U.S. patent application number 09/978697 was filed with the patent office on 2002-11-14 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, Baker, Kevin P., Botstein, David, Desnoyers, Luc, Eaton, Dan, Ferrara, Napoleone, Filvaroff, Ellen, Fong, Sherman, Gao, Wei-Qiang, Gerber, Hanspeter, Gerritsen, Mary E., Goddard, Audrey, Godowski, Paul J., Grimaldi, J. Christerpher, Gurney, Austin L., Hillan, Kenneth J., Kljavin, Ivar J., Kuo, Sophia S., Napier, Mary A., Pan, James, Paoni, Nicholas F., Roy, Margaret Ann, Shelton, David L., Stewart, Timothy A., Tumas, Daniel, Williams, P. Mickey, Wood, William I..
Application Number | 20020169284 09/978697 |
Document ID | / |
Family ID | 21909792 |
Filed Date | 2002-11-14 |
United States Patent
Application |
20020169284 |
Kind Code |
A1 |
Ashkenazi, Avi ; et
al. |
November 14, 2002 |
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) ; Baker, Kevin P.; (Darnestown, MD) ;
Botstein, David; (Belmont, CA) ; Desnoyers, Luc;
(San Francisco, CA) ; Eaton, Dan; (San Rafael,
CA) ; Ferrara, Napoleone; (San Francisco, CA)
; Filvaroff, Ellen; (San Francisco, CA) ; Fong,
Sherman; (Alameda, CA) ; Gao, Wei-Qiang;
(Foster City, CA) ; Gerber, Hanspeter; (San
Francisco, CA) ; Gerritsen, Mary E.; (San Mateo,
CA) ; Goddard, Audrey; (San Francisco, CA) ;
Godowski, Paul J.; (Burlingame, CA) ; Grimaldi, J.
Christerpher; (San Francisco, CA) ; Gurney, Austin
L.; (Belmont, CA) ; Hillan, Kenneth J.; (San
Francisco, CA) ; Kljavin, Ivar J.; (Lafayette,
CA) ; Kuo, Sophia S.; (San Francisco, CA) ;
Napier, Mary A.; (Hillsborough, CA) ; Pan, James;
(Belmont, CA) ; Paoni, Nicholas F.; (Belmont,
CA) ; Roy, Margaret Ann; (San Francisco, CA) ;
Shelton, David L.; (Oakland, 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: |
Ginger R. Dreger, Esq.
Knobbe Martens Olson & Bear
Suite 1150
201 California Street
San Francisco
CA
94111
US
|
Assignee: |
Genentech, Inc.
|
Family ID: |
21909792 |
Appl. No.: |
09/978697 |
Filed: |
October 16, 2001 |
Related U.S. Patent Documents
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Filing Date |
Patent Number |
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09978697 |
Oct 16, 2001 |
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09040220 |
Mar 17, 1998 |
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Current U.S.
Class: |
530/350 ;
435/183; 435/320.1; 435/325; 435/69.1; 536/23.1 |
Current CPC
Class: |
C07K 16/28 20130101;
A61K 38/00 20130101; G01N 33/68 20130101; C07K 2319/00 20130101;
G01N 33/57484 20130101; C07K 14/4702 20130101; C07K 14/475
20130101; G01N 2800/042 20130101; G01N 33/573 20130101; C12N
2799/027 20130101; G01N 2333/99 20130101; C07K 14/47 20130101; C07K
14/521 20130101; C07K 2319/30 20130101; A61K 48/00 20130101; C07K
14/70578 20130101; C12N 9/00 20130101; C12N 2799/026 20130101; C07K
14/70503 20130101; C07K 16/18 20130101; C07K 2317/24 20130101; C07K
14/705 20130101; C07K 14/52 20130101; C07K 14/4703 20130101; C07K
14/54 20130101 |
Class at
Publication: |
530/350 ;
435/69.1; 435/325; 536/23.1; 435/183; 435/320.1 |
International
Class: |
C07K 014/435; C07H
021/02; C07H 021/04; C12N 009/00; C12P 021/02; C12N 005/06 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 7, 1998 |
US |
PCT/US98/21141 |
Nov 20, 1998 |
US |
PCT/US98/24855 |
Jan 5, 1999 |
US |
PCT/US99/00106 |
Mar 8, 1999 |
US |
PCT/US99/05028 |
Mar 10, 1999 |
US |
PCT/US99/05190 |
May 14, 1999 |
US |
PCT/US99/10733 |
Jun 2, 1999 |
US |
PCT/US99/12252 |
Nov 30, 1999 |
US |
PCT/US99/28313 |
Dec 2, 1999 |
US |
PCT/US99/28551 |
Dec 2, 1999 |
US |
PCT/US99/28565 |
Dec 16, 1999 |
US |
PCT/US99/30095 |
Dec 30, 1999 |
US |
PCT/US99/31243 |
Dec 30, 1999 |
US |
PCT/US99/31274 |
Jan 5, 2000 |
US |
PCT/US00/00219 |
Jan 6, 2000 |
US |
PCT/US00/00277 |
Jan 6, 2000 |
US |
PCT/US00/00376 |
Feb 11, 2000 |
US |
PCT/US00/03565 |
Feb 18, 2000 |
US |
PCT/US00/04341 |
Mar 2, 2000 |
US |
PCT/US00/05841 |
Mar 21, 2000 |
US |
PCT/US00/07532 |
Feb 24, 2000 |
US |
PCT/US00/05004 |
Mar 10, 2000 |
US |
PCT/US00/06319 |
Mar 30, 2000 |
US |
PCT/US00/08439 |
May 17, 2000 |
US |
PCT/US00/13705 |
May 22, 2000 |
US |
PCT/US00/14042 |
May 30, 2000 |
US |
PCT/US00/14941 |
Jun 2, 2000 |
US |
PCT/US00/15264 |
Jul 28, 2000 |
US |
PCT/US00/20710 |
Aug 24, 2000 |
US |
PCT/US00/23328 |
Dec 1, 2000 |
US |
PCT/US00/32678 |
Dec 20, 2000 |
US |
PCT/US00/34956 |
Feb 28, 2001 |
US |
PCT/US01/06520 |
Mar 22, 2001 |
US |
PCT/US01/09552 |
May 25, 2001 |
US |
PCT/US01/17092 |
Jun 1, 2001 |
US |
PCT/US01/17800 |
Jun 20, 2001 |
US |
PCT/US01/19692 |
Jun 29, 2001 |
US |
PCT/US01/21066 |
Jul 9, 2001 |
US |
PCT/US01/21735 |
Claims
What is claimed is:
1. Isolated nucleic acid having at least 80% nucleic acid sequence
identity to a nucleotide sequence that encodes 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:7), FIG.
9 (SEQ ID NO:19), FIG. 11 (SEQ ID NO:28), FIG. 15 (SEQ ID NO:36),
FIG. 20 (SEQ ID NO:45), FIG. 22 (SEQ ID NO:52), FIG. 24 (SEQ ID
NO:59), FIG. 26 (SEQ ID NO:64), FIG. 28 (SEQ ID NO:69), FIG. 30
(SEQ ID NO:74), FIG. 33 (SEQ ID NO:85), FIG. 35 (SEQ ID NO:90),
FIG. 37 (SEQ ID NO:97), FIG. 39 (SEQ ID NO:102), FIG. 41 (SEQ ID
NO:109), FIG. 43 (SEQ ID NO:114), FIG. 45 (SEQ ID NO:119), FIG. 47
(SEQ ID NO:124), FIG. 49 (SEQ ID NO:132), FIG. 51 (SEQ ID NO:137),
FIG. 53 (SEQ ID NO:145), FIG. 55 (SEQ ID NO:150), FIG. 59 (SEQ ID
NO:157), FIG. 61 (SEQ ID NO:162), FIG. 63 (SEQ ID NO:169), FIG. 66
(SEQ ID NO:178), FIG. 68 (SEQ ID NO:183), FIG. 70 (SEQ ID NO:190),
FIG. 73 (SEQ ID NO:196), FIG. 75 (SEQ ID NO:206), FIG. 77 (SEQ ID
NO:211), FIG. 79 (SEQ ID NO:216), FIG. 81 (SEQ ID NO:221), FIG. 83
(SEQ ID NO:226), FIG. 85 (SEQ ID NO:231), FIG. 87 (SEQ ID NO:236),
FIG. 89 (SEQ ID NO:245), FIG. 91 (SEQ ID NO:254), FIG. 93 (SEQ ID
NO:259), FIG. 95 (SEQ ID NO:264), FIG. 98 (SEQ ID NO:270), FIG. 109
(SEQ ID NO:284), FIG. 118 (SEQ ID NO:296), FIG. 120 (SEQ ID
NO:301), FIG. 122 (SEQ ID NO:303), FIG. 125 (SEQ ID NO:309), FIG.
129 (SEQ ID NO:322), FIG. 132 (SEQ ID NO:330), FIG. 136 (SEQ ID
NO:337), FIG. 139 (SEQ ID NO:346), FIG. 142 (SEQ ID NO:352), FIG.
145 (SEQ ID NO:358), FIG. 147 (SEQ ID NO:363), FIG. 149 (SEQ ID
NO:370), FIG. 151 (SEQ ID NO:375), FIG. 153 (SEQ ID NO:380), FIG.
155 (SEQ ID NO:385), FIG. 157 (SEQ ID NO:390), FIG. 159 (SEQ ID
NO:395), FIG. 161 (SEQ ID NO:400), FIG. 163 (SEQ ID NO:405), FIG.
165 (SEQ ID NO:410), FIG. 167 (SEQ ID NO:415), FIG. 169 (SEQ ID
NO:420), FIG. 171 (SEQ ID NO:425), FIG. 173 (SEQ ID NO:430), FIG.
177 (SEQ ID NO:437), FIG. 179 (SEQ ID NO:442), FIG. 181 (SEQ ID
NO:447), FIG. 183 (SEQ ID NO:452), FIG. 185 (SEQ ID NO:454), FIG.
187 (SEQ ID NO:456), FIG. 190 (SEQ ID NO:459), FIG. 192 (SEQ ID
NO:464), FIG. 194 (SEQ ID NO:466), FIG. 196 (SEQ ID NO:468), FIG.
198 (SEQ ID NO:470), FIG. 200 (SEQ ID NO:472), FIG. 202 (SEQ ID
NO:477), FIG. 204 (SEQ ID NO:483), FIG. 207 (SEQ ID NO:488), FIG.
209 (SEQ ID NO:496), FIG. 211 (SEQ ID NO:498), FIG. 213 (SEQ ID
NO:506), FIG. 215 (SEQ ID NO:508), FIG. 217 (SEQ ID NO:510), FIG.
219 (SEQ ID NO:515), FIG. 222 (SEQ ID NO:523), FIG. 225 (SEQ ID
NO:526), FIG. 230 (SEQ ID NO:612), FIG. 232 (SEQ ID NO:614), FIG.
234 (SEQ ID NO:616) and FIG. 236 (SEQ ID NO:618).
2. Isolated nucleic acid having at least 80% nucleic acid sequence
identity to a nucleotide sequence selected from the group
consisting of the nucleotide sequence shown in FIG. 1 (SEQ ID
NO:1), FIG. 3 (SEQ ID NO:6), FIG. 8 (SEQ ID NO:18), FIG. 10 (SEQ ID
NO:27), FIG. 14 (SEQ ID NO:35), FIG. 19 (SEQ ID NO:44), FIG. 21
(SEQ ID NO:51), FIG. 23 (SEQ ID NO:58), FIG. 25 (SEQ ID NO:63),
FIG. 27 (SEQ ID NO:68), FIG. 29 (SEQ ID NO:73), FIG. 32 (SEQ ID
NO:84), FIG. 34 (SEQ ID NO:89), FIG. 36 (SEQ ID NO:96), FIG. 38
(SEQ ID NO:101), FIG. 40 (SEQ ID NO:108), FIG. 42 (SEQ ID NO:113),
FIG. 44 (SEQ ID NO:118), FIG. 46 (SEQ ID NO:123), FIG. 48 (SEQ ID
NO:131), FIG. 50 (SEQ ID NO:136), FIG. 52 (SEQ ID NO:144), FIG. 54
(SEQ ID NO:149), FIG. 58 (SEQ ID NO:156), FIG. 60 (SEQ ID NO:161),
FIG. 62 (SEQ ID NO:168), FIG. 65 (SEQ ID NO:177), FIG. 67 (SEQ ID
NO:182), FIG. 69 (SEQ ID NO:189), FIG. 72 (SEQ ID NO:195), FIG. 74
(SEQ ID NO:205), FIG. 76 (SEQ ID NO:210), FIG. 78 (SEQ ID NO:215),
FIG. 80 (SEQ ID NO:220), FIG. 82 (SEQ ID NO:225), FIG. 84 (SEQ ID
NO:230), FIG. 86 (SEQ ID NO:235), FIG. 88 (SEQ ID NO:244), FIG. 90
(SEQ ID NO:253), FIG. 92 (SEQ ID NO:258), FIG. 94 (SEQ ID NO:263),
FIG. 97 (SEQ ID NO:269), FIG. 108 (SEQ ID NO:283), FIG. 117 (SEQ ID
NO:295), FIG. 119 (SEQ ID NO:300), FIG. 121 (SEQ ID NO:302), FIG.
124 (SEQ ID NO:308), FIG. 128 (SEQ ID NO:321), FIG. 131 (SEQ ID
NO:329), FIG. 135 (SEQ ID NO:336), FIG. 138 (SEQ ID NO:345), FIG.
141 (SEQ ID NO:351), FIG. 144 (SEQ ID NO:357), FIG. 146 (SEQ ID
NO:362), FIG. 148 (SEQ ID NO:369), FIG. 150 (SEQ ID NO:374), FIG.
152 (SEQ ID NO:379), FIG. 154 (SEQ ID NO:384), FIG. 156 (SEQ ID
NO:389), FIG. 158 (SEQ ID NO:394), FIG. 160 (SEQ ID NO:399), FIG.
162 (SEQ ID NO:404), FIG. 164 (SEQ ID NO:409), FIG. 166 (SEQ ID
NO:414), FIG. 168 (SEQ ID NO:419), FIG. 170 (SEQ ID NO:424), FIG.
172 (SEQ ID NO:429), FIG. 176 (SEQ ID NO:436), FIG. 178 (SEQ ID
NO:441), FIG. 180 (SEQ ID NO:446), FIG. 182 (SEQ ID NO:451), FIG.
184 (SEQ ID NO:453), FIG. 186 (SEQ ID NO:455), FIG. 189 (SEQ ID
NO:458), FIG. 191 (SEQ ID NO:463), FIG. 193 (SEQ ID NO:465), FIG.
195 (SEQ ID NO:467), FIG. 197 (SEQ ID NO:469), FIG. 199 (SEQ ID
NO:471), FIG. 201 (SEQ ID NO:476), FIG. 203 (SEQ ID NO:482), FIG.
206 (SEQ ID NO:487), FIG. 208 (SEQ ID NO:495), FIG. 210 (SEQ ID
NO:497), FIG. 212 (SEQ ID NO:505), FIG. 214 (SEQ ID NO:507), FIG.
216 (SEQ ID NO:509), FIG. 218 (SEQ ID NO:514), FIG. 221 (SEQ ID
NO:522), FIG. 224 (SEQ ID NO:525), FIG. 229 (SEQ ID NO:611), FIG.
231 (SEQ ID NO:613), FIG. 233 (SEQ ID NO:615) and FIG. 235 (SEQ ID
NO:617).
3. Isolated nucleic acid having at least 80% nucleic acid sequence
identity to a nucleotide sequence selected from the group
consisting of the full-length coding sequence of the nucleotide
sequence shown in FIG. 1 (SEQ ID NO:1), FIG. 3 (SEQ ID NO:6), FIG.
8 (SEQ ID NO:18), FIG. 10 (SEQ ID NO:27), FIG. 14 (SEQ ID NO:35),
FIG. 19 (SEQ ID NO:44), FIG. 21 (SEQ ID NO:51), FIG. 23 (SEQ ID
NO:58), FIG. 25 (SEQ ID NO:63), FIG. 27 (SEQ ID NO:68), FIG. 29
(SEQ ID NO:73), FIG. 32 (SEQ ID NO:84), FIG. 34 (SEQ ID NO:89),
FIG. 36 (SEQ ID NO:96), FIG. 38 (SEQ ID NO:101), FIG. 40 (SEQ ID
NO:108), FIG. 42 (SEQ ID NO:113), FIG. 44 (SEQ ID NO:118), FIG. 46
(SEQ ID NO:123), FIG. 48 (SEQ ID NO:131), FIG. 50 (SEQ ID NO:136),
FIG. 52 (SEQ ID NO:144), FIG. 54 (SEQ ID NO:149), FIG. 58 (SEQ ID
NO:156), FIG. 60 (SEQ ID NO:161), FIG. 62 (SEQ ID NO:168), FIG. 65
(SEQ ID NO:177), FIG. 67 (SEQ ID NO:182), FIG. 69 (SEQ ID NO:189),
FIG. 72 (SEQ ID NO:195), FIG. 74 (SEQ ID NO:205), FIG. 76 (SEQ ID
NO:210), FIG. 78 (SEQ ID NO:215), FIG. 80 (SEQ ID NO:220), FIG. 82
(SEQ ID NO:225), FIG. 84 (SEQ ID NO:230), FIG. 86 (SEQ ID NO:235),
FIG. 88 (SEQ ID NO:244), FIG. 90 (SEQ ID NO:253), FIG. 92 (SEQ ID
NO:258), FIG. 94 (SEQ ID NO:263), FIG. 97 (SEQ ID NO:269), FIG. 108
(SEQ ID NO:283), FIG. 117 (SEQ ID NO:295), FIG. 119 (SEQ ID
NO:300), FIG. 121 (SEQ ID NO:302), FIG. 124 (SEQ ID NO:308), FIG.
128 (SEQ ID NO:321), FIG. 131 (SEQ ID NO:329), FIG. 135 (SEQ ID
NO:336), FIG. 138 (SEQ ID NO:345), FIG. 141 (SEQ ID NO:351), FIG.
144 (SEQ ID NO:357), FIG. 146 (SEQ ID NO:362), FIG. 148 (SEQ ID
NO:369), FIG. 150 (SEQ ID NO:374), FIG. 152 (SEQ ID NO:379), FIG.
154 (SEQ ID NO:384), FIG. 156 (SEQ ID NO:389), FIG. 158 (SEQ ID
NO:394), FIG. 160 (SEQ ID NO:399), FIG. 162 (SEQ ID NO:404), FIG.
164 (SEQ ID NO:409), FIG. 166 (SEQ ID NO:414), FIG. 168 (SEQ ID
NO:419), FIG. 170 (SEQ ID NO:424), FIG. 172 (SEQ ID NO:429), FIG.
176 (SEQ ID NO:436), FIG. 178 (SEQ ID NO:441), FIG. 180 (SEQ ID
NO:446), FIG. 182 (SEQ ID NO:451), FIG. 184 (SEQ ID NO:453), FIG.
186 (SEQ ID NO:455), FIG. 189 (SEQ ID NO:458), FIG. 191 (SEQ ID
NO:463), FIG. 193 (SEQ ID NO:465), FIG. 195 (SEQ ID NO:467), FIG.
197 (SEQ ID NO:469), FIG. 199 (SEQ ID NO:471), FIG. 201 (SEQ ID
NO:476), FIG. 203 (SEQ ID NO:482), FIG. 206 (SEQ ID NO:487), FIG.
208 (SEQ ID NO:495), FIG. 210 (SEQ ID NO:497), FIG. 212 (SEQ ID
NO:505), FIG. 214 (SEQ ID NO:507), FIG. 216 (SEQ ID NO:509), FIG.
218 (SEQ ID NO:514), FIG. 221 (SEQ ID NO:522), FIG. 224 (SEQ ID
NO:525), FIG. 229 (SEQ ID NO:611), FIG. 231 (SEQ ID NO:613), FIG.
233 (SEQ ID NO:615) and FIG. 235 (SEQ ID NO:617).
4. Isolated nucleic acid having at least 80% nucleic acid sequence
identity to the full-length coding sequence of the DNA deposited
under ATCC accession number ATCC 209791, ATCC 209786, ATCC 209788,
ATCC 209787, ATCC 209789, ATCC 209617, ATCC 209620, ATCC 209616,
ATCC 209679, ATCC 209654, ATCC 209655, ATCC 209656, ATCC 209721,
ATCC 209717, ATCC 209716, ATCC 209722, ATCC 209668, ATCC 209670,
ATCC 209718, ATCC 209784, ATCC 209703, ATCC 209808, ATCC 209810,
ATCC 209699, ATCC 209811, ATCC 209813, ATCC 209705, ATCC 209806,
ATCC 209809, ATCC 209805, ATCC 209812, ATCC 209844, ATCC 209847,
ATCC 209845, ATCC 209843, ATCC 209846, ATCC 209750, ATCC 209848,
ATCC 209851, ATCC 209754, ATCC 209747, ATCC 209861, ATCC 209862,
ATCC 209867, ATCC 209879, ATCC 209868, ATCC 209869, ATCC 209775,
ATCC 209772, ATCC 209774, ATCC 209777, ATCC 209905, ATCC 209855,
ATCC 209910, ATCC 209424, ATCC 209720, ATCC 209714, ATCC 209785,
ATCC 209911, ATCC 209669, ATCC 209704, ATCC 209702, ATCC 209701,
ATCC 209700, ATCC 209814, ATCC 209715, ATCC 209807, ATCC 209753,
ATCC 209749, ATCC 209748, ATCC 209842, ATCC 209849, ATCC 209880,
ATCC 209864, ATCC 209882, ATCC 209883, ATCC 209865, ATCC 209866,
ATCC 209857, ATCC 209870, ATCC 209859, ATCC 209653, ATCC 209389,
ATCC 209386, ATCC 203242, ATCC 203243, ATCC 209783, ATCC 209487,
ATCC 209680, 240-PTA or ATCC 209773.
5. A vector comprising the nucleic acid of any one of claims 1 to
4.
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. An isolated polypeptide having at least 80% amino acid 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:7), FIG. 9 (SEQ ID NO:19), FIG. 11 (SEQ ID
NO:28), FIG. 15 (SEQ ID NO:36), FIG. 20 (SEQ ID NO:45), FIG. 22
(SEQ ID NO:52), FIG. 24 (SEQ ID NO:59), FIG. 26 (SEQ ID NO:64),
FIG. 28 (SEQ ID NO:69), FIG. 30 (SEQ ID NO:74), FIG. 33 (SEQ ID
NO:85), FIG. 35 (SEQ ID NO:90), FIG. 37 (SEQ ID NO:97), FIG. 39
(SEQ ID NO:102), FIG. 41 (SEQ ID NO:109), FIG. 43 (SEQ ID NO:114),
FIG. 45 (SEQ ID NO:119), FIG. 47 (SEQ ID NO:124), FIG. 49 (SEQ ID
NO:132), FIG. 51 (SEQ ID NO:137), FIG. 53 (SEQ ID NO:145), FIG. 55
(SEQ ID NO:150), FIG. 59 (SEQ ID NO:157), FIG. 61 (SEQ ID NO:162),
FIG. 63 (SEQ ID NO:169), FIG. 66 (SEQ ID NO:178), FIG. 68 (SEQ ID
NO:183), FIG. 70 (SEQ ID NO:190), FIG. 73 (SEQ ID NO:196), FIG. 75
(SEQ ID NO:206), FIG. 77 (SEQ ID NO:211), FIG. 79 (SEQ ID NO:216),
FIG. 81 (SEQ ID NO:221), FIG. 83 (SEQ ID NO:226), FIG. 85 (SEQ ID
NO:231), FIG. 87 (SEQ ID NO:236), FIG. 89 (SEQ ID NO:245), FIG. 91
(SEQ ID NO:254), FIG. 93 (SEQ ID NO:259), FIG. 95 (SEQ ID NO:264),
FIG. 98 (SEQ ID NO:270), FIG. 109 (SEQ ID NO:284), FIG. 118 (SEQ ID
NO:296), FIG. 120 (SEQ ID NO:301), FIG. 122 (SEQ ID NO:303), FIG.
125 (SEQ ID NO:309), FIG. 129 (SEQ ID NO:322), FIG. 132 (SEQ ID
NO:330), FIG. 136 (SEQ ID NO:337), FIG. 139 (SEQ ID NO:346), FIG.
142 (SEQ ID NO:352), FIG. 145 (SEQ ID NO:358), FIG. 147 (SEQ ID
NO:363), FIG. 149 (SEQ ID NO:370), FIG. 151 (SEQ ID NO:375), FIG.
153 (SEQ ID NO:380), FIG. 155 (SEQ ID NO:385), FIG. 157 (SEQ ID
NO:390), FIG. 159 (SEQ ID NO:395), FIG. 161 (SEQ ID NO:400), FIG.
163 (SEQ ID NO:405), FIG. 165 (SEQ ID NO:410), FIG. 167 (SEQ ID
NO:415), FIG. 169 (SEQ ID NO:420), FIG. 171 (SEQ ID NO:425), FIG.
173 (SEQ ID NO:430), FIG. 177 (SEQ ID NO:437), FIG. 179 (SEQ ID
NO:442), FIG. 181 (SEQ ID NO:447), FIG. 183 (SEQ ID NO:452), FIG.
185 (SEQ ID NO:454), FIG. 187 (SEQ ID NO:456), FIG. 190 (SEQ ID
NO:459), FIG. 192 (SEQ ID NO:464), FIG. 194 (SEQ ID NO:466), FIG.
196 (SEQ ID NO:468), FIG. 198 (SEQ ID NO:470), FIG. 200 (SEQ ID
NO:472), FIG. 202 (SEQ ID NO:477), FIG. 204 (SEQ ID NO:483), FIG.
207 (SEQ ID NO:488), FIG. 209 (SEQ ID NO:496), FIG. 211 (SEQ ID
NO:498), FIG. 213 (SEQ ID NO:506), FIG. 215 (SEQ ID NO:508), FIG.
217 (SEQ ID NO:510), FIG. 219 (SEQ ID NO:515), FIG. 222 (SEQ ID
NO:523), FIG. 225 (SEQ ID NO:526), FIG. 230 (SEQ ID NO:612), FIG.
232 (SEQ ID NO:614), FIG. 234 (SEQ ID NO:616) and FIG. 236 (SEQ ID
NO:618).
13. An isolated polypeptide scoring at least 80% positives when
compared 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:7), FIG. 9 (SEQ ID NO:19), FIG. 11 (SEQ ID
NO:28), FIG. 15 (SEQ ID NO:36), FIG. 20 (SEQ ID NO:45), FIG. 22
(SEQ ID NO:52), FIG. 24 (SEQ ID NO:59), FIG. 26 (SEQ ID NO:64),
FIG. 28 (SEQ ID NO:69), FIG. 30 (SEQ ID NO:74), FIG. 33 (SEQ ID
NO:85), FIG. 35 (SEQ ID NO:90), FIG. 37 (SEQ ID NO:97), FIG. 39
(SEQ ID NO:102), FIG. 41 (SEQ ID NO:109), FIG. 43 (SEQ ID NO:114),
FIG. 45 (SEQ ID NO:119), FIG. 47 (SEQ ID NO:124), FIG. 49 (SEQ ID
NO:132), FIG. 51 (SEQ ID NO:137), FIG. 53 (SEQ ID NO:145), FIG. 55
(SEQ ID NO:150), FIG. 59 (SEQ ID NO:157), FIG. 61 (SEQ ID NO:162),
FIG. 63 (SEQ ID NO:169), FIG. 66 (SEQ ID NO:178), FIG. 68 (SEQ ID
NO:183), FIG. 70 (SEQ ID NO:190), FIG. 73 (SEQ ID NO:196), FIG. 75
(SEQ ID NO:206), FIG. 77 (SEQ ID NO:211), FIG. 79 (SEQ ID NO:216),
FIG. 81 (SEQ ID NO:221), FIG. 83 (SEQ ID NO:226), FIG. 85 (SEQ ID
NO:231), FIG. 87 (SEQ ID NO:236), FIG. 89 (SEQ ID NO:245), FIG. 91
(SEQ ID NO:254), FIG. 93 (SEQ ID NO:259), FIG. 95 (SEQ ID NO:264),
FIG. 98 (SEQ ID NO:270), FIG. 109 (SEQ ID NO:284), FIG. 118 (SEQ ID
NO:296), FIG. 120 (SEQ ID NO:301), FIG. 122 (SEQ ID NO:303), FIG.
125 (SEQ ID NO:309), FIG. 129 (SEQ ID NO:322), FIG. 132 (SEQ ID
NO:330), FIG. 136 (SEQ ID NO:337), FIG. 139 (SEQ ID NO:346), FIG.
142 (SEQ ID NO:352), FIG. 145 (SEQ ID NO:358), FIG. 147 (SEQ ID
NO:363), FIG. 149 (SEQ ID NO:370), FIG. 151 (SEQ ID NO:375), FIG.
153 (SEQ ID NO:380), FIG. 155 (SEQ ID NO:385), FIG. 157 (SEQ ID
NO:390), FIG. 159 (SEQ ID NO:395), FIG. 161 (SEQ ID NO:400), FIG.
163 (SEQ ID NO:405), FIG. 165 (SEQ ID NO:410), FIG. 167 (SEQ ID
NO:415), FIG. 169 (SEQ ID NO:420), FIG. 171 (SEQ ID NO:425), FIG.
173 (SEQ ID NO:430), FIG. 177 (SEQ ID NO:437), FIG. 179 (SEQ ID
NO:442), FIG. 181 (SEQ ID NO:447), FIG. 183 (SEQ ID NO:452), FIG.
185 (SEQ ID NO:454), FIG. 187 (SEQ ID NO:456), FIG. 190 (SEQ ID
NO:459), FIG. 192 (SEQ ID NO:464), FIG. 194 (SEQ ID NO:466), FIG.
196 (SEQ ID NO:468), FIG. 198 (SEQ ID NO:470), FIG. 200 (SEQ ID
NO:472), FIG. 202 (SEQ ID NO:477), FIG. 204 (SEQ ID NO:483), FIG.
207 (SEQ ID NO:488), FIG. 209 (SEQ ID NO:496), FIG. 211 (SEQ ID
NO:498), FIG. 213 (SEQ ID NO:506), FIG. 215 (SEQ ID NO:508), FIG.
217 (SEQ ID NO:510), FIG. 219 (SEQ ID NO:515), FIG. 222 (SEQ ID
NO:523), FIG. 225 (SEQ ID NO:526), FIG. 230 (SEQ ID NO:612), FIG.
232 (SEQ ID NO:614), FIG. 234 (SEQ ID NO:616) and FIG. 236 (SEQ ID
NO:618).
14. An isolated polypeptide having at least 80% amino acid sequence
identity to an amino acid sequence encoded by the full-length
coding sequence of the DNA deposited under ATCC accession number
ATCC 209791, ATCC 209786, ATCC 209788, ATCC 209787, ATCC 209789,
ATCC 209617, ATCC 209620, ATCC 209616, ATCC 209679, ATCC 209654,
ATCC 209655, ATCC 209656, ATCC 209721, ATCC 209717, ATCC 209716,
ATCC 209722, ATCC 209668, ATCC 209670, ATCC 209718, ATCC 209784,
ATCC 209703, ATCC 209808, ATCC 209810, ATCC 209699, ATCC 209811,
ATCC 209813, ATCC 209705, ATCC 209806, ATCC 209809, ATCC 209805,
ATCC 209812, ATCC 209844, ATCC 209847, ATCC 209845, ATCC 209843,
ATCC 209846, ATCC 209750, ATCC 209848, ATCC 209851, ATCC 209754,
ATCC 209747, ATCC 209861, ATCC 209862, ATCC 209867, ATCC 209879,
ATCC 209868, ATCC 209869, ATCC 209775, ATCC 209772, ATCC 209774,
ATCC 209777, ATCC 209905, ATCC 209855, ATCC 209910, ATCC 209424,
ATCC 209720, ATCC 209714, ATCC 209785, ATCC 209911, ATCC 209669,
ATCC 209704, ATCC 209702, ATCC 209701, ATCC 209700, ATCC 209814,
ATCC 209715, ATCC 209807, ATCC 209753, ATCC 209749, ATCC 209748,
ATCC 209842, ATCC 209849, ATCC 209880, ATCC 209864, ATCC 209882,
ATCC 209883, ATCC 209865, ATCC 209866, ATCC 209857, ATCC 209870,
ATCC 209859, ATCC 209653, ATCC 209389, ATCC 209386, ATCC 203242,
ATCC 203243, ATCC 209783, ATCC 209487, ATCC 209680, 240-PTA or ATCC
209773.
15. A chimeric molecule comprising a polypeptide according to any
one of claims 12 to 14 fused to a heterologous amino acid
sequence.
16. The chimeric molecule of claim 15, wherein said heterologous
amino acid sequence is an epitope tag sequence.
17. The chimeric molecule of claim 15, wherein said heterologous
amino acid sequence is a Fc region of an immunoglobulin.
18. An antibody which specifically binds to a polypeptide according
to any one of claims 12 to 14.
19. The antibody of claim 18, wherein said antibody is a monoclonal
antibody, a humanized antibody or a single-chain antibody.
20. 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:7), FIG. 9 (SEQ ID
NO:19), FIG. 11 (SEQ ID NO:28), FIG. 15 (SEQ ID NO:36), FIG. 20
(SEQ ID NO:45), FIG. 22 (SEQ ID NO:52), FIG. 24 (SEQ ID NO:59),
FIG. 26 (SEQ ID NO:64), FIG. 28 (SEQ ID NO:69), FIG. 30 (SEQ ID
NO:74), FIG. 33 (SEQ ID NO:85), FIG. 35 (SEQ ID NO:90), FIG. 37
(SEQ ID NO:97), FIG. 39 (SEQ ID NO:102), FIG. 41 (SEQ ID NO:109),
FIG. 43 (SEQ ID NO:114), FIG. 45 (SEQ ID NO:119), FIG. 47 (SEQ ID
NO:124), FIG. 49 (SEQ ID NO:132), FIG. 51 (SEQ ID NO:137), FIG. 53
(SEQ ID NO:145), FIG. 55 (SEQ ID NO:150), FIG. 59 (SEQ ID NO:157),
FIG. 61 (SEQ ID NO:162), FIG. 63 (SEQ ID NO:169), FIG. 66 (SEQ ID
NO:178), FIG. 68 (SEQ ID NO:183), FIG. 70 (SEQ ID NO:190), FIG. 73
(SEQ ID NO:196), FIG. 75 (SEQ ID NO:206), FIG. 77 (SEQ ID NO:211),
FIG. 79 (SEQ ID NO:216), FIG. 81 (SEQ ID NO:221), FIG. 83 (SEQ ID
NO:226), FIG. 85 (SEQ ID NO:231), FIG. 87 (SEQ ID NO:236), FIG. 89
(SEQ ID NO:245), FIG. 91 (SEQ ID NO:254), FIG. 93 (SEQ ID NO:259),
FIG. 95 (SEQ ID NO:264), FIG. 98 (SEQ ID NO:270), FIG. 109 (SEQ ID
NO:284), FIG. 118 (SEQ ID NO:296), FIG. 120 (SEQ ID NO:301), FIG.
122 (SEQ ID NO:303), FIG. 125 (SEQ ID NO:309), FIG. 129 (SEQ ID
NO:322), FIG. 132 (SEQ ID NO:330), FIG. 136 (SEQ ID NO:337), FIG.
139 (SEQ ID NO:346), FIG. 142 (SEQ ID NO:352), FIG. 145 (SEQ ID
NO:358), FIG. 147 (SEQ ID NO:363), FIG. 149 (SEQ ID NO:370), FIG.
151 (SEQ ID NO:375), FIG. 153 (SEQ ID NO:380), FIG. 155 (SEQ ID
NO:385), FIG. 157 (SEQ ID NO:390), FIG. 159 (SEQ ID NO:395), FIG.
161 (SEQ ID NO:400), FIG. 163 (SEQ ID NO:405), FIG. 165 (SEQ ID
NO:410), FIG. 167 (SEQ ID NO:415), FIG. 169 (SEQ ID NO:420), FIG.
171 (SEQ ID NO:425), FIG. 173 (SEQ ID NO:430), FIG. 177 (SEQ ID
NO:437), FIG. 179 (SEQ ID NO:442), FIG. 181 (SEQ ID NO:447), FIG.
183 (SEQ ID NO:452), FIG. 185 (SEQ ID NO:454), FIG. 187 (SEQ ID
NO:456), FIG. 190 (SEQ ID NO:459), FIG. 192 (SEQ ID NO:464), FIG.
194 (SEQ ID NO:466), FIG. 196 (SEQ ID NO:468), FIG. 198 (SEQ ID
NO:470), FIG. 200 (SEQ ID NO:472), FIG. 202 (SEQ ID NO:477), FIG.
204 (SEQ ID NO:483), FIG. 207 (SEQ ID NO:488), FIG. 209 (SEQ ID
NO:496), FIG. 211 (SEQ ID NO:498), FIG. 213 (SEQ ID NO:506), FIG.
215 (SEQ ID NO:508), FIG. 217 (SEQ ID NO:510), FIG. 219 (SEQ ID
NO:515), FIG. 222 (SEQ ID NO:523), FIG. 225 (SEQ ID NO:526), FIG.
230 (SEQ ID NO:612), FIG. 232 (SEQ ID NO:614), FIG. 234 (SEQ ID
NO:616) or FIG. 236 (SEQ ID NO:618), 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:7), FIG. 9 (SEQ ID NO:19), FIG. 11 (SEQ ID NO:28), FIG. 15 (SEQ
ID NO:36), FIG. 20 (SEQ ID NO:45), FIG. 22 (SEQ ID NO:52), FIG. 24
(SEQ ID NO:59), FIG. 26 (SEQ ID NO:64), FIG. 28 (SEQ ID NO:69),
FIG. 30 (SEQ ID NO:74), FIG. 33 (SEQ ID NO:85), FIG. 35 (SEQ ID
NO:90), FIG. 37 (SEQ ID NO:97), FIG. 39 (SEQ ID NO:102), FIG. 41
(SEQ ID NO:109), FIG. 43 (SEQ ID NO:114), FIG. 45 (SEQ ID NO:119),
FIG. 47 (SEQ ID NO:124), FIG. 49 (SEQ ID NO:132), FIG. 51 (SEQ ID
NO:137), FIG. 53 (SEQ ID NO:145), FIG. 55 (SEQ ID NO:150), FIG. 59
(SEQ ID NO:157), FIG. 61 (SEQ ID NO:162), FIG. 63 (SEQ ID NO:169),
FIG. 66 (SEQ ID NO:178), FIG. 68 (SEQ ID NO:183), FIG. 70 (SEQ ID
NO:190), FIG. 73 (SEQ ID NO:196), FIG. 75 (SEQ ID NO:206), FIG. 77
(SEQ ID NO:211), FIG. 79 (SEQ ID NO:216), FIG. 81 (SEQ ID NO:221),
FIG. 83 (SEQ ID NO:226), FIG. 85 (SEQ ID NO:231), FIG. 87 (SEQ ID
NO:236), FIG. 89 (SEQ ID NO:245), FIG. 91 (SEQ ID NO:254), FIG. 93
(SEQ ID NO:259), FIG. 95 (SEQ ID NO:264), FIG. 98 (SEQ ID NO:270),
FIG. 109 (SEQ ID NO:284), FIG. 118 (SEQ ID NO:296), FIG. 120 (SEQ
ID NO:301), FIG. 122 (SEQ ID NO:303), FIG. 125 (SEQ ID NO:309),
FIG. 129 (SEQ ID NO:322), FIG. 132 (SEQ ID NO:330), FIG. 136 (SEQ
ID NO:337), FIG. 139 (SEQ ID NO:346), FIG. 142 (SEQ ID NO:352),
FIG. 145 (SEQ ID NO:358), FIG. 147 (SEQ ID NO:363), FIG. 149 (SEQ
ID NO:370), FIG. 151 (SEQ ID NO:375), FIG. 153 (SEQ ID NO:380),
FIG. 155 (SEQ ID NO:385), FIG. 157 (SEQ ID NO:390), FIG. 159 (SEQ
ID NO:395), FIG. 161 (SEQ ID NO:400), FIG. 163 (SEQ ID NO:405),
FIG. 165 (SEQ ID NO:410), FIG. 167 (SEQ ID NO:415), FIG. 169 (SEQ
ID NO:420), FIG. 171 (SEQ ID NO:425), FIG. 173 (SEQ ID NO:430),
FIG. 177 (SEQ ID NO:437), FIG. 179 (SEQ ID NO:442), FIG. 181 (SEQ
ID NO:447), FIG. 183 (SEQ ID NO:452), FIG. 185 (SEQ ID NO:454),
FIG. 187 (SEQ ID NO:456), FIG. 190 (SEQ ID NO:459), FIG. 192 (SEQ
ID NO:464), FIG. 194 (SEQ ID NO:466), FIG. 196 (SEQ ID NO:468),
FIG. 198 (SEQ ID NO:470), FIG. 200 (SEQ ID NO:472), FIG. 202 (SEQ
ID NO:477), FIG. 204 (SEQ ID NO:483), FIG. 207 (SEQ ID NO:488),
FIG. 209 (SEQ ID NO:496), FIG. 211 (SEQ ID NO:498), FIG. 213 (SEQ
ID NO:506), FIG. 215 (SEQ ID NO:508), FIG. 217 (SEQ ID NO:510),
FIG. 219 (SEQ ID NO:515), FIG. 222 (SEQ ID NO:523), FIG. 225 (SEQ
ID NO:526), FIG. 230 (SEQ ID NO:612), FIG. 232 (SEQ ID NO:614),
FIG. 234 (SEQ ID NO:616) or FIG. 236 (SEQ ID NO:618), 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:7), FIG. 9 (SEQ ID NO:19), FIG. 11 (SEQ ID
NO:28), FIG. 15 (SEQ ID NO:36), FIG. 20 (SEQ ID NO:45), FIG. 22
(SEQ ID NO:52), FIG. 24 (SEQ ID NO:59), FIG. 26 (SEQ ID NO:64),
FIG. 28 (SEQ ID NO:69), FIG. 30 (SEQ ID NO:74), FIG. 33 (SEQ ID
NO:85), FIG. 35 (SEQ ID NO:90), FIG. 37 (SEQ ID NO:97), FIG. 39
(SEQ ID NO:102), FIG. 41 (SEQ ID NO:109), FIG. 43 (SEQ ID NO:114),
FIG. 45 (SEQ ID NO:119), FIG. 47 (SEQ ID NO:124), FIG. 49 (SEQ ID
NO:132), FIG. 51 (SEQ ID NO:137), FIG. 53 (SEQ ID NO:145), FIG. 55
(SEQ ID NO:150), FIG. 59 (SEQ ID NO:157), FIG. 61 (SEQ ID NO:162),
FIG. 63 (SEQ ID NO:169), FIG. 66 (SEQ ID NO:178), FIG. 68 (SEQ ID
NO:183), FIG. 70 (SEQ ID NO:190), FIG. 73 (SEQ ID NO:196), FIG. 75
(SEQ ID NO:206), FIG. 77 (SEQ ID NO:211), FIG. 79 (SEQ ID NO:216),
FIG. 81 (SEQ ID NO:221), FIG. 83 (SEQ ID NO:226), FIG. 85 (SEQ ID
NO:231), FIG. 87 (SEQ ID NO:236), FIG. 89 (SEQ ID NO:245), FIG. 91
(SEQ ID NO:254), FIG. 93 (SEQ ID NO:259), FIG. 95 (SEQ ID NO:264),
FIG. 98 (SEQ ID NO:270), FIG. 109 (SEQ ID NO:284), FIG. 118 (SEQ ID
NO:296), FIG. 120 (SEQ ID NO:301), FIG. 122 (SEQ ID NO:303), FIG.
125 (SEQ ID NO:309), FIG. 129 (SEQ ID NO:322), FIG. 132 (SEQ ID
NO:330), FIG. 136 (SEQ ID NO:337), FIG. 139 (SEQ ID NO:346), FIG.
142 (SEQ ID NO:352), FIG. 145 (SEQ ID NO:358), FIG. 147 (SEQ ID
NO:363), FIG. 149 (SEQ ID NO:370), FIG. 151 (SEQ ID NO:375), FIG.
153 (SEQ ID NO:380), FIG. 155 (SEQ ID NO:385), FIG. 157 (SEQ ID
NO:390), FIG. 159 (SEQ ID NO:395), FIG. 161 (SEQ ID NO:400), FIG.
163 (SEQ ID NO:405), FIG. 165 (SEQ ID NO:410), FIG. 167 (SEQ ID
NO:415), FIG. 169 (SEQ ID NO:420), FIG. 171 (SEQ ID NO:425), FIG.
173 (SEQ ID NO:430), FIG. 177 (SEQ ID NO:437), FIG. 179 (SEQ ID
NO:442), FIG. 181 (SEQ ID NO:447), FIG. 183 (SEQ ID NO:452), FIG.
185 (SEQ ID NO:454), FIG. 187 (SEQ ID NO:456), FIG. 190 (SEQ ID
NO:459), FIG. 192 (SEQ ID NO:464), FIG. 194 (SEQ ID NO:466), FIG.
196 (SEQ ID NO:468), FIG. 198 (SEQ ID NO:470), FIG. 200 (SEQ ID
NO:472), FIG. 202 (SEQ ID NO:477), FIG. 204 (SEQ ID NO:483), FIG.
207 (SEQ ID NO:488), FIG. 209 (SEQ ID NO:496), FIG. 211 (SEQ ID
NO:498), FIG. 213 (SEQ ID NO:506), FIG. 215 (SEQ ID NO:508), FIG.
217 (SEQ ID NO:510), FIG. 219 (SEQ ID NO:515), FIG. 222 (SEQ ID
NO:523), FIG. 225 (SEQ ID NO:526), FIG. 230 (SEQ ID NO:612), FIG.
232 (SEQ ID NO:614), FIG. 234 (SEQ ID NO:616) or FIG. 236 (SEQ ID
NO:618), lacking its associated signal peptide.
21. 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:7), FIG. 9 (SEQ ID NO:19), FIG. 11 (SEQ ID
NO:28), FIG. 15 (SEQ ID NO:36), FIG. 20 (SEQ ID NO:45), FIG. 22
(SEQ ID NO:52), FIG. 24 (SEQ ID NO:59), FIG. 26 (SEQ ID NO:64),
FIG. 28 (SEQ ID NO:69), FIG. 30 (SEQ ID NO:74), FIG. 33 (SEQ ID
NO:85), FIG. 35 (SEQ ID NO:90), FIG. 37 (SEQ ID NO:97), FIG. 39
(SEQ ID NO:102), FIG. 41 (SEQ ID NO:109), FIG. 43 (SEQ ID NO:114),
FIG. 45 (SEQ ID NO:119), FIG. 47 (SEQ ID NO:124), FIG. 49 (SEQ ID
NO:132), FIG. 51 (SEQ ID NO:131), FIG. 53 (SEQ ID NO:145), FIG. 55
(SEQ ID NO:150), FIG. 59 (SEQ ID NO:157), FIG. 61 (SEQ ID NO:162),
FIG. 63 (SEQ ID NO:169), FIG. 66 (SEQ ID NO:178), FIG. 68 (SEQ ID
NO:183), FIG. 70 (SEQ ID NO:190), FIG. 73 (SEQ ID NO:196), FIG. 75
(SEQ ID NO:206), FIG. 77 (SEQ ID NO:211), FIG. 79 (SEQ ID NO:216),
FIG. 81 (SEQ ID NO:221), FIG. 83 (SEQ ID NO:226), FIG. 85 (SEQ ID
NO:231), FIG. 87 (SEQ ID NO:236), FIG. 89 (SEQ ID NO:245), FIG. 91
(SEQ ID NO:254), FIG. 93 (SEQ ID NO:259), FIG. 95 (SEQ ID NO:264),
FIG. 98 (SEQ ID NO:270), FIG. 109 (SEQ ID NO:284), FIG. 118 (SEQ ID
NO:296), FIG. 120 (SEQ ID NO:301), FIG. 122 (SEQ ID NO:303), FIG.
125 (SEQ ID NO:309), FIG. 129 (SEQ ID NO:322), FIG. 132 (SEQ ID
NO:330), FIG. 136 (SEQ ID NO:337), FIG. 139 (SEQ ID NO:346), FIG.
142 (SEQ ID NO:352), FIG. 145 (SEQ ID NO:358), FIG. 147 (SEQ ID
NO:363), FIG. 149 (SEQ ID NO:370), FIG. 151 (SEQ ID NO:375), FIG.
153 (SEQ ID NO:380), FIG. 155 (SEQ ID NO:385), FIG. 157 (SEQ ID
NO:390), FIG. 159 (SEQ ID NO:395), FIG. 161 (SEQ ID NO:400), FIG.
163 (SEQ ID NO:405), FIG. 165 (SEQ ID NO:410), FIG. 167 (SEQ ID
NO:415), FIG. 169 (SEQ ID NO:420), FIG. 171 (SEQ ID NO:425), FIG.
173 (SEQ ID NO:430), FIG. 177 (SEQ ID NO:437), FIG. 179 (SEQ ID
NO:442), FIG. 181 (SEQ ID NO:447), FIG. 183 (SEQ ID NO:452), FIG.
185 (SEQ ID NO:454), FIG. 187 (SEQ ID NO:456), FIG. 190 (SEQ ID
NO:459), FIG. 192 (SEQ ID NO:464), FIG. 194 (SEQ ID NO:466), FIG.
196 (SEQ ID NO:468), FIG. 198 (SEQ ID NO:470), FIG. 200 (SEQ ID
NO:472), FIG. 202 (SEQ ID NO:477), FIG. 204 (SEQ ID NO:483), FIG.
207 (SEQ ID NO:488), FIG. 209 (SEQ ID NO:496), FIG. 211 (SEQ ID
NO:498), FIG. 213 (SEQ ID NO:506), FIG. 215 (SEQ ID NO:508), FIG.
217 (SEQ ID NO:510), FIG. 219 (SEQ ID NO:515), FIG. 222 (SEQ ID
NO:523), FIG. 225 (SEQ ID NO:526), FIG. 230 (SEQ ID NO:612), FIG.
232 (SEQ ID NO:614), FIG. 234 (SEQ ID NO:616) or FIG. 236 (SEQ ID
NO:618), lacking its associated signal peptide; (b) an
extracellular domnain of the polypeptide shown in FIG. 2 (SEQ ID
NO:2), FIG. 4 (SEQ ID NO:7), FIG. 9 (SEQ ID NO:19), FIG. 11 (SEQ ID
NO:28), FIG. 15 (SEQ ID NO:36), FIG. 20 (SEQ ID NO:45), FIG. 22
(SEQ ID NO:52), FIG. 24 (SEQ ID NO:59), FIG. 26 (SEQ ID NO:36),
FIG. 28 (SEQ ID NO:69), FIG. 30 (SEQ ID NO:74), FIG. 33 (SEQ ID
NO:85), FIG. 35 (SEQ ID NO:90), FIG. 37 (SEQ ID NO:97), FIG. 39
(SEQ ID NO:102), FIG. 41 (SEQ ID NO:109), FIG. 43 (SEQ ID NO:114),
FIG. 45 (SEQ ID NO:119), FIG. 47 (SEQ ID NO:124), FIG. 49 (SEQ ID
NO:132), FIG. 51 (SEQ ID NO:137), FIG. 53 (SEQ ID NO:145), FIG. 55
(SEQ ID NO:150), FIG. 59 (SEQ ID NO:157), FIG. 61 (SEQ ID NO:162),
FIG. 63 (SEQ ID NO:169), FIG. 66 (SEQ ID NO:178), FIG. 68 (SEQ ID
NO:183), FIG. 70 (SEQ ID NO:190), FIG. 73 (SEQ ID NO:196), FIG. 75
(SEQ ID NO:206), FIG. 77 (SEQ ID NO:211), FIG. 79 (SEQ ID NO:216),
FIG. 81 (SEQ ID NO:221), FIG. 83 (SEQ ID NO:226), FIG. 85 (SEQ ID
NO:231), FIG. 87 (SEQ ID NO:236), FIG. 89 (SEQ ID NO:245), FIG. 91
(SEQ ID NO:254), FIG. 93 (SEQ ID NO:259), FIG. 95 (SEQ ID NO:264),
FIG. 98 (SEQ ID NO:270), FIG. 109 (SEQ ID NO:284), FIG. 118 (SEQ ID
NO:296), FIG. 120 (SEQ ID NO:301), FIG. 122 (SEQ ID NO:303), FIG.
125 (SEQ ID NO:309), FIG. 129 (SEQ ID NO:322), FIG. 132 (SEQ ID
NO:330), FIG. 136 (SEQ ID NO:337), FIG. 139 (SEQ ID NO:346), FIG.
142 (SEQ ID NO:352), FIG. 145 (SEQ ID NO:358), FIG. 147 (SEQ ID
NO:363), FIG. 149 (SEQ ID NO:370), FIG. 151 (SEQ ID NO:375), FIG.
153 (SEQ ID NO:380), FIG. 155 (SEQ ID NO:385), FIG. 157 (SEQ ID
NO:390), FIG. 159 (SEQ ID NO:395), FIG. 161 (SEQ ID NO:400), FIG.
163 (SEQ ID NO:405), FIG. 165 (SEQ ID NO:410), FIG. 167 (SEQ ID
NO:415), FIG. 169 (SEQ ID NO:420), FIG. 171 (SEQ ID NO:425), FIG.
173 (SEQ ID NO:430), FIG. 177 (SEQ ID NO:437), FIG. 179 (SEQ ID
NO:442), FIG. 181 (SEQ ID NO:447), FIG. 183 (SEQ ID NO:452), FIG.
185 (SEQ ID NO:454), FIG. 187 (SEQ ID NO:456), FIG. 190 (SEQ ID
NO:459), FIG. 192 (SEQ ID NO:464), FIG. 194 (SEQ ID NO:466), FIG.
196 (SEQ ID NO:468), FIG. 198 (SEQ ID NO:470), FIG. 200 (SEQ ID
NO:472), FIG. 202 (SEQ ID NO:477), FIG. 204 (SEQ ID NO:483), FIG.
207 (SEQ ID NO:488), FIG. 209 (SEQ ID NO:496), FIG. 211 (SEQ ID
NO:498), FIG. 213 (SEQ ID NO:506), FIG. 215 (SEQ ID NO:508), FIG.
217 (SEQ ID NO:510), FIG. 219 (SEQ ID NO:515), FIG. 222 (SEQ ID
NO:523), FIG. 225 (SEQ ID NO:526), FIG. 230 (SEQ ID NO:612), FIG.
232 (SEQ ID NO:614), FIG. 234 (SEQ ID NO:616) or FIG. 236 (SEQ ID
NO:618), 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:7), FIG. 9 (SEQ ID NO:19), FIG. 11 (SEQ ID
NO:28), FIG. 15 (SEQ ID NO:36), FIG. 20 (SEQ ID NO:45), FIG. 22
(SEQ ID NO:52), FIG. 24 (SEQ ID NO:59), FIG. 26 (SEQ ID NO:64),
FIG. 28 (SEQ ID NO:69), FIG. 30 (SEQ ID NO:74), FIG. 33 (SEQ ID
NO:85), FIG. 35 (SEQ ID NO:90), FIG. 37 (SEQ ID NO:97), FIG. 39
(SEQ ID NO:102), FIG. 41 (SEQ ID NO:109), FIG. 43 (SEQ ID NO:114),
FIG. 45 (SEQ ID NO:119), FIG. 47 (SEQ ID NO:124), FIG. 49 (SEQ ID
NO:132), FIG. 51 (SEQ ID NO:137), FIG. 53 (SEQ ID NO:145), FIG. 55
(SEQ ID NO:150), FIG. 59 (SEQ ID NO:157), FIG. 61 (SEQ ID NO:162),
FIG. 63 (SEQ ID NO:169), FIG. 66 (SEQ ID NO:178), FIG. 68 (SEQ ID
NO:183), FIG. 70 (SEQ ID NO:190), FIG. 73 (SEQ ID NO:196), FIG. 75
(SEQ ID NO:206), FIG. 77 (SEQ ID NO:211), FIG. 79 (SEQ ID NO:216),
FIG. 81 (SEQ ID NO:221), FIG. 83 (SEQ ID NO:226), FIG. 85 (SEQ ID
NO:231), FIG. 87 (SEQ ID NO:236), FIG. 89 (SEQ ID NO:245), FIG. 91
(SEQ ID NO:254), FIG. 93 (SEQ ID NO:259), FIG. 95 (SEQ ID NO:264),
FIG. 98 (SEQ ID NO:270), FIG. 109 (SEQ ID NO:284), FIG. 118 (SEQ ID
NO:296), FIG. 120 (SEQ ID NO:301), FIG. 122 (SEQ ID NO:303), FIG.
125 (SEQ ID NO:309), FIG. 129 (SEQ ID NO:322), FIG. 132 (SEQ ID
NO:330), FIG. 136 (SEQ ID NO:337), FIG. 139 (SEQ ID NO:346), FIG.
142 (SEQ ID NO:352), FIG. 145 (SEQ ID NO:358), FIG. 147 (SEQ ID
NO:363), FIG. 149 (SEQ ID NO:370), FIG. 151 (SEQ ID NO:375), FIG.
153 (SEQ ID NO:380), FIG. 155 (SEQ ID NO:385), FIG. 157 (SEQ ID
NO:390), FIG. 159 (SEQ ID NO:395), FIG. 161 (SEQ ID NO:400), FIG.
163 (SEQ ID NO:405), FIG. 165 (SEQ ID NO:410), FIG. 167 (SEQ ID
NO:415), FIG. 169 (SEQ ID NO:420), FIG. 171 (SEQ ID NO:425), FIG.
173 (SEQ ID NO:430), FIG. 177 (SEQ ID NO:437), FIG. 179 (SEQ ID
NO:442), FIG. 181 (SEQ ID NO:447), FIG. 183 (SEQ ID NO:452), FIG.
185 (SEQ ID NO:454), FIG. 187 (SEQ ID NO:456), FIG. 190 (SEQ ID
NO:459), FIG. 192 (SEQ ID NO:464), FIG. 194 (SEQ ID NO:466), FIG.
196 (SEQ ID NO:468), FIG. 198 (SEQ ID NO:470), FIG. 200 (SEQ ID
NO:472), FIG. 202 (SEQ ID NO:477), FIG. 204 (SEQ ID NO:483), FIG.
207 (SEQ ID NO:488), FIG. 209 (SEQ ID NO:496), FIG. 211 (SEQ ID
NO:498), FIG. 213 (SEQ ID NO:506), FIG. 215 (SEQ ID NO:508), FIG.
217 (SEQ ID NO:510), FIG. 219 (SEQ ID NO:515), FIG. 222 (SEQ ID
NO:523), FIG. 225 (SEQ ID NO:526), FIG. 230 (SEQ ID NO:612), FIG.
232 (SEQ ID NO:614), FIG. 234 (SEQ ID NO:616) or FIG. 236 (SEQ ID
NO:618), lacking its associated signal peptide.
22. A method of detecting a PRO4993 polypeptide in a sample
suspected of containing a PRO4993 polypeptide, said method
comprising contacting said sample with a PRO337 polypeptide and
determining the formation of a PRO4993/PRO337 polypeptide conjugate
in said sample, wherein the formation of said conjugate is
indicative of the presence of a PRO4993 polypeptide in said
sample.
23. The method according to claim 22, wherein said sample comprises
cells suspected of expressing said PRO4993 polypeptide.
24. The method according to claim 22, wherein said PRO337
polypeptide is labeled with a detectable label.
25. The method according to claim 22, wherein said PRO337
polypeptide is attached to a solid support.
26. A method of detecting a PRO337 polypeptide in a sample
suspected of containing a PRO337 polypeptide, said method
comprising contacting said sample with a PRO4993 polypeptide and
determing the formation of a PRO4993/PRO337 polypeptide conjugate
in said sample, wherein the formation of said conjugate is
indicative of the presence of a PRO337 polypeptide in said
sample.
27. The method according to claim 26, wherein said sample comprises
cells suspected of expressing said PRO337 polypeptide.
