity/immunogenicity; these and other characteristics
may aid in defining an effective carrier.
[0881] PRO317 or PRO317 agonists or PRO317 antagonists may be
delivered by known routes of administration including but not
limited to topical creams and gels; transmucosal spray and aerosol,
transdermal patch and bandage; injectable, intravenous, and lavage
formulations; and orally administered liquids and pills,
particularly formulated to resist stomach acid and enzymes. The
particular formulation, exact dosage, and route of administration
will be determined by the attending physician and will vary
according to each specific situation.
[0882] Such determinations of administration are made by
considering multiple variables such as the condition to be treated,
the type of mammal to be treated, the compound to be administered,
and the pharmacokinetic profile of the particular treatment
compound. Additional factors which may be taken into account
include disease state (e.g. severity) of the patient, age, weight,
gender, diet, time of administration, drug combination, reaction
sensitivities, and tolerance/response to therapy. Long-acting
treatment compound formulations (such as liposomally encapsulated
PRO317 or PEGylated PRO317 or PRO317 polymeric microspheres, such
as polylactic acid-based microspheres) might be administered every
3 to 4 days, every week, or once every two weeks depending on
half-life and clearance rate of the particular treatment
compound.
[0883] 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.
Nos. 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
the uterus, for example, may necessitate delivery in a manner
different from that to another organ or tissue, such as cardiac
tissue.
[0884] Where sustained-release administration of PRO317 is desired
in a formulation with release characteristics suitable for the
treatment of any disease or disorder requiring administration of
PRO317, microencapsulation of PRO317 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. 439462; WO 97/03692, WO 96/40072, WO
96/07399; and U.S Pat. No. 5,654,010.
[0885] It is contemplated that conditions or diseases of the
uterus, endometrial tissue, or other genital tissues or cardiac
tissues may precipitate damage that is treatable with PRO317 or
PRO317 agonist where PRO317 expression is reduced in the diseased
state; or with antibodies to PRO317 or other PRO317 antagonists
where the expression of PRO317 is increased in the diseased state.
These conditions or diseases may be specifically diagnosed by the
probing tests discussed above for physiologic and pathologic
problems which affect the function of the organ.
[0886] The PRO317, PRO317 agonist, or PRO317 antagonist may be
administered to a mammal with another biologically active agent,
either separately or in the same formulation to treat a common
indication for which they are appropriate. For example, it is
contemplated that PRO317 can be administered together with EBAF-1
for those indications on which they demonstrate the same
qualitative biological effects. Alternatively, where they have
opposite effects, EBAF-1 may be administered together with an
antagonist to PRO317, such as an anti-PRO317 antibody. Further,
PRO317 may be administered together with VEGF for coronary ischemia
where such indication is warranted, or with an anti-VEGF for cancer
as warranted, or, conversely, an antagonist to PRO317 may be
administered with VEGF for coronary ischemia or with anti-VEGF to
treat cancer as warranted. These administrations would be in
effective amounts for treating such disorders.
[0887] Native PRO301 (SEQ ID NO:119) has a Blast score of 246 and
30% homology at residues 24 to 282 of FIG. 44 with A33_HUMAN, an
A33 antigen precursor. A33 antigen precursor, as explained in the
Background is a tumor-specific antigen, and as such, is a
recognized marker and therapeutic target for the diagnosis and
treatment of colon cancer. The expression of tumor-specific
antigens is often associated with the progression of neoplastic
tissue disorders. Native PRO301 (SEQ ID NO:119) and A33_HUMAN also
show a Blast score of 245 and 30% homology at residues 21 to 282 of
FIG. 44 with A33_HUMAN, the variation dependent upon how spaces are
inserted into the compared sequences. Native PRO301 (SEQ ID NO:119)
also has a Blast score of 165 and 29% homology at residues 60 to
255 of FIG. 44 with HS46KDA.sub.--1, a human coxsackie and
adenovirus receptor protein, also known as cell surface protein
HCAR. This region of PRO301 also shows a similar Blast score and
homology with HSU90716.sub.--1. Expression of such proteins is
usually associated with viral infection and therapeutics for the
prevention of such infection may be accordingly conceived. As
mentioned in the Background, the expression of viral receptors is
often associated with neoplastic tumors.
[0888] Therapeutic uses for the PRO234 polypeptides of the
invention includes treatments associated with leukocyte homing or
the interaction between leukocytes and the endothelium during an
inflammatory response. Examples include asthma, rheumatoid
arthritis, psoriasis and multiple sclerosis.
[0889] Since the PRO231 polypeptide and nucleic acid encoding it
possess sequence homology to a putative acid phosphatase and its
encoding nucleic acid, probes based upon the PRO231 nucleotide
sequence may be employed to identify other novel phosphatase
proteins. Soluble forms of the PRO231 polypeptide may be employed
as antagonists of membrane bound PRO231 activity both in vitro and
in vivo. PRO231 polypeptides may be employed in screening assays
designed to identify agonists or antagonists of the native PRO231
polypeptide, wherein such assays may take the form of any
conventional cell-type or biochemical binding assay. Moreover, the
PRO231 polypeptide may serve as a molecular marker for the tissues
in which the polypeptide is specifically expressed.
[0890] PRO229 polypeptides can be fused with peptides of interest
to determine whether the fusion peptide has an increased half-life
over the peptide of interest. The PRO229 polypeptides can be used
accordingly to increase the half-life of polypeptides of interest.
Portions of PRO229 which cause the increase in half-life are an
embodiment of the invention herein.
[0891] PRO238 can be used in assays which measure its ability to
reduce substrates, including oxygen and Aceyl-CoA, and
particularly, measure PRO238's ability to produce oxygen free
radicals. This is done by using assays which have been previously
described. PRO238 can further be used to assay for candidates which
block, reduce or reverse its reducing abilities. This is done by
performing side by side assays where candidates are added in one
assay having PRO238 and a substrate to reduce, and not added in
another assay, being the same but for the lack of the presence of
the candidate.
[0892] PRO233 polypeptides and portions thereof which have homology
to reductase may also be useful for in vivo therapeutic purposes,
as well as for various other applications. The identification of
novel reductase proteins and related molecules may be relevant to a
number of human disorders such as inflammatory disease, organ
failure, atherosclerosis, cardiac injury, infertility, birth
defects, premature aging, AIDS, cancer, diabetic complications and
mutations in general. Given that oxygen free radicals and
antioxidants appear to play important roles in a number of disease
processes, the identification of new reductase proteins and
reductase-like molecules is of special importance in that such
proteins may serve as potential therapeutics for a variety of
different human disorders. Such polypeptides may also play
important roles in biotechnological and medical research, as well
as various industrial applications. As a result, there is
particular scientific and medical interest in new molecules, such
as PRO233.
[0893] The PRO223 polypeptides of the present invention which
exhibit serine carboxypeptidease activity may be employed in vivo
for therapeutic purposes as well as for in vitro purposes. Those of
ordinary skill in the art will well know how to employ PRO223
polypeptides for such uses.
[0894] PRO235 polypeptides and portions thereof which may be
involved in cell adhesion are also useful for in vivo therapeutic
purposes, as well as for various in vitro applications. In
addition, PRO235 polypeptides and portions thereof may have
therapeutic applications in disease states which involve cell
adhesion. Given the physiological importance of cell adhesion
mechanisms in vivo, efforts are currently being under taken to
identify new, native proteins which are involved in cell adhesion.
Therefore, peptides having homology to plexin are of particular
interest to the scientific and medical communities.
[0895] Because the PRO236 and PRO262 polypeptides disclosed herein
are homologous to various known .beta.-galactosidase proteins, the
PRO236 and PRO262 polypeptides disclosed herein will find use in
conjugates of monoclonal antibodies and the polypeptide for
specific killing of tumor cells by generation of active drug from a
galactosylated prodrug (e.g., the generation of 5-fluorouridine
from the prodrug .beta.-D-galactosyl-5-fluorouridine). The PRO236
and PRO262 polypeptides disclosed herein may also find various uses
both in vivo and in vitro, wherein those uses will be similar or
identical to uses for which .beta.-galactosidase proteins are now
employed. Those of ordinary skill in the art will well know how to
employ PRO236 and PRO262 polypeptides for such uses.
[0896] PRO239 polypeptides and portions thereof which have homology
to densin may also be useful for in vivo therapeutic purposes, as
well as for various in vitro applications. In addition, PRO239
polypeptides and portions thereof may have therapeutic applications
in disease states which involve synaptic mechanisms, regeneration
or cell adhesion. Given the physiological importance of synaptic
processes, regeneration and cell adhesion mechanisms in vivo,
efforts are currently being under taken to identify new, native
proteins which are involved in synaptic machinery and cell
adhesion. Therefore, peptides having homology to densin are of
particular interest to the scientific and medical communities.
[0897] The PRO260 polypeptides described herein can be used in
assays to determine their relation to fucosidase. In particular,
the PRO260 polypeptides can be used in assays in determining their
ability to remove fucose or other sugar residues from
proteoglycans. The PRO260 polypeptides can be assayed to determine
if they have any functional or locational similarities as
fucosidase. The PRO260 polypeptides can then be used to regulate
the systems in which they are integral.
[0898] PRO263 can be used in assays wherein CD44 antigen is
generally used to determine PRO263 activity relative to that of
CD44. The results can be used accordingly.
[0899] PRO270 polypeptides and portions thereof which effect
reduction-oxidation (redox) state may also be useful for in vivo
therapeutic purposes, as well as for various in vitro applications.
More specifically, PRO270 polypeptides may affect the expression of
a large variety of genes thought to be involved in the pathogenesis
of AIDS, cancer, atherosclerosis, diabetic complications and in
pathological conditions involving oxidative stress such as stroke
and inflammation. In addition, PRO270 polypeptides and portions
thereof may affect the expression of a genes which have a role in
apoptosis. Therefore, peptides having homology to thioredoxin are
particularly desirable to the scientific and medical
communities.
[0900] PRO272 polypeptides and portions thereof which possess the
ability to bind calcium may also have numerous in vivo therapeutic
uses, as well as various in vitro applications. Therefore, peptides
having homology to reticulocalbin are particularly desirable. Those
with ordinary skill in the art will know how to employ PRO272
polypeptides and portions thereof for such purposes.
[0901] PRO294 polypeptides and portions thereof which have homology
to collagen may also be useful for in vivo therapeutic purposes, as
well as for various other applications. The identification of novel
collagens and collage-like molecules may have relevance to a number
of human disorders. Thus, the identification of new collagens and
collage-like molecules is of special importance in that such
proteins may serve as potential therapeutics for a variety of
different human disorders. Such polypeptides may also play
important roles in biotechnological and medical research as well as
various industrial applications. Given the large number of uses for
collagen, there is substantial interest in polypeptides with
homology to the collagen molecule.
[0902] PRO295 polypeptides and portions thereof which have homology
to integrin may also be useful for in vivo therapeutic purposes, as
well as for various other applications. The identification of novel
integrins and integrin-like molecules may have relevance to a
number of human disorders such as modulating the binding or
activity of cells of the immune system. Thus, the identification of
new integrins and integrin-like molecules is of special importance
in that such proteins may serve as potential therapeutics for a
variety of different human disorders. Such polypeptides may also
play important roles in biotechnological and medical research as
well as various industrial applications. As a result, there is
particular scientific and medical interest in new molecules, such
as PRO295.
[0903] As the PRO293 polypeptide is clearly a leucine rich repeat
polypeptide homologue, the peptide can be used in all applications
that the known NLRR-1 and NLRR-2 polypeptides are used. The
activity can be compared between these peptides and thus applied
accordingly.
[0904] The PRO247 polypeptides described herein can be used in
assays in which densin is used to determine the activity of PRO247
relative to densin or these other proteins. The results can be used
accordingly in diagnostics and/or therapeutic applications with
PRO247.
[0905] PRO302, PRO303, PRO304, PRO307 and PRO343 polypeptides of
the present invention which possess protease activity may be
employed both in vivo for therapeutic purposes and in vitro. Those
of ordinary skill in the art will well know how to employ the
PRO302, PRO303, PRO304, PRO307 and PRO343 polypeptides of the
present invention for such purposes.
[0906] PRO328 polypeptides and portions thereof which have homology
to GLIP and CRISP may also be useful for in vivo therapeutic
purposes, as well as for various other applications. The
identification of novel GLIP and CRISP-like molecules may have
relevance to a number of human disorders which involve
transcriptional regulation or are over expressed in human tumors.
Thus, the identification of new GLIP and CRISP-like molecules is of
special importance in that such proteins may serve as potential
therapeutics for a variety of different human disorders. Such
polypeptides may also play important roles in biotechnological and
medical research as well as in various industrial applications. As
a result, there is particular scientific and medical interest in
new molecules, such as PRO328.
[0907] Uses for PRO335, PRO331 or PRO326 including uses in
competitive assays with LIG-1, ALS and decorin to determine their
relative activities. The results can be used accordingly. PRO335,
PRO331 or PRO326 can also be used in assays where LIG-1 would be
used to determine if the same effects are incurred.
[0908] PRO332 contains GAG repeat (GKEK) at amino acidpositions
625-628 in FIG. 108 (SEQ ID NO:310). Slippage in such repeats can
be associated with human disease. Accordingly, PRO332 can use
useful for the treatment of such disease conditions by gene
therapy, i.e. by introduction of a gene containing the correct GKEK
sequence motif.
[0909] Other uses of PRO334 include use in assays in which
fibrillin or fibulin would be used to determine the relative
activity of PRO334 to fibrillin or fibulin. In particular, PRO334
can be used in assays which require the mechanisms imparted by
epidermal growth factor repeats.
[0910] Native PRO346 (SEQ ID NO:320) has a Blast score of 230,
corresponding to 27% homology between amino acid residues 21 to 343
with residues 35 to 1040 CGM6_HUMAN, a carcinoembryonic antigen
cgm6 precursor. This homology region includes nearly all but 2
N-terminal extracellular domain residues, including an
immunoglobulin superfamily homology at residues 148 to 339 of
PRO346 in addition to several transmembrane residues (340-343).
Carcinoembryonic antigen precursor, as explained in the Background
is a tumor-specific antigen, and as such, is a recognized marker
and therapeutic target for the diagnosis and treatment of colon
cancer. The expression of tumor-specific antigens is often
associated with the progression of neoplastic tissue disorders.
Native PRO346 (SEQ ID NO:320) and P_W06874, a human
carcinoembryonic antigen CEAd have a Blast score of 224 and
homology of 28% between residues 2 to 343 and 67 to 342,
respectively. This homology includes the entire extracellular
domain residues of native PRO346, minus the initiator methionine
(residues 2 to 18) as well as several transmembrane residues
(340-343).
[0911] PRO268 polypeptides which have protein disulfide isomerase
activity will be useful for many applications where protein
disulfide isomerase activity is desirable including, for example,
for use in promoting proper disulfide bond formation in
recombinantly produced proteins so as to increase the yield of
correctly folded protein. Those of ordinary skill in the art will
readily know how to employ such PRO268 polypeptides for such
purposes.
[0912] PRO330 polypeptides of the present invention which possess
biological activity related to that of the prolyl 4-hydroxylase
alpha subunit protein may be employed both in vivo for therapeutic
purposes and in vitro.
[0913] Those of ordinary skill in the art will well know how to
employ the PRO330 polypeptides of the present invention for such
purposes.
[0914] Uses of the herein disclosed molecules may also be based
upon the positive functional assay hits disclosed and described
below.
[0915] F. Anti-PRO Antibodies
[0916] The present invention further provides anti-PRO antibodies.
Exemplary antibodies include polyclonal, monoclonal, humanized,
bispecific, and heteroconjugate antibodies.
[0917] 1. Polyclonal Antibodies
[0918] The anti-PRO antibodies may comprise polyclonal antibodies.
Methods of preparing polyclonal antibodies are known to the skilled
artisan. Polyclonal antibodies can be raised in a mammal, for
example, by one or more injections of an immunizing agent and, if
desired, an adjuvant. Typically, the immunizing agent and/or
adjuvant will be injected in the mammal by multiple subcutaneous or
intraperitoneal injections. The immunizing agent may include the
PRO polypeptide or a fusion protein thereof. It may be useful to
conjugate the immunizing agent to a protein known to be immunogenic
in the mammal being immunized. Examples of such immunogenic
proteins include but are not limited to keyhole limpet hemocyanin,
serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.
Examples of adjuvants which may be employed include Freund's
complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A,
synthetic trehalose dicorynomycolate). The immunization protocol
may be selected by one skilled in the art without undue
experimentation.
[0919] 2. Monoclonal Antibodies
[0920] The anti-PRO antibodies may, alternatively, be monoclonal
antibodies. Monoclonal antibodies may be prepared using hybridoma
methods, such as those described by Kohler and Milstein, Nature
256:495 (1975). In a hybridoma method, a mouse, hamster, or other
appropriate host animal, is typically immunized with an immunizing
agent to elicit lymphocytes that produce or are capable of
producing antibodies that will specifically bind to the immunizing
agent. Alternatively, the lymphocytes may be immunized in
vitro.
[0921] The immunizing agent will typically include the PRO
polypeptide or a fusion protein thereof. Generally, either
peripheral blood lymphocytes ("PBLs") are used if cells of human
origin are desired, or spleen cells or lymph node cells are used if
non-human mammalian sources are desired. The lymphocytes are then
fused with an immortalized cell line using a suitable fusing agent,
such as polyethylene glycol, to form a hybridoma cell [Goding,
Monoclonal Antibodies: Principles and Practice, Academic Press,
(1986) pp. 59-103]. Immortalized cell lines are usually transformed
mammalian cells, particularly myeloma cells of rodent, bovine and
human origin. Usually, rat or mouse myeloma cell lines are
employed. The hybridoma cells may be cultured in a suitable culture
medium that preferably contains one or more substances that inhibit
the growth or survival of the unfused, immortalized cells. For
example, if the parental cells lack the enzyme hypoxanthine guanine
phosphoribosyl transferase (HGPRT or HPRT), the culture medium for
the hybridomas typically will include hypoxanthine, aminopterin,
and thymidine ("HAT medium"), which substances prevent the growth
of HGPRT-deficient cells.
[0922] Preferred immortalized cell lines are those that fuse
efficiently, support stable high level expression of antibody by
the selected antibody-producing cells, and are sensitive to a
medium such as HAT medium. More preferred immortalized cell lines
are murine myeloma lines, which can be obtained, for instance, from
the Salk Institute Cell Distribution Center, San Diego, Calif. and
the American Type Culture Collection, Manassas, Va. 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); Brodeur et al., Monoclonal Antibody
Production Techniques and Applications, Marcel Dekker, Inc., New
York, (1987) pp. 51-63].
[0923] The culture medium in which the hybridoma cells are cultured
can then be assayed for the presence of monoclonal antibodies
directed against PRO. Preferably, the binding specificity of
monoclonal antibodies produced by the hybridoma cells is determined
by immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay
(ELISA). Such techniques and assays are known in the art. The
binding affinity of the monoclonal antibody can, for example, be
determined by the Scatchard analysis of Munson and Pollard, Anal.
Biochem., 107:220 (1980).
[0924] After the desired hybridoma cells are identified, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods [Goding, supra]. Suitable culture media for this
purpose include, for example, Dulbecco's Modified Eagle's Medium
and RPMI-1640 medium. Alternatively, the hybridoma cells may be
grown in vivo as ascites in a mammal.
[0925] The monoclonal antibodies secreted by the subclones may be
isolated or purified from the culture medium or ascites fluid by
conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0926] The monoclonal antibodies may also be made by recombinant
DNA methods, such as those described in U.S. Pat. No. 4,816,567.
DNA encoding the monoclonal antibodies of the invention canbe
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 of the invention 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 simian COS cells, Chinese hamster ovary (CHO) cells, or
myeloma cells that do not otherwise produce immunoglobulin protein,
to obtain the synthesis of monoclonal antibodies in the recombinant
host cells. The DNA also may be modified, for example, by
substituting the coding sequence for human heavy and light chain
constant domains in place of the homologous murine sequences [U.S.
Pat. No. 4,816,567; Morrison et al., supra or by covalently joining
to the immunoglobulin coding sequence all or part of the coding
sequence for a non-immunoglobulin polypeptide. Such a
non-immunoglobulin polypeptide can be substituted for the constant
domains of an antibody of the invention, or can be substituted for
the variable domains of one antigen-combining site of an antibody
of the invention to create a chimeric bivalent antibody.
[0927] The antibodies may be monovalent antibodies. Methods for
preparing monovalent antibodies are well known in the art. For
example, one method involves recombinant expression of
immunoglobulin light chain and modified heavy chain. The heavy
chain is truncated generally at any point in the Fc region so as to
prevent heavy chain crosslinking. Alternatively, the relevant
cysteine residues are substituted with another amino acid residue
or are deleted so as to prevent crosslinking.
[0928] In vitro methods are also suitable for preparing monovalent
antibodies. Digestion of antibodies to produce fragments thereof,
particularly, Fab fragments, can be accomplished using routine
techniques known in the art.
[0929] 3. Human and Humanized Antibodies
[0930] The anti-PRO 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')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)].
[0931] 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.
[0932] Human antibodies can also be produced using various
techniques known in the art, including phage display libraries
[Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et
al., J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al.
and Boerner et al. are also available for the preparation of human
monoclonal antibodies (Cole et al., Monoclonal Antibodies and
Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J.
Immunol., 147(1):86-95 (1991)]. Similarly, human antibodies can be
made by introducing of human immunoglobulin loci into transgenic
animals, e.g., mice in which the endogenous immunoglobulin genes
have been partially or completely inactivated. Upon challenge,
human antibody production is observed, which closely resembles that
seen in humans in all respects, including gene rearrangement,
assembly, and antibody repertoire. This approach is described, for
example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825;
5,625,126; 5,633,425; 5,661,016, and in the following scientific
publications: Marks et al., Bio/Technology 10, 779-783(1992);
Lonberg et al., Nature 368856-859(1994); Morrison, Nature 368,
812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51
(1996); Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and
Huszar, Intern. Rev. Immunol. 13 65-93 (1995).
[0933] 4. Bispecific Antibodies
[0934] Bispecific antibodies are monoclonal, preferably human or
humanized, antibodies that have binding specificities for at least
two different antigens. In the present case, one of the binding
specificities is for the PRO, the other one is for any other
antigen, and preferably for a cell-surface protein or receptor or
receptor subunit.
[0935] Methods for making bispecific antibodies are known in the
art. Traditionally, the recombinant production of bispecific
antibodies is based on the co-expression of two immunoglobulin
heavy-chain/light-chain pairs, where the two heavy chains have
different specificities [Milstein and Cuello, Nature, 305:537-539
(1983)]. Because of the random assortment of immunoglobulin heavy
and light chains, these hybridomas (quadromas) produce a potential
mixture of ten different antibody molecules, of which only one has
the correct bispecific structure. The purification of the correct
molecule is usually accomplished by affinity chromatography steps.
Similar procedures are disclosed in WO 93/08829, published May 13,
1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).
[0936] Antibody variable domains with the desired binding
specificities (antibody-antigen combining sites) can be fused to
immunoglobulin constant domain sequences. The fusion preferably is
with an immunoglobulin heavy-chain constant domain, comprising at
least part of the hinge, CH2, and CH3 regions. It is preferred to
have the first heavy-chain constant region (CH1) containing the
site necessary for light-chain binding 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 organism. For further details of generating bispecific
antibodies see, for example, Suresh et al., Methods in Enzymology,
121:210 (1986).
[0937] According to another approach described in WO 96/27011, 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 CH3 region of an antibody constant 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.
[0938] Bispecific antibodies can be prepared as full length
antibodies or antibody fragments (e.g. F(ab').sub.2 bispecific
antibodies). Techniques for generating bispecific antibodies from
antibody fragments have been described in the literature. For
example, bispecific antibodies can be prepared 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.
[0939] Fab' fragments may be directly recovered from E. coli and
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.
[0940] Various technique 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
heavy-chain variable domain (V.sub.H) connected to a light-chain
variable domain (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).
[0941] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. Tutt et al.,
J. Immunol. 147:60 (1991).
[0942] Exemplary bispecific antibodies may bind to two different
epitopes on a given PRO polypeptide herein. Alternatively, an
anti-PRO polypeptide 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. CD2, CD3, CD28, or B7), 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 cellular defense mechanisms to
the cell expressing the particular PRO polypeptide. Bispecific
antibodies may also be used to localize cytotoxic agents to cells
which express a particular PRO polypeptide. These antibodies
possess a PRO-binding arm and an arm which binds a cytotoxic agent
or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA.
Another bispecific antibody of interest binds the PRO polypeptide
and further binds tissue factor (TF).
[0943] 5. Heteroconiugate Antibodies
[0944] Heteroconjugate antibodies are also within the scope of the
present invention. Heteroconjugate antibodies are composed of two
covalentlyjoined 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.
[0945] 6. Effector Function Engineering
[0946] It may be desirable to modify the antibody of the invention
with respect to effector function, so as to enhance, e.g., the
effectiveness of the antibody in treating cancer. For example,
cysteine residue(s) may be introduced into 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, 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 that 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).
[0947] 7. Immunoconiugates
[0948] The invention also pertains to immunoconjugates comprising
an antibody conjugated to a cytotoxic agent such as a
chemotherapeutic agent, 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).
[0949] 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.
[0950] 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.
[0951] In another embodiment, the antibody may be conjugated to a
"receptor" (such streptavidin) for utilization in tumor
pretargeting 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) that is conjugated to a
cytotoxic agent (e.g., a radionucleotide).
[0952] 8. Immunoliposomes
[0953] The antibodies disclosed herein may also be formulated as
immunoliposomes. 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); and U.S. Pat. Nos. 4,485,045
and 4,544,545. Liposomes with enhanced circulation time are
disclosed in U.S. Pat. No. 5,013,556.
[0954] 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 (such as Doxorubicin) is
optionally contained within the liposome. See Gabizon et al., J.
National Cancer Inst., 81(19): 1484 (1989).
[0955] 9. Pharmaceutical Compositions of Antibodies
[0956] Antibodies specifically binding a PRO 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.
[0957] If the PRO 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.
[0958] 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.
[0959] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0960] 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.
[0961] G. Uses for anti-PRO Antibodies
[0962] The anti-PRO antibodies of the invention have various
utilities. For example, anti-PRO antibodies may be used in
diagnostic assays for PRO, e.g., detecting its 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).
[0963] Anti-PRO antibodies also are useful for the affinity
purification of PRO from recombinant cell culture or natural
sources. In this process, the antibodies against PRO 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 PRO 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 PRO, which is bound to the immobilized antibody.
Finally, the support is washed with another suitable solvent that
will release the PRO from the antibody.
[0964] The following examples are offered for illustrative purposes
only, and are not intended to limit the scope of the present
invention in any way.
[0965] All patent and literature references cited in the present
specification are hereby incorporated by reference in their
entirety.
EXAMPLES
[0966] 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, Rockville, Md.
Example 1
Extracellular Domain Homology Screening to Identify Novel
Polypeptides and cDNA Encoding Therefor
[0967] 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 BLAST2 (Altschul, and
Gish, Methods in Enzymology 266: 460-80 (1996);
http://blast.wustl/edu/blast/README.html) 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.).
[0968] Using this extracellular domain homology screen, consensus
DNA sequences were assembled relative to the other identified EST
sequences. In addition, the consensus DNA sequences obtained were
often (but not always) 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.
[0969] 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 (.f) and reverse (.r) 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 (.p) sequences are typically 40-55 bp in length. In some
cases, additional oligonucleotides are synthesized when the
consensus sequence is greater than about 1-0.5 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.
[0970] 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 hemildnased 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 Encoding PRO211 and PRO217
[0971] Consensus DNA sequences were assembled as described in
Example 1 above and were designated as DNA28730 and DNA28760,
respectively. Based on these consensus sequences, oligonucleotides
were synthesized and used to identify by PCR a cDNA library that
contained the sequences of interest and for use as probes to
isolate a clone of the full-length coding sequence for the PRO211
and PRO217 polypeptides. The libraries used to isolate
DNA32292-1131 and DNA33094-1131 were fetal lung libraries. cDNA
clones were sequenced in their entirety. The entire nucleotide
sequences of PRO211 (DNA32292-1131) and PRO217 (UNQ191) are shown
in FIG. 1 (SEQ ID NO:1) and FIG. 3 (SEQ ID NO:3), respectively. The
predicted polypeptides are 353 and 379 amino acid in length,
respectively, with respective molecular weights of approximately
38,190 and 41,520 daltons.
7 The oligonucleotide sequnces used in the above procedure were the
following: 28730.p (OLI 516) (SEQ ID NO:5)
5'-AGGGAGCACGGACAGTGTGCAGATGTGGACGAGTGCTCACTAGCA-3' 28730.f (OLI
517) (SEQ ID NO:6) 5'-AGAGTGTATCTCTGGCTACGC-3' 28730.r (OLI 518)
(SEQ ID NO:7) 5'-TAAGTCCGGCACATTACAGGTC-3' 28760.p (OLI 617) (SEQ
ID NO:8) 5'-CCCACGATGTATGAATGGTGGACTTTGTGTGACTCCTGGTTTCT- GCATC-3'
28760.f (OLI 618) (SEQ ID NO:9) 5'-AAAGACGCATCTGCGAGTGTCC-3'
28760.r (OLI 619) (SEQ ID NO:10) 5'-TGCTGATTTCACACTGCTCTCCC-3'
Example 3
Isolation of cDNA Clones Encoding Human PRO230
[0972] 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 DNA30857. An EST
proprietary to Genentech was employed in the consensus assembly.
The EST is designated as DNA20088 and has the nucleotide sequence
shown in FIG. 7 (SEQ ID NO:13).
[0973] Based on the DNA30857 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 PRO230.
8 A pair of PCR primers (forward and reverse) were synthesized:
forward PCR primer 5'-TTCGAGGCCTCTGAGAAGTGGCCC-3' (SEQ ID NO:14)
reverse PCR primer 5'-GGCGGTATCTCTCTGGCCTCCC-3' (SEQ ID NO:15)
[0974] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA30857 sequence which
had the following nucleotide sequence
9 hybridization probe 5'-TTCTCCACAGCAGCTGTGGCATCCGATCGTGTCT-
CAATCCATTCTCTGGG-3' (SEQ ID NO:16)
[0975] 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 PRO230 gene
using the probe oligonucleotide and one of the PCR primers.
[0976] 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 PRO230
(herein designated as DNA33223-1136 and the derived protein
sequence for PRO230.
[0977] The entire nucleotide sequence of DNA33223-1136 is shown in
FIG. 5 (SEQ ID NO:11). Clone DNA33223-1136 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 100-103 and ending at the stop codon at
nucleotide positions 1501-1503 (FIG. 5; SEQ ID NO:11). The
predicted polypeptide precursor is 467 amino acids long FIG.
6).
Example 4
Isolation of cDNA Clones Encoding Human PRO232
[0978] 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 DNA30935. Based on
the DNA30935 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 PRO232.
10 A pair of PCR primers (forward and reverse) were synthesized:
forward PCR primer 5'-TGCTGTGCTACTCCTGCAAAGCCC-3' (SEQ ID NO: 19)
reverse PCR primer 5'-TGCACAAGTCGGTGTCACAGCACG-3' (SEQ ID
NO:20)
[0979] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA30935 sequence which
had the following nucleotide sequence
11 hybridization probe 5'-AGCAACGAGGACTGCCTGCAGGTGGAGAACTGC-
ACCCAGCTGGG-3' (SEQ ID NO:21)
[0980] 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 PRO232 gene
using the probe oligonucleotide and one of the PCR primers.
[0981] RNA for construction of the cDNA libraries was isolated from
human fetal kidney tissue.
[0982] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO232 [herein designated as
DNA34435-1140] and the derived protein sequence for PRO232.
[0983] The entire nucleotide sequence of DNA34435-1140 is shown in
FIG. 8 (SEQ ID NO:17). Clone DNA34435-1140 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 17-19 and ending at the stop codon at
nucleotide positions 359-361 (FIG. 8; SEQ ID NO:17). The predicted
polypeptide precursor is 114 amino acids long (FIG. 9). Clone
DNA34435-1140 has been deposited with ATCC on Sep. 16, 1997 and is
assigned ATCC deposit no. ATCC 209250.
[0984] Analysis of the amino acid sequence of the full-length
PRO232 suggests that it possesses 35% sequence identity with a stem
cell surface antigen from Gallus gallus.
Example 5
Isolation of cDNA Clones Encoding PRO187
[0985] A proprietary expressed sequence tag (EST) DNA database
(LIFESEQ.TM., Incyte Pharmaceuticals, Palo Alto, Calif.) was
searched and an EST (#843193) was identified which showed homology
to fibroblast growth factor (FGF-8) also known as androgen-induced
growth factor. mRNA was isolated from human fetal lung tissue using
reagents and protocols from Invitrogen, San Diego, Calif. (Fast
Track 2). The cDNA libraries used to isolate the cDNA clones were
constructed by standard methods using commercially available
reagents (e.g., Invitrogen, San Diego, Calif., Life Technologies,
Gaithersburg, Md.). 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 the cloning vector pRK5D using
reagents and protocols from Life Technologies, Gaithersburg, Md.
(Super Script Plasmid System). 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.
[0986] Several libraries from various tissue sources were screened
by PCR amplification with the following oligonucleotide probes:
12 IN843193.f (0LI315) (SEQ ID NO:24)
5'-CAGTACGTGAGGGACCAGGGCGCCATGA-3' IN843193.r (OLI 317) (SEQ ID
NQ:25) 5'-CCGGTGACCTGCACGTGCTTGCCA-3'
[0987] A positive library was then used to isolate clones encoding
the PRO187 gene using one of the above oligonucleotides and the
following oligonucleotide probe:
13 IN843193.p (OLI 316) (SEQ ID NO:26)
5'-GCGGATCTGCCGCCTGCTCANCTGGTCGGTCATGGCGCCCT-3'
[0988] A cDNA clone was sequenced in entirety. The entire
nucleotide sequence of PRO187 (DNA27864-1155) is shown in FIG. 10
(SEQ ID NO:22). Clone DNA27864-1155 contains a single open reading
frame with an apparent translational initiation site at nucleotide
position 1 (FIG. 10; SEQ ID NO:22). The predicted polypeptide
precursor is 205 amino acids long. Clone DNA27864-1155 has been
deposited with the ATCC (designation: DNA27864-1155) and is
assigned ATCC deposit no. ATCC 209375.
[0989] Based on a BLAST and FastA sequence alignent analysis (using
the ALIGN computer program) of the full-length sequence, the PRO187
polypeptide shows 74% amino acid sequence identity (Blast score
310) to human fibroblast growth factor-8 (androgen-induced growth
factor).
Example 6
Isolation of cDNA Clones Encoding PRO265
[0990] A consensus DNA sequence was assembled relative to other EST
sequences as described in Example 1 above using phrap. This
consensus sequence is herein designated DNA33679. Based on the
DNA33679 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 PRO265.
14 PCR primers (forward and reverse) were synthesized: forward PCR
primer A: 5'-CGGTCTACCTGTATGGCAACC-3' (SEQ ID NO:29); forward PCR
primer B: 5'-GCAGGACAACCAGATAAACC- AC-3' (SEQ ID NO:30); reverse
PCR primer 5'ACGCAGATTTGAGAAGGCTGTC-3' (SEQ ID NO:31)
[0991] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA33679 sequence which
had the following nucleotide sequence
15 hybridization probe 5'-TTCACGGGCTGCTCTTGCCCAGCTCTTGAAGCT-
TGAAGAGCTGCAC-3' (SEQ ID NO:32)
[0992] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with PCR primer pairs identified above. A positive
library was then used to isolate clones encoding the PRO265 gene
using the probe oligonucleotide and one of the PCR primers.
[0993] RNA for construction of the cDNA libraries was isolated from
human a fetal brain library.
[0994] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO265 [herein designated as
DNA36350-1158] (SEQ ID NO:27) and the derived protein sequence for
PRO265.