28. The method according to claim 26, wherein said PRO4993
polypeptide is labeled with a detectable label.
29. The method according to claim 26, wherein said PRO4993
polypeptide is attached to a solid support.
30. A method of detecting a PRO1559 polypeptide in a sample
suspected of containing a PRO1559 polypeptide, said method
comprising contacting said sample with a PRO725, PRO700 or PRO739
polypeptide and determining the formation of a PRO1559/PRO725,
PRO700 or PRO739 polypeptide conjugate in said sample, wherein the
formation of said conjugate is indicative of the presence of a
PRO1559 polypeptide in said sample.
31. The method according to claim 30, wherein said sample comprises
cells suspected of expressing said PRO1559 polypeptide.
32. The method according to claim 30, wherein said PRO725, PRO700
or PRO739 polypeptide is labeled with a detectable label.
33. The method according to claim 30, wherein said PRO725, PRO700
or PRO739 polypeptide is attached to a solid support.
34. A method of detecting a PRO725, PRO700 or PRO739 polypeptide in
a sample suspected of containing a PRO725, PRO700 or PRO739
polypeptide, said method comprising contacting said sample with a
PRO1559 polypeptide and determining the formation of a
PRO1559/PRO725, PRO700 or PRO739 polypeptide conjugate in said
sample, wherein the formation of said conjugate is indicative of
the presence of a PRO725, PRO700 or PRO739 polypeptide in said
sample.
35. The method according to claim 34, wherein said sample comprises
cells suspected of expressing said PRO725, PRO700 or PRO739
polypeptide.
36. The method according to claim 34, wherein said PRO1559
polypeptide is labeled with a detectable label.
37. The method according to claim 34, wherein said PRO1559
polypeptide is attached to a solid support.
38. A method of linking a bioactive molecule to a cell expressing a
PRO337 polypeptide, said method comprising contacting said cell
with a PRO4993 polypeptide that is bound to said bioactive molecule
and allowing said PRO337 and PRO4993 polypeptides to bind to one
another, thereby linking said bioactive molecules to said cell.
39. The method according to claim 38, wherein said bioactive
molecule is a toxin, a radiolabel or an antibody.
40. The method according to claim 38, wherein said bioactive
molecule causes the death of said cell.
41. A method of linking a bioactive molecule to a cell expressing a
PRO4993 polypeptide, said method comprising contacting said cell
with a PRO337 polypeptide that is bound to said bioactive molecule
and allowing said PRO4993 and PRO337 polypeptides to bind to one
another, thereby linking said bioactive molecules to said cell.
42. The method according to claim 41, wherein said bioactive
molecule is a toxin, a radiolabel or an antibody.
43. The method according to claim 41, wherein said bioactive
molecule causes the death of said cell.
44. A method of linking a bioactive molecule to a cell expressing a
PRO1559 polypeptide, said method comprising contacting said cell
with a PRO725, PRO700 or PRO739 polypeptide that is bound to said
bioactive molecule and allowing said PRO1559 and PRO725, PRO700 or
PRO739 polypeptides to bind to one another, thereby linking said
bioactive molecules to said cell.
45. The method according to claim 44, wherein said bioactive
molecule is a toxin, a radiolabel or an antibody.
46. The method according to claim 44, wherein said bioactive
molecule causes the death of said cell.
47. A method of linking a bioactive molecule to a cell expressing a
PRO725, PRO700 or PRO739 polypeptide, said method comprising
contacting said cell with a PRO1559 polypeptide that is bound to
said bioactive molecule and allowing said PRO1559 and PRO725,
PRO700 or PRO739 polypeptides to bind to one another, thereby
linking said bioactive molecules to said cell.
48. The method according to claim 47, wherein said bioactive
molecule is a toxin, a radiolabel or an antibody.
49. The method according to claim 47, wherein said bioactive
molecule causes the death of said cell.
50. A method of modulating at least one biological activity of a
cell expressing a PRO337 polypeptide, said method comprising
contacting said cell with a PRO4993 polypeptide or an anti-PRO337
antibody, whereby said PRO4993 polypeptide or said anti-PRO337
antibody binds to said PRO337 polypeptide, thereby modulating at
least one biological activity of said cell.
51. The method according to claim 50, wherein said cell is
killed.
52. A method of modulating at least one biological activity of a
cell expressing a PRO4993 polypeptide, said method comprising
contacting said cell with a PRO337 polypeptide or an anti-PRO4993
antibody, whereby said PRO337 polypeptide or said anti-PRO4993
antibody binds to said PRO4993 polypeptide, thereby modulating at
least one biological activity of said cell.
53. The method according to claim 52, wherein said cell is
killed.
54. A method of modulating at least one biological activity of a
cell expressing a PRO1559 polypeptide, said method comprising
contacting said cell with a PRO725, PRO700 or PRO739 polypeptide or
an anti-PRO1559 antibody, whereby said PRO725, PRO700 or PRO739
polypeptide or said anti-PRO1559 antibody binds to said PRO1559
polypeptide, thereby modulating at least one biological activity of
said cell.
55. The method according to claim 54, wherein said cell is
killed.
56. A method of modulating at least one biological activity of a
cell expressing a PRO725, PRO700 or PRO739 polypeptide, said method
comprising contacting said cell with a PRO1559 polypeptide or an
anti-PRO725, anti-PRO700 or anti-PRO739 antibody, whereby said
PRO1559 polypeptide or said anti-PRO725, anti-PRO700 or anti-PRO739
antibody binds to said PRO725, PRO700 or PRO739 polypeptide,
thereby modulating at least one biological activity of said
cell.
57. The method according to claim 56, 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 encoded by that DNA.
BACKGROUND OF THE INVENTION
[0002] Extracellular proteins play an important role 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.
[0003] Secreted proteins have various industrial applications,
including 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 an important
role 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. 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 interaction. The
membrane-bound proteins can also be employed for screening of
potential peptide or small molecule inhibitors of the relevant
receptor/ligand interaction. Efforts are being undertaken by both
industry and academia to identify new, native receptor proteins.
Many efforts are focused on the screening of mammalian recombinant
DNA libraries to identify the coding sequences for novel receptor
proteins.
[0006] We herein describe the identification and characterization
of novel secreted and transmembrane polypeptides and novel nucleic
acids encoding those polypeptides.
[0007] 1. PRO213
[0008] Human growth arrest-specific gene 6 (gas6) encodes a protein
that is expressed in a variety of different tissues and which has
been reported to be highly expressed during periods of serum
starvation and negatively regulated during growth induction. See
Manfioletti et al., Mol. Cell. Biol. 13(8):4976-4985 (1993) and
Stitt et al., Cell 80:661-670 (1995). Manfioletti et al. (1993),
supra, have suggested that the gas6 protein is member of the
vitamin K-dependent family of proteins, wherein the members of the
latter family of proteins (which include, for example, Protein S,
Protein C and Factor X) all play regulatory roles in the blood
coagulation pathway. Thus, it has been suggested that gas6 may play
a role in the regulation of a protease cascade relevant in growth
regulation or in the blood coagulation cascade.
[0009] Given the physiological importance of the gas6 protein,
efforts are currently being undertaken by both industry and
academia to identify new, native proteins which are homologous to
gas6. 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,
specifically those having homology to gas6. Examples of such
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 a novel polypeptide which has homology to the
gas6 polypeptide.
[0010] 2. PRO274
[0011] The 7-transmembrane ("7TM") proteins or receptors, also
referred to in the literature as G-protein coupled receptors, are
specialized proteins designed for recognition of ligands and the
subsequent signal transduction of information contained within
those ligands to the machinery of the cell. The primary purpose of
cell surface receptors is to discriinate appropriate ligands from
the various extracellular stimuli which each cell encounters, then
to activate an effector system that produces an intracellular
signal, thereby controlling cellular processes. [Dohlman, H., Ann.
Rev. Biochem., 60:653 (1991)]. The ability of 7TM receptors to bind
ligand to a recognition domain and allosterically transmit the
information to an intracellular domain is a specialized feature of
7TM proteins [Kenakin, T., Pharmacol. Rev., 48:413 (1996)]. The
gene family which encodes the 7TM receptors or G-protein linked
receptors encode receptors which recognize a large number of
ligands, including but not limited to, C5a, interleukin 8 and
related chemokines. Research in this area suggests that distinct
signals at the cell surface feed into common pathways of cell
activation. [Gerard, C. and Gerard, N., Curr. Op. Immunol., 6:140
(1994), Gerard, C. and Gerard, N., Ann. Rev. Immunol., 12:775
(1994)]. The superfamily of 7TM or G-protein coupled receptors
contains several hundred members able to recognize various messages
such as photons, ions and amino acids among others [Schwartz, T.
W., et al., H., Trends in Pharmacol. Sci., 17(6):213 (1996)].
[0012] [Dohlman, H., Ann. Rev. Biochem., 60:653 (1991)]. [Schwartz,
T. W., et al., H., Eur. J. Pharm. Sci., 2:85 (1994)]. We describe
herein the identification of a novel polypeptide (designated herein
as PRO274) which has homology to the 7 transmembrane segment
receptor proteins and the Fn54 protein.
[0013] 3. PRO300
[0014] The Diff 33 protein is over-expressed in mouse testicular
tumors. At present its role is unclear, however, it may play a role
in cancer. Given the medical importance of understanding the
physiology of cancer, efforts are currently being under taken to
identify new, native proteins which are involved in cancer. We
describe herein the identification of a novel polypeptide which has
homology to Diff 33, designated herein as PRO300.
[0015] 4. PRO284
[0016] Efforts arre currently being undertaken to identify and
characterize novel transmembrane proteins. We herein describe the
identification and characterization of a novel transmembrane
polypeptide, designated herein as PRO284.
[0017] 5. PRO296
[0018] Cancerous cells often express numerous proteins that are not
expressed in the corresponding normal cell type or are expressed at
different levels than in the corresponding normal cell type. Many
of these proteins are involved in inducing the transformation from
a normal cell to a cancerous cell or in maintaining the cancer
phenotype. As such, there is significant interest in identifying
and characterizing proteins that are expressed in cancerous cells.
We herein describe the identification and characterization of a
novel polypeptide having homology to the sarcoma-amplified protein
SAS, designated herein as PRO296.
[0019] 6. PRO329
[0020] Immunoglobulin molecules play roles in many important
mammalian physiological processes. The structure of immunoglobulin
molecules has been extensively studied and it has been well
documented that intact immunoglobulins possess distinct domains,
one of which is the constant domain or F.sub.c region of the
immunoglobulin molecule. The F.sub.c domain of an immunoglobulin,
while not being directly involved in antigen recognition and
binding, does mediate the ability of the immunoglobulin molecule,
either uncomplexed or complexed with its respective antigen, to
bind to F.sub.c receptors either circulating in the serum or on the
surface of cells. The ability of an F.sub.c domain of an
immunoglobulin to bind to an F.sub.c receptor molecule results in a
variety of important activities, including for example, in mounting
an immune response against unwanted foreign particles. As such,
there is substantial interest in identifying novel F.sub.c receptor
proteins and subunits thereof. We herein describe the
identification and characterization of a novel polypeptide having
homology to a high affinity immunoglobulin F.sub.c receptor
protein, designated herein as PRO329.
[0021] 7. PRO362
[0022] Colorectal carcinoma is a malignant neoplastic disease which
has a high incidence in the Western world, particularly in the
United States. Tumors of this type often metastasize through
lymphatic and vascular channels and result in the death of some
62,000 persons in the United States annually.
[0023] Monoclonal antibody A33 (mAbA33) is a murine immunoglobulin
that has undergone extensive preclinical analysis and localization
studies in patients inflicted with colorectal carcinoma (Welt et
al., J. Clin. Oncol. 8:1894-1906 (1990) and Welt et al., J. Clin.
Oncol. 12:1561-1571 (1994)). mAbA33 has been shown to bind to an
antigen found in and on the surface of normal colon cells and colon
cancer cells. In carcinomas originating from the colonic mucosa,
the A33 antigen is expressed homogeneously in more than 95% of the
cases. The A33 antigen, however, has not been detecting in a wide
range of other normal tissues, i.e., its expression appears to be
rather organ specific. Therefore, the A33 antigen appears to play
an important role in the induction of colorectal cancer.
[0024] Given the obvious importance of the A33 antigen in tumor
cell formation and/or proliferation, there is substantial interest
in identifying homologs of the A33 antigen. In this regard, we
herein describe the identification and characterization of a novel
polypeptide having homology to the A33 antigen protein, designated
herein as PRO362.
[0025] 8. PRO363
[0026] 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.
[0027] In light of the physiological importance of membrane-bound
proteins and speficiauy 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
reeptor 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 having homology to the cell
surface protein HCAR and to various tumor antigens including A33
and carcinoembryonic antigen, designated herein as PRO363, wherein
this polypeptide may be a novel cell surface virus receptor or
tumor antigen.
[0028] 9. PRO868
[0029] Control of cell numbers in mammals is believed to be
determined, in part, by a balance between cell proliferation and
cell death. One form of cell death, sometimes referred to as
necrotic cell death, is typically characterized as a pathologic
form of cell death resulting from some trauma or cellular injury.
In contrast, there is another, "physiologic" form of cell death
which usually proceeds in an orderly or controlled manner. This
orderly or controlled form of cell death is often referred to as
"apoptosis" [see, e.g., Barr et al., Bio/Technology, 12:487-493
(1994); Steller et al., Science, 267:1445-1449 (1995)]. Apoptotic
cell death naturally occurs in many physiological processes,
including embryonic development and clonal selection in the immune
system [Itoh et al., Cell, 66:233-243 (1991)]. Decreased levels of
apoptotic cell death have been associated with a variety of
pathological conditions, including cancer, lupus, and herpes virus
infection [Thompson, Science, 267:1456-1462 (1995)]. Increased
levels of apoptotic cell death may be associated with a variety of
other pathological conditions, including AIDS, Alzheimer's disease,
Parkinson's disease, amyotrophic lateral sclerosis, multiple
sclerosis, retinitis pigmentosa, cerebellar degeneration, aplastic
anemia, myocardial infarction, stroke, reperfusion injury, and
toxin-induced liver disease [see, Thompson, supra].
[0030] Apoptotic cell death is typically accompanied by one or more
characteristic morphological and biochemical changes in cells, such
as condensation of cytoplasm, loss of plasma membrane microvilli,
segmentation of the nucleus, degradation of chromosomal DNA or loss
of mitochondrial function. A variety of extrinsic and intrinsic
signals are believed to trigger or induce such morphological and
biochemical cellular changes [Raff, Nature, 356:397-400 (1992);
Steller, supra; Sachs et al., Blood, 82:15 (1993)]. For instance,
they can be triggered by hormonal stimuli, such as glucocorticoid
hormones for immature thymocytes, as well as withdrawal of certain
growth factors [Watanabe-Fukunaga et al., Nature, 356:314-317
(1992)]. Also, some identified oncogenes such as myc, rel, and EIA,
and tumor suppressors, like p53, have been reported to have a role
in inducing apoptosis. Certain chemotherapy drugs and some forms of
radiation have likewise been observed to have apoptosis-inducing
activity [Thompson, supra].
[0031] Various molecules, such as tumor necrosis factor-.alpha.
("TNF-.alpha."), tumor necrosis factor-.beta. ("TNF-.beta." or
"lymphotoxin-.alpha."), lynmphotoxin-.beta. ("LT-.beta."), CD30
ligand, CD27 ligand, CD40 ligand, OX-40 ligand, 4-1BB ligand, Apo-1
ligand (also referred to as Fas ligand or CD95 ligand), and Apo-2
ligand (also referred to as TRAIL) have been identified as members
of the tumor necrosis factor ("TNF") family of cytokines [See,
e.g., Gruss and Dower, Blood, 85:3378-3404 (1995); Pitti et al., J.
Biol. Chem., 271:12687-12690 (1996); Wiley et al., Immunity,
3:673-682 (1995); Browning et al., Cell, 72:847-856 (1993);
Armitage et al. Nature, 357:80-82 (1992), WO 97/01633 published
Jan. 16, 1997; WO 97/25428 published Jul. 17, 1997]. Among these
molecules, TNF-.alpha., TNF-.beta., CD30 ligand, 4-1BB ligand,
Apo-1 ligand, and Apo-2 ligand (TRAIL) have been reported to be
involved in apoptotic cell death. Both TNF-.alpha. and TNF-.beta.
have been reported to induce apoptotic death in susceptible tumor
cells [Schmid et al., Proc. Natl. Acad. Sci., 83:1881 (1986);
Dealtry et al., Eur. J. Immunol., 17:689 (1987)]. Zheng et al. have
reported that TNF-.alpha. is involved in post-stimulation apoptosis
of CD8-positive T cells [Zheng et al., Nature, 377:348-351 (1995)].
Other investigators have reported that CD30 ligand may be involved
in deletion of self-reactive T cells in the thymus [Amakawa et al.,
Cold Spring Harbor Laboratory Symposium on Programmed Cell Death,
Abstr. No. 10, (1995)].
[0032] Mutations in the mouse Fas/Apo-1 receptor or ligand genes
(called lpr and gld, respectively) have been associated with some
autoimmune disorders, indicating that Apo-1 ligand may play a role
in regulating the clonal deletion of self-reactive lymphocytes in
the periphery [Krammer et al., Curr. Op. Immunol., 6:279-289
(1994); Nagata et al., Science, 267:1449-1456 (1995)]. Apo-1 ligand
is also reported to induce post-stimulation apoptosis in
CD4-positive T lymphocytes and in B lymphocytes, and may be
involved in the elimination of activated lymphocytes when their
function is no longer needed [Krammer et al., supra; Nagata et al.,
supra]. Agonist mouse monoclonal antibodies specifically binding to
the Apo-1 receptor have been reported to exhibit cell killing
activity that is comparable to or similar to that of TNF-.alpha.
[Yonehara et al., J. Exp. Med., 169:1747-1756 (1989)].
[0033] Induction of various cellular responses mediated by such TNF
family cytokines is believed to be initiated by their binding to
specific cell receptors. Two distinct TNF receptors of
approximately 55-kDa (TNFR1) and 75-kDa (TNFR2) have been
identified [Hohman et al., J. Biol. Chem., 264:14927-14934 (1989);
Brockhaus et al., Proc. Natl. Acad. Sci., 87:3127-3131 (1990); EP
417,563, published Mar. 20, 1991] and human and mouse cDNAs
corresponding to both receptor types have been isolated and
characterized [Loetscher et al., Cell, 61:351 (1990); Schall et
al., Cell, 61:361 (1990); Smith et al., Science, 248:1019-1023
(1990); Lewis et al., Proc. Natl. Acad. Sci., 88:2830-2834 (1991);
Goodwin et al., Mol. Cell. Biol., 11:3020-3026 (1991)]. Extensive
polymorphisms have been associated with both TNF receptor genes
[see, e.g., Takao et al., Immunogenetics, 37:199-203 (1993)]. Both
TNFRs share the typical structure of cell surface receptors
including extracellular, transmembrane and intracellular regions.
The extracellular portions of both receptors are found naturally
also as soluble TNF-binding proteins [Nophar, Y. et al., EMBO J.,
9:3269 (1990); and Kohno, T. et al., Proc. Natl. Acad. Sci. U.S.A.,
87:8331 (1990)]. More recently, the cloning of recombinant soluble
TNF receptors was reported by Hale et al. [J. Cell. Biochem.
Supplement 15F, 1991, p. 113 (P424)].
[0034] The extracellular portion of type 1 and type 2 TNFRs (TNFR1
and TNFR2) contains a repetitive amino acid sequence pattern of
four cysteine-rich domains (CRDs) designated 1 through 4, starting
from the NH.sub.2-terminus. Each CRD is about 40 amino acids long
and contains 4 to 6 cysteine residues at positions which are well
conserved [Schall et al., supra; Loetscher et al., supra; Smith et
al., supra; Nophar et al., supra; Kohno et al., supra]. In TNFR1,
the approximate boundaries of the four CRDs are as follows:
CRD1--amino acids 14 to about 53; CRD2--amino acids from about 54
to about 97; CRD3--amino acids from about 98 to about 138;
CRD4--amino acids from about 139 to about 167. In TNFR2, CRD1
includes amino acids 17 to about 54; CRD2--amino acids from about
55 to about 97; CRD3--amino acids from about 98 to about 140; and
CRD4--amino acids from about 141 to about 179 [Banner et al., Cell
73:431-435 (1993)]. The potential role of the CRDs in ligand
binding is also described by Banner et al., supra.
[0035] A similar repetitive pattern of CRDs exists in several other
cell-surface proteins, including the p75 nerve growth factor
receptor (NGFR) [Johnson et al., Cell, 47:545 (1986); Radeke et
al., Nature, 325:593 (1987)], the B cell antigen CD40 [Stamenkovic
et al., EMBO J., 8:1403 (1989)], the T cell antigen OX40 [Mallet et
al., EMBO J., 9:1063 (1990)] and the Fas antigen [Yonehara et al.,
supra and Itoh et al., Cell, 66:233-243 (1991)]. CRDs are also
found in the soluble TNFR (sTNFR)-like T2 proteins of the Shope and
myxoma poxviruses [Upton et al., Virology, 160:20-29 (1987); Smith
et al., Biochem. Biophys. Res. Commun., 176:335 (1991); Upton et
al., Virology, 184:370 (1991)]. Optimal alignment of these
sequences indicates that the positions of the cysteine residues are
well conserved. These receptors are sometimes collectively referred
to as members of the TNF/NGF receptor superfamily. Recent studies
on p75NGFR showed that the deletion of CRD1 [Welcher, A. A. et al.,
Proc. Natl. Acad. Sci. USA, 88:159-163 (1991)] or a 5-amino acid
insertion in this domain [Yan, H. and Chao, M. V., J. Biol. Chem.,
266:12099-12104 (1991)] had little or no effect on NGF binding
[Yan, H. and Chao, M. V., supra]. p75 NGFR contains a proline-rich
stretch of about 60 amino acids, between its CRD4 and transmembrane
region, which is not involved in NGF binding [Peetre, C. et al.,
Eur. J. Hematol., 41:414-419(1988); Seckinger, P. et al., J. Biol.
Chem., 264:11966-11973(1989); Yan, H. and Chao, M. V., supra]. A
similar proline-rich region is found in TNFR2 but not in TNFR1.
[0036] The TNF family ligands identified to date, with the
exception of lymphotoxin-.alpha., are type II transmembrane
proteins, whose C-terminus is extracellular. In contrast, most
receptors in the TNF receptor (TNFR) family identified to date are
type I transmembrane proteins. In both the TNF ligand and receptor
families, however, homology identified between family members has
been found mainly in the extracellular domain ("ECD"). Several of
the TNF family cytokines, including TNF-.alpha., Apo-1 ligand and
CD40 ligand, are cleaved proteolytically at the cell surface; the
resulting protein in each case typically forms a homotrimeric
molecule that functions as a soluble cytokine. TNF receptor family
proteins are also usually cleaved proteolytically to release
soluble receptor ECDs that can function as inhibitors of the
cognate cytokines.
[0037] Recently, other members of the TNFR family have been
identified. Such newly identified members of the TNFR family
include CAR1, HVEM and osteoprotegerin (OPG) [Brojatsch et al.,
Cell, 87:845-855 (1996); Montgomery et al., Cell 87:427-436 (1996);
Marsters et al., J. Biol. Chem., 272:14029-14032 (1997); Simonet et
al., Cell, 89:309-319 (1997)]. Unlike other known TNFR-like
molecules, Simonet et al., supra, report that OPG contains no
hydrophobic transmembrane-spanning sequence.
[0038] Moreover, a new member of the TNF/NGF receptor family has
been identified in mouse, a receptor referred to as "GITR" for
"glucocorticoid-induced tumor necrosis factor receptor
family-related gene" [Nocentini et al., Proc. Natl. Acad. Sci. USA
94:6216-6221 (1997)]. The mouse GITR receptor is a 228 amino acid
type I transmembrane protein that is expressed in normal mouse T
lymphocytes from thymus, spleen and lymph nodes. Expression of the
mouse GITR receptor was induced in T lymphocytes upon activation
with anti-CD3 antibodies, Con A or phorbol 12-myristate 13-acetate.
It was speculated by the authors that the mouse GITR receptor was
involved in the regulation of T cell receptor-mediated cell
death.
[0039] In Marsters et al., Curr. Biol., 6:750 (1996), investigators
describe a full length native sequence human polypeptide, called
Apo-3, which exhibits similarity to the TNFR family in its
extracellular cysteine-rich repeats and resembles TNFR1 and CD95 in
that it contains a cytoplasmic death domain sequence [see also
Marsters et al., Curr. Biol., 6:1669 (1996)]. Apo-3 has also been
referred to by other investigators as DR3, wsl-1 and TRAMP
[Chinnaiyan et al., Science, 274:990 (1996); Kitson et al., Nature,
384:372 (1996); Bodmer et al., Immunity, 6:79 (1997)].
[0040] Pan et al. have disclosed another TNF receptor family member
referred to as "DR4" [Pan et al., Science, 276:111-113 (1997)]. The
DR4 was reported to contain a cytoplasmic death domain capable of
engaging the cell suicide apparatus. Pan et al. disclose that DR4
is believed to be a receptor for the ligand known as Apo-2 ligand
or TRAIL.
[0041] In Sheridan et al., Science, 277:818-821 (1997) and Pan et
al., Science, 277:815-818 (1997), another molecule believed to be a
receptor for the Apo-2 ligand (TRAIL) is described. That molecule
is referred to as DR5 (it has also been alternatively referred to
as Apo-2). Like DR4, DR5 is reported to contain a cytoplasmic death
domain and be capable of signaling apoptosis.
[0042] In Sheridan et al., supra, a receptor called DcR1 (or
alternatively, Apo-2DcR) is disclosed as being a potential decoy
receptor for Apo-2 ligand (TRAIL). Sheridan et al. report that DcR1
can inhibit Apo-2 ligand function in vitro. See also, Pan et al.,
sura, for disclosure on the decoy receptor referred to as TRID.
[0043] For a review of the TNF family of cytokines and their
receptors, see Gruss and Dower, supra.
[0044] As presently understood, the cell death program contains at
least three important elements--activators, inhibitors, and
effectors; in C. elegans, these elements are encoded respectively
by three genes, Ced4, Ced-9 and Ced-3 [Steller, Science,
267:1445(1995); Chinnaiyan et al., Science 275:1122-1126(1997);
Wang et al., Cell, 90:1-20 (1997)]. Two of the TNFR family members,
TNFR1 and Fas/Apo1 (CD95), can activate apoptotic cell death
[Chinnaiyan and Dixit, Current Biology, 6:555-562 (1996); Fraser
and Evan, Cell; 85:781-784 (1996)]. TNFR1 is also known to mediate
activation of the transcription factor, NF-.kappa.B [Tartaglia et
al., Cell 74:845-853 (1993); Hsu et al., Cell, 84:299-308 (1996)].
In addition to some ECD homology, these two receptors share
homology in their intracellular domain (ICD) in an oligomerization
interface known as the death domain [Tartaglia et al., supra;
Nagata, Cell, 88:355 (1997)]. Death domains are also found in
several metazoan proteins that regulate apoptosis, namely, the
Drosophila protein, Reaper, and the mammalian proteins referred to
as FADD/MORT1, TRADD, and RIP [Cleaveland and Ihle, Cell,
81:479-482 (1995)].
[0045] Upon ligand binding and receptor clustering, TNFR1 and CD95
are believed to recruit FADD into a death-inducing signalling
complex. CD95 purportedly binds FADD directly, while TNFR1 binds
FADD indirectly via TRADD [Chinnaiyan et al., Cell, 81:505-512
(1995); Boldin et al., J. Biol. Chem., 270:387-391 (1995); Hsu et
al., supra; Chinnaiyan et al., J. Biol. Chem., 271:4961-4965
(1996)]. It has been reported that FADD serves as an adaptor
protein which recruits the Ced-3-related protease,
MACH.alpha./FLICE (caspase 8), into the death signaling complex
[Boldin et al., Cell, 85:803-815 (1996); Muzio et al., Cell,
85:817-827 (1996)]. MACH.alpha./FLICE appears to be the trigger
that sets off a cascade of apoptotic proteases, including the
interleukin-1.beta. converting enzyme (ICE) and CPP32/Yama, which
may execute some critical aspects of the cell death programme
[Fraser and Evan, supra].
[0046] It was recently disclosed that programmed cell death
involves the activity of members of a family of cysteine proteases
related to the C. elegans cell death gene, ced-3, and to the
mammalian IL-1-converting enzyme, ICE. The activity of the ICE and
CPP32/Yama proteases can be inhibited by the product of the cowpox
virus gene, crmA [Ray et al., Cell 69:597-604 (1992); Tewari et
al., Cell, 81:801-809 (1995)]. Recent studies show that CrmA can
inhibit TNFR1- and CD95-induced cell death [Enari et al., Nature,
375:78-81 (1995); Tewari et al., J. Biol. Chem., 270:3255-3260
(1995)].
[0047] As reviewed recently by Tewari et al., TNFR1, TNFR2 and CD40
modulate the expression of proinflammatory and costimulatory
cytolines, cytoline receptors, and cell adhesionmolecules through
activation of the transcription factor, NF-.kappa.B [Tewari et al.,
Curr. Op. Genet. Develop., 6:39-44 (1996)]. NF-.kappa.B is the
prototype of a family of dimeric transcription factors whose
subunits contain conserved Rel regions [Verma et al., Genes
Develop., 9:2723-2735 (1996); Baldwin, Ann. Rev. Immunol.,
14:649-681 (1996)]. In its latent form, NF-.kappa.B is complexed
with members of the I.kappa.B inhibitor family; upon inactivation
of the I.kappa.B in response to certain stimuli, released
NF-.kappa.B translocates to the nucleus where it binds to specific
DNA sequences and activates gene transcription.
[0048] 10. PRO382
[0049] 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, including the serine
proteases which exhibit specific activity toward various
serineontainig proteins. The mammalian protease enzymes play
important roles in biological processes such as, for example,
protein digestion, activation, inactivation, or modulation of
peptide hormone activity, and alteration of the physical properties
of proteins and enzymes.
[0050] 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 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 serine protease enzymes,
designated herein as PRO382 polypeptides.
[0051] 11. PRO545
[0052] The ADAM (A Disintegrin And Metalloprotease) family of
proteins of which meltrin is a member may have an important role in
cell interactions and in modulating cellular responses. [see, for
example, Gilpin et al., J. Biol. Chem., 273(1):157-166 (1998)]. The
ADAM proteins have been implicated incarcinogenesis.
Meltrin-.alpha. (ADAM12) is a myoblast gene product reported to be
required for cell fusion. [Harris et al., J. Cell. Biochem., 67(1):
136-142 (1997), Yagami-Hiromasa et al., Nature, 377:652-656
(1995)]. The meltrins contain disintegrin and metalloprotease
domains and are implicated in cell adhesive events involved in
development, through the integrin-binding disintegrin domain, but
also have an anti-adhesive function through a zinc-dependent
metalloprotease domain. [Alfandari et al., Devel. Biol.,
182(2):314-330 (1997)]. Given the medical importance of cell fusion
and modulation of cellular responses in carcinogenesis and other
disease mechanisms, efforts are currently being under taken to
identify new, native proteins which are involved in cell fusion and
modulation of cellular responses. We describe herein the
identification of a novel polypeptide which has homology to
meltrin, designated herein as PRO545.
[0053] 12. PRO617
[0054] CD24 is a protein that is associated with the cell surface
of a variety of different cells of the mammalian immune system,
including for example, neutrophils, monocytes and some lymphocytes,
for example, B lymphocytes. CD24 has been shown to be a ligand for
the platelet-associated surface glycoprotein P-selectin (also known
as granule membrane protein-140 or GMP-140), a glycoprotein that is
constitutively synthesized in both platelets and endothelial cells
and becomes exposed on the surface of platelets when those cells
become activated. In this way, P-selectin mediates the
calcium-dependent adhesion of activated platelets and endothelial
cells to the various cells of the immune system that express one or
more ligands for the P-selectin molecule, particularly CD24. This
mechanism allows for recruitment of immune system cells to
locations where they are most needed, for example, sites of injury.
Thus, there is substantial interest in identifying novel
polypeptides that exhibit homology to the cell surface antigens of
the immune system cells. We herein describe the identification and
characterization of a novel polypeptide having homology to the CD24
protein, wherein that novel polypeptide is herein designated
PRO617.
[0055] 13. PRO700
[0056] Protein-disulfide isomerase (PDI) is a catalyst of disulfide
formation and isomerization during protein folding. It has two
catalytic sites housed in two domains homologous to thioredoxin,
one near the N terminus and the other near the C terminus. [See for
example, Gilbert H F, J. Biol. Chem., 47:29399-29402 (1997),
Mayfield K J, Science, 278:1954-1957 (1997) and Puig et al., J.
Biol. Chem., 52:32988-32994 (1997)]. PDI is useful for formation of
natural type disulfide bonds in a protein which is produced in
aprokaryotic cell. (See also, U.S. Pat. Nos. 5,700,659 and
5,700,678).
[0057] Thus, PDI and molecules related thereto are of interest,
particularly for ability to catalyze the formation of disulfide
bonds. Moreover, these molecules are generally of interest in the
study of redox reactions and related processes. PDI and related
molecules are further described in Darby, et al., Biochemistry 34,
11725-11735(1995). We herein describe the identification and
characterization of novel polypeptides having homology to protein
disulfide isomerase, designated herein as PRO700 polypeptides.
[0058] 14. PRO702
[0059] Conglutinin is a bovine serum protein that was originally
described as a vertebrate lectin protein and which belongs to the
family of C-type lectins that have four characteristic domains, (1)
an N-terminal cysteine-rich domain, (2) a collagen-like domain, (3)
a neck domain and (4) a carbohydrate recognition domain (CRD).
Recent reports have demonstrated that bovine conglutinin can
inhibit hemi gglutination by influenza A viruses as a result of
their lectin properties (Eda et al., Biochem. J. 316:43-48 (1996)).
It has also been suggested that lectins such as conglutinin can
function as immunoglobulin-independent defense molecules due to
complement-mediated mechanisms. Thus, conglutinin has been shown to
be useful for puriiying immune complexes in vitro and for removing
circulating immune complexes from patients plasma in vivo (Lin et
al., Biochem. Biophys. Res. Commun. 218:260-266 (1996)). We herein
describe the identification and characterization of a novel
polypeptide having homology to the conglutinin protein, designated
herein as PRO702.
[0060] 15. PRO703
[0061] Very-long-chain acyl-CoA synthetase ("VLCAS") is a
long-chain fatty acid transport protein which is active in the
cellular transport of long and very long chain fatty acids. [see
for example, Uchida et al., J Biochem (Tokyo) 119(3):565-571 (1996)
and Uchiyama et al., J Biol Chem 271(48):30360-30365 (1996). Given
the biological importance of fatty acid transport mechanisms,
efforts are currently being under taken to identify new, native
proteins which are involved in fatty acid transport. We describe
herein the identification of a novel polypeptide which has homology
to VLCAS, designated herein as PRO703.
[0062] 16. PRO705
[0063] The glypicans are a family of glycosylphosphatidylinositol
(GPI)-anchored proteoglycans that, by virtue of their cell surface
localization and possession of heparin sulfate chains, may regulate
the responses of cells to numerous heparin-binding growth factors,
cell adhesion molecules and extracellular matrix components.
Mutations in one glypican protein cause of syndrome of human birth
defects, suggesting that the glypicans may play an important role
in development (Litwack et al., Dev. Dyn. 211:72-87 (1998)). Also,
since the glypicans may interact with the various extracellular
matrices, they may also play important roles in wound healing
(McGrath et al., Pathol. 183:251-252 (1997)). Furthermore, since
glypicans are expressed in neurons and glioma cells, they may also
play an important role in the regulation of cell division and
survival of cells of the nervous system (Liang et al., J. Cell.
Biol. 139:851-864 (1997)). It is evident, therefore, that the
glypicans are an extremely important family of proteoglycans. There
is, therefore, substantial interest in identifying novel
polypeptides having homology to members of the glypican family. We
herein describe the identification and characterization of a novel
polypeptide having homology to K-glypican, designated herein as
PRO705.
[0064] 17. PRO708
[0065] Aryl sulfatases are enzymes that exist in a number of
different isoforms, including aryl sulfatase A (ASA), aryl
sulfatase B (ASB) and aryl sulfatase C (ASC), and that function to
hydrolyze a variety of different aromatic sulfates. Aryl sulfatases
have been isolated from a variety of different aninul tissues and
microbial sources and their structures and functions have been
extensively studied (see, e.g., Nichol and Roy, J. Biochem.
55:643-651 (1964)). ASA deficiency has been reported to be
associated with metachromatic leukodystrophy (MLD) (Giles et al.,
Prenat. Diagn. 7(4):245-252 (1987) and Herska et al., Am. J. Med.
Genet. 26(3):629-635 (1987)). Additionally, other groups have
reported that aryl sulfatases have been found in high levels in
natural killer cells of the immune system and have hypothesized a
possible role for these enzymes in NK cell-mediated cellular lysis
(see, e.g., Zucker-Franklin et al., Proc. Natl. Acad. Sci. USA
80(22):6977-6981 (1983)). Given the obvious physiological
importance of the aryl sulfatase enzymes, there is a substantial
interest in identifying novel aryl sulfatase homolog polypeptides.
We herein describe the identification and characterization of novel
polypeptides having homology to the aryl sulfatases, wherein these
novel polypeptides are herein designated PRO708 polypeptides.
[0066] 18. PRO320
[0067] Fibulin-1 is a cysteine-rich, calcium-binding extracellular
matrix (CM) component of basement membranes and connective tissue
elastic fibers and plasmaprotein, which has four isoforms, A-D,
derived from alternative splicing. Fibulin-1 is a modular
glycoprotein with amino-terminal 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. There are multiple forms of fibulin-1 that differ in
their C-terminal regions that are produced through the process of
alternative splicing of their precursor RNA. [see for example Tran
et al., Matrix Biol 15(7):479-493 (1997).]
[0068] Northern and Western blotting analysis of 16 cell lines
established from tumors formed in athymic mice and malignant cell
lines derived from patients indicate that low expression of
fibulin-1D plays a role in tumor formation and invasion. [Qing et
al., Oncogene, 18:2159-2168 (1997)]. Ovarian-cancer cells are
characterized by their ability to invade freely the peritoneal
cavity. It has been demonstrated that estradiol stimulates the
proliferation of estrogen-receptor (ER)-positive ovarian-cancer
cells, as well as expression of fibulin-1. Studies on the effect of
fibulin-1 on motility of the MDA-MB231 breast-cancer cell line,
indicated inhibition of haptotactic migration of MDA-MB231 cells,
and the authors concluded that fibulin-1 can inhibit cancer cell
motility in vitro and therefore has the potential to inhibit tumor
invasion. [Hayashido et al., Int J Cancer, 75(4):654-658
(1998)]
[0069] Thus, fibulin, and molecules related thereto are of
interest, particularly for the use of preventing cancer. Moreover,
these molecules are generally of interest in the study of
comnective tissue and attachment molecules and related mechanisms.
Fibulin and related molecules are further described in Adams, et
al., J. Mol. Biol., 272(2):226-36 (1997); Kielty and Shutteworth,
Microsc. Res. Tech., 38(4):413-27 (1997); and Child, J. Card.
Surg., 12(2Supp.):131-5 (1997).
[0070] We herein describe the identification and characterization
of novel polypeptides having homology to fibulin, designated herein
as PRO320 polypeptides.
[0071] 19. PRO324
[0072] Oxidoreductases are enzymes that catalyze a reaction in
which two molecules of a compound interact so that one molecule is
oxidized and the other is reduced, with a molecule of water
entering the reaction. There are many different types of
oxidoreductase enzymes that play very important physiological roles
in the mammalian organism. Some of the most important
oxidoreductases include, for example, lyases, lactases, cholesterol
oxidases, and the like. These enzymes play roles in such essential
processes as digestion, signal transduction, maintenance of ionic
homeostasis, and the like. As such, given that oxidoreductase
enzymes find various essential uses in the mammalian organism,
there is a substantial interest in identifying novel oxidoreductase
enzyme homologs. We herein describe the identification and
characterization of a novel polypeptide having homology to
oxidoreductases, designated herein as PRO324.
[0073] 20. PRO351
[0074] Prostasin is a novel human serine proteinase purified from
human seminal fluid. Immohistochemical locaization reveals that
prostasin is present in epithelial cells and ducts of the prostate
gland. The cDNA for prostasin has been cloned and characterized.
Southern blot analysis, following a reverse transcription
polymerase chain reaction, indicates that prostasin mRNA is
expressed in prostate, liver, salivary gland, kidney, lung,
pancreas, colon, bronchus, renal proximal tubular cells, and
prostate carcinoma LNCaP cells. Cellular localization of prostasin
mRNA was identified within epithelial cells of the human prostate
gland by in situ hybridization histochemistry. [See for example, Yu
et al., J Biol Chem. (1994) 269(29):18843-18848, and Yu et al., J
Biol Chem. (1994) 270(22):13483-13489].
[0075] Thus, prostasin, and molecules related thereto are of
interest, particularly for the study, diagnosis and treatment of
medical conditions involving the prostate. Prostasin and related
molecules are further described in Yu et al., Genomics (1996)
32(3):334-340. We herein describe the identification and
characterization of novel polypeptides having homology to
prostasin, designated herein as PRO351 polypeptides.
[0076] 21. PRO352
[0077] Butyrophilin is a milk glycoprotein that constitutes more
than 40% of the total protein associated with the fat globule
membrane in mammalian milk. Expression of butyrophilin mRNA has
been shown to correlate with the onset of milk fat production
toward the end pregnancy and is maintained throughout lactation.
Butyrophilin has been identified in bovine, murine and human (see
Taylor et al., Biochim. Biophys. Acta 1306:1-4 (1996), Ishii et
al., Biochim. Biophys. Acta 1245:285-292 (1995), Mather et al., J.
Dairy Sci. 76:3832-3850 (1993) and Banghart et al., J. Biol. Chem.
273:4171-4179 (1998)) and is a type I transmembrane protein that is
incorporated into the fat globulin membrane. It has been suggested
that butyrophilin may play a role as the principle scaffold for the
assembly of a complex with xanthine dehydrogenase/oxidase and other
proteins that function in the budding and release of milk-fat
globules from the apical surface during lactation (Banghart et al.,
supra).
[0078] Given that butyrophilin plays an obviously important role in
mammalian milk production, there is substantial interest in
identifying novel butyrophlin homologs. We herein describe the
identification and characterization of a novel polypeptide having
homology to butyrophilln, designated herein as PRO352.
[0079] 22. PRO381
[0080] The immunophilins are a family of proteins that function as
receptors for immunosuppressant drugs, such as cyclosporin A,
FK506, and rapamycin. The immunophilins occur in two separate
classes, (1) the FK506-binding proteins (FKBPs), which bind to
FK506 and rapamycin, and (2) the cyclophilins, which bind to
cyclosporin A. With regard to the PK506-binding proteins, it has
been reported that the FK506/FKBP complex functions to inhibit the
activity of the serine/threonine protein phosphatase 2B
(calcineurin), thereby providing immunosuppressant activity (Gold,
Mol. Neurobiol. 15:285-306 (1997)). It has also been reported that
the FKBP inmunophilins are found in the mammalian nervous system
and may be involved in axonal regeneration in the central nervous
system through a mechanism that is independent of the process by
which immunosuppression is achieved (Gold, supra). Thus, there is
substatial interest in identifying novel polypeptides having
homology to the FKBP immunophilins. We herein describe the
identification and characterization of a novel polypeptide having
homology to an MKBP immunophilin protein, designated herein as
PRO381.
[0081] 23. PRO386
[0082] Mammalian cell membranes perform very important functions
relating to the structural integrity and activity of various cells
and tissues. Of particular interest in membrane physiology is the
study of transmembrane ion channels which act to directly control a
variety of physiological, pharmacological and cellular processes.
Numerous ion channels have been identified including calcium (Ca),
sodium (Na) and potassium (K) channels, each of which have been
analyzed in detail to determine their roles in physiological
processes in vertebrate and insect cells.
[0083] One type of cell membrane-associated ion channel, the sodium
channel, plays an extremely important role in a cell's ability to
maintain ionic homeostasis as well as transmit intracellular and
extracelmllar signals. Voltage-gated sodium channels in brain
neurons have been shown to be complexes of a pore-forming alpha
unit with smaller beta-1 and beta-2 subunits (Isom et al., Cell
83:433-442 (1995)). Given the obvious importance of sodium channels
in cellular homeostasis and other important physiological
functions, there is a significant interest in identifying novel
polypeptides having homology to sodium channel subunits. We herein
describe the identification and characterization of a novel
polypeptide having homology to the beta-2 subunit of the rat sodium
channel, designated herein as PRO386.
[0084] 24. PRO540
[0085] Lecitin-cholesterol acyltransferase ("LCAT"), also known as
phosphatidylcholine-sterol acyltransferase is a key enzyme in the
intravascular metabolism of high density lipoproteins, specifically
in the process of cholesterol metabolism. [see, for example,
Brousseau et al., J. Lipid Res., 38(12):2537-2547 (1997), Hill et
al., Biochem. J., 294:879-884 (1993), and Drayna et al., Nature 327
(6123):632-634 (1987)]. Given the medical importance of lipid
metabolism, efforts are currently being under taken to identify
new, native proteins which are involved in lipid transport. We
describe herein the identification of a novel polypeptide which has
homology to LCAT, designated herein as PRO540.
[0086] 25. PRO615
[0087] Synaptogyrin is a synaptic vesicle protein that is uniformly
distributed in the nervous system. The cDNA encoding synaptogyrin
has been cloned and sequenced and the sequence predicts a protein
with a molecular mass of 25,900 D with four membrane-spanning
domains. Synaptogyrin has been implicated in membrane traffic to
and from the plasma membrane. Stenius et al., J. Cell. Biol.
131(6-2):1801-1809 (1995). In addition, a novel isoform of
synaptogyrin called cellugyrin exhibits sequence identity with
synaptogyrin. In rat tissues, cellugyrin and synaptogyrins are
expressed in mirror image patterns. Cellugyrin is ubiquitously
present in all tissues tested with the lowest levels in brain
tissue, whereas synaptogyrin protein is only detectable in brain.
In rat tissues, cellugyrin and synaptogyrins are expressed in
mirror image patterns. The synaptic vesicle protein synaptogyrin
may be a specialized version of a ubiquitous protein, cellugyrin,
with the two proteins sharing structural similarity but differing
in localization. This finding supports the emerging concept of
synaptic vesicles as the simplified and specialized form of a
generic trafficking organelle. [Janz et al., J. Biol. Chem.
273(5):2851-2857 (1998)]. The sequence for cellugyrin derived from
the Norway rat, Ratus norvegicus has been deposited in the Genbank
database on Dec. 23, 1997, designated accession number AF039085.
See also, Janz et al., J. Biol. Chem. 273 (1998), in press.
[0088] Given the medical importance of synaptic transmission,
efforts are currently being under taken to identify new, native
proteins that may be part of a simplified and specialized generic
trafficking organelle in the form of synaptic vesicles. We describe
herein the identification of a novel polypeptide which has homology
to synaptogyrin, designated herein as PRO615.
[0089] 26. PRO618
[0090] Enteropeptidase is a key enzyme in the intestinal digestion
cascade specifically cleaves the acidic propeptide from trypsinogen
to yield active trypsin. This cleavage initiates a cascade of
proteolytic reactions leading to the activation of many pancreatic
zymogens.
[0091] See, for example, Matsushima et al., J. Biol. Chem.
269(31):19976-19982 (1994), Kitamoto et al., Proc. Nat. Acad. Sci.,
91(16):7588-7592 (1994). Enterokinase (enteropeptidase) is a
related to mammalian serine proteases involved in digestion,
coagulation, and fibrinolysis. La Valle et al., J Biol Chem.,
268(31):23311-23317 (1993).
[0092] Given the medical importance of digestive processes, efforts
are currently being under taken to identify new, native proteins
that may be involved in digestion, coagulation, and fibrinolysis.
We describe herein the identification of a novel polypeptide which
has homology to enteropeptidase, designated herein as PRO618.
[0093] 27. PRO719
[0094] Lipoprotein lipase is a key enzyme that mediates the
hydrolysis of triglycerides and phospholipids present in
circulating plasma lipoproteins (Dugi et al., J. Biol. Chem.
270:25396-25401 (1995)). Moreover, lipoprotein lipase has been
shown to mediate the uptake of lipoproteins into cells, wherein
cellular uptake of lipoproteins is initiated by binding of
lipoprotein lipase to cell surface proteoglycans and to the low
density lipoprotein (LDL) receptor-related protein (Krapp et al.,
J. Lipid Res. 36:2362-2373 (1995)). Thus, it is clear that
lipoprotein lipase plays an extremely important role in lipoprotein
and cholesterol metabolism. There is, therefore, substantial
interest in identifying novel polypeptides that share sequence
homology and/or biological activity with lipoprotein lipase. We
herein describe the identification and characterization of a novel
polypeptide having sequence homology to lipoprotein lipase H,
designated heein as PRO719.
[0095] 28. PRO724
[0096] The low density lipoprotein (LDL) receptor is a
membrane-bound protein that plays a key role in cholesterol
homeostasis, mediating cellular uptake of lipoprotein particles by
high affinity binding to its ligands, apolipoprotein (apo) B-100
and apoE. The ligand-binding domain of the LDL receptor contains 7
cysteine-rich repeats of approximately 40 amino acids, wherein each
repeat contains 6 cysteines, which form 3 intra-repeat disulfide
bonds. These unique structural features provide the LDL receptor
with its ability to specifically interact with apo B-100 and apoE,
thereby allowing for transport of these lipoproteinparticles across
cellular membranes and metabolism of their components. Soluble
fragments containg the extracellular domain of the LDL receptor
have been shown to retain the ability to interact with its specific
lipoprotein ligands (Simmons et al., J. Biol. Chem. 272:25531-25536
(1997)). Thus, it is clear that the LDL receptor is intimately
involved in important physiological activities related to
cholesterol metabolism. As such, there is substantial interest in
identifying novel LDL receptor homolog proteins. We herein describe
the identification and characterization of a novel polypeptide
having homology to the human LDL receptor protein, designated
herein as PRO724.
[0097] 29. PRO772
[0098] Expression of the human gene A4 is enriched in the colonic
epithelium and is transcriptionally activated on differentiation of
colonic epithelial cells in vitro (Oliva et al., Arch. Biochem.
Biophys. 302:183-192 (1993) and Oliva et al., Am. J. Physiol.
272:C957-C965 (1997)). A4 cDNA contains an open reading frame that
predicts a polypeptide of approximately 17 kilodaltons in size.
Hydropathy analysis of the A4 protein revealed four putative
membrane-spanning alpha-helices. Immunocytochemical studies of
cells expressing A4 protein indicated that expression is localized
to the endoplasmic reticulum. The four membrane-spanning domains
and the biophysical characteristics of the A4 protein suggest that
it belongs to a family of integral membrane proteins called
proteolipids, some of which multimerize to form ion channels. In
fact, preliminary evidence has suggested that A4 may itself
multimerize and take on the properties of an ion channel (Oliva et
al., Am. J. Physiol. 272:C957-C965 (1997)). Given the importance of
ion channels in maintaining cellular homeostasis, there is a
significant interest in identifying novel polypeptides having
homology to known and putative ion channels. We herein describe the
identification and characterization of a novel polypeptide having
homology to the putative ion channel protein, A4, designated herein
as PRO772.
[0099] 30. PRO852
[0100] 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.
[0101] 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 PRO852 polypeptides.
[0102] 31. PRO853
[0103] 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,
hypertension and may be associated with the development of
malignancies. The levels of antioxidant enzymes, such as
reductases, which catalyze the conversion of reactive oxygen
species to water have been shown to be low in cancer cells. In
particular, malignant prostate epithelium may have lowered
expression of such antioxidant enzymes [Baker et., Prostate
32(4):229-233 (1997)]. In this regard, reductases, 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 Engman et al., Anticancer Res. (Greece), 17:4599-4605
(1997), Kelsey, et al., Br. J. Cancer, 76(7):852-4 (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 a novel prostate specific polypeptide which has
sequence similarity to reductase, designated herein as PRO853.
[0104] 32. PRO860
[0105] Neurofascin is a member of the L1 subgroup of the cellular
adhesion molecule ("CAM") family of nervous system adhesion
molecules and is involved in cellular aggregation. Cell-cell
recognition and patterning of cell contacts have a critical role in
mediating reversible assembly of a wide variety or transcellular
complexes in the nervous system. Cell interactions may be regulated
through modulation of ankyrin binding to neurofascin. See, for
example, Tuvia et al., Proc. Nat Acad. Sci., 94(24) 12957-12962
(1997). Neurofascin has been described as a member of the L1
subgroup of the immunoglobulin superfamily implicated in neurite
extension during embryonic development for which numerous isoforms
have been detected at various stages of development. See also
Hassel et al., J. Biol. Chem., 272(45) 28742-28749 (1997), Grumet.,
Cell. Tissue Res. 290(2) 423-428 (1997), Garver et al., J. Cell.
Biol., 137:703-714 (1997), and Lambert et al., J. Neurosci.,
17:7025-7-36 (1997).
[0106] Given the physiological importance of cellular adhesion
molecules and development of the nervous system in vivo, efforts
are currently being under taken to identify new, native proteins
which are involved in regulation of cellular interactions in the
nervous system. We describe herein the identification and
characterization of a novel polypeptide which has sequence
similarity to neurofascin, designated herein as PRO860.
[0107] 33. PRO846
[0108] The CMRF35 monoclonal antibody was used to identify a cell
membrane antigen, designated CMRF35, which is present on the
surface of monocytes, neutrophils, a proportion of peripheral blood
T and B lymphocytes and lymphocytic cell lines. The CMRF35 cDNA
encodes a novel integral membrane glycoprotein member of the
immunoglobulin (Ig) gene superfamily. The molecule comprises (a) a
single extracellular Ig variable domain remarkably similar to the
Fc receptor for polymeric IgA and IgM, (b) a membrane-proximal
domain containing a high proportion of proline, serine and
threonine residues that was predicted to be heavily O-glycosylated,
(c) an unusual transmembrane anchor that contained a glutamic acid
and a proline residue and (d) a short cytoplasmic tail. Transcripts
encoding the CMRF35 protein have been detected in early monocytic
cell lines, in peripheral blood T cells and in some B
lymphoblastoid cell lines, confirming the results of
immunocytological staining. Jackson et al., Eur. J. Immunol.
22(5):1157-1163 (1992). CMRF-35 molecules are differentially
expressed in hematopoietic cells, and the expression of the antigen
was shown to be markedly influenced by stimulation with mitogens
and cytokines. See, for example, Clark et al., Exp. Hematol.
25(8):759 (1997), Daish et al., Immunol. 79(1):55-63 (1993), and
Clark et al., Tissue Antigens 48:461 (1996).
[0109] Given the physiological importance of the immune system and
antigens associated with various immune system cells, efforts are
currently being under taken to identify new, native proteins which
are expressed on various cells of the immune system. We describe
herein the identification of a novel polypeptide which has sequence
similarity to CMRF35, designated herein as PRO846.
[0110] 34. PRO862
[0111] Lysozyme is a protein which is widely distributed in several
human tissues and secretions including milk, tears and saliva. It
has been demonstrated to hydrolyze linkages between
N-acetylglucosamnines. It has been demonstrated to be an inhibitor
of chemotaxis and of the production of toxic oxygen free radicals
and may also have some role in the calcification process. As such,
there is substantial interest in identifying novel polypeptides
having homology to lysozyme. We describe herein the identification
of a novel polypeptide which has sequence similarity to
lysozyme.
[0112] 35. PRO864
[0113] Wnt-4 is a secreted glycoprotein which correlates with, and
is required for, kidney tubulogenesis. Mice lacking Wnt-4 activity
fail to form pretubular cell aggregates; however, other aspects of
mesenchymal and ureteric development are unaffected. Thus, Wnt-4
appears to act as an autoinducer of the mesenchyme to epithelial
transition that underlies nephron development. Stark et al.,
Nature; 372(6507):679-683 (1994). In addition, members of the Wnt
gene family code for cysteine-rich, secreted proteins, which are
differentially expressed in the developing brain and possibly act
as intercellular signaling molecules. A Wnt gene, e.g., Wnt-1 is
known to be essential for specification of the midbrain cell fate.
Yoshioka et al., Biochem. Biophys. Res. Commun. 203(3):1581-1588
(1994). Several member of the Wnt family of secreted factors are
strongly implicated as regulators of mammary cellular growth and
differentiation. Shimizu et al., Cell Growth Differ. 8(12)
1349-1358. Wnt-4 is normally expressed in early pregnancy. Wnt-4
may therefore be a local signal driving epithelial branching in
pregnancy. Edwards P A, Biochem Soc Symp. 63:21-34 (1998). See
also, Lipschutz J H, Am. J. Kidney Dis. 31(3):383-397, (1998). We
describe herein the identification and characterizaton of a novel
polypeptide which has sequence similarity to Wnt-4, designated
herein as PRO864.
[0114] 36. PRO792
[0115] At least two cell-derived signals have been shown to be
necessary for the induction of immunoglobulin isotype switching in
B-cells. The first signal is given by either of the soluble
lymphokines, interleukin (IL)-4 or IL-13, which induce germline
epsilon transcript expression, but this alone is insufficient to
trigger secretion of immunoglobulin E (IgE). The second signal is
provided by a physical interaction between B-cells and activated
T-cells, basophils and mast cells, and it has been shown that the
CD40/CD40 ligand pairing is crucial for mediating IgE synthesis.
Additionally, amongst the numerous pairs of surface adhesion
molecules that are involved in IgE synthesis, the CD23/CD21 pair
appears to play a key role in the generation of IgE. CD23 is a
protein that is positively and negatively regulated by factors
which increase or decrease IgE production, respectively. Antibodies
to CD23 have been shown to inhibit IL-4-induced human IgE
production in vitro and to inhibit antigen-specific IgE responses
in a rat model, in an isotype selective manner (Bonnefoy et al.,
Eur. Respir. J. Suppl. 22:63S-66S (1996)). CD23 interacts with CD21
on B-cells, preferentially driving IgE production. Given that the
CD23 protein plays an extremely important role in the induction of
a mammalian IgE response, there is significant interest in
identifying novel polypeptides having homology to CD23. We herein
describe the identification and characterization of a novel
polypeptide having homology to CD23, designated herein as
PRO792.
[0116] 37. PRO866
[0117] Mindin and spondin proteins are secreted proteins that are
structurally related to one another and which have been identified
in a variety of organisms. For example, Higashijima et al., Dev
Biol. 192:211-227 (1997) have reported the identification of
spondin and mindin expression in floor plate cells in the zebrafish
embryonic axis, thereby suggesting that mindin and spondin
prtoteins play important roles in embryonic development. This same
group has reported that mindin and spondin proteins function as
extracellular matrix proteins that have a high affinity for the
basal lamina. (Id.). It has been reported that F-spondin is a
secreted protein that promotes neural adhesion and neurite
extension (Klar et al., Cell 69:95-110 (1992) and that M-spondin is
an extracellular matrix protein that localizes to muscle attachment
sites in Drosophila (Umemiya et al., Dev. Biol. 186:165-176
(1997)). Thus, there is significant inteest in identifying novel
polypeptides having homology to the mindin and spondin proteins. We
herein describe the identification and characterization of a novel
polypeptide having homology to mindin2 and mindin1, designated
herein as PRO866.
[0118] 38. PRO871
[0119] Cyclophilus are a family of proteins that bind to
cyclosporin A and possess peptidyl-prolyl cis-trans isomerase
activity (Sherry et al., Proc. Natl. Acad. Sci. USA 95:1758-1763
(1998)). In addition, cyclophilins are secreted by activated cells
and act in a cytokine-like manner, presumably via signaling through
a cell surface cyclophilin receptor. Host cell-derived cyclophilin
A has been shown to be incorporated into HIV-1 virions and its
incorporation has been shown to be essential for viral infectivity.