[0995] The entire nucleotide sequence of DNA36350-1158 is shown in
FIG. 12 (SEQ ID NO:27). Clone DNA36350-1158 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 352-354 and ending at the stop codon at
positions 2332-2334 (FIG. 12). The predicted polypeptide precursor
is 660 amino acids long (FIG. 13). Clone DNA36350-1158 has been
deposited with ATCC and is assigned ATCC deposit no. ATCC
209378.
[0996] Analysis of the amino acid sequence of the full-length
PRO265 polypeptide suggests that portions of it possess significant
homology to the fibromodulin and the fibromodulin precursor,
thereby indicating that PRO265 may be a novel member of the leucine
rich repeat family, particularly related to fibromodulin.
Example 7
Isolation of cDNA Clones Encoding Human PRO219
[0997] 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 DNA28729. Based on the
DNA28729 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 PRO219.
16 A pair of PCR primers (forward and reverse) were synthesized:
forward PCR primer 5'-GTGACCCTGGTTGTGAATACTCC-3' (SEQ ID NO:35)
reverse PCR primer 5'-ACAGCCATGGTCTATAGCTTGG-3' (SEQ ID NO:36)
[0998] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA28729 sequence which
had the following nucleotide sequence
17 hybridization probe: 5'-GCCTGTCAGTGTCCTGAGGGACACGTGCTC-
CGCAGCGATGGGAAG-3' (SEQ ID NO:37)
[0999] 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 PRO219 gene
using the probe oligonucleotide and one of the PCR primers.
[1000] RNA for construction of the cDNA libraries was isolated from
human fetal kidney tissue.
[1001] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO219 [herein designated as
DNA32290-1164] (SEQ ID NO:33) and the derived protein sequence for
PRO219.
[1002] The entire nucleotide sequence of DNA32290-1164 is shown in
FIG. 14 (SEQ ID NO:33). Clone DNA32290-1164 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 204-206 and ending at the stop codon at
nucleotide positions 2949-2951 (FIG. 14). The predicted polypeptide
precursor is 915 amino acids long (FIG. 15). Clone DNA32290-1164
has been deposited with ATCC and is assigned ATCC deposit no. ATCC
209384.
[1003] Analysis of the amino acid sequence of the full-length
PRO219 polypeptide suggests that portions of it possess significant
homology to the mouse and human matrilin-2 precursor
polypeptides.
Example 8
Isolation of cDNA Clones Encoding Human PRO246
[1004] 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.
18 A pair of PCR primers (forward and reverse) were synthesized:
forward PCR primer 5'-AGGGTCTCCAGGAGAAAGACTC-3' (SEQ ID NO:40)
reverse PCR primer 5'-ATTGTGGGCCTTGCAGACATAGAC-3' (SEQ ID
NO:41)
[1005] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA30955 sequence which
had the following nucleotide sequence
19 hybridization probe 5'-GGCCACAGCATCAAAACCTTAGAACTCAATGTA-
CTGGTTCCTCCAGCTCC-3' (SEQ ID NO:42)
[1006] 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.
[1007] 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:38) and the derived
protein sequence for PRO246.
[1008] The entire nucleotide sequence of DNA35639-1172 is shown in
FIG. 16 (SEQ ID NO:38). 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. 16). The predicted polypeptide
precursor is 390 amino acids long (FIG. 17). Clone DNA35639-1172
has been deposited with ATCC and is assigned ATCC deposit no. ATCC
209396.
[1009] 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 9
Isolation of cDNA Clones Encoding Human PRO228
[1010] 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 DNA28758. An EST
proprietary to Genentech was employed in the consensus assembly.
This EST is shown in FIG. 20 (SEQ ID NO:50) and is herein
designated as DNA21951.
[1011] Based on the DNA28758 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 PRO228.
[1012] PCR primers (forward and reverse) were synthesized:
[1013] forward PCR primer 5'-GGTAATGAGCTCCATTACAG-3' (SEQ ID
NO:51)
[1014] forward PCR primer 5'-GGAGTAGAAAGCGCATGG-3' (SEQ ID
NO:52)
[1015] forward PCR primer 5'-CACCTGATACCATGAATGGCAG-3' (SEQ ID
NO:53)
[1016] reverse PCR primer 5'-CGAGCTCGAATTAATTCG-3' (SEQ ID
NO:54)
[1017] reverse PCR primer 5'-GGATCTCCTGAGCTCAGG-3' (SEQ ID
NO:55)
[1018] reverse PCR primer 5'-CCTAGTTGAGTGATCCTTGTAAG-3' (SEQ ID
NO:56)
[1019] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA28758 sequence which
had the following nucleotide sequence
[1020] hybridization probe
[1021] 5'-ATGAGACCCACACCTCATGCCGCTGTAATCACCTGACACATTTTGCAATT-3'
(SEQ ID NO:57)
[1022] 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
PRO228 gene using the probe oligonucleotide and one of the PCR
primers.
[1023] RNA for construction of the cDNA libraries was isolated from
human fetal kidney tissue.
[1024] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO228 [herein designated as
DNA33092-1202] (SEQ ID NO:48) and the derived protein sequence for
PRO228.
[1025] The entire nucleotide sequence of DNA33092-1202 is shown in
FIG. 18 (SEQ ID NO:48). Clone DNA33092-1202 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 24-26 of SEQ ID NO:48 and ending at the stop
codon after nucleotide position 2093 of SEQ ID NO:48. The predicted
polypeptide precursor is 690 amino acids long (FIG. 19). Clone
DNA33092-1202 has been deposited with ATCC and is assigned ATCC
deposit no. ATCC 209420.
[1026] Analysis of the amino acid sequence of the full-length
PRO228 polypeptide suggests that portions of it possess significant
homology to the secretin-related proteins CD97 and EMR1 as well as
the secretin member, latrophilin, thereby indicating that PRO228
may be a new member of the secretin related proteins.
Example 10
Isolation of cDNA Clones Encoding Human PRO533
[1027] The EST sequence accession number AF007268, a murine
fibroblast growth factor (FGF-15) was used to search various public
EST databases (e.g., GenBank, Dayhoff, etc.). The search was
performed using the computer program BLAST or BLAST2 [Altschul et
al., Methods in Enzymology, 266:460480 (1996);
http://blast.wustl/edu/blast/README.html] as a comparison of the
ECD protein sequences to a 6 frame translation of the EST
sequences. The search resulted in a hit with GenBank EST AA220994,
which has been identified as stratagene NT2 neuronal precursor
937230.
[1028] Based on the Genbank EST AA220994 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. Forward and
reverse PCR primers may range from 20 to 30 nucleotides (typically
about 24), and are designed to give a PCR product of 100-1000 bp in
length. The probe sequences are typically 40-55 bp (typically about
50) in length. In order to screen several libraries for a source of
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 PCR primers.
[1029] 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 below. A positive
library was then used to isolate clones encoding the PRO533 gene
using the probe oligonucleotide and one of the PCR primers.
[1030] RNA for construction of the cDNA libraries was isolated from
human fetal retina. The cDNA libraries used to isolated the cDNA
clones were constructed by standard methods using commercially
available reagents (e.g., Invitrogen, San Diego, Calif.; Clontech,
etc.) 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.
[1031] A cDNA clone was sequenced in its entirety. The full length
nucleotide sequence of PRO533 is shown in FIG. 21 (SEQ ID NO:58).
Clone DNA49435-1219 contains a single open reading frame with an
apparent translational initiation site at nucleotide positions
459461 (FIG. 21; SEQ ID NO:58). The predicted polypeptide precursor
is 216 amino acids long. Clone DNA47412-1219 has been deposited
with ATCC and is assigned ATCC deposit no. ATCC 209480.
[1032] Based on a BLAST-2 and FastA sequence alignment analysis of
the full-length sequence, PRO533 shows amino acid sequence identity
to fibroblast growth factor (53%).
20 The oligonucleotide sequences used in the above procedure were
the following:/ FGF15.forward: 5'-ATCCGCCCAGATGGCTACAATGTGTA-3'
(SEQ ID NO:60); FGF15.probe:
5'-GCCTCCCGGTCTCCCTGAGCAGTGCCAAACAGCGGCAGTGTA-3' (SEQ ID NO:61);
FGF15.reverse: 5'-CCAGTCCGGTGACAAGCCCAAA-3' (SEQ ID NO:62).
Example 11
Isolation of cDNA Clones Encoding Human PRO245
[1033] 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.
[1034] 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.
21 A pair of PCR primers (forward and reverse) were synthesized:
forward PCR primer 5'-ATCGTTGTGAAGTTAGTGCCCC-3' (SEQ ID NO:65)
reverse PCR primer 5'-ACCTGCGATATCCAACAGAATTG-3' (SEQ ID NO:66)
[1035] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA30954 sequence which
had the following nucleotide sequence
22 hybridization probe 5'-GGAAGAGGATACAGTCACTCTGGAAGTATTAGT-
GGCTCCAGCAGTTCC-3' (SEQ ID NO:67)
[1036] 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.
[1037] 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.
[1038] The entire nucleotide sequence of DNA35638-1141 is shown in
FIG. 23 (SEQ ID NO:63). 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. 23; SEQ ID NO:63). The
predicted polypeptide precursor is 312 amino acids long (FIG. 24).
Clone DNA35638-1141 has been deposited with ATCC on Sep. 16, 1997
and is assigned ATCC deposit no. ATCC 209265.
[1039] 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 12
Isolation of cDNA Clones Encoding Human PRO220, PRO221 and
PRO227
[1040] (a) PRO220
[1041] 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 DNA28749. Based on
the DNA28749 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 PRO220.
23 A pair of PCR primers (forward and reverse) were synthesized:
forward PCR primer 5'-TCACCTGGAGCCTTTATTGGCC-3' (SEQ ID NO:74)
reverse PCR primer 5'-ATACCAGCTATAACCAGGCTGCG-3' (SEQ ID NO:75)
[1042] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA28749 sequence which
had the following nucleotide sequence:
24 hybridization probe: 5'-CAACAGTAAGTGGTTTGATGCTCTTCCAAATC-
TAGAGATTCTGATGATTGGG-3' (SEQ ID NO:76).
[1043] 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 PRO220 gene
using the probe oligonucleotide and one of the PCR primers.
[1044] 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 PRO220
[herein designated as DNA32298-1132 and the derived protein
sequence for PRO220.
[1045] The entire nucleotide sequence of DNA32298-1132 is shown in
FIG. 25 (SEQ ID NO:68). Clone DNA32298-1132 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 480-482 and ending at the stop codon at
nucleotide positions 2604-2606 (FIG. 25). The predicted polypeptide
precursor is 708 amino acids long (FIG. 26). Clone DNA32298-1132
has been deposited with ATCC and is assigned ATCC deposit no. ATCC
209257.
[1046] Analysis of the amino acid sequence of the full-length
PRO220 shows it has homology to member of the leucine rich repeat
protein superfamily, including the leucine rich repeat protein and
the neuronal leucine-rich repeat protein 1.
[1047] (b) PRO221
[1048] 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 DNA28756. Based on
the DNA28756 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 PRO221.
25 A pair of PCR primers (forward and reverse) were synthesized:
forward PCR primer 5'-CCATGTGTCTCCTCCTACAAAG-3' (SEQ ID NO:77)
reverse PCR primer 5'-GGGAATAGATGTGATCTGATTGG-3' (SEQ ID NO:78)
[1049] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA28756 sequence which
had the following nucleotide sequence:
26 hybridization probe 5'-CACCTGTAGCAATGCAAATCTCAAGGAAATACC-
TAGAGATCTTCCTCCTG-3' (SEQ ID NO:79)
[1050] 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 PRO221 gene
using the probe oligonucleotide and one of the PCR primers.
[1051] 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 PRO221
[herein designated as DNA33089-1132 and the derived protein
sequence for PRO221.
[1052] The entire nucleotide sequence of DNA33089-1132 is shown in
FIG. 27 (SEQ ID NO:70). Clone DNA33089-1132 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 179-181 and ending at the stop codon at
nucleotide positions 956-958 (FIG. 27). The predicted polypeptide
precursor is 259 amino acids long (FIG. 28). PRO221 is believed to
have a transmembrane region at amino acids 206-225. Clone
DNA33089-1132 has been deposited with ATCC and is assigned ATCC
deposit no. ATCC 209262.
[1053] Analysis of the amino acid sequence of the full-length
PRO221 shows it has homology to member of the leucine rich repeat
protein superfamily, including the SLIT protein.
[1054] (c) PRO227
[1055] 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 DNA28740. Based on
the DNA28740 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 PRO227.
[1056] A pair of PCR primers (forward and reverse) were
synthesized:
27 forward PCR primer 5'-AGCAACCGCCTGAAGCTCATCC-3' (SEQ ID NO:80)
reverse PCR primer 5'-AAGGCGCGGTGAAAGATGTAGACG-3' (SEQ ID
NO:81)
[1057] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA28740 sequence which
had the following nucleotide sequence:
28 hybridization probe 5'GACTACATGTTTCAGGACCTGTACAACCTCAAG-
TCACTGGAGGTTGGCGA-3' (SEQ ID NO:82).
[1058] 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 PRO227 gene
using the probe oligonucleotide and one of the PCR primers.
[1059] 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 PRO227
[herein designated as DNA33786-1132 and the derived protein
sequence for PRO227.
[1060] The entire nucleotide sequence of DNA33786-1132 is shown in
FIG. 29 (SEQ ID NO:72). Clone DNA33786-1132 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 33-35 and ending at the stop codon at
nucleotide positions 1893-1895 (FIG. 29). The predicted polypeptide
precursor is 620 amino acids long (FIG. 30). PRO227 is believed to
have a transmembrane region. Clone DNA33786-1132 has been deposited
with ATCC and is assigned ATCC deposit no. ATCC 209253.
[1061] Analysis of the amino acid sequence of the full-length
PRO221 shows it has homology to member of the leucine rich repeat
protein superfamily, including the platelet glycoprotein V
precursor and the human glycoprotein V.
Example 13
Isolation of cDNA Clones Encoding Human PRO258
[1062] 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.
[1063] 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.
[1064] PCR primers (forward and reverse) were synthesized:
29 forward PCR primer 5'-GCTAGGAATTCCACAGAAGCCC-3' (SEQ ID NO:85)
reverse PCR primer 5'-AACCTGGAATGTCACCGAGCTG-3' (SEQ ID NO:86)
reverse PCR primer 5'-CCTAGCACAGTGACGAGGGACTTGGC-3- ' (SEQ ID
NO:87)
[1065] Additionally, synthetic oligonucleotide hybridization probes
were constructed from the consensus DNA28740 sequence which had the
following nucleotide sequence:
30 hybridization probe 5'-AAGACACAGCCACCCTAAACTGTCAGTCTTCT-
GGGAGCAAGCCTGCAGCC-3' (SEQ ID NO:88)
5'-GCCCTGGCAGACGAGGGCGAGTACACCTGCTCAATCTTCACTATGCCTGT-3' (SEQ ID
NO:89)
[1066] 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.
[1067] 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:83) and the derived
protein sequence for PRO258.
[1068] The entire nucleotide sequence of DNA35918-1174 is shown in
FIG. 31 (SEQ ID NO:83). Clone DNA35918-1174 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 147-149 of SEQ ID NO:83 and ending at the stop
codon after nucleotide position 1340 of SEQ ID NO:83 (FIG. 31). The
predicted polypeptide precursor is 398 amino acids long (FIG. 32).
Clone DNA35918-1174 has been deposited with ATCC and is assigned
ATCC deposit no. ATCC 209402.
[1069] 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 14
Isolation of cDNA Clones Encoding Human PRO266
[1070] An expressed sequence tag database was searched for ESTs
having homology to SLIT, resulting in the identification of a
single EST sequence designated herein as T73996. Based on the
T73996 EST 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 PRO266.
[1071] A pair of PCR primers (forward and reverse) were
synthesized:
31 forward PCR primer 5'-GTTGGATCTGGGCAACAATAAC-3' (SEQ ID NO:92)
reverse PCR primer 5'-ATTGTTGTGCAGGCTGAGTTTAAG-3' (SEQ ID
NO:93)
[1072] Additionally, a synthetic oligonucleotide hybridization
probe was constructed which had the following nucleotide
sequence
32 hybridization probe 5'-GGTGGCTATACATGGATAGCAATTACCTGGAC-
ACGCTGTCCCGGG-3' (SEQ ID NO:94)
[1073] 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 PRO266 gene
using the probe oligonucleotide and one of the PCR primers.
[1074] RNA for construction of the cDNA libraries was isolated from
human fetal brain tissue. DNA sequencing of the clones isolated as
described above gave the full-length DNA sequence for PRO266
[herein designated as DNA37150-1178] (SEQ ID NO:90) and the derived
protein sequence for PRO266.
[1075] The entire nucleotide sequence of DNA37150-1178 is shown in
FIG. 33 (SEQ ID NO:90). Clone DNA37150-1178 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 167-169 and ending at the stop codon after
nucleotide position 2254 of SEQ ID NO:90. The predicted polypeptide
precursor is 696 amino acids long (FIG. 34). Clone DNA37150-1178
has been deposited with ATCC and is assigned ATCC deposit no. ATCC
209401.
[1076] Analysis of the amino acid sequence of the full-length
PRO266 polypeptide suggests that portions of it possess significant
homology to the SLIT protein, thereby indicating that PRO266 may be
a novel leucine rich repeat protein.
Example 15
Isolation of cDNA Clones Encoding Human PRO269
[1077] 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 DNA35705. Based on the
DNA35705 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 PRO269.
[1078] Forward and reverse PCR primers were synthesized:
33 forward PCR primer (.f1) 5'-TGGAAGGAGATGCGATGCCACCTG-3' (SEQ ID
NO:97) forward PCR primer (.f2) 5'-TGACCAGTGGGGAAGGACAG-- 3' (SEQ
ID NO:98) forward PCR primer (.f3) 5'-ACAGAGCAGAGGGTGCCTTG-3' (SEQ
ID NO:99) reverse PCR primer (.r1) 5'-TCAGGGACAAGTGGTGTCTCTCCC-3'
(SEQ ID NO:100) reverse PCR primer (.r2)
5'-TCAGGGAAGGAGTGTGCAGTTCTG-3' (SEQ ID NO:101)
[1079] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA35705 sequence which
had the following nucleotide sequence:
34 hybridization probe 5'-ACAGCTCCCGATCTCAGTTACTTGCATCGCGG-
ACGAAATCGGCGGTCGCT-3' (SEQ ID NO:102)
[1080] 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
PRO269 gene using the probe oligonucleotide and one of the PCR
primers.
[1081] RNA for construction of the cDNA libraries was isolated from
human fetal kidney tissue.
[1082] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO269 [herein designated as
DNA38260-1180] (SEQ ID NO:95) and the derived protein sequence for
PRO269.
[1083] The entire nucleotide sequence of DNA38260-1180 is shown in
FIG. 35 (SEQ ID NO:95). Clone DNA38260-1180 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 314-316 and ending at the stop codon at
nucleotide positions 1784-1786 (FIG. 35; SEQ ID NO:95). The
predicted polypeptide precursor is 490 amino acids long (FIG. 36).
Clone DNA38260-1180 has been deposited with ATCC and is assigned
ATCC deposit no. ATCC 209397.
[1084] Analysis of the amino acid sequence of the full-length
PRO269 suggests that portions of it possess significant homology to
the human thrombomodulin proteins, thereby indicating that PRO269
may possess one or more thrombomodulin-like domains.
Example 16
Isolation of cDNA Clones Encoding Human PRO287
[1085] 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.
[1086] A pair of PCR primers (forward and reverse) were
synthesized:
35 forward PCR primer 5'-CCGATTCATAGACCTCGAGAGT-3' (SEQ ID NO:105)
reverse PCR primer 5'-GTCAAGGAGTCCTCCACAATAC-3' (SEQ ID NO:106)
[1087] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA28728 sequence which
had the following nucleotide sequence
36 hybridization probe 5'-GTGTACAATGGCCATGCCAATGGCCAGCGCAT-
TGGCCGCTTCTGT-3' (SEQ ID NO:107)
[1088] 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.
[1089] RNA for construction of the cDNA libraries was isolated from
human fetal kidney tissue.
[1090] 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:103] and the derived protein sequence for
PRO287.
[1091] The entire nucleotide sequence of DNA39969-1185 is shown in
FIG. 37 (SEQ ID NO:103). 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. 37; SEQ ID NO:103).
[1092] The predicted polypeptide precursor is 415 amino acids long
(FIG. 38). Clone DNA39969-1185 has been deposited with ATCC and is
assigned ATCC deposit no. ATCC 209400.
[1093] 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.
[1094] 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 17
Isolation of cDNA Clones Encoding Human PRO214
[1095] A consensus DNA sequence was assembled using phrap as
described in Example 1 above. This consensus DNA sequence is
designated herein as DNA28744. Based on this 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.
[1096] 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 below. A positive
library was then used to isolate clones encoding the PRO214 gene
using the probe oligonucleotide and one of the PCR primers.
[1097] RNA for construction of the cDNA libraries was isolated from
human fetal lung tissue.
[1098] A cDNA clone was sequenced in its entirety The full length
nucleotide sequence of DNA32286-1191 is shown in FIG. 39 (SEQ ID
NO:108). DNA32286-1191 contains a single open reading frame with an
apparent translational initiation site at nucleotide position 103
(FIG. 39; SEQ ID NO:108). The predicted polypeptide precursor is
420 amino acids long (SEQ ID NO:109).
[1099] Based on a BLAST and FastA sequence alignment analysis of
the full-length sequence, PRO214 polypeptide shows amino acid
sequence identity to HT protein and/or Fibulin (49% and 38%,
respectively).
[1100] The oligonucleotide sequences used in the above procedure
were the following:
37 28744.p (OLI555) 5'-CCTGGCTATCAGCAGGTGGGCTCCAAGTGTCTCGA-
TGTGGATGAGTGTGA-3' (SEQ ID NO:110) 28744.f (OLI556)
5'-ATTCTGCGTGAACACTGAGGGC-3' (SEQ ID NO:111) 28744.r (OLI557)
5'-ATCTGCTTGTAGCCCTCGGCAC-3' (SEQ ID NO:112)
Example 18
Isolation of cDNA Clones Encoding Human PRO317
[1101] A consensus DNA sequence was assembled using phrap as
described in Example 1 above, wherein the consensus sequence is
herein designated as DNA28722. Based on this 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. The
forward and reverse PCR primers, respectively, synthesized for this
purpose were:
38 5'-AGGACTGCCATAACTTGCCTG (OLI489) (SEQ ID NO:115) and
5'-ATAGGAGTTGAAGCAGCGCTGC (OLI490) (SEQ ID NO:116).
[1102] The probe synthesized for this purpose was:
39 5'-TGTGTGGACATAGACGAGTGCCGCTACCGCTACTGCCAGCACCGC (OLI488) (SEQ
ID NO:117)
[1103] mRNA for construction of the cDNA libraries was isolated
from human fetal kidney tissue.
[1104] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification, as per Ausubel et al., Current Protocols in
Molecular Biology (1989), with the PCR primer pair identified
above. A positive library was then used to isolate clones
containing the PRO317 gene using the probe oligonucleotide
identified above and one of the PCR primers.
[1105] A cDNA clone was sequenced in its entirety. The entire
nucleotide sequence of DNA33461-1199 (encoding PRO317) is shown in
FIG. 41 (SEQ ID NO:113). Clone DNA33461-1199 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 68-70 (FIG. 41; SEQ ID NO:113). The predicted
polypeptide precursor is 366 amino acids long. The predicted signal
sequence is amino acids 1-18 of FIG. 42 (SEQ ID NO:114). There is
one predicted N-linked glycosylation site at amino acid residue
160. Clone DNA33461-1199 has been deposited with ATCC and is
assigned ATCC deposit no. ATCC 209367.
[1106] Based on BLAST" and FastA" sequence alignment analysis
(using the ALIGN.TM. computer program) of the full-length PRO317
sequence, PRO317 shows the most amino acid sequence identity to
EBAF-1 (92%). The results also demonstrate a significant homology
between human PRO317 and mouse LEFTY protein. The C-terminal end of
the PRO317 protein contains many conserved sequences consistent
with the pattern expected of a member of the TGF-superfamily.
[1107] In situ expression analysis in human tissues performed as
described below evidences that there is distinctly strong
expression of the PRO317 polypeptide in pancreatic tissue.
Example 19
Isolation of cDNA clones Encoding Human PRO301
[1108] A consensus DNA sequence designated herein as DNA35936 was
assembled using phrap as described in Example 1 above. Based on
this 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.
[1109] 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 below. A positive
library was then used to isolate clones encoding the PRO301 gene
using the probe oligonucleotide and one of the PCR primers.
[1110] RNA for construction of the cDNA libraries was isolated from
human fetal kidney.
[1111] A cDNA clone was sequenced in its entirety. The full length
nucleotide sequence of native sequence PRO301 is shown in FIG. 43
(SEQ ID NO:118). Clone DNA40628-1216 contains a single open reading
frame with an apparent translational initiation site at nucleotide
positions 52-54 (FIG. 43; SEQ ID NO:118). The predicted polypeptide
precursor is 299 amino acids long with a predicted molecular weight
of 32,583 daltons and pI of 8.29. Clone DNA40628-1216 has been
deposited with ATCC and is assigned ATCC deposit No. ATCC
209432.
[1112] Based on a BLAST and FastA sequence alignment analysis of
the full-length sequence, PRO301 shows amino acid sequence identity
to A33 antigen precursor (30%) and coxsackie and adenovirus
receptor protein (29%).
[1113] The oligonucleotide sequences used in the above procedure
were the following:
40 OLI2162 (35936.f1) 5'-TCGCGGAGCTGTGTTCTGTTTCCC-3' (SEQ ID
NO:120) OLI2163 (35936.p1)
5'-TGATCGCGATGGGGACAAAGGCGCAAGCTCGAGAGGAAACTGTTGTGCCT-3' (SEQ ID
NO:121) OLI2164 (35936.f2) 5'-ACACCTGGTTCAAAGATGGG-3' (SEQ ID
NO:122) OLI2165 (35936.r1) 5'-TAGGAAGAGTTGCTGAAGGCACGG-3' (SEQ ID
NO:123) OLI2166 (35936.f3) 5'-TTGCCTTACTCAGGTGCTAC-3' (SEQ ID
NO:124) OLI2167 (35936.r2) 5'-ACTCAGCAGTGGTAGGAAAG-3' (SEQ ID
NO:125)
Example 20
Isolation of cDNA Clones Encoding Human PRO224
[1114] A consensus DNA sequence assembled relative to the other
identified EST sequences as described in Example 1, wherein the
consensus sequence is designated herein as DNA30845. Based on the
DNA30845 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 PRO224.
[1115] A pair of PCR primers (forward and reverse) were
synthesized:
41 forward PCR primer 5'-AAGTTCCAGTGCCGCACCAGTGGC-3' (SEQ ID
NO:128) reverse PCR primer 5'-TTGGTTCCACAGCCGAGCTCGTCG-3- ' (SEQ ID
NO:129)
[1116] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA30845 sequence which
had the following nucleotide sequence
42 hybridization probe 5'-GAGGAGGAGTGCAGGATTGAGCCATGTACCCA-
GAAAGGGCAATGCCCACC-3' (SEQ ID NO:130)
[1117] 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 PRO224 gene
using the probe oligonucleotide and one of the PCR primers.
[1118] RNA for construction of the cDNA libraries was isolated from
human fetal liver tissue.
[1119] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO224 [herein designated as
DNA33221-1133] and the derived protein sequence for PRO224.
[1120] The entire nucleotide sequence of DNA33221-1133 is shown in
FIG. 45 (SEQ ID NO:126). Clone DNA33221-1133 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 33-35 and ending at the stop codon at
nucleotide positions 879-899 (FIG. 45; SEQ ID NO:126). The start of
a transmembrane region begins at nucleotide position 777. The
predicted polypeptide precursor is 282 amino acids long (FIG. 46).
Clone DNA33221-1133 has been deposited with ATCC and is assigned
ATCC deposit no. ATCC 209263.
[1121] Analysis of the amino acid sequence of the full-length
PRO224 suggests that it has homology to very low-density
lipoprotein receptors, apolipoprotein E receptor and chicken oocyte
receptors P95. Based on a BLAST and FastA sequence alignment
analysis of the full-length sequence, PRO224 has amino acid
identity to portions of these proteins in the range from 28% to
45%, and overall identity with these proteins in the range from 33%
to 39%.
Example 21
Isolation of cDNA Clones Encoding Human PRO222
[1122] 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 DNA28771. Based on
the DNA28771 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 PRO222.
[1123] A pair of PCR primers (forward and reverse) were
synthesized:
43 forward-PCR primer 5'-ATCTCCTATCGCTGCTTTCCCGG-3' (SEQ ID NO:133)
reverse PCR primer 5'-AGCCAGGATCGCAGTAAAACTCC-3' (SEQ ID
NO:134)
[1124] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA28771 sequence which
had the following nucleotide sequence:
44 hybridization probe 5'-ATTTAAACTTGATGGGTCTGCGTATCTTGAGT-
GCTTACAAAACCTTATCT-3' (SEQ ID NO:135)
[1125] 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 PRO222 gene
using the probe oligonucleotide and one of the PCR primers.
[1126] RNA for construction of the cDNA libraries was isolated from
human fetal kidney tissue.
[1127] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO222 [herein designated as
DNA33107-1135] and the derived protein sequence for PRO222.
[1128] The entire nucleotide sequence of DNA33107-1135 is shown in
FIG. 47 (SEQ ID NO:131). Clone DNA33107-1135 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 159-161 and ending at the stop codon at
nucleotide positions 1629-1631 (FIG. 47; SEQ ID NO:131). The
predicted polypeptide precursor is 490 amino acids long (FIG. 48).
Clone DNA33107-1135 has been deposited with ATCC and is assigned
ATCC deposit no. ATCC 209251.
[1129] Based on a BLAST and FastA sequence alignment analysis of
the full-length sequence, PRO222 shows amino acid sequence identity
to mouse complement factor h precursor (25-26%), complement
receptor (27-29%), mouse complement C3b receptor type 2 long form
precursor (2547%) and human hypothetical protein kiaa0247
(40%).
Example 22
Isolation of cDNA clones Encoding PRO234
[1130] A consensus DNA sequence was assembled (DNA30926) using
phrap as described in Example 1 above. Based on this 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.
[1131] RNA for the construction of the cDNA libraries was isolated
using standard isolation protocols, e.g., Ausubel et al., Current
Protocols in Molecular Biology, from tissue or cell line sources or
it was purchased from commercial sources (e.g., Clontech). The cDNA
libraries used to isolate the cDNA clones were constructed by
standard methods (e.g., Ausubel et al.) using commercially
available reagents (e.g., Invitrogen). This library was derived
from 22 week old fetal brain tissue.
[1132] A cDNA clone was sequenced in its entirety. The entire
nucleotide sequence of PRO234 is shown in FIG. 49 (SEQ ID NO:136).
The predicted polypeptide precursor is 382 amino acids long and has
a calculated molecular weight of approximately 43.1 kDa.
[1133] The oligonucleotide sequences used in the above procedure
were the following:
45 30926.p (OLI826) (SEQ ID NO:138): 5'-GTTCATTGAAAACCTCTTGCCATCT
GATGGTGACTTCTGGATTGGGCTCA-3' 30926.f (OLI827) (SEQ ID NO:139):
5'-AAGCCAAAGAAGCCTGCAGGA- GGG-3' 30926.r (OLI828) (SEQ ID NO:140):
5'-CAGTCCAAGCATAAAGGTCCTGGC-3'
Example 23
Isolation of cDNA Clones Encoding Human PRO231
[1134] 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.
[1135] Three PCR primers (two forward and one reverse) were
synthesized:
46 forward PCR primer 1 5'-CCAACTACCAAAGCTGCTGGAGCC-3' (SEQ ID
NO:143) forward PCR primer 2 5'-GCAGCTCTATTACCACGGGAAGGA- -3' (SEQ
ID NO:144) reverse PCR primer 5'-TCCTTCCCGTGGTAATAGAGCTGC-3' (SEQ
ID NO:145)
[1136] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA30933 sequence which
had the following nucleotide sequence
47 hybridization probe 5'-GGCAGAGAACCAGAGGCCGGAGGAGACTGCCTC-
TTTACAGCCAGG-3' (SEQ ID NO:146)
[1137] 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.
[1138] RNA for construction of the cDNA libraries was isolated from
human fetal liver tissue.
[1139] 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.
[1140] The entire nucleotide sequence of DNA34434-1139 is shown in
FIG. 51 (SEQ ID NO:141). 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. 51; SEQ ID NO:141). The
predicted polypeptide precursor is 428 amino acids long (FIG. 52).
Clone DNA34434-1139 has been deposited with ATCC on Sep. 16, 1997
and is assigned ATCC deposit no. ATCC 209252.
[1141] 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 24
Isolation of cDNA Clones Encoding Human PRO229
[1142] 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 DNA28762. Based on the
DNA28762 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 PRO229.
[1143] A pair of PCR primers (forward and reverse) were
synthesized:
48 forward PCR primer 5'-TTCAGCTCATCACCTTCACCTGCC-3' (SEQ ID
NO:149) reverse PCR primer 5'-GGCTCATACAAAATACCACTAGGG-3- ' (SEQ ID
NO:150)
[1144] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA28762 sequence which
had the following nucleotide sequence
49 hybridization probe 5'-GGGCCTCCACCGCTGTGAAGGGCGGGTGGAGGT-
GGAACAGAAAGGCCAGT-3' (SEQ ID NO:151)
[1145] 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 PRO229 gene
using the probe oligonucleotide and one of the PCR primers.
[1146] RNA for construction of the cDNA libraries was isolated from
human fetal liver tissue.
[1147] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO229 [herein designated as
DNA33100-1159] (SEQ ID NO:147) and the derived protein sequence for
PRO229.
[1148] The entire nucleotide sequence of DNA33100-1159 is shown in
FIG. 53 (SEQ ID NO:147). Clone DNA33100-1159 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 98-100 and ending at the stop codon at
nucleotide positions 1139-1141 (FIG. 53). The predicted polypeptide
precursor is 347 amino acids long (FIG. 54). Clone DNA33100-1159
has been deposited with ATCC and is assigned ATCC deposit no.ATCC
209377.
[1149] Analysis of the amino acid sequence of the full-length
PRO229 polypeptide suggests that portions of it possess significant
homology to antigen wc1.1, M130 antigen and CD6.
Example 25
Isolation of cDNA Clones Encoding Human PRO238
[1150] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described above in Example 1. This
consensus sequence is herein designated DNA30908. Based on the
DNA30908 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 PRO238.
[1151] PCR primers (forward and reverse) were synthesized:
50 forward PCR primer 1 5'-GGTGCTAAACTGGTGCTCTGTGGC-3' (SEQ ID
NO:154) forward PCR primer 2 5'-CAGGGCAAGATGAGCATTCC-3' (SEQ ID
NO:155) reverse PCR primer 5'-TCATACTGTTCCATCTCGGCACGC-3' (SEQ ID
NO:156)
[1152] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA30908 sequence which
had the following nucleotide sequence
51 hybridization probe 5'-AATGGTGGGGCCCTAGAAGAGCTCATCAGAGAA-
CTCACCGCTTCTCATGC-3' (SEQ ID NO:157)
[1153] 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 PRO238 gene
using the probe oligonucleotide and one of the PCR primers.
[1154] RNA for construction of the cDNA libraries was isolated from
human fetal liver tissue.
[1155] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO238 and the derived
protein sequence for PRO238.
[1156] The entire nucleotide sequence of DNA35600-1162 is shown in
FIG. 55 (SEQ ID NO:152). Clone DNA35600-1162 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 134-136 and ending prior to the stop codon at
nucleotide positions 1064-1066 (FIG. 55). The predicted polypeptide
precursor is 310 amino acids long (FIG. 56). Clone DNA35600-1162
has been deposited with ATCC and is assigned ATCC deposit no. ATCC
209370.
[1157] Analysis of the amino acid sequence of the full-length
PRO238 polypeptide suggests that portions of it possess significant
homology to reductase, particularly oxidoreductase, thereby
indicating that PRO238 may be a novel reductase.