Thus, one or more the cyclophilins may be directly associated with
HIV-1 infectivity. Given the obvious importance of the cyclophilin
proteins, there is substantial interest in identifying novel
polypeptides which have sequence homology to one or more of the
cyclophilin proteins. We herein describe the identification and
characterization of a novel polypeptide having homology to
cyclophilin-like protein CyP-60, designated herein as PRO871.
[0120] 39. PRO873
[0121] Enzymatic proteins play important roles in the chemical
reactions involved in the digestion of foods, the biosynthesis of
macromolecules, the controlled release and utilization of chemical
energy, and other processes necessary to sustain life. Enzymes have
also been shown to play important roles in combating various
diseases and disorders. For example, liver carboxylesterases have
been reported to assist in sensitizing human tumor cells to the
cancer prodrugs. Danks et al., report that stable expression of the
cDNA encoding a carboxylesterase in Rh30 human rhabdomyosarcoma
cells increased the sensitivity of the cells to the CPT-11 cancer
prodrug 8.1-fold. Cancer Res. (1998) 58(l):20-22. The authors
propose that this prodrug/enzyme combination could be exploited
therapeutically in a manner analogous to approaches currently under
investigation with the combinations of ganciclovir/herpes simplex
virus thymidine kinase and 5-fluorocytosine/cytosine deaminase. van
Pelt et al. demonstrated that a 55 kD human liver carboxylesterase
inhibits the invasion of Plasmodium falciparum malaria sporozoites
into primary human hepatocytes in culture. J Hepatol (1997)
27(4):688-698.
[0122] Carboxylesterases have also been found to be of importance
in the detoxification of drugs, pesticides and other xenobiotics.
Purified human liver carboxylesterases have been shown to be
involved in the metabolism of various drugs including cocaine and
heroin. Prindel et al. describe the purification and cloning of a
broad substrate specificity human liver carboxylesterase which
catalyzes the hydrolysis of cocaine and heroin and which may play
an important role in the degradation of these drugs in human
tissues. J. Biol. Chem. (1997) 6:272(23):14769-14775. Brzenzinski
et al. describe a spectrophotometric competitive inhibition assay
used to identify drug or environmental esters that are metabolized
by carboxylesterases. Drug Metab Dispos (1997) 25(9):
1089-1096.
[0123] In light of the important physiological roles played by
carboxylesterases, efforts are being undertaken by both industry
and academia to identify new, native carboxylesterase homologs. We
herein describe the identification and characterization of a novel
polypeptide having homology to carboxylesterase, designated herein
as PRO873.
[0124] 40. PRO940
[0125] CD33 is a cell-surface protein that is a member of the
sialoadhesin family of proteins that are capable of mediating
sialic-acid dependent binding with distinct specificities for both
the type of sialic acid and its linkage to subterminal sugars. CD33
is specifically expressed in early myeloid and some monocyte cell
lineages and has been shown to be strongly associated with various
myeloid tumors including, for example, acute non-lymphocytic
leukemia (ANLL). As such, CD33 has been suggested as a potential
target for the treatment of cancers associated with high level
expression of the protein. There is, therefore, significant
interest in the identification of novel polypeptides having
homology to CD33. In fact, one CD33 homolog (designated CD33L) has
already been identified and described (see Takei et al., Cytogenet.
Cell Genet. 78:295-300 (1997)). We herein describe the
identification of another novel polypeptide having homology to
CD33, designated herein as PRO940. The novel polypeptide described
herein also exhibits significant homology to the human OB binding
proteins designated HSU71382.sub.--1 and HSU71383.sub.--1 in the
Dayhoff database (version 35.45 SwissProt 35).
[0126] 41. PRO941
[0127] Cadherins are a large family of transmembrane proteins.
Cadherins comprise a family of calcium-dependent glycoproteins that
function in mediating cell-cell adhesion in virally all solid
tissues of multicellular organisms. At least cadherins 1-13 as well
as types B, E, EP, M, N, P and R have been identified and
characterized. Among the functions cadherins are known for, with
some exceptions, are that cadherins participate in cell aggregation
and are associated with cell-cell adhesion sites. Recently, it has
been reported that while all cadherins share multiple repeats of a
cadherin specific motif believed to correspond to folding of
extracellular domains, members of the cadherin superfamily have
divergent structures and, possibly, functions. In particular it has
been reported that members of the cadherin superfamily are involved
in signal transduction. See, Suzuki, J. Cell Biochem.,
61(4):531-542 (1996). Cadherins are further described in Tanihara
et al., J. Cell Sci., 107(6):1697-1704 (1994), Aberle et al., J.
Cell Biochem., 61(4):514-523 (1996) and Tanihara et al., Cell
Adhes. Commun., 2(1):15-26 (1994). We herein describe the
identification and characterization of a novel polypeptide having
homology to a cadherin protein, designated herein as PRO941.
[0128] 42. PRO944
[0129] Clostridium perfringens enterotoxin (CPE) is considered to
be the virulence factor responsible for causing the symptoms of C.
perfringens type A food poisoning and may also be involved in other
human and veterinary illnesses (McClane, Toxicon. 34:1335-1343
(1996)). CPE carries out its adverse cellular functions by binding
to an approximately 50 kD cell surface receptor protein designated
the Clostridium perfringens enterotoxin receptor (CPE-R) to form an
approximately 90,000 kD complex on the surface of the cell. cDNAs
encoding the CPE-R protein have been identified characterized in
both human and mouse (Katahira et al., J. Cell Biol. 136:1239-1247
(1997) and Katahira et al., J. Biol. Chem. 272:26652-26658 (1997)).
Since the CPE toxin has been reported to cause a variety of
illnesses in mammalian hosts and those illnesses are initiated by
binding of the CPE toxin to the CPE-R, there is significant
interest in identifying novel CPE-R homologs. We herein describe
the identification and characterization of a novel polypeptide
having homology to the CPE-R, designated herein as PRO944.
[0130] 43. PRO983
[0131] Membrane-bound proteins include not only cell-surface
membrane-bound proteins, but also proteins that are found on the
surface of intracellular vesicles. These vesicles are involved in
exocytosis, which is the fusion of secretory vesicles with the
cellular plasma membrane, and have two main functions. One is the
discharge of the vesicle contents into the extracellular space, and
the second is the incorporation of new proteins and lipids into the
plasma membrane itself. Exocytosis can be either constitutive or
regulated. All eukaryotic cells exhibit constitutive exocytosis,
which is marked by the immediate fusion of the secretory vesicle
after formation. In contrast, regulated exocytosis results in the
accumulation of the secretory vesicles that fuse with the plasma
membrane upon receipt of an appropriate signal by
vesicle-associated membrane proteins. Usually, this signal is an
increase in the cytosolic free Ca.sup.2+ concentration. However,
regulated exocytosis that is independent of Ca.sup.2+ has been
reported (see, e.g. Fujita-Yoshigaki et al. J. Biol. Chem. (1996)
31:271(22):13130-13134). Regulated exocytosis is crucial to many
specialized cells, including neurons (neurotransmitter release from
synaptic vesicles), adrenal chromaffin cells (adrenaline
secretion), pancreatic acinar cells (digestive enzyme secretion),
pancreatic .beta.-cells (insulin secretion), mast cells (histamine
secretion), mammary cells (milk protein secretion), sperm (enzyme
secretion), egg cells (creation of fertiization envelope) and
adipocytes (insertion of glucose transporters into the plasma
membrane).
[0132] Disorders involving exocytosis are known. For example,
inflammatory mediator release from mast cells leads to a variety of
disorders, including asthma. Similarly, Chediak-Higashi Syndrome
(CHS) is a rare autosomal recessive disease in which neutrophils,
monocytes and lymphocytes contain giant cytoplasmic granules.
Accordingly, the proteins involved in exocytosis are of paramount
interest and efforts are being undertaken by both industry and
academia to identify new, vesicle-associated proteins. For example,
Skehel et al. identified a 33-kilodalton membrane protein in
Aplysia, termed VAP-33, which is required for the exocytosis of
neurotransmitter. Science (1995) 15:269(5230):1580-1583, and
Neuropharmacology (1995) 34(11):1379-1385. Many efforts are focused
on the screening of mammalian recombinant DNA libraries to identiiy
the coding sequences for novel vesicle- associated membrane
proteins. It is an object of the invention to provide proteins
having homology to the vesicle associated protein, VAP-33,
designated herein as PRO983.
[0133] 44. PRO1057
[0134] 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 mammala protease
enzymes play important roles inmany 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.
[0135] 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 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 PRO1057 polypeptides.
[0136] 45. PRO1071
[0137] Thrombospondin-1 is a trimeric high molecular weight
glycoprotein that is released from platelet alpha-granules in
response to thrombin stimulation and that is also a transient
component of the extracellular matrix in developing and repairing
tissues (Adams, Int. J. Biochem. Cell Biol. 29:861-865 (1997) and
Qian et al., Proc. Soc. Exp. Biol. Med. 212:199-207 (1996)). A
variety of factors regulate thrombospondin expression and the
protein is degraded by both extracellular and intracellular routes.
Thrombospondin-1 functions as acell adhesion molecule and also
modulates cell movement, cell proliferation, neurite outgrowth and
angiogenesis. As such, there is substantial interest in identifying
novel polypeptides having homology to thrombospondin. We herein
describe the identification and characterization of a novel
polypeptide having homology to thrombospondin, designated herein as
PRO1071.
[0138] 46. PRO1072
[0139] 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,
hypertension and may be associated with the development of
malignancies. The levels of antioxidant enzymes, such as
reductases, which catalyze the conversion of reactive oxygen
species to water have been shown to be low in cancer cells. In
particular, malignant prostate epithelium may have lowered
expression of such antioxidant enzymes [Baker et al., Prostate
32(4):229-233 (1997)]. In this regard, reductases, 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 Engman et al., Anticancer Res. (Greece), 17:4599-4605
(1997), Kelsey, et al., Br. J. Cancer, 76(7):852-854 (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 a novel polypeptide which has sequence similarity
to reductase enzymes, desiignated herein as PRO1072.
[0140] 47. PRO1075
[0141] 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.
[0142] 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 identily the coding sequences for novel protein
disulfide isomerase homologs. 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 a novel polypeptide having homology
to protein disulfide isomerase, designated herein as PRO1075.
[0143] 48. PRO181
[0144] In Drosophila, the dorsal-ventral polarity of the egg
chamber depends on the localization of the oocyte nucleus and the
gurken RNA to the dorsal-anterior corner of the oocyte. Gurken
protein presumably acts as a ligand for the drosophila EGF receptor
(torpedo/DER) expressed in the somatic follicle cells surrounding
the oocyte. Cornichon is a gene required in the germline for
dorsal-ventral signaling (Roth et al., Cell 81:967-978 (1995)).
Cornichon, gurken and torpedo also function in an earlier signaling
event that establishes posterior follicle cell fates and specifies
the anterior-posterior polarity of the egg chamber. Mutations in
any or all of these genes prevent the formation of a correctly
polarized microtubule cytoskeleton required for proper localization
of the anterior and posterior determinants bicoid and oskar and for
the asymmetric positioning of the oocyte nucleus. Thus, it is clear
that the cornichon gene product plays an important role in early
development. We herein describe the identification and
characterization of a novel polypeptide having homology to the
cornichon protein, designated herein as PRO181.
[0145] 49. PRO195
[0146] Efforts arre currently being undertaken to identify and
characterize novel transmembrane proteins. We herein describe the
identification and characterization of a novel transmembrane
polypeptide, designated herein as PRO195.
[0147] 50. PRO865
[0148] Efforts arre currently being undertaken to identify and
characterize novel secreted proteins. We herein describe the
identification and characterization of a novel secreted
polypeptide, designated herein as PRO865.
[0149] 51. PRO827
[0150] VLA-2 is an cell-surface integrin protein that has been
identified and characterized in a number of mammalian organisms,
including both mouse and human. VLA-2 has been shown to be a
receptor on the surface of cells for echovirus-1 (EV-1) which
mediates infection of VLA-2-expressing cells by EV-1 (Zhang et al.,
Virology 235(2):293-301 (1997) and Bergelson et al., Science
255:1718-1720 (1992)). VLA-2 has also been shown to mediate the
interaction of collagen with endothelium during in vitro vascular
tube formation (Jackson et al., Cell Biol. Int. 18(9):859-867
(1994)). Various other integrin proteins that share various degrees
of amino acid sequence homology with VLA-2 have been identified and
characterized in a variety of mammalian organism. These integrins
have been reported to play important roles in a variety of
different physiological functions. Therefore, there is significant
interest in identifying novel polypeptides having homology to one
or more of the integrin proteins. We herein describe the
identification and characterization of a novel polypeptide having
homology to VLA-2 integrin protein, designated herein as
PRO827.
[0151] 52. PRO1114
[0152] Many important cytokine proteins have been identified and
characterized and shown to signal through specific cell surface
receptor complexes. For example, the class II cytokine receptor
family (CRF2) includes the interferon receptors, the interleukin-10
receptor and the tissue factor CRFB4 (Spencer et al., J. Exp. Med.
187:571-578 (1998) and Kotenko et al., EMBO J. 16:5894-5903
(1997)). Thus, the multitude of biological activities exhibited by
the various cytokine proteins is absolutely dependent upon the
presence of cytokine receptor proteins on the surface of target
cells. There is, therefore, a significant interest in identifying
and characterizing novel polypeptides having homology to one or
more of the cytokine receptor family. We herein describe the
identification and characterization of a novel polypeptide having
homology to cytokine receptor family-4 proteins, designated herein
as PRO1117.
[0153] Interferons (IFNs) encompass a large family of secreted
proteins occurring in vertebrates. Although they were originally
named for their antiviral activity, growing evidence supports a
critical role for IFNs in cell growth and differentiation
(Jaramillo et al., Cancer Investigation 13(3):327-338 (1995)). IFNs
belong to a class of negative growth factors having the ability to
inhibit the growth of a wide variety of cells with both normal and
transformed phenotypes. IFN therapy has been shown to be beneficial
in the treatnent of human malignancies such as Karposi's sarcoma,
chronic myelogenous leukemia, non-Hodgkin's lymphoma, and hairy
cell leukemia as well as in the treatment of infectious diseases
such as hepatitis B (Gamliel et al., Scanning Microscopy
2(1):485-492 (1988), Einhorn et al., Med. Oncol. & Tumor
Pharmacother. 10:25-29 (1993), Ringenberg et al., Missouri Medicine
85(1):21-26 (1988), Saracco et al., Journal of Gastroenterology and
Hepatology 10:668-673 (1995), Gonzalez-Mateos et al.,
Hepato-Gastroenterology 42:893-899 (1995) and Malaguarnera et al.,
Pharmacotherapy 17(5):998-1005 (1997)).
[0154] Interferons can be classified into two major groups based
upon their primary sequence. Type I interferons, IFN-.alpha. and
IFN-.beta., are encoded by a superfamlly of intronless genes
consisting of the IFN-.alpha. gene family and a single IFN-.beta.
gene that are thought to have arisen from a common ancestral gene.
Type I interferons may be produced by most cell types. Type II IFN,
or IFN-.gamma., is restricted to lymphocytes (T cells and natural
killer cells) and is stimulated by nonspecific T cell activators or
specific antigens in vivo.
[0155] Although both type I and type II IFNs produce similar
antiviral and antiproliferative effects, they act on distinct cell
surface receptors, wherein the binding is generally species
specific (Langer et al., Immunol. Today 9:393-400 (1988)). Both
IFN-.alpha. and IFN-.beta. bind competitively to the same high
affinity type I receptor, whereas IFN-.gamma. binds to a distinct
type II receptor. The presence and number of IFN receptors on the
surface of a cell does not generally reflect the sensitivity of the
cell to IFN, although it is clear that the effects of the IFN
protein is mediated through binding to a cell surface interferon
receptor. As such, the identification and characterization of novel
interferon receptor proteins is of extreme interest.
[0156] We herein describe the identification and characterization
of novel interferon receptor polypeptides, designated herein as
"PRO1114 interferon receptor" polypeptides. Thus, the PRO1114
polypeptides of the present invention represents a novel cell
surface interferon receptor.
[0157] 53. PRO237
[0158] Carbonic anhydrase is an enzymatic protem that which aids
carbon dioxide transport and release in the mammalian blood system
by catalyzing the synthesis (and the dehydration) of carbonic acid
from (and to) carbon dioxide and water. Thus, the actions of
carbonic anhydrase are essential for a variety of important
physiological reactions in the mammal. As such, there is
significant interest in the identification and characterization of
novel polypeptides having homology to carbonic anhydrase. We herein
describe the identification and characterization of a novel
polypeptide having homology to carbonic anhydrase, designated
herein as PRO237.
[0159] 54. PRO541
[0160] Numerous trypsin inhibitory proteins have been identified
and characterized (see, e.g., Yamakawa et al., Biochim. Biophys.
Acta 1395:202-208 (1998) and Mizuki et al., Mammalian Genome
3:274-280 (1992)). Trypsin inhibitor proteins play important roles
in a variety of different physiological and biological pathways and
are specifically involved in such processes as the regulation of
protein degradation, digestion, and the like. Given the important
roles played by such enzymatic proteins, there is significant
interest in identifying and characterizing novel polypeptides
having homology to known trypsin inhibitor proteins. We herein
describe the identification and characterization of a novel
polypeptide having homology to a trypsin inhibitor protein,
designated herein as PRO541.
[0161] 55. PRO273
[0162] Leukocytes include monocytes, macrophages, basophils, and
eosinophils and play an important role in the immune response.
These cells are important in the mechanisms initiated by T and/or B
lymphocytes and secrete a range of cytokines which recruit and
activate other inflammatory cells and contribute to tissue
destruction.
[0163] Thus, investigation of the regulatory processes by which
leukocytes move to their appropriate destination and interact with
other cells is critical. Currently, leukocytes are thought to move
from the blood to injured or inflamed tissues by robing along the
endothelial cells of the blood vessel wall. This movement is
mediated by transient interactions between selectins and their
ligands. Next, the leukocyte must move through the vessel wall and
into the tissues. This diapedesis and extravasation step involves
cell activation which promotes a more stable leukocyte-endothelial
cell interaction, again mediated by integrins and their
ligands.
[0164] Chemokines are a large family of structurally related
polypeptide cytokines. These molecules stimulate leukocyte movement
and may explain leukocyte traffcking in different inflammatory
situations. Chemokines mediate the expression of particular
adhesion molecules on endothelial cells, and they produce
chemoattractants which activate specific cell tppes. In addition,
the chemokines stimulate proliferation and regulate activation of
specific cell types. In both of these activities, chemokines
demonstrate a high degree of target cell specificity.
[0165] The chemokine family is divided into two subfamilies based
on whether two amino terminal cysteine residues are immediately
adjacent (C-C) or separated by one amino acid (C-X-C). Chemolines
of the C-X-C family generally activate neutrophils and fibroblasts
while the C-C chemokines act on a more diverse group of target
cells including monocytes/macrophages, basophils, eosinophils and T
lymphocytes. The known chemokines of both subfamilies are
synthesized by many diverse cell types as reviewed in Thomson A.
(1994) The Cytokine Handbook, 2 d Ed. Academic Press, N.Y.
Chemokines are also reviewed in Schall T J (1994) Chemotactic
Cytokines: Targets for Therapeutic Development. International
Business Communications, Southborough Mass. pp 180-270; and in Paul
W E (1993) Fundamental Immunology, 3rd Ed. Raven Press, N.Y. pp
822-826.
[0166] Known chemokines of the C-X-C subfamily include macrophage
inflammatory proteins alpha and beta (MIP-1 and MIP-2 ),
interleukin-8 (IL-8), and growth regulated protein (GRO-alpha axid
beta).
[0167] MIP-2 was first identified as a 6 kDa heparin binding
protein secreted by the mouse macrophage cell line RAW 264.7 upon
stimulation with lipopolysaccharide (LPS). MIP-2 is a member of the
C-X-C (or CXC) subfamily of chemokines. Mouse MIP-2 is chemotactic
for human neutrophils and induces local neutrophil infiltration
when injected into the foot pads of mice. Rat MIP-2 shows 86% amino
acid homology to the mouse MIP-2 and is chemotactic for rat
neutrophils but does not stimulate migration of rat alveolar
macrophages or human peripheral blood eosinophils or lymphocytes.
In addition, the rat MIP-2 has been shown to stimulate
proliferation of rat alveolar epithelial cells but not
fibroblasts.
[0168] Current techniques for diagnosis of abnormalities in
inflamed or diseased issues mainly rely on observation of clinical
symptoms or serological analyses of body tissues or fluids for
hormones, polypeptides or various metabolites. Problems exist with
these diagnostic techniques. First, patients may not manifest
clinical symptoms at early stages of disease. Second, serological
tests do not always differentiate between invasive diseases and
genetic syndromes. Thus, the identification of expressed chemokines
is important to the development of new diagnostic techniques,
effective therapies, and to aid in the understanding of molecular
pathogenesis.
[0169] To date, chemokines have been implicated in at least the
following conditions: psoriasis, inflammatory bowel disease, renal
disease, arthritis, immune-mediated alopecia, stroke, encephalitis,
MS, hepatitis, and others. In addition, non-ELR-containing
chemokines have been implicated in the inhibition of angiogenesis,
thus indicating that these chemokines have a rule in tumor
vascularization and tumorigenesis.
[0170] Therefore it is the object of this invention to identify
polypeptides and nucleic acids encoding the same which have
sequence identity and similarity with cytokine-induced neutrophil
chemoattractants, MIP-1, MIP-2, and other related proteins. The
efforts of this object are provided herein.
[0171] 56. PRO701
[0172] Beta neurexins and neuroligins are plasma membrane proteins
that are displayed on the neuronal cell surface. Neuroligin 1 is
enriched in synaptic plasma membranes and acts as a splice
site-specific ligand for beta neurexins as described in Ichtchenko,
et al., Cell, 81(3):435-443 (1995). The extracellular sequence of
neuroligin 1 is composed of a catalytically inactive esterase
domain homologous to acetylcholinesterase. Neuroligin 2 and 3 are
similar in structure and sequence to neuroligin 1. All neuroligins
contain an N-terminal hydrophobic sequence with the characteristics
of a cleaved signal peptide followed by a large esterase homology
domain, a highly conserved single transmembrane region, and a short
cytoplasmic domain. The three neuroligins are alternatively spliced
at the same position and are expressed at high levels only in the
brain. Tight binding of the three neuroligins to beta neurexins is
observed only for beta neurexins lacking an insert in splice site
4. Thus, neuroligins constitute a multigene family of
brain-specific proteins with distinct isoforms that may have
overlapping functions in mediating recognition processes between
neurons, see Ichtchenko, et al., J. Biol. Chem., 271(5):2676-2682
(1996). Moreover, neurexins and neuroligins have been reported as
functioning as adhesion molecules in a Ca.sup.2+ dependent reaction
that is regulated by alternative splicing of beta neurexins, i.e.,
see Nguyen and Sudhof, J. Biol. Chem., 272(41):26032-26039 (1997).
Given the foregoing, membrane bound proteins are of interest. More
generally, membrane-bound proteins and receptors can play an
important role 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. 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.
[0173] 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 interaction. The
membrane-bound proteins can also be employed for screening of
potential peptide or small molecule inhibitors of the relevant
receptor/ligand interaction.
[0174] Efforts are being undertaken by both industry and academia
to identify new, native membrane-bound receptor proteins,
particularly those having sequence identity and/or similarity with
neuroligins 1, 2 and 3. 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)]. The results of such
efforts are provided herein.
[0175] 57. PRO704
[0176] VIP36 is localized to the Golgi apparatus and the cell
surface, and belongs to a family of legume lectin homologues in the
animnal secretory pathway that might be involved in the trafficking
of glycoproteins, glycolipids, or both. It is further believed that
VIP36 binds to sugar residues of glycosphingolipids and/or
gycosylphosphatidyl-inositol anchors and might provide a link
between the extracellular/luminal face of glycolipid rafts and the
cytoplasmic protein segregation machinery. Further regarding VIP36,
it is believed that there is a signal at its C-terminus that
matches an internalization consensus sequence which confers its
ability to cycle between the plasma membrane and Golgi. See,
Fiedler, et al, EMBO J., 13(7):1729-1740 (1994); Fiedler and
Simons, J. Cell Sci., 109(1):271-276 (1996); Itin, et al., MBO J.,
14(10):2250-2256 (1995). It is believed that VIP36 is either the
same as or very closely related to the human GP36b protein. VIP36
and/or GP36b are of interest.
[0177] More generally, vesicular, cytoplasmic, 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.
[0178] Secreted proteins have various industrial applications,
including use as pharmaceuticals, diagnostics, biosensors and
bioreactors. In fact, 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-bound
proteins, also have potential as therapeutic or diagnostic agents.
Receptor immunoadhesins, for instance, can be employed as
therapeutic agents to block receptor-ligand interaction.
Membrane-bound proteins can also be employed for screening
ofpotential peptide or small molecule inhibitors of the relevant
receptor/ligand interaction. 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. 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.
[0179] Efforts are being undertaken by both industry and academia
to identify new, native vesicular, cytoplasmic, secreted and
membrane-bound receptor proteins, particularly those having
sequence identity and/or similarity with VIP36. 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. Examnples 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)].
[0180] 58. PRO706
[0181] Acid phophatase proteins are secreted proteins which
dephophorylate terminal phosphate groups under acidic pH
conditions. Acid phophatases contain a RHGXRXP amino acid sequence,
which is predicted to be mechanistically significant. Acid
phosphatases may have important functions in the diagnosis and
treatment of human diseases. For example, prostatic acid
phosphatase is a secreted protein uniquely expressed in prostatic
tissue and prostate cancer. The level of prostatic acid phosphatase
is a potential prognostic factor for local and biochemical control
in prostate cancer patients treated with radiotherapy, as described
in Lankford et al., Int. J. Radiat. Oncol. Biol. Phys. 38(2):
327-333 (1997). Research suggests that a cellular immune response
to prostatic acid phosphatase may mediate destructive autoimmune
prostatitis, and that xenogeneic forms of prostatic acid
phosphatase may prove useful for immunotherapy of prostate cancer.
See Fong et al., J. Immunol. 169(7): 3113-3117(1997). Seminal
prostatic acid phosphatase levels correlate significantly with very
low sperm levels (oligospermia) in individuals over 35, see Singh
et al., Singapore Med. J. 37(6): 598-599 (1996). Thus, prostatic
acid phosphatase has been implicated in a variety of human
diseases, and may have an important function in diagnosis and
therapy of these diseases. A series of aminobenzylphosphatic acid
compounds are highly potent inhibitors of prostatic acid
phosphatase, as described in Beers et al., Bioorg. Med. Chem.
4(10): 1693-1701 (1996).
[0182] More generally, extracellular proteins play an important
role 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.
[0183] Secreted proteins have various industrial applications,
including 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, particularly those having sequence identity with
prostate acid phosphatase precursor and lysosomal acid phosphatase
precursor and in some cases, those having identity with DNA found
in fetal heart. 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)].
[0184] 59. PRO707
[0185] Cadherins are a large family of transmembrane proteins. At
least cadherins 1-13 as well as types B, E, EP, M, N, P and R have
been characterized. Among the functions cadherins are known for,
with some exceptions, cadherins participate in cell aggregation and
are associated with cell-cell adhesion sites. Cadherins are further
described in Tanihara, et al., J. Cell Sci., 107(6):1697-1704
(1994) and Tanihara, et al., Cell Adhes. Commun., 2(1):15-26
(1994). Moreover, it has been reported that some members of the
cadherin superfamily are involved in general cell-cell interaction
processes including transduction. See, Suzuki, J. Cell Biochem.,
61(4):531-542 (1996). Therefore, novel members of the cadherin
superfamily are of interest.
[0186] More generally, all novel proteins are of interest,
including membrane-bound proteins. Membrane-bound proteins and
receptors can play an important role 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. 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 adlesin 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.
[0187] 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 interaction. The
membrane-bound proteins can also be employed for screening of
potential peptide or small molecule inhibitors of the relevant
receptor/ligand interaction.
[0188] Efforts are being undertaken by both industry and academia
to identify new, native secreted and membrane-bound receptor
proteins, particularly membrane bound proteins having identity with
cadherins. The results of such efforts are provided herein.
[0189] 60. PRO322
[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, including the serine
proteases which exhibit specific activity toward various
serine-containing proteins. The mammalian protease enzymes play
important roles in biological processes such as, for example,
protein digestion, activation, inactivation, or modulation of
peptide hormone activity, and alteration of the physical properties
of proteins and enzymes.
[0191] Neuropsin is a novel serine protease whose mRNA is expressed
in the central nervous system. Mouse neuropsin has been cloned, and
studies have shown that it is involved in the hippocampal
plasticity. Neuropsin has also been indicated as associated with
extracellular matrix modifications and cell migrations. See,
generally, Chen, et al., Neurosci., 7(2):5088-5097 (1995) and Chen,
et al., J. Histochem. Cytochem., 46:313-320 (1998).
[0192] Efforts are being undertaken by both industry and academia
to identify new, native membrane-bound or secreted proteins,
particularly those having homology to neuropsin, serineprotease,
neurosin and trypsinogen. 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)].
[0193] 61. PRO526
[0194] Protein-protein interactions include those involved with
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.
[0195] 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):415-421 (October 1994).
[0196] A study has been reported on leucine-rich proteoglycans
which serve as tissue organizers, orienting and ordering collagen
fibrnls 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 factor .beta. 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
counective tissue, skin and bone; in promoting body growth in
humans and animals; and in stimulating other growth-related
processes. The acid labile subunit (ALS) of IGF is also of interest
in that it increases the half-life of IGF and is part of the IGF
complex in vivo. ALS is further described in Leong and Baxter, Mol.
Endocrinol., 6(6):870-876 (1992); Baxter, J. Biol. Chem.,
264(20):11843-11848 (1989); and Khosravi, et al., J. Clin.
Endocrinol. Metab., 82(12):3944-3951 (1997).
[0197] Another protein which has been reported to have leucine-rich
repeats is the SLIT protein which has been reported to be useful in
treating neurodegenerative 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. Also of 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).
[0198] 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
identity or similarity to known proteins having leucine rich
repeats such as ALS. 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)].
[0199] 62. PRO531
[0200] Cadherins are a large family of transmembrane proteins.
Cadherins comprise a family of calcium-dependent glycoproteins that
function in mediating cell-cell adhesion in virtually all solid
tissues of multicellular organisms. At least cadherins 1-13 as well
as types B, E, EP, M, N, P and R have been characterized. Among the
functions cadherins are known for, with some exceptions, cadherins
participate in cell aggregation and are associated with cell-cell
adhesion sites. Recently, it has been reported that while all
cadherins share multiple repeats of a cadherin specific motif
believed to correspond to folding of extracellular domains, members
of the cadherin superfamily have divergent structures and,
possibly, functions. In particular it has been reported that
members of the cadherin superfamily are involved in signal
transduction. See, Suzuki, J. Cell Biochem., 61(4):531-542 (1996).
Cadherins are further described in Tanihara, et al., J. Cell Sci.,
107(6):1697-1704 (1994), Aberle, et al., J. Cell Biochem.,
61(4):514-523 (1996) and Tanihara, et al., Cell Adhes. Commun.,
2(1):15-26 (1994).
[0201] Protocadherins are members of the cadherin superfamily which
are highly expressed in the brain. In some studies, protocadherins
have shown cell adhesion activity. See, Sano, et al., EMBO J.,
12(6):2249-2256 (1993). However, studies have also shown that some
protocadherins, such as protocadlerin 3 (also referred to as Pcdh3
or pc3), do not show strong calcium dependent cell aggregation
activity. See, Sago, et al., Genomics, 29(3):631-640 (1995) for
this study and further characteristics of Pcdh3.
[0202] Therefore, novel members of the cadlerin superfamily are of
interest. More generally, all membrane-bound proteins and receptors
are of interest. Such proteins can play an important role 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. 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.
[0203] 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 interaction. The
membrane-bound proteins can also be employed for screening of
potential peptide or small molecule inhibitors of the relevant
receptor/ligand interaction.
[0204] Efforts are therefore being undertaken by both industry and
academia to identify new, native membrane bound proteins,
particular those having sequence identity with protocadherins,
especially 3 and 4. Many efforts are focused on the screening of
mamian recombinant DNA libraries to identify the coding sequences
for novel membrane-bound proteins. Provided herein are the results
of such efforts.
[0205] 63. PRO534
[0206] 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. Protein disulfide isomerase and related proteins are
further described in Laboissiere, et al., J. Biol. Chem.,
270(47:28006-28009 (1995); Jeenes, et al., Gene, 193(2):151-156
(1997; Koivunen, et al., Genomics, 42(3):397-404 (1997); and
Desilva, et al., DNA Cell Biol., 15(1):9-16 (1996). These studies
indicate the importance of the identification of protein disultide
related proteins.
[0207] More generally, and also of interest are all novel
membrane-bound proteins and receptors. Such proteins can play an
important role 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. 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.
[0208] 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 interaction. The
membrane-bound proteins can also be employed for screening of
potential peptide or small molecule inhibitors of the relevant
receptor/ligand interaction.
[0209] Given the importance of membrane bound proteins, efforts are
under way to identity novel membrane bound proteins. Moreover,
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
sequence identity with 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 sequence identity with protein disulfide
isomerase and the nucleic acids encoding the same.
[0210] 64. PRO697
[0211] Secreted frizzled related proteins (sFRPs) are related to
the frizzled family of transmembrane receptors. The sFRPs are
approximately 30 kDa in size, and each contains a putative signal
sequence, a frizzled-like cysteine-rich domain, and a conserved
hydrophilic carboxy-terminal domain. It has been reported that
sFRPs may function to modulate Wnt signaling, or function as
ligands for certain receptors. Rattner, et al., PNAS USA,
94(7):2859-2863 (1997). Therefore, sFRPs and proteins having
sequence identity and/or similarity to sFRPs are of interest.
[0212] Another secreted protein of interest is any member of the
family of secreted apoptosis-related proteins (SARPs). Expression
of SARPs modifies the intracellular levels of beta-catenin,
suggesting that SARPs interfere with the Wnt-frizzled proteins
signaling pathway. Melkonyan, et al., PNAS USA, 94(25):13636-13641
(1997). Therefore, SARPs and proteins having sequence identity
and/or similarity to SARPs are of interest.
[0213] In addition to sFRPs and SARPs, many extracellular proteins
are of interest. Extracellular proteins play an important role 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.
[0214] Secreted proteins have various industrial applications,
including 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.
[0215] Efforts are being undertaken by both industry and academia
to identify new, native secreted proteins, particularly those
having sequence identity or similarity with sFRP-2 and SARP-1. 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)].
[0216] 65. PRO717
[0217] Efforts are being undertaken by both industry and academia
to identify new, native transmembrane receptor proteins. Many
efforts are focused on the screening of mammalian recombinant DNA
libraries to identify the coding sequences for novel receptor
proteins. The results of such efforts are provided herein.
[0218] 66. PRO731
[0219] Cadherins are a large family of transmembrane proteins.
Cadherins comprise a family of calcium-dependent glycoproteins that
function in mediating cell-cell adhesion in virtully all solid
tissues of multicellular organisms. At least cadherins 1-13 as well
as types B, E, EP, M, N, P and Rhave been characterized. Among the
functions cadherins are known for, with some exceptions, cadherins
participate in cell aggregation and are associated with ceh-cell
adhesion sites. Recently, it has been reported that while all
cadherins share multiple repeats of a cadherin specific motif
believed to correspond to folding of extracellular domains, members
of the cadherin superfamily have divergent structures and,
possibly, functions. In particular it has been reported that
members of the cadherin superfamily are involved in signal
transduction. See, Suzuki, J. Cell Biochem., 61(4):531-542 (1996).
Cadherins are further described in Tanihara, et al., J. Cell Sci.,
107(6):1697-1704 (1994), Aberle, et al., J. Cell Biochem.,
61(4):514-523 (1996) and Tanihara, et al., Cell Adhes. Commun.,
2(1):15-26 (1994).
[0220] Protocadherins are members of the cadherin superfamily which
are highly expressed in the brain. In some studies, protocadherins
have shown cell adhesion activity. See, Sano, et al., EMBO J.,
12(6):2249-2256 (1993). However, studies have also shown that some
protocadherins, such as protocadherin 3 (also referred to as Pcdh3
or pc3), do not show strong calcium dependent cell aggregation
activity. See, Sago, et al., Genomics, 29(3):631-640 (1995) for
this study and further characteristics of Pcdh3.
[0221] Therefore, novel members of the cadherin superfamily are of
interest. More generally, all membrane-bound proteins and receptors
are of interest. Such proteins can play an important role 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. 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.
[0222] 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 interaction. The
membrane-bound proteins can also be employed for screening of
potential peptide or small molecule inhibitors of the relevant
receptor/ligand interaction.
[0223] Efforts are therefore being undertaken by both industry and
academia to identify new, native membrane bound proteins,
particular those having sequence identity with protocadherins,
especially 4, 68, 43, 42, 3 and 5. Many efforts are focused on the
screening of mammalian recombinant DNA libraries to identify the
coding sequences for novel membrane-bound proteins. Provided herein
are the results of such efforts.
[0224] 67. PRO218
[0225] Efforts are being undertaken by both industry and academia
to identify new, native membrane bound proteins, particularly those
having sequence identity with membrane regulator proteins. Many
efforts are focused on the screening of mammalian recombinant DNA
libraries to identify the coding sequences for novel receptor
proteins.
[0226] 68. PRO768
[0227] 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.
[0228] Of particular interest is H36-alpha 7, an integrin alpha
chain that is developmentally regulated during myogenesis as
described in Song, et al., J. Cell Biol., 117(3):643-657 (1992).
The expression pattern of the laminin-binding alpha 7 beta 1
integrin is developmentally regulated in skeletal, cardiac, and
smooth muscle. Ziober, et al., Mol. Biol. Cell, 8(9):1723-1734
(1997). It has been reported that expression of the alpha 7-X1/X 2
integrin is a novel mechanism that regulates receptor affinty
states in a cell-specific context and may modulate
integrin-dependent events during muscle development and repair. Id.
It has further been reported that laminins promote the locomotion
of skeletal myoblasts via the alpha 7 integrin receptor. In
particular it was reported that alpha 7 beta 1 receptor can promote
myoblast adhesion and motility on a restricted number of laminin
isoforms and may be important in myogenic precursor recruitment
during regeneration and differentiation. Yao, et al., J. Cell Sci.,
109(13):3139-3150 (1996). Spliced variants of integrin alpha 7 are
also described in Leung, et al., Biochem. Biophys. Res. Commun.,
243(1):317-325 (1998) and Fornaro and Languino, Matrix Biol.,
16(4):185-193 (1997). Moreover, it has been reported that absence
of integrin alpha 7 causes a form of muscular dystrophy. Thus
integrins, particularly those related to integrin 7 and related
molecules, are of interest.
[0229] In addition to the interest of integrins, more generally,
all membrane-bound proteins and receptors are of interest since
such proteins can play an important role 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. Such
membrane-bound proteins and cell receptors include, but are not
limited to, cytokine receptors, receptor kicnases, 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.
[0230] 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 interaction. The
membrane-bound proteins can also be employed for screening of
potential peptide or small molecule inhibitors of the relevant
receptor/ligand interaction.
[0231] Therefore, efforts are being undertaken by both industry and
academia to identify new, native receptor proteins. Many efforts
are focused on the screening of mammalian recombinant DNA libraries
to identify the coding sequences for novel receptor proteins. The
results of such efforts, particularly those focused on identifying
new polypeptides having sequence identity with integrins, are
provided herein.
[0232] 69. PRO771
[0233] Testican is a multidomain testicular proteoglycan which is
expressed in numerous tissue types including, but not limited to
neuromuscular tissue, the brain and reproductive tissues. Testican
resembles modulators of cell social behavior such as the regulation
of cell shape, adhesion, migration and proliferation. [Bonnet, F.
et al., J. Biol. Chem., 271(8):4373 (1996), Perin, J. P. et al.,
EXS (Switzerland), 70:191 (1994), Alliel, P. M., et al, Eur. J.
Biochem., 214(1):346 (1993), Charbonnier, F., et al., C. R. Seances
Soc. Biol. Fil. (France), 191(1):127 (1997)]. Among other reasons,
since testican has been implicated in neuronal processes and may be
associated with the growth of connective tissue, testican and
related molecules are of interest.
[0234] More generally, all extracellular proteins are of interest.
Extracellular proteins play an important role 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.
[0235] Secreted proteins have various industrial applications,
including 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)]. The results of such efforts,
particularly those focused on identifying molecules having identity
and/or similarity with testican are of interest.
[0236] 70. PRO733
[0237] T1/ST2 is a receptor-like molecule homologous to the type I
interleukin-1 receptor, believed to be involved in cell signaling.
The T1/ST2 receptor and/or putative ligands are further described
in Gayle, et al., J. Biol. Chem., 271(10):5784-5789 (1996), Kumar,
et al., J. Biol. Chem., 270(46):27905-27913 (1995), and Mitcham, et
al., J. Biol. Chem., 271(10):5777-5783 (1996). These proteins, and
proteins related thereto are of interest.
[0238] More generally all membrane-bound proteins and receptors are
of interest since they can play an important role 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. 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.
[0239] 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 interaction. The
membrane-bound proteins can also be employed for screening of
potential peptide or small molecule inhibitors of the relevant
receptor/ligand interaction.
[0240] Efforts are being undertaken by both industry and academia
to identify new, native receptor proteins. Many efforts are focused
on the screening of mammalian recombinant DNA libraries to identify
the coding sequences for novel receptor proteins. The results of
such efforts are provided herein.
[0241] 71. PRO162
[0242] Pancreatitis-associated protein (PAP) is a secretory protein
that is overexpressed by the pancreas during acute pancreatitis.
Serum PAP concentrations have been shown to be abnormally high in
patients with acute pancreatitis. Pezzilli et al., Am. J.
Gastroenterol., 92(10):1887-1890 (1997).
[0243] PAP is synthesized by the pancreas due to pancreatic
inflammation and has been shown to be a good serum marker for
injury of the pancreas. In addition, serum PAP levels appear to
strongly correlate with creatinine clearance measurements. In
patients with a pancreas-kidney transplantation, PAP may prove to
be a useful biological and histological marker of pancreatic graft
rejection. Van der Pijl et al., Transplantation, 63(7):995-1003
(1997). Further, PAP has been shown to be useful in screening
neonates for cystic fibrosis. In fact, PAP may discriminate cystic
fibrosis neonates with better specificity than the current
immunoreactive trypsis assay. Iovanna et al., C. R. Acad. Aci. III,
317(6):561-564.
[0244] Secreted proteins such as PAP have various industrial
applications, including 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.
[0245] 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)]. The results of such
efforts are presented herein.
[0246] 72. PRO788
[0247] Anti-neoplastic urinary protein (ANUP) was identified as the
major protein present in a fraction of human urine which exhibits
antiproliferative activity against human tumor cell lines without
affecting the growth of several normal diploid cell lines or tumor
cells of mouse or hamster origin. Sloane et al., Biochem. J.,
234(2):355-362 (1986).
[0248] ANUP is a unique cytokine that has been found in human
granulocytes. The N-terminal amino acid sequence has been shown to
be unique. A synthetic peptide corresponding to the first nine
residues, with Cys at positions 4 and 7, was found to be an
anti-tumor agent in vitro. Ridge and Sloane, Cytokine, 8(1): 1-5
(1996).
[0249] Secreted proteins such as ANUP have various industrial
applications, including 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)].
[0250] 73. PRO1008
[0251] Dickkopf-1 (dkk-1) is a member of a family of secreted
proteins and functions in head induction. Dkk-1 is an inducer of
Spemann organizer in amphibian embryos. Glinka, et al., Nature,
391(6665):357-362 (1998). Dkk-1 is a potent antagonist of Wnt
signalling, suggesting that dkk genes encode a family of secreted
Wnt inhibitors. Thus, dkk-1 family members and related molecules
are of interest.
[0252] More generally, all extracellular proteins are of interest
since they can play an important role 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.
[0253] Secreted proteins have various industrial applications,
including pharmaceuticals, diagnostics, biosensors and bioreactors.
Most protein drugs available at present, such as thrombolytic
agents, interferons, interlerkins, 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.
[0254] Efforts are being undertaken by both industry and academia
to identify new, native secreted proteins, particularly those
related to dkk-1. 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)]. The results of such efforts to identify
molecules related to dkk-1 are provided herein.
[0255] 74. PRO1012
[0256] 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 renatrration 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. Protein disulfide isomerase and related proteins are
further described in Laboissiere, et al., J. Biol. Chem.,
270(47:28006-28009 (1995); Jeenes, et al., Gene, 193(2):151-156
(1997; Koivunen, et al., Genomics, 42(3):397-404 (1997); and
Desilva, et al., DNA Cell Biol., 15(1):9-16 (1996). These studies
indicate the importance of the identification of protein disulfide
related proteins.
[0257] More generally, the identification of all extracellular and
membrane-bound proteins is of interest since they 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.
[0258] Secreted proteins have various industrial applications,
including use as pharmaceuticals, diagnostics, biosensors and
bioreactors. In fact, 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-bound
proteins, also have potential as therapeutic or diagnostic agents.
Receptor immunoadhesins, for instance, can be employed as
therapeutic agents to block receptor-ligand interaction.
Membrane-bound proteins can also be employed for screening of
potential peptide or small molecule inhibitors of the relevant
receptor/ligand interaction. 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. 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.
[0259] Of particular interest are cellular proteins having
endoplasmic reticulum (ER) retention signals. These proteins are
retained in the cell and function closely with endoplasmic
reticulum in protein production. Such proteins have been described
previously, i.e., see Shorrosh and Dixon, Plant J., 2(1):51-58
(1992).
[0260] Efforts are being undertaken by both industry and academia
to identify new, native secreted and membrane-bound receptor
proteins, and in particular, cellular proteins having ER retension
signals. 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)]. The results of such
efforts, particularly the identification of novel polypeptides and
nucleic acids encoding the same, which have sequence identity and
similarity to protein disulfide isomerase are presented herein.
[0261] 75. PRO1014
[0262] 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):852-4 (1997); Friedrich and Weiss, J.
Theor. Biol., 187(4):529-40 (1997) and Pieulle, et al., J.
Bacteriol., 179(18):5684-92 (1997).
[0263] In addition to reductases in particular, novel polypeptides
are generally of interest. Extracellular proteins play an important
role 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.
[0264] Secreted proteins have various industrial applications,
including 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)]. The results of such efforts,
particularly those identifying polypeptides having sequence
identity with reductases, and the nucleic acids encoding the same,
are presented herein.
[0265] 76. PRO1017
[0266] Enzymatic proteins play important roles in the chemical
reactions involved in the digestion of foods, the biosynthesis of
macromolecules, the controlled release and utilization of chemical
energy, and other processes necessary to sustain life.
Sulfotransferases are enzymes which transfer sulfate from a sulfate
donor to acceptor substrates, particularly those contaning terminal
glucoronic acid. The HNK-1 carbohydrate epitope is expressed on
several neural adhesion glycoproteins and a glycolipid, and is
involved in cell interactions. The glucuronyltransferase and
sulfotransferase are considered to be the key enzymes in the
biosynthesis of this epitope because the rest of the structure
occurs often in glycoconjugates. HNK-1 sulfotransfererase is
further described in Bakker, H., et al., J. Biol. Chem.,
272(47):29942-29946 (1997).
[0267] In addition to HNK-1 sulfotransfererase, and novel proteins
related thereto, all novel proteins are of interest. 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,
andhormones) 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.
[0268] Secreted proteins have various industrial applications,
including use as pharmaceuticals, diagnostics, biosensors and
bioreactors. In fact, 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-bound
proteins, also have potential as therapeutic or diagnostic agents.
Receptor immunoadhesins, for instance, can be employed as
therapeutic agents to block receptor-ligand interaction.
Membrane-bound proteins can also be employed for screening of
potential peptide or small molecule inhibitors of the relevant
receptor/ligand interaction. 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. 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.
[0269] Efforts are being undertaken by both industry and academia
to identify new, native secreted and membrane-bound receptor
proteins, particularly those having sequence identity with HNK-1
sulfotransferase. 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)]. The results of such
efforts are provided herein.
[0270] 77. PRO474
[0271] Enzymatic proteins play important roles in the chemical
reactions involved in the digestion of foods, the biosynthesis of
macromolecules, the controlled release and utilization of chemical
energy, and other processes necessary to sustain life. Glucose
dehydrogenase functions in the oxidation of glucose to gluconate to
generate metabolically useful energy. The regulation of the
PQQ-linked glucose dehydrogenase in different organisms is reviewed
in Neijssel, et al., Antonie Van Leeuwenhoek, 56(1):51-61 (1989).
Glucose dehydrogenase functions as an auxiliary energy generating
mechanism, because it is maximally synthesized under conditions of
energy stress. In addition to molecules related to glucose
dehydrogenase, all novel proteins are of interest. 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.
[0272] Secreted proteins have various industrial applications,
including use as pharmaceuticals, diagnostics, biosensors and
bioreactors. In fact, 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-bound
proteins, also have potential as therapeutic or diagnostic agents.
Receptor immunoadhesins, for instance, can be employed as
therapeutic agents to block receptor-ligand interaction.
Membrane-bound proteins can also be employed for screening of
potential peptide or small molecule inhibitors of the relevant
receptor/ligand interaction. 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. 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.
[0273] Efforts are being undertaken by both industry and academia
to identify new, native secreted and membrane-bound receptor
proteins, and particularly cellular proteins and those related to
dehydrogenase or oxidoreductase. 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)].
The results of such efforts are presented herein.
[0274] 78. PRO1031
[0275] It has been reported that the cytokine interleukin 17
(IL-17) stimulates epithelial, endothelial, and fibroblastic cells
to secrete cytokines such as IL-6, IL-8, and
granulocyte-colony-stimulating factor, as well as prostaglandin E2.
Moreover, it has been shown that when cultured in the presence of
IL-17, fibroblasts could sustain proliferation of CD34+
preferential maturation into neutrophils. Thus it has been
suggested that IL-17 constitutes an early initiator of the T
cell-dependent inflammatory reaction and/or an element of the
cytokine network that bridges the immune system to hematopoiesis.
See, Yao, et al., J. Immunol., 155(12):5483-5486 (1995); Fossiez,
et al., J. Exp. Med., 183(6):2593-2603 (1996); Kennedy, et al., J.
Interferon Cytokine Res., 16(8):611-617 (1996). Thus, proteins
related to IL-17 are of interest.
[0276] More generally, all novel proteins are of interest.
Extracellular proteins play an important role 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.
[0277] Secreted proteins have various industrial applications,
including 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.
[0278] Efforts are being undertaken by both industry and academia
to identify new, native secreted proteins, particularly those
related to IL-17. 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)]. The results of such efforts are
presented herein.
[0279] 79. PRO938
[0280] 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. Protein disulfide isomerase and related proteins are
further described in Laboissiere, et al., J. Biol. Chem.,
270(47):28006-28009 (1995); Jeenes, et al., Gene, 193(2):151-156
(1997); Koivunen, et al., Genomics, 42(3):397-404 (1997); Desilva,
et al., DNA Cell Biol., 15(1):9-16 (1996); Freedman, et al. Trends
in Biochem. Sci. 19:331-336 (1994); Bulleid, N.J. Advances in Prot.
Chem. 44:125-50 (1993); and Noiva, R., Prot. Exp. and Purification
5:1-13 (1994). These studies indicate the importance of the
identification of protein disulfide related proteins.
[0281] More generally, and also of interest are all novel
membrane-bound proteins and receptors. Such proteins can play an
importantrole 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. 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.
[0282] 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 interaction. The
membrane-bound proteins can also be employed for screening of
potential peptide or small molecule inhibitors of the relevant
receptor/ligand interaction.
[0283] Given the importance of membrane bound proteins, efforts are
under way to identity novel membrane bound proteins. Moreover,
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
sequence identity with 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.
[0284] We herein describe the identification and characterization
of a novel polypeptide having homology to protein disulfide
isomerase.
[0285] 80. PRO1082
[0286] The low density lipoprotein (LDL) receptor is a
membrane-bound protein that plays a key role in cholesterol
homeostasis, mediating cellular uptake of lipoprotein particles by
high affinity binding to its ligands, apolipoprotein (apo) B-100
and apoE. The ligand-binding domain of the LDL receptor contains 7
cysteine-rich repeats of approximately 40 amino acids, wherein each
repeat contains 6 cysteines, which form 3 intra-repeat disulfide
bonds. These unique structural features provide the LDL receptor
with its ability to specifically interact with apo B-100 and apoE,
thereby allowing for transport of these lipoprotein particles
across cellular membranes and metabolism of their components.
Soluble fragments containing the extracellular domain of the LDL
receptor have been shown to retain the ability to interact with its
specific lipoprotein ligands (Simmons et al., J. Biol. Chem.
272:25531-25536 (1997)). LDL receptors are further described in
Javitt, FASEB J., 9(13):1378-1381 (1995) and Herz and Willnow, Ann.
NY Acad. Sci., 737:14-19 (1994). Thus, proteins having sequence
identity with LDL receptors are of interest.
[0287] More generally, all membrane-bound proteins and receptors
can play an important role 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. 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. Of particular interest
are membrane bound proteins that have type II transmembrane
domains.
[0288] 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 interaction. The
membrane-bound proteins can also be employed for screening of
potential peptide or small molecule inhibitors of the relevant
receptor/ligand interaction.
[0289] Efforts are thus being undertaken by both industry and
academia to identify new, native proteins, particularly membrane
bound proteins including type II transmembrane bound proteins. Many
efforts are focused on the screening of mammalian recombinant DNA
libraries to identify the coding sequences for novel receptor
proteins. The results of such efforts are provided herein.
[0290] 81. PRO1083
[0291] Of particular interest are membrane bound proteins that
belong to the seven transmembrane (7TM) receptor superfamily.
Examples of these receptors include G-protein coupled receptors
such as ion receptors. Another example of a 7TM receptor
superfamily member is described in Osterhoff, et al., DNA Cell
Biol., 16(4):379-389 (1997).
[0292] 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 interaction. The
membrane-bound proteins can also be employed for screening of
potential peptide or small molecule inhibitors of the relevant
receptor/ligand interaction.
[0293] Efforts are being undertaken by both industry and academia
to identify new, native receptor proteins. Many efforts are focused
on the screening of mammalian recombinant DNA libraries to identify
the coding sequences for novel receptor proteins. The results of
such efforts are presented herein.
[0294] 82. PRO200
[0295] Polypeptides involved in survival, proliferation and/or
differentiation of cells are of interest. Polypeptides known to be
involved in the survival, proliferation and/or differentiation of
cells include VEGF and members of the bone morphogenetic protein
family. Therefore, novel polypeptides which are related to either
VEGF or the bone morphogenetic protein are of interest.
[0296] The heparin-binding endothelial cell-growth factor, VEGF,
was identified and purified from media conditioned by bovine
pituitary follicular or folliculo-stellate cells over several years
ago. See Ferrara et al., Biophys. Res. Comm. 161, 851 (1989). VEGF
is a naturally occurring compound that is produced in follicular or
folliculo-stellate cells (FC), a morphologically well characterized
population of granular cells. The FC are stellate cells that send
cytoplasmic processes between secretory cells.
[0297] VEGF is expressed in a variety of tissues as multiple
homodimeric forms (121, 165, 189 and 206 amino acids per monomer)
resulting from alternative RNA splicing. VEGF.sub.121 is a soluble
mitogen that does not bind heparin; the longer forms of VEGF bind
heparin with progressively higher affinity. The heparin-binding
forms of VEGF can be cleaved in the carboxy terminus by plasmin to
release (a) diffusible form(s) of VEGF. Amino acid sequencing of
the carboxy terminal peptide identified after plasmin cleavage is
Arg.sub.110-Ala.sub.111. Amino terminal "core" protein, VEGF
(1-110) isolated as ahomodimer, binds neutralizing monoclonal
antibodies (4.6.1 and 2E3) and soluble forms of FMS-like tyrosine
kinase (FLT-1), kinase domain region (KDR) and fetal liver kinase
(FLK) receptors with similar affinity compared to the intact
VEGF.sub.165 homodimer.
[0298] As noted, VEGF contains two domains that are responsible
respectively for binding to the KDR and FLT-1 receptors. These
receptors exist only on endothelial (vascular) cells. As cells
become depleted in oxygen, because of trauma and the like, VEGF
production increases in such cells which then bind to the
respective receptors in order to signal ultimate biological effect.
The signal then increases vascular permeability and the cells
divide and expand to form new vascular pathways--vasculogenesis and
angiogenesis.
[0299] Thus, VEGF is useful for treating conditions in which a
selected action on the vascular endothelial cells, in the absence
of excessive tissue growth, is important, for example, diabetic
ulcers and vascular injuries resulting from trauma such as
subcutaneous wounds. Being a vascular (artery and venus)
endothelial cell growth factor, VEGF restores cells that are
damaged, a process referred to as vasculogenesis, and stimulates
the formulation of new vessels, a process referred to as
angiogenesis.
[0300] VEGF would also find use in the restoration of vasculature
after a myocardial infarct, as well as other uses that can be
deduced. In this regard, inhibitors of VEGF are sometimes
desirable, particularly to mitigate processes such as angiogenesis
and vasculogenesis in cancerous cells.
[0301] Regarding the bone morphogenetic protein family, members of
this family have been reported as being involved in the
differentiation of cartilage and the promotion of vascularization
and osteoinduction in preformed hydroxyapatite. Zou, et al., Genes
Dev. (U.S.), 11(17):2191 (1997); Levine, et al., Ann. Plast. Surg.,
39(2):158 (1997). A number of related bone morphogenetic proteins
have been identified, all members of the bone morphogenetic protein
(BMP) family. Bone morphogenetic native and mutant proteins,
nucleic acids encoding therefor, related compounds including
receptors, host cells and uses are further described in at least:
U.S. Pat. Nos. 5,670,338; 5,454,419; 5,661,007; 5,637,480;
5,631,142; 5,166,058; 5,620,867; 5,543,394; 4,877,864; 5,013,649;
55,106,748; and 5,399,677. Of particular interest are proteins
having homology with bone morphogenetic protein 1, a procollagen
C-proteinase that plays key roles in regulating matrix
deposition.
[0302] The present invention is predicated upon research intended
to identify novel polypeptides which are related to VEGF and the
BMP family, and in particular, polypeptides which have a role in
the survival, proliferation and/or differentiation of cells. While
the novel polypeptides are not expected to have biological activity
identical to the known polypeptides to which they have homology,
the known polypeptide biological activities can be used to
determine the relative biological activities of the novel
polypeptides. In particular, the novel polypeptides described
herein can be used in assays which are intended to determine the
ability of a polypeptide to induce survival, proliferation or
differentiation of cells. In turn, the results of these assays can
be used accordingly, for diagnostic and therapeutic purposes. The
results of such research is the subject of the present
invention.
[0303] 83. PRO285 and PRO286
[0304] The cloning of the Toll gene of Drosophila, a maternal
effect gene that plays a central role in the establishment of the
embryonic dorsal-ventral pattern, has been reported by Hashimoto et
al., Cell 52, 269-279 (1988). The Drosophila Toll gene encodes an
integral membrane protein with an extracytoplasmic domain of 803
amino acids and a cytoplasmic domain of 269 amino acids. The
extracytoplasmic domain has a potential membrane-spanning segment,
and contains multiple copies of a leucine-rich segment, a
structural motif found in many transmembrane proteins. The Toll
protein controls dorsal-ventral patterning in Drosophila embryos
and activates the transcription factor Dorsal upon binding to its
ligand Sptzle. (Morisato and Anderson, Cell 76, 677-688 (1994).) In
adult Drosophila, the Toll/Dorsal signaling pathway participates in
the anti-fungal immune response. (Lenaitre et al., Cell 86, 973-983
(1996).)
[0305] A human homologue of the Drosophila Toll protein has been
described by Medhhitov et al., Nature 388, 394-397 (1997). This
human Toll, just as Drosophila Toll, is a type I transmembrane
protein, with an extracellular domain consisting of 21 tandemnly
repeated leucine-rich motifs (leucine-rich region--LRR), separated
by a non-LRR region, and a cytoplasmic domain homologous to the
cytoplasmic domain of the human interleukin-1 (IL-1) receptor. A
constitutively active mutant of the human Toll transfected into
human cell lines was shown to be able to induce the activation of
NF-.kappa.B and the expression of NF-.kappa.B-controlled genes for
the inflammatory cytokines IL-1, IL-6 and IL-8, as well as the
expression of the constimulatory molecule B7.1, which is required
for the activation of native T cells. It has been suggested that
Toll functions in vertebrates as a non-clonal receptor of the
immune system, which can induce signals for activating both an
innate and an adaptive immune response in vertebrates. The human
Toll gene reported by Medzhitov et al., supra was most strongly
expressed in spleen and peripheral blood leukocytes (PBL), and the
authors suggested that its expression in other tissues may be due
to the presence of macrophages and dendritic cells, in which it
could act as an early-warning system for infection. The public
GenBank database contains the following Toll sequences: Toll1
(DNAX#HSU88540-1, which is identical with the random sequenced
full-length cDNA#HUMRSC786-1); Toll2 (DNAX# HSU88878-1); Toll3
(DNAX# HSU88879-1); and Toll4 (DNAX# HSU88880-1, which is identical
with the DNA sequence reported by Medzhitov et al., supra). A
partial Toll sequence (Toll5) is available from GenBank under DNAX#
HSU88881-1.
[0306] Further human homologues of the Drosophila Toll protein,
designated as Toll-like receptors (huTLRs1-5) were recently cloned
and shown to mirror the topographic structure of the Drosophila
counterpart (Rock et al., Proc. Natl. Acad. Sci. USA 95, 588-593
[1998]). Overexpression of a constitutively active mutant of one
human TLR (Toll-protein homologue--Medzhitov et al., supra;
TLR4--Rock et al., supra) leads to the activation of NF-.kappa.B
and induction of the inflarmmatory cytokines and constimulatory
molecules. Medzhitov et al., supra.
[0307] 84. PRO213-1, PRO1330 and PRO1449
[0308] Cancer is characterized by the increase in the number of
abnormal, or neoplastic, cells derived from a normal tissue which
proliferate to form a tumor mass, the invasion of adjacent tissues
by these neoplastic tumor cells, and the generation of malignant
cells which eventually spread via the blood or lymphatic system to
regional lymph nodes and to distant sites (metastasis). In a
cancerous state a cell proliferates under conditions in which
normal cells would not grow. Cancer manifests itself in a wide
variety of forms, characterized by different degrees of
invasiveness and aggressiveness.
[0309] Alteration of gene expression is intimately related to the
uncontrolled cell growth and de-differentiation which are a common
feature of all cancers. The genomes of certain well studied tumors
have been found to show decreased expression of recessive genes,
usually referred to as tumor suppression genes, which would
normally function to prevent malignant cell growth, and/or
overexpression of certain dominant genes, such as oncogenes, that
act to promote malignant growth. Each of these genetic changes
appears to be responsible for importing some of the traits that, in
aggregate, represent the full neoplastic phenotype (Hunter, Cell
64, 1129 [1991]; Bishop, Cell 64, 235-248 [1991]).
[0310] A well known mechanism of gene (e.g. oncogene)
overexpression in cancer cells is gene amplification. This is a
process where in the chromosome of the ancestral cell multiple
copies of a particular gene are produced. The process involves
unscheduled replication of the region of chromosome comprising the
gene, followed by recombination of the replicated segments back
into the chromosome (Alitalo et al., Adv. Cancer Res. 47, 235-281
[1986]). It is believed that the overexpression of the gene
parallels gene amplification, i.e. is proportionate to the number
of copies made.
[0311] Proto-oncogenes that encode growth factors and growth factor
receptors have been identified to play important roles in the
pathogenesis of various human malignancies, including breast
cancer. For example, it has been found that the human ErbB2 gene
(erbB2, also known as her2, or c-erbB-2), which encodes a 185-kd
transmembrane glycoprotein receptor (p185HER2; HER2) related to the
epidermal growth factor receptor (EGFR), is overexpressed in about
25% to 30% of human breast cancer (Slamon et al., Science
235:177-182 [1987]; Slamon et al., Science 244:707-712 [1989]).
[0312] It has been reported that gene amplification of a
protooncogene is an event typically involved in the more malignant
forms of cancer, and could act as a predictor of clinical outcome
(Schwab et al., Genes Chromosomes Cancer 1, 181-193 [1990]; Alitalo
et al., supra). Thus, erbB2 overexpression is commonly regarded as
a predictor of a poor prognosis, especially in patients with
primary disease that involves axillary lymph nodes (Slamon et al.,
[1987] and [1989], supra; Ravdin and Chamness, Gene 159:19-27
[1995]; and Hynes and Stern, Biochem Biophys Acta 1198: 165-184
[1994]), and has been linked to sensitivity and/or resistance to
hormone therapy and chemotherapeutic regimens, including CMF
(cyclophosphamide, methotrexate, and fluoruracil) and
anthracyclines (Baselga et al., Oncology 11 (3 Suppl 1): 43-48
[1997]). However, despite the association of erbB2 overexpression
with poor prognosis, the odds of HER2-positive patients responding
clinically to treatment with taxanes were greater than three times
those of HER2-negative patients (Ibid). A recombinant humanized
anti-ErbB2 (antih-HER2) monoclonal antibody (a humanized version of
the murine anti-ErbB2 antibody 4D5, referred to as rhuMAb HER2 or
Herceptin 7o) has been clinically active in patients with
ErbB2-overexpressing metastatic breast cancers that had received
extensive prior anticancer therapy. (Baselga et al., J. Clin.
Oncol. 14:737-744 [1996]).
[0313] The protein Notch and its homologues are key regulatory
receptors in determining the cell fate in various development
processes. The protein Notch-4, also known as int-3 oncogene, was
originally identified as a frequent target in mouse mammary tumor
virus (MMVS). Notch-4 is believed to be a transgene which affects
the differentiation capacity of stem cells and leads to neoplastic
proliferation in epithelial cells. Shirayoshi et al., Genes Cells
2(3): 213-224 (1997). During embryogenesis, the expression of
Notch-4 was detected in endothelial cells of blood vessels forming
tissues such as the dorsal aorta, intersegmental vessels, yolk sac
vessels, cephalic vessels, heart, vessels in branchial arches, and
capillary plexuses. Notch-4 expression in these tissues was also
associated with flk-1, the major regulatory gene of vasculogenesis
and angiogenesis. Notch-4 is also upregulated in vitro during the
differentiation of endothelial stem cell. The endothelial cell
specific expression pattern of Notch-4, as well as its structural
similarity to Notch suggest that Notch-4 is an endothelial cell
specific homologue of Notch and that it may play a role in
vaculogenesis and angiogenesis.
[0314] 85. PRO298
[0315] Efforts are being undertaken by both industry and academia
to identify new, native receptor proteins. Many 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 and characterization of novel
transmembrane polypeptides, designated herein as PRO298
polypeptides.
[0316] 86. PRO337
[0317] Neuronal development in higher vertebrates is characterized
by processes that must successfully navigate distinct cellular
environment en route to their synaptic targets. The result is a
functionally precise formation of neural circuits. The precision is
believed to result form mechanisms that regulate growth cone
pathfinding and target recognition, followed by latter refinement
and remodeling of such projections by events that require neuronal
activity, Goodman and Shatz, Cell/Neuron [Suppl.] 72(10): 77-98
(1993). It is further evident that different neurons extend nerve
fibers that are biochemically distinct and rely on specific
guidance cues provided by cell-cell, cell-matrix, and chemotrophic
interactions to reach their appropriate synaptic targets, Goodman
et al., supra.
[0318] One particular means by which diversity of the neuronal cell
surface may be generated is through differential expression of cell
surface proteins referred to as cell adhesion molecules (CAMs).
Neuronally expressed CAMs have been implicated in diverse
developmental processes, including migration of neurons along
radial glial cells, providing permissive or repulsive substrates
for neurite extension, and in promoting the selective fasciculation
of axons in projectional pathways. Jessel, Neuron 1: 3-13 (1988);
Edelman and Crossin, Annu. Rev. Biochem. 60: 155-190 (1991).
Interactions between CAMs present on the growth cone membrane and
molecules on opposing cell membranes or in the extracellular matrix
are thought to provide the specific guidance cues that direct nerve
fiber outgrowth along appropriate projectional pathways. Such
interactions are likely to result in the activation of various
second messenger systems within the growth cone that regulate
neurite outgrowth. Doherty and Walsh, Curr. Opin Neurobiol. 2:
595-601 (1992).
[0319] In higher vertebrates, most neural CAMs have been found to
be members of three major structural families of proteins: the
integrins, the cadherins, and the immunoglobulin gene superfamily
(IgSF). Jessel, supra.; Takeichi, Annu. Rev. Biochem. 59: 237-252
(1990); Reichardt and Tomaselli, Annu. Rev. Neurosci. 14: 531-570
(1991). Cell adhesion molecules of the IgSF (or Ig-CAMs), in
particular, constitute a large family of proteins frequently
implicated in neural cell interactions and nerve fiber outgrowth
during development, Salzer and Colman, Dev. Neurosci. 11: 377-390
(1989); Brummendorf and Rathjen, J. Neurochem. 61: 1207-1219
(1993). However, the majority of mammalian Ig-CAMs appear to be too
widely expressed to specify tnavigational pathways or synaptic
targets suggesting that other CAMs, yet to be identified, have role
in these more selective interactions of neurons.
[0320] Many of the known neural Ig-CAMs have been found to be
attached to the plasma membrane via a glycosylphosphatidylinositol
(GPI) anchor. Additionally, many studies have implicated
GPI-anchored proteins in providing specific guidance cues during
the outgrowth on neurons in specific pathways. In studies of the
grasshopper nervous system, treatment of embryos with
phosphatidylinositol-specific phopholipase C (PIPLC), which
selectively removes GPI-anchored proteins from the surfaces of
cells, resulted in misdirection and faulty navigation among subsets
of pioneering growth cones, as well as inhibited migratory patterns
of a subset of early neurons, Chang et al., Devel. 114: 507-519
(1992). The projection of retinal fibers to the optic tectum
appears to depend, in part, on a 33 kDa GPI-anchored protein,
however, the precise nature of this protein is unknown. Stahl et
al., Neuron 5: 735-743 (1990).
[0321] The expression of various GPI-anchored proteins has been
characterized amongst the different populations of primary rat
neurons amongst dorsal root ganglion, sympathetic neurons of the
cervical ganglion, sympathetic neurons of the superior cervical
ganglion, and cerebellar granule neurons. Rosen et al., J. Cell
Biol. 117: 617-627 (1992). In contrast to the similar pattern of
total membrane protein expression by these different types of
neurons, striking differences were observed in the expression of
GPI-anchored proteins between these neurons. Recently, a 65 kDa
protein band known as neurotrimin was discovered and found to be
differentially expressed by primary neurons (Rosen et al., supra),
and restricted to the nervous system and found to be the most
abundant and earliest expressed of the GPI-anchored species in the
CNS. Struyk et al., J. Neuroscience 15(3): 2141-2156 (1995). The
discovery of neurotrimin has further lead to the identification of
a family of IgSF members, each containing three Ig-like domains
that share significant amino acid identity, now termed IgLON.
Struyk et al., supra; Pimenta et al., Gene 170(2): 189-95
(1996).
[0322] Additional members of the IgLON subfamily include opiate
binding cell adhesion molecule (OBCAM), Schofield et al., EMBO J.
8: 489-495 (1989); limbic associated membrane protein (LAMP),
Pimenta et al., supra; CEPU-1; GP55, Wilson et al., J. Cell Sci.
109: 3129-3138 (1996); Eur. J. Neurosci. 9(2): 334-41 (1997); and
AvGp50, Hancox et al., Brain Res. Mol. Brain Res. 44(2): 273-85
(1997).
[0323] While the expression of neurotrimln appears to be
widespread, it does appear to correlated with the development of
several neural circuits. For example, between E18 and P10,
neurotimin mRNA expression within the forebrain is maintained at
high levels in neurons of the developing thalamus, cortical
subplate, and cortex, particularly laminae V and VI (with less
intense expression in II, II, and IV, and minimal expression in
lamina I). Cortical subplate neurons may provide an early,
temporary scaffold for the ingrowing thalamic afferents en route to
their final synaptic targets in the cortex. Allendoerfer and Shatz,
Annu. Rev. Neurosci. 17: 185-218 (1994). Conversely, subplate
neurons have been suggested to be required for cortical neurons
from layer V to select VI to grow into the thalamus, and neurons
from layer V to select their targets in the colliculus, pons, and
spinal cord (McConnell et al., J. Neurosci. 14: 1892-1907 (1994).
The high level expression of neurotrimin in many of these
projections suggests that it could be involved in their
development.
[0324] In the hindbrain, high levels of neurotrimin message
expression were observed witin the pontine nucleus and by the
internal granule cells and Purkinje cells of the cerebellum. The
pontine nucleus received afferent input from a variety of sources
including corticopontine fibers of layer V, and is a major source
of afferent input, via mossy fibers, to the granule cells which, in
turn, are a major source of afferent input via parallel fibers to
Purkinje cells. [Palay and Chan-Palay, The cerebellar cortex:
cytology and organization. New York: Springer (1974]. High level
expression of neurotrimin these neurons again suggests potential
involvement in the establishment of these circuits.
[0325] Neurotrimin also exhibits a graded expression pattern in the
early postnatal striatum. Increased neurotrimin expression is found
overlying the dorsolateral striatum of the rat, while lesser
hybridization intensity is seen overlying the ventromedial
striatum. Struyk et al., supra. This region of higher neurotrimin
hybridization intensity does not correspond to a
cytoarchitecturally differentiable region, rather it corresponds to
the primary area of afferent input from layer VI of the
contralateral sensormotor cortex (Gerfen, Nature 311: 461-464
(1984); Donoghue and Herkenham, Brain Res. 365: 397-403 (1986)).
The ventromedial striatum, by contrast, receives the majority of
its afferent input from the perirhinal and association cortex. It
is noteworthy that a complementary graded pattern of LAMP
expression, has been observed within the striatium, with highest
expression in ventromedial regions, and lowest expression
dorsolaterally. Levitt, Science 223: 299-301 (1985); Chesselet et
al., Neuroscience 40: 725-733 (1991).
[0326] 87. PRO403
[0327] Type II transmembrane proteins, also known as single pass
transmembrane proteins have an N-terminal portion lodged in the
cytoplasm while the C-terminal portion is exposed to the
extracellular domain.
[0328] Endothelin is a family of vasoconstrictor peptides about
which much activity has been focused to better understand its basic
pharmacological, biochemical and molecular biological features,
including the presence and structure of isopeptides and their genes
(endothelin-1, -2 and 3), regulation of gene expression,
intracellular processing, specific endothelin converting enzymes
(ECE), receptor subtypes (ET-A and ET-B), intracellular signal
transduction following receptor activation, etc.
[0329] The endothelin (ET) family of peptides have potent vascular,
cardiac and renal actions which may be of pathophysiological
importance in many human disease states. ET-1 is expressed as an
inactive 212 amino acid prepropeptide. The prepropeptide is first
cleaved at Arg52-Cys53 and Arg92-Ala93 and then the carboxy
terminal Lys91 and Arg92 are trimmed from the protein to generate
the propeptide big ET-1.
[0330] Endothelin is generated from inactive intermediates, the big
endothelins, by a unique processing event catalyzed by the zinc
metalloprotease, endothelin converting enzyme (ECE). ECE was
recently cloned, and its structure was shown to be a single pass
transmembrane protein with a short intracellular N-terminal and a
long extracellular C-terminal that contains the catalytic domain
and numerous N-glycosylation sites. ECEs cleave the endothelin
propeptide between Trp73 and Val74 producing the active peptide,
ET, which appears to function as a local rather than a circulating
hormone (Rubanyi, G. M. & Polokoff, M. A., Pharmachological
Reviews 46: 325-415 (1994). Thus ECE activity is apotential site of
regulation of endothelin production and apossible target for
therapeutic intervention in the endothelin system. By blocking ECE
activity, it is possible stop the production of ET-1 by inhibiting
the conversion of the relatively inactive precursor, big ET-1, to
the physiologically active form.
[0331] Endothelins may play roles in the pathophysiology of a
number of disease states including: 1) cardiovascular diseases
(vasospasm, hypertension, myocardial ischemia; reperfusion injury
and acute myochardial infarction, stroke (cerebral ischemia),
congestive heart failure, shock, atherosclerosis, vascular
thickening), 2) kidney disease (acute and chronic renal failure,
glomerulonephritis, cirrhosis); 3) lung disease (bronchial asthma,
pulmonary hypertension); 4) gastrointestinal disorders (gastric
ulcer, inflammatory bowel diseases); 5) reproductive disorders
(premature labor, dysmenorhea, preeclampsia) and 6) carcinogenesis.
Rubanyi & Polokoff, supra.
SUMMARY OF THE INVENTION
[0332] 1. PRO213
[0333] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as "PRO213".
[0334] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO213 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO213 polypeptide having amino acid residues 1 to 295 of FIG. 2
(SEQ ID NO:2), 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.
[0335] In another embodiment, the invention provides isolated
PRO213 polypeptide. In particular, the invention provides isolated
native sequence PRO213 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 295 of
FIG. 2 (SEQ ID NO:2).
[0336] 2. PRO274
[0337] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as "PRO274".
[0338] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO274 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO274 polypeptide having amino acid residues 1 to 492 of FIG. 4
(SEQ ID NO:7), 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. The isolated
nucleic acid sequence may comprise the cDNA insert of the
DNA39987-1184 vector deposited on Apr. 21, 1998 as ATCC 209786
which includes the nucleotide sequence encoding PRO274.
[0339] In another embodiment, the invention provides isolated
PRO274 polypeptide. In particular, the invention provides isolated
native sequence PRO274 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 492 of
FIG. 4 (SEQ ID NO:7). An additional embodiment of the present
invention is directed to an isolated extracellular domain of a
PRO274 polypeptide. Optionally, the PRO274 polypeptide is obtained
or is obtainable by expressing the polypeptide encoded by the cDNA
insert of the DNA39987-1184 vector deposited on Apr. 21, 1998 as
ATCC 209786.
[0340] In another embodiment, the invention provides three
expressed sequence tags (EST) comprising the nucleotide sequences
of SEQ ID NO:8 (herein designated as DNA17873), SEQ ID NO:9 (herein
designated as DNA36157) and SEQ ID NO:10 (herein designated as
DNA28929) (see FIG. 5-7, respactively).
[0341] 3. PRO300
[0342] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as "PRO300".
[0343] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO300 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO300 polypeptide having amino acid residues 1 to 457 of FIG. 9
(SEQ ID NO:19), 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. The isolated
nucleic acid sequence may comprise the cDNA insert of the
DNA40625-1189 vector deposited on Apr. 21, 1998 as ATCC 209788
which includes the nucleotide sequence encoding PRO300.
[0344] In another embodiment, the invention provides isolated
PRO300 polypeptide. In particular, the invention provides isolated
native sequence PRO300 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 457 of
FIG. 9 (SEQ ID NO:19). An additional embodiment of the present
invention is directed to an isolated extracellular domain of a
PRO300 polypeptide. Optionally, the PRO300 polypeptide is obtained
or is obtainable by expressing the polypeptide encoded by the cDNA
insert of the DNA40625-1189 vector deposited on Apr. 21, 1998 as
ATCC 209788.
[0345] 4. PRO284
[0346] Applicants have identified a cDNA clone that encodes a novel
transmembrane polypeptide, wherein the polypeptide is designated in
the present application as "PRO284".
[0347] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO284 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO284 polypeptide having amino acid residues 1 to 285 of FIG. 11
(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. In other aspects,
the isolated nucleic acid comprises DNA encoding the PRO284
polypeptide having amino acid residues about 25 to 285 of FIG. 11
(SEQ ID NO:28) or 1 or about 25 to X of FIG. 11 (SEQ ID NO:28),
where X is any amino acid from 71 to 80 of FIG. 11 (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. The isolated nucleic acid
sequence may comprise the cDNA insert of the DNA23318-1211 vector
deposited on Apr. 21, 1998 as ATCC 209787 which includes the
nucleotide sequence encoding PRO284.
[0348] In another embodiment, the invention provides isolated
PRO284 polypeptide. In particular, the invention provides isolated
native sequence PRO284 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 285 of
FIG. 11 (SEQ ID NO:28). Additional embodiments of the present
invention are directed to isolated PRO284 polypeptides comprising
amino acids about 25 to 285 of FIG. 11 (SEQ ID NO:28) or 1 or about
25 to X of FIG. 11 (SEQ ID NO:28), where X is any amino acid from
71 to 80 of FIG. 11 (SEQ ID NO:28). Optionally, the PRO284
polypeptide is obtained or is obtainable by expressing the
polypeptide encoded by the cDNA insert of the DNA23318-1211 vector
deposited on Apr. 21, 1998 as ATCC 209787.
[0349] In another embodiment, the invention provides an expressed
sequence tag (EST) designated herein as DNA12982 which comprises
the nucleotide sequence of SEQ ID NO:29.
[0350] In another embodiment, the invention provides an expressed
sequence tag (EST) designated herein as DNA15886 which comprises
the nucleotide sequence of SEQ ID NO:30.
[0351] 5. PRO296
[0352] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to the sarcoma-amplified protein SAS,
wherein the polypeptide is designated in the present application as
"PRO296".
[0353] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO296 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO296 polypeptide having amino acid residues 1 to 204 of FIG. 15
(SEQ ID NO:36), 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 other aspects,
the isolated nucleic acid comprises DNA encoding the PRO296
polypeptide having amino acid residues about 35 to 204 of FIG. 15
(SEQ ID NO:36) or amino acid 1 or about 35 to X of FIG. 15 (SEQ ID
NO:36), where X is any amino acid from 42 to 51 of FIG. 15 (SEQ ID
NO:36), 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. The isolated nucleic
acid sequence may comprise the cDNA insert of the DNA39979-1213
vector deposited on Apr. 21, 1998 as ATCC 209789 which includes the
nucleotide sequence encoding PRO296.
[0354] In another embodiment, the invention provides isolated
PRO296 polypeptide. In particular, the invention provides isolated
native sequence PRO296 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 204 of
FIG. 15 (SEQ ID NO:36). Additional embodiments of the present
invention are directed to PRO296 polypeptides comprising amino
acids about 35 to 204 of FIG. 15 (SEQ ID NO:36) or amino acid 1 or
about 35 to X of FIG. 15 (SEQ ID NO:36), where X is any amino acid
from 42 to 51 of FIG. 15 (SEQ ID NO:36). Optionally, the PRO296
polypeptide is obtained or is obtainable by expressing the
polypeptide encoded by the cDNA insert of the DNA39979-1213 vector
deposited on Apr. 21, 1998 as ATCC 209789.
[0355] In another embodiment, the invention provides an expressed
sequence tag (EST) designated herein as DNA23020 comprising the
nucleotide sequence of SEQ ID NO:37.
[0356] In another embodiment, the invention provides an expressed
sequence tag (EST) designated herein as DNA21971 comprising the
nucleotide sequence of SEQ ID NO:38.
[0357] In another embodiment, the invention provides an expressed
sequence tag (EST) designated herein as DNA29037 comprising the
nucleotide sequence of SEQ ID NO:39.
[0358] 6. PRO329
[0359] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to a high affnity immunoglobulin
F.sub.c receptor, wherein the polypeptide is designated in the
present application as "PRO329".
[0360] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO329 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO329 polypeptide having amino acid residues 1 to 359 of FIG. 20
(SEQ ID NO:45), 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. The isolated
nucleic acid sequence may comprise the cDNA insert of the
DNA40594-1233 vector deposited on Feb. 5, 1998 as ATCC 209617 which
includes the nucleotide sequence encoding PRO329.
[0361] In another embodiment, the invention provides isolated
PRO329 polypeptide. In particular, the invention provides isolated
native sequence PRO329 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 359 of
FIG. 20 (SEQ ID NO:45). Optionally, the PRO329 polypeptide is
obtained or is obtainable by expressing the polypeptide encoded by
the cDNA insert of the DNA40594-1233 vector deposited on Feb. 5,
1998 as ATCC 209617.
[0362] 7. PRO362
[0363] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to A33 antigen and HCAR membrane-bound
protein, wherein the polypeptide is designated in the present
application as "PRO362".
[0364] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO362 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO362 polypeptide having amino acid residues 1 to 321 of FIG. 22
(SEQ ID NO:52), 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 PRO362
polypeptide having amino acid residues 1 to X of FIG. 22 (SEQ ID
NO:52) where X is any amino acid from amino acid 271 to 280, 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. The isolated nucleic acid sequence may
comprise the cDNA insert of the DNA45416-1251 vector deposited on
Feb. 5, 1998 as ATCC 209620 which includes the nucleotide sequence
encoding PRO362.
[0365] In another embodiment, the invention provides isolated
PRO362 polypeptide. In particular, the invention provides isolated
native sequence PRO362 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 321 of
FIG. 22 (SEQ ID NO:52). An additional embodiment of the present
invention is directed to an isolated extracellular domain of a
PRO362 polypeptide comprising amino acids 1 to X of the amino acid
sequence shown in FIG. 22 (SEQ ID NO:52), wherein X is any amino
acid from amino acid 271 to 280. Optionally, the PRO362 polypeptide
is obtained or is obtainable by expressing the polypeptide encoded
by the cDNA insert of the DNA45416-1251 vector deposited on Feb. 5,
1998 as ATCC 209620.
[0366] 8. PRO363
[0367] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to the cell surface receptor protein
HCAR, wherein the polypeptide is designated in the present
application as "PRO363".
[0368] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO363 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO363 polypeptide having amino acid residues 1 to 373 of FIG. 24
(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 isolated nucleic acid comprises DNA encoding a PRO363
extracellular domain polypeptide having amino acid residues 1 to X
of FIG. 24 (SEQ ID NO:59) where X is any amino acid from amino acid
216 to amino acid 225, 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. The
isolated nucleic acid sequence may comprise the cDNA insert of the
DNA45419-1252 vector deposited on Feb. 5, 1998 as ATCC 209616 which
includes the nucleotide sequence encoding PRO363.
[0369] In another embodiment, the invention provides isolated
PRO363 polypeptide. In particular, the invention provides isolated
native sequence PRO363 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 373 of
FIG. 24 (SEQ ID NO:59). An additional embodiment of the present
invention is directed to an isolated extracellular domain of a
PRO363 polypeptide, wherein that extracellular domain may comprise
amino acids 1 to X of the sequence shown in FIG. 24 (SEQ ID NO:59),
where X is any amino acid from amino acid 216 to 225. Optionally,
the PRO363 polypeptide is obtained or is obtainable by expressing
the polypeptide encoded by the cDNA insert of the DNA45419-1252
vector deposited on Feb. 5, 1998 as ATCC 209616.
[0370] 9. PRO868
[0371] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to tumor necrosis factor receptor,
wherein the polypeptide is designated in the present application as
"PRO868".
[0372] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO868 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO868 polypeptide having amino acid residues 1 to 655 of FIG. 26
(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. In another
aspect, the isolated nucleic acid comprises DNA encoding the PRO868
polypeptide having amino acid residues 1 to X of FIG. 26 (SEQ ID
NO:64), where X is any amino acid from amino acid 343 to 352 of the
sequence shown in FIG. 26 (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. In yet another aspect, the isolated nucleic acid
comprises DNA encoding the PRO868 polypeptide having amino acid
residues X to 655 of FIG. 26 (SEQ ID NO:64), where X is any amino
acid from amino acid 371 to 380 of the sequence shown in FIG. 26
(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. The isolated
nucleic acid sequence may comprise the cDNA insert of the
DNA52594-1270 vector deposited on Mar. 17, 1998 as ATCC 209679
which includes the nucleotide sequence encoding PRO868.
[0373] In another embodiment, the invention provides isolated
PRO868 polypeptide. In particular, the invention provides isolated
native sequence PRO868 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 655 of
FIG. 26 (SEQ ID NO:64). In another aspect, the isolated PRO868
polypeptide comprises amino acid residues 1 to X of FIG. 26 (SEQ ID
NO:64), where X is any amino acid from amino acid 343 to 352 of the
sequence shown in FIG. 26 (SEQ ID NO:64). In yet another aspect,
the PRO868 polypeptide comprises amino acid residues X to 655 of
FIG. 26 (SEQ ID NO:64), where X is any amino acid from amino acid
371 to 380 of the sequence shown in FIG. 26 (SEQ ID NO:64).
Optionally, the PRO868 polypeptide is obtained or is obtainable by
expressing the polypeptide encoded by the cDNA insert of the
DNA52594-1270 vector deposited on Mar. 17, 1998 as ATCC 209679.
[0374] 10. PRO382
[0375] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to serine proteases, wherein the
polypeptide is designated in the present application as
"PRO382".
[0376] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO382 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO382 polypeptide having amino acid residues 1 to 453 of FIG. 28
(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. The isolated
nucleic acid sequence may comprise the cDNA insert of the
DNA45234-1277 vector deposited on Mar. 5, 1998 as ATCC 209654 which
includes the nucleotide sequence encoding PRO382.
[0377] In another embodiment, the invention provides isolated
PRO382 polypeptide. In particular, the invention provides isolated
native sequence PRO382 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 453 of
FIG. 28 (SEQ ID NO:69). An additional embodiment of the present
invention is directed to an isolated extracelular domain of a
PRO382 polypeptide, with or without the signal peptide. Optionally,
the PRO382 polypeptide is obtained or is obtainable by expressing
the polypeptide encoded by the cDNA insert of the DNA45234-1277
vector deposited on Mar. 5, 1998 as ATCC 209654.
[0378] 11. PRO545
[0379] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to meltrin, wherein the polypeptide is
designated in the present application as "PRO545".
[0380] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO545 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO545 polypeptide having amino acid residues 1 to 735 of FIG. 30
(SEQ ID NO:74), 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. The isolated
nucleic acid sequence may comprise the cDNA insert of the vector
deposited on Mar. 5, 1998 as ATCC 209655 which includes the
nucleotide sequence encoding PRO545.
[0381] In another embodiment, the invention provides isolated
PRO545 polypeptide. In particular, the invention provides isolated
native sequence PRO545 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 735 of
FIG. 30 (SEQ ID NO:74). An additional embodiment of the present
invention is directed to an isolated extraceliular domain of a
PRO545 polypeptide. Optionally, the PRO545 polypeptide is obtained
or is obtainable by expressing the polypeptide encoded by the cDNA
insert of the vector deposited on Mar. 5, 1998 as ATCC 209655.
[0382] In another embodiment, the invention provides an expressed
sequence tag (EST) designated herein as DNA13217 comprising the
nucleotide sequence of SEQ ID NO:75 (FIG. 31).
[0383] 12. PRO617
[0384] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to CD24, wherein the polypeptide is
designated in the present application as "PRO617".
[0385] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO617 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO617 polypeptide having amino acid residues 1 to 67 of FIG. 33
(SEQ ID NO:85), 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. The isolated
nucleic acid sequence may comprise the cDNA insert of the
DNA48309-1280 vector deposited on Mar. 5, 1998 as ATCC 209656 which
includes the nucleotide sequence encoding PRO617.
[0386] In another embodiment, the invention provides isolated
PRO617 polypeptide. In particular, the invention provides isolated
native sequence PRO617 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 67 of FIG.
33 (SEQ ID NO:85). Optionally, the PRO617 polypeptide is obtained
or is obtainable by expressing the polypeptide encoded by the cDNA
insert of the DNA48309-1280 vector deposited on Mar. 5, 1998 as
ATCC 209656.
[0387] 13. PRO700
[0388] Applicants have identified a cDNA clone that encodes a novel
polypeptide having sequence similarity to protein disulfide
isomerase, wherein the polypeptide is designated in the present
application as "PRO700".
[0389] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO700 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO700 polypeptide having amino acid residues 1 to 432 of FIG. 35
(SEQ ID NO:90), 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 PRO700
polypeptide having amino acid residues from about 34 to 432 of FIG.
35 (SEQ ID NO:90), 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. The
isolated nucleic acid sequence may comprise the cDNA insert of the
vector deposited on Mar. 31, 1998 as ATCC 209721 which includes the
nucleotide sequence encoding PRO700.
[0390] In another embodiment, the invention provides isolated
PRO700 polypeptide. In particular, the invention provides isolated
native sequence PRO700 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 432 of
FIG. 35 (SEQ ID NO:90). In another embodiment, the invention
provides an isolated PRO700 polypeptide absent the signal sequence,
which includes an amino acid sequence comprising residues from
about 34 to 432 of FIG. 35 (SEQ ID NO:90). Optionally, thle PRO700
polypeptide is obtained or is obtainable by expressing the
polypeptide encoded by the cDNA insert of the vector deposited on
Mar. 31, 1998 as ATCC 209721.
[0391] 14. PRO702
[0392] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to conglutinin, wherein the polypeptide
is designated in the present application as "PRO702".
[0393] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO702 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO702 polypeptide having amino acid residues 1 to 277 of FIG. 37
(SEQ ID NO:97), 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 PRO702
polypeptide having amino acid residues 26 to 277 of FIG. 37 (SEQ ID
NO:97), 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. The isolated nucleic
acid sequence may comprise the cDNA insert of the DNA50980-1286
vector deposited on Mar. 31, 1998 as ATCC 209717 which includes the
nucleotide sequence encoding PRO702.
[0394] In another embodiment, the invention provides isolated
PRO702 polypeptide. In particular, the invention provides isolated
native sequence PRO702 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 277 of
FIG. 37 (SEQ ID NO:97). An additional embodiment of the present
invention is directed to an isolated PRO702 polypeptide comprising
amino acid residues 26 to 277 of FIG. 37 (SEQ ID NO:97).
Optionally, the PRO702 polypeptide is obtained or is obtainable by
expressing the polypeptide encoded by the cDNA insert of the
DNA50980-1286 vector deposited on Mar. 31, 1998 as ATCC 209717.
[0395] 15. PRO703
[0396] Applicants have identified a cDNA clone that encodes a novel
polypeptide having sequence similarity to VLCAS, wherein the
polypeptide is designated in the present application as
"PRO703".
[0397] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO703 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO703 polypeptide having amino acid residues 1 to 730 of FIG. 39
(SEQ ID NO:102), 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 PRO703
polypeptide having amino acid residues from about 43 to 730 of FIG.
39 (SEQ ID NO:102), 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. The
isolated nucleic acid sequence may comprise the cDNA insert of the
DNA50913-1287 vector deposited on Mar. 31, 1998 as ATCC 209716
which includes the nucleotide sequence encoding PRO703.
[0398] In another embodiment, the invention provides isolated
PRO703 polypeptide. In particular, the invention provides isolated
native sequence PRO703 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 730 of
FIG. 39 (SEQ ID NO:102). In another embodiment, the invention
provides an isolated PRO703 polypeptide absent the signal sequence,
which includes an amino acid sequence comprising residues from
about 43 to 730 of FIG. 30 (SEQ ID NO:102). Optionally, the PRO730
polypeptide is obtained or is obtainable by expressing the
polypeptide encoded by the cDNA insert of the DNA50913-1287 vector
deposited on Mar. 31, 1998 as ATCC 209716.
[0399] 16. PRO705
[0400] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to K-glypican, wherein the polypeptide
is designated in the present application as "PRO705".
[0401] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO705 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO705 polypeptide having amino acid residues 1 to 555 of FIG. 41
(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 isolated nucleic acid comprises DNA encoding the PRO705
polypeptide having amino acid residues about 24 to 555 of FIG. 41
(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. The isolated
nucleic acid sequence may comprise the cDNA insert of the
DNA50914-1289 vector deposited on Mar. 31, 1998 as ATCC 209722
which includes the nucleotide sequence encoding PRO705.
[0402] In another embodiment, the invention provides isolated
PRO705 polypeptide. In particular, the invention provides isolated
native sequence PRO705 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 555 of
FIG. 41 (SEQ ID NO:109). An additional embodiment of the present
invention is directed to an isolated PRO705 polypeptide comprising
amino acid residues about 24 to 555 of FIG. 41 (SEQ ID NO:109).
Optionally, the PRO705 polypeptide is obtained or is obtainable by
expressing the polypeptide encoded by the cDNA insert of the
DNA50914-1289 vector deposited on Mar. 31, 1998 as ATCC 209722.
[0403] 17. PRO708
[0404] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to the aryl sulfatases, wherein the
polypeptide is designated in the present application as
"PRO708".
[0405] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO708 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO708 polypeptide having amino acid residues 1 to 515 of FIG. 43
(SEQ ID NO:114), 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. The isolated
nucleic acid sequence may comprise the cDNA insert of the
DNA48296-1292 vector deposited on Mar. 11, 1998 as ATCC 209668
which includes the nucleotide sequence encoding PRO708.
[0406] In another embodiment, the invention provides isolated
PRO708 polypeptide. In particular, the invention provides isolated
native sequence PRO708 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 515 of
FIG. 43 (SEQ ID NO:114). Another embodiment is directed to a PRO708
polypeptide comprising residues 38-515 of the amino acid sequence
shown in FIG. 43 (SEQ ID NO:114). Optionally, the PRO708
polypeptide is obtained or is obtainable by expressing the
polypeptide encoded by the cDNA insert of the DNA48296-1292 vector
deposited on Mar. 11, 1998 as ATCC 209668.
[0407] 18. PRO320
[0408] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to fibulin, wherein the polypeptide is
designated in the present application as "PRO320".
[0409] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO320 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO320 polypeptide having amino acid residues 1 to 338 of FIG. 45
(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. The isolated
nucleic acid sequence may comprise the cDNA insert of the vector
deposited on Mar. 11, 1998 as ATCC 209670 which includes the
nucleotide sequence encoding PRO320.
[0410] In another embodiment, the invention provides isolated
PRO320 polypeptide. In particular, the invention provides isolated
native sequence PRO320 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 338 of
FIG. 45 (SEQ ID NO:119). Optionally, the PRO320 polypeptide is
obtained or is obtainable by expressing the polypeptide encoded by
the cDNA insert of the vector deposited on Mar. 11, 1998 as ATCC
209670.
[0411] 19. PRO324
[0412] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to oxidoreductases, wherein the
polypeptide is designated in the present application as
"PRO324".
[0413] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO324 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO324 polypeptide having amino acid residues 1 to 289 of FIG. 47
(SEQ ID NO:124), 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 other aspects,
the isolated nucleic acid comprises DNA encoding the PRO324
polypeptide having amino acid residues 1 or about 32 to X of FIG.
47 (SEQ ID NO:124), where X is any amino acid from 131 to 140, 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. The isolated nucleic acid
sequence may comprise the cDNA insert of the DNA36343-1310 vector
deposited on Mar. 30, 1998 as ATCC 209718 which includes the
nucleotide sequence encoding PRO324.
[0414] In another embodiment, the invention provides isolated
PRO324 polypeptide. In particular, the invention provides isolated
native sequence PRO324 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 289 of
FIG. 47 (SEQ ID NO:124). The invention also provides isolated
PRO324 polypeptide comprising residues 1 or about 32 to X of FIG.
47 (SEQ ID NO:124), wherein X is any amino acid from about 131-140.
Optionally, the PRO324 polypeptide is obtained or is obtainable by
expressing the polypeptide encoded by the cDNA insert of the
DNA36343-1310 vector deposited on Mar. 30, 1998 as ATCC 209718.
[0415] 20. PRO351
[0416] Applicants have identified a cDNA clone that encodes a novel
polypeptide having sequence similarity to prostasin, wherein the
polypeptide is designated in the present application as
"PRO351".
[0417] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO351 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO351 polypeptide having amino acid residues 1 to 571 of FIG. 49
(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. In another
aspect, the isolated nucleic acid comprises DNA encoding the PRO351
polypeptide having amino acid residues about 16 to 571 of FIG. 49
(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. The isolated
nucleic acid sequence may comprise the cDNA insert of the
DNA40571-1315 vector deposited on Apr. 21, 1998 as ATCC 209784
which includes the nucleotide sequence encoding PRO351.
[0418] In another embodiment, the invention provides isolated
PRO351 polypeptide. In particular, the invention provides isolated
native sequence PRO351 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 571 of
FIG. 49 (SEQ ID NO:132). In another embodiment, the invention
provides an isolated PRO351 polypeptide absent the signal sequence,
which includes an amino acid sequence comprising residues from
about 16 to 571 of FIG. 49 (SEQ ID NO:132). Optionally, the PRO351
polypeptide is obtained or is obtainable by expressing the
polypeptide encoded by the cDNA insert of the DNA40571-1315 vector
deposited on Apr. 21, 1998 as ATCC 209784.
[0419] 21. PRO352
[0420] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to butyrophilin, wherein the
polypeptide is designated in the present application as
"PRO352".
[0421] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO352 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO352 polypeptide having amino acid residues 1 to 316 of FIG. 51
(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 other aspects,
the isolated nucleic acid comprises DNA encoding the PRO352
polypeptide having amino acid residues of about 29 to 316 of FIG.
51 (SEQ ID NO:137), or 1 or about 29 to X of FIG. 51, where X is
any amino acid from 246 to 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. The isolated nucleic acid sequence may comprise the
cDNA insert of the DNA41386-1316 vector deposited on Mar. 26, 1998
as ATCC 209703 which includes the nucleotide sequence encoding
PRO352.
[0422] In another embodiment, the invention provides isolated
PRO352 polypeptide. In particular, the invention provides isolated
native sequence PRO352 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 316 of
FIG. 51 (SEQ ID NO:137). In other embodiments, the invention
provides isolated PRO352 polypeptide comprising residues about 29
to 316 of FIG. 51 (SEQ ID NO:137) and 1 or about 29 to X of FIG. 51
(SEQ ID NO:137), wherein X is any amino acid from 246 to 255.
Optionally, the PRO352 polypeptide is obtained or is obtainable by
expressing the polypeptide encoded by the cDNA insert of the
DNA41386-1316 vector deposited on Mar. 26, 1998 as ATCC 209703.
[0423] 22. PRO381
[0424] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to immunophilin proteins, wherein the
polypeptide is designated in the present application as
"PRO381".
[0425] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO381 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO381 polypeptide having amino acid residues 1 to 211 of FIG. 53
(SEQ ID NO:145), 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 PRO381
polypeptide having amino acid residues about 21 to 211 of FIG. 53
(SEQ ID NO:145), 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. The isolated
nucleic acid sequence may comprise the cDNA insert of the
DNA44194-1317 vector deposited on Apr. 28, 1998 as ATCC 209808
which includes the mncleotide sequence encoding PRO381.
[0426] In another embodiment, the invention provides isolated
PRO381 polypeptide. In particular, the invention provides isolated
native sequence PRO381 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 211 of
FIG. 53 (SEQ ID NO:145). Another embodiment is directed to a PRO381
plypeptide comprising amino acids about 21 to 211 of FIG. 53 (SEQ
ID NO:145). Optionally, the PRO381 polypeptide is obtained or is
obtainable by expressing the polypeptide encoded by the cDNA insert
of the DNA44194-1317 vector deposited on Apr. 28, 1998 as ATCC
209808.
[0427] 23. PRO386
[0428] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to the beta-2 subunit of a sodium
channel, wherein the polypeptide is designated in the present
application as "PRO386".
[0429] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO386 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO386 polypeptide having amino acid residues 1 to 215 of FIG. 55
(SEQ ID NO:150), 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 PRO386
polypeptide having amino acid residues about 21 to 215 of FIG. 55
(SEQ ID NO:150) or 1 or about 21 to X, where X is any amino acid
from 156 to 165 of FIG. 55 (SEQ ID NO:150), 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. The isolated nucleic acid sequence may comprise the
cDNA insert of the DNA45415-1318 vector deposited on Apr. 28, 1998
as ATCC 209810 which includes the nucleotide sequence encoding
PRO386.
[0430] In another embodiment, the invention provides isolated
PRO386 polypeptide. In particular, the invention provides isolated
native sequence PRO386 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 215 of
FIG. 55 (SEQ ID NO:150). Other embodiments of the present invention
are directed to PRO386 polypeptides comprising amino acids about 21
to 215 of FIG. 55 (SEQ ID NO:150) and 1 or about 21 to X of FIG. 55
(SEQ ID NO:150), wherein X is any amino acid from 156 to 165 of
FIG. 55 (SEQ ID NO:150). Optionally, the PRO386 polypeptide is
obtained or is obtainable by expressing the polypeptide encoded by
the cDNA insert of the DNA45415-1318 vector deposited on Apr. 28,
1998 as ATCC 209810.
[0431] In another embodiment, the invention provides an expressed
sequence tag (EST) comprising the nucleotide sequence of SEQ ID
NO:151 which corrsponds to an EST designated herein as
DNA23350.
[0432] In another embodiment, the invention provides an expressed
sequence tag (EST) comprising the nucleotide sequence of SEQ ID
NO:152 which corrsponds to an EST designated herein as
DNA23536.
[0433] 24. PRO540
[0434] Applicants have identified a cDNA clone that encodes a novel
polypeptide having sequence similarity to LCAT, wherein the
polypeptide is designated in the present application as
"PRO540".
[0435] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO540 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO540 polypeptide having amino acid residues 1 to 412 of FIG. 59
(SEQ ID NO:157), 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 PRO540
polypeptide having amino acid residues about 29 to 412 of FIG. 59
(SEQ ID NO:157), 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. The isolated
nucleic acid sequence may comprise the cDNA insert of the
DNA44189-1322 vector deposited on Mar. 26, 1998 as ATCC 209699
which includes the nucleotide sequence encoding PRO540.
[0436] In another embodiment, the invention provides isolated
PRO540 polypeptide. In particular, the invention provides isolated
native sequence PRO540 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 412 of
FIG. 59 (SEQ ID NO:157). The invention also provides isolated
PRO540 polypeptide, which in one embodiment, includes an amino acid
sequence comprising residues about 29 to 412 of FIG. 59 (SEQ ID
NO:157). Optionally, the PRO540 polypeptide is obtained or is
obtainable by expressing the polypeptide encoded by the cDNA insert
of the DNA44189-1322 vector deposited on Mar. 26, 1998 as ATCC
209699.
[0437] 25. PRO615
[0438] Applicants have identified a cDNA clone that encodes a novel
polypeptide having sequence similarity to synaptogyrin, wherein the
polypeptide is designated in the present application as
"PRO615".
[0439] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO615 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO615 polypeptide having amino acid residues 1 to 224 of FIG. 61
(SEQ ID NO:162), 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 PRO615
polypeptide having amino acid residues X to 224 of FIG. 61 (SEQ ID
NO:162), where X is any amino acid from 157 to 166, 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. The isolated nucleic acid sequence may
comprise the cDNA insert of the DNA48304-1323 vector deposited on
Apr. 28, 1998 as ATCC 209811 which includes the nucleotide sequence
encoding PRO615.
[0440] In another embodiment, the invention provides isolated
PRO615 polypeptide. In particular, the invention provides isolated
native sequence PRO615 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 224 of
FIG. 61 (SEQ ID NO:162). An additional embodiment of the present
invention is directed to an isolated extracellular domain of a
PRO615 polypeptide which comprises amino acid residues X to 224 of
FIG. 61 (SEQ ID NO:162), where X is any amino acid from 157 to 166
of FIG. 61 (SEQ ID NO:162). Optionally, the PRO615 polypeptide is
obtained or is obtainable by expressing the polypeptide encoded by
the cDNA insert of the DNA48304-1323 vector deposited on Apr. 28,
1998 as ATCC 209811.
[0441] 26. PRO618
[0442] Applicants have identified a cDNA clone that encodes a novel
polypeptide having sequence similarity to enteropeptidase, wherein
the polypeptide is designated in the present application as
"PRO618".
[0443] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO618 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO618 polypeptide having amino acid residues 1 to 802 of FIG. 63
(SEQ ID NO:169), 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 an
isolated extracellular domain of a PRO618 polypeptide having amino
acid residues X to 802 of FIG. 63 (SEQ ID NO:169), where X is any
amino acid from 63 to 72 of FIG. 63 (SEQ ID NO:169), 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. The isolated nucleic acid sequence may
comprise the cDNA insert of the DNA49152-1324 vector deposited on
Apr. 28, 1998 as ATCC 209813 which includes the nucleotide sequence
encoding PRO618.
[0444] In another embodiment, the invention provides isolated
PRO618 polypeptide. In particular, the invention provides isolated
native sequence PRO618 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 802 of
FIG. 63 (SEQ ID NO:169). An additional embodiment of the present
invention is directed to an isolated extracellular domain of a
PRO618 polypeptide comprising amino acid X to 802 where X is any
amino acid from 63 to 72 of FIG. 63 (SEQ ID NO:169). Optionally,
the PRO618 polypeptide is obtained or is obtainable by expressing
the polypeptide encoded by the cDNA insert of the DNA49152-1324
vector deposited on Apr. 28, 1998 as ATCC 209813.
[0445] In another embodiment, the invention provides an expressed
sequence tag (EST) comprising the nucleotide sequence of SEQ ID
NO:170, designated herein as DNA35597 (see FIG. 64).
[0446] 27. PRO719
[0447] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to lipoprotein lipase H, wherein the
polypeptide is designated in the present application as
"PRO719".
[0448] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO719 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO719 polypeptide having amino acid residues 1 to 354 of FIG. 66
(SEQ ID NO:178), 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 PRO719
polypeptide having amino acid residues about 17 to 354 of FIG. 66
(SEQ ID NO:178), 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. The isolated
nucleic acid sequence may comprise the cDNA insert of the
DNA49646-1327 vector deposited on Mar. 26, 1998 as ATCC 209705
which includes the nucleotide sequence encoding PRO719.
[0449] In another embodiment, the invention provides isolated
PRO719 polypeptide. In particular, the invention provides isolated
native sequence PRO719 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 354 of
FIG. 66 (SEQ ID NO:178). In another embodiment, the invention
provides isolated PRO719 polypeptide which comprises residues about
17 to 354 of FIG. 66 (SEQ ID NO:178). Optionally, the PRO719
polypeptide is obtained or is obtainable by expressing the
polypeptide encoded by the cDNA insert of the DNA49646-1327 vector
deposited on Mar. 26, 1998 as ATCC 209705.
[0450] 28. PRO724
[0451] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to the LDL receptor, wherein the
polypeptide is designated in the present application as
"PRO724".
[0452] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO724 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO724 polypeptide having amino acid residues 1 to 713 of FIG. 68
(SEQ ID NO:183), 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 a soluble
PRO724 polypeptide having amino acid residues 1 to X of FIG. 68
(SEQ ID NO:183) where X is any amino acid from amino acid 437 to
446, 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. The above two
polypeptides may either possess or not possess the signal peptide.
The isolated nucleic acid sequence may comprise the cDNA insert of
the DNA49631-1328 vector deposited on Apr. 28, 1998 as ATCC 209806
which includes the nucleotide sequence encoding PRO724.
[0453] In another embodiment, the invention provides isolated
PRO724 polypeptide. In particular, the invention provides isolated
native sequence PRO724 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 713 of
FIG. 68 (SEQ ID NO:183). In another embodiment, the invention
provides isolated soluble PRO724 polypeptide. In particular, the
invention provides isolated soluble PRO724 polypeptide, which in
one embodiment, includes an amino acid sequence comprising residues
1 to X of FIG. 68 (SEQ ID NO:183), where X is any amino acid from
437 to 446 of the sequence shown in FIG. 68 (SEQ ID NO:183).
Optionally, the PRO724 polypeptide is obtained or is obtainable by
expressing the polypeptide encoded by the cDNA insert of the
DNA49631-1328 vector deposited on Apr. 28, 1998 as ATCC 209806.
[0454] 29. PRO772
[0455] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to A4 protein, wherein the polypeptide
is designated in the present application as "PRO772".
[0456] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO772 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO772 polypeptide having amino acid residues 1 to 152 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. In other aspects,
the isolated nucleic acid comprises DNA encoding the PRO772
polypeptide having amino acid residues 1 to X of FIG. 70 (SEQ ID
NO:190), where X is any amino acid from 21 to 30 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. The isolated
nucleic acid sequence may comprise the cDNA insert of the
DNA49645-1347 vector deposited on Apr. 28, 1998 as ATCC 209809
which includes the nucleotide sequence encoding PRO772.
[0457] In another embodiment, the invention provides isolated
PRO772 polypeptide. In particular, the invention provides isolated
native sequence PRO772 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 152 of
FIG. 70 (SEQ ID NO:190). Additional embodiments of the present
invention are directed to PRO772 polypeptides comprising amino
acids 1 to X of FIG. 70 (SEQ ID NO:190), where X is any amino acid
from 21 to 30 of FIG. 70 (SEQ ID NO:190). Optionally, the PRO772
polypeptide is obtained or is obtainable by expressing the
polypeptide encoded by the cDNA insert of the DNA49645-1347 vector
deposited on Apr. 28, 1998 as ATCC 209809.
[0458] In another embodiment, the invention provides an expressed
sequence tag (EST) designated herein as DNA43509 comprising the
nucleotide sequence of SEQ ID NO:191 (FIG. 71).
[0459] 30. PRO852
[0460] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to various protease enzymes, wherein
the polypeptide is designated in the present application as
"PRO852".
[0461] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO852 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO852 polypeptide having amino acid residues 1 to 518 of FIG. 73
(SEQ ID NO:196), 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 other aspects,
the isolated nucleic acid comprises DNA encoding the PRO852
polypeptide having amino acid residues about 21 to 518 of FIG. 73
(SEQ ID NO:196) or 1 or about 21 to X of FIG. 73 (SEQ ID NO:196)
where X is any amino acid from amino acid 461 to amino acid 470 of
FIG. 73 (SEQ ID NO:196), 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.
The isolated nucleic acid sequence may comprise the cDNA insert of
the DNA45493-1349 vector deposited on Apr. 28, 1998 as ATCC 209805
which includes the nucleotide sequence encoding PRO852.
[0462] In another embodiment, the invention provides isolated
PRO852 polypeptide. In particular, the invention provides isolated
native sequence PRO852 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 518 of
FIG. 73 (SEQ ID NO:196). In other embodiments, the PRO852 comprises
amino acids about 21 to amino acid 518 of FIG. 73 (SEQ ID NO:196)
or amino acids 1 or about 21 to X of FIG. 73 (SEQ ID NO:196), where
X is any amino acid from amino acid 461 to amino acid 470 of FIG.
73 (SEQ ID NO:196). Optionally, the PRO852 polypeptide is obtained
or is obtainable by expressing the polypeptide encoded by the cDNA
insert of the DNA45493-1349 vector deposited on Apr. 28, 1998 as
ATCC 209805.
[0463] 31. PRO853
[0464] Applicants have identified a cDNA clone that encodes a novel
polypeptide having sequence simllarity to reductase, wherein the
polypeptide is designated in the present application as
"PRO853".
[0465] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO853 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO853 polypeptide having amino acid residues 1 to 377 of FIG. 75
(SEQ ID NO:206), 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 PRO853
polypeptide having amino acid residues about 17 to 377 of FIG. 75
(SEQ ID NO:206), 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. The isolated
nucleic acid sequence may comprise the cDNA insert of the
DNA48227-1350 vector deposited on Apr. 28, 1998 as ATCC 209812
which includes the nucleotide sequence encoding PRO853.
[0466] In another embodiment, the invention provides isolated
PRO853 polypeptide. In particular, the invention provides isolated
native sequence PRO853 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 377 of
FIG. 75 (SEQ ID NO:206). In another embodiment, the invention
provides an isolated PRO853 polypeptide absent the signal sequence,
which includes an amino acid sequence comprising residues from
about 17 to 377 of FIG. 75 (SEQ ID NO:206). Optionally, the PRO853
polypeptide is obtained or is obtainable by expressing the
polypeptide encoded by the cDNA insert of the DNA48227-1350 vector
deposited on Apr. 28, 1998 as ATCC 209812.
[0467] 32. PRO860
[0468] Applicants have identified a cDNA clone that encodes a novel
polypeptide having sequence similarity to neurofascin, wherein the
polypeptide is designated in the present application as
"PRO860".
[0469] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO860 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO860 polypeptide having amino acid residues 1 to 985 of FIG. 77
(SEQ ID NO:211), 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 PRO860
polypeptide having amino acid residues 1 to X of FIG. 77 (SEQ ID
NO:211), where X is any amino acid from 443-452 of FIG. 77 (SEQ ID
NO:211), 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. The isolated
nucleic acid sequence may comprise the cDNA insert of the
DNA41404-1352 vector deposited on May 6, 1998 as ATCC 209844 which
includes the nucleotide sequence encoding PRO860.
[0470] In another embodiment, the invention provides isolated
PRO860 polypeptide. In particular, the invention provides isolated
native sequence PRO860 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 985 of
FIG. 77 (SEQ ID NO:211). In another embodiment, the invention
provides an isolated PRO860 polypeptide which includes an amino
acid sequence comprising residues 1 to X of FIG. 77 (SEQ ID
NO:211), where X is any amino acid residue from 443 to 452 of FIG.
77 (SEQ ID NO:211). Optionally, the PRO860 polypeptide is obtained
or is obtainable by expressing the polypeptide encoded by the cDNA
insert of the DNA41404-1352 vector deposited on May 6, 1998 as ATCC
209844.
[0471] 33. PRO846
[0472] Applicants have identified a cDNA clone that encodes a novel
polypeptide having sequence similarity to CMRF35, wherein the
polypeptide is designated in the present application as
"PRO846".
[0473] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO846 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO846 polypeptide having amino acid residues 1 to 332 of FIG. 79
(SEQ ID NO:216), 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 other aspects,
the isolated nucleic acid comprises DNA encoding the PRO846
polypeptide having amino acid residues about 18 to 332 of FIG. 79
(SEQ ID NO:216) or 1 or about 18 to X of SEQ ID NO:216, where X is
any amino acid from 243 to 252 of FIG. 79 (SEQ ID NO:216), 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. The isolated nucleic acid sequence may
comprise the cDNA insert of the DNA44196-1353 vector deposited on
May 6, 1998 as ATCC 209847 which includes the nucleotide sequence
encoding PRO846.
[0474] In another embodiment, the invention provides isolated
PRO846 polypeptide. In particular, the invention provides isolated
native sequence PRO846 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 332 of
FIG. 79 (SEQ ID NO:216). In other embodiments, the invention
provides an isolated PRO846 polypeptide absent the signal sequence,
which includes an amino acid sequence comprising residues from
about 18 to 332 of FIG. 79 (SEQ ID NO:216). Additional embodiments
of the present invention are directed to an isolated PRO846
polypeptide comprising amino acid 1 or about 18 to X of FIG. 79
(SEQ ID NO:216), where X is any amino acid from 243 to 252 of FIG.
79 (SEQ ID NO:216). Optionally, the PRO846 polypeptide is obtained
or is obtainable by expressing the polypeptide encoded by the cDNA
insert of the DNA44196-1353 vector deposited on May 6, 1998 as ATCC
209847.
[0475] 34. PRO862
[0476] Applicants have identified a cDNA clone that encodes a novel
polypeptide having sequence similarity to lysozyme, wherein the
polypeptide is designated in the present application as
"PRO862".
[0477] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising ]DNA encoding a PRO862
polypeptide. In one aspect, the isolated nucleic acid comprises DNA
encoding the PRO862 polypeptide having amino acid residues 1 to 146
of FIG. 81 (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.
In another aspect, the isolated nucleic acid comprises DNA encoding
the PRO862 polypeptide having amino acid residues about 19 to 146
of FIG. 81 (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.
The isolated nucleic acid sequence may comprise the cDNA insert of
the DNA52187-1354 vector deposited on May 6, 1998 as ATCC 209845
which includes the nucleotide sequence encoding PRO862.
[0478] In another embodiment, the invention provides isolated
PRO862 polypeptide. In particular, the invention provides isolated
native sequence PRO862 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 146 of
FIG. 81 (SEQ ID NO:221). In another embodiment, the invention
provides an isolated PRO862 polypeptide absent the signal sequence,
which includes an amino acid sequence comprising residues from
about 19 to 146 of FIG. 81 (SEQ ID NO:221). Optionally, the PRO862
polypeptide is obtained or is obtainable by expressing the
polypeptide encoded by the cDNA insert of the DNA52187-1354 vector
deposited on May 6, 1998 as ATCC 209845.
[0479] 35. PRO864
[0480] Applicants have identified a cDNA clone that encodes a novel
polypeptide having sequence similarity to Wnt-4, wherein the
polypeptide is designated in the present application as
"PRO864".