Example 26
Isolation of cDNA Clones Encoding Human PRO233
[1158] 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. The EST databases included
public EST databases (e.g., GenBank) and a proprietary EST DNA
database (LIFESEQ.TM., Incyte Pharmaceuticals, Palo Alto, Calif.).
The search was performed using the computer program BLAST or BLAST2
(Altshul et al., Methods in Enzymology 266:460480 (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.;
http://bozeman.mbt.washington.edu/phrap.docs/phrap.html).
[1159] An expressed sequence tag (EST) was identified by the EST
database search and a consensus DNA sequence was assembled relative
to other EST sequences using phrap. This consensus sequence is
herein designated DNA30945. Based on the DNA30945 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 PRO233.
[1160] Forward and reverse PCR primers were synthesized:
52 forward PCR primer 5'-GGTGAAGGCAGAAATTGGAGATG-3' (SEQ ID NO:160)
reverse PCR primer 5'-ATCCCATGCATCAGCCTGTTTACC-3' (SEQ ID
NO:161)
[1161] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA30945 sequence which
had the following nucleotide sequence
53 hybridization probe 5'-GCTGGTGTAGTCTATACATCAGATTTGTTTGCT-
ACACAAGATCCTCAG-3' (SEQ ID NO:162)
[1162] 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 PRO233 gene
using the probe oligonucleotide.
[1163] RNA for construction of the cDNA libraries was isolated from
human fetal brain tissue.
[1164] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO233 [herein designated as
DNA34436-1238] (SEQ ID NO:158) and the derived protein sequence for
PRO233.
[1165] The entire nucleotide sequence of DNA34436-1238 is shown in
FIG. 57 (SEQ ID NO:158). Clone DNA34436-1238 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 101-103 and ending at the stop codon at
nucleotide positions 1001-1003 (FIG. 57). The predicted polypeptide
precursor is 300 amino acids long (FIG. 58). The full-length PRO233
protein shown in FIG. 58 has an estimated molecular weight of about
32,964 daltons and a p1 of about 9.52. Clone DNA34436-1238 has been
deposited with ATCC and is assigned ATCC deposit no. ATCC
209523.
[1166] Analysis of the amino acid sequence of the full-length
PRO233 polypeptide suggests that portions of it possess significant
homology to reductase proteins, thereby indicating that PRO233 may
be a novel reductase.
Example 27
Isolation of cDNA Clones Encoding Human PRO223
[1167] 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 DNA30836. Based on the
DNA30836 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 PRO223.
[1168] PCR primer pairs (one forward and two reverse) were
synthesized:
54 forward PCR primer 5'-TTCCATGCCACCTAAGGGAGACTC-3' (SEQ ID
NO:165) reverse PCR primer 1 5'-TGGATGAGGTGTGCAATGGCTGGC- -3' (SEQ
ID NO:166) reverse PCR primer 2 5'-AGCTCTCAGAGGCTGGTCATAGGG-3' (SEQ
ID NO:167)
[1169] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA30836 sequence which
had the following nucleotide sequence
55 hybridization probe 5'-GTCGGCCCTTTCCCAGGACTGAACATGAAGAGT-
TATGCCGGCTTCCTCAC-3' (SEQ ID NO:168)
[1170] 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 PRO223 gene
using the probe oligonucleotide and one of the PCR primers.
[1171] RNA for construction of the cDNA libraries was isolated from
human fetal liver tissue.
[1172] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO223 [herein designated as
DNA33206-1165] (SEQ ID NO:163) and the derived protein sequence for
PRO223.
[1173] The entire nucleotide sequence of DNA33206-1165 is shown in
FIG. 59 (SEQ ID NO:163). Clone DNA33206-1165 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 97-99 and ending at the stop codon at
nucleotide positions 1525-1527 (FIG. 59). The predicted polypeptide
precursor is 476 amino acids long (FIG. 60). Clone DNA33206-1165
has been deposited with ATCC and is assigned ATCC deposit no. ATCC
209372.
[1174] Analysis of the amino acid sequence of the full-length
PRO223 polypeptide suggests that it possesses significant homology
to various serine carboxypeptidase proteins, thereby indicating
that PRO223 may be a novel serine carboxypeptidase.
Example 28
Isolation of cDNA Clones Encoding Human PRO235
[1175] 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 "DNA30927". Based on the
DNA30927 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 PRO235.
[1176] A pair of PCR primers (forward and reverse) were
synthesized:
56 forward PCR primer 5'-TGGAATACCGCCTCCTGCAG-3' (SEQ ID NO:171)
reverse PCR primer 5'-CTTCTGCCCTTTGGAGAAGATGGC-3' (SEQ ID
NO:172)
[1177] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA30927 sequence which
had the following nucleotide sequence
57 hybridization probe 5'-GGACTCACTGGCCCAGGCCTTCAATATCACCA-
GCCAGGACGAT-3' (SEQ ID NO:173)
[1178] 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 PRO235 gene
using the probe oligonucleotide and one of the PCR primers.
[1179] RNA for construction of the cDNA libraries was isolated from
human fetal liver tissue.
[1180] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO235 [herein designated as
DNA35558-1167] (SEQ ID NO:169) and the derived protein sequence for
PRO235.
[1181] The entire nucleotide sequence of DNA35558-1167 is shown in
FIG. 61 (SEQ ID NO:169). Clone DNA35558-1167 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 667-669 and ending at the stop codon at
nucleotide positions 2323-2325 (FIG. 61). The predicted polypeptide
precursor is 552 amino acids long (FIG. 62). Clone DNA35558-1167
has been deposited with ATCC and is assigned ATCC deposit no.
209374.
[1182] Analysis of the amino acid sequence of the full-length
PRO235 polypeptide suggests that portions of it possess significant
homology to the human, mouse and Xenopus plexin protein, thereby
indicating that PRO235 may be a novel plexin protein.
Example 29
Isolation of cDNA Clones Encoding Human PRO236 and Human PRO262
[1183] Consensus DNA sequences were assembled relative to other EST
sequences using phrap as described in Example 1 above. These
consensus sequences are herein designated DNA30901 and DNA30847.
Based on the DNA30901 and DNA30847 consensus sequences,
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 and PRO262, respectively.
[1184] Based upon the DNA30901 consensus sequence, a pair of PCR
primers (forward and reverse) were synthesized:
58 forward PCR primer 5'-TGGCTACTCCAAGACCCTGGCATG-3' (SEQ ID
NO:178) reverse PCR primer 5'-TGGACAAATCCCCTTGCTCAGCCC-3- ' (SEQ ID
NO:179)
[1185] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA30901 sequence which
had the following nucleotide sequence
59 hybridization probe 5'-GGGCTTCACCGAAGCAGTGGACCTTTATTTTG-
ACCACCTGATGTCCAGGG-3' (SEQ ID NO:180)
[1186] Based upon the DNA30847 consensus sequence, a pair of PCR
primers (forward and reverse) were synthesized:
60 forward PCR primer 5'-CCAGCTATGACTATGATGCACC-3' (SEQ ID NO:181)
reverse PCR primer 5'-TGGCACCCAGAATGGTGTTGGCTC-3' (SEQ ID
NO:182)
[1187] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA30847 sequence which
had the following nucleotide sequence
61 hybridization probe 5'-CGAGATGTCATCAGCAAGTTCCAGGAAGTTCC-
TTTGGGACCTTTACCTCC-3' (SEQ ID NO:183)
[1188] 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 and
PRO262 genes using the probe oligonucleotides and one of the PCR
primers.
[1189] RNA for construction of the cDNA libraries was isolated from
human fetal lung tissue for PRO236 and human fetal liver tissue for
PRO262.
[1190] 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:174), the derived protein sequence for
PRO236, the full-length DNA sequence for PRO262 [herein designated
as DNA36992-1168] (SEQ ID NO:176) and the derived protein sequence
for PRO262.
[1191] The entire nucleotide sequence of DNA35599-1168 is shown in
FIG. 63 (SEQ ID NO:174). 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. 63). The predicted polypeptide
precursor is 636 amino acids long (FIG. 64). Clone DNA35599-1168
has been deposited with ATCC and is assigned ATCC deposit no. ATCC
209373.
[1192] The entire nucleotide sequence of DNA36992-1168 is shown in
FIG. 65 (SEQ ID NO:176). Clone DNA36992-1168 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 240-242 and ending at the stop codon at
nucleotide positions 2202-2204 (FIG. 65). The predicted polypeptide
precursor is 654 amino acids long (FIG. 66). Clone DNA36992-1168
has been deposited with ATCC and is assigned ATCC deposit no. ATCC
209382.
[1193] Analysis of the amino acid sequence of the full-length
PRO236 and PRO262 polypeptides suggests that portions of those
polypeptides possess significant homology to .beta.-galactosidase
proteins derived from various sources, thereby indicating that
PRO236 and PRO262 may be novel .beta.-galactosidase homologs.
Example 30
Isolation of cDNA Clones Encoding Human PRO239
[1194] 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 DNA30909. Based on the
DNA30909 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 PRO239.
[1195] A pair of PCR primers (forward and reverse) were
synthesized:
62 forward PCR primer 5'-CCTCCCTCTATTACCCATGTC-3' (SEQ ID NO:186)
reverse PCR primer 5'-GACCAACTTTCTCTGGGAGTGAGG-3' (SEQ ID
NO:187)
[1196] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA30909 sequence which
had the following nucleotide sequence
63 hybridization probe 5'-GTCACTTTATTTCTCTAACAACAAGCTCGAAT-
CCTTACCAGTGGCAG-3' (SEQ ID NO:188)
[1197] 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 PRO239 gene
using the probe oligonucleotide and one of the PCR primers.
[1198] RNA for construction of the cDNA libraries was isolated from
human fetal lung tissue.
[1199] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO239 [herein designated as
DNA34407-1169] (SEQ ID NO:184) and the derived protein sequence for
PRO239.
[1200] The entire nucleotide sequence of DNA34407-1169 is shown in
FIG. 67 (SEQ ID NO:184). Clone DNA34407-1169 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 72-74 and ending at the stop codon at
nucleotide positions 1575-1577 (FIG. 67). The predicted polypeptide
precursor is 501 amino acids long (FIG. 68). Clone DNA34407-1169
has been deposited with ATCC and is assigned ATCC deposit no.ATCC
209383.
[1201] Analysis of the amino acid sequence of the full-length
PRO239 polypeptide suggests that portions of it possess significant
homology to the densin protein, thereby indicating that PRO239 may
be a novel molecule in the densin family.
Example 31
Isolation of cDNA Clones Encoding Human PRO257
[1202] 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 DNA28731. Based on the
DNA28731 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 PRO257.
[1203] A pair of PCR primers (forward and reverse) were
synthesized:
64 forward PCR primer 5'-TCTCTATTCCAAACTGTGGCG-3' (SEQ ID NO:191)
reverse PCR primer 5'-TTTGATGACGATTCGAAGGTGG-3' (SEQ ID NO:192)
[1204] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA28731 sequence which
had the following nucleotide sequence
65 hybridization probe 5'-GGAAGGATCCTTCACCAGCCCCAATTACCCAA-
AGCCGCATCCTGAGC-3' (SEQ ID NO:193)
[1205] 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 PRO257 gene
using the probe oligonucleotide and one of the PCR primers.
[1206] RNA for construction of the cDNA libraries was isolated from
human fetal kidney tissue.
[1207] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO257 [herein designated as
DNA35841-1173 (SEQ ID NO:189) and the derived protein sequence for
PRO257.
[1208] The entire nucleotide sequence of DNA35841-1173 is shown in
FIG. 69 (SEQ ID NO:189). Clone DNA35841-1173 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 964-966 and ending at the stop codon at
nucleotide positions 2785-2787 (FIG. 69). The predicted polypeptide
precursor is 607 amino acids long (FIG. 70). Clone DNA35841-1173
has been deposited with ATCC and is assigned ATCC deposit no. ATCC
209403.
[1209] Analysis of the amino acid sequence of the full-length
PRO257 polypeptide suggests that portions of it possess significant
homology to the ebnerin protein, thereby indicating that PRO257 may
be a novel protein member related to the ebnerin protein.
Example 32
Isolation of cDNA Clones Encoding Human PRO260
[1210] 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 DNA30834. Based on the
DNA30834 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 PRO260.
[1211] PCR primers (forward and two reverse) were synthesized:
66 forward PCR primer: 5'-TGGTTTGACCAGGCCAAGTTCGG-3' (SEQ ID
NO:196); reverse PCR primer A: 5'-GGATTCATCCTCAAGGAAGAGC- GG-3'
(SEQ ID NO:197); and reverse PCR primer B:
5'-AACTTGCAGCATCAGCCACTCTGC-3' (SEQ ID NO:198)
[1212] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA30834 sequence which
had the following nucleotide sequence:
67 hybridization probe 5'-TTCCGTGCCCAGCTTCGGTAGCGAGTGGTTCTG-
GTGGTATTGGCA-3' (SEQ ID NO:199)
[1213] 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 PRO260 gene
using the probe oligonucleotide and one of the PCR primers.
[1214] RNA for construction of the cDNA libraries was isolated from
human fetal kidney tissue.
[1215] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO260 [herein designated as
DNA33470-1175] (SEQ ID NO:194) and the derived protein sequence for
PRO260.
[1216] The entire nucleotide sequence of DNA33470-1175 is shown in
FIG. 71 (SEQ ID NO:194). Clone DNA33470-1175 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 67-69 and ending at the stop codon 1468-1470
(see FIG. 71). The predicted polypeptide precursor is 467 amino
acids long (FIG. 72). Clone DNA33470-1175 has been deposited with
ATCC and is assigned ATCC deposit no. ATCC 209398.
[1217] Analysis of the amino acid sequence of the full-length
PRO260 polypeptide suggests that portions of it possess significant
homology to the alpha-1-fucosidase precursor, thereby indicating
that PRO260 may be a novel fucosidase.
Example 33
Isolation of cDNA Clones Encoding Human PRO263
[1218] 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 DNA30914. Based on the
DNA30914 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 PRO263.
[1219] PCR primers (tow forward and one reverse) were
synthesized:
68 forward PCR primer 1: 5'-GAGCTTTCCATCCAGGTGTCATGC-3' (SEQ ID
NO:202); forward PCR primer 2: 5'-GTCAGTGACAGTACCTACTCGG- -3 (SEQ
ID NO:203); reverse PCR primer: 5'-TGGAGCAGGAGGAGTAGTAGTAGG-3' (SEQ
ID NO:204)
[1220] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA30914 sequence which
had the following nucleotide sequence:
69 hybridization probe 5'-AGGAGGCCTGTAGGCTGCTGGGACTAAGTTTGG-
CCGGCAAGGACCAAGTT-3' (SEQ ID NO:205)
[1221] 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 PRO263 gene
using the probe oligonucleotide and one of the PCR primers.
[1222] RNA for construction of the cDNA libraries was isolated from
human fetal liver tissue.
[1223] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO263 [herein designated as
DNA34431-1177] (SEQ ID NO:200) and the derived protein sequence for
PRO263.
[1224] The entire nucleotide sequence of DNA34431-1177 is shown in
FIG. 73 (SEQ ID NO:200). Clone DNA34431-1177 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 160-162 of SEQ ID NO:200 and ending at the
stop codon after the nucleotide at position 1126-1128 of SEQ ID
NO:200 (FIG. 73). The predicted polypeptide precursor is 322 amino
acids long (FIG. 74). Clone DNA34431-1177 has been deposited with
ATCC and is assigned ATCC deposit no. ATCC 209399.
[1225] Analysis of the amino acid sequence of the full-length
PRO263 polypeptide suggests that portions of it possess significant
homology to CD44 antigen, thereby indicating that PRO263 may be a
novel cell surface adhesion molecule.
Example 34
Isolation of cDNA Clones Encoding Human PRO270
[1226] 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 DNA35712. Based on
the DNA35712 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 PRO270. Forward and reverse PCR
primers were synthesized:
70 forward PCR primer (.f1) 5'-GCTTGGATATTCGCATGGGCCTAC-3' (SEQ ID
NO:208) forward PCR primer (.f2) 5'-TGGAGACAATATCCCTGAGG- -3' (SEQ
ID NO:209) reverse PCR primer (.r1) 5'-AACAGTTGGCCACAGCATGGCAGG-3'
(SEQ ID NO:210)
[1227] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA35712 sequence which
had the following nucleotide sequence
71 hybridization probe 5'-CCATTGATGAGGAACTAGAACGGGACAAGAGGG-
TCACTTGGATTGTGGAG-3' (SEQ ID NO:211)
[1228] 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 PRO270 gene
using the probe oligonucleotide and one of the PCR primers.
[1229] RNA for construction of the cDNA libraries was isolated from
human fetal lung tissue.
[1230] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO270 [herein designated as
DNA39510-1181] (SEQ ID NO:206) and the derived protein sequence for
PRO270.
[1231] The entire nucleotide sequence of DNA39510-1181 is shown in
FIG. 75 (SEQ ID NO:206). Clone DNA39510-1181 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 3-5 and ending at the stop codon at nucleotide
positions 891-893 (FIG. 75; SEQ ID NO:206). The predicted
polypeptide precursor is 296 amino acids long (FIG. 76). Clone
DNA39510-1181 has been deposited with ATCC and is assigned ATCC
deposit no. ATCC 209392.
[1232] Analysis of the amino acid sequence of the full-length
PRO270 suggests that portions of it possess significant homology to
the thioredoxin-protein, thereby indicating that the PRO270 protein
may be a novel member of the thioredoxin family.
Example 35
Isolation of cDNA Clones Encoding Human PRO271
[1233] 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 DNA35737. Based on the
DNA35737 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 PRO271.
[1234] Forward and reverse PCR primers were synthesized:
72 forward-PCR primer 1 5'-TGCTTCGCTACTGCCCTC-3' (SEQ ID NO:214)
forward PCR primer 2 5'-TTCCCTTGTGGGTTGGAG-3' (SEQ ID NO:215)
forward PCR primer 3 5'-AGGGCTGGAAGCCAGTTC-3' (SEQ ID NO:216)
reverse PCR primer 1 5'-AGCCAGTGAGGAAATGCG-3' (SEQ ID NO:217)
reverse PCR primer 2 5'-TGTCCAAAGTACACACACCTGAGG-3 (SEQ ID
NO:218)
[1235] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA35737 sequence which
had the following nucleotide sequence
73 hybridization probe 5'-GATGCCACGATCGCCAAGGTGGGACAGCTCTTT-
GCCGCCTGGAAG-3' (SEQ ID NO:219)
[1236] 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 PRO271 gene
using the probe oligonucleotide and one of the PCR primers.
[1237] RNA for construction of the cDNA libraries was isolated from
human fetal brain tissue.
[1238] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO271 [herein designated as
DNA39423-1182] (SEQ ID NO:212) and the derived protein sequence for
PRO271.
[1239] The entire nucleotide sequence of DNA39423-1182 is shown in
FIG. 77 (SEQ ID NO:212). Clone DNA39423-1182 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 101-103 and ending at the stop codon at
nucleotide positions 1181-1183 (FIG. 77). The predicted polypeptide
precursor is 360 amino acids long (FIG. 78). Clone DNA39423-1182
has been deposited with ATCC and is assigned ATCC deposit no. ATCC
209387.
[1240] Analysis of the amino acid sequence of the full-length
PRO271 polypeptide suggests that it possess significant homology to
the proteoglycan link protein, thereby indicating that PRO271 may
be a link protein homolog.
Example 36
Isolation of cDNA Clones Encoding Human PRO272
[1241] 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 DNA36460. Based on the
DNA36460 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 PRO272.
[1242] Forward and reverse PCR primers were synthesized:
74 forward PCR primer (.f1) 5'-CGCAGGCCCTCATGGCCAGG-3' (SEQ ID
NO:222) forward PCR primer (.f2) 5'-GAAATCCTGGGTAATTGG-3- ' (SEQ ID
NO:223) reverse PCR primer 5'-GTGCGCGGTGCTCACAGCTCATC-3' (SEQ ID
NO:224)
[1243] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA36460 sequence which
had the following nucleotide sequence
75 hybridization 5-'CCCCCCTGAGCGACGCTCCCCCATGATGACGCCCACGGG-
AACTTC-3' (SEQ ID NO:225)
[1244] 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
PRO272 gene using the probe oligonucleotide and one of the PCR
primers.
[1245] RNA for construction of the cDNA libraries was isolated from
human fetal lung tissue.
[1246] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO272 [herein designated as
DNA40620-1183] (SEQ ID NO:220) and the derived protein sequence for
PRO272.
[1247] The entire nucleotide sequence of DNA40620-1183 is shown in
FIG. 79 (SEQ ID NO:220). Clone DNA40620-1183 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 35-37 and ending at the stop codon at
nucleotide positions 1019-1021 (FIG. 79). The predicted polypeptide
precursor is 328 amino acids long (FIG. 80). Clone DNA40620-1183
has been deposited with ATCC and is assigned ATCC deposit no. ATCC
209388.
[1248] Analysis of the amino acid sequence of the full-length
PRO272 polypeptide suggests that portions of it possess significant
homology to the human and mouse reticulocalbin proteins,
respectively, thereby indicating that PRO272 may be a novel
reticulocalbin protein.
Example 37
Isolation of cDNA Clones Encoding Human PRO294
[1249] 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 DNA35731. Based on the
DNA35731 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 PRO294.
[1250] Forward and reverse PCR primers were synthesized:
76 forward PCR primer (.f1) 5'-TGGTCTCGCACACCGATC-3' (SEQ ID
NO:228) forward PCR primer (.f2) 5'-CTGCTGTCCACAGGGGAG-3- ' (SEQ ID
NO:229) forward PCR primer (.f3) 5'-CCTTGAAGCATACTGCTC-3' (SEQ ID
NO:230) forward PCR primer (.f4) 5'-GAGATAGCAATTTCCGCC-3' (SEQ ID
NO:231) reverse PCR primer (.r1) 5'-TTCCTCAAGAGGGCAGCC-3' (SEQ ID
NO:232) reverse PCR primer (.r2) 5'-CTTGGCACCAATGTCCGAGATTTC-3'
(SEQ ID NO:233)
[1251] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA35731 sequence which
had the following nucleotide sequence
77 hybridization probe 5'-GCTCTGAGGAAGGTGACGCGCGGGGCCTCCGA-
ACCCTTGGCCTTG-3' (SEQ ID NO:234)
[1252] 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
PRO294 gene using the probe oligonucleotide and one of the PCR
primers.
[1253] RNA for construction of the cDNA libraries was isolated from
human fetal brain tissue. DNA sequencing of the clones isolated as
described above gave the full-length DNA sequence for PRO294
[herein designated as DNA40604-1187] (SEQ ID NO:226) and the
derived protein sequence for PRO294.
[1254] The entire nucleotide sequence of DNA40604-1187 is shown in
FIG. 81 (SEQ ID NO:226). Clone DNA40604-1187 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 396-398 and ending at the stop codon at
nucleotide positions 2046-2048 (FIG. 81). The predicted polypeptide
precursor is 550 amino acids long (FIG. 82). Clone DNA40604-1187
has been deposited with ATCC and is assigned ATCC deposit no.
209394.
[1255] Analysis of the amino acid sequence of the full-length
PRO294 polypeptide suggests that portions of it possess significant
homology to portions of various collagen proteins, thereby
indicating that PRO294 may be collagen-like molecule.
Example 38
Isolation of cDNA Clones Encoding Human PRO295
[1256] 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 DNA35814. Based on the
DNA35814 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 PRO295.
[1257] Forward and reverse PCR primers were synthesized:
78 forward PCR primer (.f1) 5'-GCAGAGCGGAGATGCAGCGGCTTG-3 (SEQ ID
NO:238) forward PCR primer (.f2) 5'-CCCAGCATGTACTGCCAG-3- ' (SEQ ID
NO:239) forward PCR primer (.f3) 5'-TTGGCAGCTTCATGGAGG-3' (SEQ ID
NO:240) forward PCR primer (.f4) 5'-CCTGGGCAAAAATGCAAC-3' (SEQ ID
NO:241) reverse PCR primer (.r1) 5'-CTCCAGCTCCTGGCGCACCTCCTC-3'
(SEQ ID NO:242)
[1258] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA35814 sequence which
had the following nucleotide sequence
79 hybridization probe 5'-GGCTCTCAGCTACCGCGCAGGAGCGAGGCCAC-
CCTCAATGAGATG-3' (SEQ ID NO:243)
[1259] 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
PRO295 gene using the probe oligonucleotide and one of the PCR
primers.
[1260] RNA for construction of the cDNA libraries was isolated from
human fetal lung tissue.
[1261] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO295 [herein designated as
DNA38268-1188] (SEQ ID NO:235) and the derived protein sequence for
PRO295.
[1262] The entire nucleotide sequence of DNA38268-1188 is shown in
FIG. 83 (SEQ ID NO:235). Clone DNA38268-1188 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 153-155 and ending at the stop codon at
nucleotide positions 1202-1204 (FIG. 83). The predicted polypeptide
precursor is 350 amino acids long (FIG. 84). Clone DNA38268-1188
has been deposited with ATCC and is assigned ATCC deposit no.
209421.
[1263] Analysis of the amino acid sequence of the full-length
PRO295 polypeptide suggests that portions of it possess significant
homology to the integrin proteins, thereby indicating that PRO295
may be a novel integrin.
Example 39
Isolation of cDNA Clones Encoding Human PRO293
[1264] 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. The EST databases included
public EST databases (e.g., GenBank) and a proprietary EST DNA
database (LIFESEQ.TM., Incyte Pharmaceuticals, Palo Alto, Calif.).
The search was performed using the computer program BLAST or BLAST2
(Altshul et al., Methods in Enzymology 266:460480 (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.;
http://bozeman.mbt.washington.edu/phrap.docs/phrap.html).
[1265] Based on an expression tag sequence designated herein as
T08294 identified in the above analysis, 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 PRO293.
[1266] A pair of PCR primers (forward and reverse) were
synthesized:
80 forward PCR primer 5'-AACAAGGTAAGATGCCATCCTG-3' (SEQ ID NO:246)
reverse PCR primer 5'-AAACTTGTCGATGGAGACCAGCTC-3' (SEQ ID
NO:247)
[1267] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the expression sequence tag which had
the following nucleotide sequence
81 hybridization probe 5'-AGGGGCTGCAAAGCCTGGAGAGCCTCTCCTTC-
TATGACAACCAGC-3' (SEQ ID NO:248)
[1268] 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 PRO293 gene
using the probe oligonucleotide and one of the PCR primers.
[1269] RNA for construction of the cDNA libraries was isolated from
human fetal brain tissue.
[1270] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO293 [herein designated as
DNA37151-1193] (SEQ ID NO:244) and the derived protein sequence for
PRO293.
[1271] The entire nucleotide sequence of DNA37151-1193 is shown in
FIG. 85 (SEQ ID NO:244). Clone DNA37151-1193 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 881-883 and ending at the stop codon after
nucleotide position 3019 of SEQ ID NO:244, FIG. 85). The predicted
polypeptide precursor is 713 amino acids long (FIG. 86). Clone
DNA37151-1193 has been deposited with ATCC and is assigned ATCC
deposit no. ATCC 209393.
[1272] Analysis of the amino acid sequence of the full-length
PRO293 polypeptide suggests that portions of it possess significant
homology to the NLRR proteins, thereby indicating that PRO293 may
be a novel NLRR protein.
Example 40
Isolation of cDNA Clones Encoding Human PRO247
[1273] 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 DNA33480. Based on the
DNA33480 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 PRO247.
[1274] A pair of PCR primers (forward and reverse) were
synthesized:
82 forward PCR primer 5'-CAACAATGAGGGCACCAAGC-3' (SEQ ID NO:251)
reverse PCR primer 5'-GATGGCTAGGTTCTGGAGGTTCTG-3' (SEQ ID
NO:252)
[1275] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the DNA33480 expression sequence tag
which had the following nucleotide sequence
83 hybridization probe 5'-CAACCTGCAGGAGATTGACCTCAAGGACAACA-
ACCTCAAGACCATCG-3' (SEQ ID NO:253)
[1276] 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 PRO247 gene
using the probe oligonucleotide and one of the PCR primers.
[1277] RNA for construction of the cDNA libraries was isolated from
human fetal brain tissue.
[1278] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO247 [herein designated as
DNA35673-1201] (SEQ ID NO:249) and the derived protein sequence for
PRO247.
[1279] The entire nucleotide sequence of DNA35673-1201 is shown in
FIG. 89 (SEQ ID NO:249). Clone DNA35673-1201 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 80-82 of SEQ ID NO:249 and ending at the stop
codon after nucleotide position 1717 of SEQ ID NO:249 (FIG. 89).
The predicted polypeptide precursor is 546 anino acids long (FIG.
88). Clone DNA35673-1201 has been deposited with ATCC and is
assigned ATCC deposit no. 209418.
[1280] Analysis of the amino acid sequence of the full-length
PRO247 polypeptide suggests that portions of it possess significant
homology to the densin molecule and KIAA0231, thereby indicating
that PRO247 may be a novel leucine rich repeat protein.
Example 41
Isolation of cDNA Clones Encoding Human PRO302. PRO303. PRO304.
PRO307 and PRO343
[1281] Consensus DNA sequences were assembled relative to other EST
sequences using phrap as described in Example 1 above. These
consensus sequences are herein designated DNA35953, DNA35955,
DNA35958, DNA37160 and DNA30895. Based on the DNA35953 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 PRO302.
[1282] PCR primers (forward and reverse) were synthesized:
84 forward PCR primer 1 5'-GTCCGCAAGGATGCCTACATGTTC-3' (SEQ ID
NO:264) forward PCR primer 2 5'-GCAGAGGTGTCTAAGGTTG-3' (SEQ ID
NO:265) reverse PCR primer 5'-AGCTCTAGACCAATGCCAGCTTCC-3' (SEQ ID
NO:266)
[1283] Also, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA35953 sequence which had the
following nucleotide sequence
85 hybridization probe 5'-GCCACCAACTCCTGCAAGAACTTCTCAGAACT-
GCCCCTGGTCATG-3' (SEQ ID NO:267)
[1284] 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
PRO302 gene using the probe oligonucleotide and one of the PCR
primers.
[1285] RNA for construction of the cDNA libraries was isolated from
human fetal kidney tissue (LIB228).
[1286] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO302 [herein designated as
DNA40370-1217] (SEQ ID NO:254) and the derived protein sequence for
PRO302.
[1287] The entire nucleotide sequence of DNA40370-1217 is shown in
FIG. 89 (SEQ ID NO:254). Clone DNA40370-1217 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 34-36 and ending at the stop codon at
nucleotide positions 1390-1392 (FIG. 89). The predicted polypeptide
precursor is 452 amino acids long (FIG. 90). Various unique aspects
of the PRO302 protein are shown in FIG. 90. Clone DNA40370-1217 has
been deposited with the ATCC on Nov. 21, 1997 and is assigned ATCC
deposit no. ATCC 209485.
[1288] Based on the DNA35955 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 PRO303.
[1289] A pair of PCR primers (forward and reverse) were
synthesized:
86 forward PCR primer 5'-GGGGAATTCACCCTATGACATTGCC-3' (SEQ ID
NO:268) reverse PCR primer 5'-GAATGCCCTGCAAGCATCAACTGG-3- ' (SEQ ID
NO:269)
[1290] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA35955 sequence which
had the following nucleotide sequence:
87 hybridization probe 5'-GCACCTGTCACCTACACTAAACACATCCAGCCC-
ATCTGTCTCCAGGCCTC-3' (SEQ ID NO:270)
[1291] 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
PRO303 gene using the probe oligonucleotide and one of the PCR
primers.
[1292] RNA for construction of the cDNA libraries was isolated from
human fetal lung tissue (L1125).
[1293] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO303 [herein designated as
DNA42551-1217] (SEQ ID NO:256) and the derived protein sequence for
PRO303.
[1294] The entire nucleotide sequence of DNA42551-1217 is shown in
FIG. 91 (SEQ ID NO:256). Clone DNA42551-1217 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 20-22 and ending at the stop codon at
nucleotide positions 962-964 (FIG. 91). The predicted polypeptide
precursor is 314 amino acids long (FIG. 92). Various unique aspects
of the PRO303 protein are shown in FIG. 92. Clone DNA42551-1217 has
been deposited on Nov. 21, 1997 with the ATCC and is assigned ATCC
deposit no. ATCC 209483.
[1295] Based on the DNA35958 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 PRO304.
88 Pairs of PCR primers (forward and reverse) were synthesized:
forward PCR primer 1 5'-GCGGAAGGGCAGAATGGGACTCCAAG-3' (SEQ ID
NO:271) forward PCR primer 2 5'-CAGCCCTGCCACATGTGC-3' (SEQ ID
NO:272) forward PCR primer 3 5'-TACTGGGTGGTCAGCAAC-3' (SEQ ID
NO:273) reverse PCR primer 5'-GGCGAAGAGCAGGGTGAGACCCCG-3' (SEQ ID
NO:274)
[1296] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA35958 sequence which
had the following nucleotide sequence
89 hybridization probe: 5'-GCCCTCATCCTCTCTGGCAAATGCAGTTACAG-
CCCGGAGCCCGAC-3' (SEQ ID NO:275)
[1297] 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
PRO304 gene using the probe oligonucleotide and one of the PCR
primers.
[1298] RNA for construction of the cDNA libraries was isolated from
22 week human fetal brain tissue (LIB153).
[1299] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO304 [herein designated as
DNA39520-1217] (SEQ ID NO:258) and the derived protein sequence for
PRO304.
[1300] The entire nucleotide sequence of DNA39520-1217 is shown in
FIG. 93 (SEQ ID NO:258). Clone DNA39520-1217 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 34-36 and ending at the stop codon at
nucleotide positions 1702-1704 (FIG. 93). The predicted polypeptide
precursor is 556 amino acids long (FIG. 94). Various unique aspects
of the PRO304 protein are shown in FIG. 94. Clone DNA39520-1217 has
been deposited with ATCC on Nov. 21, 1997 and is assigned ATCC
deposit no. ATCC 209482.
[1301] Based on the DNA37160 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 PRO307.
90 Pairs of PCR primers (forward and reverse) were synthesized:
forward PCR primer 1 5'-GGGCAGGGATTCCAGGGCTCC-3' (SEQ ID NO:276)
forward PCR primer 2 5'-GGCTATGACAGCAGGTTC-3' (SEQ ID NO:277)
forward PCR primer 3 5'-TGACAATGACCGACCAGG-3' (SEQ ID NO:278)
reverse PCR primer 5'-GCATCGCATTGCTGGTAGAGCAAG-3' (SEQ ID
NO:279)
[1302] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA37160 sequence which
had the following nucleotide sequence
91 hybridization probe 5'-TTACAGTGCCCCCTGGAAACCCACTTGGCCTGC-
ATACCGCCTCCC-3' (SEQ ID NO:280)
[1303] 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
PRO307 gene using the probe oligonucleotide and one of the PCR
primers.
[1304] RNA for construction of the cDNA libraries was isolated from
human fetal liver tissue (LIB229).
[1305] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO307 [herein designated as
DNA41225-1217] (SEQ ID NO:260) and the derived protein sequence for
PRO307.
[1306] The entire nucleotide sequence of DNA41225-1217 is shown in
FIG. 95 (SEQ ID NO:260). Clone DNA41225-1217 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 92-94 and ending at the stop codon at
nucleotide positions 1241-1243 (FIG. 95). The predicted polypeptide
precursor is 383 amino acids long (FIG. 96). Various unique aspects
of the PRO307 protein are shown in FIG. 96. Clone DNA41225-1217 has
been deposited with ATCC on Nov. 21, 1997 and is assigned ATCC
deposit no. ATCC 209491.
[1307] Based on the DNA30895 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 PRO343.
92 A pair of PCR primers (forward and reverse) were synthesized:
forward PCR primer 5'-CGTCTCGAGCGCTCCATACAGTTCCCTTGCCCCA-3' (SEQ ID
NO:281) reverse PCR primer
5'-TGGAGGGGGAGCGGGATGCTTGTCTGGGCGACTCCGGGGGCCCCCTCATGT-
GCCAGGTGGA-3' (SEQ ID NO:282)
[1308] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA30895 sequence which
had the following nucleotide sequence
93 hybridization probe 5'-CCCTCAGACCCTGCAGAAGCTGAAGGTTCCTA-
TCATCGACTCGGAAGTCTGCAGCCATCTG (SEQ ID NO:283)
TACTGGCGGGGAGCAGGACAGGGACCCATCACTGAGGACATGCTGTGTGCCGGCTACT-3'
[1309] 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
PRO343 gene using the probe oligonucleotide and one of the PCR
primers.