[0481] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO864 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO864 polypeptide having amino acid residues 1 to 351 of FIG. 83
(SEQ ID NO:226), 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 PRO864
polypeptide having amino acid residues about 23 to 351 of FIG. 83
(SEQ ID NO:226), 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. The isolated
nucleic acid sequence may comprise the cDNA insert of the
DNA48328-1355 vector deposited on May 6, 1998 as ATCC 209843 which
includes the nucleotide sequence encoding PRO864.
[0482] In another embodiment, the invention provides isolated
PRO864 polypeptide. In particular, the invention provides isolated
native sequence PRO864 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 351 of
FIG. 83 (SEQ ID NO:226). In another embodiment, the invention
provides an isolated PRO864 polypeptide absent the signal sequence,
which includes an amino acid sequence comprising residues from
about 23 to 351 of FIG. 83 (SEQ ID NO:226). Optionally, the PRO864
polypeptide is obtained or is obtainable by expressing the
polypeptide encoded by the cDNA insert of the DNA48328-1355 vector
deposited on May 6, 1998 as ATCC 209843.
[0483] 36. PRO792
[0484] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to CD23, wherein the polypeptide is
designated in the present application as "PRO792".
[0485] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO792 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO792 polypeptide having amino acid residues 1 to 293 of FIG. 85
(SEQ ID NO:231), 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 PRO792
polypeptide having amino acid residues X to 293 of FIG. 85 (SEQ ID
NO:231) where X is any amino acid from 50 to 59 of FIG. 85 (SEQ ID
NO:231), 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. The isolated
nucleic acid sequence may comprise the cDNA insert of the
DNA56352-1358 vector deposited on May 6, 1998 as ATCC 209846 which
includes the nucleotide sequence encoding PRO792.
[0486] In another embodiment, the invention provides isolated
PRO792 polypeptide. In particular, the invention provides isolated
native sequence PRO792 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 293 of
FIG. 85 (SEQ ID NO:231). An additional embodiment of the present
invention is directed to PRO792 polypeptide comprising amino acids
X to 293 of FIG. 85 (SEQ ID NO:231), where X is any amino acid from
50 to 59 of FIG. 85 (SEQ ID NO:231). Optionally, the PRO792
polypeptide is obtained or is obtainable by expressing the
polypeptide encoded by the cDNA insert of the DNA56352-1358 vector
deposited on May 6, 1998 as ATCC 209846.
[0487] 37. PRO866
[0488] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to mindin and spondin proteins, wherein
the polypeptide is designated in the present application as
"PRO866".
[0489] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO866 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO866 polypeptide having amino acid residues 1 to 331 of FIG. 87
(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. In another
aspect, the isolated nucleic acid comprises DNA encoding the PRO866
polypeptide having amino acid residues about 27 to 229 of FIG. 87
(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. The isolated
nucleic acid sequence may comprise the cDNA insert of the
DNA53971-1359 vector deposited on Apr. 7, 1998 as ATCC 209750 which
includes the nucleotide sequence encoding PRO866.
[0490] In another embodiment, the invention provides isolated
PRO866 polypeptide. In particular, the invention provides isolated
native sequence PRO866 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 331 of
FIG. 87 (SEQ ID NO:236). Another embodiment of the present
invention is directed to PRO866 polypeptides comprising amino acids
about 27 to 331 of FIG. 87 (SEQ ID NO:236). Optionally, the PRO866
polypeptide is obtained or is obtainable by expressing the
polypeptide encoded by the cDNA insert of the DNA53971-1359 vector
deposited on Apr. 7, 1998 as ATCC 209750.
[0491] 38. PRO871
[0492] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to CyP-60, wherein the polypeptide is
designated in the present application as "PRO871".
[0493] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO871 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO871 polypeptide having amino acid residues 1 to 472 of FIG. 89
(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. In other aspects,
the isolated nucleic acid comprises DNA encoding the PRO871
polypeptide having amino acid residues about 22 to 472 of FIG. 89
(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. The isolated
nucleic acid sequence may comprise the cDNA insert of the
DNA50919-1361 vector deposited on May 6, 1998 as ATCC 209848 which
includes the nucleotide sequence encoding PRO871.
[0494] In another embodiment, the invention provides isolated
PRO871 polypeptide. In particular, the invention provides isolated
native sequence PRO871 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 472 of
FIG. 89 (SEQ ID NO:245). An additional embodiment of the present
invention is directed to PRO871 polypeptides comprising amino acids
about 22 to 472 of FIG. 89 (SEQ ID NO:245). Optionally, the PRO871
polypeptide is obtained or is obtainable by expressing the
polypeptide encoded by the cDNA insert of the DNA50919-1361 vector
deposited on May 6, 1998 as ATCC 209848.
[0495] 39. PRO873
[0496] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to carboxylesterase, wherein the
polypeptide is designated in the present application as
"PRO873".
[0497] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO873 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO873 polypeptide having amino acid residues 1 to 545 of FIG. 91
(SEQ ID NO:254), 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 other aspects,
the isolated nucleic acid comprises DNA encoding the PRO873
polypeptide having amino acid residues about 30 to about 545 of
FIG. 91 (SEQ ID NO:254), 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.
The isolated nucleic acid sequence may comprise the cDNA insert of
the DNA44179-1362 vector deposited on May 6, 1998 as ATCC 209851
which includes the nucleotide sequence encoding PRO873.
[0498] In another embodiment, the invention provides isolated
PRO873 polypeptide. In particular, the invention provides isolated
native sequence PRO873 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 545 of
FIG. 91 (SEQ ID NO:254). Additional embodiments of the present
invention are directed to PRO873 polypeptides comprising amino
acids about 30 to about 545 of FIG. 91 (SEQ ID NO:254). Optionally,
the PRO873 polypeptide is obtained or is obtainable by expressing
the polypeptide encoded by the cDNA insert of the DNA44179-1362
vector deposited on May 6, 1998 as ATCC 209851.
[0499] 40. PRO940
[0500] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to CD33 and OB binding protein-2,
wherein the polypeptide is designated in the present application as
"PRO940".
[0501] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO940 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO940 polypeptide having amino acid residues 1 to 544 of FIG. 93
(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. In other aspects,
the isolated nucleic acid comprises DNA encoding the PRO940
polypeptide having amino acid residues about 16 to 544 of FIG. 93
(SEQ ID NO:259) or 1 or about 16 to X of FIG. 93 (SEQ ID NO:259),
where X is any amino acid from 394 to 403 of FIG. 93 (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. The isolated
nucleic acid sequence may comprise the cDNA insert of the
DNA54002-1367 vector deposited on Apr. 7, 1998 as ATCC 209754 which
includes the nucleotide sequence encoding PRO940.
[0502] In another embodiment, the invention provides isolated
PRO940 polypeptide. In particular, the invention provides isolated
native sequence PRO940 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 544 of
FIG. 93 (SEQ ID NO:259). Other embodiments of the present invention
are directed to PRO940 polypeptides comprising amino acids about 16
to 544 of FIG. 93 (SEQ ID NO:259) or 1 or about 16 to X of FIG. 93
(SEQ ID NO:259), where X is any amino acid from 394 to 403 of FIG.
93 (SEQ ID NO:259). Optionally, the PRO940 polypeptide is obtained
or is obtainable by expressing the polypeptide encoded by the cDNA
insert of the DNA54002-1367 vector deposited on Apr. 7, 1998 as
ATCC 209754.
[0503] 41. PRO941
[0504] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to a cadherin protein, wherein the
polypeptide is designated in the present application as
"PRO941".
[0505] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO941 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO941 polypeptide having amino acid residues 1 to 772 of FIG. 95
(SEQ ID NO:264), 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 other aspects,
the isolated nucleic acid comprises DNA encoding the PRO941
polypeptide having amino acid residues about 22 to 772 of FIG. 95
(SEQ ID NO:264) or 1 or about 22 to X of FIG. 95 (SEQ ID NO:264),
where X is any amino acid from 592 to 601 of FIG. 95 (SEQ ID
NO:264), 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. The isolated
nucleic acid sequence may comprise the cDNA insert of the
DNA53906-1368 vector deposited on Apr. 7, 1998 as ATCC 209747 which
includes the nucleotide sequence encoding PRO941.
[0506] In another embodiment, the invention provides isolated
PRO941 polypeptide. In particular, the invention provides isolated
native sequence PRO941 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 772 of
FIG. 95 (SEQ ID NO:264). Additional embodiments of the present
invention are directed to PRO941 polypeptides which comprise amino
acid about 21 to 772 of FIG. 95 (SEQ ID NO:264) or 1 or about 22 to
X of FIG. 95 (SEQ ID NO:264), where X is any amino acid from 592 to
601 of FIG. 95 (SEQ ID NO:264). Optionally, the PRO941 polypeptide
is obtained or is obtainable by expressing the polypeptide encoded
by the cDNA insert of the DNA53906-1368 vector deposited on Apr. 7,
1998 as ATCC 209747.
[0507] In another embodiment, the invention provides an expressed
sequence tag (EST) designated herein as DNA6415 comprising the
nucleotide sequence of FIG. 96 (SEQ ID NO:265).
[0508] 42. PRO944
[0509] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to Clostridium perfringens enterotoxin
receptor (CPE-R), wherein the polypeptide is designated in the
present application as "PRO944".
[0510] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO944 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO944 polypeptide having amino acid residues 1 to 211 of FIG. 98
(SEQ ID NO:270), 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 other aspects,
the isolated nucleic acid comprises DNA encoding the PRO944
polypeptide having amino acid residues about 22 to 229 of FIG. 98
(SEQ ID NO:270) or amino acid 1 or about 22 to X of FIG. 98 (SEQ ID
NO:270) where X is any amino acid from 77 to 80 of FIG. 98 (SEQ ID
NO:270), 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. The isolated
nucleic acid sequence may comprise the cDNA insert of the
DNA52185-1370 vector deposited on May 14, 1998 as ATCC 209861 which
includes the nucleotide sequence encoding PRO944.
[0511] In another embodiment, the invention provides isolated
PRO944 polypeptide. In particular, the invention provides isolated
native sequence PRO944 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 211 of
FIG. 98 (SEQ ID NO:270). Additional embodiments of the present
invention are directed to PRO944 polypeptides comprising amino
acids about 22 to 211 of FIG. 98 (SEQ ID NO:270) or amino acid 1 or
about 22 to X of FIG. 98 (SEQ ID NO:270), where X is any amino acid
from 77 to 86 of FIG. 98 (SEQ ID NO:270). Optionally, the PRO944
polypeptide is obtained or is obtainable by expressing the
polypeptide encoded by the cDNA insert of the DNA52185-1370 vector
deposited on May 14, 1998 as ATCC 209861.
[0512] In another embodiment, the invention provides an expressed
sequence tag (EST) designated herein as DNA14007 comprising the
nucleotide sequence of FIG. 99 (SEQ ID NO:271).
[0513] In another embodiment, the invention provides an expressed
sequence tag (EST) designated herein as DNA12733 comprising the
nucleotide sequence of FIG. 100 (SEQ ID NO:272).
[0514] In another embodiment, the invention provides an expressed
sequence tag (EST) designated herein as DNA12746 comprising the
nucleotide sequence of FIG. 101 (SEQ ID NO:273).
[0515] In another embodiment, the invention provides an expressed
sequence tag (EST) designated herein as DNA12834 comprising the
nucleotide sequence of FIG. 102 (SEQ ID NO:274).
[0516] In another embodiment, the invention provides an expressed
sequence tag (EST) designated herein as DNA12846 comprising the
nucleotide sequence of FIG. 103 (SEQ ID NO:275).
[0517] In another embodiment, the invention provides an expressed
sequence tag (EST) designated herein as DNA13104 comprising the
nucleotide sequence of FIG. 104 (SEQ ID NO:276).
[0518] In another embodiment, the invention provides an expressed
sequence tag (EST) designated herein as DNA13259 comprising the
nucleotide sequence of FIG. 105 (SEQ ID NO:277).
[0519] In another embodiment, the invention provides an expressed
sequence tag (EST) designated herein as DNA13959 comprising the
nucleotide sequence of FIG. 106 (SEQ ID NO:278).
[0520] In another embodiment, the invention provides an expressed
sequence tag (EST) designated herein as DNA13961 comprising the
nucleotide sequence of FIG. 107 (SEQ ID NO:279).
[0521] 43. PRO983
[0522] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to a vesicle associated protein,
VAP-33, wherein the polypeptide is designated in the present
application as "PRO983".
[0523] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO983 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO983 polypeptide having amino acid residues 1 to 243 of FIG. 109
(SEQ ID NO:284), 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 other aspects,
the isolated nucleic acid comprises DNA encoding the PRO983
polypeptide having amino acid residue 1 to X of FIG. 109 (SEQ ID
NO:284) where X is any amino acid from 219 to 228 of FIG. 109 (SEQ
ID NO:284), 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. The isolated
nucleic acid sequence may comprise the cDNA insert of the
DNA53977-1371 vector deposited on May 14, 1998 as ATCC 209862 which
includes the nucleotide sequence encoding PRO983.
[0524] In another embodiment, the invention provides isolated
PRO983 polypeptide. In particular, the invention provides isolated
native sequence PRO983 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 243 of
FIG. 109 (SEQ ID NO:284). Additional embodiments of the present
invention are directed to PRO983 polypeptides comprising amino acid
1 to X of FIG. 109 (SEQ ID NO:284), where X is any amino acid from
219 to 228 of FIG. 109 (SEQ ID NO:284). Optionally, the PRO983
polypeptide is obtained or is obtainable by expressing the
polypeptide encoded by the cDNA insert of the DNA53977-1371 vector
deposited on May 14, 1998 as ATCC 209862.
[0525] In another embodiment, the invention provides an expressed
sequence tag (EST) designated herein as DNA17130 comprising the
nucleotide sequence of FIG. 110 (SEQ ID NO:285).
[0526] In another embodiment, the invention provides an expressed
sequence tag (EST) designated herein as DNA23466 comprising the
nucleotide sequence of FIG. 111 (SEQ ID NO:286).
[0527] In another embodiment, the invention provides an expressed
sequence tag (EST) designated herein as DNA26818 comprising the
nucleotide sequence of FIG. 112 (SEQ ID NO:287).
[0528] In another embodiment, the invention provides an expressed
sequence tag (EST) designated herein as DNA37618 comprising the
nucleotide sequence of FIG. 113 (SEQ ID NO:288).
[0529] In another embodiment, the invention provides an expressed
sequence tag (EST) designated herein as DNA41732 comprising the
nucleotide sequence of FIG. 114 (SEQ ID NO:289).
[0530] In another embodiment, the invention provides an expressed
sequence tag (EST) designated herein as DNA45980 comprising the
nucleotide sequence of FIG. 115 (SEQ ID NO:290).
[0531] In another embodiment, the invention provides an expressed
sequence tag (EST) designated herein as DNA46372 comprising the
nucleotide sequence of FIG. 116 (SEQ ID NO:291).
[0532] 44. PRO1057
[0533] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to proteases, wherein the polypeptide
is designated in the present application as "PRO1057".
[0534] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO1057
polypeptide. In one aspect, the isolated nucleic acid comprises DNA
encoding the PRO1057 polypeptide having amino acid residues 1 to
413 of FIG. 118 (SEQ ID NO:296), 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 other aspects, the isolated nucleic acid comprises
DNA encoding the PRO1057 polypeptide having amino acid residues
about 17 to 413 of FIG. 118 (SEQ ID NO:296), 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. The isolated nucleic acid sequence may comprise the
cDNA insert of the DNA57253-1382 vector deposited on May 14, 1998
as ATCC 209867 which includes the nucleotide sequence encoding
PRO1057.
[0535] In another embodiment, the invention provides isolated
PRO1057 polypeptide. In particular, the invention provides isolated
native sequence PRO1057 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 413 of
FIG. 118 (SEQ ID NO:296). Additional embodiments of the present
invention are directed to PRO1057 polypeptides comprising amino
acids about 17 to 413 of FIG. 118 (SEQ ID NO:296). Optionally, the
PRO1057 polypeptide is obtained or is obtainable by expressing the
polypeptide encoded by the cDNA insert of the DNA57253-1382 vector
deposited on May 14, 1998 as ATCC 209867.
[0536] 45. PRO1071
[0537] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to thrombospondin, wherein the
polypeptide is designated in the present application as
"PRO1071".
[0538] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO1071
polypeptide. In one aspect, the isolated nucleic acid comprises DNA
encoding the PRO1071 polypeptide having amino acid residues 1 to
525 of FIG. 120 (SEQ ID NO:301), 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 other aspects, the isolated nucleic acid comprises
DNA encoding the PRO1071 polypeptide having amino acid residues
about 26 to 525 of FIG. 120 (SEQ ID NO:301), 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. The isolated nucleic acid sequence may comprise the
cDNA insert of the DNA58847-1383 vector deposited on May 20, 1998
as ATCC 209879 which includes the nucleotide sequence encoding
PRO1071.
[0539] In another embodiment, the invention provides isolated
PRO1071 polypeptide. In particular, the invention provides isolated
native sequence PRO1071 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 525 of
FIG. 120 (SEQ ID NO:301). Additional embodiments of the present
invention are directed to PRO1071 polypeptides comprising amino
acids about 26 to 525 of FIG. 120 (SEQ ID NO:301). Optionally, the
PRO1071 polypeptide is obtained or is obtainable by expressing the
polypeptide encoded by the cDNA insert of the DNA58847-1383 vector
deposited on May 20, 1998 as ATCC 209879.
[0540] 46. PRO1072
[0541] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to reductase proteins, wherein the
polypeptide is designated in the present application as
"PRO1072".
[0542] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO1072
polypeptide. In one aspect, the isolated nucleic acid comprises DNA
encoding the PRO1072 polypeptide having amino acid residues 1 to
336 of FIG. 122 (SEQ ID NO:303), 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 other aspects, the isolated nucleic acid comprises
DNA encoding the PRO1072 polypeptide having amino acid residues
about 22 to 336 of FIG. 122 (SEQ ID NO:303), 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. The isolated nucleic acid sequence may comprise the
cDNA insert of the DNA58747-1384 vector deposited on May 14, 1998
as ATCC 209868 which includes the nucleotide sequence encoding
PRO1072.
[0543] In another embodiment, the invention provides isolated
PRO1072 polypeptide. In particular, the invention provides isolated
native sequence PRO1072 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 336 of
FIG. 122 (SEQ ID NO:303). Additional embodiments of the present
invention are directed to PRO1072 polypeptides comprising amino
acids about 22 to 336 of FIG. 122 (SEQ ID NO:303). Optionally, the
PRO1072 polypeptide is obtained or is obtainable by expressing the
polypeptide encoded by the cDNA insert of the DNA58747-1384 vector
deposited on May 14, 1998 as ATCC 209868.
[0544] In another embodiment, the invention provides an expressed
sequence tag (EST) designated herein as DNA40210 comprising the
nucleotide sequence of FIG. 123 (SEQ ID NO:304).
[0545] 47. PRO1075
[0546] 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
"PRO1075".
[0547] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO1075
polypeptide. In one aspect, the isolated nucleic acid comprises DNA
encoding the PRO1075 polypeptide having amino acid residues 1 to
406 of FIG. 125 (SEQ ID NO:309), 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 other aspects, the isolated nucleic acid comprises
DNA encoding the PRO1075 polypeptide having amino acid residues
about 30 to 406 of FIG. 125 (SEQ ID NO:309), 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. The isolated nucleic acid sequence may comprise the
cDNA insert of the DNA57689-1385 vector deposited on May 14, 1998
as ATCC 209869 which includes the nucleotide sequence encoding
PRO1075.
[0548] In another embodiment, the invention provides isolated
PRO1075 polypeptide. In particular, the invention provides isolated
native sequence PRO1075 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 406 of
FIG. 125 (SEQ ID NO:309). Additional embodiments of the present
invention are directed to PRO1075 polypeptides comprising amino
acids about 30 to 406 of FIG. 125 (SEQ ID NO:309). Optionally, the
PRO1075 polypeptide is obtained or is obtainable by expressing the
polypeptide encoded by the cDNA insert of the DNA57689-1385 vector
deposited on May 14, 1998 as ATCC 209869.
[0549] In another embodiment, the invention provides an expressed
sequence tag (EST) designated herein as DNA13059 comprising the
nucleotide sequence of FIG. 126 (SEQ ID NO:310).
[0550] In another embodiment, the invention provides an expressed
sequence tag (EST) designated herein as DNA19463 comprising the
nucleotide sequence of FIG. 127 (SEQ ID NO:311).
[0551] 48. PRO181
[0552] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to the cornichon protein, wherein the
polypeptide is designated in the present application as
"PRO181".
[0553] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO181 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO181 polypeptide having amino acid residues 1 to 144 of FIG. 129
(SEQ ID NO:322), 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 other aspects,
the isolated nucleic acid comprises DNA encoding the PRO181
polypeptide having amino acid residues about 21 to 144 of FIG. 129
(SEQ ID NO:322) or amino acid 1 or about 21 to X of FIG. 129 (SEQ
ID NO:322) where X is any amino acid from 52 to 61 of FIG. 129 (SEQ
ID NO:322), 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. The isolated
nucleic acid sequence may comprise the cDNA insert of the
DNA23330-1390 vector deposited on Apr. 14, 1998 as ATCC 209775
which includes the nucleotide sequence encoding PRO181.
[0554] In another embodiment, the invention provides isolated
PRO181 polypeptide. In particular, the invention provides isolated
native sequence PRO181 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 144 of
FIG. 129 (SEQ ID NO:322). Additional embodiments of the present
invention are directed to PRO181 polypeptides comprising amino
acids about 21 to 144 of FIG. 129 (SEQ ID NO:322) or amino acid 1
or about 21 to X of FIG. 129 (SEQ ID NO:322), where X is any amino
acid from 52 to 61 of FIG. 129 (SEQ ID NO:322). Optionally, the
PRO181 polypeptide is obtained or is obtainable by expressing the
polypeptide encoded by the cDNA insert of the DNA23330-1390 vector
deposited on Apr. 14, 1998 as ATCC 209775.
[0555] In another embodiment, the invention provides an expressed
sequence tag (EST) designated herein as DNA13242 comprising the
nucleotide sequence of FIG. 130 (SEQ ID NO:323).
[0556] 49. PRO195
[0557] Applicants have identified a cDNA clone that encodes a novel
transmembrane polypeptide, wherein the polypeptide is designated in
the present application as "PRO195".
[0558] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO195 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO195 polypeptide having amino acid residues 1 to 323 of FIG. 132
(SEQ ID NO:330), 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 other aspects,
the isolated nucleic acid comprises DNA encoding the PRO195
polypeptide having amino acid residues about 32 to 323 of FIG. 132
(SEQ ID NO:330) or amino acid 1 or about 32 to X of FIG. 132 (SEQ
ID NO:330) where X is any amino acid from 236 to 245 of FIG. 132
(SEQ ID NO:330), 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. The isolated
nucleic acid sequence may comprise the cDNA insert of the
DNA26847-1395 vector deposited on Apr. 14, 1998 as ATCC 209772
which includes the nucleotide sequence encoding PRO195.
[0559] In another embodiment, the invention provides isolated
PRO195 polypeptide. In particular, the invention provides isolated
native sequence PRO195 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 323 of
FIG. 132 (SEQ ID NO:330). Additional embodiments of the present
invention are directed to PRO195 polypeptides comprising amino
acids about 32 to 323 of FIG. 132 (SEQ ID NO:330) or amino acid 1
or about 32 to X of FIG. 132 (SEQ ID NO:330), where X is any amino
acid from 236 to 245 of FIG. 132 (SEQ ID NO:330). Optionally, the
PRO195 polypeptide is obtained or is obtainable by expressing the
polypeptide encoded by the cDNA insert of the DNA26847-1395 vector
deposited on Apr. 14, 1998 as ATCC 209772.
[0560] In another embodiment, the invention provides an expressed
sequence tag (EST) comprising the nucleotide sequence of FIG. 133
(SEQ ID NO:331), herein designated DNA15062.
[0561] In another embodiment, the invention provides an expressed
sequence tag (EST) comprising the nucleotide sequence of FIG. 134
(SEQ ID NO:332), herein designated DNA13199.
[0562] 50. PRO865
[0563] Applicants have identified a cDNA clone that encodes a novel
secreted polypeptide, wherein the polypeptide is designated in the
present application as "PRO865".
[0564] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO865 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO865 polypeptide having amino acid residues 1 to 468 of FIG. 136
(SEQ ID NO:337), 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 other aspects,
the isolated nucleic acid comprises DNA encoding the PRO865
polypeptide having amino acid residues about 24 to 229 of FIG. 136
(SEQ ID NO:337), 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. The isolated
nucleic acid sequence may comprise the cDNA insert of the
DNA53974-1401 vector deposited on Apr. 14, 1998 as ATCC 209774
which includes the nucleotide sequence encoding PRO865.
[0565] In another embodiment, the invention provides isolated
PRO865 polypeptide. In particular, the invention provides isolated
native sequence PRO865 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 468 of
FIG. 136 (SEQ ID NO:337). An additional embodiment of the present
invention is directed to a PRO865 polypeptide comprising amino
acids about 24 to 468 of FIG. 136 (SEQ ID NO:337). Optionally, the
PRO865 polypeptide is obtained or is obtainable by expressing the
polypeptide encoded by the cDNA insert of the DNA53974-1401 vector
deposited on Apr. 14, 1998 as ATCC 209774.
[0566] In another embodiment, the invention provides an expressed
sequence tag (EST) comprising the nucleotide sequence of FIG. 137
(SEQ ID NO:338), herein designated as DNA37642.
[0567] 51. PRO827
[0568] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to integrin proteins, wherein the
polypeptide is designated in the present application as
"PRO827".
[0569] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO827 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO827 polypeptide having amino acid residues 1 to 124 of FIG. 139
(SEQ ID NO:346), 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 other aspects,
the isolated nucleic acid comprises DNA encoding the PRO827
polypeptide having amino acid residues about 23 to 124 of FIG. 139
(SEQ ID NO:346), 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. The isolated
nucleic acid sequence may comprise the cDNA insert of the
DNA57039-1402 vector deposited on Apr. 14, 1998 as ATCC 209777
which includes the nucleotide sequence encoding PRO827.
[0570] In another embodiment, the invention provides isolated
PRO827 polypeptide. In particular, the invention provides isolated
native sequence PRO827 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 124 of
FIG. 139 (SEQ ID NO:346). An additional embodiment of the present
invention is directed to a PRO827 polypeptide comprising amino
acids about 23 to 124 of FIG. 139 (SEQ ID NO:346). Optionally, the
PRO827 polypeptide is obtained or is obtainable by expressing the
polypeptide encoded by the cDNA insert of the DNA57039-1402 vector
deposited on Apr. 14, 1998 as ATCC 209777.
[0571] 52. PRO1114
[0572] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to cytokine receptor family-4 proteins,
wherein the polypeptide is designated in the present application as
"PRO1114".
[0573] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO1114
polypeptide. In one aspect, the isolated nucleic acid comprises DNA
encoding the PRO1114 polypeptide having amino acid residues 1 to
311 of FIG. 142 (SEQ ID NO:352), 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 other aspects, the isolated nucleic acid comprises
DNA encoding the PRO1114 polypeptide having amino acid residues
about 30 to 311 of FIG. 142 (SEQ ID NO:352) or amino acid 1 or
about 30 to X of FIG. 142 (SEQ ID NO:352), where X is any amino
acid from 225 to 234 of FIG. 142 (SEQ ID NO:352), 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. The isolated nucleic acid sequence may
comprise the cDNA insert of the DNA57033-1403 vector deposited on
May 27, 1998 as ATCC 209905 which includes the nucleotide sequence
encoding PRO1114.
[0574] In another embodiment, the invention provides isolated
PRO1114 polypeptide. In particular, the invention provides isolated
native sequence PRO1114 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 311 of
FIG. 142 (SEQ ID NO:352). Additional embodiments of the present
invention are directed to PRO1114 polypeptides comprising amino
acids about 30 to 311 of FIG. 142 (SEQ ID NO:352) or amino acid 1
or about 30 to X of FIG. 142 (SEQ ID NO:352), where X is any amino
acid from 225 to 234 of FIG. 142 (SEQ ID NO:352). Optionally, the
PRO1114 polypeptide is obtained or is obtainable by expressing the
polypeptide encoded by the cDNA insert of the DNA57033-1403 vector
deposited on May 27, 1998 as ATCC 209905.
[0575] In another embodiment, the invention provides an expressed
sequence tag (EST) designated herein as DNA48466 comprising the
nucleotide sequence of FIG. 143 (SEQ ID NO:353).
[0576] A cDNA clone (DNA57033-1403) has been identified that
encodes a novel interferon receptor polypeptide, designated in the
present application as "PRO1114 interferon receptor".
[0577] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO1114 interferon
receptor polypeptide.
[0578] In one aspect, the isolated nucleic acid comprises DNA
having at least about 80% sequence identity, preferably at least
about 85% sequence identity, more preferably at least about 90%
sequence identity, most preferably at least about 95% sequence
identity to (a) a DNA molecule encoding a PRO1114 interferon
receptor polypeptide having the sequence of amino acid residues
from about 1 or about 30 to about 311, inclusive of FIG. 142 (SEQ
ID NO:352), or (b) the complement of the DNA molecule of (a).
[0579] In another aspect, the invention concerns an isolated
nucleic acid molecule encoding a PRO1114 interferon receptor
polypeptide comprising DNA hybridizing to the complement of the
nucleic acid between about nucleotides 250 or about 337 and about
1182, inclusive, of FIG. 141 (SEQ ID NO:351). Preferably,
hybridization occurs under stringent hybridization and wash
conditions.
[0580] In a further aspect, the invention concerns an isolated
nucleic acid molecule comprising DNA having at least about 80%
sequence identity, preferably at least about 85% sequence identity,
more preferably at least about 90% sequence identity, most
preferably at least about 95% sequence identity to (a) a DNA
molecule encoding the same mature polypeptide encoded by the human
protein cDNA in ATCC Deposit No. 209905 (DNA57033-1403) or (b) the
complement of the nucleic acid molecule of (a). In a preferred
embodiment, the nucleic acid comprises a DNA encoding the same
mature polypeptide encoded by the human protein cDNA in ATCC
Deposit No. 209905 (DNA57033-1403).
[0581] In still a further aspect, the invention concerns an
isolated nucleic acid molecule comprising (a) DNA encoding a
polypeptide having at least about 80% sequence identity, preferably
at least about 85% sequence identity, more preferably at least
about 90% sequence identity, most preferably at least about 95%
sequence identity to the sequence of amino acid residues 1 or about
30 to about 311, inclusive of FIG. 142 (SEQ ID NO:352), or (b) the
complement of the DNA of (a).
[0582] In a further aspect, the invention concerns an isolated
nucleic acid molecule having at least 10 nucleotides and produced
by hybridizing a test DNA molecule under stringent conditions with
(a) a DNA molecule encoding a PRO1114 interferon receptor
polypeptide having the sequence of amino acid residues from 1 or
about 30 to about 311, inclusive of FIG. 142 (SEQ ID NO:352), or
(b) the complement of the DNA molecule of (a), and, if the DNA
molecule has at least about an 80% sequence identity, prefereably
at least about an 85% sequence identity, more preferably at least
about a 90% sequence identity, most preferably at least about a 95%
sequence identity to (a) or (b), isolating the test DNA
molecule.
[0583] In a specific aspect, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO1114 interferon
receptor polypeptide, with or without the N-terminal signal
sequence and/or the initiating methionine, and its soluble, i.e.,
transmembrane domain deleted or inactivated variants, or is
complementary to such encoding nucleic acid molecule. The signal
peptide has been tentatively identified as extending from about
amino acid position 1 to about amino acid position 29 in the
sequence of FIG. 142 (SEQ ID NO:352). The transmembrane domain has
been tentatively identified as extending from about amino acid
position 230 to about amino acid position 255 in the PRO1114
interferon receptor amino acid sequence (FIG. 142, SEQ ID
NO:352).
[0584] In another aspect, the invention concerns an isolated
nucleic acid molecule comprising (a) DNA encoding a polypeptide
scoring at least about 80% positives, preferably at least about 85%
positives, more preferably at least about 90% positives, most
preferably at least about 95% positives when compared with the
amino acid sequence of residues 1 or about 30 to about 311,
inclusive of FIG. 142 (SEQ ID NO:352), or (b) the complement of the
DNA of (a).
[0585] Another embodiment is directed to fragments of a PRO1114
interferon receptor polypeptide coding sequence that may find use
as hybridization probes. Such nucleic acid fragments are from about
20 to about 80 nucleotides in length, preferably from about 20 to
about 60 nucleotides in length, more preferably from about 20 to
about 50 nucleotides in length and most preferably from about 20 to
about 40 nucleotides in length and may be derived from the
nucleotide sequence shown in FIG. 141 (SEQ ID NO:351).
[0586] In another embodiment, the invention provides a vector
comprising DNA encoding PRO1114 interferon receptor or its
variants. The vector may comprise any of the isolated nucleic acid
molecules hereinabove identified.
[0587] A host cell comprising such a vector is also provided. By
way of example, the host cells may be CHO cells, E. coli, or yeast.
A process for producing PRO1114 interferon receptor polypeptides is
further provided and comprises culturing host cells under
conditions suitable for expression of PRO1114 interferon receptor
and recovering PRO1114 interferon receptor from the cell
culture.
[0588] In another embodiment, the invention provides isolated
PRO1114 interferon receptor polypeptide encoded by any of the
isolated nucleic acid sequences hereinabove identified.
[0589] In a specific aspect, the invention provides isolated native
sequence PRO1114 interferon receptor polypeptide, which in certain
embodiments, includes an amino acid sequence comprising residues 1
or about 30 to about 311 of FIG. 142 (SEQ ID NO:352).
[0590] In another aspect, the invention concerns an isolated
PRO1114 interferon receptor polypeptide, comprising an amino acid
sequence having at least about 80% sequence identity, preferably at
least about 85% sequence identity, more preferably at least about
90% sequence identity, most preferably at least about 95% sequence
identity to the sequence of amino acid residues 1 or about 30 to
about 311, inclusive of FIG. 142 (SEQ ID NO:352).
[0591] In a further aspect, the invention concerns an isolated
PRO1114 interferon receptor polypeptide, comprising an amino acid
sequence scoring at least about 80% positives, preferably at least
about 85% positives, more preferably at least about 90% positives,
most preferably at least about 95% positives when compared with the
amino acid sequence of residues 1 or about 30 to about 311,
inclusive of FIG. 142 (SEQ ID NO:352).
[0592] In yet another aspect, the invention concerns an isolated
PRO1114 interferon receptor polypeptide, comprising the sequence of
amino acid residues 1 or about 30 to about 311, inclusive of FIG.
142 (SEQ ID NO:352), or a fragment thereof sufficient to provide a
binding site for an anti-PRO1114 interferon receptor antibody.
Preferably, the PRO1114 interferon receptor fragment retains a
qualitative biological activity of a native PRO1114 interferon
receptor polypeptide.
[0593] In a still further aspect, the invention provides a
polypeptide produced by (i) hybridizing a test DNA molecule under
stringent conditions with (a) a DNA molecule encoding a PRO1114
interferon receptor polypeptide having the sequence of amino acid
residues from about 1 or about 30 to about 311, inclusive of FIG.
142 (SEQ ID NO:352), or (b) the complement of the DNA molecule of
(a), and if the test DNA molecule has at least about an 80%
sequence identity, preferably at least about an 85% sequence
identity, more preferably at least about a 90% sequence identity,
most preferably at least about a 95% sequence identity to (a) or
(b), (ii) culturing a host cell comprising the test DNA molecule
under conditions suitable for expression of the polypeptide, and
(iii) recovering the polypeptide from the cell culture.
[0594] In another embodiment, the invention provides chimeric
molecules comprising a PRO1114 interferon receptor polypeptide
fused to a heterologous polypeptide or amino acid sequence. An
example of such a chimeric molecule comprises a PRO1114 interferon
receptor polypeptide fused to an epitope tag sequence or a Fc
region of an immunoglobulin.
[0595] In another embodiment, the invention provides an antibody
which specifically binds to a PRO1114 interferon receptor
polypeptide. Optionally, the antibody is a monoclonal antibody.
[0596] In yet another embodiment, the invention concerns agonists
and antagonists of a native PRO1114 interferon receptor
polypeptide. In a particular embodiment, the agonist or antagonist
is an anti-PRO1114 interferon receptor antibody.
[0597] In a further embodiment, the invention concerns a method of
identifying agonists or antagonists of a native PRO1114 interferon
receptor polypeptide by contacting the native PRO1114 interferon
receptor polypeptide with a candidate molecule and monitoring a
biological activity mediated by said polypeptide.
[0598] In a still further embodiment, the invention concerns a
composition comprising a PRO1114 interferon receptor polypeptide,
or an agonist or antagonist as hereinabove defined, in combination
with a pharmaceutically acceptable carrier.
[0599] 53. PRO237
[0600] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to carbonic anhydrase, wherein the
polypeptide is designated in the present application as
"PRO237".
[0601] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO237 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO237 polypeptide having amino acid residues 1 to 328 of FIG. 145
(SEQ ID NO:358), 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 other aspects,
the isolated nucleic acid comprises DNA encoding the PRO237
polypeptide having amino acid residues about 24 to 328 of FIG. 145
(SEQ ID NO:358) or amino acid 1 or about 24 to X of FIG. 145 (SEQ
ID NO:358), where X is any amino acid from 172 to 181 of FIG. 145
(SEQ ID NO:358), 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. The isolated
nucleic acid sequence may comprise the cDNA insert of the
DNA34353-1428 vector deposited on May 12, 1998 as ATCC 209855 which
includes the nucleotide sequence encoding PRO237.
[0602] In another embodiment, the invention provides isolated
PRO237 polypeptide. In particular, the invention provides isolated
native sequence PRO237 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 328 of
FIG. 145 (SEQ ID NO:358). Additional embodiments of the present
invention are directed to PRO237 polypeptides comprising amino
acids about 24 to 328 of FIG. 145 (SEQ ID NO:358) or amino acid 1
or about 24 to X of FIG. 145 (SEQ ID NO:358), where X is any amino
acid from 172 to 181 of FIG. 145 (SEQ ID NO:358). Optionally, the
PRO237 polypeptide is obtained or is obtainable by expressing the
polypeptide encoded by the cDNA insert of the DNA34353-1428 vector
deposited on May 12, 1998 as ATCC 209855.
[0603] 54. PRO541
[0604] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to a trypsin inhibitor protein, wherein
the polypeptide is designated in the present application as
"PRO541".
[0605] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO541 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO541 polypeptide having amino acid residues 1 to 500 of FIG. 147
(SEQ ID NO:363), 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 other aspects,
the isolated nucleic acid comprises DNA encoding the PRO541
polypeptide having amino acid residues about 21 to 500 of FIG. 147
(SEQ ID NO:363), 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. The isolated
nucleic acid sequence may comprise the cDNA insert of the
DNA45417-1432 vector deposited on May 27, 1998 as ATCC 209910 which
includes the nucleotide sequence encoding PRO541.
[0606] In another embodiment, the invention provides isolated
PRO541 polypeptide. In particular, the invention provides isolated
native sequence PRO541 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 500 of
FIG. 147 (SEQ ID NO:363). Additional embodiments of the present
invention are directed to PRO541 polypeptides comprising amino
acids about 21 to 500 of FIG. 147 (SEQ ID NO:363). Optionally, the
PRO541 polypeptide is obtained or is obtainable by expressing the
polypeptide encoded by the cDNA insert of the DNA45417-1432 vector
deposited on May 27, 1998 as ATCC 209910.
[0607] 55. PRO273
[0608] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as "PRO273".
[0609] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO273 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO273 polypeptide having amino acid residues 1 through 111 of FIG.
149 (SEQ ID NO:370), 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.
[0610] In another embodiment, the invention provides isolated
PRO273 polypeptide. In particular, the invention provides isolated
native sequence PRO273 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 through 111
of FIG. 149 (SEQ ID NO:370).
[0611] 56. PRO701
[0612] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to neuroligins 1, 2, and 3, wherein the
polypeptide is designated in the present application as
"PRO701".
[0613] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO701 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO701 polypeptide having amino acid residues 1 through 816 of FIG.
151 (SEQ ID NO:375), or is complementary to such encoding nucleic
acid sequence, and remains stably bound to it under at least
moderate, and optionally, under high strirgency conditions. The
isolated nucleic acid sequence may comprise the cDNA insert of the
vector deposited with the ATCC on Mar. 31, 1998 which includes the
nucleotide sequence encoding PRO701.
[0614] In another embodiment, the invention provides isolated
PRO701 polypeptide. In particular, the invention provides isolated
native sequence PRO701 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 through 816
of FIG. 151 (SEQ ID NO:375). An additional embodiment of the
present invention is directed to an isolated extracellular domain
of a PRO701 polypeptide. Optionally, the PRO701 polypeptide is
obtained or is obtainable by expressing the polypeptide encoded by
the cDNA insert of the vector deposited with the ATCC on Mar. 31,
1998.
[0615] 57. PRO704
[0616] Applicants have identified a cDNA clone that encodes a novel
polypeptide having sequence identity with VIP36, wherein the
polypeptide is designated in the present application as
"PRO704".
[0617] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO704 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO704 polypeptide having amino acid residues 1 through 348 of FIG.
153 (SEQ ID NO:380), 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. The
isolated nucleic acid sequence may comprise the cDNA insert of the
vector deposited on Mar. 31, 1998 with the ATCC as DNA50911-1288,
which includes the nucleotide sequence encoding PRO704.
[0618] In another embodiment, the invention provides isolated
PRO704 polypeptide. In particular, the invention provides isolated
native sequence PRO704 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 through 348
of FIG. 153 (SEQ ID NO:380). An additional embodiment of the
present invention is directed to an isolated extracellular domain
of a PRO704 polypeptide. Optionally, the PRO704 polypeptide is
obtained or is obtainable by expressing the polypeptide encoded by
the cDNA insert of the vector deposited on Mar. 31, 1998 with the
ATCC as DNA50911-1288.
[0619] 58. PRO706
[0620] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to prostatic acid phosphatase precursor
and lysosomal acid phosphatase precursor, wherein the polypeptide
is designated in the present application as "PRO706".
[0621] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO706 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO706 polypeptide having amino acid residues 1 through 480 of FIG.
155 (SEQ ID NO:385), 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. The
isolated nucleic acid sequence may comprise the cDNA insert of the
vector deposited on Apr. 21, 1998 with the ATCC as DNA48329-1290
which includes the nucleotide sequence encoding PRO706.
[0622] In another embodiment, the invention provides isolated
PRO706 polypeptide. In particular, the invention provides isolated
native sequence PRO706 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 through 480
of FIG. 155 (SEQ ID NO:385), or comprising residues 19 through 480
of FIG. 155 (SEQ ID NO:385). Optionally, the PRO706 polypeptide is
obtained or is obtainable by expressing the polypeptide encoded by
the cDNA insert of the vector deposited on Apr. 21, 1998 with the
ATCC as DNA48329-1290.
[0623] 59. PRO707
[0624] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to cadherins, particularly cadherin
FIB3, wherein the polypeptide is designated in the present
application as "PRO707".
[0625] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO707 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO707 polypeptide having amino acid residues 1 to 916 of FIG. 157
(SEQ ID NO:390), 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. The isolated
nucleic acid sequence may comprise the cDNA insert of the vector
deposited on May 27, 1998 with the ATCC as DNA48306-1291 which
includes the nucleotide sequence encoding PRO707.
[0626] In another embodiment, the invention provides isolated
PRO707 polypeptide. In particular, the invention provides isolated
native sequence PRO707 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 916 of
FIG. 157 (SEQ ID NO:390). An additional embodiment of the present
invention is directed to an isolated extracellular domain of a
PRO707 polypeptide. Optionally, the PRO707 polypeptide is obtained
or is obtainable by expressing the polypeptide encoded by the cDNA
insert of the vector deposited on May 27, 1998 with the ATCC as
DNA48306-1291.
[0627] 60. PRO322
[0628] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to neuropsin, wherein the polypeptide
is designated in the present application as "PRO322".
[0629] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO322 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO322 polypeptide having amino acid residues 1 or 24 through 260
of FIG. 159 (SEQ ID NO:395), 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.
The isolated nucleic acid sequence may comprise the cDNA insert of
the vector deposited on Mar. 11, 1998 as ATCC no. 209669 which
includes the nucleotide sequence encoding PRO322.
[0630] In another embodiment, the invention provides isolated
PRO322 polypeptide. In particular, the invention provides isolated
native sequence PRO322 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 or 24 through
260 of FIG. 159 (SEQ ID NO:395). An additional embodiment of the
present invention is directed to an isolated extracellular domain
of a PRO322 polypeptide. Optionally, the PRO322 polypeptide is
obtained or is obtainable by expressing the polypeptide encoded by
the cDNA insert of the vector deposited on Mar. 11, 1998 as ATCC
no. 209669.
[0631] 61. PRO526
[0632] Applicants have identified a cDNA clone that encodes a novel
polypeptide having sequence identity with ALS, wherein the
polypeptide is designated in the present application as
"PRO526".
[0633] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO526 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO526 polypeptide having amino acid residues 1 to 473 of FIG. 161
(SEQ ID NO:400), 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. The isolated
nucleic acid sequence may comprise the cDNA insert of the vector
deposited on Mar. 26, 1998 with the ATCC as DNA44184-1319 which
includes the nucleotide sequence encoding PRO526.
[0634] In another embodiment, the invention provides isolated
PRO526 polypeptide. In particular, the invention provides isolated
native sequence PRO526 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 473 of
FIG. 161 (SEQ ID NO:400). Optionally, the PRO526 polypeptide is
obtained or is obtainable by expressing the polypeptide encoded by
the cDNA insert of the vector deposited on Mar. 26, 1998 with the
ATCC as DNA44184-1319 which includes the nucleotide sequence
encoding PRO526.
[0635] 62. PRO531
[0636] Applicants have identified a cDNA clone that encodes a novel
polypeptide having sequence identity with protocadherins, wherein
the polypeptide is designated in the present application as
"PRO531".
[0637] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO531 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO531 polypeptide having amino acid residues 1 to 789 of FIG. 163
(SEQ ID NO:405), 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. The isolated
nucleic acid sequence may comprise the cDNA insert of the vector
deposited on Mar. 26, 1998 as DNA48314-1320 which includes the
nucleotide sequence encoding PRO531.
[0638] In another embodiment, the invention provides isolated
PRO531 polypeptide. In particular, the invention provides isolated
native sequence PRO531 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 789 of
FIG. 163 (SEQ ID NO:405). An additional embodiment of the present
invention is directed to an isolated extracellular domain of a
PRO531 polypeptide. Optionally, the PRO531 polypeptide is obtained
or is obtainable by expressing the polypeptide encoded by the cDNA
insert of the vector deposited on Mar. 26, 1998 as
DNA48314-1320.
[0639] 63. PRO534
[0640] Applicants have identified a cDNA clone that encodes a novel
polypeptide having sequence identity with disulfide isomerase
(sometimes referred to herein as protein disulfide isomerase),
wherein the polypeptide is designated in the present application as
"PRO534".
[0641] In one embodiment, the invention provides an isolated
nucleic acid molecule comprsing DNA encoding a PRO534 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO534 polypeptide having amino acid residues 1 to 360 of FIG. 165
(SEQ ID NO:410), 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. The isolated
nucleic acid sequence may comprise the cDNA insert of the vector
deposited on Mar. 26, 1998 as DNA48333-1321 which includes the
nucleotide sequence encoding PRO534.
[0642] In another embodiment, the invention provides isolated
PRO534 polypeptide. In particular, the invention provides isolated
native sequence PRO534 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 360 of
FIG. 165 (SEQ ID NO:410). An additional embodiment of the present
invention is directed to an isolated extracellular domain of a
PRO534 polypeptide. Optionally, the PRO534 polypeptide is obtained
or is obtainable by expressing the polypeptide encoded by the cDNA
insert of the vector deposited on Mar. 26, 1998 as
DNA48333-1321.
[0643] 64. PRO697
[0644] Applicants have identified a cDNA clone that encodes a novel
polypeptide having sequence identity with sFRPs, wherein the
polypeptide is designated in the present application as
"PRO697".
[0645] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO697 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO697 polypeptide having amino acid residues 1 through 295 of FIG.
167 (SEQ ID NO:415), 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. The
isolated nucleic acid sequence may comprise the cDNA insert of the
vector deposited with the ATCC on Mar. 26, 1998 as DNA50920-1325
which includes the nucleotide sequence encoding PRO697.
[0646] In another embodiment, the invention provides isolated
PRO697 polypeptide. In particular, the invention provides isolated
native sequence PRO697 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 through 295
of FIG. 167 (SEQ ID NO:415). Optionally, the PRO697 polypeptide is
obtained or is obtainable by expressing the polypeptide encoded by
the cDNA insert of the vector deposited with the ATCC on Mar. 26,
1998 as DNA50920-1325.
[0647] 65. PRO717
[0648] Applicants have identified a cDNA clone that encodes a novel
12 transmembrane polypeptide, wherein the polypeptide is designated
in the present application as "PRO717".
[0649] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO717 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO717 polypeptide having amino acid residues 1 through 560 of FIG.
169 (SEQ ID NO:420), or is complementary to such encoding nuleic
acid sequence, and remains stably bound to it under at least
moderate, and optionally, under high stringency conditions. The
isolated nucleic acid sequence may comprise the cDNA insert of the
vector deposited on Apr. 28, 1998 with the ATCC as DNA50988-1326
which includes the nucleotide sequence encoding PRO717.
[0650] In another embodiment, the invention provides isolated
PRO717 polypeptide. In particular, the invention provides isolated
native sequence PRO717 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 through 560
of FIG. 169 (SEQ ID NO:420). An additional embodiment of the
present invention is directed to an isolated extracellular domain
of a PRO717 polypeptide. Optionally, the PRO717 polypeptide is
obtained or is obtainable by expressing the polypeptide encoded by
the cDNA insert of the vector deposited on Apr. 28, 1998 with the
ATCC as DNA50988-1326.
[0651] 66. PRO731
[0652] Applicants have identified a cDNA clone that encodes a novel
polypeptide having sequence identity with protocadherin 4, wherein
the polypeptide is designated in the present application as
"PRO731".
[0653] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO731 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO731 polypeptide having amino acid residues 1 through 1184 of
FIG. 171 (SEQ ID NO:425), 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.
The isolated nucleic acid sequence may comprise the cDNA insert of
the vector deposited on Mar. 31, 1998 with the ATCC as
DNA48331-1329 which includes the nucleotide sequence encoding
PRO731.
[0654] In another embodiment, the invention provides isolated
PRO731 polypeptide. In particular, the invention provides isolated
native sequence PRO731 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 through 1184
of FIG. 171 (SEQ ID NO:425). An additional embodiment of the
present invention is directed to an isolated extracellular domain
of a PRO731 polypeptide. Optionally, the PRO731 polypeptide is
obtained or is obtainable by expressing the polypeptide encoded by
the cDNA insert of the vector deposited on Mar. 31, 1998 with the
ATCC as DNA48331-1329.
[0655] 67. PRO218
[0656] Applicants have identified a cDNA clone that encodes a novel
multi-transmembrane protein having sequence identity with membrane
regulator proteins, wherein the polypeptide is designated in the
present application as "PRO218".
[0657] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO218 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO218 polypeptide having amino acid residues 1 through 455 of FIG.
173 (SEQ ID NO:430), 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. The
isolated nucleic acid sequence may comprise the cDNA insert of the
vector deposited on Apr. 28, 1998 with the ATCC as DNA30867-1335
which includes the nucleotide sequence encoding PRO218.
[0658] In another embodiment, the invention prcvides isolated
PRO218 polypeptide. In particular, the invention provides isolated
native sequence PRO218 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 through 455
of FIG. 173 (SEQ ID NO:430). Optionally, the PRO218 polypeptide is
obtained or is obtainable by expressing the polypeptide encoded by
the cDNA insert of the vector deposited on Apr. 28, 1998 with the
ATCC as DNA30867-1335.
[0659] In another embodiment, the invention provides an expressed
sequence tag (EST) sequence comprising the nucleotide sequence of
FIG. 174 (SEQ ID NO:431), designated herein as DNA14472.
[0660] In another embodiment, the invention provides an expressed
sequence tag (EST) sequence comprising the nucleotide sequence of
FIG. 175 (SEQ ID NO:432), designated herein as DNA15846.
[0661] 68. PRO768
[0662] Applicants have identified a cDNA clone that encodes a novel
polypeptide having sequence identity with integrins, wherein the
polypeptide is designated in the present application as
"PRO768".
[0663] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO768 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO768 polypeptide having amino acid residues 1 through 1141 of
FIG. 177 (SEQ ID NO:437), 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.
The isolated nucleic acid sequence may comprise the cDNA insert of
the vector deposited on Apr. 6, 1998 as DNA55737-1345 which
includes the nucleotide sequence encoding PRO768.
[0664] In another embodiment, the invention provides isolated
PRO768 polypeptide. In particular, the invention provides isolated
native sequence PRO768 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 through 1141
of FIG. 177 (SEQ ID NO:437). An additional embodiment of the
present invention is directed to an isolated extracellular domain
of a PRO768 polypeptide. Optionally, the PRO768 polypeptide is
obtained or is obtainable by expressing the polypeptide encoded by
the cDNA insert of the vector deposited on Apr. 6, 1998 as
DNA55737-1345.
[0665] 69. PRO771
[0666] Applicants have identified a cDNA clone that encodes a novel
polypeptide having sequence identity with testican, wherein the
polypeptide is designated in the present application as
"PRO771".
[0667] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO771 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO771 polypeptide having amino acid residues 1 through 436 of FIG.
179 (SEQ ID NO:442), 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. The
isolated nucleic acid sequence may comprise the cDNA insert of the
vector deposited on Apr. 7, 1998 with the ATCC as DNA49829-1346
which includes the nucleotide sequence encoding PRO771.
[0668] In another embodiment, the invention provides isolated
PRO771 polypeptide. In particular, the invention provides isolated
native sequence PRO771 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 through 436
of FIG. 179 (SEQ ID NO:442). Optionally, the PRO771 polypeptide is
obtained or is obtainable by expressing the polypeptide encoded by
the cDNA insert of the vector deposited on Apr. 7, 1998 with the
ATCC as DNA49829-1346.
[0669] 70. PRO733
[0670] Applicants have identified a cDNA clone that encodes a novel
polypeptide having sequence identity with the T1/ST2 receptor
binding protein, wherein the polypeptide is designated in the
present application as "PRO733".
[0671] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO733 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO733 polypeptide having amino acid residues 1 through 229 of FIG.
181 (SEQ ID NO:447), 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. The
isolated nucleic acid sequence may comprise the cDNA insert of the
vector deposited on Apr. 7, 1998 with the ATCC as DNA52196-1348
which includes the nucleotide sequence encoding PRO733.
[0672] In another embodiment, the invention provides isolated
PRO733 polypeptide. In particular, the invention provides isolated
native sequence PRO733 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 through 229
of FIG. 181 (SEQ ID NO:447). An additional embodiment of the
present invention is directed to an isolated extracellular domain
of a PRO733 polypeptide. Optionally, the PRO733 polypeptide is
obtained or is obtainable by expressing the polypeptide encoded by
the cDNA insert of the vector deposited on Apr. 7, 1998 as
DNA52196-1348.
[0673] 71. PRO162
[0674] Applicants have identified a cDNA clone that encodes a novel
polypeptide having sequence identity with pancreatitis-associated
protein, wherein the polypeptide is designated in the present
application as "PRO162".
[0675] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO162 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO162 polypeptide having amino acid residues 1 through 175 of FIG.
183 (SEQ ID NO:452), 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. The
isolated nucleic acid sequence may comprise the cDNA insert of the
vector deposited on May 6, 1998 with the ATCC as DNA56965-1356
which includes the nucleotide sequence encoding PRO162.
[0676] In another embodiment, the invention provides isolated
PRO162 polypeptide. In particular, the invention provides isolated
native sequence PRO162 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 through 175
of FIG. 183 (SEQ ID NO:452). Optionally, the PRO162 polypeptide is
obtained or is obtainable by expressing the polypeptide encoded by
the cDNA insert of the vector deposited on May 6, 1998 with the
ATCC as DNA56965-1356.
[0677] 72. PRO788
[0678] Applicants have identified a cDNA clone that encodes a novel
polypeptide having sequence identity with anti-neoplastic urinary
protein, wherein the polypeptide is designated in the present
application as "PRO788".
[0679] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO788 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO788 polypeptide having amino acid residues 1 through 125 of FIG.
185 (SEQ ID NO:454), 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. The
isolated nucleic acid sequence may comprise the cDNA insert of the
vector deposited on May 6, 1998 with the ATCC as DNA56405-1357
which includes the nucleotide sequence encoding PRO788.
[0680] In another embodiment, the invention provides isolated
PRO788 polypeptide. In particular, the invention provides isolated
native sequence PRO788 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 through 125
of FIG. 185 (SEQ ID NO:454). An additional embodiment of the
present invention is directed to an isolated extracellular domain
of a PRO788 polypeptide. Optionally, the PRO788 polypeptide is
obtained or is obtainable by expressing the polypeptide encoded by
the cDNA insert of the vector deposited on May 6, 1998 with the
ATCC as DNA56405-1357.
[0681] 73. PRO1008
[0682] Applicants have identified a cDNA clone that encodes a novel
polypeptide having sequence identity with dickkopf-1 (dkk-1),
wherein the polypeptide is designated in the present application as
"PRO1008".
[0683] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO1008
polypeptide. In one aspect, the isolated nucleic acid comprises DNA
encoding the PRO1008 polypeptide having amino acid residues 1
through 266 of FIG. 187 (SEQ ID NO:456), 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. The isolated nucleic acid sequence may comprise the
cDNA insert of the vector deposited on May 20, 1998 with the ATCC
as DNA57530-1375 which includes the nucleotide sequence encoding
PRO1008.
[0684] In another embodiment, the invention provides isolated
PRO1008 polypeptide. In particular, the invention provides isolated
native sequence PRO1008 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 through 266
of FIG. 187 (SEQ ID NO:456). Optionally, the PRO1008 polypeptide is
obtained or is obtainable by expressing the polypeptide encoded by
the cDNA insert of the vector deposited on May 20, 1998 with the
ATCC as DNA57530-1375.
[0685] In another embodiment, the invention provides an expressed
sequence tag (EST) designated herein as DNA16508 comprising the
nucleotide sequence of FIG. 188 (SEQ ID NO:457).
[0686] 74. PRO1012
[0687] Applicants have identified a cDNA clone that encodes a novel
polypeptide having sequence identity with disulfide isomerase and
phospholipase C, wherein the polypeptide is designated in the
present application as "PRO1012".
[0688] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO1012
polypeptide. In one aspect, the isolated nucleic acid comprises DNA
encoding the PRO1012 polypeptide having amino acid residues 1
through 747 of FIG. 190 (SEQ ID NO:459), 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. The isolated nucleic acid sequence may comprise the
cDNA insert of the vector deposited on May 14, 1998 with the ATCC
as DNA56439-1376, which includes the nucleotide sequence encoding
PRO1012.
[0689] In another embodiment, the invention provides isolated
PRO1012 polypeptide. In particular, the invention provides isolated
native sequence PRO1012 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 through 747
of FIG. 190 (SEQ ID NO:459). Optionally, the PRO1012 polypeptide is
obtained or is obtainable by expressing the polypeptide encoded by
the cDNA insert of the vector deposited on May 14, 1998 with the
ATCC as DNA56439-1376.
[0690] 75. PRO1014
[0691] Applicants have identified a cDNA clone that encodes a novel
polypeptide having sequence identity with reductase, wherein the
polypeptide is designated in the present application as
"PRO1014".
[0692] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO1014
polypeptide. In one aspect, the isolated nucleic acid comprises DNA
encoding the PRO1014 polypeptide having amino acid residues 1
through 300 of FIG. 192 (SEQ ID NO:464), 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. The isolated nucleic acid sequence may comprise the
cDNA insert of the vector deposited on May 20, 1998 as
DNA56409-1377 with the ATCC which includes the nucleotide sequence
encoding PRO1014.
[0693] In another embodiment, the invention provides isolated
PRO1014 polypeptide. In particular, the invention provides isolated
native sequence PRO1014 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 through 300
of FIG. 192 (SEQ ID NO:464). Optionally, the PRO1014 polypeptide is
obtained or is obtainable by expressing the polypeptideencoded by
the cDNA insert of the vector deposited on May 20, 1998 as
DNA56409-1377 with the ATCC.
[0694] 76. PRO1017
[0695] Applicants have identified a cDNA clone that encodes a novel
polypeptide having sequence identity with HNK-1 sulfotransferase,
wherein the polypeptide is designated in the present application as
"PRO1017".
[0696] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO1017
polypeptide. In one aspect, the isolated nucleic acid comprises DNA
encoding the PRO1017 polypeptide having amino acid residues 1
through 414 of FIG. 194 (SEQ ID NO:466), 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. The isolated nucleic acid sequence may comprise the
cDNA insert of the vector deposited on May 20, 1998 with the ATCC
as DNA56112-1379 which includes the nucleotide sequence encoding
PRO1017.
[0697] In another embodiment, the invention provides isolated
PRO1017 polypeptide. In particular, the invention provides isolated
native sequence PRO1017 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 through 414
of FIG. 194 (SEQ ID NO:466). Optionally, the PRO1017 polypeptide is
obtained or is obtainable by expressing the polypeptide encoded by
the cDNA insert of the vector deposited on May 20, 1998 with the
ATCC as DNA56112-1379.
[0698] 77. PRO474
[0699] Applicants have identified a cDNA clone that encodes a novel
polypeptide having sequence identity with dehydrogenase, wherein
the polypeptide is designated in the present application as
"PRO474".
[0700] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO474 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO474 polypeptide having amino acid residues 1 through 270 of FIG.
196 (SEQ ID NO:468), 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. The
isolated nucleic acid sequence may comprise the cDNA insert of the
vector deposited on May 14, 1998 with the ATCC as DNA56045-1380
which includes the nucleotide sequence encoding PRO474.
[0701] In another embodiment, the invention provides isolated
PRO474 polypeptide. In particular, the invention provides isolated
native sequence PRO474 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 through 270
of FIG. 196 (SEQ ID NO:468). Optionally, the PRO474 polypeptide is
obtained or is obtainable by expressing the polypeptide encoded by
the cDNA insert of the vector deposited on May 14, 1998 with the
ATCC as DNA56045-1380.
[0702] 78. PRO1031
[0703] Applicants have identified a cDNA clone that encodes a novel
polypeptide having sequence identity with IL-17, wherein the
polypeptide is designated in the present application as
"PRO1031".
[0704] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO1031
polypeptide. In one aspect, the isolated nucleic acid comprises DNA
encoding the PRO1031 polypeptide having amino acid residues 1
through 180 of FIG. 198 (SEQ ID NO:470), 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. The isolated nucleic acid sequence may comprise the
cDNA insert of the vector deposited on May 14, 1998 with the ATCC
as DNA59294-1381 which includes the nucleotide sequence encoding
PRO1031.
[0705] In another embodiment, the invention provides isolated
PRO1031 polypeptide. In particular, the inventionprovides isolated
native sequence PRO1031 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 through 180
of FIG. 198 (SEQ ID NO:470). Optionally, the PRO1031 polypeptide is
obtained or is obtainable by expressing the polypeptide encoded by
the cDNA insert of the vector deposited on May 14, 1998 with the
ATCC as DNA59294-1381.
[0706] 79. PRO938
[0707] Applicants have identified a cDNA clone that encodes a novel
polypeptide having sequence identity to protein disulfide
isomerase, wherein the polypeptide is designated in the present
application as "PRO938".
[0708] In one embodiment, the inventionprovides an isolated nucleic
acid molecule comprising DNA encoding a PRO938 polypeptide. In one
aspect, the isolated nucleic acid comprises DNA encoding the PRO938
polypeptide having amino acid residues 1 to 349 of FIG. 200 (SEQ ID
NO:472), 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 other aspects,
the isolated nucleic acid comprises DNA encoding the PRO938
polypeptide having amino acid residues about 23 to 349 of FIG. 200
(SEQ ID NO:472) or amino acid 1 or about 23 to X of FIG. 200 (SEQ
ID NO:472), where X is any amino acid from 186 to 195 of FIG. 200
(SEQ ID NO:472), 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. The isolated
nucleic acid sequence may comprise the cDNA insert of the
DNA56433-1406 vector deposited on May 12, 1998, as ATCC Accession
No. 209857 which includes the nucleotide sequence encoding
PRO938.
[0709] In another embodiment, the invention provides isolated
PRO938 polypeptide. In particular, the invention provides isolated
native sequence PRO938 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 349 of
FIG. 200 (SEQ ID NO:472). Additional embodiments of the present
invention are directed to PRO938 polypeptides comprising amino
acids about 23 to 349 of FIG. 200 (SEQ ID NO:472) or amino acid 1
or about 23 to X of FIG. 200 (SEQ ID NO:472), where X is any amino
acid from 186 to 195 of FIG. 200 (SEQ ID NO:472). Optionally, the
PRO938 polypeptide is obtained or is obtainable by expressing the
polypeptide encoded by the cDNA insert of the DNA56433-1406 vector
deposited on May 12, 1998, as ATCC Accession No. 209857.
[0710] 80. PRO1082
[0711] Applicants have identified a cDNA clone that encodes a novel
polypeptide having sequence identity with a lectin-like oxidized
LDL receptor, wherein the polypeptide is designated in the present
application as "PRO1082".
[0712] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO1082
polypeptide. In one aspect, the isolated nucleic acid comprises DNA
encoding the PRO1082 polypeptide having amino acid residues 1
through 201 of FIG. 202 (SEQ ID NO:477), 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. The isolated nucleic acid sequence may comprise the
cDNA insert of the vector deposited on May 14, 1998 with the ATCC
as DNA53912-1457 which includes the nucleotide sequence encoding
PRO1082.
[0713] In another embodiment, the invention provides isolated
PRO1082 polypeptide. In particular, the invention provides isolated
native sequence PRO1082 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 through 201
of FIG. 202 (SEQ ID NO:477). An additional embodiment of the
present invention is directed to an isolated domain of a PRO1082
polypeptide, excluding the transmembrane domain. Optionally, the
PRO1082 polypeptide is obtained or is obtainable by expressing the
polypeptide encoded by the cDNA insert of the vector deposited on
May 14, 1998 with the ATCC as DNA53912-1457.
[0714] 81. PRO1083
[0715] Applicants have identified a cDNA clone that encodes a novel
polypeptide having sequence identity with a 7TM receptor,
latrophilin-related protein 1, and a macrophage restricted cell
surface glycoprotein, wherein the polypeptide is designated in the
present application as "PRO1083".
[0716] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO1083
polypeptide. In one aspect, the isolated nucleic acid comprises DNA
encoding the PRO1083 polypeptide having amino acid residues 1
through 693 of FIG. 204 (SEQ ID NO:483), 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. The isolated nucleic acid sequence may comprise the
cDNA insert of the vector deposited on May 12, 1998 with the ATCC
as DNA50921-1458 which includes the nucleotide sequence encoding
PRO1083.
[0717] In another embodiment, the invention provides isolated
PRO1083 polypeptide. In particular, the invention provides isolated
native sequence PRO1083 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 through 693
of FIG. 204 (SEQ ID NO:483). An additional embodiment of the
present invention is directed to an isolated extracellular domain
of a PRO1083 polypeptide. Optionally, the PRO1083 polypeptide is
obtained or is obtainable by expressing the polypeptide encoded by
the cDNA insert of the vector deposited on May 12, 1998 with the
ATCC as DNA50921-1458.
[0718] In another embodiment, the invention provides an expressed
sequence tag (EST) designated herein as DNA24256 which comprises
the nucleotide sequence of FIG. 205 (SEQ ID NO:484).
[0719] 82. PRO200
[0720] The objects of this invention, as defined generally supra,
are achieved at least in part by the provision of a novel
polypeptide, VEGF-E also herein designated PRO200, (SEQ ID NO:488)
and the nucleic acid encoding therefor, SEQ ID NO:487, residues 259
through 1293.
[0721] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a VEGF-E polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
VEGF-E polypeptide having amino acid residues 1 through 345 of FIG.
207 (SEQ ID NO:488), or is complementary to such encoding nucleic
acid sequence, and remains stably bound to it under low stringency
conditions. In another embodiment, variants are provided wherein
the VEGF-E nucleic acid has single or multiple deletions,
substitutions, insertions, truncations or combinations thereof.
[0722] In another embodiment, the invention provides isolated
VEGF-E polypeptide. In particular, the invention provides an
isolated native sequence VEGF-E polypeptide, which in one
embodiment, includes an amino acid sequence comprising residues 1
through 345 of FIG. 207 (SEQ ID NO:488). In another embodiment,
variants are provided wherein the VEGF-E polypeptide has single or
multiple deletions, substitiutions, insertions, truncations or
combinations thereof.
[0723] In yet further embodiments, the present invention is
directed to compositions useful for treating indications where
proliferation, survival and/or differentiation of cells is desired,
comprising a therapeutically effective amount of a VEGF-E
polypeptide hereof in admixture with a pharmaceutically acceptable
carrier.
[0724] The invention further includes associated embodiments of
VEGF-E such as modified VEGF-E polypeptides and modified variants
which have the same biological applications as VEGF-E, and
pharmaceutical compositions incorporating same. Inhibitors of
VEGF-E are also provided.
[0725] 83. PRO285 and PRO286
[0726] Applicants have identified two novel cDNA clones that encode
novel human Toll polypeptides, designated in the present
application as PRO285 (encoded by DNA40021-1154) and PRO286
(encoded by DNA42663-1154).
[0727] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising a DNA encoding a polypeptide
having at least about 80% sequence identity, preferably at least
about 85% sequence identity, more preferably at least about 90%
sequence identity, most preferably at least about 95% sequence
identity to (a) a DNA molecule encoding a PRO285 polypeptide having
amino acid residues 27 to 839 of FIG. 209 (SEQ ID NO:496); or (b)
to a DNA molecule encoding a PRO286 polypeptide having amino acid
residues 27 to 825 of FIG. 211 (SEQ ID NO:498) or (c) the
complement of the DNA molecule of (a) or (b). The complementary DNA
molecule preferably remains stably bound to such encoding nucleic
acid sequence under at least moderate, and optionally, under high
stringency conditions.
[0728] In a further embodiment, the isolated nucleic acid molecule
comprises a polynucleotide that has at least about 90%, preferably
at least about 95% sequence identity with a polynucleotide encoding
a polypeptide comprising the sequence of amino acids 1 to 839 of
FIG. 209 (SEQ ID NO:496); or at least about 90%, preferably at
least about 95% sequence identity with a polynucleotide encoding a
polypeptide comprising the sequence of amino acids 1 to 1041 of
FIG. 211 (SEQ ID NO:498).
[0729] In a specific embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding native or variant
PRO285 and PRO286 polypeptides, with or without the N-terminal
signal sequence, and with or without the transmembrane regions of
the respective full-length sequences. In one aspect, the isolated
nucleic acid comprises DNA encoding a mature, full-length native
PRO285 or PRO286 polypeptide having amino acid residues 1 to 1049
of FIG. 209 (SEQ ID NO:496) and 1 to 1041 of FIG. 211 (SEQ ID
NO:498), or is complementary to such encoding nucleic acid
sequence. In another aspect, the invention concerns an isolated
nucleic acid molecule that comprises DNA encoding a native PRO285
or PRO286 polypeptide without an N-terminal signal sequence, or is
complementary to such encoding nucleic acid sequence. In yet
another embodiment, the invention concerns nucleic acid encoding
transmembrane-domain deleted or inactivated forms of the
full-length native PRO285 or PRO286 proteins.
[0730] In another embodiment, the invention the isolated nucleic
acid molecule comprises the clone (DNA40021-1154) deposited on Oct.
17, 1997, under ATCC number 209389; or the clone (DNA42663-1154)
deposited on Oct. 17, 1997, under ATCC number 209386.
[0731] In yet another embodiment, the invention provides a vector
comprising DNA encoding PRO285 and PRO286 polypeptides, or their
variants. Thus, the vector may comprise any of the isolated nucleic
acid molecules hereinabove defined.
[0732] In another embodiment, the invention provides isolated
PRO285 and PRO286 polypeptides. In particular, the invention
provides isolated native sequence PRO285 and PRO286 polypeptides,
which in one embodiment, include the amino acid sequences
comprising residues 1 to 1049 and 1 to 1041 of FIGS. 209 and 211
(SEQ ID NOS:496 and 498), respectively. The invention also provides
for variants of the PRO285 and PRO286 polypeptides which are
encoded by any of the isolated nucleic acid molecules hereinabove
defined. Specific variants include, but are not limited to,
deletion (truncated) variants of the full-length native sequence
PRO285 and PRO286 polypeptides which lack the respective N-terminal
signal sequences and/or have their respective transmembrane and/or
cytoplasmic domains deleted or inactivated.
[0733] The invention also specifically includes antibodies with
dual specificities, e.g., bispecific antibodies binding more than
one Toll polypeptide.
[0734] In yet another embodiment, the invention concerns agonists
and antagonists of the native PRO285 and PRO286 polypeptides. In a
particular embodiment, the agonist or antagonist is an anti-PRO285
or anti-PRO286 antibody.
[0735] In a further embodiment, the invention concerns screening
assays to identify agonists or antagonists of the native PRO285 and
PRO286 polypeptides.
[0736] In a still firther embodiment, the invention concerns a
composition comprising a PRO285 or PRO286 polypeptide, or an
agonist or antagonist as hereinabove defined, in combination with a
pharmaceutically acceptable carrier.
[0737] The invention further concerns a composition comprising an
antibody specifically binding a PRO285 or PRO286 polypeptide, in
combination with a pharmaceutically acceptable carrier.
[0738] The invention also concerns a method of treating septic
shock comprising administering to a patient an effective amount of
an antagonist of a PRO285 or PRO286 polypeptide. In a specific
embodiment, the antagonist is a blocking antibody specifically
binding a native PRO285 or PRO286 polypeptide.
[0739] 84. PRO213-1, PRO1330 and PRO1449
[0740] The present invention concerns compositions and methods for
the diagnosis and treatment of neoplastic cell growth and
proliferation in mammals, including humans. The present invention
is based on the identification of genes that are amplified in the
genome of tumor cells. Such gene amplification is expected to be
associated with the overexpression of the gene product and
contribute to tumorigenesis. Accordingly, the proteins encoded by
the amplified genes are believed to be useful targets for the
diagnosis and/or treatment (including prevention) of certain
cancers, and may act as predictors of the prognosis of tumor
treatment.
[0741] In one embodiment, the present invention provides an
isolated nucleic acid molecule comprising DNA encoding a PRO213-1,
PRO1330 and/or PRO1449 polypeptide. In one aspect, the isolated
nucleic acid comprises DNA encoding the PRO213-1, PRO1330 and/or
PRO1449 polypeptide having amino acid residues 1 to 295 of FIG. 213
(SEQ ID NO:506), 20 to 273 of FIG. 215 (SEQ ID NO:508) and 20 to
273 of FIG. 217 (SEQ ID NO:510), 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. The isolated nucleic acid sequence may comprise the
cDNA insert of the vector designated as DNA30943-1163 (ATCC 209791)
deposited on Apr. 21, 1998; DNA64907-1163-1 (ATCC 203242) deposited
on Sep. 9, 1998 and/or DNA64908-1163-1 (ATCC 203243) deposited on
Sep. 9, 1998.
[0742] In another embodiment, the present invention comprises an
isolated nucleic acid molecule having at least about 80% sequence
identity, preferably at least about 85% sequence identity, more
preferably at least about 90% sequence identity, most preferably at
least about 95% sequence identity to (a) a DNA molecule encoding a
PRO213-1, PRO1330 and/or PRO1449 polypeptide having amino acid
residues 1 to 295 of FIG. 213 (SEQ ID NO:506), 20 to 273 of FIG.
215 (SEQ ID NO:508) and 20 to 273 of FIG. 217 (SEQ ID NO:510),
respectively; or (b) the complement of the DNA molecule of (a).
[0743] In another embodiment, the invention provides an isolated
PRO213-1, PRO1330 and/or PRO1449 polypeptide. In particular, the
invention provides isolated native sequence PRO213-1, PRO1330
and/or PRO1449 polypeptide, which in one embodiment, includes an
amino acid sequence comprising residues 1 to 295 of FIG. 213 (SEQ
ID NO:506), 20 to 273 of FIG. 215 (SEQ ID NO:508) or 20 to 273 of
FIG. 217 (SEQ ID NO:510), respectively. Optionally, the PRO213-1,
PRO1330 and/or PRO1449 polypeptide is obtained or obtainable by
expressing the polypeptide encoded by the cDNA insert of the
DNA30943-1163 (ATCC 209791), DNA64907-1163-1 (ATCC 203242) or
DNA64908-1163-1 (ATCC 203243).
[0744] In another aspect, the invention provides an isolated
PRO213-1, PRO1330, and/or PRO1449 polypeptide, comprising an amino
acid sequence having at least about 80% sequence identity,
preferably at least about 85% sequence identity, more preferably at
least about 95% sequence identity to amino acid residues 1 to 295
of FIG. 213 (SEQ ID NO:506), 20 to 273 of FIG. 215 (SEQ ID NO:508)
or 20 to 273 of FIG. 217 (SEQ ID NO:510), inclusive.
[0745] In yet another embodiment, the invention provides an
isolated PRO213-1, PRO1330, and/or PRO1449 polypeptide, comprising
the amino acid residues 1 to 295 of FIG. 213 (SEQ ID NO:506), 20 to
273 of FIG. 215 (SEQ ID NO:508) or 20 to 273 of FIG. 217 (SEQ ID
NO:510), or a fragment thereof sufficient to provide a binding site
for an anti-PRO213-1, anti-PRO1330 and/or anti-PRO1449 antibody.
Preferably, the PRO213-1, PRO1330, and/or PRO1449 fragment retains
a qualitative biological activity of a native PRO213-1, PRO1330,
and/or PRO1449 polypeptide.
[0746] In a further aspect, the invention concerns an isolated
PRO213-1, PRO1330, and/or PRO1449 polypeptide, comprising an amino
acid sequence scoring at least about 80% positives, preferably at
least about 85% positives, more preferably at least about 90%
positives, most preferably at least about 95% positives when
compared with the amino acid sequence of residues 1 to 295 of FIG.
213 (SEQ ID NO:506), 20 to 273 of FIG. 215 (SEQ ID NO:508) and 20
to 273 of FIG. 217 (SEQ ID NO:510), respectively.
[0747] In still a further aspect, the invention provides a
polypeptide produced by (i) hybridizing a test DNA molecule under
stringent conditions with: (a) a DNA molecule encoding a PRO213-1,
PRO1330, and/or PRO1449 polypeptide having the amino acid residues
from 1 to 295 of FIG. 213 (SEQ ID NO:506), 20 to 273 of FIG. 215
(SEQ ID NO:508) and 20 to 273 of FIG. 217 (SEQ ID NO:510),
respectively; or the complement of the DNA molecule of (a), and if
said test DNA molecule has at least about an 80% sequence identity
to (a) or (b), (ii) culturing a host cell comprising said test DNA
molecule under conditions suitable for the expression of said
polypeptide, and (iii) recovering said polypeptide from the cell
culture.
[0748] In one embodiment, the present invention concerns an
isolated antibody which binds a PRO213-1, PRO1330 and/or PRO1449
polypeptide. In one aspect, the antibody induces death of a cell
overexpressing a PRO213-1, PRO1330 and/or PRO1449 polypeptide. In
another aspect, the antibody is a monoclonal antibody, which
preferably has nonhuman complementarity determining region (CDR)
residues and human framework region (FR) residues. The antibody may
be labeled and may be immobilized on a solid support. In a further
aspect, the antibody is an antibody fragrnent, a single-chain
antibody, or an anti-idiotypic antibody.
[0749] In another embodiment, the invention concerns a composition
comprising an antibody which binds a PRO213-1, PRO1330 and/or
PRO1449 polypeptide in admixture with a pharmaceutically acceptable
carrier. In one aspect, the composition comprises a therapeutically
effective amount of the antibody. In another aspect, the
composition comprises a further active ingredient, which may, for
example, be a further antibody or a cytotoxic or chemotherapeutic
agent. Preferably, the composition is sterile.
[0750] In a further embodiment, the inventionconcerns nucleic acid
encoding an anti-PRO213-1, anti-PRO1330 and/or anti-PRO1449
antibody, and vectors and recombinant host cells comprising such
nucleic acid.
[0751] The invention further concerns antagonists and agonists of a
PRO213-1, PRO1330 and/or PRO1449 polypeptide that inhibit one or
more of the functions or activities of the PRO213-1, PRO1330 and/or
PRO1449 polypeptide.
[0752] In a further embodiment, the invention concerns isolated
nucleic acid molecules that hybridize to the complement of the
nucleic acid molecules encoding the PRO213-1, PRO1330 and/or
PRO1449 polypeptides. The nucleic acid preferably is DNA, and
hybridization preferably occurs under stringent conditions. Such
nucleic acid molecules can act as antisense molecules of the
amplified genes identified herein, which, in turn, can find use in
the modulation of the respective amplified genes, or as antisense
primers in amplification reactions. Furthermore, such sequences can
be used as part of ribozyme and/or triple helix sequence which, in
turn, may be used in regulation of the amplified genes.
[0753] In another embodiment, the invention concerns a method for
determining the presence of a PRO213-1, PRO1330 and/or PRO1449
polypeptide comprising exposing a cell suspected of containing the
PRO213-1, PRO1330 and/or PRO1449 polypeptide to an anti-PRO213-1,
PRO1330 and/or PRO1449 antibody and determining binding of the
antibody to the cell.
[0754] In yet another embodiment, the present invention concerns a
method of diagnosing tumor in a mammal, comprising detecting the
level of expression of a gene encoding a PRO213-1, PRO1330 and/or
PRO1449 polypeptide (a) in a test sample of tissue cells obtained
from the manmal, and (b) in a control sample of known normal tissue
cells of the same cell type, wherein a higher expression level in
the test sample indicates the presence of tumor in the mammal from
which the test tissue cells were obtained.
[0755] In another embodiment, the present invention concerns a
method of diagnosing tumor in a mammal, comprising (a) contacting
an anti-PRO213-1, anti-PRO1330 and/or anti-PRO1449 antibody with a
test sample of tissue cells obtained from the mammal, and (b)
detecting the formation of a complex between the anti-PRO213-1,
anti-PRO1330 and/or anti-PRO1449 antibody and the PRO213-1, PRO1330
and/or PRO1449 polypeptide in the test sample. The detection may be
qualitative or quantitative, and may be performed in comparison
with monitoring the complex formation in a control sample of known
normal tissue cells of the same cell type. A larger quantity of
complexes formed in the test sample indicates the presence of tumor
in the mammal from which the test tissue cells were obtained. The
antibody preferably carries a detectable label. Complex formation
can be monitored, for example, by light microscopy, flow cytometry,
fluorimetry, or other techniques known in the art. The test sample
is usually obtained from an individual suspected to have neoplastic
cell growth or proliferation (e.g. cancerous cells).
[0756] In another embodiment, the present invention concerns a
cancer diagnostic kit, comprising an anti-PRO213-1, anti-PRO1330
and/or anti-PRO1449 antibody and a carrier (e.g. a buffer) in
suitable packaging. The kit preferably contains instructions for
using the antibody to detect the PRO213-1, PRO1330 and/or PRO1449
polypeptide.
[0757] In yet another embodiment, the invention concerns a method
for inhibiting the growth of tumor cells comprising exposing a cell
which overexpresses a PRO213-1, PRO1330 and/or PRO1449 polypeptide
to an effective amount of an agent inhibiting the expression and/or
activity of the PRO213-1, PRO1330 and/or PRO1449 polypeptide. The
agent preferably is an anti-PRO213-1, anti-PRO1330 and/or
anti-PRO1449 antibody, a small organic and inorganic molecule,
peptide, phosphopeptide, antisense or ribozyme molecule, or a
triple helix molecule. In a specific aspect, the agent, e.g.
anti-PRO213-1, anti-PRO1330 and/or anti-PRO1449 antibody induces
cell death. In a further aspect, the tumor cells are further
exposed to radiation treatment and/or a cytotoxic or
chemotherapeutic agent.
[0758] In a further embodiment, the invention concerns an article
of manufacture, comprising:
[0759] a) a container;
[0760] b) a label on the container; and
[0761] c) a composition comprising an active agent contained within
the container; wherein the composition is effective for inhibiting
the growth of tumor cells, the label on the container indicates
that the composition can be used for treating conditions
characterized by overexpression of a PRO213-1, PRO1330 and/or
PRO1449 polypeptide, and the active agent in the composition is an
agent inhibiting the expression and/or activity of the PRO213-1,
PRO1330 and/or PRO1449 polypeptide. In a preferred aspect, the
active agent is an anti-PRO213-1, anti-PRO1330 and/or anti-PRO1449
antibody.
[0762] In yet a further embodiment, the invention provides a method
for identilying a compound capable of inhibiting the expression
and/or activity of a PRO213-1, PRO1330 and/or PRO1449 polypeptide,
comprising contacting a candidate compound with a PRO213-1, PRO1330
and/or PRO1449 polypeptide under conditions and for a time
sufficient to allow these two components to interact. In a specific
aspect, either the candidate compound or the PRO213-1, PRO1330
and/or PRO1449 polypeptide is immobilized on a solid support. In
another aspect, the non-immobilized component carries a detectable
label.
[0763] 85. PRO298
[0764] Applicants have identified a cDNA clone that encodes a novel
polypeptide. The DNA is designated in the present application as
"DNA39975-1210", encoding a novel multi-transmnembrane protein,
referred to as "PRO298".
[0765] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA having at least about 80%,
preferably at least about 85%, more preferably at least about 90%,
most preferably at least about 95% sequence identity to (a) a DNA
molecule encoding PRO298, comprising the sequence of amino acids 1
to 364 of FIG. 219 (SEQ ID NO:515), or (b) the complement of the
DNA molecule of (a). In one aspect, the isolated nucleic acid
comprises DNA encoding a PRO298 polypeptide having amino acid
residues 1 to 364 of FIG. 219 (SEQ ID NO:515), 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.
[0766] In a further embodiment, the invention concerns an isolated
nucleic acid molecule comprising DNA having at least an 80%
sequence identity to (a) a DNA molecule encoding the same mature
polypeptide encoded by the human protein cDNA in ATCC Deposit No.
209783 (DNA9975-1210), or (b) the complement of the DNA molecule of
(a).
[0767] In a still further embodiment, the invention concerns
nucleic acid which comprises a DNA molecule encoding the same
mature polypeptide encoded by the human protein cDNA in ATCC
Deposit No. 209783 (DNA9975-1210).
[0768] In another embodiment, the invention provides isolated
PRO298 polypeptide. In particular, the invention provides isolated
native sequence PRO298 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 364 of
FIG. 219 (SEQ ID NO:515).
[0769] In another embodiment, the invention provides an expressed
sequence tag (EST) designated DNA26832 comprising the nucleotide
sequence of FIG. 220 (SEQ ID NO:516).
[0770] 86. PRO337
[0771] Applicants have identified a cDNA clone (DNA43316-1237) that
encodes a novel polypeptide, designated in the present application
as "PRO337".
[0772] 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 PRO337 polypeptide comprising the
sequence of amino acids 1 to 344 of FIG. 222 (SEQ ID NO:523), 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
(including 96, 97, 98 and 99%) sequence identity with a polypeptide
having amino acid residues 1 to 344 of FIG. 222 (SEQ ID NO:523).
Preferably, the highest degree of sequence identity occurs within
the immunoglobulin and major histocompatibility domains (amino
acids 113 to 130 of FIG. 222, SEQ ID NO:523).
[0773] In a further embodiment, the isolated nucleic acid molecule
comprises DNA encoding a neurotrimin polypeptide having amino acid
residues 1 to 344 of FIG. 222 (SEQ ID NO:523), 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 DNA43316-1237, deposited
with the ATCC under accession number ATCC 209487, alternatively the
coding sequence of clone DNA43316-1237, deposited under accession
number ATCC 209487.
[0774] In yet another embodiment, the invention provides isolated
PRO337 polypeptide. In particular, the invention provides isolated
native sequence PRO337 polypeptide, which in one embodiment,
inchlides an amino acid sequence comprising residues 1 to 344 of
FIG. 222 (SEQ ID NO:523). Native PRO337 polypeptides with or
without the native signal sequence (amino acids 1 to about 28 in
FIG. 222 (SEQ ID NO:523), and with or without the initiating
methionine are specifically included. Alternatively, the invention
provides a PRO337 polypeptide encoded by the nucleic acid deposited
under accession number ATCC 209487.
[0775] In yet another embodiment, the invention provides an
expressed sequence tag (EST) comprising the nucleotide sequences
identified in FIG. 223 as DNA42301 (SEQ ID NO:524).
[0776] 87. PRO403
[0777] Applicants have identified a cDNA clone (DNA55800-1263) that
encodes a novel polypeptide, designated in the present application
as "PRO403".
[0778] 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 PRO403 polypeptide comprising the
sequence of amino acids 1 to 736 of FIG. 225 (SEQ ID NO:526), 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 736 of
FIG. 225 (SEQ ID NO:526). Preferably, the highest degree of
sequence identity occurs within: (1) the putative N-glycosylatation
sites (amino acid residues 132, 136, 177, 237, 282, 349, 505, 598
and 606; (2) Cys residues conserved with the Kell blood group
protein family (amino acid residues 65, 70, 88 and 96) and the
putative zinc binding motif (amino acid residues 570-579).
[0779] In a further embodiment, the isolated nucleic acid molecule
comprises DNA encoding a PRO403 polypeptide having amino acid
residues 1 to 736 of FIG. 225 (SEQ ID NO:526), 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 DNA55800-1263, deposited
with the ATCC under accession number ATCC 209680, alternatively the
coding sequence of clone DNA55800-1263, deposited under accession
number ATCC 209680.
[0780] In yet another embodiment, the invention provides isolated
PRO403 polypeptide. In particular, the invention provides isolated
native sequence PRO403 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 736 of
FIG. 225 (SEQ ID NO:526). Native PRO403 polypeptides with or the
initiating methionine are specifically included. Alternatively, the
invention provides a PRO403 polypeptide encoded by the nucleic acid
deposited under accession number ATCC 209680.
[0781] In yet another embodiment, the invention provides an
expressed sequence tag (EST) and other sequence fragments
comprising the nucleotide sequences identified herein as DNA34415
(FIG. 226; SEQ ID NO:527); DNA49830 (FIG. 227; SEQ ID NO:528) and
DNA49831 (FIG. 228; SEQ ID NO:529).
[0782] 88. Additional Embodiments
[0783] 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.
[0784] 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.
[0785] 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.
[0786] In yet other embodiments, the invention provides
oligonucleotide probes useful for isolating genomic and cDNA
nucleotide sequences or as antisense probes, wherein those probes
may be derived from any of the above or below described nucleotide
sequences.
[0787] In other embodiments, the invention provides an isolated
nucleic acid molecule comprising a nucleotide sequence that encodes
a PRO polypeptide.
[0788] 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, an extracellular domain of a
transmembrane protein, with or without the signal peptide, as
disclosed herein or any other specifically defined fragment of the
full-length amino acid sequence as disclosed herein, or (b) the
complement of the DNA molecule of (a).
[0789] 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, the
coding sequence of an extracellular domain of a transmembrane PRO
polypeptide, with or without the signal peptide, as disclosed
herein or the coding sequence of any other specifically defined
fragment of the full-length amino acid sequence as disclosed
herein, or (b) the complement of the DNA molecule of (a).
[0790] 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).
[0791] Another aspect the invention provides an isolated nucleic
acid molecule comprising a nucleotide sequence encoding a PRO
polypeptide which is either transmembrane domnain-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.
[0792] 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, for encoding
fragments of a PRO polypeptide that may optionally encode a
polypeptide comprising a binding site for an anti-PRO antibody or
as antisense oligonucleotide probes. Such nucleic acid fragments
are usually at least about 20 nucleotides in length, preferably at
least about 30 nucleotides in length, more 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
coding 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.
[0793] In another embodiment, the invention provides isolated PRO
polypeptide encoded by any of the isolated nucleic acid sequences
hereinabove identified.
[0794] 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 leastabout 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, an extracellular domain of
a transmembrane protein, with or without the signal peptide, as
disclosed herein or any other specifically defined fragment of the
full-length amino acid sequence as disclosed herein.
[0795] 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 9 4% 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.
[0796] 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, an extracellular
domain of a transmembrane protein, with or without the signal
peptide, as disclosed herein or any other specifically defined
fragment of the full-length amino acid sequence as disclosed
herein.
[0797] 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.
[0798] 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.
[0799] 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.
[0800] 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.
[0801] 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.
[0802] 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
[0803] FIG. 1 shows a nucleotide sequence (SEQ ID NO:1) of a native
sequence PRO213 cDNA, wherein SEQ ID NO:1 is a clone designated
herein as "UNQ187" and/or "DNA30943-1163".
[0804] 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.
[0805] FIG. 3 shows a nucleotide sequence (SEQ ID NO:6) of a native
sequence PRO274 cDNA, wherein SEQ ID NO:6 is a clone designated
herein as "UNQ241" and/or "DNA39987-1184".
[0806] FIG. 4 shows the amino acid sequence (SEQ ID NO:7) derived
from the coding sequence of SEQ ID NO:6 shown in FIG. 3.
[0807] FIG. 5 shows an EST nucleotide sequence designated herein as
DNA17873 (SEQ ID NO:8).
[0808] FIG. 6 shows an EST nucleotide sequence designated herein as
DNA36157 (SEQ ID NO:9).
[0809] FIG. 7 shows an EST nucleotide sequence designated herein as
DNA28929 (SEQ ID NO:10).
[0810] FIG. 8 shows a nucleotide sequence (SEQ ID NO:18) of a
native sequence PRO300 cDNA, wherein SEQ ID NO:18 is a clone
designated herein as "UNQ263" and/or "DNA40625-1189".
[0811] FIG. 9 shows the amino acid sequence (SEQ ID NO:19) derived
from the coding sequence of SEQ ID NO:18 shown in FIG. 8.
[0812] FIG. 10 shows a nucleotide sequence (SEQ ID NO:27) of a
native sequence PRO284 cDNA, wherein SEQ ID NO:27 is a clone
designated herein as "UNQ247" and/or "DNA23318-1211".
[0813] FIG. 11 shows the amino acid sequence (SEQ ID NO:28) derived
from the coding sequence of SEQ ID NO:27 shown in FIG. 10.
[0814] FIG. 12 shows an EST nucleotide sequence designated herein
as DNA12982 (SEQ ID NO:29).
[0815] FIG. 13 shows an EST nucleotide sequence designated herein
as DNA15886 (SEQ ID NO:30).
[0816] FIG. 14 shows a nucleotide sequence (SEQ ID NO:35) of a
native sequence PRO296 cDNA, wherein SEQ ID NO:35 is a clone
designated herein as "UNQ260" and/or "DNA39979-1213".
[0817] FIG. 15 shows the amino acid sequence (SEQ ID NO:36) derived
from the coding sequence of SEQ ID NO:35 shown in FIG. 14.
[0818] FIG. 16 shows an EST nucleotide sequence designated herein
as DNA23020 (SEQ ID NO:37).
[0819] FIG. 17 shows an EST nucleotide sequence designated herein
as DNA21971 (SEQ ID NO:38).
[0820] FIG. 18 shows an EST nucleotide sequence designated herein
as DNA29037 (SEQ ID NO:39).
[0821] FIG. 19 shows a nucleotide sequence (SEQ ID NO:44) of a
native sequence PRO329 cDNA, wherein SEQ ID NO:44 is a clone
designated herein as "UNQ291" and/or "DNA40594-1233".
[0822] FIG. 20 shows the amino acid sequence (SEQ ID NO:45) derived
from the coding sequence of SEQ ID NO:44 shown in FIG. 19.
[0823] FIG. 21 shows a nucleotide sequence (SEQ ID NO:51) of a
native sequence PRO362 cDNA, wherein SEQ ID NO:51 is a clone
designated herein as "UNQ317" and/or "DNA45416-1251".
[0824] FIG. 22 shows the amino acid sequence (SEQ ID NO:52) derived
from the coding sequence of SEQ ID NO:51 shown in FIG. 21.
[0825] FIG. 23 shows a nucleotide sequence (SEQ ID NO:58) of a
native sequence PRO363 cDNA, wherein SEQ ID NO:58 is a clone
designated herein as "UNQ318" and/or "DNA45419-1252".
[0826] FIG. 24 shows the amino acid sequence (SEQ ID NO:59) derived
from the coding sequence of SEQ ID NO:58 shown in FIG. 23.
[0827] FIG. 25 shows a nucleotide sequence (SEQ ID NO:63) of a
native sequence PRO868 cDNA, wherein SEQ ID NO:63 is a clone
designated herein as "UNQ437" and/or "DNA52594-1270".
[0828] FIG. 26 shows the amino acid sequence (SEQ ID NO:64) derived
from the coding sequence of SEQ ID NO:63 shown in FIG. 25.
[0829] FIG. 27 shows a nucleotide sequence (SEQ ID NO:68) of a
native sequence PRO382 cDNA, wherein SEQ ID NO:68 is a clone
designated herein as "UNQ323" and/or "DNA45234-1277".
[0830] FIG. 28 shows the amino acid sequence (SEQ ID NO:69) derived
from the coding sequence of SEQ ID NO:68 shown in FIG. 27.
[0831] FIG. 29 shows anucleotide sequence (SEQ ID NO:73) of a
native sequence PRO545 cDNA, wherein SEQ ID NO:73 is a clone
designated herein as "UNQ346" and/or "DNA49624-1279".
[0832] FIG. 30 shows the amino acid sequence (SEQ ID NO:74) derived
from the coding sequence of SEQ ID NO:73 shown in FIG. 29.
[0833] FIG. 31 shows an EST nucleotide sequence designated herein
as DNA13217 (SEQ ID NO:75).
[0834] FIG. 32 shows a nucleotide sequence (SEQ ID NO:84) of a
native sequence PRO617 cDNA, wherein SEQ ID NO:84 is a clone
designated herein as "UNQ353" and/or "DNA48309-1280".
[0835] FIG. 33 shows the amino acid sequence (SEQ ID NO:85) derived
from the coding sequence of SEQ ID NO:84 shown in FIG. 32.
[0836] FIG. 34 shows a nucleotide sequence (SEQ ID NO:89) of a
native sequence PRO700 cDNA, wherein SEQ ID NO:89 is a clone
designated herein as "UNQ364" and/or "DNA46776-1284".
[0837] FIG. 35 shows the amino acid sequence (SEQ ID NO:90) derived
from the coding sequence of SEQ ID NO:89 shown in FIG. 34.
[0838] FIG. 36 shows a nucleotide sequence (SEQ ID NO:96) of a
native sequence PRO702 cDNA, wherein SEQ ID NO:96 is a clone
designated herein as "UNQ366" and/or "DNA50980-1286".
[0839] FIG. 37 shows the amino acid sequence (SEQ ID NO:97) derived
from the coding sequence of SEQ ID NO:96 shown in FIG. 36.
[0840] FIG. 38 shows a nucleotide sequence (SEQ ID NO:101) of a
native sequence PRO703 cDNA, wherein SEQ ID NO:101 is a clone
designated herein as "UNQ367" and/or "DNA50913-1287".
[0841] FIG. 39 shows the amino acid sequence (SEQ ID NO:102)
derived from the coding sequence of SEQ ID NO:101 shown in FIG.
38.
[0842] FIG. 40 shows a nucleotide sequence (SEQ ID NO:108) of a
native sequence PRO705 cDNA, wherein SEQ ID NO:108 is a clone
designated herein as "UNQ369" and/or "DNA50914-1289".
[0843] FIG. 41 shows the amino acid sequence (SEQ ID NO:109)
derived from the coding sequence of SEQ ID NO:108 shown in FIG.
40.
[0844] FIGS. 42A-B show a nucleotide sequence (SEQ ID NO:113) of a
native sequence PRO708 cDNA, wherein SEQ ID NO:113 is a clone
designated herein as "UNQ372" and/or "DNA48296-1292".
[0845] FIG. 43 shows the amino acid sequence (SEQ ID NO:114)
derived from the coding sequence of SEQ ID NO:113 shown in FIGS.
42A-B.
[0846] FIG. 44 shows a nucleotide sequence (SEQ ID NO:118) of a
native sequence PRO320 cDNA, wherein SEQ ID NO:118 is a clone
designated herein as "UNQ281" and/or "DNA32284-1307".
[0847] FIG. 45 shows the amino acid sequence (SEQ ID NO:119)
derived from the coding sequence of SEQ ID NO:118 shown in FIG.
44.
[0848] FIG. 46 shows a nucleotide sequence (SEQ ID NO:123) of a
native sequence PRO324cDNA, wherein SEQ ID NO:123 is a clone
designated herein as "UNQ285" and/or "DNA36343-1310".
[0849] FIG. 47 shows the amino acid sequence (SEQ ID NO:124)
derived from the coding sequence of SEQ ID NO:123 shown in FIG.
46.
[0850] FIG. 48 shows a nucleotide sequence (SEQ ID NO:131) of a
native sequence PRO351 cDNA, wherein SEQ ID NO:131 is a clone
designated herein as "UNQ308" and/or "DNA40571-1315".
[0851] FIG. 49 shows the amino acid sequence (SEQ ID NO:132)
derived from the coding sequence of SEQ ID NO:131 shown in FIG.
48.
[0852] FIG. 50 shows a nucleotide sequence (SEQ ID NO:136) of a
native sequence PRO352 cDNA, wherein SEQ ID NO:136 is a clone
designated herein as "UNQ309" and/or "DNA41386-1316".
[0853] FIG. 51 shows the amino acid sequence (SEQ ID NO:137)
derived from the coding sequence of SEQ ID NO:136 shown in FIG.
50.
[0854] FIG. 52 shows a nucleotide sequence (SEQ ID NO:144) of a
native sequence PRO381 cDNA, wherein SEQ ID NO:144 is a clone
designated herein as "UNQ322" and/or "DNA44194-1317".
[0855] FIG. 53 shows the amino acid sequence (SEQ ID NO:145)
derived from the coding sequence of SEQ ID NO:144 shown in FIG.
52.
[0856] FIG. 54 shows a nucleotide sequence (SEQ ID NO:149) of a
native sequence PRO386 cDNA, wherein SEQ ID NO:149 is a clone
designated herein as "UNQ326" and/or "DNA45415-1318".
[0857] FIG. 55 shows the amino acid sequence (SEQ ID NO:150)
derived from the coding sequence of SEQ ID NO:149 shown in FIG.
54.
[0858] FIG. 56 shows an EST nucleotide sequence designated herein
as DNA23350 (SEQ ID NO:151).
[0859] FIG. 57 shows an EST nucleotide sequence designated herein
as DNA23536 (SEQ ID NO:152).
[0860] FIG. 58 shows a nucleotide sequence (SEQ ID NO:156) of a
native sequence PRO540 cDNA, wherein SEQ ID NO:156 is a clone
designated herein as "UNQ341" and/or "DNA44189-1322".
[0861] FIG. 59 shows the amino acid sequence (SEQ ID NO:157)
derived from the coding sequence of SEQ ID NO:156 shown in FIG.
58.
[0862] FIG. 60 shows a nucleotide sequence (SEQ ID NO:161) of a
native sequence PRO615 cDNA, wherein SEQ ID NO:161 is a clone
designated herein as "UNQ352" and/or "DNA48304-1323".
[0863] FIG. 61 shows the amino acid sequence (SEQ ID NO:162)
derived from the coding sequence of SEQ ID NO:161 shown in FIG.
60.
[0864] FIG. 62 shows a nucleotide sequence (SEQ ID NO:168) of a
native sequence PRO618 cDNA, wherein SEQ ID NO:168 is a clone
designated herein as "UNQ354" and/or "DNA49152-1324".
[0865] FIG. 63 shows the amino acid sequence (SEQ ID NO:169)
derived from the coding sequence of SEQ ID NO:168 shown in FIG.
62.
[0866] FIG. 64 shows an EST nucleotide sequence designated herein
as DNA35597 (SEQ ID NO:170).
[0867] FIG. 65 shows a nucleotide sequence (SEQ ID NO:177) of a
native sequence PRO719 cDNA, wherein SEQ ID NO:177 is a clone
designated herein as "UNQ387" and/or "DNA49646-1327".
[0868] FIG. 66 shows the amino acid sequence (SEQ ID NO:178)
derived from the coding sequence of SEQ ID NO:177 shown in FIG.
65.
[0869] FIG. 67 shows a nucleotide sequence (SEQ ID NO:182) of a
native sequence PRO724 cDNA, wherein SEQ ID NO:182 is a clone
designated herein as "UNQ389" and/or "DNA49631-1328".
[0870] FIG. 68 shows the amino acid sequence (SEQ ID NO:183)
derived from the coding sequence of SEQ ID NO:182 shown in FIG.
67.
[0871] FIG. 69 shows a nucleotide sequence (SEQ ID NO:189) of a
native sequence PRO772 cDNA, wherein SEQ ID NO:189 is a clone
designated herein as "UNQ410" and/or "DNA49645-1347".
[0872] 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.
[0873] FIG. 71 shows an EST nucleotide sequence designated herein
as DNA43509 (SEQ ID NO:191).
[0874] FIG. 72 shows a nucleotide sequence (SEQ ID NO:195) of a
native sequence PRO852 cDNA, wherein SEQ ID NO:195 is a clone
designated herein as "UNQ418" and/or "DNA45493-1349".
[0875] FIG. 73 shows the amino acid sequence (SEQ ID NO:196)
derived from the coding sequence of SEQ ID NO:195 shown in FIG.
72.
[0876] FIG. 74 shows a nucleotide sequence (SEQ ID NO:205) of a
native sequence PRO853 cDNA, wherein SEQ ID NO:205 is a clone
designated herein as "UNQ419" and/or "DNA48227-1350".
[0877] FIG. 75 shows the amino acid sequence (SEQ ID NO:206)
derived from the coding sequence of SEQ ID NO:205 shown in FIG.
74.
[0878] FIG. 76 shows a nucleotide sequence (SEQ ID NO:210) of a
native sequence PRO860 cDNA, wherein SEQ ID NO:210 is a clone
designated herein as "UNQ421" and/or "DNA41404-1352".
[0879] FIG. 77 shows the amino acid sequence (SEQ ID NO:211)
derived from the coding sequence of SEQ ID NO:210 shown in FIG.
76.
[0880] FIG. 78 shows a nucleotide sequence (SEQ ID NO:215) of a
native sequence PRO846 cDNA, wherein SEQ ID NO:215 is a clone
designated herein as "UNQ422" and/or "DNA44196-1353".
[0881] FIG. 79 shows the amino acid sequence (SEQ ID NO:216)
derived from the coding sequence of SEQ IID NO:215 shown in FIG.
78.
[0882] FIG. 80 shows a nucleotide sequence (SEQ ID NO:220) of a
native sequence PRO862 cDNA, wherein SEQ ID NO:220 is a clone
designated herein as "UNQ424" and/or "DNA52187-1354".
[0883] FIG. 81 shows the amino acid sequence (SEQ ID NO:221)
derived from the coding sequence of SEQ ID NO:220 shown in FIG.
80.
[0884] FIG. 82 shows a nucleotide sequence (SEQ ID NO:225) of a
native sequence PRO864 cDNA, wherein SEQ ID NO:225 is a clone
designated herein as "UNQ426" and/or "DNA48328-1355".
[0885] FIG. 83 shows the amino acid sequence (SEQ ID NO:226)
derived from the coding sequence of SEQ ID NO:225 shown in FIG.
82.
[0886] FIG. 84 shows a nucleotide sequence (SEQ ID NO:230) of a
native sequence PRO792 cDNA, wherein SEQ ID NO:230 is a clone
designated herein as "UNQ431" and/or "DNA56352-1358".
[0887] FIG. 85 shows the amino acid sequence (SEQ ID NO:231)
derived from the coding sequence of SEQ ID NO:230 shown in FIG.
84.
[0888] FIG. 86 shows a nucleotide sequence (SEQ ID NO:235) of a
native sequence PRO866 cDNA, wherein SEQ ID NO:235 is a clone
desiged herein as "UNQ435" and/or "DNA53971-1359".
[0889] FIG. 87 shows the amino acid sequence (SEQ ID NO:236)
derived from the coding sequence of SEQ ID NO:235 shown in FIG.
86.
[0890] FIG. 88 shows a nucleotide sequence (SEQ ID NO:244) of a
native sequence PRO871 cDNA, wherein SEQ ID NO:244 is a clone
designated herein as "UNQ438" and/or "DNA50919-1361".
[0891] FIG. 89 shows the amino acid sequence (SEQ ID NO:245)
derived from the coding sequence of SEQ ID NO:244 shown in FIG.
88.
[0892] FIG. 90 shows a nucleotide sequence (SEQ ID NO:253) of a
native sequence PRO873 cDNA, wherein SEQ ID NO:253 is a clone
designated herein as "UNQ440" and/or "DNA44179-1362".
[0893] FIG. 91 shows the amino acid sequence (SEQ ID NO:254)
derived from the coding sequence of SEQ ID NO:253 shown in FIG.
90.
[0894] FIG. 92 shows a nucleotide sequence (SEQ ID NO:258) of a
native sequence PRO940 cDNA, wherein SEQ ID NO:258 is a clone
designated herein as "UNQ477" and/or "DNA54002-1367".
[0895] FIG. 93 shows the amino acid sequence (SEQ ID NO:259)
derived from the coding sequence of SEQ ID NO:258 shown in FIG.
92.
[0896] FIG. 94 shows a nucleotide sequence (SEQ ID NO:263) of a
native sequence PRO941 cDNA, wherein SEQ ID NO:263 is a clone
designated herein as "UNQ478" and/or "DNA53906-1368".
[0897] FIG. 95 shows the amino acid sequence (SEQ ID NO:264)
derived from the coding sequence of SEQ ID NO:263 shown in FIG.
94.
[0898] FIG. 96 shows an EST nucleotide sequence designated herein
as DNA6415 (SEQ ID NO:265).
[0899] FIG. 97 shows a nucleotide sequence (SEQ ID NO:269) of a
native sequence PRO944 cDNA, wherein SEQ ID NO:269 is a clone
designated herein as "UNQ481" and/or "DNA52185-1370".
[0900] FIG. 98 shows the amino acid sequence (SEQ ID NO:270)
derived from the coding sequence of SEQ ID NO:269 shown in FIG.
97.
[0901] FIG. 99 shows an EST nucleotide sequence designated herein
as DNA14007 (SEQ ID NO:271).
[0902] FIG. 100 shows an EST nucleotide sequence designated herein
as DNA12773 (SEQ ID NO:272).
[0903] FIG. 101 shows an EST nucleotide sequence designated herein
as DNA12746 (SEQ ID NO:273).
[0904] FIG. 102 shows an EST nucleotide sequence designated herein
as DNA12834 (SEQ ID NO:274).
[0905] FIG. 103 shows an EST nucleotide sequence designated herein
as DNA12846 (SEQ ID NO:275).
[0906] FIG. 104 shows an EST nucleotide sequence designated herein
as DNA13104 (SEQ ID NO:276).
[0907] FIG. 105 shows an EST nucleotide sequence designated herein
as DNA13259 (SEQ ID NO:277).
[0908] FIG. 106 shows an EST nucleotide sequence designated herein
as DNA13959 (SEQ ID NO:278).
[0909] FIG. 107 shows an EST nucleotide sequence designated herein
as DNA13961 (SEQ ID NO:279).
[0910] FIG. 108 shows a nucleotide sequence (SEQ ID NO:283) of a
native sequence PRO983 cDNA, wherein SEQ ID NO:283 is a clone
designated herein as "UNQ484" and/or "DNA53977-1371".
[0911] FIG. 109 shows the amino acid sequence (SEQ ID NO:284)
derived from the coding sequence of SEQ ID NO:283 shown in FIG.
108.
[0912] FIG. 110 shows an EST nucleotide sequence designated herein
as DNA17130 (SEQ ID NO:285).
[0913] FIG. 111 shows an EST nucleotide sequence designated herein
as DNA23466 (SEQ ID NO:286).
[0914] FIG. 112 shows an EST nucleotide sequence designated herein
as DNA26818 (SEQ ID NO:287).
[0915] FIG. 113 shows an EST nucleotide sequence designated herein
as DNA37618 (SEQ ID NO:288).
[0916] FIG. 114 shows an EST nucleotide sequence designated herein
as DNA41732 (SEQ ID NO:289).
[0917] FIG. 115 shows an EST nucleotide sequence designated herein
as DNA45980 (SEQ ID NO:290).
[0918] FIG. 116 shows an EST nucleotide sequence designated herein
as DNA46372 (SEQ ID NO:291).
[0919] FIG. 117 shows a nucleotide sequence (SEQ ID NO:295) of a
native sequence PRO1057 cDNA, wherein SEQ ID NO:295 is a clone
designated herein as "UNQ522" and/or "DNA57253-1382".
[0920] FIG. 118 shows the amino acid sequence (SEQ ID NO:296)
derived from the coding sequence of SEQ ID NO:295 shown in FIG.
117.
[0921] FIG. 119 shows a nucleotide sequence (SEQ ID NO:300) of a
native sequence PRO1071 cDNA, wherein SEQ ID NO:300 is a clone
designated herein as "UNQ528" and/or "DNA58847-1383".
[0922] FIG. 120 shows the amino acid sequence (SEQ ID NO:301)
derived from the coding sequence of SEQ ID NO:300 shown in FIG.
119.
[0923] FIG. 121 shows a nucleotide sequence (SEQ ID NO:302) of a
native sequence PRO1072 cDNA, wherein SEQ ID NO:302 is a clone
designated herein as "UNQ529" and/or "DNA58747-1384".
[0924] FIG. 122 shows the amino acid sequence (SEQ ID NO:303)
derived from the coding sequence of SEQ ID NO:302 shown in FIG.
121.
[0925] FIG. 123 shows an EST nucleotide sequence designated herein
as DNA40210 (SEQ ID NO:304).
[0926] FIG. 124 shows a nucleotide sequence (SEQ ID NO:308) of a
native sequence PRO1075 cDNA, wherein SEQ ID NO:308 is a clone
designated herein as "UNQ532" and/or "DNA57689-1385".
[0927] FIG. 125 shows the amino acid sequence (SEQ ID NO:309)
derived from the coding sequence of SEQ ID NO:308 shown in FIG.
124.
[0928] FIG. 126 shows an EST nucleotide sequence designated herein
as DNA13059 (SEQ ID NO:310).
[0929] FIG. 127 shows an EST nucleotide sequence designated herein
as DNA19463 (SEQ ID NO:311).
[0930] FIG. 128 shows a nucleotide sequence (SEQ ID NO:321) of a
native sequence PRO181 cDNA, wherein SEQ ID NO:321 is a clone
designated herein as "UNQ155" and/or "DNA23330-1390".
[0931] FIG. 129 shows the amino acid sequence (SEQ ID NO:322)
derived from the coding sequence of SEQ ID NO:321 shown in FIG.
128.
[0932] FIG. 130 shows an EST nucleotide sequence designated herein
as DNA13242 (SEQ ID NO:323).
[0933] FIG. 131 shows a nucleotide sequence (SEQ ID NO:329) of a
native sequence PRO195 cDNA, wherein SEQ ID NO:329 is a clone
designated herein as "UNQ169" and/or "DNA26847-1395".
[0934] FIG. 132 shows the amino acid sequence (SEQ ID NO:330)
derived from the coding sequence of SEQ ID NO:329 shown in FIG.
131.
[0935] FIG. 133 shows an EST nucleotide sequence designated herein
as DNA15062 (SEQ ID NO:331).
[0936] FIG. 134 shows an EST nucleotide sequence designated herein
as DNA13199 (SEQ ID NO:332).
[0937] FIG. 135 shows a nucleotide sequence (SEQ ID NO:336) of a
native sequence PRO865 cDNA, wherein SEQ ID NO:336 is a clone
designated herein as "UNQ434" and/or "DNA53974-1401".
[0938] FIG. 136 shows the amino acid sequence (SEQ ID NO:337)
derived from the coding sequence of SEQ ID NO:336 shown in FIG.
135.
[0939] FIG. 137 shows an EST nucleotide sequence designated herein
as DNA37642 (SEQ ID NO:338).
[0940] FIG. 138 shows a nucleotide sequence (SEQ ID NO:345) of a
native sequence PRO827 cDNA, wherein SEQ ID NO:345 is a clone
designated herein as "UNQ468" and/or "DNA57039-1402".
[0941] FIG. 139 shows the amino acid sequence (SEQ ID NO:346)
derived from the coding sequence of SEQ ID NO:345 shown in FIG.
138.
[0942] FIG. 140 shows an EST nucleotide sequence designated herein
as DNA47751 (SEQ ID NO:347).
[0943] FIG. 141 shows a nucleotide sequence (SEQ ID NO:351) of a
native sequence PRO11 14 cDNA, wherein SEQ ID NO:351 is a clone
designated herein as "UNQ557" and/or "DNA57033-1403".
[0944] FIG. 142 shows the amino acid sequence (SEQ ID NO:352)
derived from the coding sequence of SEQ ID NO:351 shown in FIG.
141.
[0945] FIG. 143 shows an EST nucleotide sequence designated herein
as DNA48466 (SEQ ID NO:353).
[0946] FIG. 144 shows a nucleotide sequence (SEQ ID NO:357) of a
native sequence PRO237 cDNA, wherein SEQ ID NO:357 is a clone
designated herein as "UNQ211" and/or "DNA34353-1428".
[0947] FIG. 145 shows the amino acid sequence (SEQ ID NO:358)
derived from the coding sequence of SEQ ID NO:357 shown in FIG.
144.
[0948] FIG. 146 shows a nucleotide sequence (SEQ ID NO:362) of a
native sequence PRO541 cDNA, wherein SEQ ID NO:362 is a clone
designated herein as "UNQ342" and/or "DNA45417-1432".
[0949] FIG. 147 shows the amino acid sequence (SEQ ID NO:363)
derived from the coding sequence of SEQ ID NO:362 shown in FIG.
146.
[0950] FIG. 148 shows a nucleotide sequence (SEQ ID NO:369) of a
native sequence PRO273 cDNA, wherein SEQ ID NO:369 is a clone
designated herein as "UNQ240" and/or "DNA39523-1192".
[0951] FIG. 149 shows the amino acid sequence (SEQ ID NO:370)
derived from the coding sequence of SEQ ID NO:369 shown in FIG.
148.
[0952] FIG. 150 shows a nucleotide sequence (SEQ ID NO:374) of a
native sequence PRO701 cDNA, wherein SEQ ID NO:374 is a clone
designated herein as "UNQ365" and/or "DNA44205-1285".
[0953] FIG. 151 shows the amino acid sequence (SEQ ID NO:375)
derived from the coding sequence of SEQ ID NO:374 shown in FIG.
150.
[0954] FIG. 152 shows a nucleotide sequence (SEQ ID NO:379) of a
native sequence PRO704 cDNA, wherein SEQ ID NO:379 is a clone
designated herein as "UNQ368" and/or "DNA50911-1288".
[0955] FIG. 153 shows the amino acid sequence (SEQ ID NO:380)
derived from the coding sequence of SEQ ID NO:379 shown in FIG.
152.
[0956] FIG. 154 shows a nucleotide sequence (SEQ ID NO:384) of a
native sequence PRO706 cDNA, wherein SEQ ID NO:384 is a clone
designated herein as "UNQ370" and/or "DNA48329-1290".
[0957] FIG. 155 shows the amino acid sequence (SEQ ID NO:385)
derived from the coding sequence of SEQ ID NO:384 shown in FIG.
154.
[0958] FIG. 156 shows a nucleotide sequence (SEQ ID NO:389) of a
native sequence PRO707 cDNA, wherein SEQ ID NO:389 is a clone
designated herein as "UNQ371" and/or "DNA48306-1291".