[1310] RNA for construction of the cDNA libraries was isolated from
human fetal lung tissue (LIB26).
[1311] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO343 [herein designated as
DNA43318-1217] (SEQ ID NO:262) and the derived protein sequence for
PRO343.
[1312] The entire nucleotide sequence of DNA43318-1217 is shown in
FIG. 97 (SEQ ID NO:262). Clone DNA43318-1217 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 53-55 and ending at the stop codon at
nucleotide positions 1004-1006 (FIG. 97). The predicted polypeptide
precursor is 317 amino acids long (FIG. 98). Various unique aspects
of the PRO343 protein are shown in FIG. 98. Clone DNA43318-1217 has
been deposited with ATCC on Nov. 21, 1997 and is assigned ATCC
deposit no. ATCC 209481.
Example 42
Isolation of cDNA Clones Encoding Human PRO328
[1313] 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.
94 Forward and reverse PCR primers were synthesized: forward PCR
primer 5'-TCCTGCAGTTTCCTGATGC-3' (SEQ ID NO:286) reverse PCR primer
5'-CTCATATTGCACACCAGTAATTCG-3- ' (SEQ ID NO:287)
[1314] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA35615 sequence which
had the following nucleotide sequence
95 hybridization probe 5'-ATGAGGAGAAACGTTTGATGGTGGAGCTGCACA-
ACCTCTACCGGG-3' (SEQ ID NO:288)
[1315] 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.
[1316] RNA for construction of the cDNA libraries was isolated from
human fetal kidney tissue.
[1317] 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:284) and the derived protein sequence for
PRO328.
[1318] The entire nucleotide sequence of DNA40587-1231 is shown in
FIG. 99 (SEQ ID NO:284). 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. 99). The predicted polypeptide
precursor is 463 amino acids long (FIG. 100). Clone DNA40587-1231
has been deposited with ATCC and is assigned ATCC deposit no. ATCC
209438.
[1319] 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 43
Isolation of cDNA Clones Encoding Human PRO335, PRO331 or
PRO326
[1320] 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 DNA36685. Based on the
DNA36685 consensus sequence, and Incyte EST sequence no. 2228990,
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
PRO335, PRO331 or PRO326.
96 Forward and reverse PCR primers were synthesized: forward PCR
primer 5'-GGAACCGAATCTCAGCTA-3' (SEQ ID NO:295) forward PCR primer
5'-CCTAAACTGAACTGGACCA-3' (SEQ ID NO:296) forward PCR primer
5'-GGCTGGAGACACTGAACCT-3' (SEQ ID NO:297) forward PCR primer
5'-ACAGCTGCACAGCTCAGAACAGTG-3' (SEQ ID NO:298) reverse PCR primer
5'-CATTCCCAGTATAAAAATTTTC-3' (SEQ ID NO:299) reverse PCR primer
5'-GGGTCTTGGTGAATGAGG-3' (SEQ ID NO:300) reverse PCR primer
5'-GTGCCTCTCGGTTACCACCAATGG-3' (SEQ ID NO:301)
[1321] Additionally, a synthetic oligonucleotide hybridization
probe was constructed for the determination of PRO335 which had the
following nucleotide sequence
97 hybridization probe 5'-GCGGCCACTGTTGGACCGAACTGTAACCAAGGG-
AGAAACAGCCGTCCTAC-3' (SEQ ID NO:302)
[1322]
98 Forward and reverse PCR primers were synthesized for the
determination of PRO331: forward PCR primer
5'-GCCTTTGACAACCTTCAGTCACTAGTGG-3' (SEQ ID NO:303) reverse PCR
primer 5'-CCCCATGTGTCCATGACTGTTCCC-3' (SEQ ID NO:304)
[1323] Additionally, a synthetic oligonucleotide hybridization
probe was constructed for the determination of PRO331 which bad the
following nucleotide sequence
99 hybridization probe 5'-TACTGCCTCATGACCTCTTCACTCCCTTGCATC-
ATCTTAGAGCGG-3' (SEQ ID NO:305)
[1324]
100 Forward and reverse PCR primers were synthesized for the
determi- nation of PRO326: forward PCR primer
5'-ACTCCAAGGAAATCGGATCCGTTC-3' (SEQ ID NO:306) reverse PCR primer
5'-TTAGCAGCTGAGGATGGGCACAAC-3' (SEQ ID NO:307)
[1325] Additionally, a synthetic oligonucleotide hybridization
probe was constructed for the determination of PRO331 which had the
following nucleotide sequence
101 hybridization probe: 5'-GCCTTCACTGGTTTGGATGCATTGGAGCATC-
TAGACCTGAGTGACAACGC-3' (SEQ ID NO:308)
[1326] 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
PRO335, PRO331 or PRO326 gene using the probe oligonucleotide and
one of the PCR primers.
[1327] RNA for construction of the cDNA libraries was isolated from
human fetal kidney tissue (PRO335 and PRO326) and human fetal brain
(PRO331).
[1328] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO335, PRO331 or PRO326
[herein designated as SEQ ID NOS:289, 291 and 293, respectively;
see FIGS. 101, 103 and 105, respectively], and the derived protein
sequence for PRO335, PRO331 or PRO326 (see FIGS. 102, 104 and 106,
respectively; SEQ ID NOS:290, 292 and 294, respectively).
[1329] The entire nucleotide sequences are shown in FIGS. 101, 103
and 105, deposited with the ATCC on Jun. 2, 1998, Nov. 7, 1997 and
Nov. 21, 1997, respectively.
[1330] Analysis of the amino acid sequence of the full-length
PRO335, PRO331 or PRO326 polypeptide suggests that portions of it
possess significant homology to the LIG-1 protein, thereby
indicating that PRO335, PRO331 and PRO326 may be a novel
LIG-1-related protein.
Example 44
Isolation of cDNA clones Encoding Human PRO332
[1331] Based upon an ECD homology search performed as described in
Example 1 above, a consensus DNA sequence designated herein as
DNA36688 was assembled. Based on the DNA36688 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 PRO332.
102 A pair of PCR primers (forward and reverse) were synthesized:
5'-GCATTGGCCGCGAGACTTTGCC-3' (SEQ ID NO:311)
5'-GCGGCCACGGTCCTTGGAAATG-3' (SEQ ID NO:312)
[1332] A probe was also synthesized:
103 5'-TGGAGGAGCTCAACCTCAGCTACAACCGCATCACCAGCCCACAGG-3' (SEQ ID
NO:313)
[1333] 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 PRO332 gene
using the probe oligonucleotide and one of the PCR primers.
[1334] RNA for construction of the cDNA libraries was isolated from
a human fetal liver library (LIB229).
[1335] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for DNA40982-1235 and the derived
protein sequence for PRO332.
[1336] The entire nucleotide sequence of DNA40982-1235 is shown in
FIG. 107 (SEQ ID NO:309). Clone DNA40982-1235 contains a single
open reading frame (with an apparent translational initiation site
at nucleotide positions 342-344, as indicated in FIG. 107). The
predicted polypeptide precursor is 642 amino acids long, and has a
calculated molecular weight of 72,067 (p1: 6.60). Clone
DNA40982-1235 has been deposited with ATCC and is assigned ATCC
deposit no. ATCC 209433.
[1337] Based on a BLAST and FastA sequence alignment analysis of
the full-length sequence, PRO332 shows about 30-40% amino acid
sequence identity with a series of known proteoglycan sequences,
including, for example, fibromodulin and fibromodulin precursor
sequences of various species (FMOD_BOVIN, FMOD CHICK, FMOD_RAT,
FMOD_MOUSE, FMOD_HUMAN, P_R36773), osteomodulin sequences
(AB0001141, AB0078481), decorin sequences (CFU83141.sub.--1,
OCU033941, P_R42266, P_R42267, P_R42260, P_R89439), keratan sulfate
proteoglycans (BTU48360.sub.--1, AF022890.sub.--1), corneal
proteoglycan (AF022256.sub.--1), and bone/cartilage proteoglycans
and proteoglycane precursors (PGS1_BOVIN, PGS2_MOUSE,
PGS2_HUMAN).
Example 45
Isolation of cDNA clones Encoding Human PRO334
[1338] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described in Example 1 above. Based on the
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 PRO334.
104 Forward and reverse PCR primers were synthesized for the
determi- nation of PRO334: forward PCR primer
5'-GATGGTTCCTGCTCAAGTGCCCTG-3' (SEQ ID NO:316) reverse PCR primer
5'-TTGCACTTGTAGGACCCACGTACG-3' (SEQ ID NO:317)
[1339] Additionally, a synthetic oligonucleotide hybridization
probe was constructed for the determination of PRO334 which had the
following nucleotide sequence
105 hybridization probe 5'-CTGATGGGAGGACCTGTGTAGATGTTGATGAA-
TGTGCTACAGGAAGAGCC-3' (SEQ ID NO:318)
[1340] 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 PRO334 gene
using the probe oligonucleotide and one of the PCR primers.
[1341] Human fetal kidney 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.
[1342] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO334 [herein designated as
DNA41379-1236] (SEQ ID NO:314) and the derived protein sequence for
PRO334.
[1343] The entire nucleotide sequence of DNA41379-1236 (also
referred to as UNQ295) is shown in FIG. 109 (SEQ ID NO:314). Clone
DNA41379-1236 contains a single open reading frame with an apparent
translational initiation site at nucleotide positions 203-205 and
ending at the stop codon at nucleotide positions 1730-1732 (FIG.
109). The predicted polypeptide precursor is 509 amino acids long
(FIG. 110). Clone DNA41379-1236 has been deposited with ATCC and is
assigned ATCC deposit no. ATCC 209488.
[1344] Analysis of the amino acid sequence of the full-length
PRO334 polypeptide suggests that portions of it possess significant
homology to the fibulin and fibrillin proteins, thereby indicating
that PRO334 may be a novel member of the EGF protein family.
Example 46
Isolation of cDNA Clones Encoding Human PRO346
[1345] A consensus DNA sequence was identified using phrap as
described in Example 1 above. Specifically, this consensus sequence
is herein designated DNA38240. Based on the DNA38240 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 PRO346 coding
sequence.
[1346] RNA for construction of the cDNA libraries was isolated from
human fetal liver. The cDNA libraries used to isolated the cDNA
clones were constructed by standard methods using commercially
available reagents (e.g., Invitrogen, San Diego, Calif.; Clontech,
etc.) 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.
[1347] A cDNA clone was sequenced in entirety. The entire
nucleotide sequence of DNA44167-1243 is shown in FIG. 111 (SEQ ID
NO:319). Clone DNA44167-1243 contains a single open reading frame
with an apparent translational initiation site at nucleotide
positions 64-66 (FIG. 1 11; SEQ ID NO:319). The predicted
polypeptide precursor is 450 amino acids long. Clone DNA44167-1243
has been deposited with ATCC and is assigned ATCC deposit no. ATCC
209434 (designation DNA44167-1243).
[1348] Based on a BLAST, BLAST-2 and FastA sequence alignment
analysis (using the ALIGN computer program) of the full-length
sequence, PRO346 shows amino acid sequence identity to
carcinoembryonic antigen (28%).
[1349] The oligonucleotide sequences used in the above procedure
were the following: OL12691 (38240.f1)
106 5'-GATCCTGTCACAAAGCCAGTGGTGC-3' (SEQ ID NO:321) OLI2693
(38240.r1) 5'-CACTGACAGGGTTCCTCACCCAGG-3' (SEQ ID NO:322) OLI2692
(38240.p1) 5'-CTCCCTCTGGGCTGTGGAGTATGTGGGG- AACATGACCCTGACATG-3'
(SEQ ID NO:323)
Example 47
Isolation of cDNA Clones Encoding Human PRO268
[1350] 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 DNA35698. Based on the
DNA35698 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 PRO268.
107 Forward and reverse PCR primers were synthesized: forward PCR
primer 1 5'-TGAGGTGGGCAAGCGGCGAAATG-3' (SEQ ID NO:326) forward PCR
primer 2 5'-TATGTGGATCAGGACGTGCC-- 3' (SEQ ID NO:327) forward PCR
primer 3 5'-TGCAGGGTTCAGTCTAGATTG-3' (SEQ ID NO:328) reverse PCR
primer 5'-TTGAAGGACAAAGGCAATCTGCCAC-3' (SEQ ID NO:329)
[1351] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA35698 sequence which
had the following nucleotide sequence
108 hybridization probe: 5'-GGAGTCTTGCAGTTCCCCTGGCAGTCCTGGT-
GCTGTTGCTTTGGG-3' (SEQ ID NO:330)
[1352] 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 PRO268 gene
using the probe oligonucleotide and one of the PCR primers.
[1353] RNA for construction of the cDNA libraries was isolated from
human fetal lung tissue.
[1354] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO268 [herein designated as
DNA39427-1179] (SEQ ID NO:324) and the derived protein sequence for
PRO268.
[1355] The entire nucleotide sequence of DNA39427-1179 is shown in
FIG. 113 (SEQ ID NO:324). Clone DNA39427-1179 contains a single
open reading frame with an apparent translational initiation site
at nucleotide positions 13-15 and ending at the stop codon at
nucleotide positions 853-855 (FIG. 113). The predicted polypeptide
precursor is 280 amino acids long (FIG. 114). Clone DNA39427-1179
has been deposited with ATCC and is assigned ATCC deposit no. ATCC
209395.
[1356] Analysis of the amino acid sequence of the full-length
PRO268 polypeptide suggests that it possess significant homology to
protein disulfide isomerase, thereby indicating that PRO268 may be
a novel protein disulfide isomerase.
Example 48
Isolation of cDNA Clones Encoding Human PRO330
[1357] 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 DNA35730. Based on the
DNA35730 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 PRO330.
109 Forward and reverse PCR primers were synthesized: forward PCR
primer 1 5'-CCAGGCACAATTTCCAGA-3' (SEQ ID NO:333) forward PCR
primer 2 5'-GGACCCTTCTGTGTGCCAG-3' (SEQ ID NO:334) reverse PCR
primer 1 5'-GGTCTCAAGAACTCCTGTC-3' (SEQ ID NO:335) reverse PCR
primer 2 5'-ACACTCAGCATTGCCTGGTACTTG-3' (SEQ ID NO:336)
[1358] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus sequence which had the
following nucleotide sequence
110 hybridization probe 5'-GGGCACATGACTGACCTGATTTATGCAGAGAA-
AGAGCTGGTGCAG-3' (SEQ ID NO:337)
[1359] 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 PRO330 gene
using the probe oligonucleotide and one of the PCR primers.
[1360] RNA for construction of the cDNA libraries was isolated from
human fetal liver tissue.
[1361] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO330 [herein designated as
DNA40603-1232] (SEQ ID NO:331) and the derived protein sequence for
PRO330.
[1362] The entire nucleotide sequence of DNA40603-1232 is shown in
FIG. 115 (SEQ ID NO:331). Clone DNA40603-1232 contains a single
open reading frame with an apparent translational initiation site
at nucleotide positions 167-169 and ending at the stop codon at
nucleotide positions 1766-1768 (FIG. 115). The predicted
polypeptide precursor is 533 amino acids long (FIG. 116). Clone
DNA40603-1232 has been deposited with ATCC and is assigned ATCC
deposit no.ATCC 209486 on Nov. 21, 1997.
[1363] Analysis of the amino acid sequence of the full-length
PRO330 polypeptide suggests that portions of it possess significant
homology to the mouse prolyl 4-hydroxylase alpha subunit protein,
thereby indicating that PRO330 may be a novel prolyl 4-hydroxylase
alpha subunit polypeptide.
Example 49
Isolation of cDNA Clones Encoding Human PRO310
[1364] 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 DNA40553. Based on the
DNA40553 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 PRO310.
111 Forward and reverse PCR primers were synthesized: forward PCR
primer 1 5'-TCCCCAAGCCGTTCTAGACGCGG-3' (SEQ ID NO:342) forward PCR
primer 2 5'-CTGGTTCTTCCTTGCACG-3' (SEQ ID NO:343) reverse PCR
primer 5'-GCCCAAATGCCCTAAGGCGGTATACCCC-3' (SEQ ID NO:344)
[1365] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus sequence which had the
following nucleotide sequence
112 hybridization probe 5'-GGGTGTGATGCTTGGAAGCATTTTCTGTGCTT-
TGATCACTATGCTAGGAC-3' (SEQ ID NO:345)
[1366] 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 PRO310 gene
using the probe oligonucleotide and one of the PCR primers.
[1367] RNA for construction of the cDNA libraries was isolated from
human fetal liver tissue.
[1368] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO310 [herein designated as
DNA43046-1225 (SEQ ID NO:340) and the derived protein sequence for
PRO310 (SEQ ID NO:341).
[1369] The entire nucleotide sequence of DNA43046-1225 is shown in
FIG. 119 (SEQ ID NO:340). Clone DNA43046-1225 contains a single
open reading frame with an apparent translational initiation site
at nucleotide positions 81-83 and ending at the stop codon at
nucleotide positions 1035-1037 (FIG. 119). The predicted
polypeptide precursor is 318 amino acids long (FIG. 120) and has a
calculated molecular weight of approximately 36,382 daltons. Clone
DNA43046-1225 has been deposited with ATCC and is assigned ATCC
deposit no. ATCC 209484.
[1370] Analysis of the amino acid sequence of the full-length
PRO310 polypeptide suggests that portions of it possess homology to
C. elegans proteins and to fringe, thereby indicating that PRO310
may be involved in development.
Example 50
Isolation of cDNA clones Encoding Human PRO339
[1371] An expressed sequence tag (EST) DNA database (LIFESEQ.TM.,
Incyte Pharmaceuticals, Palo Alto, Calif.) was searched and ESTs
were identified. An assembly of Incyte clones and a consensus
sequence was formed using phrap as described in Example 1
above.
[1372] Forward and reverse PCR primers were synthesized based upon
the assembly-created consensus sequence:
113 forward PCR primer 1 5'-GGGATGCAGGTGGTGTCTCATGGGG-3' (SEQ ID
NO:346) forward PCR primer 2 5'-CCCTCATGTACCGGCTCC-3' (SEQ ID
NO:347) forward PCR primer 3 5'-GTGTGACACAGCGTGGGC-3' (SEQ ID
NO:43) forward PCR primer 4 5'-GACCGGCAGGCTTCTGCG-3' (SEQ ID NO:44)
reverse PCR primer 1 5'-CAGCAGCTTCAGCCACCAGGAGTGG-3' (SEQ ID NO:45)
reverse PCR primer 2 5'-CTGAGCCGTGGGCTGCAGTCTCGC-3' (SEQ ID
NO:46)
[1373] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus sequence which had the
following nucleotide sequence
114 hybridization probe 5'-CCGACTACGACTGGTTCTTCATCATGCAGGAT-
GACACATATGTGC-3' (SEQ ID NO:47)
[1374] 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
PRO339 gene using the probe oligonucleotide and one of the PCR
primers.
[1375] RNA for construction of the cDNA libraries was isolated from
human fetal liver tissue.
[1376] A cDNA clone was sequenced in entirety. The entire
nucleotide sequence of DNA43466-1225 is shown in FIG. 117 (SEQ ID
NO:338). Clone DNA43466-1225 contains a single open reading frame
with an apparent translational initiation site at nucleotide
positions 333-335 and ending at the stop codon found at nucleotide
positions 2649-2651 (FIG. 117; SEQ ID NO:338). The predicted
polypeptide precursor is 772 amino acids long and has a calculated
molecular weight of approximately 86,226 daltons. Clone
DNA43466-1225 has been deposited with ATCC and is assigned ATCC
deposit no. ATCC 209490.
[1377] Based on a BLAST and FastA sequence alignment analysis
(using the ALIGN computer program) of the full-length sequence,
PRO339 has homology to C. elegans proteins and collagen-like
polymer sequences as well as to fringe, thereby indicating that
PRO339 may be involved in development or tissue growth.
Example 51
Isolation of cDNA Clones Encoding Human PRO244
[1378] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described in Example 1 above. Based on
this 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 PRO244.
115 A pair of PCR primers (forward and reverse) were synthesized:
5'-TTCAGCTTCTGGGATGTAGGG-3' (30923.f1) (SEQ ID NO:378)
5'-TATTCCTACCATTTCACAAATCCG-3- ' (30923.r1) (SEQ ID NO:379) A probe
was also synthesized:
5'-GGAGGACTGTGCCACCATGAGAGACTCTTCAAACCCAAGGCAAAATTGG-3' (30923.p1)
(SEQ ID NO:380)
[1379] 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 PRO244 gene
using the probe oligonucleotide and one of the PCR primers.
[1380] RNA for construction of the cDNA libraries was isolated from
a human fetal kidney library. DNA sequencing of the clones isolated
as described above gave the full-length DNA sequence and the
derived protein sequence for PRO244.
[1381] The entire nucleotide sequence of PRO244 is shown in FIG.
121 (SEQ ID NO:376). Clone DNA35668-1171 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 106-108 (FIG. 121). The predicted polypeptide
precursor is 219 amino acids long. Clone DNA35668-1171 has been
deposited with ATCC (designated as DNA35663-1171) and is assigned
ATCC deposit no. ATCC209371. The protein has a cytoplasmic domain
(aa 1-20), a transmembrane domain (aa 2146), and an extracellular
domain (aa 47-219), with a C-lectin domain at aa 55-206.
[1382] Based on a BLAST and FastA sequence alignment analysis of
the full-length sequence, PRO244 shows notable amino acid sequence
identity to hepatic lectin gallus gallus (43%), HIC hp120-binding
C-type lectin (42%), macrophage lectin 2 (HUMHML2-1, 41%), and
sequence PR32188 (44%).
Example 52
Use of PRO Polypeptide-Encoding Nucleic Acid as Hybridization
Probes
[1383] The following method describes use of a nucleotide sequence
encoding a PRO polypeptide as a hybridization probe.
[1384] DNA comprising the coding sequence of of a PRO polypeptide
of interest as disclosed herein may be employed as a probe or used
as a basis from which to prepare probes to screen for homologous
DNAs (such as those encoding naturally-occurring variants of the
PRO polypeptide) in human tissue cDNA libraries or human tissue
genomic libraries.
[1385] Hybridization and washing of filters containing either
library DNAs is performed under the following high stringency
conditions. Hybridization of radiolabeled PRO polypeptide-encoding
nucleic acid-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.
[1386] DNAs having a desired sequence identity with the DNA
encoding full-length native sequence PRO polypeptide can then be
identified using standard techniques known in the art.
Example 53
Expression of PRO Polypeptides in E. coli
[1387] This example illustrates preparation of an unglycosylated
form of a desired PRO polypeptide by recombinant expression in E.
coli.
[1388] The DNA sequence encoding the desired PRO 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 specific PRO
polypeptide coding region, lambda transcriptional terminator, and
an argU gene.
[1389] 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.
[1390] 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.
[1391] 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 PRO polypeptide can then be purified using
a metal chelating column under conditions that allow tight binding
of the protein.
[1392] PRO187, PRO317, PRO301, PRO224 and PRO238 were successfully
expressed in E. coli in apoly-His tagged form, using the following
procedure. The DNA encoding PRO187, PRO317, PRO301, PRO224 or
PRO238 was initially amplified using selected PCR primers. The
primers contained 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 were then ligated into an
expression vector, which was used to transform an E. coil host
based on strain 52 (W3110 fuhA(tonA) Ion galE rpoHts(htpRts)
clpP(lacIq). Transformants were first grown in LB containing 50
mg/ml carbenicillin at 30.degree. C. with shaking until an O.D.600
of 3-5 was reached. Cultures were then diluted 50-100 fold into
CRAP media (prepared by mixing 3.57 g (NH.sub.4)2SO4, 0.71 g sodium
citrate-2H.sub.2O, 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
were removed to verify expression by SDS-PAGE analysis, and the
bulk culture is centrifuged to pellet the cells. Cell pellets were
frozen until purification and refolding.
[1393] E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets)
was resuspended in 10 volumes (wlv) 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 was stirred overnight at 4.degree. C. This step
results in a denatured protein with all cysteine residues blocked
by sulfitolization. The solution was centrifuged at 40,000 rpm in a
Beckman Ultracentifuge for 30 min. The supernatant was 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.
Depending the clarified extract was loaded onto a 5 ml Qiagen
Ni-NTA metal chelate column equilibrated in the metal chelate
column buffer. The column was washed with additional buffer
containing 50 mM imidazole (Calbiochem, Utrol grade), pH 7.4. The
protein was eluted with buffer containing 250 mM imidazole.
Fractions containing the desired protein were pooled and stored at
4.degree. C. Protein concentration was estimated by its absorbance
at 280 nm using the calculated extinction coefficient based on its
amino acid sequence.
[1394] The proteins were refolded by diluting 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 were chosen so that the final protein
concentration was between 50 to 100 micrograms/ml. The refolding
solution was stirred gently at 4.degree. C. for 12-36 hours. The
refolding reaction was 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 was filtered through a
0.22 micron filter and acetonitrile was added to 2-10% final
concentration. The refolded protein was 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 were analyzed on SDS polyacrylamide
gels and fractions containing homogeneous refolded protein were
pooled. Generally, the properly refolded species of most proteins
are eluted at the lowest conceni rations 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.
[1395] Fractions containing the desired folded PRO187, PRO317,
PRO301, PRO224 and PRO238 proteins, respectively, were pooled and
the acetonitrile removed using a gentle stream of nitrogen directed
at the solution. Proteins were 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 54
Expression of PRO Polypeptides in Mammalian Cells
[1396] This example illustrates preparation of a glycosylated form
of a desired PRO polypeptide by recombinant expression in mammalian
cells.
[1397] The vector, pRK5 (see EP 307,247, published Mar. 15, 1989),
is employed as the expression vector. Optionally, the PRO
polypeptide-encoding DNA is ligated into pRK5 with selected
restriction enzymes to allow insertion of the PRO polypeptide DNA
using ligation methods such as described in Sambrook et al., supra.
The resulting vector is called pRK5-PRO polypeptide.
[1398] In one embodiment, 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-PRO polypeptide 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.
[1399] 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 PRO polypeptide. The cultures containing transfected
cells may undergo further incubation (in serum free medium) and the
medium is tested in selected bioassays.
[1400] In an alternative technique, PRO polypeptide 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-PRO polypeptide 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 PRO polypeptide can then be concentrated and
purified by any selected method, such as dialysis and/or column
chromatography.
[1401] In another embodiment, PRO polypeptides can be expressed in
CHO cells. The pRK5-PRO polypeptide 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 PRO 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 PRO polypeptide can then be
concentrated and purified by any selected method.
[1402] Epitope-tagged PRO polypeptide may also be expressed in host
CHO cells. The PRO polypeptide 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 PRO polypeptide 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 PRO polypeptide can then be concentrated and
purified by any selected method, such as by Ni.sup.2--chelate
affinity chromatography. PRO211, PRO217, PRO230, PRO219, PRO245,
PRO221, PRO258, PRO301, PRO224, PRO222, PRO234, PRO229, PRO223,
PRO328 and PRO332 were successfully expressed in CHO cells by both
a transient and a stable expression procedure. In addition, PRO232,
PRO265, PRO246, PRO228, PRO227, PRO220, PRO266, PRO269, PRO287,
PRO214, PRO231, PRO233, PRO238, PRO244, PRO235, PRO236, PRO262,
PRO239, PRO257, PRO260, PRO263, PRO270, PRO271, PRO272, PRO294,
PRO295, PRO293, PRO247, PRO303 and PRO268 were successfully
transiently expressed in CHO cells.
[1403] Stable expression in CHO cells was performed using the
following procedure. The proteins were expressed as an IgG
construct (immunoadhesin), in which the coding sequences for the
soluble forms (e.g. extracellular domains) of the respective
proteins were fused to an IgG1 constant region sequence containing
the hinge, CH2 and CH2 domains and/or is a poly-His tagged
form.
[1404] Following PCR amplification, the respective DNAs were
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.
[1405] Twelve micrograms of the desired plasmid DNA were introduced
into approximately 10 million CHO cells using commercially
available transfection reagents Superfect.RTM. (Quiagen),
Dosper.RTM. or Fugene.RTM. (Boehringer Mannheim). The cells were
grown and 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.
[1406] The ampules containing the plasmid DNA were thawed by
placement into water bath and mixed by vortexing. The contents were
pipetted into a centrifuge tube containing 10 mLs of media and
centrifuged at 1000 rpm for 5 minutes. The supernatant was
aspirated and the cells were 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 were then aliquoted into a 100 mL spinner
containing 90 mL of selective media. After 1-2 days, the cells were
transferred into a 250 mL spinner filled with 150 mL selective
growth medium and incubated at 37.degree. C. After another 2-3
days, a 250 mL, 500 mL and 2000 mL spinners were seeded with
3.times.10.sup.5 cells/mL. The cell media was 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
was actually used. 3L production spinner is seeded at
1.2.times.10.sup.6 cells/mL. On day 0, the cell number pH were
determined. On day 1, the spinner was sampled and sparging with
filtered air was commenced. On day 2, the spinner was 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). Throughout the
production, pH was adjusted as necessary to keep at around 7.2.
After 10 days, or until viability dropped below 70%, the cell
culture was harvested by centrifugtion 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.
[1407] For the poly-His tagged constructs, the proteins were
purified using a Ni-NTA column (Qiagen). Before purification,
imidazole was added to the conditioned media to a concentration of
5 mM. The conditioned media was 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 was washed with additional equilibration
buffer and the protein eluted with equilibration buffer containing
0.25 M imidazole. The highly purified protein was 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.
[1408] Immunoadhesin (Fc containing) constructs of were purified
from the conditioned media as follows. The conditioned medium was
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 was washed extensively with equilibration buffer before
elution with 100 mM citric acid, pH 3.5. The eluted protein was
immediately neutralized by collecting 1 ml fractions into tubes
containing 275 .mu.L of 1 M Tris buffer, pH 9. The highly purified
protein was subsequently desalted into storage buffer as described
above for the poly-His tagged proteins. The homogeneity was
assessed by SDS polyacrylamide gels and by N-terminal amino acid
sequencing by Edman degradation.
[1409] PRO211, PRO217, PRO230, PRO232, PRO187, PRO265, PRO219,
PRO246, PRO228, PRO533, PRO245, PRO221, PRO227, PRO220, PRO258,
PRO266, PRO269, PRO287, PRO214, PRO317, PRO301, PRO224, PRO222,
PRO234, PRO231, PRO229, PRO233, PRO238, PRO223, PRO235, PRO236,
PRO262, PRO239, PRO257, PRO260, PRO263, PRO270, PRO271, PRO272,
PRO294, PRO295, PRO293, PRO247, PRO304, PRO302, PRO307, PRO303,
PRO343, PRO328, PRO326, PRO331, PRO332, PRO334, PRO346, PRO268,
PRO330, PRO310 and PRO339 were also successfully transiently
expressed in COS cells.
Example 55
Expression of PRO Polypeptides in Yeast
[1410] The following method describes recombinant expression of a
desired PRO polypeptide in yeast.
[1411] First, yeast expression vectors are constructed for
intracellular production or secretion of PRO polypeptides from the
ADH2/GAPDH promoter. DNA encoding a desired PRO polypeptide, a
selected signal peptide and the promoter is inserted into suitable
restriction enzyme sites in the selected plasmid to direct
intracellular expression of the PRO polypeptide. For secretion, DNA
encoding the PRO polypeptide can be cloned into the selected
plasmid, together with DNA encoding the ADH2/GAPDH promoter, the
yeast alpha-factor secretory signal/leader sequence, and linker
sequences (if needed) for expression of the PRO polypeptide.
[1412] Yeast cells, such as yeast strain AB 110, 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.
[1413] Recombinant PRO polypeptide 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 the PRO polypeptide
may further be purified using selected column chromatography
resins.
Example 56
Expression of PRO Polypeptides in Baculovirus-Infected Insect
Cells
[1414] The following method describes recombinant expression of PRO
polypeptides in Baculovirus-infected insect cells.
[1415] The desired PRO polypeptide is fused upstream of an epitope
tag contained with 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 PRO polypeptide or the desired portion of
the PRO polypeptide (such as the sequence encoding the
extracellular domain of a transmembrane protein) 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.
[1416] 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 is performed as described by O'Reilley et al.,
Baculovirus expression vectors: A laboratory Manual, Oxford: Oxford
University Press (1994).
[1417] Expressed poly-his tagged PRO polypeptide 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% NP40; 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 PRO
polypeptide are pooled and dialyzed against loading buffer.
[1418] Alternatively, purification of the IgG tagged (or Fc tagged)
PRO polypeptide can be performed using known chromatography
techniques, including for instance, Protein A or protein G column
chromatography. PRO211, PRO217, PRO230, PRO187, PRO265, PRO246,
PRO228, PRO533, PRO245, PRO221, PRO220, PRO258, PRO266, PRO269,
PRO287, PRO214, PRO301, PRO224, PRO222, PRO234, PRO231, PRO229,
PRO235, PRO239, PRO257, PRO272, PRO294, PRO295, PRO328, PRO326,
PRO331, PRO334, PRO346 and PRO310 were successfully expressed in
baculovirus infected Sf9 or high5 insect cells. While the
expression was actually performed in a 0.5-2 L scale, it can be
readily scaled up for larger (e.g. 8 L) preparations. The proteins
were expressed as an IgG construct (immunoadhesin), in which the
protein extracellular region was fused to an IgG1 constant region
sequence containing the hinge, CH2 and CH3 domains and/or in
poly-His tagged forms.
[1419] Following PCR amplification, the respective coding sequences
were subcloned into a baculovirus expression vector (pb.PH.IgG for
IgG fusions and pb.PH.His.c for poly-His tagged proteins), and the
vector and Baculogold.RTM. baculovirus DNA (Pharmingen) were
co-transfected into 105 Spodoptera frugiperda ("Sf9") cells (ATCC
CRL 1711), using Lipofectin (Gibco BRL). pb.PH.IgG and pb.PH.His
are modifications of the commercially available baculovirus
expression vector pVL1393 (Pharmingen), with modified polylinker
regions to include the His or Fc tag sequences. The cells were
grown in Hink's TNM-FH medium supplemented with 10% FBS (Hyclone).
Cells were incubated for 5 days at 28.degree. C. The supernatant
was harvested and subsequently used for the first viral
amplification by infecting Sf9 cells in Hink's TNM-FH medium
supplemented with 10% FBS at an approximate multiplicity of
infection (MO1) of 10. Cells were incubated for 3 days at
28.degree. C. The supernatant was harvested and the expression of
the constructs in the baculovirus expression vector was determined
by batch binding of 1 ml of supernatant to 25 mL of Ni-NTA beads
(QIAGEN) for histidine tagged proteins or Protein-A Sepharose CL-4B
beads (Pharmacia) for IgG tagged proteins followed by SDS-PAGE
analysis comparing to a known concentration of protein standard by
Coomassie blue staining.
[1420] The first viral amplification supernatant was used to infect
a spinner culture (500 ml) of Sf9 cells grown in ESF-921 medium
(Expression Systems LLC) at an approximate MOI of 0.1. Cells were
incubated for 3 days at 28.degree. C. The supernatant was harvested
and filtered. Batch binding and SDS-PAGE analysis was repeated, as
necessary, until expression of the spinner culture was
confirmed.
[1421] The conditioned medium from the transfected cells (0.5 to 3
L) was harvested by centrifugation to remove the cells and filtered
through 0.22 micron filters. For the poly-His tagged constructs,
the protein construct were purified using a Ni-NTA column (Qiagen).
Before purification, imidazole was added to the conditioned media
to a concentration of 5 mM. The conditioned media were 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 was washed with
additional equilibration buffer and the protein eluted with
equilibration buffer containing 0.25 M imidazole. The highly
purified protein was 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.