[0959] FIG. 157 shows the amino acid sequence (SEQ ID NO:390)
derived from the coding sequence of SEQ ID NO:389 shown in FIG.
156.
[0960] FIG. 158 shows a nucleotide sequence (SEQ ID NO:394) of a
native sequence PRO322 cDNA, wherein SEQ ID NO:394 is a clone
designated herein as "UNQ283" and/or "DNA48336-1309".
[0961] FIG. 159 shows the amino acid sequence (SEQ ID NO:395)
derived from the coding sequence of SEQ ID NO:394 shown in FIG.
158.
[0962] FIG. 160 shows a nucleotide sequence (SEQ ID NO:399) of a
native sequence PRO526 cDNA, wherein SEQ ID NO:399 is a clone
designated herein as "UNQ330" and/or "DNA44184-1319".
[0963] FIG. 161 shows the amino acid sequence (SEQ ID NO:400)
derived from the coding sequence of SEQ ID NO:399 shown in FIG.
160.
[0964] FIG. 162 shows a nucleotide sequence (SEQ ID NO:404) of a
native sequence PRO531 cDNA, wherein SEQ ID NO:404 is a clone
designated herein as "UNQ332" and/or "DNA48314-1320".
[0965] FIG. 163 shows the amino acid sequence (SEQ ID NO:405)
derived from the coding sequence of SEQ ID NO:404 shown in FIG.
162.
[0966] FIG. 164 shows a nucleotide sequence (SEQ ID NO:409) of a
native sequence PRO534 cDNA, wherein SEQ ID NO:409 is a clone
designated herein as "UNQ335" and/or "DNA48333-1321".
[0967] FIG. 165 shows the amino acid sequence (SEQ ID NO:410)
derived from the coding sequence of SEQ ID NO:409 shown in FIG.
164.
[0968] FIG. 166 shows a nucleotide sequence (SEQ ID NO:414) of a
native sequence PRO697 cDNA, wherein SEQ ID NO:414 is a clone
designated herein as "UNQ361" and/or "DNA50920-1325".
[0969] FIG. 167 shows the amino acid sequence (SEQ ID NO:415)
derived from the coding sequence of SEQ ID NO:414 shown in FIG.
166.
[0970] FIG. 168 shows a nucleotide sequence (SEQ ID NO:419) of a
native sequence PRO717 cDNA, wherein SEQ ID NO:419 is a clone
designated herein as "UNQ385" and/or "DNA50988-1326".
[0971] FIG. 169 shows the amino acid sequence (SEQ ID NO:420)
derived from the coding sequence of SEQ ID NO:419 shown in FIG.
168.
[0972] FIG. 170 shows a nucleotide sequence (SEQ ID NO:424) of a
native sequence PRO731 cDNA, wherein SEQ ID NO:424 is a clone
designated herein as "UNQ395" and/or "DNA48331-1329".
[0973] FIG. 171 shows the amino acid sequence (SEQ ID NO:425)
derived from the coding sequence of SEQ ID NO:424 shown in FIG.
170.
[0974] FIG. 172 shows a nucleotide sequence (SEQ ID NO:429) of a
native sequence PRO218 cDNA, wherein SEQ ID NO:429 is a clone
designated herein as "UNQ192" and/or "DNA30867-1335".
[0975] FIG. 173 shows the amino acid sequence (SEQ ID NO:430)
derived from the coding sequence of SEQ ID NO:429 shown in FIG.
172.
[0976] FIG. 174 shows an EST nucleotide sequence designated herein
as DNA14472 (SEQ ID NO:431).
[0977] FIG. 175 shows an EST nucleotide sequence designated herein
as DNA15846 (SEQ ID NO:432).
[0978] FIG. 176 shows a nucleotide sequence (SEQ ID NO:436) of a
native sequence PRO768 cDNA, wherein SEQ ID NO:436 is a clone
designated herein as "UNQ406" and/or "DNA55737-1345".
[0979] FIG. 177 shows the amino acid sequence (SEQ ID NO:437)
derived from the coding sequence of SEQ ID NO:436 shown in FIG.
176.
[0980] FIG. 178 shows a nucleotide sequence (SEQ ID NO:441) of a
native sequence PRO771 cDNA, wherein SEQ ID NO:441 is a clone
designated herein as "UNQ409" and/or "DNA49829-1346".
[0981] FIG. 179 shows the amino acid sequence (SEQ ID NO:442)
derived from the coding sequence of SEQ ID NO:441 shown in FIG.
178.
[0982] FIG. 180 shows a nucleotide sequence (SEQ ID NO:446) of a
native sequence PRO733 cDNA, wherein SEQ ID NO:446 is a clone
designated herein as "UNQ411" and/or "DNA52196-1348".
[0983] FIG. 181 shows the amino acid sequence (SEQ ID NO:447)
derived from the coding sequence of SEQ ID NO:446 shown in FIG.
180.
[0984] FIG. 182 shows a nucleotide sequence (SEQ ID NO:451) of a
native sequence PRO162 cDNA, wherein SEQ ID NO:451 is a clone
designated herein as "UNQ429" and/or "DNA56965-1356".
[0985] FIG. 183 shows the amino acid sequence (SEQ ID NO:452)
derived from the coding sequence of SEQ ID NO:451 shown in FIG.
182.
[0986] FIG. 184 shows a nucleotide sequence (SEQ ID NO:453) of a
native sequence PRO788 cDNA, wherein SEQ ID NO:453 is a clone
designated herein as "UNQ430" and/or "DNA56405-1357".
[0987] FIG. 185 shows the amino acid sequence (SEQ ID NO:454)
derived from the coding sequence of SEQ ID NO:453 shown in FIG.
184.
[0988] FIG. 186 shows a nucleotide sequence (SEQ ID NO:455) of a
native sequence PRO1008 cDNA, wherein SEQ ID NO:455 is a clone
designated herein as "UNQ492" and/or "DNA57530-1375".
[0989] FIG. 187 shows the amino acid sequence (SEQ ID NO:456)
derived from the coding sequence of SEQ ID NO:455 shown in FIG.
186.
[0990] FIG. 188 shows an EST nucleotide sequence designated herein
as DNA16508 (SEQ ID NO:457).
[0991] FIG. 189 shows a nucleotide sequence (SEQ ID NO:458) of a
native sequence PRO1012 cDNA, wherein SEQ ID NO:458 is a clone
designated herein as "UNQ495" and/or "DNA56439-1376".
[0992] FIG. 190 shows the amino acid sequence (SEQ ID NO:459)
derived from the coding sequence of SEQ ID NO:458 shown in FIG.
189.
[0993] FIG. 191 shows a nucleotide sequence (SEQ ID NO:463) of a
native sequence PRO1014 cDNA, wherein SEQ ID NO:463 is a clone
designated herein as "UNQ497" and/or "DNA56409-1377".
[0994] FIG. 192 shows the amino acid sequence (SEQ ID NO:464)
derived from the coding sequence of SEQ ID NO:463 shown in FIG.
191.
[0995] FIG. 193 shows a nucleotide sequence (SEQ ID NO:465) of a
native sequence PRO1017 cDNA, wherein SEQ ID NO:465 is a clone
designated herein as "UNQ500" and/or "DNA56112-1379".
[0996] FIG. 194 shows the amino acid sequence (SEQ ID NO:466)
derived from the coding sequence of SEQ ID NO:465 shown in FIG.
193.
[0997] FIG. 195 shows a nucleotide sequence (SEQ ID NO:467) of a
native sequence PRO474 cDNA, wherein SEQ ID NO:467 is a clone
designated herein as "UNQ502" and/or "DNA56045-1380".
[0998] FIG. 196 shows the amino acid sequence (SEQ ID NO:468)
derived from the coding sequence of SEQ ID NO:467 shown in FIG.
195.
[0999] FIG. 197 shows a nucleotide sequence (SEQ ID NO:469) of a
native sequence PRO1031 cDNA, wherein SEQ ID NO:469 is a clone
designated herein as "UNQ516" and/or "DNA59294-1381".
[1000] FIG. 198 shows the amino acid sequence (SEQ ID NO:470)
derived from the coding sequence of SEQ ID) NO:469 shown in FIG.
197.
[1001] FIG. 199 shows a nucleotide sequence (SEQ ID NO:471) of a
native sequence PRO938 cDNA, wherein SEQ ID NO:471 is a clone
designated herein as "UNQ475" and/or "DNA56433-1406".
[1002] FIG. 200 shows the amino acid sequence (SEQ ID NO:472)
derived from the coding sequence of SEQ ID NO:471 shown in FIG.
199.
[1003] FIG. 201 shows a nucleotide sequence (SEQ ID NO:476) of a
native sequence PRO1082 cDNA, wherein SEQ ID NO:476 is a clone
designated herein as "UNQ539" and/or "DNA53912-1457".
[1004] FIG. 202 shows the amino acid sequence (SEQ ID NO:477)
derived from the coding sequence of SEQ ID NO:476 shown in FIG.
201.
[1005] FIG. 203 shows a nucleotide sequence (SEQ ID NO:482) of a
native sequence PRO1083 cDNA, wherein SEQ ID NO:482 is a clone
designated herein as "UNQ540" and/or "DNA50921-1458".
[1006] FIG. 204 shows the amino acid sequence (SEQ ID NO:483)
derived from the coding sequence of SEQ ID NO:482 shown in FIG.
203.
[1007] FIG. 205 shows an EST nucleotide sequence designated herein
as DNA24256 (SEQ ID NO:484).
[1008] FIG. 206 shows a nucleotide sequence (SEQ ID NO:487) of a
native sequence PRO200 cDNA, wherein SEQ ID NO:487 is a clone
designated herein as "UNQ174" and/or "DNA29101-1122".
[1009] FIG. 207 shows the amino acid sequence (SEQ ID NO:488)
derived from the coding sequence of SEQ ID NO:487 shown in FIG.
206.
[1010] FIG. 208 shows a nucleotide sequence (SEQ ID NO:495) of a
native sequence PRO285 cDNA, wherein SEQ ID NO:495 is a clone
designated herein as "DNA40021-1154".
[1011] FIG. 209 shows the amino acid sequence (SEQ ID NO:496)
derived from the coding sequence of SEQ IID NO:495 shown in FIG.
208.
[1012] FIG. 210 shows a nucleotide sequence (SEQ ID NO:497) of a
native sequence PRO286 cDNA, wherein SEQ ID NO:497 is a clone
designated herein as "DNA42663-1154".
[1013] FIG. 211 shows the amino acid sequence (SEQ ID NO:498)
derived from the coding sequence of SEQ ID NO:497 shown in FIG.
210.
[1014] FIG. 212 shows a nucleotide sequence (SEQ ID NO:505) of a
native sequence PRO213-1 cDNA, wherein SEQ ID NO:505 is a clone
designated herein as "DNA30943-1-1163-1".
[1015] FIG. 213 shows the amino acid sequence (SEQ ID NO:506)
derived from the coding sequence of SEQ ID NO:505 shown in FIG.
212.
[1016] FIG. 214 shows a nucleotide sequence (SEQ ID NO:507) of a
native sequence PRO1330 cDNA, wherein SEQ ID NO:507 is a clone
designated herein as "DNA64907-1163-1".
[1017] FIG. 215 shows the amino acid sequence (SEQ ID NO:508)
derived from the coding sequence of SEQ ID NO:507 shown in FIG.
214.
[1018] FIG. 216 shows a nucleotide sequence (SEQ ID NO:509) of a
native sequence PRO1449 cDNA, wherein SEQ ID NO:509 is a clone
designated herein as "DNA64908-1163-1".
[1019] FIG. 217 shows the amino acid sequence (SEQ ID NO:510)
derived from the coding sequence of SEQ ID NO:509 shown in FIG.
216.
[1020] FIG. 218 shows a nucleodide sequence (SEQ ID NO:514) of a
native sequence PRO298 cDNA, wherein SEQ ID NO:514 is a clone
designated herein as "UNQ261" and/or "DNA39975-1210".
[1021] FIG. 219 shows the amino acid sequence (SEQ ID NO:515)
derived from the coding sequence of SEQ ID NO:514 shown in FIG.
218.
[1022] FIG. 220 shows an EST nucleotide sequence designated herein
as DNA26832 (SEQ ID NO:516).
[1023] FIG. 221 shows a nucleotide sequence (SEQ ID NO:522) of a
native sequence PRO337 cDNA, wherein SEQ ID NO:522 is a clone
designated herein as "DNA43316-1237".
[1024] FIG. 222 shows the amino acid sequence (SEQ ID NO:523)
derived from the coding sequence of SEQ ID NO:522 shown in FIG.
221.
[1025] FIG. 223 shows an EST nucleotide sequence designated herein
as DNA42301 (SEQ ID NO:524).
[1026] FIG. 224 shows a nucleotide sequence (SEQ ID NO:525) of a
native sequence PRO403 cDNA, wherein SEQ ID NO:525 is a clone
designated herein as "DNA55800-1263".
[1027] FIG. 225 shows the amino acid sequence (SEQ ID NO:526)
derived from the coding sequence of SEQ ID NO:525 shown in FIG.
224.
[1028] FIG. 226 shows an EST nucleotide sequence designated herein
as DNA34415 (SEQ ID NO:527).
[1029] FIG. 227 shows an EST nucleotide sequence designated herein
as DNA49830 (SEQ ID NO:528).
[1030] FIG. 228 shows an EST nucleotide sequence designated herein
as DNA49831 (SEQ ID NO:529).
[1031] FIG. 229 shows a nucleotide sequence (SEQ ID NO:611) of a
native sequence PRO4993 cDNA, wherein SEQ ID NO:611 is a clone
designated herein as "DNA94832-2659".
[1032] FIG. 230 shows the amino acid sequence (SEQ ID NO:612)
derived from the coding sequence of SEQ ID NO:611 shown in FIG.
229.
[1033] FIG. 231 shows a nucleotide sequence (SEQ ID NO:613) of a
native sequence PRO1559 cDNA, wherein SEQ ID NO:613 is a clone
designated herein as "DNA68886".
[1034] FIG. 232 shows the amino acid sequence (SEQ ID NO:614)
derived from the coding sequence of SEQ ID NO:613 shown in FIG.
231.
[1035] FIG. 233 shows a nucleotide sequence (SEQ ID NO:615) of a
native sequence PRO725 cDNA, wherein SEQ ID NO:615 is a clone
designated herein as "DNA52758-1399".
[1036] FIG. 234 shows the amino acid sequence (SEQ ID NO:616)
derived from the coding sequence of SEQ ID NO:615 shown in FIG.
233.
[1037] FIG. 235 shows a nucleotide sequence (SEQ ID NO:617) of a
native sequence PRO739 cDNA, wherein SEQ ID NO:617 is a clone
designated herein as "DNA52756".
[1038] FIG. 236 shows the amino acid sequence (SEQ ID NO:618)
derived from the coding sequence of SEQ ID NO:617 shown in FIG.
235.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[1039] I. Definitions
[1040] 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 flier 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.
[1041] 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.
[1042] 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.
[1043] 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. 14:4683-4690 (1986)). Moreover, it is also
recognized that, in some cases, cleavage of a signal sequence from
a secreted polypeptide is not entirely uniform, resulting in more
than one secreted species. These mature polypeptides, where the
signal peptide is cleaved within no more than about 5 amino acids
on either side of the C-terminal boundary of the signal peptide as
identified herein, and the polynucleotides encoding them, are
contemplated by the present invention.
[1044] "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 wil 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. Ordirily, 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.
[1045] "Percent (%) amino acid sequence identity" with respect to
the PRO polypeptide sequences identified herein is defined as the
percentage of amino acid residues in a candidate sequence that are
identical with the amino acid residues in the specific PRO
polypeptide sequence, after aligning the sequences and introducing
gaps, if necessary, to achieve the maximum percent sequence
identity, and not considering any conservative substitutions as
part of the sequence identity. Alignment for purposes of
determining percent amino acid sequence identity can be achieved in
various ways that are within the skill in the art, for instance,
using publicly available computer software such as BLAST, BLAST-2,
ALIGN or Megalign (DNASTAR) software. Those skilled in the art can
determine appropriate parameters for measuring alignment, including
any algorithms needed to achieve maximal alignment over the full
length of the sequences being compared. For purposes herein,
however, % amino acid sequence identity values are generated using
the sequence comparison computer program ALIGN-2, wherein the
complete source code for the ALIGN-2 program is provided in Table 1
below. The ALIGN-2 sequence comparison computer program was
authored by Genentech, Inc. and the source code shown in Table 1
below has been filed with user documentation in the U.S. Copyright
Office, Washington D.C., 20559, where it is registered under U.S.
Copyright Registration No. TXU510087. The ALIGN-2 program is
publicly available through Genentech, Inc., South San Francisco,
Calif. or may be compiled from the source code provided in Table 1
below. The ALIGN-2 program should be compiled for use on a UNIX
operating system, preferably digital UNIX V4.0D. All sequence
comparison parameters are set by the ALIGN-2 program and do not
vary.
[1046] In situations where ALIGN-2 is employed for amino acid
sequence comparisons, the % amino acid sequence identity of a given
amino acid sequence A to, with, or against a given amino acid
sequence B (which can alternatively be phrased as a given amino
acid sequence A that has or comprises a certain % amino acid
sequence identity to, with, or against a given amino acid sequence
B) is calculated as follows:
100 times the fraction X/Y
[1047] 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.
[1048] 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.
[1049] 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=15/5,
multi-pass e-value=0.01, constant for nulti-pass=25, dropoff for
final gapped alignment=25 and scoring matrix=BLOSUM62.
[1050] 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 X/Y
[1051] 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.
[1052] "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.
[1053] 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.
[1054] "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.
[1055] In situations where ALIGN-2 is employed for nucleic acid
sequence comparisons, the % nucleic acid sequence identity of a
given nucleic acid sequence C to, with, or against a given nucleic
acid sequence D (which can alternatively be phrased as a given
nucleic acid sequence C that has or comprises a certain % nucleic
acid sequence identity to, with, or against a given nucleic acid
sequence D) is calculated as follows:
100 times the fraction W/Z
[1056] 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.
[1057] 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 266:460-480
(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 % 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.
[1058] 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.
[1059] 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 W/Z
[1060] 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.
[1061] 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.
[1062] 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.
[1063] 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.
[1064] 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 X/Y
[1065] 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.
[1066] "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.
[1067] 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.
[1068] 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 nbosome binding site.
Eukaryotic cells are known to utilize promoters, polyadenylation
signals, and enhancers.
[1069] 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.
[1070] 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.
[1071] "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).
[1072] "Stringent conditions" or "high stringency conditions", as
defined herein, may be identified by those that: (1) employ low
ionic strength and high temperature for washing, for example 0.015
M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl
sulfate at 50.degree. C.; (2) employ during hybridization a
denaturing agent, such as formamide, for example, 50% (v/v)
formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%
polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with
750 mM sodium chloride, 75 mM sodium citrate at 42.degree. C.; or
(3) employ 50% formamide, 5.times.SSC (0.75 M NaCl, 0.075 M sodium
citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium
pyrophosphate, 5.times.Denhardt's solution, sonicated salmon sperm
DNA (50 .mu.g/ml), 0.1% SDS, and 10% dextran sulfate at 42.degree.
C., with washes at 42.degree. C. in 0.2.times.SSC (sodium
chloride/sodium citrate) and 50% formamide at 55.degree. C.,
followed by a high-stringency wash consisting of 0.1.times.SSC
containing EDTA at 55.degree. C.
[1073] "Moderately stringent conditions" may be identified as
described by Sambrook et al., Molecular Cloning: A Laboratory
Manual, New York: Cold Spring Harbor Press, 1989, and include the
use of washing solution and hybridization conditions (e.g.,
temperature, ionic strength and % SDS) less stringent that those
described above. An example of moderately stringent conditions is
overnight incubation at 37.degree. C. in a solution comprising: 20%
formamide, 5.times.SSC (150 mM NaCl, 15 mM trisodium citrate), 50
mM sodium phosphate (pH 7.6), 5.times.Denhardt's solution, 10%
dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA,
followed by washing the filters in 1.times.SSC at about
37-50.degree. C. The skilled artisan will recognize how to adjust
the temperature, ionic strength, etc. as necessary to accommodate
factors such as probe length and the like.
[1074] 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).
[1075] 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 inmunoadhesins
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 IgG4 subtypes, IgA (including IgA-1 and
IgA-2), IgE, IgD or IgM.
[1076] "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.
[1077] 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.
[1078] "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.
[1079] "Chronic" administration refers to administration of the
agent(s) in a continuous mode as opposed to an acute mode, so as to
maintain the initial therapeutic effect (activity) for an extended
period of time. "Intermittent" administration is treatment that is
not consecutively done without interruption, but rather is cyclic
in nature.
[1080] "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.
[1081] Administration "in combination with" one or more further
therapeutic agents includes simultaneous (concurrent) and
consecutive administration in any order.
[1082] "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..
[1083] "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.
[1084] 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.
[1085] "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.
[1086] 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 CH1 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.
[1087] 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.
[1088] 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., IgGI, IgG2, IgG3, IgG4, IgA, and
IgA2.
[1089] Single-chain Fv" or "sFv" antibody fragments comprise the VH
and VL 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).
[1090] 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 93/11161; and Hollinger et al., Proc. Natl.
Acad. Sci. USA, 90:6444-6448 (1993).
[1091] An "isolated" antibody is one which has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials which would interfere with diagnostic or therapeutic uses
for the antibody, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. 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.
[1092] 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.
[1093] 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.
[1094] 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.
[1095] A "small molecule" is defined herein to have a molecular
weight below about 500 Daltons.
[1096] As used herein, "vascular endothelial cell growth factor-E,"
or "VEGF-E," refers to a mammalian growth factor as described
herein, including the human amino acid sequence of FIG. 207, a
sequence which has homology to VEGF and bone morphogenetic protein
1 and which includes complete conservation of all VEGF cysteine
residues, which have been shown to be required for biological
activity of VEGF. VEGF-E expression includes expression in human
fetal bone, thymus, and the gastrointestinal tract. The biological
activity of native VEGF-E is shared by any analogue or variant
thereof that is capable of promoting selective growth and/or
survival of umbilical vein endothelial cells, induces proliferation
of pluripotent fibroblast cells, induces immediate early gene c-fos
in human endothelhal cell lines and causes myocyte hypertrophy in
cardiac cells, or which possesses an immune epitope that is
immunologically cross-reactive with an antibody raised against at
least one epitope of the corresponding native VEGF-E. The human
VEGF-E herein is active on rat and mouse cells indicating
conservation across species. Moreover, the VEGF-E herein is
expressed at the growth plate region and has been shown to embrace
fetal myocytes.
[1097] As used herein, "vascular endothelial cell growth factor,"
or "VEGF," refers to a mammalian growth factor as defined in U.S.
Pat. No. 5,332,671. The biological activity of native VEGF is
shared by any analogue or variant thereof that is capable of
promoting selective growth of vascular endothelial cells but not of
bovine corneal endothelial cells, lens epithelial cells, adrenal
cortex cells, BHK-21 fibroblasts, or keratinocytes, or that
possesses an immune epitope that is immunologically cross-reactive
with an antibody raised against at least one epitope of the
corresponding native VEGF.
[1098] The terms "VEGF-E polypeptide" and "VEGF-E" when used herein
encompass native sequence VEGF-E polypeptide and VEGF-E polypeptide
variants (which are further defined herein). The VEGF-E
polypeptides 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.
[1099] Inhibitors of VEGF-E include those which reduce or inhibit
the activity or expression of VEGF-E and includes antisense
molecules.
[1100] The abbreviation "KDR" refers to the kinase domain region of
the VEGF molecule. VEGF-E has no homology with VEGF in this
domain.
[1101] The abbreviation "FLT-1" refers to the FMS-like tyrosine
kinase binding domain which is known to bind to the corresponding
FLT-1 receptor. VEGF-E has no homology with VEGF in this
domain.
[1102] "Toll receptor2", "TLR2" and "huTLR2" are used
interchangeably, and refer to a human Toll receptor designated as
"HuTLR2" by Rock et al., Proc. Natl. Acad. Sci. USA 95, 588-593
(1998).
[1103] The term "lipopolysaccharide" or "LPS" is used herein as a
synonym of "endotoxin." Lipopolysaccharides (LPS) are
characteristic components of the outer membrane of Gram-negative
bacteria, e.g., Escherichia coli. They consist of a polysaccharide
part and a fat called lipid A. The polysaccharide, which varies
from one bacterial species to another, is made up of the O-specific
chain (built from repeating units of three to eight sugars) and the
two-part core. Lipid A virtully always includes two glucosamine
sugars modified by phosphate and a variable number of fatty acids.
For further information see, for example, Rietschel and Brade,
Scientific American August 1992, 54-61.
[1104] The term "septic shock" is used herein in the broadest
sense, including all definitions disclosed in Bone, Ann. Intern
Med. 114, 332-333 (1991). Specifically, septic shock starts with a
systemic response to infection, a syndrome called sepsis. When this
syndrome results in hypotension and organ dysfunction, it is called
septic shock. Septic shock may be initiated by gram-positive
organisms and fungi, as well as endotoxin-containing Gram-negative
organisms. Accordingly, the present definition is not limited to
"endotoxin shock."
[1105] The phrases "gene amplification" and "gene duplication" are
used interchangeably and refer to a process by which multiple
copies of a gene or gene fragment are formed in a particular cell
or cell line. The duplicated region (a stretch of amplified DNA) is
often referred to as "amplicon". Usually, the amount of the
messenger RNA (mRNA) produced, i.e., the level of gene expression,
also increases in the proportion of the number of copies made of
the particular gene expressed.
[1106] "Tumor", as used herein, refers to all neoplastic cell
growth and proliferation, whether malignant or benign, and all
pre-cancerous and cancerous cells and tissues. The terms "cancer"
and "cancerous" refer to or describe the physiological condition in
mammals that is typically characterized by unregulated cell growth.
Examples of cancer include but are not limited to, carcinoma,
lymphoma, blastoma, sarcoma, and leukemia. More particular examples
of such cancers include breast cancer, prostate cancer, colon
cancer, squamous cell cancer, small-cell lung cancer, non-small
cell lung cancer, gastrointestinal cancer, pancreatic cancer,
glioblastoma, cervical cancer, ovarian cancer, liver cancer,
bladder cancer, hepatoma, colorectal cancer, endometrial carcinoma,
salivary gland carcinoma, kidney cancer, vulval cancer, thyroid
cancer, hepatic carcinoma and various types of head and neck
cancer.
[1107] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents the function of cells and/or
causes destruction of cells. The term is intended to include
radioactive isotopes (e.g. I131, I125, Y90 and Re186),
chemotherapeutic agents, and toxins such as enzymatically active
toxins of bacterial, fungal, plant or animal origin, or fragments
thereof.
[1108] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer. Examples of chemotherapeutic agents
include adriamycin, doxorubicin, epirubicin, 5-fluorouracil,
cytosine arabinoside ("Ara-C"), cyclophosphamide, thiotepa,
busulfan, cytoxin, taxoids, e.g. paclitaxel (Taxol, Bristol-Myers
Squibb Oncology, Princeton, N.J.), and doxetaxel (raxotere<<,
Rhone-Poulenc Rorer, Antony, France), toxotere, methotrexate,
cisplatin, melphalan, vinblastine, bleomycin, etoposide,
ifosfamide, mitomycin C, mitoxantrone, vincristine, vinorelbine,
carboplatin, teniposide, daunomycin, carminomycin, aminopterin,
dactinomycin, mitomycins, esperamicins (see U.S. Pat. No.
4,675,187), melphalan and other related nitrogen mustards. Also
included in this definition are hormonal agents that act to
regulate or inhibit hormone action on tumors such as tamoxifen and
onapristone.
[1109] A "growth inhibitory agent" when used herein refers to a
compound or composition which inhibits growth of a cell, especially
cancer cell overexpressing any of the genes identified herein,
either in vitro or in vivo. Thus, the growth inhibitory agent is
one which significantly reduces the percentage of cells
overexpressing such genes in S phase. Examples of growth inhibitory
agents include agents that block cell cycle progression (at a place
other than S phase), such as agents that induce G1 arrest and
M-phase arrest. Classical M-phase blockers include the vincas
(vincristine and vinblastine), taxol, and topo II inhibitors such
as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.
Those agents that arrest G1 also spill over into S-phase arrest,
for example, DNA alkylating agents such as tamoxifen, prednisone,
dacarbazine, mechlorethamine, cisplatin, methotrexate,
5-fluorouracil, and ara-C. Further information can be found in The
Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1,
entitled "Cell cycle regulation, oncogens, and antineoplastic
drugs" by Murakami et al. (W B Saunders: Philadelphia, 1995),
especially p. 13.
[1110] "Doxorubicin" is an athracycline antibiotic.
[1111] The term "cytokine" is a generic term for proteins released
by one cell population which act on another cell as intercellular
mediators. Examples of such cytokines are lymphokines, monokines,
and traditional polypeptide hormones. Included among the cytohines
are growth hormone such as human growth hormone, N-methionyl human
growth hormone, and bovine growth hormone; parathyroid hormone;
thyroxme; insulin; proinsulin; relaxin; prorelaxin; and the like.
As used herein, the term cytohine includes proteins from natural
sources or from recombinant cell culture and biologically active
equivalents of the native sequence cytokines.
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,-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 */ #deflne JMPS
1024 /* max jmps in an path */ #define MX 4 /* save if there's at
least MX-1 bases since last jmp */ #define DMAT 3 /* value of
matching bases */ #define DMIS 0 /* penalty for mismatched bases */
#define DINS0 8 /* penalty for a gap */ #define DINS1 1 /* penalty
per base */ #define PINS0 8 /* penalty for a gap */ #define PINS1 4
/* penalty per residue */ struct jmp { short n[MAXJMP]; /* size of
jmp (neg for dely) */ unsigned short x[MAXJMP]; /* base no. of jmp
in seq x */ }; /* limits seq to 2 16 -1 */ struct diag { int score;
/* score at last jmp */ long offset; /* offset of prev block */
short ijmp; /* current jmp index */ struct jmp jp; /* list of jmps
*/ }; struct path { int spc; /* number of leading spaces */ short
n[JMPS]; /* size of jmp (gap) */ int x[JMPS]; /* loc of jmp (last
elem before gap) */ }; char *ofile; /* output file name */ char
*namex[2]; /* seq names: getseqs() */ char *prog; /* prog name for
err msgs */ char *seqx[2]; /* seqs: getseqs() */ int dmax; /* best
diag: nw() */ int dmax0; /* final diag */ int dna; /* set if dna:
main() */ int endgaps; /* set if penalizing end gaps */ int gapx,
gapy; /* total gaps in seqs */ int len0, len1; /* seq lens */ int
ngapx, ngapy; /* total size of gaps */ int smax; /* max score: nw()
*/ int *xbm; /* bitmap for matching */ long offset; /* current
offset in jmp file */ struct diag *dx; /* holds diagonals */ struct
path pp[2]; /* holds path for seqs */ char *calloc(), *malloc(),
*index(), *strcpy(); char *getseq(), *g_calloc(); /*
Needleman-Wunsch alignment program * * usage: progs file1 file2 *
where file1 and file2 are two dna or two protein sequences. * The
sequences can be in upper- or lower-case an may contain ambiguity *
Any lines beginning with `;`, `>` or `<` are ignored * Max
file length is 65535 (limited by unsigned short x in the jmp
struct) * A sequence with 1/3 or more of its elements ACGTU is
assumed to be DNA * Output is in the file "align.out" * * The
program may create a tmp file in /tmp to hold info about traceback.
* Original version developed under BSD 4.3 on a vax 8650 */
#include "nw.h" #include "day.h" static _dbval[26] = {
1,14,2,13,0,0,4,11,0,0,12,0,3,15,0,0,0,5,6,8,8,7,9,0,10,0 }; static
_pbval[26] = { 1, 2.vertline.(1<<(`D`-`A`)).vertlin-
e.(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 alignment, return best score: main() * dna: values in
Fitch and Smith, PNAS, 80, 1382-1386, 1983 * pro: PAM 250 values *
When scores are equal, we prefer mismatches to any gap, prefer * a
new gap to extending an ongoing gap, and prefer a gap in seqx * to
a gap in seq y. */ nw() nw { char *px, *py; /* seqs and ptrs */ int
*ndely, *dely; /* keep track of dely */ int ndelx, delx; /* keep
track of delx */ int *tmp; /* for swapping 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] =
-ndely[yy]; dx[id].jp.x[ij] = xx; dx[id].score = dely[yy]; } if (xx
== len0 && yy < len1) { /* last col */ if (endgaps)
col1[yy] -= ins0+ins1*(len1-yy); if (col1[yy] > smax) { smax =
col1[yy]; dmax = id; } } } if (endgaps && xx < len0)
col1[yy-1] -= ins0+ins1*(len0-xx); if (col1[yy-1] > smax) { smax
= col1[yy-1]; dmax = id; } tmp = col0; col0 = col1; col1 = tmp; }
(void) free((char *)ndely); (void) free((char *)dely); (void)
free((char *)col0); (void) free((char *)col1); } /* * * print() --
only routine visible outside this module * * static: * getmat() --
trace back best path, count matches: print() * pr_align() -- print
alignment of described in array p[]: print() * dumpblock() -- dump
a block of lines with numbers, stars: pr_align() * nums() -- put
out a number line: dumpblock() * putline() -- put out a line (name,
[num], seq, [num]): dumpblock() * stars() - -put a line of stars:
dumpblock() * stripname() -- strip any path and prefix from a
seqname */ #include "nw.h" #define SPC 3 #define P_LINE 256 /*
maximum output line */ #define P_SPC 3 /* space between name or num
and seq */ extern _day[26][26]; int olen; /* set output line length
*/ FILE *fx; /* output file */ print() print { int lx, ly,
firstgap, lastgap; /* overlap */ if ((fx = fopen(ofile, "w")) == 0)
{ fprintf(stderr,"%s: can't write %s.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(); }
/* * 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);
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[_LINE]; /* set by stars() */ /* * print alignment of described
in struct
path pp [] */ static pr_align() pr_align { int nn; /* char count */
int more; register i; for(i = 0, lmax = 0; i < 2; i++) { nn =
stripname(namex[i]); if (nn > lmax) lmax = nn; nc[i] = 1; ni[i]
= 1; siz[i] = ij[i] = 0; ps[i] = seqx[i]; po[i] = out[i]; } for (nn
= nm = 0, more = 1; more;) { ...pr_align for (i = more = 0; i <
2; i++) { /* * do we have more of this sequence? */ if (!*ps[i])
continue; more++; if (pp[i].spc) { /* leading space */ *po[i]++ =
``; pp[i].spc-; } else if (siz[i]) { /* in a gap */ *po[i]++ = `-`;
siz[i]-; } else { /* we're putting a seq element */ *po[i] =
*ps[i]; if (islower(*ps[i])) *ps[i] = toupper(*ps[i]); po[i]++;
ps[i]++; /* * are we at next gap for this seq? */ if( ni[i] ==
pp[i].x[ij[i]]) { /* * we need to merge all gaps * at this location
*/ siz[i] = pp[i].n[ij[i]++]; while (ni[i] == pp[i].x[ij[i]])
siz[i] += pp[i].n[ij[i]++]; } ni[i]++; } } if (++nn == olen
.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`; ...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 .backslash.= 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) putline int ix; {
...putline int i; register char *px; for (px = namex[ix], i = 0;
*px && *px != `:`; px++, i++) (void) putc(*px, fx); for(;i
< lmax+P_SPC; i++) (void) putc(` `, fx); /* these count from 1:
* ni[] is current element (from 1) * nc[] is number at start of
current line */ for (px = out[ix]; *px; px++) (void)
putc(*px&0x7F, fx); (void) putc(`.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`; } /* * strip path or prefix
from pn, return len: pr_align() */ static stripname(pn) stripname
char *pn; /* file name (may be path) */ { register char *px, *py;
py = 0; for (px = pn; *px; px++) if(*px == `/`) py = px + 1; if
(py) (void) strcpy(pn, py); return(strlen(pn)); } /* * cleanup() --
cleanup any tmp file * getseq() -- read in seq, set dna, len,
maxlen * g_calloc() -- calloc() with error checkin * readjmps() --
get the good jmps, from tmp file if necessary * writejmps() --
write a filled array of jmps to a tmp file: nw() */ #include "nw.h"
#include <sys/file.h> char *jname = "/tmp/homgXXXXXX"; /* tmp
file for jmps */ FILE *fj; int cleanup(); /* cleanup tmp file */
long lseek(); /* * remove any tmp file if we blow */ cleanup(i)
cleanup int i; { if (fj) (void) unlink(jname); exit(i); } /* *
read, return ptr to seq, set dna, len, maxlen * skip lines starting
with `;`, `<`, or `>` * seq in upper or lower case */ char *
getseq(file, len) getseq char *file; /* file name */ int *len; /*
seq len */ { char line[1024], *pseq; register char *px, *py; int
natgc, tlen; FILE *fp; if ((fp = fopen(file, "r")) == 0) {
fprintf(stderr, "%s: can't read %s.backslash.n", prog, file);
exit(1); } tlen = natgc = 0; while (fgets(line, 1024, fp)) { 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`; ...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-)
; ...readjmps if (j < 0 && dx[dmax].offset &&
fj) { (void) lseek(fd, dx[dmax].offset, 0); (void) read(fd, (char
*)&dx[dmax].jp, sizeof(struct jmp)); (void) read(fd, (char
*)&dx[dmax].offset, sizeof(dx[dmax].offset)); dx[dmax].ijmp =
MAXJMP-1; } else break } if (i >= JMPS) { fprintf(stderr, "%s:
too many gaps in alignment.backslash.n", prog); cleanup(1); } if (j
>= 0) { siz = dx[dmax].jp.n[j]; xx = dx[dmax].jp.x[j]; dmax +=
siz; if (siz < 0) { /* gap in second seq */ pp[1].n[i1] = -siz;
xx += siz; /* id = xx - yy + len1 - 1 */ pp[1].x[i1] = xx - dmax +
len1 - 1; gapy++; ngapy -= siz; /* ignore MAXGAP when doing endgaps
*/ siz = (-siz < MAXGAP .vertline..vertline. endgaps)? -siz :
MAXGAP; i1++; } else if (siz > 0) { /*gap in first seq*/
pp[0].n[i0] = siz; pp[0].x[i0] = xx; gapx++; ngapx += siz; /*
ignore MAXGAP when doing endgaps */ siz = (siz < MAXGAP
.vertline..vertline. endgaps)? siz : MAXGAP; i0++; } } else break;
} /* reverse the order of jmps */ for (j = 0, i0-; j < i0; j++,
i0-) { i = pp[0].n[j]; pp[0].n[j] = pp[0].n[i0]; pp[0].n[i0] = i; i
= pp[0].x[j]; pp[0].x[j] = pp[0].x[i0]; pp[0].x[i0] = i; } for (j =
0, i1-; j < i1; j++, i1-) { i = pp[1].n[j]; pp[1].n[j] =
pp[1].n[i1]; pp[1].n[i1] = i; i = pp[1].x[j]; pp[1].x[j] =
pp[1].x[i1]; pp[1].x[i1] = i; } if (fd >= 0) (void) close(fd);
if (fj) { (void) unlink(jname); fj = 0; offset = 0; } } /* * write
a filled jmp struct offset of the prev one (if any): nw() */
writejmps(ix) writejmps int ix; { char *mktemp(); if (!fj) { if
(mktemp(jname) < 0) { fprintf(stderr, "%s: can't mktemp()
%s.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); }
[1112]
2TABLE 2 PRO XXXXXXXXXXXXXXX (Length = 15 amino acids) Comparison
Protein XXXXXYYYYYYY (Length = 12 amino acids) % 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%
[1113]
3TABLE 3 PRO XXXXXXXXXX (Length = 10 amino acids) Comparison
Protein XXXXXYYYYYYZZYZ (Length = 15 amino acids) % 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%
[1114]
4 TABLE 4 PRO-DNA NNNNNNNNNNNNNN (Length = 14 nucleotides)
Comparison DNA NNNNNNLLLLLLLLLL (Length = 16 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) = 6 divided by 14 = 42.9%
[1115]
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%
[1116] II. Compositions and Methods of the Invention
[1117] A. Full-Length PRO Polypeptides
[1118] 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.
[1119] 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
tiine.
[1120] 1. Full-length PRO213 Polypeptides
[1121] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO213. In particular, Applicants have
identified and isolated cDNA encoding a PRO213 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 PRO213 polypeptide has significant homology with the
human growth arrest-specific 6 (gas6) protein. Accordingly, it is
presently believed that PRO213 polypeptide disclosed in the present
application may have the same or simular activity as does the gas6
protein.
[1122] 2. Full-length PRO274 Polypeptides
[1123] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO274. In particular, Applicants have
identified and isolated cDNA encoding a PRO274 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 PRO274 polypeptide have significant
homology with the 7 transmembrane segment receptor proteins and
Fn54 protein. Accordingly, it is presently believed that PRO274
polypeptide disclosed in the present application is a newly
identified member of the 7 transmembrane segment receptor protein
and/or Fn54 protein family.
[1124] 3. Full-length PRO300 Polypeptides
[1125] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO300. In particular, Applicants have
identified and isolated cDNA encoding a PRO300 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 PRO300 polypeptide have significant
homology with the human Diff 33 protein. Accordingly, it is
presently believed that PRO300 polypeptide disclosed in the present
application is a newly identified member of the Diff 33 family.
[1126] 4. Full-length PRO284 Polypeptides
[1127] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO284. In particular, Applicants have
identified and isolated cDNA encoding a PRO284 polypeptide, as
disclosed in further detail in the Examples below. To Applicants
present knowledge, the UNQ247 (DNA23318-1211) nucleotide sequence
encodes a novel factor; using BLAST and FastA sequence alignment
computer programs, no sequence identities to any known proteins
were revealed.
[1128] 5. Full-length PRO296 Polypeptides
[1129] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO296. In particular, Applicants have
identified and isolated cDNA encoding a PRO296 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
the PRO296 polypeptide has significant smiularity to the
sarcoma-amplified SAS protein. Accordingly, it is presently
believed that PRO296 polypeptide disclosed in the present
application is a newly identified SAS protein homolog.
[1130] 6. Full-length PRO329 Polypeptides
[1131] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO329. In particular, Applicants have
identified and isolated cDNA encoding a PRO329 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
the PRO329 polypeptide has significant similarity to a high
affinity immunoglobulin F.sub.c receptor. Accordingly, it is
presently believed that PRO329 polypeptide disclosed in the present
application is a newly identified F.sub.c receptor homolog.
[1132] 7. Full-length PRO362 Polypeptides
[1133] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO362. In particular, Applicants have
identified and isolated cDNA encoding a PRO362 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
the PRO362 polypeptide has significant similarity to the A33
antigen protein as well as the HCAR protein and the NRCAM related
cell adhesion molecule. Accordingly, it is presently believed that
PRO362 polypeptide disclosed in the present application is a newly
A33 antigen and HCAR protein homolog.
[1134] 8. Full-length PRO363 Polypeptides
[1135] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO363. In particular, Applicants have
identified and isolated cDNA encoding a PRO363 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
the PRO363 polypeptide has significant similarity to the cell
surface protein HCAR. Accordingly, it is presently believed that
PRO363 polypeptide disclosed in the present application is a newly
HCAR homolog.
[1136] 9. Full-length PRO868 Polypeptides
[1137] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO868. In particular, Applicants have
identified and isolated cDNA encoding a PRO868 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
the PRO868 polypeptide has significant similarity to the tumor
necrosis factor receptor. Accordingly, it is presently believed
that PRO868 polypeptide disclosed in the present application is a
newly identified member of the tumor necrosis factor receptor
family of proteins.
[1138] 10. Full-length PRO382 Polypeptides
[1139] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO382. In particular, Applicants have
identified and isolated cDNA encoding a PRO382 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
the native PRO382 polypeptide shares significant homology with
various serine protease proteins. Applicants have also found that
the DNA encoding the PRO382 polypeptide shares significant homology
with nucleic acid encoding various serine protease proteins.
Accordingly, it is presently believed that PRO382 polypeptide
disclosed in the present application is a newly identified serine
protease homolog.
[1140] 11. Full-length PRO545 Polypeptides
[1141] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO545. In particular, Applicants have
identified and isolated cDNA encoding a PRO545 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 PRO545 polypeptide have significant
homology with the sequences identified designated as; human
metaloproteinase ("P_W01825"), mouse meltrin alpha("S60257"),
metalloprotease-disintegrin meltrin-alpha ("GEN13695"), ADAM 13
-Xenopus laevis ("XLU66003.sub.--1"), mouse meltrin beta
("S60258"), rabbit metalloprotease-disintegrin meltrin-beta,
("GEN13696"), human meltrin S ("AF023477.sub.--1"), human meltrin
precursor ("AF023476.sub.--1"), human ADAM 21 ("AF029900.sub.--1"),
and human ADAM 20 (AF029899.sub.--1"), thereby indicating that
PRO545 may be a novel meltrin protein. Accordingly, it is presently
believed that the PRO545 polypeptide disclosed in the present
application is a newly identified member of the meltrin family and
possesses the cellular adhesiveness typical of the meltrin proteins
which comprise both metalloprotease and disintegrin domains.
[1142] 12. Full-length PRO617 Polypeptides
[1143] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO617. In particular, Applicants have
identified and isolated cDNA encoding a PRO617 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
the PRO617 polypeptide shares significant homology with the CD24
protein. Applicants have also found that the DNA encoding the
PRO617 polypeptide has significant homology with DNA encoding the
CD24 protein. Accordingly, it is presently believed that PRO617
polypeptide disclosed in the present application is a newly
identified CD24 homolog.
[1144] 13. Full-length PRO700 Polypeptides
[1145] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO700. In particular, Applicants have
identified and isolated cDNA encoding a PRO700 polypeptide, as
disclosed in further detail in the Examples below. Analysis of the
amino acid sequence of the full-length PRO700 polypeptide using
BLAST and FastA sequence alignment computer programs, suggests that
various portions of the PRO700 polypeptide possess significant
sequence similarity to various protein disulfide isomerases. More
specifically, an analysis of the Dayhoff database (version 35.45
SwissProt 35) evidenced significant sequence similarity between the
PRO700 amino acid sequence and the following Dayhoff sequences;
polypeptide with protein disulfide isomerase activity, designated
as ("P_P80664"), human PDI, designated as ("P_R51696"), human PDI,
designated as (P_R5297"), probable protein disulfide isomerase
er-60 precursor, designated as ("ER60_SCHMA"), protein disulfide
isomerase precursor--Drosophila melanogaster, designated as
("PDI_DROME"), protein disulfide-isomerase precursor--Nicotiana
tabaccum, designated as ("NTPDIGENE.sub.--1"), protein disulfide
isomerase--Onchocerca volvulus, designated as ("OVU12440.sub.--1"),
human probable protein disulfide isomerase p5 precursor, designated
as ("ERP5_HUMAN"), human protein disulfide isomerase-related
protein 5, ("HSU79278.sub.--1"), and protein disulfide isomerase
precursor/prolyl 4-hydroxy, ("PDI_HUMAN"), thereby indicating that
PRO700 may be a novel protein disulfide isomerase. Accordingly, it
is presently believed that PRO700 polypeptide disclosed in the
present application is a newly identified member of the protein
disulfide isomerase family and possesses the ability to catalyze
the formation of disulfide bonds typical of the protein disulfide
isomerase family.
[1146] 14. Full-length PRO702 Polypeptides
[1147] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO702. In particular, Applicants have
identified and isolated cDNA encoding a PRO702 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
the PRO702 polypeptide has significant similarity to the
conglutinin protein. Accordingly, it is presently believed that
PRO702 polypeptide disclosed in the present application is a newly
identified conglutinin homolog.
[1148] 15. Full-length PRO703 Polypeptides
[1149] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO703. In particular, Applicants have
identified and isolated cDNA encoding a PRO703 polypeptide, as
disclosed in further detail in the Examples below. Analysis of the
amino acid sequence of the full-length PRO703 polypeptide using
BLAST and FastA sequence alignment computer programs, suggests that
various portions of the PRO703 polypeptide possess significant
sequence similarity to the VLCAS protein, thereby indicating that
PRO703 may be a novel VLCAS protein. More specifically, an analysis
of the Dayhoff database (version 35.45 SwissProt 35) evidenced
significant sequence similarity between the PRO703 amino acid
sequence and the following Dayhoff sequences, human mRNA for
very-long-chain acyl-CoA, ("D88308"), rat mRNA for very-long-chain
acyl-CoA synthetase, ("D85100"), Mus musculus fatty acid transport
protein, ("MMU15976"), human very-long-chain acyl-CoA synthetase,
("D88308.sub.--1"), Mus musculus very-long-chain acyl-CoA
synthetase, ("AF033031.sub.--1"), very-long-chain acyl-CoA
synthetase--Rattus, ("D85100.sub.--1"), rat long-chain fatty acid
transport protein, ("FATP_RAT"), mouse long-chain fatty acid
transport protein, ("FATP_MOUSE"), probable long-chain fatty acid
transport protein, ("FAT1_YEAST"), and fatty acid transporter
protein, ("CHY15839.sub.--2"), thereby indicating that PRO703 may
be a novel VLCAS. Accordingly, it is presently believed that PRO703
polypeptide disclosed in the present application is a newly
identified member of the VLCAS family and possesses the ability to
facilitate the cellular transport of long and very long chain fatty
acids typical of the VLCAS family.
[1150] 16. Full-length PRO705 Polypeptides
[1151] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO705. In particular, Applicants have
identified and isolated cDNA encoding a PRO705 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
the PRO705 polypeptide has significant similarity to the K-glypican
protein. Accordingly, it is presently believed that PRO705
polypeptide disclosed in the present application is a newly
identified member of the glypican family of proteoglycan
proteins.
[1152] 17. Full-length PRO708 Polypeptides
[1153] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO708. In particular, Applicants have
identified and isolated cDNA encoding a PRO708 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
the PRO708 polypeptide has significant homology with the aryl
sulfatase proteins. Applicants have also found that the DNA
encoding the PRO708 polypeptide has significant homology with DNA
encoding the aryl sulfatase proteins. Accordingly, it is presently
believed that PRO708 polypeptide disclosed in the present
application is a newly identified aryl sulfatase homolog.
[1154] 18. Full-length PRO320 Polypeptides
[1155] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO320. In particular, Applicants have
identified and isolated cDNA encoding a PRO320 polypeptide, as
disclosed in further detail in the Examples below. Analysis of the
amino acid sequence of the full-length PRO320 polypeptide using
BLAST and FastA sequence alignment computer programs, suggests that
various portions of the PRO320 polypeptide have significant
homology to the fibulin protein. More specifically, an analysis of
the Dayhoff database (version 35.45 SwissProt 35) evidenced
significant homology between the PRO320 amino acid sequence and the
following Dayhoff sequences, human fibulin-2 precursor, designated
"FBL2_HUMAN", human fibulin-1 isoform a precursor, designated
"FBLA_HUMAN", ZK783.1--Caenorhabditis elegans, designated
"CELZK783.sub.--1", human-notch2, designated "HSU77493.sub.--1",
Nel protein precursor--rattus norvegicus, designated "NEL_RAT", Mus
musculus cell surface protein, designated "D32210.sub.--1", mouse
(fragment) Notch B protein, designated "A49175",
C50H2.3a--Caenorhabditis elegans, designated "CEC50H2.sub.--3",
MEC-9L--Caenorhabditis elegans, designated "CEU33933.sub.--1", and
Mus musculus notch 4, designated "10MMMHC29N7.sub.--2", thereby
indicating that PRO320 may be a novel fibulin or fibulin-like
protein. Accordingly, it is presently believed that PRO320
polypeptide disclosed in the present application is a newly
identified member of the fibulin family and possesses biological
activity typical of the fibulin family.
[1156] 19. Full-length PRO324 Polypeptides
[1157] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO324. In particular, Applicants have
identified and isolated cDNA encoding a PRO324 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
the PRO324 polypeptide has significant similarity to
oxidoreductases. Accordingly, it is presently believed that PRO324
polypeptide disclosed in the present application is a newly
identified oxidoreductase homolog.
[1158] 20. Full-length PRO351 Polypeptides
[1159] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO351. In particular, Applicants have
identified and isolated cDNA encoding a PRO351 polypeptide, as
disclosed in further detail in the Examples below. Analysis of the
amino acid sequence of the full-length PRO351 polypeptide using
BLAST and FastA sequence alignment computer programs, suggests that
various portions of the PRO351 polypeptide possess significant
sequence similarity to the prostasin protein, thereby indicating
that PRO351 may be a novel prostasin protein. More specifically, an
analysis of the Dayhoff database (version 35.45 SwissProt 35)
evidenced significant sequence similarity between the PRO351 amino
acid sequence and the following Dayhoff sequences,
"AC003965.sub.--1", "CELC07G1.sub.--7", "GEN12917", "HEPS_HUMAN",
"GEN14584", "MCT6_MOUSE", "HSU75329.sub.--1", "PLMN_ERIEU",
"TRYB_HUMAN", and "P_W22987". Accordingly, it is presently believed
that PRO351 polypeptide disclosed in the present application is a
newly identified member of the prostasin family and possesses
properties and activities typical of the prostasin family.
[1160] 21. Full-length PRO352 Polypeptides
[1161] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO352. In particular, Applicants have
identified and isolated cDNA encoding a PRO352 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
the PRO352 polypeptide has significant similarity to the
butyrophilin protein. Accordingly, it is presently believed that
PRO352 polypeptide disclosed in the present application is a newly
identified butyrophilin homolog.
[1162] 22. Full-length PRO381 Polypeptides
[1163] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO381. In particular, Applicants have
identified and isolated cDNA encoding a PRO381 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
the PRO381 polypeptide has significant similarity to immunophilin
proteins. Accordingly, it is presently believed that PRO381
polypeptide disclosed in the present application is a newly
identified FKBP immunophilin homolog.
[1164] 23. Full-length PRO386 Polypeptides
[1165] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO386. In particular, Applicants have
identified and isolated cDNA encoding a PRO386 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
the PRO386 polypeptide has significant similarity to the beta-2
subunit of a sodium channel protein. Accordingly, it is presently
believed that PRO386 polypeptide disclosed in the present
application is homolog of a beta-2 subunit of a sodium channel
expressed in mammalian cells.
[1166] 24. Full-length PRO540 Polypeptides
[1167] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO540. In particular, Applicants have
identified and isolated cDNA encoding a PRO540 polypeptide, as
disclosed in further detail in the Examples below. Analysis of the
amino acid sequence of the full-length PRO540 polypeptide using
BLAST and FastA sequence alignment computer programs, suggests that
various portions of the PRO540 polypeptide possess significant
sequence similarity to the LCAT protein, thereby indicating that
PRO540 may be a novel LCAT protein. More specifically, an analysis
of the Dayhoff database (version 35.45 SwissProt 35) evidenced
significant sequence similarity between the PRO540 amino acid
sequence and the following Dayhoff sequences,
phosphatidylcholine-sterol acyltransferase, designated
"LCAT_HUMAN", hypothetical 75.4 kd protein, designated
"YN84_YEAST", Bacillus lichenifomris esterase, designated
"BLU35855.sub.--1", macrotetrolide resistance
protein--Streptomyces, designated "JH0655", T-cell receptor delta
chain precursor, designated "C30583", Rhesus kringle 2, designated
"P_W07551", RAGE-1 ORF5, designated "HSU46191.sub.--3", human Ig
kappa chain VKIII-JK3, designated "HSU07466.sub.--1", and
Alstroemeria inodora reverse transcriptase, designated
"AL1223606.sub.--1". Accordingly, it is presently believed that
PRO540 polypeptide disclosed in the present application is a newly
identified member of the LCAT protein family and possesses lipid
transport capability typical of the LCAT family.
[1168] 25. Full-length PRO615 Polypeptides
[1169] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO615. In particular, Applicants have
identified and isolated cDNA encoding a PRO615 polypeptide, as
disclosed in further detail in the Examples below. Analysis of the
amino acid sequence of the full-length PRO615 polypeptide using
BLAST and FastA sequence alignment computer programs, suggests that
various portions of the PRO615 polypeptide possess significant
sequence similarity to the human synaptogyrin protein, thereby
indicating that PRO615 may be a novel synaptogyrin protein. More
specifically, an analysis of the Dayhoff database (version 35.45
SwissProt 35) evidenced significant sequence similarity between the
PRO615 amino acid sequence and the following Dayhoff sequences,
"AF039085.sub.--1", "RNU39549.sub.--1", "CELT08A9.sub.--8",
"FSU62028.sub.--1", "S73645", "Y348_MYCPN", "AC000103.sub.--5", "",
"RT12_LEITA", and "EBVLMP218.sub.--1". Accordingly, it is presently
believed that PRO615 polypeptide disclosed in the present
application is a newly identified member of the synaptogyrin family
and possesses activity and properties typical of the synaptogyrin
family.
[1170] 26. Full-length PRO618 Polypeptides
[1171] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO618. In particular, Applicants have
identified and isolated cDNA encoding a PRO618 polypeptide, as
disclosed in further detail in the Examples below. Analysis of the
amino acid sequence of the full-length PRO618 polypeptide using
BLAST and FastA sequence alignment computer programs, suggests that
various portions of the PRO618 polypeptide possess significant
sequence similarity to the enteropeptidase protein, thereby
indicating that PRO618 may be a novel enteropeptidase. More
specifically, an analysis of the Dayhoff database (version 35.45
SwissProt 35) evidenced significant sequence similarity between the
PRO618 amino acid sequence and the following Dayhoff sequences,
"P_W22987", "KAL_HUMAN", "AC003965.sub.--1", "GEN12917",
"ENTK_HUMAN", "FA1_HUMAN", "HSU75329.sub.--1", "P_W22986", and
"PLMN_HORSE". Accordingly, it is presently believed thatPRO618
polypeptide disclosed in the present application is a newly
identified member of the enteropeptidase family and possesses
catalytic activity typical of the enteropeptidase family.
[1172] 27. Full-length PRO719 Polypeptides
[1173] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO719. In particular, Applicants have
identified and isolated cDNA encoding a PRO719 polypeptide, as
disclosed in further detail in the Examnples below. Using BLAST and
FastA sequence aligmnent computer programs, Applicants found that
the PRO719 polypeptide has significant similarity to the
lipoprotein lipase H protein. Accordingly, it is presently believed
that PRO719 polypeptide disclosed in the present application is a
newly identified lipoprotein lipase H homolog.
[1174] 28. Full-length PRO724 Polypeptides
[1175] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO724. In particular, Applicants have
identified and isolated cDNA encoding a PRO724 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
the PRO724 polypeptide has significant similarity to the human low
density lipoprotein (LDL) receptor protein. Accordingly, it is
presently believed that PRO724 polypeptide disclosed in the present
application is a newly identified LDL receptor homolog.
[1176] 29. Full-length PRO772 Polypeptides
[1177] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO772. In particular, Applicants have
identified and isolated cDNA encoding a PRO772 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
the PRO772 polypeptide has significant similarity to the human A4
protein. Accordingly, it is presently believed that PRO772
polypeptide disclosed in the present application is a newly
identified A4 protein homolog.
[1178] 30. Full-length PRO852 Polypeptides
[1179] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO852. In particular, Applicants have
identified and isolated cDNA encoding a PRO852 polypeptide, as
disclosed in furter detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
the PRO852 polypeptide has significant similarity to various
protease proteins. Accordingly, it is presently believed that
PRO852 polypeptide disclosed in the present application is a newly
identified protease enzyme homolog.
[1180] 31. Full-length PRO853 Polypeptides
[1181] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO853. In particular, Applicants have
identified and isolated cDNA encoding a PRO853 polypeptide, as
disclosed in furter detail in the Examples below. Analysis of the
amino acid sequence of the full-length PRO853 polypeptide using
BLAST and FastA sequence alignment computer programs, suggests that
various portions of the PRO853 polypeptide possess significant
sequence similarity to the reductase protein, thereby indicating
that PRO853 may be a novel reductase. More specifically, an
analysis of the Dayhoff database (version 35.45 SwissProt 35)
evidenced significant sequence similarity between the PRO853 amino
acid sequence and the following Dayhoff sequences, "P_W03198",
"CEC15H11.sub.--6", "MTV030.sub.--12", "P_W15759", "S42651",
"ATAC00234314", "MTV022.sub.--13", "SCU43704.sub.--1",
"CELE04F6.sub.--7", and "ALFA.sub.--1". Accordingly, it is
presently believed that PRO853 polypeptide disclosed in the present
application is a newly identified member of the reductase family
and possesses the antioxidant enzymatic activity typical of the
reductase family.