[1422] Immunoadhesin (Fc containing) constructs of proteins were
purified from the conditioned media as follows. The conditioned
media were 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 was washed extensively with equilibration
buffer before elution with 100 mM citric acid, pH 3.5. The eluted
protein was immediately neutralized by collecting 1 ml fractions
into tubes containing 275 mL of 1 M Tris buffer, pH 9. The highly
purified protein was subsequently desalted into storage buffer as
described above for the poly-His tagged proteins. The homogeneity
of the proteins was verified by SDS polyacrylamide gel (PEG)
electrophoresis and N-terminal amino acid sequencing by Edman
degradation.
Example 57
Preparation of Antibodies that Bind to PRO Polypeptides
[1423] This example illustrates preparation of monoclonal
antibodies which can specifically bind to a PRO polypeptide.
[1424] 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 PRO polypeptide,
fusion proteins containing the PRO polypeptide, and cells
expressing recombinant PRO polypeptide on the cell surface.
Selection of the immunogen can be made by the skilled artisan
without undue experimentation.
[1425] Mice, such as Balb/c, are immunized with the PRO polypeptide
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-PRO polypeptide antibodies.
[1426] After a suitable antibody titer has been detected, the
animals "positive" for antibodies can be injected with a final
intravenous injection of PRO polypeptide. 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 P3.times.63AgU.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.
[1427] The hybridoma cells will be screened in an ELISA for
reactivity against the PRO polypeptide. Determination of "positive"
hybridoma cells secreting the desired monoclonal antibodies against
the PRO polypeptide is within the skill in the art.
[1428] The positive hybridoma cells can be injected
intraperitoneally into syngeneic Balb/c mice to produce ascites
containing the anti-PRO polypeptide 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 58
Chimeric PRO Polypeptides
[1429] PRO polypeptides may be expressed as chimeric proteins with
one or more additional polypeptide domains added to facilitate
protein purification. Such purification facilitating domains
include, but are not limited to, metal chelating peptides such as
histidine-tryptophan modules that allow purification on immobilized
metals, protein A domains that allow purification on immobilized
immunoglobulin, and the domain utilized in the FLAGS.TM.
extension/affinity purification system (Immunex Corp., Seattle
Wash.). The inclusion of a cleavable linker sequence such as Factor
XA or enterokinase (Invitrogen, San Diego Calif.) between the
purification domain and the PRO polypeptide sequence may be useful
to facilitate expression of DNA encoding the PRO polypeptide.
Example 59
Purification of PRO Polypeptides Using Specific Antibodies
[1430] Native or recombinant PRO polypeptides may be purified by a
variety of standard techniques in the art of protein purification.
For example, pro-PRO polypeptide, mature PRO polypeptide, or
pre-PRO polypeptide is purified by immunoaffinity chromatography
using antibodies specific for the PRO polypeptide of interest. In
general, an immunoaffinity column is constructed by covalently
coupling the anti-PRO polypeptide antibody to an activated
chromatographic resin.
[1431] 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.
[1432] Such an immunoaffinity column is utilized in the
purification of PRO polypeptide by preparing a fraction from cells
containing PRO 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 PRO polypeptide containing a signal sequence
may be secreted in useful quantity into the medium in which the
cells are grown.
[1433] A soluble PRO polypeptide-containing preparation is passed
over the immunoaffinity column, and the column is washed under
conditions that allow the preferential absorbance of PRO
polypeptide (e.g., high ionic strength buffers in the presence of
detergent). Then, the column is eluted under conditions that
disrupt antibody/PRO 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 PRO polypeptide is
collected.
Example 60
Drug Screening
[1434] This invention is particularly useful for screening
compounds by using PRO polypeptides or binding fragment thereof in
any of a variety of drug screening techniques. The PRO 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 PRO 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 PRO polypeptide or a
fragment and the agent being tested. Alternatively, one can examine
the diminution in complex formation between the PRO polypeptide and
its target cell or target receptors caused by the agent being
tested.
[1435] Thus, the present invention provides methods of screening
for drugs or any other agents which can affect a PRO
polypeptide-associated disease or disorder. These methods comprise
contacting such an agent with an PRO polypeptide or fragment
thereof and assaying (I) for the presence of a complex between the
agent and the PRO polypeptide or fragment, or (ii) for the presence
of a complex between the PRO polypeptide or fragment and the cell,
by methods well known in the art. In such competitive binding
assays, the PRO polypeptide or fragment is typically labeled. After
suitable incubation, free PRO 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 PRO polypeptide or to interfere with the PRO
polypeptide/cell complex.
[1436] 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 PRO polypeptide, the peptide test compounds are reacted with
PRO polypeptide and washed. Bound PRO polypeptide is detected by
methods well known in the art. Purified PRO 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.
[1437] This invention also contemplates the use of competitive drug
screening assays in which neutralizing antibodies capable of
binding PRO polypeptide specifically compete with a test compound
for binding to PRO 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 PRO
polypeptide.
Example 61
Rational Drug Design
[1438] The goal of rational drug design is to produce structural
analogs of biologically active polypeptide of interest (i.e., a PRO
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
PRO polypeptide or which enhance or interfere with the function of
the PRO polypeptide in vivo (cf., Hodgson, Bio/Technology, 9:
19-21(1991)).
[1439] In one approach, the three-dimensional structure of the PRO
polypeptide, or of an PRO 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 PRO 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 PRO
polypeptide may be gained by modeling based on the structure of
homologous proteins. In both cases, relevant structural information
is used to design analogous PRO 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).
[1440] 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.
[1441] By virtue of the present invention, sufficient amounts of
the PRO polypeptide may be made available to perform such
analytical studies as X-ray crystallography. In addition, knowledge
of the PRO 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.
Example 62
Diagnostic Test Using PRO317 Polypeptide-Specific Antibodies
[1442] Particular anti-PRO317 polypeptide antibodies are useful for
the diagnosis of prepathologic conditions, and chronic or acute
diseases such as gynecological diseases or ischemic diseases which
are characterized by differences in the amount or distribution of
PRO317. PRO317 has been found to be expressed in human kidney and
is thus likely to be associated with abnormalities or pathologies
which affect this organ. Further, since it is so closely related to
EBAF-1, it is likely to affect the endometrium and other genital
tissues. Further, due to library sources of certain ESTs, it
appears that PRO317 may be involved as well in forming blood
vessels and hence to be a modulator of angiogenesis.
[1443] Diagnostic tests for PRO317 include methods utilizing the
antibody and a label to detect PRO317 in human body fluids,
tissues, or extracts of such tissues. The polypeptide and
antibodies of the present invention may be used with or without
modification. Frequently, the polypeptide and antibodies will be
labeled by joining them, either covalently or noncovalently, with a
substance which provides for a detectable signal. A wide variety of
labels and conjugation techniques are known and have been reported
extensively in both the scientific and patent literature. Suitable
labels include radionuclides, enzymes, substrates, cofactors,
inhibitors, fluorescent agents, chemiluminescent agents, magnetic
particles, and the like. Patents teaching the use of such labels
include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;
4,277,437; 4,275,149; and 4,366,241. Also, recombinant
immunoglobulins may be produced as shown in U.S. Pat. No.
4,816,567.
[1444] A variety of protocols for measuring soluble or
membrane-bound PRo317, using either polyclonal or monoclonal
antibodies specific for that PRO317, are known in the art. Examples
include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay
(RIA), radioreceptor assay (RRA), and fluorescent activated cell
sorting (FACS). A two-site monoclonal-based immunoassay utilizing
monoclonal antibodies reactive to two non-interfering epitopes on
PRO317 is preferred, but a competitive binding assay may be
employed. These assays are described, among other places, in Maddox
et al. J. Exp. Med., 158:1211 (1983).
Example 63
Identification of PRO317 Receptors
[1445] Purified PRO317 is useful for characterization and
purification of specific cell surface receptors and other binding
molecules. Cells which respond to PRO317 by metabolic changes or
other specific responses are likely to express a receptor for
PRO317. Such receptors include, but are not limited to, receptors
associated with and activated by tyrosine and serine/threonine
kinases. See Kolodziejczyk and Hall, supra, for a review on known
receptors for the TGF-superfamily. Candidate receptors for this
superfamily fall into two primary groups, termed type I and type II
receptors. Both types are serine/threonine kinases. Upon activation
by the appropriate ligand, type I and type II receptors physically
interact to form hetero-oligomers and subsequently activate
intracellular signaling cascades, ultimately regulating gene
transcription and expression. In addition, TGF-binds to a third
receptor class, type III, a membrane-anchored proteoglycan lacking
the kinase activity typical of signal transducing molecules.
[1446] PRO317 receptors or other PRO317-binding molecules may be
identified by interaction with radiolabeled PRO317. Radioactive
labels may be incorporated into PRO317 by various methods known in
the art. A preferred embodiment is the labeling of primary amino
groups in PRO317 with .sup.125I Bolton-Hunter reagent (Bolton and
Hunter, Biochem. J., 133:529 (1973)), which has been used to label
other polypeptides without concomitant loss of biological activity
(Hebert et al., J. Biol. Chem., 266:18989 (1991); McColl et al., J.
Immunol., 150:4550-4555 (1993)). Receptor-bearing cells are
incubated with labeled PRO317. The cells are then washed to removed
unbound PRO317, and receptor-bound PRO317 is quantified. The data
obtained using different concentrations of PRO317 are used to
calculate values for the number and affinity of receptors.
[1447] Labeled PRO317 is useful as a reagent for purification of
its specific receptor. In one embodiment of affinity purification,
PRO317 is covalently coupled to a chromatography column.
Receptor-bearing cells are extracted, and the extract is passed
over the column. The receptor binds to the column by virtue of its
biological affinity for PRO317. The receptor is recovered from the
column and subjected to N-terminal protein sequencing. This amino
acid sequence is then used to design degenerate oligonucleotide
probes for cloning the receptor gene.
[1448] In an alternative method, mRNA is obtained from
receptor-bearing cells and made into a cDNA library. The library is
transfected into a population of cells, and those cells expressing
the receptor are selected using fluorescently labeled PRO317. The
receptor is identified by recovering and sequencing recombinant DNA
from highly labeled cells.
[1449] In another alternative method, antibodies are raised against
the surface of receptor bearing cells, specifically monoclonal
antibodies. The monoclonal antibodies are screened to identify
those which inhibit the binding of labeled PRO317. These monoclonal
antibodies are then used in affinity purification or expression
cloning of the receptor.
[1450] Soluble receptors or other soluble binding molecules are
identified in a similar manner. Labeled PRO317 is incubated with
extracts or other appropriate materials derived from the uterus.
After incubation, PRO317 complexes larger than the size of purified
PRO317 are identified by a sizing technique such as size-exclusion
chromatography or density gradient centrifugation and are purified
by methods known in the art. The soluble receptors or binding
protein(s) are subjected to N-terminal sequencing to obtain
information sufficient for database identification, if the soluble
protein is known, or for cloning, if the soluble protein is
unknown.
Example 64
Determination of PRO317-Induced Cellular Response
[1451] The biological activity of PRO317 is measured, for example,
by binding of an PRO317 of the invention to an PRO317 receptor. A
test compound is screened as an antagonist for its ability to block
binding of PRO317 to the receptor. A test compound is screened as
an agonist of the PRO317 for its ability to bind an PRO317 receptor
and influence the same physiological events as PRO317 using, for
example, the KIRA-ELISA assay described by Sadick et al.,
Analytical Biochemistry, 235:207-214 (1996) in which activation of
a receptor tyrosine kinase is monitored by immuno-capture of the
activated receptor and quantitation of the level of ligand-induced
phosphorylation. The assay may be adapted to monitor PRO317-induced
receptor activation through the use of an PRO317 receptor-specific
antibody to capture the activated receptor. These techniques are
also applicable to other PRO polypeptides described herein.
Example 65
Use of PRO224 for Screening Compounds
[1452] PRO224 is expressed in a cell stripped of membrane proteins
and capable of expressing PRO224. Low density lipoproteins having a
detectable label are added to the cells and incubated for a
sufficient time for endocytosis. The cells are washed. The cells
are then analysed for label bound to the membrane and within the
cell after cell lysis. Detection of the low density lipoproteins
within the cell determines that PRO224 is within the family of low
density lipoprotein receptor proteins. Members found within this
family are then used for screening compounds which affect these
receptors, and particularly the uptake of cholesterol via these
receptors.
Example 66
Ability of PRO Polypeptides to Inhibit Vascular Endothelial Growth
Factor (VEGF) Stimulated Proliferation of Endothelial Cell Growth
(Assay 9)
[1453] The ability of various PRO polypeptides to inhibit VEGF
stimulated proliferation of endothelial cells was tested.
Polypeptides testing positive in this assay are useful for
inhibiting endothelial cell growth in mammals where such an effect
would be beneficial, e.g., for inhibiting tumor growth.
[1454] Specifically, bovine adrenal cortical capillary endothelial
cells (ACE) (from primary culture, maximum of 12-14 passages) were
plated in 96-well plates at 500 cells/well per 100 microliter.
Assay media included low glucose DMEM, 10% calf serum, 2 mM
glutamine, and 1.times. penicillin/streptomycin/fungizone. Control
wells included the following: (1) no ACE cells added; (2) ACE cells
alone; (3) ACE cells plus 5 ng/ml FGF; (4) ACE cells plus 3 ng/ml
VEGF; (5) ACE cells plus 3 ng/ml VEGF plus 1 ng/ml TGF-beta; and
(6) ACE cells plus 3 ng/ml VEGF plus 5 ng/ml LIF. The test samples,
poly-his tagged PRO polypeptides (in 100 microliter volumes), were
then added to the wells (at dilutions of 1%, 0.1% and 0.01%,
respectively). The cell cultures were incubated for 6-7 days at
37.degree. C./5% CO.sub.2. After the incubation, the media in the
wells was aspirated, and the cells were washed 1.times. with PBS.
An acid phosphatase reaction mixture (100 microliter; 0.1M sodium
acetate, pH 5.5, 0.1% Triton X-100, 10 mM p-nitrophenyl phosphate)
was then added to each well. After a 2 hour incubation at
37.degree. C., the reaction was stopped by addition of 10
microliters 1N NaOH. Optical density (OD) was measured on a
microplate reader at 405 umn.
[1455] The activity of PRO polypeptides was calculated as the
percent inhibition of VEGF (3 ng/ml) stimulated proliferation (as
determined by measuring acid phosphatase activity at OD 405 nm)
relative to the cells without stimulation. TGF-beta was employed as
an activity reference at 1 ng/ml, since TGF-beta blocks 70-90% of
VEGF-stimulated ACE cell proliferation. The results are indicative
of the utility of the PRO polypeptides in cancer therapy and
specifically in inhibiting tumor angiogenesis. Numerical values
(relative inhibition) are determined by calculating the percent
inhibition of VEGF stimulated proliferation by the PRO polypeptides
relative to cells without stimulation and then dividing that
percentage into the percent inhibition obtained by TGF-.beta. at 1
nglml which is known to block 70-90% of VEGF stimulated cell
proliferation. The results are considered positive if the PRO
polypeptide exhibits 30% or greater inhibition of VEGF stimulation
of endothelial cell growth (relative inhibition 30% or
greater).
[1456] The following polypeptides tested positive in this assay:
PRO211, PRO217, PRO187, PRO219, PRO246, PRO228, PRO245, PRO221,
PRO258, PRO301, PRO224, PRO272, PRO328, PRO331, PRO224, PRO328,
PRO272, PRO301, PRO331 and PRO214.
Example 67
Retinal Neuron Survival (Assay 52)
[1457] This example demonstrates that certain PRO polypeptides have
efficacy in enhancing the survival of retinal neuron cells and,
therefore, are useful for the therapeutic treatment of retinal
disorders or injuries including, for example, treating sight loss
in mammals due to retinitis pigmentosum, AMD, etc.
[1458] Sprague Dawley rat pups at postnatal day 7 (mixed
population: glia and retinal neuronal types) are killed by
decapitation following CO.sub.2 anesthesia and the eyes are removed
under sterile conditions. The neural retina is dissected away from
the pigment epithelium and other ocular tissue and then dissociated
into a single cell suspension using 0.25% trypsin in Ca.sup.2+,
Mg.sup.2++-free PBS. The retinas are incubated at 37.degree. C. for
7-10 minutes after which the trypsin is inactivated by adding 1 ml
soybean trypsin inhibitor. The cells are plated at 100,000 cells
per well in 96 well plates in DMEM/F12 supplemented with N2 and
with or without the specific test PRO polypeptide. Cells for all
experiments are grown at 37.degree. C. in a water saturated
atmosphere of 5% CO.sub.2. After 2-3 days in culture, cells are
stained with calcein AM then fixed using 4% paraformaldehyde and
stained with DAPI for determination of total cell count. The total
cells (fluorescent) are quantified at 20.times. objective
magnification using CCD camera and NIH image software for
MacIntosh. Fields in the well are chosen at random.
[1459] The effect of various concentration of PRO polypeptides are
reported herein where percent survival is calculated by dividing
the total number of calcein AM positive cells at 2-3 days in
culture by the total number of DAPI-labeled cells at 2-3 days in
culture. Anything above 30% survival is considered positive.
[1460] The following PRO polypeptides tested positive in this assay
using polypeptide concentrations within the range of 0.01% to 1.0%
in the assay: PRO220 and PRO346.
Example 68
Rod Photoreceptor Cell Survival (Assay 56)
[1461] This assay shows that certain polypeptides of the invention
act to enhance the survival/proliferation of rod photoreceptor
cells and, therefore, are useful for the therapeutic treatment of
retinal disorders or injuries including, for example, treating
sight loss in mammals due to retinitis pigmentosum, AMD, etc.
Sprague Dawley rat pups at 7 day postnatal (mixed population: glia
and retinal neuronal cell types) are killed by decapitation
following CO.sub.2 anesthesis and the eyes are removed under
sterile conditions. The neural retina is dissected away form the
pigment epithelium and other ocular tissue and then dissociated
into a single cell suspension using 0.25% trypsin in Ca.sup.2+,
Mg.sup.2+-free PBS. The retinas are incubated at 37.degree. C. for
7-10 minutes after which the trypsin is inactivated by adding 1 ml
soybean trypsin inhibitor. The cells are plated at 100,000 cells
per well in 96 well plates in DMEM/F12 supplemented with N.sub.2.
Cells for all experiments are grown at 37.degree. C. in a water
saturated atmosphere of 5% CO.sub.2. After 2-3 days in culture,
cells are fixed using 4% paraformaldehyde, and then stained using
CellTracker Green CMFDA. Rho 4D2 (ascites or IgG 1: 100), a
monoclonal antibody directed towards the visual pigment rhodopsin
is used to detect rod photoreceptor cells by indirect
immunofluorescence. The results are calculated as % survival: total
number of calcein--rhodopsin positive cells at 2-3 days in culture,
divided by the total number of rhodopsin positive cells at time 2-3
days in culture. The total cells (fluorescent) are quantified at
20.times. objective magnification using a CCD camera and NIH image
software for MacIntosh. Fields in the well are chosen at
random.
[1462] The following polypeptides tested positive in this assay:
PRO220 and PRO346.
Example 69
Induction of Endothelial Cell Apoptosis (Assay 73)
[1463] The ability of PRO polypeptides to induce apoptosis in
endothelial cells was tested in human venous umbilical vein
endothelial cells (HUVEC, Cell Systems). A positive test in the
assay is indicative of the usefulness of the polypeptide in
therapeutically treating tumors as well as vascular disorders where
inducing apoptosis of endothelial cells would be beneficial.
[1464] The cells were plated on 96-well microtiter plates (Amersham
Life Science, cytostar-T scintillating microplate, RPNQ160,
sterile, tissue-culture treated, individually wrapped), in 10%
serum (CSG-medium, Cell Systems), at a density of 2.times.10.sup.4
cells per well in a total volume of 100 .mu.l. On day 2, test
samples containing the PRO polypeptide were added in triplicate at
dilutions of 1%, 0.33% and 0.11%. Wells without cells were used as
a blank and wells with cells only were used as a negative control.
As a positive control 1:3 serial dilutions of 50 .mu.l of a
3.times. stock of staurosporine were used. The ability of the PRO
polypeptide to induce apoptosis was determined by processing of the
96 well plates for detection of Annexin V, a member of the calcium
and phospholipid binding proteins, to detect apoptosis.
[1465] 0.2 ml Annexin V--Biotin stock solution (100 .mu.g/ml) was
diluted in 4.6 ml 2.times. Ca.sup.2+ binding buffer and 2.5% BSA
(1:25 dilution). 50 .mu.l of the diluted Annexin V--Biotin solution
was added to each well (except controls) to a final concentration
of 1.0 .mu.g/ml. The samples were incubated for 10-15 minutes with
Annexin-Biotin prior to direct addition of .sup.35S-Streptavidin.
.sup.35S-Streptavidin was diluted in 2.times. Ca.sup.2+ Binding
buffer, 2.5% BSA and was added to all wells at a final
concentration of 3.times.10.sup.4 cpm/well. The plates were then
sealed, centrifuged at 1000 rpm for 15 minutes and placed on
orbital shaker for 2 hours. The analysis was performed on a 1450
Microbeta Trilux (Wallac). Percent above background represents the
percentage amount of counts per minute above the negative controls.
Percents greater than or equal to 30% above background are
considered positive.
[1466] The following PRO polypeptides tested positive in this
assay: PRO228, PRO217 and PRO301.
Example 70
PDB12 Cell Inhibition (Assay 40)
[1467] This example demonstrates that various PRO polypeptides have
efficacy in inhibiting protein production by PDB12 pancreatic
ductal cells and are, therefore, useful in the therapeutic
treatment of disorders which involve protein secretion by the
pancreas, including diabetes, and the like.
[1468] PDB12 pancreatic ductal cells are plated on fibronectin
coated 96 well plates at 1.5.times.10.sup.3 cells per well in 100
.mu.L/180 .mu.L of growth media. 100 .mu.L of growth media with the
PRO polypeptide test sample or negative control lacking the PRO
polypeptide is then added to well, for a final volume of 200 .mu.L.
Controls contain growth medium containing a protein shown to be
inactive in this assay. Cells are incubated for 4 days at
37.degree. C. 20 .mu.L of Alamar Blue Dye (AB) is then added to
each well and the flourescent reading is measured at 4 hours post
addition of AB, on a microtiter plate reader at 530 .mu.m
excitation and 590 nm emission. The standard employed is cells
without Bovine Pituitary Extract (BPE) and with various
concentrations of BPE. Buffer or CM controls from unknowns are run
2 times on each 96 well plate.
[1469] These assays allow one to calculate a percent decrease in
protein production by comparing the Alamar Blue Dye calculated
protein concentration produced by the PRO polypeptide-treated cells
with the Alamar Blue Dye calculated protein concentration produced
by the negative control cells. A percent decrease in protein
production of greater than or equal to 25% as compared to the
negative control cells is considered positive.
[1470] The following polypeptides tested positive in this assay:
PRO211, PRO287, PRO301 and PRO293.
Example 71
Stimulation of Adult Heart Hypertrophy (Assay 2)
[1471] This assay is designed to measure the ability of various PRO
polypeptides to stimulate hypertrophy of adult heart. PRO
polypeptides testing positive in this assay would be expected to be
useful for the therapeutic treatment of various cardiac
insufficiency disorders.
[1472] Ventricular myocytes freshly isolated from adult (250 g)
Sprague Dawley rats are plated at 2000 cell/well in 180 .mu.l
volume. Cells are isolated and plated on day 1, the PRO
polypeptide-containing test samples or growth medium only (negative
control) (20 .mu.l volume) is added on day 2 and the cells are then
fixed and stained on day 5. After staining, cell size is visualized
wherein cells showing no growth enhancement as compared to control
cells are given a value of 0.0, cells showing small to moderate
growth enhancement as compared to control cells are given a value
of 1.0 and cells showing large growth enhancement as compared to
control cells are given a value of 2.0. Any degree of growth
enhancement as compared to the negative control cells is considered
positive for the assay.
[1473] The following PRO polypeptides tested positive inthis assay:
PRO287, PRO301, PRO293 and PRO303.
Example 72
PDB12 Cell Proliferation (Assay 29)
[1474] This example demonstrates that various PRO polypeptides have
efficacy in inducing proliferation of PDB 12 pancreatic ductal
cells and are, therefore, useful in the therapeutic treatment of
disorders which involve protein secretion by the pancreas,
including diabetes, and the like.
[1475] PDB12 pancreatic ductal cells are plated on fibronectin
coated 96 well plates at 1.5.times.10.sup.3 cells per well in 100
.mu.L/180 .mu.L of growth media. 100 .mu.L of growth media with the
PRO polypeptide test sample or negative control lacking the PRO
polypeptide is then added to well, for a final volume of 200 .mu.L.
Controls contain growth medium containing a protein shown to be
inactive in this assay. Cells are incubated for 4 days at
37.degree. C. 20 .mu.L of Alamar Blue Dye (AB) is then added to
each well and the flourescent reading is measured at 4 hours post
addition of AB, on a microtiter plate reader at 530 .mu.m
excitation and 590 nm emission. The standard employed is cells
without Bovine Pituitary Extract (BPE) and with various
concentrations of BPE. Buffer or growth medium only controls from
unknowns are run 2 times on each 96 well plate.
[1476] Percent increase in protein production is calculated by
comparing the Alamar Blue Dye calculated protein concentration
produced by the PRO polypeptide-treated cells with the Alamar Blue
Dye calculated protein concentration produced by the negative
control cells. A percent increase in protein production of greater
than or equal to 25% as compared to the negative control cells is
considered positive.
[1477] The following PRO polypeptides tested positive in this
assay: PRO301 and PRO303.
Example 73
Enhancement of Heart Neonatal Hypertrophy (Assay 1)
[1478] This assay is designed to measure the ability of PRO
polypeptides to stimulate hypertrophy of neonatal heart. PRO
polypeptides testing positive in this assay are expected to be
useful for the therapeutic treatment of various cardiac
insufficiency disorders.
[1479] Cardiac myocytes from 1-day old Harlan Sprague Dawley rats
were obtained. Cells (180 .mu.l at 7.5.times.10.sup.4/ml, serum
<0.1%, freshly isolated) are added onday 1 to 96-well plates
previously coated with DMEM/F12+4% FCS. Test samples containing the
test PRO polypeptide or growth medium only (hegative control) (20
.mu.l/well) are added directly to the wells on day 1. PGF (20
.mu.l/well) is then added on day 2 at final concentration of
10.sup.-6 M. The cells are then stained on day 4 and visually
scored on day 5, wherein cells showing no increase in size as
compared to negative controls are scored 0.0, cells showing a small
to moderate increase in size as compared to negative controls are
scored 1.0 and cells showing a large increase in size as compared
to negative controls are scored 2.0. A positive result in the assay
is a score of 1.0 or greater.
[1480] The following polypeptides tested positive in this assay:
PRO224 and PRO231.
Example 74
Stimulatory Activity in Mixed Lymphocvte Reaction (MLR) Assay
(Assay 24)
[1481] This example shows that certain polypeptides of the
invention are active as a stimulator of the proliferation of
stimulated T-lymphocytes. Compounds which stimulate proliferation
of lymphocytes are useful therapeutically where enhancement of an
immune response is beneficial. A therapeutic agent may take the
form of antagonists of the polypeptide of the invention, for
example, murine-human chimeric, humanized or human antibodies
against the polypeptide.
[1482] The basic protocol for this assay is described in Current
Protocols in Immunology, unit 3.12; edited by J E Coligan, A M
Kruisbeek, D H Marglies, E M Shevach, W Strober, National Insitutes
of Health, Published by John Wiley & Sons, Inc.
[1483] More specifically, in one assay variant, peripheral blood
mononuclear cells (PBMC) are isolated from mammalian individuals,
for example a human volunteer, by leukopheresis (one donor will
supply stimulator PBMCs, the other donor will supply responder
PBMCs). If desired, the cells are frozen in fetal bovine serum and
DMSO after isolation. Frozen cells may be thawed overnight in assay
media (37.degree. C., 5% CO.sub.2) and then washed and resuspended
to 3.times.10.sup.6 cells/ml of assay media (RPMI; 10% fetal bovine
serum, 1% penicillin/streptomycin, 1% glutamine, 1% HEPES, 1%
non-essential amino acids, 1% pyruvate). The stimulator PBMCs are
prepared by irradiating the cells (about 3000 Rads).
[1484] The assay is prepared by plating in triplicate wells a
mixture of:
[1485] 100:1 of test sample diluted to 1% or to 0.1%,
[1486] 50:1 of irradiated stimulator cells, and
[1487] 50:1 of responder PBMC cells.
[1488] 100 microliters of cell culture media or 100 microliter of
CD4-IgG is used as the control. The wells are then incubated at
37.degree. C., 5% CO.sub.2 for 4 days. On day 5, each well is
pulsed with tritiated thymidine (1.0 mC/well; Amersham). After 6
hours the cells are washed 3 times and then the uptake of the label
is evaluated.
[1489] In another variant of this assay, PBMCs are isolated from
the spleens of Balb/c mice and C57B6 mice. The cells are teased
from freshly harvested spleens in assay media (RPMI; 10% fetal
bovine serum, 1% penicillin/streptomycin, 1% glutamine, 1% HEPES,
1% non-essential amino acids, 1% pyruvate) and the PBMCs are
isolated by overlaying these cells over Lympholyte M (Organon
Teknika), centrifuging at 2000 rpm for 20 minutes, collecting and
washing the mononuclear cell layer in assay media and resuspending
the cells to 1.times.10.sup.7 cells/ml of assay media. The assay is
then conducted as described above.
[1490] Positive increases over control are considered positive with
increases of greater than or equal to 180% being preferred.
However, any value greater than control indicates a stimulatory
effect for the test protein.
[1491] The following PRO polypeptides tested positive in this
assay: PRO245, PRO269, PRO217, PRO301, PRO266, PRO335, PRO331,
PRO533 and PRO326.
Example 75
Pericvte c-Fos Induction (Assay 93)
[1492] This assay shows that certain polypeptides of the invention
act to induce the expression of c-fos in pericyte cells and,
therefore, are useful not only as diagnostic markers for particular
types of pericyte-associated tumors but also for giving rise to
antagonists which would be expected to be useful for the
therapeutic treatment of pericyte-associated tumors. Specifically,
on day 1, pericytes are received from VEC Technologies and all but
5 ml of media is removed from flask. On day 2, the pericytes are
trypsinized, washed, spun and then plated onto 96 well plates. On
day 7, the media is removed and the pericytes are treated with 100
.mu.l of PRO polypeptide test samples and controls (positive
control=DME+5% serum+1-PDGF at 500 ng/ml; negative control=protein
32). Replicates are averaged and SD/CV are determined. Fold
increase over Protein 32 (buffer control) value indicated by
chemiluminescence units (RLU) luminometer reading verses frequency
is plotted on a histogram. Two-fold above Protein 32 value is
considered positive for the assay. ASY Matrix: Growth media=low
glucose DMEM=20% FBS+1.times. pen strep+1.times. fungizone. Assay
Media=low glucose DMEM+5% FBS.
[1493] The following polypeptides tested positive in this assay:
PRO214, PRO219, PRO221 and PRO224.
Example 76
Ability of PRO Polypeptides to Stimulate the Release of
Proteoglvcans from Cartilage (Assay
[1494] The ability of various PRO polypeptides to stimulate the
release of proteoglycans from cartilage tissue was tested as
follows.
[1495] The metacarphophalangeal joint of 4-6 month old pigs was
aseptically dissected, and articular cartilage was removed by free
hand slicing being careful to avoid the underlying bone. The
cartilage was minced and cultured in bulk for 24 hours in a
humidified atmosphere of 95% air, 5% CO.sub.2 in serum free (SF)
media (DME/F12 1:1) woth 0.1% BSA and 100 U/ml penicillin and 100
.mu.g/ml streptomycin. After washing three times, approximately 100
mg of articular cartilage was aliquoted into micronics tubes and
incubated for an additional 24 hours in the above SF media. PRO
polypeptides were then added at 1% either alone or in combination
with 18 ng/ml interleukin-1.alpha., a known stimulator of
proteoglycan release from cartilage tissue. The supernatant was
then harvested and assayed for the amount of proteoglycans using
the 1,9-dimethyl-methylene blue (DMB) calorimetric assay (Farndale
and Buttle, Biochem. Biophys. Acta 883:173-177 (1985)). A positive
result in this assay indicates that the test polypeptide will find
use, for example, in the treatment of sports-related joint
problems, articular cartilage defects, osteoarthritis or rheumatoid
arthritis.
[1496] When various PRO polypeptides were tested in the above
assay, the polypeptides demonstrated a marked ability to stimulate
release of proteoglycans from cartilage tissue both basally and
after stimulation with interleukin-1.alpha. and at 24 and 72 hours
after treatment, thereby indicating that these PRO polypeptides are
useful for stimulating proteoglycan release from cartilage tissue.
As such, these PRO polypeptides are useful for the treatment of
sports-related joint problems, articular cartilage defects,
osteoarthritis or rheumatoid arthritis. The polypeptides testing
positive in this assay are: PRO211.
Example 77
Skin Vascular Permeability Assay (Assay 64)
[1497] This assay shows that certain polypeptides of the invention
stimulate an immune response and induce inflammation by inducing
mononuclear cell, eosinophil and PMN infiltration at the site of
injection of the animal. Compounds which stimulate an immune
response are useful therapeutically where stimulation of an immune
response is beneficial. This skin vascular permeability assay is
conducted as follows. Hairless guinea pigs weighing 350 grams or
more are anesthetized with ketamine (75-80 mg/Kg) and 5 mg/Kg
xylazine intramuscularly (IM). A sample of purified polypeptide of
the invention or a conditioned media test sample is injected
intradermally onto the backs of the test animals with 100 .mu.l per
injection site. It is possible to have about 10-30, preferably
about 16-24, injection sites per animal. One .mu.l of Evans blue
dye (1% in physiologic buffered saline) is injected intracardially.
Blemishes at the injection sites are then measured (mm diameter) at
1 hr and 6 hr post injection. Animals were sacrificed at 6 hrs
after injection. Each skin injection site is biopsied and fixed in
formalin. The skins are then prepared for histopathologic
evaluation. Each site is evaluated for inflammatory cell
infiltration into the skin. Sites with visible inflammatory cell
inflammation are scored as positive. Inflammatory cells may be
neutrophilic, eosinophilic, monocytic or lymphocytic. At least a
minimal perivascular infiltrate at the injection site is scored as
positive, no infiltrate at the site of injection is scored as
negative.
[1498] The following polypeptides tested positive in this assay:
PRO245, PRO217, PRO326, PRO266, PRO272, PRO301, PRO331 and
PRO335.
Example 78
Enhancement of Heart Neonatal Hypertrophy Induced by F2a (Assay
37)
[1499] This assay is designed to measure the ability of PRO
polypeptides to stimulate hypertrophy of neonatal heart. PRO
polypeptides testing positive in this assay are expected to be
useful for the therapeutic treatment of various cardiac
insufficiency disorders.
[1500] Cardiac myocytes from 1-day old Harlan Sprague Dawley rats
were obtained. Cells (180 .mu.l at 7.5.times.10.sup.4/ml, serum
<0.1%, freshly isolated) are added on day 1 to 96-well plates
previously coated with DMEM/F12+4% FCS. Test samples containing the
test PRO polypeptide (20 .mu.l/well) are added directly to the
wells on day 1. PGF (20 .mu.l/well) is then added on day 2 at a
final concentration of 10-6 M. The cells are then stained on day 4
and visually scored on day 5. Visual scores are based on cell size,
wherein cells showing no increase in size as compared to negative
controls are scored 0.0, cells showing a small to moderate increase
in size as compared to negative controls are scored 1.0 and cells
showing a large increase in size as compared to negative controls
are scored 2.0. A score of 1.0 or greater is considered
positive.
[1501] No PBS is included, since calcium concentration is critical
for assay response. Plates are coated with DMEM/F12 plus 4% FCS
(200 .mu.l/well). Assay media included: DMEM/F12 (with 2.44 gm
bicarbonate), 10 .mu.g/ml transferrin, 1 .mu.g/ml insulin, 1
.mu.g/ml aprotinin, 2 mmol/L glutamine, 100 U/ml penicillin G, 100
.mu.g/ml streptomycin. Protein buffer containing mannitol (4%) gave
a positive signal (score 3.5) at 1/10 (0.4%) and 1/100 (0.04%), but
not at 1/1000 (0.004%). Therefore the test sample buffer containing
mannitol is not run.
[1502] The following PRO polypeptides tested positive in this
assay: PRO224.
Example 79
Inhibitory Activity in Mixed Lymphocyte Reaction (MLR) Assay (Assay
67)
[1503] This example shows that one or more of the polypeptides of
the invention are active as inhibitors of the proliferation of
stimulated T-lymphocytes. Compounds which inhibit proliferation of
lymphocytes are useful therapeutically where suppression of an
immune response is beneficial.
[1504] The basic protocol for this assay is described in Current
Protocols in Immunology, unit 3.12; edited by J E Coligan, A M
Kruisbeek, D H Marglies, E M Shevach, W Strober, National Insitutes
of Health, Published by John Wiley & Sons, Inc.
[1505] More specifically, in one assay variant, peripheral blood
mononuclear cells (PBMC) are isolated from mammalian individuals,
for example a human volunteer, by leukopheresis (one donor will
supply stimulator PBMCs, the other donor will supply responder
PBMCs). If desired, the cells are frozen in fetal bovine serum and
DMSO after isolation. Frozen cells may be thawed overnight in assay
media (37.degree. C., 5% CO.sub.2) and then washed and resuspended
to 3.times.10.sup.6 cells/ml of assay media (RPMI; 10% fetal bovine
serum, 1% penicillin/streptomycin, 1% glutamine, 1% HEPES, 1%
non-essential amino acids, 1% pyruvate). The stimulator PBMCs are
prepared by irradiating the cells (about 3000 Rads).
[1506] The assay is prepared by plating in triplicate wells a
mixture of:
[1507] 100:1 of test sample diluted to 1% or to 0.1%,
[1508] 50:1 of irradiated stimulator cells, and
[1509] 50:1 of responder PBMC cells.
[1510] 100 microliters of cell culture media or 100 microliter of
CD4-IgG is used as the control. The wells are then incubated at
37.degree. C., 5% CO.sub.2 for 4 days. On day 5, each well is
pulsed with tritiated thymidine (1.0 mC/well; Amersham). After 6
hours the cells are washed 3 times and then the uptake of the label
is evaluated.
[1511] In another variant of this assay, PBMCs are isolated from
the spleens of Balb/c mice and C57B6 mice. The. cells are teased
from freshly harvested spleens in assay media (RPMI; 10% fetal
bovine serum, 1% penicillin/streptomycin, 1% glutamine, 1% HEPES,
1% non-essential amino acids, 1% pyruvate) and the PBMCs are
isolated by overlaying these cells over Lympholyte M (Organon
Teknika), centrifuging at 2000 rpm for 20 minutes, collecting and
washing the mononuclear cell layer in assay media and resuspending
the cells to 1.times.10.sup.7 cells/ml of assay media. The assay is
then conducted as described above.
[1512] Any decreases below control is considered to be a positive
result for an inhibitory compound, with decreases of less than or
equal to 80% being preferred. However, any value less than control
indicates an inhibitory effect for the test protein.
[1513] The following polypeptide tested positive in this assay:
PRO235, PRO245 and PRO332.
Example 80
Induction of Endothelial Cell Apoptosis (ELISA) (Assay 109)
[1514] The ability of PRO polypeptides to induce apoptosis in
endothelial cells was tested in human venous umbilical vein
endothelial cells (HUVEC, Cell Systems) using a 96-well format, in
0% serum media supplemented with 100 ng/ml VEGF, 0.1% BSA, 1.times.
penn/strep. A positive result in this assay indicates the
usefulness of the polypeptide for therapeutically treating any of a
variety of conditions associated with undesired endothelial cell
growth including, for example, the inhibition of tumor growth. The
96-well plates used were manufactured by Falcon (No. 3072). Coating
of 96 well plates were prepared by allowing gelatinization to occur
for >30 minutes with 100 .mu.l of 0.2% gelatin in PBS solution.
The gelatin mix was aspirated thoroughly before plating HUVEC cells
at a final concentration of 2.times.10.sup.4 cells/ml in 10% serum
containing medium -100 .mu.l volume per well. The cells were grown
for 24 hours before adding test samples containing the PRO
polypeptide of interest.
[1515] To all wells, 100 .mu.l of 0% serum media (Cell Systems)
complemented with 100 ng/ml VEGF, 0. 1% BSA, 1.times. penn/strep
was added. Test samples containing PRO polypeptides were added in
triplicate at dilutions of 1%, 0.33% and 0.11%. Wells without cells
were used as a blank and wells with cells only were used as a
negative control. As a positive control, 1:3 serial dilutions of 50
of a 3.times. stock of staurosporine were used. The cells were
incubated for 24 to 35 hours prior to ELISA.
[1516] ELISA was used to determine levels of apoptosis preparing
solutions according to the Boehringer Manual [Boehringer, Cell
Death Detection ELISA plus, Cat No. 1 920 685]. Sample
preparations: 96 well plates were spun down at 1 krpm for 10
minutes (200 g); the supernatant was removed by fast inversion,
placing the plate upside down on a paper towel to remove residual
liquid. To each well, 200 .mu.l of 1.times. Lysis buffer was added
and incubation allowed at room temperature for 30 minutes without
shaking. The plates were spun down for 10 minutes at 1 krpm, and 20
.mu.l of the lysate (cytoplasmic fraction) was transferred into
streptavidin coated MTP. 80 .mu.l of immunoreagent mix was added to
the 20 .mu.l lystate in each well. The MTP was covered with
adhesive foil and incubated at room tempearature for 2 hours by
placing it on an orbital shaker (200 rpm). After two hours, the
supernatant was removed by suction and the wells rinsed three times
with 250 .mu.l of 1.times. incubation buffer per well (removed by
suction). Substrate solution was added (100 .mu.l) into each well
and incubated on an orbital shaker at room temperature at 250 rpm
until color development was sufficient for a photometric analysis
(approx. after 10-20 minutes). A 96 well reader was used to read
the plates at 405 nm, reference wavelength, 492 nm. The levels
obtained for PIN 32 (control buffer) was set to 100%. Samples with
levels >130% were considered positive for induction of
apoptosis.
[1517] The following PRO polypeptides tested positive in this
assay: PRO235.
Example 81
Human Venous Endothelial Cell Calcium Flux Assay (Assay 68)
[1518] This assay is designed to determine whether PRO polypeptides
of the present invention show the ability to stimulate calcium flux
in human umbilical vein endothelial cells (HUVEC, Cell Systems).
Calcium influx is a well documented response upon binding of
certain ligands to their receptors. A test compound that results in
a positive response in the present calcium influx assay can be said
to bind to a specific receptor and activate a biological signaling
pathway in human endothelial cells. This could ultimately lead, for
example, to endothelial cell division, inhibition of endothelial
cell proliferation, endothelial tube formation, cell migration,
apoptosis, etc.
[1519] Human venous umbilical vein endothelial cells (HUVEC, Cell
Systems) in growth media (50:50 without glycine, 1% glutamine, 10
mM Hepes, 10% FBS, 10 ng/mi bFGF), were plated on 96-well
microtiter ViewPlates-96 (Packard Instrument Company Part #6005182)
microtiter plates at a cell density of 2.times.10.sup.4 cells/well.
The day after plating, the cells were washed three times with
buffer (HBSS plus 10 MM Hepes), leaving 100 .mu.l/well. Then 100
.mu.l/well of 8 .mu.M Fluo-3 (2.times.) was added. The cells were
incubated for 1.5 hours at 37.degree. C./5% CO.sub.2. After
incubation, the cells were then washed 3.times. with buffer
(described above) leaving 100 .mu.l/well. Test samples of the PRO
polypeptides were prepared on different 96-well plates at 5.times.
concentration in buffer. The positive control corresponded to 50
.mu.M ionomycin (5.times.); the negative control corresponded to
Protein 32. Cell plate and sample plates were run on a FLIPR
(Molecular Devices) machine. The FLIPR machine added 25 .mu.l of
test sample to the cells, and readings were taken every second for
one minute, then every 3 seconds for the next three minutes.
[1520] The fluorescence change from baseline to the maximum rise of
the curve (A change) was calculated, and replicates averaged. The
rate of fluorescence increase was monitored, and only those samples
which had a A change greater than 1000 and a rise within 60
seconds, were considered positive.
[1521] The following PRO polypeptides tested positive in the
present assay: PRO245.
Example 82
Fibroblast (BHK-21) Proliferation (Assay 98)
[1522] This assay shows that certain PRO polypeptides of the
invention act to induce proliferation of mammalian fibroblast cells
in culture and, therefore, function as useful growth factors in
mammalian systems. The assay is performed as follows. BHK-21
fibroblast cells plated in standard growth medium at 2500
cells/well in a total volume of 100 .mu.l. The PRO polypeptide,
P-FGF (positive control) or nothing (negative control) are then
added to the wells in the presence of 1 .mu.g/ml of heparin for a
total final volume of 200 .mu.l. The cells are then incubated at
37.degree. C. for 6 to 7 days. After incubation, the media is
removed, the cells are washed with PBS and then an acid phosphatase
substrate reaction mixture (100 .mu.l/well) is added. The cells are
then incubated at 37.degree. C. for 2 hours. 10 .mu.l per well of
1N NaOH is then added to stop the acid phosphatase reaction. The
plates are then read at OD 405 nm. A positive in the assay is acid
phosphatase activity which is at least 50% above the negative
control.
[1523] The following PRO polypeptide tested positive in this assay:
PRO258.
Example 83
Inhibition of Heart Adult Hypertrophy (Assay 42)
[1524] This assay is designed to measure the inhibition of heart
adult hypertrophy. PRO polypeptides testing positive in this assay
may find use in the therapeutic treatment of cardiac disorders
associated with cardiac hypertrophy. Ventricular myocytes are
freshly isolated from adult (250 g) Harlan Sprague Dawley rats and
the cells are plated at 2000/well in 180 .mu.l volume. On day two,
test samples (20 .mu.l) containing the test PRO polypeptide are
added. On day five, the cells are fixed and then stained. An
increase in ANP message can also be measured by PCR from cells
after a few hours. Results are based on a visual score of cell
size: 0=no inhibition, -1=small inhibition, -2=large inhibition. A
score of less than 0 is considered positive. Activity reference
corresponds to phenylephrin (PE) at 0.1 mM, as a positive control.
Assay media included: M199 (modified)-glutamine free, NaHCO.sub.3,
phenol red, supplemented with 100 nM insulin, 0.2% BSA, 5 mM
cretine, 2 mM L-carnitine, 5 mM taurine, 100 U/ml penicillin G, 100
.mu.g/ml streptomycin (CCT medium). Only inner 60 wells are used in
96 well plates. Of these, 6 wells are reserved for negative and
positive (PE) controls.
[1525] The following PRO polypeptides provided a score of less than
0 in the above assay: PRO269.
Example 84
Induction of c-fos in Endothelial Cells (Assay 34)
[1526] This assay is designed to determine whether PRO polypeptides
show the ability to induce c-fos in endothelial cells. PRO
polypeptides testing positive in this assay would be expected to be
useful for the therapeutic treatment of conditions or disorders
where angiogenesis would be beneficial including, for example,
wound healing, and the like (as would agonists of these PRO
polypeptides). Antagonists of the PRO polypeptides testing positive
in this assay would be expected to be useful for the therapeutic
treatment of cancerous tumors.
[1527] Human venous umbilical vein endothelial cells (HUVEC, Cell
Systems) in growth media (50% Ham's F12 w/o GHT: low glucose, and
50% DMEM without glycine: with NaHCO3, 1% glutamine, 10 mM HEPES,
10% FBS, 10 ng/ml bFGF) were plated on 96-well microtiter plates at
a cell density of 1.times.10.sup.4 cells/well. The day after
plating, the cells were starved by removing the growth media and
treating the cells with 100 .mu.l/well test samples and controls
(positive control=growth media; negative control=Protein 32
buffer=10 mM HEPES, 140 mM NaCl, 4% (w/v) mannitol, pH 6.8). The
cells were incubated for 30 minutes at 37.degree. C., in 5%
CO.sub.2. The samples were removed, and the first part of the bDNA
kit protocol (Chiron Diagnostics, cat. #6005-037) was followed,
where each capitalized reagent/buffer listed below was available
from the kit.
[1528] Briefly, the amounts of the TM Lysis Buffer and Probes
needed for the tests were calculated based on information provided
by the manufacturer. The appropriate amounts of thawed Probes were
added to the TM Lysis Buffer. The Capture Hybridization Buffer was
warmed to room temperature. The bDNA strips were set up in the
metal strip holders, and 100 .mu.l of Capture Hybridization Buffer
was added to each b-DNA well needed, followed by incubation for at
least 30 minutes. The test plates with the cells were removed from
the incubator, and the media was gently removed using the vacuum
manifold. 100 .mu.l of Lysis Hybridization Buffer with Probes were
quickly pipetted into each well of the microtiter plates. The
plates were then incubated at 55.degree. C. for 15 minutes. Upon
removal from the incubator, the plates were placed on the vortex
mixer with the microtiter adapter head and vortexed on the #2
setting for one minute. 80 .mu.l of the lysate was removed and
added to the bDNA wells containing the Capture Hybridization
Buffer, and pipetted up and down to mix. The plates were incubated
at 53.degree. C. for at least 16 hours.
[1529] On the next day, the second part of the bDNA kit protocol
was followed. Specifically, the plates were removed from the
incubator and placed on the bench to cool for 10 minutes. The
volumes of additions needed were calculated based upon information
provided by the manufacturer. An Amplifier Working Solution was
prepared by making a 1:100 dilution of the Amplifier Concentrate
(20 fm/.mu.l) in AL Hybridization Buffer. The hybridization mixture
was removed from the plates and washed twice with Wash A. 50 .mu.l
of Amplifier Working Solution was added to each well and the wells
were incubated at 53.degree. C. for 30 minutes. The plates were
then removed from the incubator and allowed to cool for 10 minutes.
The Label Probe Working Solution was prepared by making a 1:100
dilution of Label Concentrate (40 pmoles/.mu.l) in AL Hybridization
Buffer. After the 10-minute cool-down period, the amplifier
hybridization mixture was removed and the plates were washed twice
with Wash A. 50 .mu.l of Label Probe Working Solution was added to
each well and the wells were incubated at 53.degree. C. for 15
minutes. After cooling for 10 minutes, the Substrate was warmed to
room temperature. Upon addition of 3 .mu.l of Substrate Enhancer to
each ml of Substrate needed for the assay, the plates were allowed
to cool for 10 minutes, the label hybridization mixture was
removed, and the plates were washed twice with Wash A and three
times with Wash D. 50 .mu.l of the Substrate Solution with Enhancer
was added to each well. The plates were incubated for 30 minutes at
37.degree. C. and RLU was read in an appropriate luminometer.
[1530] The replicates were averaged and the coefficient of
variation was determined. The measure of activity of the fold
increase over the negative control (Protein 32/HEPES buffer
described above) value was indicated by chemiluminescence units
(RLU). The results are considered positive if the PRO polypeptide
exhibits at least a two-fold value over the negative buffer
control. Negative control=1.00 RLU at 1.00% dilution. Positive
control=8.39 RLU at 1.00% dilution.
[1531] The following PRO polypeptides tested positive in this
assay: PRO287.
Example 85
Guinea Pig Vascular Leak (Assays 32 and 51)
[1532] This assay is designed to determine whether PRO polypeptides
of the present invention show the ability to induce vascular
permeability. Polypeptides testing positive in this assay are
expected to be useful for the therapeutic treatment of conditions
which would benefit from enhanced vascular permeability including,
for example, conditions which may benefit from enhanced local
immune system cell infiltration.
[1533] Hairless guinea pigs weighing 350 grams or more were
anesthetized with Ketamine (75-80 mg/kg) and 5 mg/kg Xylazine
intramuscularly. Test samples containing the PRO polypeptide or a
physiological buffer without the test polypeptide are injected into
skin on the back of the test animals with 100 .mu.l per injection
site intradermally. There were approximately 16-24 injection sites
per animal. One ml of Evans blue dye (1% in PBS) is then injected
intracardially. Skin vascular permeability responses to the
compounds (i.e., blemishes at the injection sites of injection) are
visually scored by measuring the diameter (in mm) of blue-colored
leaks from the site of injection at 1 and 6 hours post
administration of the test materials. The mm diameter of blueness
at the site of injection is observed and recorded as well as the
severity of the vascular leakage. Blemishes of at least 5 mm in
diameter are considered positive for the assay when testing
purified proteins, being indicative of the ability to induce
vascular leakage or permeability. A response greater than 7 mm
diameter is considered positive for conditioned media samples.
Human VEGF at 0.1 .mu.g/100 .mu.l is used as a positive control,
inducing a response of 15-23 mm diameter.
[1534] The following PRO polypeptides tested positive in this
assay: PRO302 and PRO533.
Example 86
Detection of Endothelial Cell Apoptosis (FACS) (Assay 96)
[1535] The ability of PRO polypeptides of the present invention to
induce apoptosis in endothelial cells was tested in human venous
umbilical vein endothelial cells (HUVEC, Cell Systems) in
gelatinized T175 flasks using HUVEC cells below passage 10. PRO
polypeptides testing positive in this assay are expected to be
useful for therapeutically treating conditions where apoptosis of
endothelial cells would be beneficial including, for example, the
therapeutic treatment of tumors.
[1536] On day one, the cells were split [420,000 cells per
gelatinized 6 cm dishes-(11.times.10.sup.3 cells/cm.sup.2 Falcon,
Primaria)] and grown in media containing serum (CS-C, Cell System)
overnight or for 16 hours to 24 hours.
[1537] On day 2, the cells were washed 1.times. with 5 ml PBS; 3 ml
of 0% serum medium was added with VEGF (100 ng/ml); and 30 .mu.l of
the PRO test compound (final dilution 1%) or 0% serum medium
(negative control) was added. The mixtures were incubated for 48
hours before harvesting.
[1538] The cells were then harvested for FACS analysis. The medium
was aspirated and the cells washed once with PBS. 5 ml of 1.times.
trypsin was added to the cells in a T-175 flask, and the cells were
allowed to stand until they were released from the plate (about
5-10 minutes). Trypsinization was stopped by adding 5 ml of growth
media. The cells were spun at 1000 rpm for 5 minutes at 4.degree.
C. The media was aspirated and the cells were resuspended in 10 ml
of 10% serum complemented medium (Cell Systems), 5 .mu.l of
Annexin-FITC (BioVison) added and chilled tubes were submitted for
FACS. A positive result was determined to be enhanced apoptosis in
the PRO polypeptide treated samples as compared to the negative
control.
[1539] The following PRO polypeptides tested positive in this
assay: PRO331.
Example 87
Induction of c-fos in Cortical Neurons (Assay 83)
[1540] This assay is designed to determine whether PRO polypeptides
show the ability to induce c-fos in cortical neurons. PRO
polypeptides testing positive in this assay would be expected to be
useful for the therapeutic treatment of nervous system disorders
and injuries where neuronal proliferation would be beneficial.
[1541] Cortical neurons are dissociated and plated in growth medium
at 10,000 cells per well in 96 well plates. After aproximately 2
cellular divisions, the cells are treated for 30 minutes with the
PRO polypeptide or nothing (negative control). The cells are then
fixed for 5 minutes with cold methanol and stained with an antibody
directed against phosphorylated CREB. mRNA levels are then
calculated using chemiluminescence. A positive in the assay is any
factor that results in at least a 2-fold increase in c-fos message
as compared to the negative controls.
[1542] The following PRO polypeptides tested positive in this
assay: PRO229 and PRO269.
Example 88
Stimulation of Endothelial Tube Formation (Assay 85)
[1543] This assay is designed to determine whether PRO polypeptides
show the ability to promote endothelial vacuole and lumen formation
in the absence of exogenous growth factors. PRO polypeptides
testing positive in this assay would be expected to be useful for
the therapeutic treatment of disorders where endothelial vacuole
and/or lumen formation would be beneficial including, for example,
where the stimulation of pinocytosis, ion pumping, vascular
permeability and/or junctional formation would be beneficial.
[1544] HUVEC cells (passage <8 from primary) are mixed with type
I rat tail collagen (final concentration 2.6 mg/ml) at a density of
6.times.10.sup.5 cells per ml and plated at 50 .mu.l per well of
M199 culture media supplemented with 1% FBS and 1 .mu.M 6-FAM-FITC
dye to stain the vacuoles while they are forming and in the
presence of the PRO polypeptide. The cells are then incubated at
37.degree. C./5% CO.sub.2 for 48 hours, fixed with 3.7% formalin at
room temperature for 10 minutes, washed 5 times with M199 medium
and then stained with Rh-Phalloidin at 4.degree. C. overnight
followed by nuclear staining with 4 .mu.M DAPI. A positive result
in the assay is when vacuoles are present in greater than 50% of
the cells.
[1545] The following PRO polypeptides tested positive in this
assay: PRO230.
Example 89
Detection of Polypeptides That Affect Glucose and/or FFA Uptake in
Skeletal Muscle (Assay 106)
[1546] This assay is designed to determine whether PRO polypeptides
show the ability to affect glucose or FFA uptake by skeletal muscle
cells. PRO polypeptides testing positive in this assay would be
expected to be useful for the therapeutic treatment of disorders
where either the stimulation or inhibition of glucose uptake by
skeletal muscle would be beneficial including, for example,
diabetes or hyper- or hypo-insulinemia.
[1547] In a 96 well format, PRO polypeptides to be assayed are
added to primary rat differentiated skeletal muscle, and allowed to
incubate overnight. Then fresh media with the PRO polypeptide and
+/-insulin are added to the wells. The sample media is then
monitored to determine glucose and FFA uptake by the skeletal
muscle cells. The insulin will stimulate glucose and FFA uptake by
the skeletal muscle, and insulin in media without the PRO
polypeptide is used as a positive control, and a limit for scoring.
As the PRO polypeptide being tested may either stimulate or inhibit
glucose and FFA uptake, results are scored as positive in the assay
if greater than 1.5 times or less than 0.5 times the insulin
control.
[1548] The following PRO polypeptides tested positive as either
stimulators or inhibitors of glucose and/or FFA uptake in this
assay: PRO187, PRO211, PRO221, PRO222, PRO224, PRO230, PRO239,
PRO231, PRO245, PRO247, PRO258, PRO269, PRO328 and PRO533.
Example 90
Rod Photoreceptor Cell Survival Assay (Assay 46)
[1549] This assay shows that certain polypeptides of the invention
act to enhance the survival/proliferation of rod photoreceptor
cells and, therefore, are useful for the therapeutic treatment of
retinal disorders or injuries including, for example, treating
sight loss in mammals due to retinitis pigmentosum, AMD, etc.
[1550] Sprague Dawley rat pups (postnatal day 7, mixed population:
glia and netinal neural cell types) are killed by decapitation
following CO.sub.2 anesthesia and the eyes removed under sterile
conditions. The neural retina is dissected away from the pigment
epithelium and other ocular tissue and then dissociated into a
single cell suspension using 0.25% trypsin in Ca.sup.2+,
Mg.sup.2+-free PBS. The retinas are incubated at 37.degree. C. in
this solution for 7-10 minutes after which the trypsin is
inactivated by adding 1 ml soybean trypsin inhibitor. The cells are
plated at a density of approximately 10, 000 cells/ml into 96 well
plates in DMEM/F12 supplemented with N.sub.2. Cells for all
experiments are grown at 37.degree. C. in a water saturated
atmosphere of 5% CO.sub.2. After 7-10 days in culture, the cells
are stained using calcein AM or CellTracker Green CMFDA and then
fixed using 4% paraformaldehyde. Rho 4D2 (ascities or IgG 1: 100)
monoclonal antibody directed towards the visual pigment rhodopsin
is used to detect rod photoreceptor cells by indirect
immunofluorescence. The results are calculated as % survival: total
number of calcein--rhodopsin positive cells at 7-10 days in
culture, divided by the total number of rhodopsin positive cells at
time 7-10 days in culture. The total cells (fluorescent) are
quantified at 20.times. objective magnification using a CCD camera
and NIH image software for MacIntosh. Fields in the well are chosen
at random.
[1551] The following polypeptides tested positive in this assay:
PRO245.
Example 91
In Vitro Antitumor Assay (Assay 161)
[1552] The antiproliferative activity of various PRO polypeptides
was determined in the investigational, disease-oriented in vitro
anti-cancer drug discovery assay of the National Cancer Institute
(NCI), using a sulforhodamine B (SRB) dye binding assay essentially
as described by Skehan et al., J. Natl. Cancer Inst. 82:1107-1112
(1990). The 60 tumor cell lines employed in this study ("the NCI
panel"), as well as conditions for their maintenance and culture in
vitro have been described by Monks et al., J. Natl. Cancer Inst.
83:757-766 (1991). The purpose of this screen is to initially
evaluate the cytotoxic and/or cytostatic activity of the test
compounds against different types of tumors (Monks et al., supra;
Boyd, Cancer: Princ. Pract. Oncol. Update 3(10):1-12 [1989]).
[1553] Cells from approximately 60 human tumor cell lines were
harvested with trypsin/EDTA (Gibco), washed once, resuspended in
IMEM and their viability was determined. The cell suspensions were
added by pipet (100 .mu.L volume) into separate 9-well microtiter
plates. The cell density for the 6-day incubation was less than for
the 2-day incubation to prevent overgrowth. Inoculates were allowed
a preincubation period of 24 hours at 37.degree. C. for
stabilization. Dilutions at twice the intended test concentration
were added at time zero in 100 .mu.L aliquots to the microtiter
plate wells (1:2 dilution). Test compounds were evaluated at five
half-log dilutions (1000 to 100,000-fold). Incubations took place
for two days and six days in a 5% CO.sub.2 atmosphere and 100%
humidity.
[1554] After incubation, the medium was removed and the cells were
fixed in 0.1 ml of 10% trichloroacetic acid at 40.degree. C. The
plates were rinsed five times with deionized water, dried, stained
for 30 minutes with 0.1 ml of 0.4% sulforhodamine B dye (Sigma)
dissolved in 1% acetic acid, rinsed four times with 1% acetic acid
to remove unbound dye, dried, and the stain was extracted for five
minutes with 0.1 ml of 10 mM Tris base
[tris(hydroxymethyl)aminomethane], pH 10.5. The absorbance (OD) of
sulforhodamine B at 492 nm was measured using a
computer-interfaced, 96-well microtiter plate reader.
[1555] A test sample is considered positive if it shows at least
50% growth inhibitory effect at one or more concentrations. PRO
polypeptides testing positive in this assay are shown in Table 7,
where the abbreviations are as follows:
[1556] NSCL:=non-small cell lung carcinoma
[1557] CNS=central nervous system
116TABLE 7 Test compound Tumor Cell Line Type Cell Line Designation
PRO211 NSCL HOP62 PRO211 Leukemia RPMI-8226 PRO211 Leukemia HL-60
(TB) PRO211 NSCL NCI-H522 PRO211 CNS SF-539 PRO211 Melanoma LOX
IMVI PRO211 Breast MDA-MB-435 PRO211 Leukemia MOLT-4 PRO211 CNS
U251 PRO211 Breast MCF7 PRO211 Leukemia HT-60 (TB) PRO211 Leukemia
MOLT-4 PRO211 NSCL EKVX PRO211 NSCL NCI-H23 PRO211 NSCL NCI-H322M
PRO211 NSCL NCI-H460 PRO211 Colon HCT-116 PRO211 Colon HT29 PRO211
CNS SF-268 PRO211 CNS SF-295 PRO211 CNS SNB-19 PRO211 CNS U251
PRO211 Melanoma LOX IMVI PRO211 Melanoma SK-MEL-5 PRO211 Melanoma
UACC-257 PRO211 Melanoma UACC-62 PRO211 Ovarian OVCAR-8 PRO211
Renal RXF 393 PRO211 Breast MCF7 PRO211 Breast NCI/ADR-REHS 578T
PRO2l1 Breast T-47D PRO211 Leukemia HL-60 (TB) PRO211 Leukemia SR
PRO211 NSCL NCI-H23 PRO211 Colon HCT-116 PRO211 Melanoma LOX-IMVI
PRO211 Melanoma SK-MEL-5 PRO211 Breast T-47D PRO228 Leukemia MOLT-4
PRO228 NSCL EKVX PRO228 Colon KM12 PRO228 Melanoma UACC-62 PRO228
Ovarian OVCAR-3 PRO228 Renal TK10 PRO228 Renal SN12C PRO228 Breast
MCF7 PRO228 Leukemia CCRF-CEM PRO228 Leukemia HL-60 (TB) PRO228
Colon COLO 205 PRO228 Colon HCT-15 PRO228 Colon KM12 PRO228 CNS
SF-268 PRO228 CNS SNB-75 PRO228 Melanoma LOX-IMVI PRO228 Melanoma
SK-MEL2 PRO228 Melanoma UACC-257 PRO228 Ovarian IGROV1 PRO228
Ovarian OVCAR-4 PRO228 Ovarian OVCAR-5 PRO228 Ovarian OVCAR-8
PRO228 Renal 786-0 PRO228 Renal CAKI-1 PRO228 Renal RXF 393 PRO228
Renal TK-10 PRO228 Renal UO-31 PRO228 Prostate PC-3 PRO228 Prostate
DU-145 PRO228 Breast MCF7 PRO228 Breast NCI/ADR-REHS 578T PRO228
Breast MDA-MB-435MDA-N PRO228 Breast T-47D PRO219 Leukemia SR
PRO219 NSCL NCI-H5222 PRO219 Breast MCF7 PRO219 Leukemia K-562;
RPMI-8226 PRO219 NSCL HOP-62; NCI-H322M PRO219 NSCL NCI-H460 PRO219
Colon HT29; KM12; HCT-116 PRO219 CNS SF-539; U251 PRO219 Prostate
DU-145 PRO219 Breast MDA-N PRO219 Ovarian IGROV1 PRO219 NSCL
NCI-H226 PRO219 Leukemia MOLT-4 PRO219 NSCL A549/ATCC; EKVX;
NCI-H23 PRO219 Colon HCC-2998 PRO219 CNS SF-295; SNB-19 PRO219
Melanoma SK-MEL-2; SK-MEL-5 PRO219 Melanoma UACC-257; UACC-62
PRO219 Ovarian OCAR-4; SK-OV-3 PRO219 Renal 786-0; ACHN; CAKI-1;
SN12C PRO219 Renal TK-10; UO-31 PRO219 Breast NCI/ADR-RES; BT-549;
T-47D PRO219 Breast MDA-MB-435 PRO221 Leukemia CCRF-CEM PRO221
Leukemia MOLT-4 PRO221 NSCL HOP-62 PRO221 Breast MDA-N PRO221
Leukemia RPMI-8226; SR PRO221 NSCL NCI-H460 PRO221 Colon HCC-2998
PRO221 Ovarian IGROV1 PRO221 Renal TK-10 PRO221 Breast MCF7 PRO221
Leukemia K-562 PRO221 Breast MDA-MB-435 PRO224 Ovarian OVCAR-4
PRO224 Renal RXF 393 PRO224 Prostate DU-145 PRO224 NSCL HOP-62;
NCI-H322M PRO224 Melanoma LOX IMVI PRO224 Ovarian OVCAR-8 PRO224
Leukemia SR PRO224 NSCL NCI-H460 PRO224 CNS SF-295 PRO224 Leukemia
RPMI-8226 PRO224 Breast BT-549 PRO224 Leukemia CCRF-CEM; LH-60 (TB)
PRO224 Colon HCT-116 PRO224 Breast MDA-MB-435 PRO224 Leukemia HL-60
(TB) PRO224 Colon HCC-2998 PRO224 Prostate PC-3 PRO224 CNS U251
PRO224 Colon HCT-15 PRO224 CNS SF-539 PRO224 Renal ACHN PRO328
Leukemia RPMI-8226 PRO328 NSCL A549/ATCC; EKVX; HOP-62 PRO328 NSCL
NCI-H23; NCI-H322M PRO328 Colon HCT-15; KM12 PRO328 CNS SF-295;
SF-539; SNB-19; U251 PRO328 Melanoma M14; UACC-257; UCAA-62 PRO328
Renal 786-0; ACHN PRO328 Breast MCF7 PRO328 Leukemia SR PRO328
Colon NCI-H23 PRO328 Melanoma SK-MEL-5 PRO328 Prostate DU-145
PRO328 Melanoma LOX IMVI PRO328 Breast MDA-MB-435 PRO328 Ovarian
OVCAR-3 PRO328 Breast T-47D PRO301 NSCL NCI-H322M PRO301 Leukemia
MOLT-4; SR PRO301 NSCL A549/ATCC; EKVX; PRO301 NSCL NCI-H23;
NCI-460; NCI-H226 PRO301 Colon COLO 205; HCC-2998; PRO301 Colon
HCT-15; KM12; HT29; PRO301 Colon HCT-116 PRO301 CNS SF-268; SF-295;
SNB-19 PRO301 Melanoma MALME-3M; SK-MEL-2; PRO301 Melanoma
SK-MEL-5; UACC-257 PRO301 Melanoma UACC-62 PRO301 Ovarian IGROV1;
OVCAR-4 PRO301 Ovarian OVCAR-5 PRO301 Ovarian OVCAR-8; SK0OV-3
PRO301 Renal ACHN;CAKI-1; TK-10; UO-31 PRO301 Prostate PC-3; DU-145
PRO301 Breast NCI/ADR-RES; HS 578T PRO301 Breast MDA-MB-435; MDA-N;
T-47D PRO301 Melanoma M14 PRO301 Leukemia CCRF-CEM; HL-60(TB);
K-562 PRO301 Leukemia RPMI-8226 PRO301 Melanoma LOX IMVI PRO301
Renal 786-0; SN12C PRO301 Breast MCF7; MDA-MB-231/ATCC PRO301
Breast BT-549 PRO301 NSCL HOP-62 PRO301 CNS SF-539 PRO301 Ovarian
OVCAR-3 PRO326 NSCL NCI-H322M PRO326 CNS SF295 PRO326 CNS ST539
PRO326 CNS U251
[1558] The results of these assays demonstrate that the positive
testing PRO polypeptides are useful for inhibiting neoplastic
growth in a nunber of different tumor cell types and may be used
therapeutically therefor. Antibodies against these PRO polypeptides
are useful for affinity purification of these useful polypeptides.
Nucleic acids encoding these PRO polypeptides are useful for the
recombinant preparation of these polypeptides.
Example 92
Gene Amplification
[1559] This example shows that certain PRO polypeptide-encoding
genes are amplified in the genome of certain human lung, colon
and/or breast cancers and/or cell lines. Amplification is
associated with overexpression of the gene product, indicating that
the polypeptides are useful targets for therapeutic intervention in
certain cancers such as colon, lung, breast and other cancers and
diagnostic determination of the presence of those cancers.
Therapeutic agents may take the form of antagonists of the PRO
polypeptide, for example, murine-human chimeric, humanized or human
antibodies against a PRO polypeptide.
[1560] The starting material for the screen was genomic DNA
isolated from a variety cancers. The DNA is quantitated precisely,
e.g., fluorometrically. As a negative control, DNA was isolated
from the cells of ten normal healthy individuals which was pooled
and used as assay controls for the gene copy in healthy individuals
(not shown). The 5' nuclease assay (for example, TaqMan.TM.) and
real-time quantitative PCR (for example, ABI Prizm 7700 Sequence
Detection Systems (Perkin Elmer, Applied Biosystems Division,
Foster City, Calif.)), were used to find genes potentially
amplified in certain cancers. The results were used to determine
whether the DNA encoding the PRO polypeptide is over-represented in
any of the primary lung or colon cancers or cancer cell lines or
breast cancer cell lines that were screened. The primary lung
cancers were obtained from individuals with tumors of the type and
stage as indicated in Table 8. An explanation of the abbreviations
used for the designation of the primary tumors listed in Table 8
and the primary tumors and cell lines referred to throughout this
example are given below.
[1561] The results of the TaqMan.TM. are reported in delta (A) Ct
units. One unit corresponds to 1 PCR cycle or approximately a
2-fold amplification relative to normal, two units corresponds to
4-fold, 3 units to 8-fold amplification and so on. Quantitation was
obtained using primers and a TaqMan.TM. fluorescent probe derived
from the PRO polypeptide-encoding gene. Regions of the PRO
polypeptide-encoding gene which are most likely to contain unique
nucleic acid sequences and which are least likely to have spliced
out introns are preferred for the primer and probe derivation,
e.g., 3'-untranslated regions. The sequences for the primers and
probes (forward, reverse and probe) used for the PRO polypeptide
gene amplification analysis were as follows:
117 PRO187 (DNA27864-1155) 27864.tm.p:
5'-GCAGATTTTGAGGACAGCCACCTCCA-3' (SEQ ID NO: 381) 27864.tm.f:
5'-GGCCTTGCAGACAACCGT-3' (SEQ ID NO: 382) 27864.tm.r:
5'-CAGACTGAGGGAGATCCGAGA-3' (SEQ ID NO: 383) 27864.tm.p2:
5'-CAGCTGCCCTTCCCCAACCA-3' (SEQ ID NO: 384) 27864.tm.f2:
5'-CATCAAGCGCCTCTACCA-3' (SEQ ID NO: 385) 27864.tm.r2:
5'-CACAAACTCGAACTGCTTCTG-3' (SEQ ID NO: 386) PRO214
(DNA32286-1191): 32286.3utr-5: 5'-GGGCCATCACAGCTCCCT-3' (SEQ ID NO:
387) 32286.3utr-3b: 5'-GGGATGTGGTGAACACAGAACA-3' (SEQ ID NO: 388)
32286.3utr-probe: 5'-TGCCAGCTGCATGCTGCCAGTT-3- ' (SEQ ID NO: 389)
PRO211 (DNA32292-1131): 32292.3utr-5: 5'-CAGAAGGATGTCCCGTGGAA-3'
(SEQ ID NO: 390) 32292.3utr-3: 5'-GCCGCTGTCCACTGCAG-3' (SEQ ID NO:
391) 32292.3utr-probe.rc: 5'-GACGGCATCCTCAGGGCCACA-3' (SEQ ID NO:
392) PRO230 (DNA33223-1136): 33223.tm.p3:
5'-ATGTCCTCCATGCCCACGCG-3' (SEQ ID NO: 393) 33223.tm.f3:
5'-GAGTGCGACATCGAGAGCTT-3' (SEQ ID NO: 394) 33223.tm.r3:
5'-CCGCAGCCTCAGTGATGA-3' (SEQ ID NO: 395) 33223.3utr-5:
5'-GAAGAGCACAGCTGCAGATCC-3- ' (SEQ ID NO: 396) 33223.3utr-3:
5'-GAGGTGTCCTGGCTTTGGTAGT-3' (SEQ ID NO: 397) 33223.3utr-probe:
5'-CCTCTGGCGCCCCCACTCAA-3' (SEQ ID NO: 398) PRO317 (DNA33461-1199):
33461.tm.f: 5'-CCAGGAGAGCTGGCGATG-3' (SEQ ID NO: 399) 33461.tm.r:
5'-GCAAATTCAGGGCTCACTAGAGA-3' (SEQ ID NO: 400) 33461.tm.p:
5'-CACAGAGCATTTGTCCATCAGCAGTTCAG-3' (SEQ ID NO: 401) PRO246
(DNA35639-1172): 35639.3utr-5: 5'-GGCAGAGACTTCCAGTCACTGA-3' (SEQ ID
NO: 402) 35639.3utr-3: 5'-GCCAAGGGTGGTGTTAGATAGG-3' (SEQ ID NO:
403) 35639.3utr-probe: 5'-CAGGCCCCCTTGATCTGTACCCCA-3' (SEQ ID NO:
404) PRO533 (DNA49435-1219): 49435.tm.f: 5'-GGGACGTGCTTCTACAAGAA-
CAG-3' (SEQ ID NO: 405) 49435.tm.r:
5'-CAGGCTTACAATGTTATGATCAGACA-3' (SEQ ID NO: 406) 49435.tm.p:
5'-TATTCAGAGTTTTCCATTGGCAGTGCCAGTT-3' (SEQ ID NO: 407) PRO343
(DNA43318-1217): 43318.tm.f1 5'-TCTACATCAGCCTCTCTGCGC-3' (SEQ ID
NO: 408) 43318.tm.p1 5'-CGATCTTCTCCACCCAGGAGCGG-3' (SEQ ID NO: 409)
43318.tm.r1 5'-GGAGCTGCACCCCTTGC-3' (SEQ ID NO: 237) PRO232
(DNA34435-1140): 34435.3utr-5: 5'-GCCAGGCCTCACATTCGT-3' (SEQ ID NO:
410) DNA34435.3utr-probe: 5'-CTCCCTGAATGGCAGCCTGAGCA-3' (SEQ ID NO:
411) DNA34435.3utr-3: 5'-AGGTGTTTATTAAGGGCCTAC- GCT-3' (SEQ ID NO:
412) PRO269 (DNA38260-1180): 38260.tm.f: 5'-CAGAGCAGAGGGTGCCTTG-3'
(SEQ ID NO: 413 3826O.tm.p: 5'-TGGCGGAGTCCCCTCTTGGCT-3' (SEQ ID NO:
414) 38260.tm.r: 5'-CCCTGTTTCCCTATGCATCACT-3' (SEQ ID NO: 415)
PRO304 (DNA39520-1217): 39520.tm.f: 5'-TCAACCCCTGACCCTTTCCT- A-3'
(SEQ ID NO: 416) 39520.tm.p: 5'-GGCAGGGGACAAGCCATCTCTCCT-3' (SEQ ID
NO: 417) 39520.tm.r: 5'-GGGACTGAACTGCCAGCTTC-3' (SEQ ID NO: 418)
PRO339 (DNA43466-1225): 43466.tm.f1: 5'-GGGCCCTAACCTCATTACCTTT-3'
(SEQ ID NO: 419) 43466.tm.p1: 5'-TGTCTGCCTCAGCCCCAGGAAGG-3' (SEQ ID
NO: 420) 43466.tm.r1: 5'-TCTGTCCACCATCTTGCCTTG-3' (SEQ ID NO:
421)
[1562] 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 amplification in real time. Two
oligonucleotide primers (forward [.f] and reverse [.r]) are used to
generate an amplicon typical of a PCR reaction. A third
oligonucleotide, or probe (.p), 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 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.
[1563] The 5' nuclease procedure is run on a real-time quantitative
PCR device such as the ABI Prism 7700TM 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.
[1564] 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
.DELTA.Ct 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 DNA results to
normal human DNA results.
[1565] Table 8 describes the stage, T stage and N stage of various
primary tumors which were used to screen the PRO polypeptide
compounds of the invention.
118TABLE 8 Primary Lung and Colon Tumor Profiles Primary Tumor
Stage Stage Other Stage Dukes Stage T Stage N Stage Human lung
tumor AdenoCa (SRCC724) [LT1] IIA T1 N1 Human lung tumor SqCCa
(SRCC725) [LT1a] IIB T3 N0 Human lung tumor AdenoCa (SRCC726) [LT2]
IB T2 N0 Human lung tumor AdenoCa (SRCC727) [LT3] IIIA T1 N2 Human
lung tumor AdenoCa (SRCC728) [LT4] IB T2 N0 Human lung tumor SqCCa
(SRCC729) [LT6] IB T2 N0 Human lung tumor Aden/SqCCa (SRCC730)
[LT7] IA T1 N0 Human lung tumor AdenoCa (SRCC731) [LT9] IB T2 N0
Human lung tumor SqCCa (SRCC732) [LT10] IIB T2 N1 Human lung tumor
SqCCa (SRCC733) [LT11] IIA T1 N1 Human lung tumor AdenoCa (SRCC734)
[LT12] IV T2 N0 Human lung tumor AdenoSqCCa (SRCC735)[LT13] IB T2
N0 Human lung tumor SqCCa (SRCC736) [LT15] IB T2 N0 Human lung
tumor SqCCa (SRCC737) [LT16] IB T2 N0 Human lung tumor SqCCa
(SRCC738) [LT17] IIB T2 N1 Human lung tumor SqCCa (SRCC739) [LT18]
IB T2 N0 Human lung tumor SqCCa (SRCC740) [LT19] IB T2 N0 Human
lung tumor LCCa (SRCC741) [LT21] IIB T3 N1 Human lung AdenoCa
(SRCC811) [LT22] 1A T1 N0 Human colon AdenoCa (SRCC742) [CT2] M1 D
pT4 N0 Human colon AdenoCa (SRCC743) [CT3] B pT3 N0 Human colon
AdenoCa (SRCC744) [CT8] B T3 N0 Human colon AdenoCa (SRCC745) [CT
10] A pT2 N0 Human colon AdenoCa (SRCC746) [CT12] MO, R1 B T3 N0
Human colon AdenoCa (SRCC747) [CT14] pMO, RO B pT3 pN0 Human colon
AdenoCa (SRCC748) [CT15] M1, R2 D T4 N2 Human colon AdenoCa
(SRCC749) [CT16] pMO B pT3 pN0 Human colon AdenoCa (SRCC750) [CT17]
C1 pT3 pN1 Human colon AdenoCa (SRCC751) [CT1] MO, R1 B pT3 N0
Human colon AdenoCa (SRCC752) [CT4] B pT3 M0 Human colon AdenoCa
(SRCC753) [CT5] G2 C1 pT3 pN0 Human colon AdenoCa (SRCC754) [CT6]
pMO, RO B pT3 pN0 Human colon AdenoCa (SRCC755) [CT7] G1 A pT2 pN0
Human colon AdenoCa (SRCC756) [CT9] G3 D pT4 pN2 Human colon
AdenoCa (SRCC757) [CT11] B T3 N0 Human colon AdenoCa (SRCC758)
[CT18] MO, RO B pT3 pN0
[1566] DNA Preparation:
[1567] DNA was prepared from cultured cell lines, primary tumors,
normal human blood. The isolation was performed using purification
kit, buffer set and protease and all from Quiagen, according to the
manufacturer's instructions and the description below.
[1568] Cell Culture Lysis:
[1569] Cells were washed and trypsinized at a concentration of
7.5.times.10.sup.8 per tip and pelleted by centrifuging at 1000 rpm
for 5 minutes at 4.degree. C., followed by washing again with 1/2
volume of PBS recentrifugation. The pellets were washed a third
time, the suspended cells collected and washed 2.times. with PBS.
The cells were then suspended into 10 ml PBS. Buffer C1 was
equilibrated at 4.degree. C. Qiagen protease #19155 was diluted
into 6.25 ml cold ddH.sub.2O to a final concentration of 20 mg/ml
and equilibrated at 4.degree. C. 10 ml of G2 Buffer was prepared by
diluting Qiagen RNAse A stock (100 mglml) to a final concentration
of 200 .mu.g/ml.
[1570] Buffer C1 (10 ml, 4.degree. C.) and ddH.sub.2O (40 ml,
4.degree. C.) were then added to the 10 ml of cell suspension,
mixed by inverting and incubated on ice for 10 minutes. The cell
nuclei were pelleted by centrifuging in a Beckman swinging bucket
rotor at 2500 rpm at 4.degree. C. for 15 minutes. The supernatant
was discarded and the nuclei were suspended with a vortex into 2 ml
Buffer C1 (at 4.degree. C.) and 6 ml ddH.sub.2O, followed by a
second 4.degree. C. centrifugation at 2500 rpm for 15 minutes. The
nuclei were then resuspended into the residual buffer using 200
.mu.l per tip. G2 buffer (10 ml) was added to the suspended nuclei
while gentle vortexing was applied. Upon completion of buffer
addition, vigorous vortexing was applied for 30 seconds.
Quiagenprotease (200 .mu.l, prepared as indicated above) was added
and incubated at 50.degree. C. for 60 minutes. The incubation and
centrifugation was repeated until the lysates were clear (e.g.,
incubating additional 30-60 minutes, pelleting at 3000.times. g for
10 min., 4.degree. C.).
[1571] Solid Human Tumor Sample Preparation and Lysis:
[1572] Tumor samples were weighed and placed into 50 ml conical
tubes and held on ice. Processing was limited to no more than 250
mg tissue per preparation (1 tip/preparation). The protease
solution was freshly prepared by diluting into 6.25 ml cold
ddH.sub.2O to a final concentration of 20 mg/ml and stored at
4.degree. C. G2 buffer (20 ml) was prepared by diluting DNAse A to
a final concentration of 200 mg/ml (from 100 mg/ml stock). The
tumor tissue was homogenated in 19 ml G2 buffer for 60 seconds
using the large tip of the polytron in a laminar-flow TC hood in
order to avoid inhalation of aerosols, and held at room
temperature. Between samples, the polytron was cleaned by spinning
at 2.times.30 seconds each in 2L ddH.sub.2O, followed by G2 buffer
(50 ml). If tissue was still present on the generator tip, the
apparatus was disassembled and cleaned.
[1573] Quiagen protease (prepared as indicated above, 1.0 ml) was
added, followed by vortexing and incubation at 50.degree. C. for 3
hours. The incubation and centrifugation was repeated until the
lysates were clear (e.g., incubating additional 30-60 minutes,
pelleting at 3000.times. g for 10 min., 4.degree. C.).
[1574] Human Blood Preparation and Lysis:
[1575] Blood was drawn from healthy volunteers using standard
infectious agent protocols and citrated into 10 ml samples per tip.
Quiagen protease was freshly prepared by dilution into 6.25 ml cold
ddH.sub.2O to a final concentration of 20 mg/ml and stored at
4.degree. C. G2 buffer was prepared by diluting RNAse A to a final
concentration of 200 .mu.g/ml from 100 mg/ml stock. The blood (10
ml) was placed into a 50 ml conical tube and 10 ml Cl buffer and 30
ml ddH.sub.2O (both previously equilibrated to 4.degree. C.) were
added, and the components mixed by inverting and held on ice for 10
minutes. The nuclei were pelleted with a Beckman swinging bucket
rotor at 2500 rpm, 4.degree. C. for 15 minutes and the supernatant
discarded. With a vortex, the nuclei were suspended into 2 ml C1
buffer (4.degree. C.) and 6 ml ddH.sub.2O (4.degree. C.). Vortexing
was repeated until the pellet was white. The nuclei were then
suspended into the residual buffer using a 200 .mu.l tip. G2 buffer
(10 ml) were added to the suspended nuclei while gently vortexing,
followed by vigorous vortexing for 30 seconds. Quiagen protease was
added (200 .mu.l) and incubated at 50.degree. C. for 60 minutes.
The incubation and centrifugation was repeated until the lysates
were clear (e.g., incubating additional 30-60 minutes, pelleting at
3000.times. g for 10 min., 4.degree. C.).
[1576] Purification of Cleared Lysates:
[1577] (1) Isolation of Genomic DNA:
[1578] Genomic DNA was equilibrated (I sample per maxi tip
preparation) with 10 ml QBT buffer. QF elution buffer was
equilibrated at 50.degree. C. The samples were vortexed for 30
seconds, then loaded onto equilibrated tips and drained by gravity.
The tips were washed with 2.times.15 ml QC buffer. The DNA was
eluted into 30 ml silanized, autoclaved 30 ml Corex tubes with 15
ml QF buffer (50.degree. C.). Isopropanol (10.5 ml) was added to
each sample, the tubes covered with parafm and mixed by repeated
inversion until the DNA precipitated. Samples were pelleted by
centrifugation in the SS-34 rotor at 15,000 rpm for 10 minutes at
4.degree. C. The pellet location was marked, the supernatant
discarded, and 10 ml 70% ethanol (4.degree. C.) was added. Samples
were pelleted again by centrifugation on the SS-34 rotor at 10,000
rpm for 10 minutes at 4.degree. C. The pellet location was marked
and the supernatant discarded. The tubes were then placed on their
side in a drying rack and dried 10 minutes at 37.degree. C., taking
care not to overdry the samples.
[1579] After drying, the pellets were dissolved into 1.0 ml TE (pH
8.5) and placed at 50.degree. C. for 1-2 hours. Samples were held
overnight at 4.degree. C. as dissolution continued. The DNA
solution was then transferred to 1.5 ml tubes with a 26 gauge
needle on a tuberculin syringe. The transfer was repeated 5.times.
in order to shear the DNA. Samples were then placed at 50.degree.
C. for 1-2 hours.
[1580] (2) Quantitation of Genomic DNA and Preparation for Gene
Amplification Assay:
[1581] The DNA levels in each tube were quantified by standard
A.sub.260, A.sub.280 spectrophotometry on a 1:20 dilution (5 .mu.l
DNA+95 .mu.l ddH.sub.2O) using the 0.1 ml quartz cuvetts in the
Beckman DU640 spectrophotometer. A.sub.260/A.sub.280 ratios were in
the range of 1.8-1.9. Each DNA samples was then diluted further to
approximately 200 ng/ml in TE (pH 8.5). If the original material
was highly concentrated (about 700 ng/.mu.l), the material was
placed at 50.degree. C. for several hours until resuspended.
[1582] Fluorometric DNA quantitation was then performed on the
diluted material (20-600 ng/ml) using the manufacturer's guidelines
as modified below. This was accomplished by allowing a Hoeffer DyNA
Quant 200 fluorometer to warm-up for about 15 minutes. The Hoechst
dye working solution (#H33258, 10 .mu.l, prepared within 12 hours
of use) was diluted into 100 ml 1.times. TNE buffer. A 2 ml cuvette
was filled with the fluorometer solution, placed into the machine,
and the machine was zeroed. pGEM 3Zf(+) (2 .mu.l, lot #360851026)
was added to 2 ml of fluorometer solution and calibrated at 200
units. An additional 2 .mu.l of pGEM 3Zf(+) DNA was then tested and
the reading confirmed at 400+/-10 units. Each sample was then read
at least in triplicate. When 3 samples were found to be within 10%
of each other, their average was taken and this value was used as
the quantification value.
[1583] The fluorometricly determined concentration was then used to
dilute each sample to 10 ng/.mu.l in ddH.sub.2O. This was done
simultaneously on all template samples for a single TaqMan.TM.
plate assay, and with enough material to run 500-1000 assays. The
samples were tested in triplicate with Taqman.TM. primers and probe
both B-actin and GAPDH on a single plate with normal human DNA and
no-template controls. The diluted samples were used provided that
the CT value of normal human DNA subtracted from test DNA was +/-1
Ct. The diluted, lot-qualified genomic DNA was stored in 1.0 ml
aliquots at -80.degree. C. Aliquots which were subsequently to be
used in the gene amplification assay were stored at 4.degree. C.
Each 1 ml aliquot is enough for 8-9 plates or 64 tests.
[1584] Gene Amplification Assay:
[1585] The PRO polypeptide compounds of the invention were screened
in the following primary tumors and the resulting .DELTA.Ct values
greater than or equal to 1.0 are reported in Table 9 below.
119TABLE 9 .DELTA.Ct values in lung and colon primary tumors and
cell line models Primary Tumors PRO PRO PRO PRO PRO PRO PRO PRO PRO
PRO PRO PRO of Cell lines 187 533 214 343 211 230 246 317 232 269
304 339 LT7 1.52 1.04 1.08 LT13 2.74 1.85 2.71 1.88 3.42 1.63 1.90
1.27 1.29 1.04 2.98 1.83 2.23 2.26 3.22 1.68 2.24 2.44 2.84 2.93
2.15 2.75 2.53 1.82 LT3 1.57 1.97 1.06 1.86 1.17 LT4 1.17 1.18 LT9
1.42 1.04 1.80 1.03 LT12 2.70 1.38 2.23 1.51 2.86 1.54 2.54 2.40
1.14 1.15 1.26 2.90 1.49 1.50 1.27 2.96 2.47 1.74 2.27 2.92 1.25
2.68 2.28 1.34 LT30 1.67 2.13 1.36 LT21 1.26 1.09 1.50 LT1-a 1.02
1.18 1.29 LT6 1.93 LT10 1.96 1.07 2.57 LT11 1.09 1.67 1.00 2.05
1.32 3.43 2.20 1.14 1.51 1.39 1.80 1.89 1.14 1.41 2.33 1.54 1.02
LT15 3.75 1.77 3.62 2.44 4.32 2.11 2.06 1.86 1.36 1.34 3.92 1.58
1.30 2.16 4.47 1.56 2.76 3.49 3.64 1.63 2.94 3.56 3.32 2.68 LT16
2.10 1.66 1.70 1.25 1.15 1.55 1.00 2.04 1.08 1.83 1.33 LT17 1.32
1.93 1.15 1.85 1.26 2.68 2.29 1.35 1.42 1.68 1.63 1.87 2.30 1.39
1.69 2.03 1.30 1.10 1.33 1.30 LT18 1.17 1.04 LT19 4.05 1.67 2.09
3.82 2.42 4.05 1.91 2.51 1.21 1.60 1.15 3.99 1.98 2.55 4.92 1.68
2.03 4.93 1.16 3.78 4.76 HF-000840 1.58 Calu-1 1.08 SW900 1.86 CT2
3.56 2.49 1.95 1.42 2.75 3.49 2.36 CT3 2.06 1.15 1.34 CT8 1.01 1.48
1.29 1.58 CT10 1.81 1.84 1.88 1.00 1.88 1.49 CT12 1.81 1.74 1.13
CT14 1.82 2.48 2.33 1.36 1.72 1.24 CT15 1.63 2.06 1.33 1.41 1.04
CT16 1.95 1.78 1.40 CT17 2.04 2.40 1.74 CT1 1.24 1.22 1.27 1.25
2.41 1.34 1.46 1.14 CT4 1.36 1.77 1.33 1.32 1.10 1.17 2.05 1.42
1.02 CT5 2.96 1.56 2.68 1.76 2.27 1.33 1.59 2.99 2.76 1.64 2.39 CT6
1.10 1.33 1.01 1.14 CT7 1.40 1.66 1.39 1.00 CT9 1.39 1.16 1.09 1.24
1.13 CT11 2.22 2.05 1.55 2.01 1.75 1.48 1.92 2.26 1.85 1.83 1.12
HF000539 1.57 SW620 1.14 HF000611 4.64 HF000733 1.93 2.33 HF000716
1.68 2.82 CT18 1.29
[1586] Summary
[1587] Because amplification of the various DNA's as described
above occurs in various tumors, it is likely associated with tumor
formation and/or growth. As a result, antagonists (e.g.,
antibodies) directed against these polypeptides would be expected
to be useful in cancer therapy.
Example 94
Detection of PRO Polypeptides That Affect Glucose or FFA Uptake by
Primary Rat Adipocytes (Assay 94)
[1588] This assay is designed to determine whether PRO polypeptides
show the ability to affect glucose or FFA uptake by adipocyte
cells. PRO polypeptides testing positive in this assay would be
expected to be useful for the therapeutic treatment of disorders
where either the stimulation or inhibition of glucose uptake by
adipocytes would be beneficial including, for example, obesity,
diabetes or hyper- or hypo-insulinemia.
[1589] In a 96 well format, PRO polypeptides to be assayed are
added to primary rat adipocytes, and allowed to incubate overnight.
Samples are taken at 4 and 16 hours and assayed for glycerol,
glucose and FFA uptake. After the 16 hour incubation, insulin is
added to the media and allowed to incubate for 4 hours. At this
time, a sample is taken and glycerol, glucose and FFA uptake is
measured. Media containing insulin without the PRO polypeptide is
used as a positive reference control. As the PRO polypeptide being
tested may either stimulate or inhibit glucose and FFA uptake,
results are scored as positive in the assay if greater than 1.5
times or less than 0.5 times the insulin control.
[1590] The following PRO polypeptides tested positive as
stimulators of glucose and/or FFA uptake in this assay: PRO221,
PRO235, PRO245, PRO295, PRO301 and PRO332.
[1591] The following PRO polypeptides tested positive as inhibitors
of glucose and/or FFA uptake in this assay: PRO214, PRO219, PRO228,
PRO222, PRO231 and PRO265.
Example 95
Chondrocyte Re-differentiation Assay (Assay 110)
[1592] This assay shows that certain polypeptides of the invention
act to induce redifferentiation of chondrocytes, therefore, are
expected to be useful for the treatment of various bone and/or
cartilage disorders such as, for example, sports injuries and
arthritis. The assay is performed as follows. Porcine chondrocytes
are isolated by overnight collagenase digestion of articulary
cartilage of metacarpophalangeal joints of 4-6 month old female
pigs. The isolated cells are then seeded at 25,000 cells/cm.sup.2
in Ham F-12 containing 10% FBS and 4 .mu.g/ml gentamycin. The
culture media is changed every third day and the cells are then
seeded in 96 well plates at 5,000 cells/well in 100 .mu.l of the
same media without serum and 100 .mu.l of the test PRO polypeptide,
5 nM staurosporin (positive control) or medium alone (negative
control) is added to give a final volume of 200 L/well. After 5
days of incubation at 37.degree. C., a picture of each well is
taken and the differentiation state of the chondrocytes is
determined. A positive result in the assay occurs when the
redifferentiation of the chondrocytes is determined to be more
similar to the positive control than the negative control.
[1593] The following polypeptide tested positive inthis assay:
PRO214, PRO219, PRO229, PRO222, PRO224, PRO230, PRO257, PRO272 and
PRO301.
Example 96
Fetal Hemoglobin Induction in an Erythroblastic Cell Line (Assay
107)
[1594] This assay is useful for screening PRO polypeptides for the
ability to induce the switch from adult hemoglobin to fetal
hemoglobin in an erythroblastic cell line. Molecules testing
positive in this assay are expected to be useful for
therapeutically treating various mammalian hemoglobin-associated
disorders such as the various thalassemias. The assay is performed
as follows. Erythroblastic cells are plated in standard growth
medium at 1000 cells/well in a 96 well format. PRO polypeptides are
added to the growth medium at a concentration of 0.2% or 2% and the
cells are incubated for 5 days at 37.degree. C. As a positive
control, cells are treated with 100 .mu.M hemin and as a negative
control, the cells are untreated. After 5 days, cell lysates are
prepared and analyzed for the expression of gamma globin (a fetal
marker). A positive in the assay is a gamma globin level at least
2-fold above the negative control.
[1595] The following polypeptides tested positive in this assay:
PRO221 and PRO245.
Example 97
Mouse Kidney Mesangial Cell Proliferation Assay (Assay 92)
[1596] This assay shows that certain polypeptides of the invention
act to induce proliferation of mammalian kidney mesangial cells
and, therefore, are useful for treating kidney disorders associated
with decreased mesangial cell function such as Berger disease or
other nephropathies associated with Schonlein-Henoch purpura,
celiac disease, dermatitis herpetiformis or Crohn disease. The
assay is performed as follows. On day one, mouse kidney mesangial
cells are plated on a 96 well plate in growth media (3:1 mixture of
Dulbecco's modified Eagle's medium and Ham's F12 medium, 95% fetal
bovine serum, 5% supplemented with 14 mM HEPES) and grown
overnight. On day 2, PRO polypeptides are diluted at 2
concentrations(1% and 0.1%) in serum-free medium and added to the
cells. Control samples are serum-free medium alone. On day 4, 20
.mu.l of the Cell Titer 96 Aqueous one solution reagent (Progema)
was added to each well and the colormetric reaction was allowed to
proceed for 2 hours. The absorbance (OD) is then measured at 490
nm. A positive in the assay is anything that gives an absorbance
reading which is at least 15% above the control reading.
[1597] The following polypeptide tested positive in this assay:
PRO227.
Example 98
Proliferation of Rat Utricular Supporting Cells (Assay 54)
[1598] This assay shows that certain polypeptides of the invention
act as potent mitogens for inner ear supporting cells which are
auditory hair cell progenitors and, therefore, are useful for
inducing the regeneration of auditory hair cells and treating
hearing loss in mammals. The assay is performed as follows. Rat
UEC-4 utricular epithelial cells are aliquoted into 96 well plates
with a density of 3000 cells/well in 200 .mu.l of serum-containing
medium at 33.degree. C. The cells are cultured overnight and are
then switched to serun-free medium at 37.degree. C. Various
dilutions of PRO polypeptides (or nothing for a control) are then
added to the cultures and the cells are incubated for 24 hours.
After the 24 hour incubation,.sup.3H-thymidine (1 .mu.Ci/well) is
added and the cells are then cultured for an additional 24 hours.
The cultures are then washed to remove unincorporated radiolabel,
the cells harvested and Cpm per well determined. Cpm of at least
30% or greater in the PRO polypeptide treated cultures as compared
to the control cultures is considered a positive in the assay.
[1599] The following polypeptides tested positive in this assay:
PRO310 and PRO346.
Example 99
Chondrocyte Proliferation Assay (Assay 111)
[1600] This assay is designed to determine whether PRO polypeptides
of the present invention show the ability to induce the
proliferation and/or redifferentiation of chondrocytes in culture.
PRO polypeptides testing positive in this assay would be expected
to be useful for the therapeutic treatment of various bone and/or
cartilage disorders such as, for example, sports injuries and
arthritis.
[1601] Porcine chondrocytes are isolated by overnight collagenase
digestion of articular cartilage of the metacarpophalangeal joint
of 4-6 month old female pigs. The isolated cells are then seeded at
25,000 cells/cm.sup.2 in Ham F-12 containing 10% FBS and 4 .mu.g/ml
gentamycin. The culture media is changed every third day and the
cells are reseeded to 25,000 cells/cm.sup.2 every five days. On day
12, the cells are seeded in 96 well plates at 5,000 cells/well in
100 .mu.l of the same media without serum and 100 .mu.l of either
serum-free medium (negative control), staurosporin (final
concentration of 5 nM; positive control) or the test PRO
polypeptide are added to give a final volume of 200 .mu.l/well.
After 5 days at 37.degree. C., 20 .mu.l of Alamar blue is added to
each well and the plates are incubated for an additional 3 hours at
37.degree. C. The fluorescence is then measured in each well
(Ex:530 nm; Em: 590 nm). The fluorescence of a plate containing 200
.mu.l of the serum-free medium is measured to obtain the
background. A positive result in the assay is obtained when the
fluorescence of the PRO polypeptide treated sample is more like
that of the positive control than the negative control.
[1602] The following PRO polypeptides tested positive in this
assay: PRO219, PRO222, PRO317, PRO257, PRO265, PRO287, PRO272 and
PRO533.
Example 100
Inhibition of Heart Neonatal Hypertrophy Induced by LIF+ET-1 (Assay
74)
[1603] This assay is designed to determine whether PRO polypeptides
of the present invention show the ability to inhibit neonatal heart
hypertrophy induced by LIF and endothelin-1 (ET-1). A test compound
that provides a positive response in the present assay would be
useful for the therapeutic treatment of cardiac insufficiency
diseases or disorders characterized or associated with an undesired
hypertrophy of the cardiac muscle.
[1604] Cardiac myocytes from 1-day old Harlan Sprague Dawley rats
(180 .mu.l at 7.5.times.10.sup.4/ml, serum <0.1, freshly
isolated) are introduced on day 1 to 96-well plates previously
coated with DMEM/F12+4%FCS. Test PRO polypeptide samples or growth
medium alone (negative control) are then added directly to the
wells on day 2 in 20 .mu.l volume. LIF+ET-1 are then added to the
wells on day 3. The cells are stained after an additional 2 days in
culture and are then scored visually the next day. A positive in
the assay occurs when the PRO polypeptide treated myocytes are
visually smaller on the average or less numerous than the untreated
myocytes.
[1605] The following PRO polypeptides tested positive in this
assay: PRO238.
Example 101
Tissue Expression Distribution
[1606] Oligonucleotide probes were constructed from some of the PRO
polypeptide-encoding nucleotide sequences shown in the accompanying
figures for use in quantitative PCR amplification reactions. The
oligonucleotide probes were chosen so as to give an approximately
200-600 base pair amplified fragment from the 3' end of its
associated template in a standard PCR reaction. The oligonucleotide
probes were employed in standard quantitative PCR amplification
reactions with cDNA libraries isolated from different human adult
and/or fetal tissue sources and analyzed by agarose gel
electrophoresis so as to obtain a quantitative determination of the
level of expression of the PRO polypeptide-encoding nucleic acid in
the various tissues tested. Knowledge of the expression pattern or
the differential expression of the PRO polypeptide-encoding nucleic
acid in various different human tissue types provides a diagnostic
marker useful for tissue typing, with or without other
tissue-specific markers, for determining the primary tissue source
of a metastatic tumor, and the like. These assays provided the
following results.
120 Tissues With Tissues Lacking DNA Molecule Significant
Expression Significant Expression DNA34436-1238 lung, placenta,
brain testis DNA35557-1137 lung, kidney, brain placenta
DNA35599-1168 kidney, brain liver, placenta DNA35668-1171 liver,
lung, kidney placenta, brain DNA36992-1168 liver, lung, kidney,
brain placenta DNA39423-1182 kidney, brain liver DNA40603-1232
liver brain, kidney, lung DNA40604-1187 liver brain, kidney, lung
DNA41379-1236 lung, brain liver DNA33206-1165 heart, spleen,
dendrocytes substantia nigra, hippocampus, cartilage, prostate,
HUVEC DNA34431-1177 spleen, HUVEC, cartilage, brain, colon tumor,
heart, uterus prostate, THP-1 macrophages DNA41225-1217 HUVEC,
uterus, colon spleen, brain, heart, tumor, cartilage, IM-9
lymphoblasts prostate
Example 102
In situ Hybridization
[1607] 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.
[1608] 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, paraffm-embedded human tissues were sectioned,
deparaffinized, deproteinated in proteinase K (20 glml) for 15
minutes at 37.degree. C., and further processed for in situ
hybridization as described by Lu and Gillett, supra. A [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.
[1609] .sup.33P-Riboprobe Synthesis
[1610] 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:
[1611] 2.0 .mu.l 5.times. transcription buffer
[1612] 1.0 .mu.l DTT (100 mM)
[1613] 2.0 .mu.l NTP mix (2.5 mM: 10 .mu.; each of 10 mM GTP, CTP
& ATP+10 .mu.l H.sub.2O)
[1614] 1.0 UTP (50 .mu.M)
[1615] 1.0 .mu.l Rnasin
[1616] 1.0 .mu.l DNA template (1 .mu.g)
[1617] 1.0 l.sup.1 H.sub.2O
[1618] 1.0 .mu.l RNA polymerase (for PCR products T3 AS, T7=S,
usually)
[1619] 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.
[1620] The probe was run on a TBE/urea gel. 1-3 .mu.l of the probe
or 5 al 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
gel 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.
[1621] .sup.33P-Hybridization
[1622] A. Pretreatment of Frozen Sections
[1623] 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.
[1624] B. Pretreatment of Paraffin-embedded Sections
[1625] The slides were deparaffmized, 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.
[1626] C. Prehybridization
[1627] The slides were laid out in a plastic box lined with Box
buffer (4.times. SSC, 50% formamide)--saturated filter paper. The
tissue was covered with 50 .mu.l of hybridization buffer (3.75 g
Dextran Sulfate+6 ml SQ H.sub.2O), vortexed and heated in the
microwave for 2 minutes with the cap loosened. After cooling on
ice, 18.75 ml formamide, 3.75 ml 20.times. SSC and 9 ml SQ H.sub.2O
were added, the tissue was vortexed well, and incubated at
42.degree. C. for 1-4 hours.
[1628] D. Hybridization
[1629] 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.
[1630] E. Washes
[1631] 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,
V.sub.f=4L), 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, V.sub.f=4L).
[1632] F. Oligonucleotides
[1633] In situ analysis was performed on a variety of DNA sequences
disclosed herein. The oligonucleotides employed for these analyses
are as follows.
121 (1) DNA33094-1131 (PR0217) p1 5'-GGATTCTAATACGACTCACTA-
TAGGGCTCAGAAAAGCGCAACAGAGAA-3' (SEQ ID NO:348) p2
5'-CTATGAAATTAACCCTCACTAAAGGGATGTCTTCCATGCCAACCTTC-3' (SEQ ID
NO:349) (2) DNA33223-1136 (PR0230) p1 5'-GGATTCTAATACGACTCACTA-
TAGGGCGGCGATGTCCACTGGGGCTAC-3' (SEQ ID NO:350) P2
5'-CTATGAAATTAACCCTCACTAAAGGGACGAGGAAGATGGGCGGATGGT-3' (SEQ ID
NO:351) (3) DNA34435-1140 (PR0232) p1 5'-GGATTCTAATACGACTCACT-
ATAGGGCACCCACGCGTCCGGCTGCTT-3' (SEQ ID NO:352) p2
5'-CTATGAAATTAACCCTCACTAAAGGGACGGGGGACACCACGGACCAGA-3' (SEQ ID
NO:353) (4) DNA35639-1172 (PR0246) p1 5'-GGATTCTAATACGACTCACT-
ATAGGGCTTGCTGCGGTTTTTGTTCCTG-3' (SEQ ID NO:354) p2
5'-CTATGAAATTAACCCTCACTAAAGGGAGCTGCCGATCCCACTGGTATT-3' (SEQ ID
NO:355) (5) DNA49435-1219 (PR0533) p1 5'-GGATTCTAATACGACTCACT-
ATAGGGCGGATCCTGGCCGGCCTCTG-3' (SEQ ID NO:356) P2
5'-CTATGAAATTAACCCTCACTAAAGGGAGCCCGGGCATGGTCTCAGTTA-3' (SEQ ID
NO:357) (6) DNA35638-1141 (PR0245) p1 5'-GGATTCTAATACGACTCACT-
ATAGGGCGGGAAGATGGCGAGGAGGAG-3' (SEQ ID NO:358) p2
5'-CTATGAAATTAACCCTCACTAAAGGGACCAAGGCCACAAACGGAAATC-3' (SEQ ID
NO:359) (7) DNA33089-1132 (PR0221) p1 5'-GGATTCTAATACGACTCACT-
ATAGGGCTGTGCTTTCATTCTGCCAGTA-3' (SEQ ID NO:360) p2
5'-CTATGAAATTAACCCTCACTAAAGGGAGGGTACAATTAAGGGGTGGAT-3' (SEQ ID
NO:361) (8) DNA35918-1174 (PR0258) p1 5'-GGATTCTAATACGACTCACT-
ATAGGGCCCGCCTCGCTCCTGCTCCTG-3' (SEQ ID NO:362) p2
5'-CTATGAAATTAACCCTCACTAAAGGGAGGATTGCCGCGACCCTCACAG-3' (SEQ ID
NO:363) (9) DNA32286-1191 (PR0214) p1 5'-GGATTCTAATACGACTCACT-
ATAGGGCCCCTCCTGCCTTCCCTGTCC-3' (SEQ ID NO:364) p2
5'-CTATGAAATTAACCCTCACTAAAGGGAGTGGTGGCCGCGATTATCTGC-3' (SEQ ID
NO:365) (10) DNA33221-1133 (PR0224) p1
5'-GGATTCTAATACGACTCACTATAGGGCGCAGCGATGGCAGCGATGAGG-3' (SEQ ID
NO:366) P2 5'-CTATGAAATTAACCCTCACTAAAGGGACAGACGGGGCAGAGGGAGTG-3'
(SEQ ID NO:367) (11) DNA35557-1137 (PR0234) p1
5'-GGATTCTAATACGACTCACTATAGGGCCAGGAGGCGTGAGGAGAAAC-3' (SEQ ID
NO:368) p2 5'-CTATGAAATTAACCCTCACTAAAGGGAAAGACATGTCATCGGGAGTGG-3'
(SEQ ID NO:369) (12) DNA33LOO-1159 (PR0229) p1
5'-GGATTCTAATACGACTCACTATAGGGCCGGGTGGAGGTGGAACAGAAA-3' (SEQ ID
NO:370) P2 5'-CTATGAAATTAACCCTCACTAAAGGGACACAGACAGAGCCCCATACGC-3'
(SEQ ID NO:371) (13) DNA34431-1177 (PR0263) p1
5'-GGATTCTAATACGACTCACTATAGGGCCAGGGAAATCCGGATGTCTC-3' (SEQ ID
NO:372) p2 5'-CTATGAAATTAACCCTCACTAAAGGGAGTAAGGGGATGCCACCGAGTA-3'
(SEQ ID NO:373) (14) DNA38268-1188 (PR0295) p1
5'-GGATTCTAATACGACTCACTATAGGGCCAGCTACCCGCAGGAGGAGG-3' (SEQ ID
NO:374) p2 5'-CTATGAAATTAACCCTCACTAAAGGGATCCCAGGTGATGAGGTCCAGA-3'
(SEQ ID NO:375)
[1634] G. Results
[1635] In situ analysis was performed on a variety of DNA sequences
disclosed herein. The results from these analyses are as
follows.
[1636] (1) DNA33094-1131 (PRO217)
[1637] Highly distinctive expression pattern, that does not
indicate an obvious biological function. In the human embryo it was
expressed in outer smooth muscle layer of the GI tract, respiratiry
cartilage, branching respiratory epithelium, osteoblasts, tendons,
gonad, in the optic nerve head and developing dermis. In the adult
expression was observed in the epidermal pegs of the chimp tongue,
the basal epithelial/myoepithelial cells of the prostate and
urinary bladder. Also expressed in the alveolar lining cells of the
adult lung, mesenchymal cells juxtaposed to erectile tissue in the
penis and the cerebral cortex (probably glial cells). In the
kidney, expression was only seen in disease, in cells surrounding
thyroidized renal tubules.
[1638] Human fetal tissues examined (E12-E16 weeks) include:
Placenta, umbilical cord, liver, kidney, adrenals, thyroid, lungs,
heart, great vessels, oesophagus, stomach, small intestine, spleen,
thymus, pancreas, brain, eye, spinal cord, body wall, pelvis and
lower limb.
[1639] Adult human tissues examined: Kidney (normal and end-stage),
adrenal, myocardium, aorta, spleen, lymph node, gall bladder,
pancreas, lung, skin, eye (inc. retina), prostate, bladder, liver
(normal, cirrhotic, acute failure).
[1640] Non-human primate tissues examined:
[1641] (a) Chimp Tissues: Salivary gland, stomach, thyroid,
parathyroid, skin, thymus, ovary, lymph node.
[1642] (b) Rhesus Monkey Tissues: Cerebral cortex, hippocampus,
cerebellum, penis.
[1643] (2) DNA33223-1136 (PRO230)
[1644] Sections show an intense signal associated with arterial and
venous vessels in the fetus. In arteries the signal appeared to be
confined to smooth-muscle/pericytic cells. The signal is also seen
in capillary vessels and in glomeruli. It is not clear whether or
not endothelial cells are expressing this mRNA. Expression is also
observed in epithelial cells in the fetal lens. Strong expression
was also seen in cells within placental trophoblastic villi, these
cells lie between the trophoblast and the fibroblast-like cells
that express HGF--uncertain histogenesis. In the adult, there was
no evidence of expression and the wall of the aorta and most
vessels appear to be negative. However, expression was seen over
vascular channels in the normal prostate and in the epithelium
lining the gallbladder. Insurers expression was seen in the vessels
of the soft-tissue sarcoma and a renal cell carcinoma. In summary,
this is a molecule that shows relatively specific vascular
expression in the fetus as well as in some adult organs. Expression
was also observed in the fetal lens and the adult gallbladder.
[1645] In a secondary screen, vascular expression was observed,
similar to that observed above, seen in fetal blocks. Expression is
on vascular smooth muscle, rather than endothelium. Expression also
seen in smooth muscle of the developing oesophagus, so as reported
previously, this molecule is not vascular specific. Expression was
examined in 4 lung and 4 breast carcinomas. Substantial expression
was seen in vascular smooth muscle of at least 3/4 lung cancers and
2/4 breast cancers. In addition, in one breast carcinoma,
expression was observed in peritumoral stromal cells of uncertain
histogenesis (possibly myofibroblasts). No endothelial cell
expression was observed in this study.
[1646] (3) DNA34435-1140 (PRO232)
[1647] Strong expression in prostatic epithelium and bladder
epithelium, lower level of expression in bronchial epithelium. High
background/low level expression seen in a number of sites,
including among others, bone, blood, chondrosarcoma, adult heart
and fetal liver. It is felt that this level of signal represents
background, partly because signal at this level was seen over the
blood. All other tissues negative.
[1648] Human fetal tissues examined (E12-E16 weeks) include:
Placenta, umbilical cord, liver, kidney, adrenals, thyroid, lungs,
heart, great vessels, oesophagus, stomach, small intestine, spleen,
thymus, pancreas, brain, eye, spinal cord, body wall, pelvis,
testis and lower limb.
[1649] Adult human tissues examined: Kidney (normal and end-stage),
adrenal, spleen, lymph node, pancreas, lung, eye (inc. retina),
bladder, liver (normal, cirrhotic, acute failure).
[1650] Non-human primate tissues examined:
[1651] Chimp Tissues: adrenal
[1652] Rhesus Monkey Tissues: Cerebral cortex, hippocampus
[1653] In a secondary screen, expression was observed in the
epithelium of the prostate, the superficial layers of the
urethelium of the urinary bladder, the urethelium lining the renal
pelvis and the urethelium of the ureter (1 out of 2 experiments).
The urethra of a rhesus monkey was negative; it is unclear whether
this represents a true lack of expression by the urethra, or if it
is the result of a failure of the probe to cross react with rhesus
tissue. The findings in the prostate and bladder are similar to
those previously described using an isotopic detection technique.
Expression of the mRNA for this antigen is NOT prostate epithelial
specific. The antigen may serve as a useful marker for urethelial
derived tissues. Expression in the superficial, post-mitotic cells,
of the urinary tract epithelium also suggest that it is unlikely to
represent a specific stem cell marker, as this would be expected to
be expressed specifically in basal epithelium.
[1654] (4) DNA35639-1172 (PRO246)
[1655] Strongly expressed in fetal vascular endothelium, including
tissues of the CNS. Lower level of expression in adult vasculature,
including the CNS. Not obviously expressed at higher levels in
tumor vascular endothelium. Signal also seen over bone matrix and
adult spleen, not obviously cell associated, probably related to
non-specific background at these sites.
[1656] Human fetal tissues examined (E12-E16 weeks) include:
Placenta, umbilical cord, liver, kidney, adrenals, thyroid, lungs,
heart, great vessels, oesophagus, stomach, small intestine, spleen,
thymus, pancreas, brain, eye, spinal cord, body wall, pelvis,
testis and lower limb.
[1657] Adult human tissues examined: Kidney (normal and end-stage),
adrenal, spleen, lymph node, pancreas, lung, eye (inc. retina),
bladder, liver (normal, cirrhotic, acute failure).
[1658] Non-human primate tissues examined:
[1659] Chimp Tissues: adrenal
[1660] Rhesus Monkey Tissues: Cerebral cortex, hippocampus
[1661] (5) DNA49435-1219 (PRO533)
[1662] Moderate expression over cortical neurones in the fetal
brain. Expression over the inner aspect of the fetal retina,
possible expression in the developing lens. Expression over fetal
skin, cartilage, small intestine, placental villi and umbilical
cord. In adult tissues there is an extremely high level of
expression over the gallbladder epithelium. Moderate expression
over the adult kidney, gastric and colonic epithelia. Low-level
expression was observed over many cell types in many tissues, this
may be related to stickiness of the probe, these data should
therefore be interpreted with a degree of caution.
[1663] Human fetal tissues examined (E12-E16 weeks) include:
Placenta, umbilical cord, liver, kidney, adrenals, thyroid, lungs,
heart, great vessels, oesophagus, stomach, small intestine, spleen,
thymus, pancreas, brain, eye, spinal cord, body wall, pelvis,
testis and lower limb.
[1664] Adult human tissues examined: Kidney (normal and end-stage),
adrenal, spleen, lymph node, pancreas, lung, eye (inc. retina),
bladder, liver (normal, cirrhotic, acute failure).
[1665] Non-human primate tissues examined:
[1666] Chimp Tissues: adrenal
[1667] Rhesus Monkey Tissues: Cerebral cortex, hippocampus,
cerebellum.
[1668] (6) DNA35638-1141 (PRO245)
[1669] Expression observed in the endothelium lining a subset of
fetal and placental vessels. Endothelial expression was confined to
these tissue blocks. Expression also observed over intermediate
trophoblast cells of placenta. Expression also observed tumor
vasculature but not in the vasculature of normal tissues of the
same type. All other tissues negative.
[1670] Fetal tissues examined (E12-E16 weeks) include: Placenta,
umbilical cord, liver, kidney, adrenals, thyroid, lungs, heart,
great vessels, oesophagus, stomach, small intestine, spleen,
thymus, pancreas, brain, eye, spinal cord, body wall, pelvis and
lower limb.
[1671] Adult tissues examined: Liver, kidney, adrenal, myocardium,
aorta, spleen, lymph node, pancreas, lung, skin, cerebral cortex
(rm), hippocampus(rm), cerebellum(rm), penis, eye, bladder,
stomach, gastric carcinoma, colon, colonic carcinoma, thyroid
(chimp), parathyroid (chimp) ovary (chimp) and chondrosarcoma.
Acetominophen induced liver injury and hepatic cirrhosis
[1672] (7) DNA33089-1132 (PRO221)
[1673] Specific expression over fetal cerebral white and grey
matter, as well as over neurones in the spinal cord. Probe appears
to cross react with rat. Low level of expression over cerebellar
neurones in adult rhesus brain. All other tissues negative.
[1674] Fetal tissues examined (E12-E16 weeks) include: Placenta,
umbilical cord, liver, kidney, adrenals, thyroid, lungs, heart,
great vessels, oesophagus, stomach, small intestine, spleen,
thymus, pancreas, brain, eye, spinal cord, body wall, pelvis and
lower limb.
[1675] Adult tissues examined: Liver, kidney, adrenal, myocardium,
aorta, spleen, lymph node, pancreas, lung, skin, cerebral cortex
(rm), hippocampus(rm), cerebellum(rm), penis, eye, bladder,
stomach, gastric carcinoma, colon, colonic carcinoma and
chondrosarcoma. Acetominophen induced liver injury and hepatic
cirrhosis
[1676] (8) DNA35918-1174 (PRO258)
[1677] Strong expression in the nervous system. In the rhesus
monkey brain expression is observed in cortical, hippocampal and
cerebellar neurones. Expression over spinal neurones in the fetal
spinal cord, the developing brain and the inner aspects of the
fetal retina. Expression over developing dorsal root and autonomic
ganglia as well as enteric nerves. Expression observed over
ganglion cells in the adult prostate. In the rat, there is strong
expression over the developing hind brain and spinal cord. Strong
expression over interstitial cells in the placental villi. All
other tissues were negative.
[1678] Fetal tissues examined (E12-E16 weeks) include: Placenta,
umbilical cord, liver, kidney, adrenals, thyroid, lungs, heart,
great vessels, oesophagus, stomach, small intestine, spleen,
thymus, pancreas, brain, eye, spinal cord, body wall, pelvis and
lower limb.
[1679] Adult tissues examined: Liver, kidney, renal cell carcinoma,
adrenal, aorta, spleen, lymph node, pancreas, lung, myocardium,
skin, cerebral cortex (rm), hippocampus(rm), cerebellum(rm),
bladder, prostate, stomach, gastric carcinoma, colon, colonic
carcinoma, thyroid (chimp), parathyroid (chimp) ovary (chimp) and
chondrosarcoma. Acetominophen induced liver injury and hepatic
cirrhosis.
[1680] (9) DNA32286-1191 (PRO214)
[1681] Fetal tissue: Low level throughout mesenchyme. Moderate
expression in placental stromal cells in membranous tissues and in
thyroid. Low level expression in cortical neurones. Adult tissue:
all negative.
[1682] Fetal tissues examined (E12-E16 weeks) include: Placenta,
umbilical cord, liver, kidney, adrenals, thyroid, lungs, heart,
great vessels, oesophagus, stomach, small intestine, spleen,
thymus, pancreas, brain, eye, spinal cord, body wall, pelvis and
lower limb.
[1683] Adult tissues examined include: Liver, kidney, adrenal,
myocardium, aorta, spleen, lymph node, pancreas, lung and skin.
[1684] (10) DNA33221-1133 (PRO224)
[1685] Expression limited to vascular endothelium in fetal spleen,
adult spleen, fetal liver, adult thyroid and adult lymph node
(chimp). Additional site of expression is the developing spinal
ganglia. All other tissues negative.
[1686] Human fetal tissues examined (E12-E16 weeks) include:
Placenta, umbilical cord, liver, kidney, adrenals, thyroid, lungs,
heart, great vessels, oesophagus, stomach, small intestine, spleen,
thymus, pancreas, brain, eye, spinal cord, body wall, pelvis and
lower limb.
[1687] Adult human tissues examined: Kidney (normal and end-stage),
adrenal, myocardium, aorta, spleen, lymph node, pancreas, lung,
skin, eye (inc. retina), bladder, liver (normal, cirrhotic, acute
failure).
[1688] Non-human primate tissues examined:
[1689] Chimp Tissues: Salivary gland, stomach, thyroid,
parathyroid, skin, thymus, ovary, lymph node.
[1690] Rhesus Monkey Tissues: Cerebral cortex, hippocampus,
cerebellum, penis.
[1691] (11) DNA35557-1137 (PRO234)
[1692] Specific expression over developing motor neurones in
ventral aspect of the fetal spinal cord (will develop into ventral
horns of spinal cord). All other tissues negative. Possible role in
growth, differentiation and/or development of spinal motor
neurons.
[1693] Fetal tissues examined (E12-E16 weeks) include: Placenta,
umbilical cord, liver, kidney, adrenals, thyroid, lungs, heart,
great vessels, oesophagus, stomach, small intestine, spleen,
thymus, pancreas, brain, eye, spinal cord, body wall, pelvis and
lower limb.
[1694] Adult tissues examined: Liver, kidney, adrenal, myocardium,
aorta, spleen, lymph node, pancreas, lung, skin, cerebral cortex
(rm), hippocampus(rm), cerebellum(rm), penis, eye, bladder,
stomach, gastric carcinoma, colon, colonic carcinoma and
chondrosarcoma. Acetominophen induced liver injury and hepatic
cirrhosis
[1695] (12) DNA33100-1159 (PRO229)
[1696] Striking expression in mononuclear phagocytes (macrophages)
of fetal and adult spleen, liver, lymph node and adult thymus (in
tingible body macrophages). The highest expression is in the
spleen. All other tissues negative. Localisation and homology are
entirely consistent with a role as a scavenger receptor for cells
of the reticuloendothelial system. Expression also observed in
placental mononuclear cells.
[1697] Human fetal tissues examined (E12-E16 weeks) include:
Placenta, umbilical cord, liver, kidney, adrenals, thyroid, lungs,
heart, great vessels, oesophagus, stomach, small intestine, spleen,
thymus, pancreas, brain, eye, spinal cord, body wall, pelvis and
lower limb.
[1698] Adult human tissues examined: Kidney (normal and end-stage),
adrenal, myocardium, aorta, spleen, lymph node, gall bladder,
pancreas, lung, skin, eye (inc. retina), prostate, bladder, liver
(normal, cirrhotic, acute failure).
[1699] Non-human primate tissues examined:
[1700] Chimp Tissues: Salivary gland, stomach, thyroid,
parathyroid, skin, thymus, ovary, lymph node.
[1701] Rhesus Monkey Tissues: Cerebral cortex, hippocampus,
cerebellum, penis.
[1702] (13) DNA34431-1177 (PRO263)
[1703] Widepread expression in human fetal tissues and placenta
over mononuclear cells, probably macrophages +/-lymphocytes. The
cellular distribution follows a perivascular pattern in many
tissues. Strong expression also seen in epithelial cells of the
fetal adrenal cortex. All adult tissues were negative.
[1704] Fetal tissues examined (E12-E16 weeks) include: Placenta,
umbilical cord, liver, kidney, adrenals, thyroid, lungs, heart,
great vessels, oesophagus, stomach, small intestine, spleen,
thymus, pancreas, brain, eye, spinal cord, body wall, pelvis and
lower limb.
[1705] Adult tissues examined: Liver, kidney, adrenal, spleen,
lymph node, pancreas, lung, skin, cerebral cortex (rm),
hippocampus(rm), cerebellum(rm), bladder, stomach, colon and
colonic carcinoma. Acetominophen induced liver injury and hepatic
cirrhosis.
[1706] A secondary screen evidenced expression over stromal
mononuclear cells probably histiocytes.
[1707] (14) DNA38268-1188 (PRO295)
[1708] High expression over ganglion cells in human fetal spinal
ganglia and over large neurones in the anterior horns of the
developing spinal cord. In the adult there is expression in the
chimp adrenal medulla (neural), neurones of the rhesus monkey brain
(hippocampus [+++] and cerebral cortex) and neurones in ganglia in
the normal adult human prostate (the only section that contains
ganglion cells, expression in this cell type is presumed NOT to be
confined to the prostate). All other tissues negative.
[1709] Human fetal tissues examined (E12-E16 weeks) include:
Placenta, umbilical cord, liver, kidney, adrenals, thyroid, lungs,
great vessels, stomach, small intestine, spleen, thymus, pancreas,
brain, eye, spinal cord, body wall, pelvis, testis and lower
limb.
[1710] Adult human tissues examined: Kidney (normal and end-stage),
adrenal, spleen, lymph node, pancreas, lung, eye (inc. retina),
bladder, liver (normal, cirrhotic, acute failure).
[1711] Non-human Primate tissues examined:
[1712] Chimp Tissues: adrenal
[1713] Rhesus Monkey Tissues: Cerebral cortex, hippocampus,
cerebellum.
Example 103
Isolation of cDNA clones Encoding Human PRO1868
[1714] 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 DNA49803. Based up an
observed homology between the DNA49803 consensus sequence and an
EST sequence contained within the Incyte EST clone no. 2994689,
Incyte EST clone no. 2994689 was purchased and its insert obtained
and sequenced. The sequence of that insert is shown in FIG. 123 and
is herein designated DNA77624-2515.
[1715] The entire nucleotide sequence of DNA77624-2515 is shown in
FIG. 123 (SEQ ID NO:422). Clone DNA77624-2515 contains a single
open reading frame with an apparent translational initiation site
at nucleotide positions 51-53 and ending at the stop codon at
nucleotide positions 981-983 (FIG. 123). The predicted polypeptide
precursor is 310 amino acids long (FIG. 124). The full-length
PRO1868 protein shown in FIG. 124 has an estimated molecular weight
of about 35,020 daltons and a pI of about 7.90. Analysis of the
full-length PRO1868 sequence shown in FIG. 124 (SEQ ID NO:423)
evidences the presence of the following: a signal peptide from
about amino acid 1 to about amino acid 30, a transmembrane domain
from about amino acid 243 to about amino acid 263, potential
N-glycosylation sites from about amino acid 104 to about amino acid
107 and from about amino acid 192 to about amino acid 195, a cAMP-
and cGMP-dependent protein kinase phosphorylation site from about
amino acid 107 to about amino acid 110, casein kinase II
phosphorylation sites from about amino acid 106 to about amino acid
109 and from about amino acid 296 to about amino acid 299, a
tyrosine kinase phosphorylation site from about amino acid 69 to
about amino acid 77 and potential N-myristolation sites from about
amino acid 26 to about amino acid 31, from about amino acid 215 to
about amino acid 220, from about amino acid 226 to about amino acid
231, from about amino acid 243 to about amino acid 248, from about
amino acid 244 to about amino acid 249 and from about amino acid
262 to about amino acid 267. Clone DNA77624-2515 has been deposited
with ATCC on Dec. 22, 1998 and is assigned ATCC deposit no.
203553.
[1716] 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. 124 (SEQ ID NO:423), evidenced
significant homology between the PRO1868 amino acid sequence and
the following Dayhoff sequences: HGS_RC75, P_W61379, A33_HUMAN,
P_W14146, P_W14158, AMAL_DROME, P_R77437, I38346, NCM2_HUMAN and
PTPD_HUMAN.
Example 104
Identification of Receptor/Ligand Interactions
[1717] In this assay, various PRO polypeptides are tested for
ability to bind to a panel of potential receptor molecules for the
purpose of identifying receptor/ligand interactions. The
identification of a ligand for a known receptor, a receptor for a
known ligand or a novel receptor/ligand pair is useful for a
variety of indications including, for example, targeting bioactive
molecules (linked to the ligand or receptor) to a cell known to
express the receptor or ligand, use of the receptor or ligand as a
reagent to detect the presence of the ligand or receptor in a
composition suspected of containing the same, wherein the
composition may comprise cells suspected of expressing the ligand
or receptor, modulating the growth of or another biological or
immunological activity of a cell known to express or respond to the
receptor or ligand, modulating the immune response of cells or
toward cells that express the receptor or ligand, allowing the
preparaion of agonists, antagonists and/or antibodies directed
against the receptor or ligand which will modulate the growth of or
a biological or immunological activity of a cell expressing the
receptor or ligand, and various other indications which will be
readily apparent to the ordinarily skilled artisan.
[1718] The assay is performed as follows. A PRO polypeptide of the
present invention suspected of being a ligand for a receptor is
expressed as a fusion protein containing the Fc domain of human IgG
(an immunoadhesin). Receptor-ligand binding is detected by allowing
interaction of the immunoadhesin polypeptide with cells (e.g. Cos
cells) expressing candidate PRO polypeptide receptors and
visualization of bound immunoadhesin with fluorescent reagents
directed toward the Fc fusion domain and examination by microscope.
Cells expressing candidate receptors are produced by transient
transfection, in parallel, of defined subsets of a library of cDNA
expression vectors encoding PRO polypeptides that may function as
receptor molecules. Cells are then incubated for 1 hour in the
presence of the PRO polypeptide immunoadhesin being tested for
possible receptor binding. The cells are then washed and fixed with
paraformaldehyde. The cells are then incubated with fluorescent
conjugated antibody directed against the Fc portion of the PRO
polypeptide immunoadhesin (e.g. FITC conjugated goat anti-human-Fc
antibody). The cells are then washed again and examined by
microscope. A positive interaction is judged by the presence of
fluorescent labeling of cells transfected with cDNA encoding a
particular PRO polypeptide receptor or pool of receptors and an
absence of similar fluorescent labeling of similarly prepared cells
that have been transfected with other cDNA or pools of cDNA. If a
defined pool of cDNA expression vectors is judged to be positive
for interaction with a PRO polypeptide immunoadhesin, the
individual cDNA species that comprise the pool are tested
individually (the pool is "broken down") to determine the specific
cDNA that encodes a receptor able to interact with the PRO
polypeptide immunoadhesin.
[1719] In another embodiment of this assay, an epitope-tagged
potential ligand PRO polypeptide (e.g. 8 histidine "His" tag) is
allowed to interact with a panel of potential receptor PRO
polypeptide molecules that have been expressed as fusions with the
Fc domain of human IgG (immunoadhesins). Following a 1 hour
co-incubation with the epitope tagged PRO polypeptide, the
candidate receptors are each immunoprecipitated with protein A
beads and the beads are washed. Potential ligand interaction is
determined by western blot analysis of the immunoprecipitated
complexes with antibody directed towards the epitope tag. An
interaction is judged to occur if a band of the anticipated
molecular weight of the epitope tagged protein is observed in the
western blot analysis with a candidate receptor, but is not
observed to occur with the other members of the panel of potential
receptors.
[1720] Using these assays, the following receptor/ligand
interactions have been herein identified: PRO245 binds to
PRO1868.
[1721] Deposit of Material
[1722] The following materials have been deposited with the
American Type Culture Collection, 12301 Parklawn Drive, Rockville,
Md., USA (ATCC):
122 Material ATCC Dep. No. Deposit Date DNA32292-1131 ATCC 209258
September 16, 1997 DNA33094-1131 ATCC 209256 September 16, 1997
DNA33223-1136 ATCC 209264 September 16, 1997 DNA34435-1140 ATCC
209250 September 16, 1997 DNA27864-1155 ATCC 209375 October 16,
1997 DNA36350-1158 ATCC 209378 October 16, 1997 DNA32290-1164 ATCC
209384 October 16, 1997 DNA35639-1172 ATCC 209396 October 17, 1997
DNA33092-1202 ATCC 209420 October 28, 1997 DNA49435-1219 ATCC
209480 November 21, 1997 DNA35638-1141 ATCC 209265 September 16,
1997 DNA32298-1132 ATCC 209257 September 16, 1997 DNA33089-1132
ATCC 209262 September 16, 1997 DNA33786-1132 ATCC 209253 September
16, 1997 DNA35918-1174 ATCC 209402 October 17, 1997 DNA37150-1178
ATCC 209401 October 17, 1997 DNA38260-1180 ATCC 209397 October 17,
1997 DNA39969-1185 ATCC 209400 October 17, 1997 DNA32286-1191 ATCC
209385 October 16, 1997 DNA33461-1199 ATCC 209367 October 15, 1997
DNA40628-1216 ATCC 209432 November 7, 1997 DNA33221-1133 ATCC
209263 September 16, 1997 DNA33107-1135 ATCC 209251 September 16,
1997 DNA35557-1137 ATCC 209255 September 16, 1997 DNA34434-1139
ATCC 209252 September 16, 1997 DNA33100-1159 ATCC 209373 October
16, 1997 DNA35600-1162 ATCC 209370 October 16, 1997 DNA34436-1238
ATCC 209523 December 10, 1997 DNA33206-1165 ATCC 209372 October 16,
1997 DNA35558-1167 ATCC 209374 October 16, 1997 DNA35599-1168 ATCC
209373 October 16, 1997 DNA36992-1168 ATCC 209382 October 16, 1997
DNA34407-1169 ATCC 209383 October 16, 1997 DNA35841-1173 ATCC
209403 October 17, 1997 DNA33470-1175 ATCC 209398 October 17, 1997
DNA34431-1177 ATCC 209399 October 17, 1997 DNA39510-1181 ATCC
209392 October 17, 1997 DNA39423-1182 ATCC 209387 October 17, 1997
DNA40620-1183 ATCC 209388 October 17, 1997 DNA40604-1187 ATCC
209394 October 17, 1997 DNA38268-1188 ATCC 209421 October 28, 1997
DNA37151-1193 ATCC 209393 October 17, 1997 DNA35673-1201 ATCC
209418 October 28, 1997 DNA40370-1217 ATCC 209485 November 21, 1997
DNA42551-1217 ATCC 209483 November 21, 1997 DNA39520-1217 ATCC
209482 November 21, 1997 DNA41225-1217 ATCC 209491 November 21,
1997 DNA43318-1217 ATCC 209481 November 21, 1997 DNA40587-1231 ATCC
209438 November 7, 1997 DNA41338-1234 ATCC 209927 June 2, 1998
DNA40981-1234 ATCC 209439 November 7, 1997 DNA37140-1234 ATCC
209489 November 21, 1997 DNA40982-1235 ATCC 209433 November 7, 1997
DNA41379-1236 ATCC 209488 November 21, 1997 DNA44167-1243 ATCC
209434 November 7, 1997 DNA39427-1179 ATCC 209395 October 17, 1997
DNA40603-1232 ATCC 209486 November 21, 1997 DNA43466-1225 ATCC
209490 November 21, 1997 DNA43046-1225 ATCC 209484 November 21,
1997 DNA35668-1171 ATCC 209371 October 16, 1997 DNA77624-2515 ATCC
203553 December 22, 1998
[1723] These deposit 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).
[1724] 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.
[1725] The foregoing written specification is considered to be
sufficient to enable one skilled in the art to practice the
invention. The present invention is not to be limited in scope by
the construct deposited, since the deposited embodiment is intended
as a single illustration of certain aspects of the invention and
any constructs that are functionally equivalent are within the
scope of this invention. The deposit of material herein does not
constitute an admission that the written description herein
contained is inadequate to enable the practice of any aspect of the
invention, including the best mode thereof, nor is it to be
construed as limiting the scope of the claims to the specific
illustrations that it represents. Indeed, various modifications of
the invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description and fall within the scope of the appended claims.
Appendix A
[1726] I hereby claim benefit under Title 35, United States Code
.sctn.119(e) of any United States Provisional application listed
below:
123 Provisional Application Serial Number Filing Date 60/059,115
September 17, 1997 60/059,184 September 17, 1997 60/059,122
September 17, 1997 60/059,117 September 17, 1997 60/059,113
September 17, 1997 60/059,121 September 17, 1997 60/059,119
September 17, 1997 60/059,263 September 18, 1997 60/059,266
September 18, 1997 60/062,125 October 15, 1997 60/062,287 October
17, 1997 60/062,285 October 17, 1997 60/063,486 October 21, 1997
60/062,816 October 24, 1997 60/062,814 October 24, 1997 60/063,127
October 24, 1997 60/063,120 October 24, 1997 60/063,121 October 24,
1997 60/063,045 October 24, 1997 60/063,128 October 24, 1997
60/063,329 October 27, 1997 60/063,327 October 27, 1997 60/063,549
October 28, 1997
Appendix A
[1727] I hereby claim benefit under Title 35, United States Code
.sctn.119(e) of any United States Provisional application listed
below:
124 60/063,541 October 28, 1997 60/063,550 October 28, 1997
60/063,542 October 28, 1997 60/063,544 October 28, 1997 60/063,564
October 28, 1997 60/063,734 October 29, 1997 60/063,738 October 29,
1997 60/063,704 October 29, 1997 60/063,435 October 29, 1997
60/064,215 October 29, 1997 60/063,735 October 29, 1997 60/063,732
October 29, 1997 60/064,103 October 31, 1997 60/063,870 October 31,
1997 60/064,248 November 3, 1997 60/064,809 November 7, 1997
60/065,846 November 11, 1997 60/065,186 November 12, 1997
60/065,846 November 17, 1997 60/065,693 November 18, 1997
60/066,120 November 21, 1997 60/066,364 November 21, 1997
60/066,772 November 24, 1997 60/066,466 November 24, 1997
Appendix A
[1728] I hereby claim benefit under Title 35, United States Code
.sctn.119(e) of any United States Provisional application listed
below:
125 60/066,770 November 24, 1997 60/066,511 November 24, 1997
60/066,453 November 24, 1997 60/066,840 November 25, 1997
60/069,425 December 12, 1997 60/088,026 June 4, 1998 60/099,803
September 10, 1998 60/100,262 September 14, 1998 60/100,858
September 17, 1998 60/104,080 October 13, 1998 60/109,304 November
20, 1998 60/113,296 December 22, 1998 60/143,048 July 7, 1999
60/145,698 July 26, 1999 60/146,222 July 28, 1999
Appendix B
[1729] I hereby claim benefit under Title 35, United States Code
.sctn.120 of any United States and PCT International application
listed below:
126 PCT/US Ser. No. Filed Status PCT/US98/18824 September 10, 1998
Abandoned PCT/US98/19177 September 14, 1998 Abandoned
PCT/US98/19330 September 16, 1998 Abandoned PCT/US98/19437
September 17, 1998 Abandoned PCT/US98/25108 December 1, 1998
Abandoned PCT/US99/20594 September 8, 1999 Pending PCT/US99/20944
September 13, 1999 Pending PCT/US99/21090 September 15, 1999
Pending PCT/US99/21547 September 15, 1999 Pending PCT/US99/23089
October 5, 1999 Pending PCT/US99/28214 November 29, 1999 Pending
PCT/US99/28313 November 30, 1999 Pending PCT/US99/28301 December 1,
1999 Abandoned PCT/US99/28564 December 2, 1999 Pending
PCT/US99/28565 December 2, 1999 Pending PCT/US99/30095 December 16,
1999 Pending PCT/US99/30999 December 20, 1999 Pending
PCT/US99/30911 December 20, 1999 Pending PCT/US00/00219 January 5,
2000 Pending PCT/US00/03565 February 11, 2000 Pending
PCT/US00/04414 February 22, 2000 Pending PCT/US00/05004 February
24, 2000 Pending PCT/US00/05841 March 2, 2000 Pending
PCT/US00/07377 March 20, 2000 Pending PCT/US00/08439 March 30, 2000
Pending PCT/US00/14042 May 22, 2000 Pending PCT/US00/15264 June 2,
2000 Pending PCT/US00/20710 July 28, 2000 Pending PCT/US00/23328
August 24, 2000 Pending
[1730]
Sequence CWU 0
0
* * * * *
References