[1182] 32. Full-length PRO860 Polypeptides
[1183] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO860. In particular, Applicants have
identified and isolated cDNA encoding a PRO860 polypeptide, as
disclosed in further detail in the Examples below. Analysis of the
amino acid sequence of the full-length PRO860 polypeptide using
BLAST and FastA sequence alignment computer programs, suggests that
various portions of the PRO860 polypeptide possess significant
sequence similarity to the neurofascin protein, thereby indicating
that PRO860 may be a novel neurofascin. More specifically, an
analysis of the Dayhoff database (version 35.45 SwissProt 35)
evidenced significant sequence similarity between the PRO860 amino
acid sequence and the following Dayhoff sequences,
"AF040990.sub.--1", "AF041053.sub.--1", "CELZK377.sub.--2",
"RNU81035.sub.--1", "D86983.sub.--1", "S26180", "MMBIG2A.sub.--1",
"S46216", and "RNU687261.sub.--1". Accordingly, it is presently
believed that PRO860 polypeptide disclosed in the present
application is a newly identified member of the neurofascin family
and possesses the cellular adhesion properties typical of the
neurofascin family.
[1184] 33. Full-length PRO846 Polypeptides
[1185] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO846. In particular, Applicants have
identified and isolated cDNA encoding a PRO846 polypeptide, as
disclosed in further detail in the Examples below. Analysis of the
amino acid sequence of the full-length PRO846 polypeptide using
BLAST and FastA sequence alignment computer programs, suggests that
various portions of the PRO846 polypeptide possess significant
sequence similarity to the CMRF35 protein, thereby indicating that
PRO846 may be a novel CMRF35 protein. More specifically, an
analysis of the Dayhoff database (version 35.45 SwissProt 35)
evidenced significant sequence sliilarity between the PRO846 amino
acid sequence and the following Dayhoff sequences,"CM35_HUMAN",
"AF035963.sub.--1", "PIGR_RABIT", "AF043724.sub.--1",
"RNU89744.sub.--1", "A52091.sub.--1", "S48841", "ELK06A9.sub.--3",
and "AF049588.sub.--1". Accordingly, it is presently believed that
PRO846 polypeptide disclosed in the present application is a newly
identified member of the CMRF35 protein family and possesses
properties typical of the CMRF35 protein family.
[1186] 34. FUll-Ienoth PRO862 Polypeptides
[1187] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO862. In particular, Applicants have
identified and isolated cDNA encoding a PRO862 polypeptide, as
disclosed in further detail in the Examples below. Analysis of the
amino acid sequence of the full-length PRO862 polypeptide using
BLAST and FastA sequence alignment computer programs, suggests that
various portions of the PRO862 polypeptide possess significant
sequence similarity to the lysozyme protein, thereby indicating
that PRO862 may be a novel lysozyme protein. More specifically, an
analysis of the Dayhoff database (version 35.45 SwissProt 35)
evidenced significant sequence similarity between the PRO862 amino
acid sequence and the following Dayhoff sequences, "P_P90343", and
"LYC_HUMAN. Accordingly, it is presently believed that PRO862
polypeptide disclosed in the present application is a newly
identified member of the lysozyme family and possesses catalytic
activity typical of the lysozyme family.
[1188] 35. Full-length PRO864 Polypeptides
[1189] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO864. In particular, Applicants have
identified and isolated cDNA encoding a PRO864 polypeptide, as
disclosed in further detail in the Examples below. Analysis of the
amino acid sequence of the full-length PRO864 polypeptide using
BLAST and FastA sequence alignment computer programs, suggests that
various portions of the PRO864 polypeptide possess significant
sequence similarity to tme Wnt-4 protein, thereby indicating that
PRO864 may be a novel Wnt-4 protein. More specifically, an analysis
of the Dayhoff database (version 35.45 SwissProt 35) evidenced
significant sequence similarity between the PRO864 amino acid
sequence and the following Dayhoff sequences, "WNT4_MOUSE",
"WNT3_MOUSE", "WN5A_HUMAN", "WN7B_MOUSE", "WN3A_MOUSE",
"XLU66288.sub.--1", "WN13_HUMAN", "WN5B_ORYLA", "WNT2_MOUSE", and
"WN7A_MOUSE". Accordingly, it is presently believed that PRO864
polypeptide disclosed in the present application is a newly
identified member of the Wnt-4 protein family and possesses
properties typical of the Wnt-4 protein family.
[1190] 36. Full-length PRO792 Polypeptides
[1191] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO792. In particular, Applicants have
identified and isolated cDNA encoding a PRO792 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found matthe
PRO792 polypeptide has significant similarity to the CD23 protein.
Accordingly, it is presently believed that PRO792 polypeptide
disclosed in the present application is a newly identified CD23
homolog.
[1192] 37. Full-length PRO866 Polypeptides
[1193] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO866. In particular, Applicants have
identified and isolated cDNA encoding a PRO866 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
the PRO866 polypeptide has significant similarity to various mindin
and spondin proteins. Accordingly, it is presently believed that
PRO866 polypeptide disclosed in the present application is a newly
identified mindin/spondin homolog.
[1194] 38. Full-enpth PRO871 Polypeptides
[1195] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO871. In particular, Applicants have
identified and isolated cDNA encoding a PRO871 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
the PRO871 polypeptide has significant similarity to the CyP-60
protein. Accordingly, it is presently believed that PRO871
polypeptide disclosed in the present application is a newly
identified member of the cyclophilin protein family and possesses
activity typical of the cyclophilin protein family.
[1196] 39. Full-length PRO873 Polypeptides
[1197] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO873. In particular, Applicants have
identified and isolated cDNA encoding a PRO873 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
the PRO873 polypeptide has significant similarity to a human liver
carboxylesterase. Accordingly, it is presently believed that PRO873
polypeptide disclosed in the present application is a newly
identified member of the carboxylesterase family and possesses
enzymatic activity typical of the carboxylesterase family.
[1198] 40. Full-length PRO940 Polypeptides
[1199] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO940. In particular, Applicants have
identified and isolated cDNA encoding a PRO940 polypeptide, as
disclosed in firher detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
the PRO940 polypeptide has significant simllarity to CD33 and the
OB binding protein-2. Accordingly, it is presently believed that
PRO940 polypeptide disclosed in the present application is a newly
CD33 and/or OB binding protein-2 homolog.
[1200] 41. Full-length PRO941 Polypeptides
[1201] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO941. In particular, Applicants have
identified and isolated cDNA encoding a PRO941 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
the PRO941 polypeptide has significant similarity to one or more
cadherin proteins. Accordingly, it is presently believed that
PRO941 polypeptide disclosed in the present application is a newly
identified cadherin homolog.
[1202] 42. Full-length PRO944 Polypeptides
[1203] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO944. In particular, Applicants have
identified and isolated cDNA encoding a PRO944 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
the PRO944 polypeptide has significant similarity to the CPE-R cell
surface protein. Accordingly, it is presently believed that PRO944
polypeptide disclosed in the present application is a newly
identified CPE-R homolog.
[1204] 43. Full-length PRO983 Polypeptides
[1205] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO983. In particular, Applicants have
identified and isolated cDNA encoding a PRO983 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
the PRO983 polypeptide has significant similarity to the
vesicle-associated protein, VAP-33. Accordingly, it is presently
believed that PRO983 polypeptide disclosed in the present
application is a newly identified member of the vesicle-associated
membrane protein family and possesses activity typical of
vesicle-associated membrane proteins.
[1206] 44. Full-length PRO1057 Polypeptides
[1207] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO1057. In particular, Applicants have
identified and isolated cDNA encoding a PRO1057 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
the PRO1057 polypeptide has significant similarity to various
protease proteins. Accordingly, it is presently believed that
PRO1057 polypeptide disclosed in the present application is a newly
identified protease homolog.
[1208] 45. Full-length PRO1071 Polypeptides
[1209] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO1071. In particular, Applicants have
identified and isolated EDNA encoding a PRO1071 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
the PRO1071 polypeptide has significant similarity to the
thrombospondin protein. Accordingly, it is presently believed that
PRO1071 polypeptide disclosed in the present application is a newly
identified thrombospondin homolog.
[1210] 46. Full-length PRO1072 Polypeptides
[1211] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO1072. In particular, Applicants have
identified and isolated cDNA encoding a PRO1072 polypeptide, as
disclosed in firther detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
the PRO1072 polypeptide has significant similarity to various
reductase proteins. Accordingly, it is presently believed that
PRO1072 polypeptide disclosed in the present application is a newly
identified member of the reductase protein family.
[1212] 47. Full-length PRO1075 Polypeptides
[1213] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO1075. In particular, Applicants have
identified and isolated cDNA encoding a PRO1075 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
the PRO1075 polypeptide has significant similarity to protein
disulfide isomerase. Accordingly, it is presently believed that
PRO1075 polypeptide disclosed in the present application is a newly
identified member of the protein disulfide isomerase family and
possesses activity typical of that family.
[1214] 48. Full-length PRO181 Polypeptides
[1215] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO181. In particular, Applicants have
identified and isolated cDNA encoding a PRO181 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
the PRO181 polypeptide has significant similarity to the cornichon
protein. Accordingly, it is presently believed that PRO181
polypeptide disclosed in the present application is a newly
identified cornichon homolog.
[1216] 49. Full-length PRO195 Polypeptides
[1217] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO195. In particular, Applicants have
identified and isolated cDNA encoding a PRO195 polypeptide, as
disclosed in further detail in the Examples below. The
PRO195-encoding clone was isolated from a human fetal placenta
lhbrary using a trapping technique which selects for nucleotide
sequences encoding secreted proteins. To Applicants present
knowledge, the UNQ169 (DNA26847-1395) nucleotide sequence encodes a
novel factor; using BLAST and FastA sequence alignment computer
programs, no sequence identities to any known proteins were
revealed.
[1218] 50. Full-length PRO865 Polypeptides
[1219] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO865. In particular, Applicants have
identified and isolated cDNA encoding a PRO865 polypeptide, as
disclosed in further detail in the Exanples below. The
PRO865-encoding clone was isolated from a human fetal kidney
library using a trapping technique which selects for nucleotide
sequences encoding secreted proteins. Thus, the PRO865-encoding
clone may encode a secreted factor. To Applicants present
knowledge, the UNQ434 (DNA53974-1401) nucleotide sequence encodes a
novel factor; using BLAST and FastA sequence alignment computer
programs, no sequence identities to any known proteins were
revealed.
[1220] 51. Full-length PRO827 Polypeptides
[1221] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO827. In particular, Applicants have
identified and isolated cDNA encoding a PRO827 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
the PRO827 polypeptide has significant similarity to VLA-2 and
various other integrin proteins. Accordingly, it is presently
believed that PRO827 polypeptide disclosed in the present
application is a novel integrin protein or splice variant
thereof.
[1222] 52. Full-length PRO1114 Polypeptides
[1223] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO1114. In particular, Applicants have
identified and isolated cDNA encoding a PRO1114 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
the PRO1114 polypeptide has significant similarity to the cytokine
receptor family of proteins. Accordingly, it is presently believed
that PRO1114 polypeptide disclosed in the present application is a
newly identified member of the cytokine receptor family of proteins
and possesses activity typical of that family.
[1224] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO1114 interferon receptor (UNQ557). In
particular, cDNA encoding a PRO1114 interferon receptor polypeptide
has 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 DNA57033-1403 as well
as all further native homologues and variants included in the
foregoing definition of PRO1114 interferon receptor, will be
referred to as "PRO1114 interferon receptor", regardless of their
origin or mode of preparation.
[1225] Using the WU-BLAST2 sequence alignment computer program, it
has been found that a full-length native sequence PRO1114
interferon receptor polypeptide (shown in FIG. 142 and SEQ ID
NO:352) has sequence identity with the other known interferon
receptors. Accordingly, it is presently believed that PRO1114
interferon receptor possesses activity typical of other interferon
receptors.
[1226] 53. Full-length PRO237 Polypeptides
[1227] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO237. In particular, Applicants have
identified and isolated cDNA encoding a PRO237 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
the PRO237 polypeptide has significant similarity to carbonic
anhydrase. Accordingly, it is presently believed that PRO237
polypeptide disclosed in the present application is a newly
identified carbonic anhydrase homolog.
[1228] 54. Full-length PRO541 Polypeptides
[1229] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO541. In particular, Applicants have
identified and isolated cDNA encoding a PRO541 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
the PRO541 polypeptide has significant similarity to a trypsin
inhibitor protein. Accordingly, it is presently believed that
PRO541 polypeptide disclosed in the present application is a newly
identified member of the trypsin inhibitor protein family.
[1230] 55. Full-length PRO273 Polypeptides
[1231] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO273. In particular, Applicants have
identified and isolated cDNA encoding a PRO273 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 PRO273 polypeptide have significant
sequence identity with various chemokines. Accordingly, it is
presently believed that PRO273 polypeptide disclosed in the present
application is a newly identified member of the chemokine family
and possesses activity typical of the chemokine family.
[1232] 56. Full-length PRO701 Polypeptides
[1233] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO701. In particular, Applicants have
identified and isolated cDNA encoding a PRO701 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 PRO701 polypeptide have significant
homology with the neuroligins 1, 2 and 3 and esterases including
carboxyesterases and acytlcholinesterases. Accordingly, it is
presently believed that PRO701 polypeptide disclosed in the present
application is a newly identified member of the neuroligin family
and is involved in mediating recognition processes between neurons
and/or functions as a cell adhesin molecule as is typical of
neuroligins.
[1234] 57. Full-length PRO704 Polypeptides
[1235] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO704. In particular, Applicants have
identified and isolated cDNA encoding a PRO704 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 PRO704 polypeptide have significant
homology with the VIP36 and GP36b. Accordingly, it is presently
believed that PRO704 polypeptide disclosed in the present
application is a newly identified member of the vesicular integral
membrane protein family and possesses the ability to bind to sugars
and cycle between the plasma membrane and the Golgi typical of this
family.
[1236] 58. Full-length PRO706 Polypeptides
[1237] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO706. In particular, Applicants have
identified and isolated cDNA encoding a PRO706 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 PRO706 polypeptide have sequence identity
with the human prostatic acid phosphatase precursor and the human
lysosomal acid phosphatase precursor. Accordingly, it is presently
believed that PRO706 polypeptide disclosed in the present
application is a newly identified member of the human prostatic
acid phosphatase precursor family and possesses phosphatase typical
of the acid phosphatase family.
[1238] 59. Full-length PRO707 Polypeptides
[1239] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO707. In particular, Applicants have
identified and isolated cDNA encoding a PRO707 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 PRO707 polypeptide have significant
homology with cadherins, particularly cadherin FIB3 found in
fibroblasts. Accordingly, it is presently believed that PRO707
polypeptide disclosed in the present application is a newly
identified member of the cadherin family and possesses cell
interaction signaling typical of the cadherin family.
[1240] 60. Full-length PRO322 Polypeptides
[1241] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO322. In particular, Applicants have
identified and isolated cDNA encoding a PRO322 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 PRO322 polypeptide have significant
homology with human neuropsin, serine protease, neurosin and
trypsinogen. Accordingly, it is presently believed that PRO322
polypeptide disclosed in the present application is a newly
identified member of the serine protease family and possesses
protease activity typical of this family. It is also believed that
PRO322 is involved in hippocampal plasticity and is associated with
extracellular matrix modifications and cell migrations.
[1242] 61. Full-length PRO526 Polypeptides
[1243] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO526. In particular, Applicants have
identified and isolated cDNA encoding a PRO526 polypeptide, as
disclosed in farther detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
various portions of the PRO526 polypeptide have significant
homology with the acid labile subunit of the insulin-like growth
factor complex (ALS), as well carboxypeptidase, SLIT, and platelet
glycoprotein V. Accordingly, it is presently believed that PRO526
polypeptide disclosed in the present application is a newly
identified member of the leucine-repeat rich superfamily, and
possesses protein-protein interaction capabilities typical of this
family.
[1244] 62. Full-length PRO531 Polypeptides
[1245] The present inventionprovides newly identified and
isolatednucleotide sequences encoding polypeptides referred to in
the present application as PRO531. In particular, Applicants have
identified and isolated cDNA encoding a PRO531 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 PRO531 polypeptide have significant
sequence identity and similarity with members of the cadherin
superfamily, particularly, protocadherin 3. Accordingly, it is
presently believed that PRO531 polypeptide disclosed in the present
application is a newly identified member of the cadherin
superfamily, and is a protocadherin. PRO531 is a transmembrane
protein which has extracellular cadherin motifs. PRO531 is believed
to be involved in cell-cell activity, in particular, cell
signaling.
[1246] 63. Full-length PRO534 Polypeptides
[1247] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO534. In particular, Applicants have
identified and isolated cDNA encoding a PRO534 polypeptide, as
disclosed in fturther detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
various portions of the PRO534 polypeptide have significant
identity or similarity with the putative disulfide isomerase erp38
precursor and thioredoxin c-3. Accordingly, it is presently
believed that PRO534 polypeptide disclosed in the present
application is a newly identified member of the disulfide isomerase
family and possesses the ability to recognize and unscramble either
intermediate or incorrect folding patterns typical of this
family.
[1248] 64. Full-length PRO697 Polypeptides
[1249] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO697. In particular, Applicants have
identified and isolated cDNA encoding a PRO697 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 PRO697 polypeptide have significant
identity or similarity with sFRP-2, sFRP-1 and SARP-1, -2 and -3.
Accordingly, it is presently believed that PRO697 polypeptide
disclosed in the present application is a newly identified member
of the sFRP family and possesses activity related to the Wnt signal
pathway.
[1250] 65. Full-length PRO717 Polypeptides
[1251] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO717. In particular, Applicants have
identified and isolated cDNA encoding a PRO717 polypeptide, as
disclosed in further detail in the Examples below. To Applicants
present knowledge, the UNQ385 (DNA50988-1326) nucleotide sequence
encodes a novel factor; using BLAST and FastA sequence alignment
computer programs, no significant sequence identities to any known
human proteins were revealed.
[1252] 66. Full-length PRO731 Polypeptides
[1253] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO73 1. In particular, Applicants have
identified and isolated cDNA encoding a PRO731 polypeptide, as
disclosed in frther detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
various portions of the PRO731 polypeptide have significant
homology with the protocadherins 4, 68, 43, 42, 3, and 5.
Accordingly, it is presently believed that PRO731 polypeptide
disclosed in the present application is a newly identified member
of the protocadherin family and possesses cell-cell aggregation or
signaling activity or signal transduction involvement typical of
this family.
[1254] 67. Full-length PRO218 Polypeptides
[1255] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO218. In particular, Applicants have
identified and isolated cDNA encoding a PRO218 polypeptide, as
disclosed in further detail in the Examples below. The
PRO218-encoding clone was isolated from a human fetal kidney
library. To Applicants present knowledge, the UNQ192
(DNA30867-1335) nucleotide sequence encodes a novel factor; using
BLAST and FastA sequence alignment computer programs, no
significant sequence identities to any known proteins were
revealed. Some sequence identity was found with membrane regulator
proteins, indicating that PRO218 may function as a membrane
regulator.
[1256] 68. Full-length PRO768 Polypeptides
[1257] The present invention provides newly ideniffied and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO768. In particular, Applicants have
identified and isolated cDNA encoding a PRO768 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 PRO768 polypeptide have significant
homology with integrins, including integrin 7 and 6. Accordingly,
it is presently believed that PRO768 polypeptide disclosed in the
present application is a newly identified member of the integrin
family, either a homologue or a splice variant of integrin 7, and
is involved with cell adhesion and communication between muscle
cells and the extracellular matrix.
[1258] 69. Full-length PRO771 Polypeptides
[1259] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO771. In particular, Applicants have
identified and isolated cDNA encoding a PRO771 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 PRO771 polypeptide have significant
sequence identity and similarity with testican. Accordingly, it is
presently believed that PRO771 polypeptide disclosed in the present
application is a newly identified member of the testican family and
possesses cell signaling, binding, or adhesion properties, typical
of this family.
[1260] 70. Full-length PRO733 Polypeptides
[1261] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO733. hn particular, Applicants have
identified and isolated cDNA encoding a PRO733 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 PRO733 polypeptide have significant
sequence identity with the T1/ST receptor binding protein.
Accordingly, it is presently believed that PRO733 polypeptide
disclosed in the present application is a newly identified member
of the interleukin-like family binding proteins which may be a
cytoline and which may be involved in cell signaling. It is
believed that PRO733 is an ApoAIV homologue.
[1262] 71. Full-length PRO162 Polypeptides
[1263] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO162. In particular, Applicants have
identified and isolated cDNA encoding a PRO162 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 PRO162 polypeptide have significant
homology with human pancreatitis-associated protein (PAP).
Applicants have also found that the DNA encoding the PRO162
polypeptide has significant homology with bovine lithostathine
precursor and bovine pancreatic thread protein (PTP). Accordingly,
it is presently believed that PRO162 polypeptide disclosed in the
present application is a newly identified member of the
pancreatitis-associated protein family and possesses activity
typical of the pancreatitis-associated protein family.
[1264] 72. Full-length PRO788 Polypeptides
[1265] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO788. In particular, Applicants have
identified and isolated cDNA encoding a PRO788 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 PRO788 polypeptide have significant
homology with the anti-neoplastic urinary protein. Applicants have
also found that the DNA encoding the PRO788 polypeptide has
significant homology with human E48 antigen, human component B
protein, and human prostate stem cell antigen. Accordingly, it is
presently believed that PRO788 polypeptide disclosed in the present
application is a newly identified member of the anti-neoplastic
urinary protein family and possesses anti-neoplastic activity
typical of the anti-neoplastic urinary protein family.
[1266] 73. Full-length PRO1008 Polypeptides
[1267] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO1008. In particular, Applicants have
identified and isolated cDNA encoding a PRO1008 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 PRO1008 polypeptide have significant
sequence identity and similarity with mouse dkk-1 (mdkk-1).
Accordingly, it is presently believed that PRO1008 polypeptide
disclosed in the present application is a newly identified member
of the dkk-1 family and possesses head inducing activity typical of
this family.
[1268] 74. Full-length PRO1012 Polypeptides
[1269] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO1012. In particular, Applicants have
identified and isolated cDNA encoding a PRO1012 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 PRO1012 polypeptide have sequence identity
with disulfide isomerase. Accordingly, it is presently believed
that PRO1012 polypeptide disclosed in the present application is a
newly identified member of the ER retained protein family and
possesses activity related to the processing, production and/or
folding of polypeptides typical of the disulfide isomerase
family.
[1270] 75. Full-length PRO1014 Polypeptides
[1271] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO1014. In particular, Applicants have
identified and isolated cDNA encoding a PRO1014 polypeptide, as
disclosed in frrther detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
various portions of the PRO1014 polypeptide have sequence identity
with reductase and dehydrogenase. Accordingly, it is presently
believed that PRO1014 polypeptide disclosed in the present
application is a newly identified member of the reductase super
family and possesses reduction capabilities typical of this
family.
[1272] 76. Full-length PRO1017 Polypeptides
[1273] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO1017. In particular, Applicants have
identified and isolated cDNA encoding a PRO1017 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 PRO1017 polypeptide have sequence identity
with HNK-1 sulfotransferase. Accordingly, it is presently believed
that PRO1017 polypeptide disclosed in the present application is a
newly identified member of the HNK-1 sulfotransferase family and is
involved with the synthesis of HNK-1 carbohydrate epitopes typical
of this family.
[1274] 77. Full-length PRO474 Polypeptides
[1275] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO474. In particular, Applicants have
identified and isolated cDNA encoding a PRO474 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 PRO474 polypeptide have sequence identity
with dehydrogenase, glucose dehydrogenase and oxidoreductase.
Accordingly, it is presently believed that PRO474 polypeptide
disclosed in the present application is a newly identified member
of the dehydrogenase family and is involved in the oxidation of
glucose.
[1276] 78. Full-length PRO1031 Polypeptides
[1277] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO1031. In particular, Applicants have
identified and isolated cDNA encoding a PRO1031 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 PRO1031 polypeptide have sequence identity
with IL-17 and CTLA-8. Accordingly, it is presently believed that
PRO1031 polypeptide disclosed in the present application is a newly
identified member of the cytoline family and thus may be involved
in inflammation and/or the immune system.
[1278] 79. Full-length PRO938 Polypeptides
[1279] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO938. In particular, Applicants have
identified and isolated cDNA encoding a PRO938 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
the PRO938 polypeptide has significant simllarity to protein
disulfide isomerase. Accordingly, it is presently believed that
PRO938 polypeptide disclosed in the present application is a newly
identified member of the thioredoxin family proteins and possesses
activity typical of protein disulfide isomerase.
[1280] 80. Full-Ienth PRO1082 Polypeptides
[1281] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO1082. In particular, Applicants have
identified and isolated cDNA encoding a PRO1082 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 PRO1082 polypeptide have sequence identity
with a lectin-like oxidized LDL receptor appearing in the database
as AB010710.sub.--1". Accordingly, it is presently believed that
PRO1082 polypeptide disclosed in the present application is a newly
identified member of the LDL receptor family.
[1282] 81. Full-length PRO1083 Polypeptides
[1283] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO1083. In particular, Applicants have
identified and isolated cDNA encoding a PRO1083 polypeptide, as
disclosed infurther detail in the Examples below. The
PRO1083-encoding clone was isolated from a human fetal kidney
library using a trapping technique which selects for nucleotide
sequences encoding secreted proteins. To Applicants present
knowledge, the UNQ540 (DNA50921-1458) nucleotide sequence encodes a
novel factor; using BLAST and FastA sequence alignment computer
programs, some sequence identity with a 7TM receptor, latrophilin
related protein 1 and a macrophage restricted cell surface
glycoprotein was shown. The kinase phosphorylation site and
G-coupled receptor domain shown in FIG. 204 indicate that PRO1083
is a novel member of the 7TM receptor superfamily.
[1284] 82. Full-length PRO200 Polyeptides
[1285] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as VEGF-E. In particular, Applicants have
identified and isolated cDNA encoding a VEGF-E polypeptide, as
disclosed in further detail in the Examples below. Using BLAST
sequence alignment computer programs, Applicants found that the
VEGF-E polypeptide has significant homology with VEGF and bone
morphogenetic protein 1. Inparticular, the cDNA sequence of VEGF-E
exhibits 24% amino acid similarity with VEGF and has structural
conservation. In addition, VEGF-E contains a N-terminal half which
is not present in VEGF and that has 28% homology to bone
morphogenetic protein 1.
[1286] 83. Full-length PRO285 and PRO286 Polypeptides
[1287] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO285 and PRO286 In particular, Applicants
have identified and isolated cDNAs encoding PRO285 and PRO286
polypeptides, as disclosed in further detail in the Examples below.
Using BLAST and FastA sequence alignment computer programs,
Applicants found that the coding sequences of PRO285 and PRO286 are
highly homologous to DNA sequences HSU88540.sub.--1,
HSU88878.sub.--1, HSU88879.sub.--1, HSU88880.sub.--1, and
HSU88881.sub.--1 in the GenBank database.
[1288] Accordingly, it is presently believed that the PRO285 and
PRO286 proteins disclosed in the present application are newly
identified human homologues of the Drosophila protein Toll, and are
likely to play an important role in adaptive immunity. More
specifically, PRO285 and PRO286 may be involved in inflammation,
septic shock, and response to pathogens, and play possible roles in
diverse medical conditions that are aggravated by immune response,
such as, for example, diabetes, ALS, cancer, rheumatoid arhritis,
and ulcers. The role of PRO285 and PRO286 as pathogen pattern
recognition receptors, sensing the presence of conserved molecular
structures present on microbes, is further supported by the data
disclosed in the present application, showing that a known human
Toll-like receptor, TLR2 is a direct mediator of LPS signaing.
[1289] 84. Full-length PRO213-1, PRO1330 and PRO1449
Polypeptides
[1290] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO213-1, PRO1330 and/or PRO1449. In
particular, cDNA encoding a PRO213-1, PRO1330 and/or PRO1449
polypeptide has 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
DNA30943-1163-1, DNA64907-1163-1 and DNA64908-1163-1 as well as all
further native homologues and variants included in the foregoing
definition of PRO213-1, PRO1330 and/or PRO1449, will be referred to
as "PRO213-1, PRO1330 and/or PRO1449", regardless of their origin
or mode of preparation. 85. Full-Ienh PRO298 Polypeptides The
present invention provides newly identified and isolated nucleotide
sequences encoding polypeptides referred to in the present
application as PRO298. (It is noted that PRO298 is an arbitrary
designation of a protein encoded by the nucleic acid shown in FIG.
218, SEQ ID NO:514, and having the amino acid sequence shown in
FIG. 219, SEQ ID NO:515. Further proteins having the same amino
acid sequence but expressed in different rounds of expression, may
be given different "PRO" numbers.) In particular, Applicants have
identified and isolated cDNA encoding a PRO298 polypeptide, as
disclosed in furter detail in the Examples below. Using BLASTX
2.Oa8MP-WashU computer program, socring parameters: T=12, S=68,
S2=36, Matrix: BLOSUM62, Applicants found that the PRO298 protein
specifically disclosed herein shows a limited (27-38%) sequence
identity with the following sequences found in the GenBank
database: S59392 (probable membrane protein YLR246w--yeast); S58154
hypothetical protein SPAC2F7.10--yeast); CELF33D11.sub.--9
(F33D11.9b--Caenorhabditis elegans); YO41_CAEEL (hypothetical 68.7
kd protein zk757.1); CEAC3.sub.--5 (AC3.4--Caenorhabditis elegans);
S52691 (probable transmembrane protein YDR126w--yeast);
ATT12H17.sub.--14 (protein--Arabidopsis thaliana); S55963 (probable
membrane protein YNL326c--yeast); CELC43H6.sub.--2
(C43H6.7--Caenorhabditis elegans); TMO18A10.sub.--14
(A_TMO18A10.8--Arabinosa thaliana).
[1291] 86. Full-length PRO337 Polypeptides
[1292] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO337. In particular, Applicants have
identified and isolated cDNA encoding a PRO337 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST,
BLAST-2 and FastA sequence alignment computer programs, Applicants
found that a full-length native sequence PRO337 has 97% amino acid
sequence identity with rat neurotrimiu, 85% sequence identity with
chicken CEPU, 73% sequence identity with chicken G55, 59% homology
with human LAMP and 84% homology with human OPCAM. Accordingly, it
is presently believed that PRO337 disclosed in the present
application is a newly identified member of the IgLON sub family of
the immunoglobulin superfamily and may possess neurite growth and
differentiation potentiating properties.
[1293] 87. Full-length PRO403 Polypeptides
[1294] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO403. In particular, Applicants have
identified and isolated cDNA encoding a PRO403 polypeptide, as
disclosed in further detail in the Examples below. Using a BLAST,
BLAST-2 and FastA sequence alignment computer programs, Applicants
found that a full-length native sequence PRO403 has 94% identity to
bovine ECE-2 and 64% identity to human ECE-1. Accordingly is
presently believed that PRO403 is a new member of the ECE protein
family and may posses ability to catalyze the production of active
endothelin.
[1295] B. PRO Polypeptide Variants
[1296] 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.
[1297] Variations in the native full-length sequence PRO or in
various domains of the PRO described herein, can be made, for
examnple, 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 a mino 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.
[1298] 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.
[1299] 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.
[1300] 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.
6 TABLE 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
[1301] 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:
[1302] (1) hydrophobic: norleucine, met, ala, val, leu, ile;
[1303] (2) neutral hydrophilic: cys, ser, thr;
[1304] (3) acidic: asp, glu;
[1305] (4) basic: asn, gin, his, lys, arg;
[1306] (5) residues that influence chain orientation: gly, pro;
and
[1307] (6) aromatic: trp, tyr, phe.
[1308] 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.
[1309] 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.
[1310] 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 sidechain beyond the
betaarbon 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.
[1311] C. Modifications of PRO
[1312] 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 crosslinling PRO to a water-insoluble support matrix or surface
for use in the method for purifying anti-PRO antibodies, and
vice-versa. Commonly used crosslinling agents include, e.g.,
1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,
N-hydroxysuccinimide esters, for example, esters with
4azidosalicylic 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- )dithiolpropioimidate.
[1313] 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 a-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.
[1314] 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.
[1315] 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 O-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.
[1316] 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).
[1317] 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. Biothys., 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).
[1318] 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.
[1319] 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.
[1320] 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)1;
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)].
[1321] In an alternative embodiment, the chimeric molecule may
comprse 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, C112 and CH3
regions of an IgGI molecule. For the production of immunoglobulin
fusions see also U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.
[1322] D. Preparation of PRO
[1323] 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.
[1324] 1. Isolation of DNA Encoding PRO
[1325] 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).
[1326] 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 Laboratory 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)].
[1327] 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.
[1328] Sequences identified in such library screening methods can
be compared and aligned to other known sequences deposited and
available in public databases such as Genfank 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.
[1329] 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.
[1330] 2. Selection and Transformation of Host Cells
[1331] 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.
[1332] 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 89J05859 published Jun. 29, 1989. For
mammalian cells without such cell walls, the calcium phosphate
precipitation method of Graham and van der Eb, Virology, 52:456-457
(1978) can be employed. General aspects of mammalian cell host
system transfections have been described in U.S. Pat. No.
4,399,216. Transformations into yeast are typically carried out
according to the method of Van Solingen et al., J. Bact.,
130:946(1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA),
76:3829 (1979). However, other methods for introducing DNA into
cells, such as by nuclear microinjection, electroporation,
bacterial protoplast fusion with intact cells, or polycations,
e.g., polybrene, polyornithine, may also be used. For various
techniques for transforming mammalian cells, see Keown et al.,
Methods in Enzymology, 185:527-537 (1990) and Mansour et al.,
Nature, 336:348-352 (1988).
[1333] Suitable host cells for cloning or expressing the DNA in the
vectors herein include prokaryote, yeast, or higher eukaryote
cells. Suitable prokaryotes include but are not limited to
eubacteria, such as Gram-negative or Gram-positive organisms, for
example, Enterobacteriaceae such as E. coli. Various E. coli
strains are publicly available, such as E. coli K12 strain MM294
(ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli strain W3110
(ATCC 27,325) and K5 772 (ATCC 53,635). Other suitable prokaryotic
host cells include Enterobacteriaceae such as Eschenchia, 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. subtiils and B.
lichenifonnis (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 W3 110 is one particularly preferred host or parent host
because it is a common host strain for recombinant DNA product
fermentations. Preferably, the host cell secretes minimal amounts
of proteolytic enzymes. For example, strain W3 110 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 W3 110 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 ompT kan.sup.r; E.
coli W3110 strain 37D6, which has the complete genotype tona ptr3
phoA E15 (argF-lac)169 degP onipT rbs7 ilvG kan.sup.r; E. coli
W3110 strain 40B4, which is strain 37D6 with a non-kanamycin
resistant degP deletion mutation; and an E. coli strain having
mutant periplasmic protease disclosed in U.S. Pat. No. 4,946,783
issued Aug. 7, 1990. Alternatively, in vitro methods of cloning,
e.g., PCR or other nucleic acid polymerase reactions, are
suitable.
[1334] 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 2 May 1985); Kyveromyces hosts (U.S.
Pat. No. 4,943,529; Fleer et al., Bio/Technologv, 9:968-975(1991))
such as, e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et
al., J. Bacteriol., 154(2):737-742 [1983]), K. fragilis (ATCC
12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178),
K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906; Van den
Berg et al., Bio/Technology, 8:135 (1990)), K. thernotolerans, and
K. mairxianus; yarrowla (EP 402,226); Pichia pastoris (EP 183,070;
Sreekrishna et al., J. Basic Microbiol., 28:265-278 [1988]);
Candida; Trichoderma reesia (EP 244,234); Neurospora crassa (Case
et al., Proc. Natl. Acad. Sci. USA, 76:5259-5263 [1979]);
Schwannionmyces such as Schwanniomyces occidentalis (EP 394,538
published Oct. 31, 1990); and filamentous fungi such as, e.g.,
Neurospora, Penicillium, Tolypocladium (WO 91/00357published Jan.
10, 1991), andAspergilfsshosts such as A. nidulans (Ballance et
al., Biochem. Biophys. Res. Commun., 112:284-289 [1983]; Tilburn et
al., Gene, 26:205-221 [19831; 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, Saccharonyces, 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).
[1335] 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 subdloned for growth in suspension culture, Graham et
al., J. Gen Virol., 36:59 (1977)); Chinese hamster ovary
cellsl-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, BB 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.
[1336] 3. Selection and Use of a Replicable Vector
[1337] The nucleic acid (e.g., cDNA or genomic DNA) encoding PRO
may be inserted into a repilcable 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.
[1338] 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, peniciulinase, 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 Saccharonyces and Kluyverongces a-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.
[1339] Both expression and cloning vectors contain a nucleic acid
sequence that enables the vector to replicate in one or more
selected host cells. Such sequences are well known for a variety of
bacteria, yeast, and viruses. The origin of replication from the
plasmid pBR322 is suitable for most Gram-negative bacteria, the
2.mu. plasmid origin is suitable for yeast, and various viral
origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for
clonfing vectors in mammalian cells.
[1340] 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.
[1341] 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 tryptoplaan, for example, ATCC No. 44076 or PEP1 [Jones,
Genetics, 85:12 (1977)].
[1342] Expression and cloning vectors usually contain a promoter
operably linked to the PRO-encoding nucleic acid sequence to direct
MnRNA 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.
[1343] Examples of suitable promoting sequences for use with yeast
hosts include the promoters for 3-phosphoglycerate kinase [Hitzeman
et al., J. Biol. Chem., 255:2073 (1980)] or other glycolytic
enzymes [Hess et al., J. Adv. Enzyme Reg., 7:149 (1968); Holland,
Biochemistry, 17:4900 (1978)], such as enolase,
glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate
decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate
isomerase, phosphoglucose isomerase, and glucokinase.
[1344] 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.
[1345] 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
retrovirms, 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.
[1346] 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.
[1347] 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 transcnred as polyadenylated
fragments in the untranslated portion of the mRNA encoding PRO.
[1348] 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, 281:4046 (1979); EP 117,060; and EP
117,058.
[1349] 4. Detectine Gene Amplification/Expression
[1350] 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 rmIomas,
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.
[1351] Gene expression, alternatively, may be measured by
immunological methods, such as innnunohistochemical 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.
[1352] 5. Purification of Polypeptide
[1353] 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.
[1354] 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.
[1355] E. Uses for PRO
[1356] 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.
[1357] 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 natually-occurrig 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 .sup.35S, or
enzymatic labels such as alkaline phosphatase coupled to the probe
via avidin/biotin coupling systems. Labeled probes having a
sequence complementary to that of the 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.
[1358] Any EST sequences disclosed in the present application may
similarly be employed as probes, using the methods disclosed
herein.
[1359] 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).
[1360] 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.
[1361] 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.
[1362] 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, DCTSB and DCTSC (see WO 90/13641).
[1363] 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
91104753. 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.
[1364] Alternatively, a sense or an antisense oligonucleotide may
be introduced into a cell contaimng the target nucleic acid
sequence by formation of an oligonucleotide-lipid complex, as
described in WO 90110448. The sense or antisense
oligonucleotide-lipid complex is preferably dissociated witiithe
cell by an endogenous lipase.
[1365] Antisense or sense RNA or DNA molecules are generally at
least about 5 bases in length, about 10 bases in length, about 15
bases in length, about 20 bases in length, about 25 bases in
length, about 30 bases in length, about 35 bases in length, about
40 bases in length, about 45 bases in length, about 50 bases in
length, about 55 bases in length, about 60 bases in length, about
65 bases in length, about 70 bases in length, about 75 bases in
length, about 80 bases in length, about 85 bases in length, about
90 bases in length, about 95 bases in length, about 100 bases in
length, or more.
[1366] The probes may also be employed in PCR techniques to
generate a pool of sequences for identification of closely related
PRO coding sequences.
[1367] 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.
[1368] 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.
[1369] Nucleic acids which encode PRO or its modified forms can
also be used to generate either transgenic animals or "knock out"
animals which, in turn, are useful in the development and screening
of therapeutically useful reagents. A transgenic animal (e.g., a
mouse or rat) is an animal having cells that contain a transgene,
which transgene was introduced into the animal or an ancestor of
the animal at a prenatal, e.g., an embryonic stage. A transgene is
a DNA which is integrated into the genome of a cell from which a
transgenic animal develops. In one embodiment, cDNA encoding PRO
can be used to clone genomic DNA encoding PRO in accordance with
established techniques and the genornic 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.
[1370] 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 animl. 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.
[1371] 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 83:4143-4146 [1986]). The
oligonucleotides can be modified to enhance their uptake, e.g. by
substituting their negatively charged phosphodiester groups by
uncharged groups.
[1372] 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, DEAEdextran, the calcium phosphate
precipitation method, etc. The currently preferred in vivo gene
transfer techniques include transfection with viral (typically
retroviral) vectors and viral coat protein-liposome mediated
transfection (Dzau et al., Trends in Biotechnology 11, 205-210
[1993]). In some situations it is desirable to provide the nucleic
acid source with an agent that targets the target cells, such as an
antibody specific for a cell surface membrane protein or the target
cell, a ligand for a receptor on the target cell, etc. Where
liposomes are employed, proteins which bind to a cell surface
membrane protein associated with endocytosis may be used for
targeting and/or to facilitate uptake, e.g. capsid proteins or
fragments thereof tropic for a particular cell type, antibodies for
proteins which undergo internalization in cycling, proteins that
target intracellular localization and enhance intracellular
half-life. The technique of receptor-mediated endocytosis is
described, for example, by Wu et al., J. Biol. Chem. 262, 4429-4432
(1987); and Wagner et al., Proc. Natl. Acad. Sci. USA 87, 3410-3414
(1990). For review of gene marking and gene therapy protocols see
Anderson et al., Science 256, 808-813 (1992).
[1373] 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.
[1374] 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.
[1375] 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.
[1376] 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 usefwl 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,
glutarine, asparagine, arginine or lysine; monosaccharides,
disaccharides and other carbohydrates including glucose, mannose,
or dextins; chelating agents such as EDTA; sugar alcohols such as
mannitol or sorbitol; salt-forming counterions such as sodium;
and/or nonionic surfactints such as TWEEN.TM., PLURONICS.TM. or
PEG.
[1377] 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.
[1378] Therapeutic compositions herein generally are placed into a
container havng a sterile access port, for example, an intravenous
solution bag or vial having a stopper pierceable by a hypodermic
injection needle.
[1379] 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.
[1380] 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.
[1381] 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.
[1382] 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 successfilly 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 Adiuvant 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.
[1383] The sustained-release formulations of these proteins were
developed using poly-lactic-coglycolic acid (PLGA) polymer due to
its biocompatibility and wide range of biodegradable properties.
The degradation products of PLGA, lactic and glycolic acids, can be
cleared quickly within the human body. Moreover, the degradability
of this polymer can be adjusted from months to years depending on
its molecular weight and composition. Lewis, "Controlled release of
bioactive agents from lactide/glycolide polymer," in: M. Chasin and
R. Langer (Eds.), Biodegradable Polymers as Drug Delivery Systems
(Marcel Dekker: New York, 1990), pp. 1-41.
[1384] 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.
[1385] 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.
[1386] 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.
[1387] 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.
[1388] 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 GALA, 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 GAL4A, and another, in which candidate activating proteins are
fused to the activation domain. The expression of a GAL1-lacZ
reporter gene under control of a GAL4activated 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.
[1389] 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.
[1390] 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.
[1391] 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.
[1392] 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.
[1393] 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.
[1394] 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 r 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.
[1395] 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.
[1396] Ribozymes are enzymatic RNA molecules capable of catalyzing
the specific cleavage of RNA. Ribozymes act by sequence-specific
hybridization to the complementary target RNA, followed by
endonucleolytic cleavage. Specific ribozyme cleavage sites within a
potential RNA target can be identified by known techniques. For
further details see, e.g., Rossi, Current Biology, 4:469-471(1994),
and PCT publication No. WO 97/33551 published Sep. 18, 1997).
[1397] 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 flrder
details see, e.g., PCT publication No. WO 97/33551, supra.
[1398] 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.
[1399] PRO213 polypeptides and portions thereof which possess the
ability to regulate the growth induction cascade and/or the blood
coagulation cascade may also be employed for such purposes both in
vivo therapy and in vitro. Those of ordinary skill in the art will
well know how to employ PRO213 polypeptides for such uses.
[1400] PRO274 polypeptides and portions thereof which have homology
to 7TM protein and Fn54 may also be useful for in vivo therapeutic
purposes, as well as for various other applications. The
identification of novel 7TM protein and Fn54-like molecules may
have relevance to a number of human disorders which involve
recognition of ligands and the subsequent signal transduction of
information contained within those ligands in order to control
cellular processes. Thus, the identification of new 7TM protein and
Fn54like 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
PRO274.
[1401] PRO300 polypeptides and portions thereof which have homology
to Diff 33 may also be useful for in vivo therapeutic purposes, as
well as for various other applications. The identification of novel
Diff 33-ike molecules may have relevance to a number of human
disorders such as the physiology of cancer. Thus, the
identification of new Diff 33-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 PRO300.
[1402] PRO296 polypeptides of the present invention which possess
biological activity related to that of the sarcoma-amplified SAS
protein 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 PRO296 polypeptides of the present invention for such
purposes.
[1403] PRO329 polypeptides of the present invention which possess
biological activity related to that of immunoglobulin F.sub.c
receptor protein or subunit thereof 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 PRO329 polypeptides of the
present invention for such purposes.
[1404] PRO362 polypeptides of the present invention which possess
biological activity related to that of the A33 antigen protein,
HCAR protein or the NRCAM related cell adhesion molecule may be
employed both in vivo for therapeutic purposes and in vitro.
[1405] PRO363 polypeptides of the present invention which possess
biological activity related to that of the cell surface HCAR
protein 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 PRO363 polypeptides of the present invention for such
purposes. Specifically, extracellular domains derived from the
PRO363 polypeptides may be employed therapeutically in vivo for
lessening the effects of viral infection.
[1406] PRO868 polypeptides of the present invention which possess
biological activity related to that of the tumor necrosis factor
protein 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 PRO868 polypeptides of the present invention for such
purposes.
[1407] PRO382 polypeptides of the present invention which possess
biological activity related to that of the serine protease proteins
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
PRO382 polypeptides of the present invention for such purposes.
[1408] PRO545 polypeptides and portions thereof which have homology
to meltrin may also be useful for in vivo therapeutic purposes, as
well as for various other applications. The identification of novel
molecules associated with cellular adhesion may be relevant to a
number of human disorders. Given that the meltrin proteins may play
an important role in a number of disease processes, the
identification of new meltrin proteins and meltrin-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 PRO545.
[1409] PRO617 polypeptides of the present invention which possess
biological activity related to that of the CD24 protein 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
PRO617 polypeptides of the present invention for such purposes.
[1410] PRO700 polypeptides and portions thereof which have homology
to protein disulfide isomerase may also be useful for in vivo
therapeutic purposes, as well as for various other applications.
The identification of novel protein disulfide isomerases and
related molecules may be relevant to a number ofhuman disorders.
Given that formation of disulfide bonds and protein folding play
important roles in a number of biological processes, the
identification of new protein disulfide isomerases and protein
disulfide isomerase-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 PRO700.
[1411] PRO702 polypeptides of the present invention which possess
biological activity related to that of the conglutinin protein 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
PRO702 polypeptides of the present invention for such purposes.
PRO702 polypeptides having conglutimn activity would be expected to
be capable of inhibiting haemagglutinin activity by influenza
viruses and/or function as immunoglobulin-independent defense
molecules as a result of a complement-mediated mechanism.
[1412] PRO703 polypeptides of the present invention which possess
biological activity related to that of the VLCAS protein 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
PRO703 polypeptides of the present invention for such purposes.
[1413] PRO703 polypeptides and portions thereof which have homology
to VLCAS may also be useful for in vivo therapeutic purposes, as
well as for various other applications. The identification of novel
VLCAS proteins and related molecules may be relevant to a number of
human disorders. Thus, the identification of new VLCAS proteins and
VLCAS protein-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
PRO703.
[1414] PRO705 polypeptides of the present invention which possess
biological activity related to that of the K-glypican protein 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
PRO705 polypeptides of the present invention for such purposes.
[1415] PRO708 polypeptides of the present invention which possess
biological activity related to that of the aryl sulfatase proteins
may be employed both in iWvo for therapeutic purposes and in vitro.
Those of ordinary skill in the art will well know how to employ the
PRO708 polypeptides of the present invention for such purposes.
[1416] PRO320 polypeptides of the present invention which possess
biological activity related to that of the fibulin protein may be
employed both in vivo for therapeutic purposes and in vitro. Those
of ordinary skll in the art will well know how to employ the PRO320
polypeptides of the present invention for such purposes.
[1417] PRO320 polypeptides and portions thereof which have homology
to fibulin may also be useful for in vivo therapeutic purposes, as
well as for various other applications. The identification of novel
fibulin proteins and related molecules may be relevant to a number
of human disorders such as cancer or those involving connective
tissue, attachment molecules and related mechanisms. Thus, the
identification of new fibulin proteins and fibulin protein-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 PRO320.
[1418] PRO324 polypeptides of the present invention which possess
biological activity related to that of oxidoreductases 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
PRO324 polypeptides of the present invention for such purposes.
[1419] PRO351 polypeptides of the present invention which possess
biological activity related to that of the prostasin protein 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
PRO351 polypeptides of the present invention for such purposes.
[1420] PRO351 polypeptides and portions thereof which have homology
to prostasin may also be useful for in vivo therapeutic purposes,
as well as for various other applications. The identification of
novelprostasin proteins and related molecules may be relevant to a
number of human disorders. Thus, the identification of new
prostasin proteins and prostasin-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 PRO351.
[1421] PRO352 polypeptides of the present invention which possess
biological activity related to that of the butyrophilin protein 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
PRO352 polypeptides of the present invention for such purposes.
[1422] PRO381 polypeptides of the present invention which possess
biological activity related to that of one or more of the FKPB
immunophtlin proteins may be employed both in vivo for therapeutic
purposes and in vitro, for example for enhancing immunosuppressant
activity and/or for axonal regeneration. Those of ordinary skill in
the art will well know how to employ the PRO381 polypeptides of the
present invention for such purposes.
[1423] PRO386 polypeptides of the present invention which possess
biological activity related to that of the beta-2 subunit of a
sodium channel expressed in mammalian cells may be employed both in
vivo for therapeutic purposes and in vitro. Those of ordinary skill
in the art wilt well know how to employ the PRO386 polypeptides of
the present invention for such purposes.
[1424] PRO540 polypeptides of the present invention which possess
biological activity related to that of the LCAT protein may be
employed both in vivo for therapeutic purposes and in vitro. Those
of ordinary skidU in the art will well know how to employ the
PRO540 polypeptides of the present invention for such purposes.
[1425] PRO615 polypeptides of the present invention which possess
biological activity related to that of the synaptogyrin protein may
be employed both in viva for therapeutic purposes and in vitro.
Those of ordinary skill in the art will well know how to employ the
PRO615 polypeptides of the present invention for such purposes.
[1426] PRO615 polypeptides and portions thereof which have homology
to synaptogyrin may also be useful for in vivo therapeutic
purposes, as well as for various other applications. The
identification of novel synaptogyrin proteins and related molecules
may be relevant to a number of human disorders. Thus, the
identification of new synaptogyrin proteins and synaptogyrin-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 PRO615.
[1427] PRO618 polypeptides of the present invention which possess
biological activity related to that of an enteropeptidase 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
PRO618 polypeptides of the present invention for such purposes.
[1428] PRO618 polypeptides and portions thereof which have homology
to enteropeptidase may also be useful for in vivo therapeutic
purposes, as well as for various other applications. The
identification of novel enteropeptidase proteins and related
molecules may be relevant to a number of human disorders. Thus, the
identification of new enteropeptidase proteins and
enteropeptidase-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
PRO618.
[1429] PRO719 polypeptides of the present invention which possess
biological activity related to that of the lipoprotein lipase H
protein 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 PRO719 polypeptides of the present invention for such
purposes.
[1430] PRO724 polypeptides of the present invention which possess
biological activity related to that of the human LDL receptor
protein 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 PRO724 polypeptides of the present invention for such
purposes.
[1431] PRO772 polypeptides of the present invention which possess
biological activity related to that of the human A4 protein 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
PRO772 polypeptides of the present invention for such purposes.
[1432] PRO852 polypeptides of the present invention which possess
biological activity related to that of certain protease protein may
be employed both in vivo for therapeutic purposes and in vitro.
Those of ordiary skfl in the art will well know how to employ the
PRO852 polypeptides of the present invention for such purposes.
[1433] PRO853 polypeptides of the present invention which possess
biological activity related to that of the reductase protein 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
PRO853 polypeptides of the present invention for such purposes.
[1434] PRO853 polypeptides and portions thereof which have homology
to reductase proteins may also be useful for in vivo therapeutic
purposes, as well as for various other applications. 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 PRO853.
[1435] PRO860 polypeptides of the present invention which possess
biological activity related to that of the neurofascin protein 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
PRO860 polypeptides of the present invention for such purposes.
[1436] PRO860 polypeptides and portions thereof which have homology
to neurofascin may also be useflA for in vivo therapeutic purposes,
as well as for various other applications. The identification of
novel neurofascin proteins and related molecules may be relevant to
a number of human disorders which involve cellular adhesion. Thus,
the identification of new neurofascin proteins and neurofascin
protein-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
PRO860.
[1437] PRO846 polypeptides of the present invention which possess
biological activity related to that of the CMRF35 protein 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
PRO846 polypeptides of the present invention for such purposes.
[1438] PRO846 polypeptides and portions thereof which have homology
to the CMRF35 protein may also be useful for in vivo therapeutic
purposes, as well as for various other applications. The
identification of novel CMRF35 protein and related molecules may be
relevant to a number of human disorders. Thus, the identification
of new CMRF35 protein and CMRF35 protein-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 inbiotechnological and
medical research as well as various industrial applications. As a
result, there is particular scientific and medical interest in new
molecules, such as PRO846.
[1439] PRO862 polypeptides of the present invention which possess
biological activity related to that of the lysozyme protein 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
PRO862 polypeptides of the present invention for such purposes.
[1440] PRO862 polypeptides and portions thereof which have homology
to the lysozyme protein may also be useful for in vivo therapeutic
purposes, as well as for various other applications. The
identification of novel lysozyme proteins and related molecules may
be relevant to a number of human disorders. Thus, the
identification of new lysozymes and lysozyme-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 PRO862.
[1441] PRO864 polypeptides of the present invention which possess
biological activity related to that of the Wnt-4 protein 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
PRO864 polypeptides of the present invention for such purposes.
[1442] PRO864 polypeptides and portions thereof which have homology
to the Wnt-4 protein may also be useful for in vivo therapeutic
purposes, as well as for various other applications. The
identification of novel Wnt-4 proteins and related molecules may be
relevant to a number of human disorders. Thus, the identification
of new Wnt-4 protein and Wnt-4 protein-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 PRO864.
[1443] PRO792 polypeptides of the present invention which possess
biological activity related to that of the CD23 protein 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
PRO792 polypeptides of the present invention for such purposes.
[1444] PRO866 polypeptides of the present invention which possess
biological activity related to that of mindin and/or spondin
protein 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 PRO866 polypeptides of the present invention for such
purposes.
[1445] PRO871 polypeptides of the present invention which possess
biological activity related to that of the cyclophilin protein
family 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 PRO871 polypeptides of the present invention for such
purposes.
[1446] PRO873 polypeptides of the present invention which possess
biological activity related to that of carboxylesterases may be
employed both in vivo for therapeutic purposes and in vitro. For
example, they be used in conjunction with prodrugs to convert the
prodrug to its active form (see Danks et al.,supra). They may be
used to inhibit parasite infection (see van Pelt et al., supra).
Methods for employ the PRO873 polypeptides of the present invention
for these, and other purposes will be readily apparent to those of
ordinary skill in the art.
[1447] PRO940 polypeptides of the present invention which possess
biological activity related to that of the CD33 protein and/or OB
binding protein-2 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 PRO940 polypeptides of the present invention
for such purposes.
[1448] PRO941 polypeptides of the present invention which possess
biological activity related to that of a cadherin protein may be
employed both in vivo for therapeutic purposes and in vitro. Those
of ordinary skill in the art wil well know how to employ the PRO941
polypeptides of the present invention for such purposes.
[1449] PRO944 polypeptides of the present invention which possess
biological activity related to that of the CPE-R protein 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
PRO944 polypeptides of the present invention for such purposes.
PRO944 polypeptides of the present invention that function to bind
to Clostridium perfringens enterotoxin (CPE) may find use for
effectively treating infection by the CPE endotoxin.
[1450] PRO983 polypeptides of the present invention which possess
biological activity related to that of the vesicle-associated
membrane protein, VAP-33, may be employed both in vivo for
therapeutic purposes and in vitro. Those of ordinary skiu in the
art will wel know how to employ the PRO983 polypeptides of the
present invention for such purposes.
[1451] PRO1057 polypeptides of the present invention which possess
biological activity related to that of protease proteins may be
employed both in vivo for therapeutic purposes and in vitro. Those
of ordiary skill in the art will well know how to employ the
PRO1057 polypeptides of the present invention for such
purposes.
[1452] PRO1071 polypeptides of the present invention which possess
biological activity related to that of the thrombospondin protein
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
PRO1071 polypeptides of the present invention for such
purposes.
[1453] PRO1072 polypeptides of the present invention which possess
biological activity related to that of reductase proteins may be
employed both in vivo for therapeutic purposes and in vitro. Those
of ordinary skill in the art will wel know how to employ the
PRO1072 polypeptides of the present invention for such
purposes.
[1454] PRO1075 polypeptides of the present invention which possess
biological activity related to that of protein disulfide isomerase
may be employed both in vivo for therapeutic purposes and in vitro.
Those of ordinary skill in the art will wel know how to employ the
PRO1075 polypeptides of the present invention for such
purposes.
[1455] PRO181 polypeptides of the present invention which possess
biological activity related to that of the cornichon protein may be
employed both in vivo for therapeutic purposes and in vitro. Those
of ordinary skill in the art wil well know how to employ the PRO181
polypeptides of the present invention for such purposes.
[1456] PRO827 polypeptides of the present invention which possess
biological activity related to that of various integrin proteins
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
PRO827 polypeptides of the present invention for such purposes.
[1457] PRO1114 polypeptides of the present invention which possess
biological activity related to that of the cytokine receptor family
of proteins 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 PRO I114 polypeptides of the present invention for
such purposes.
[1458] In addition to the above, the PRO1114 interferon receptor
polypeptides may be employed in applications, both in vivo and in
vitro, where the ability to bind to an interferon ligand is
desired. Such applications will be well within the skill level in
the art.
[1459] PRO237 polypeptides of the present invention which possess
biological activity related to that of the carbonic anhydrase
protein 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 PRO237 polypeptides of the present invention for such
purposes.
[1460] PRO541 polypeptides of the present invention which possess
biological activity related to that of a trypsin inhibitor protein
may be employed both in vivo for therapeutic purposes and in vitro.
Those of ordiary skill in the art will well know how to employ the
PRO541 polypeptides of the present invention for such purposes.
[1461] PRO273 polypeptides can be used in assays that other
chemokines would be used in to perform comparative assays. The
results can be used accordingly.
[1462] PRO701 polypeptides of the present invention which possess
biological activity related to that of the neuroligin family 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
PRO701 polypeptides of the present invention for such purposes.
[1463] PRO701 can be used in assays with neurons and its activity
thereon can be compared with that of neuroligins 1, 2 and 3. The
results can be applied accordingly.
[1464] PRO704 polypeptides of the present invention which possess
biological activity related to that of vesicular integral membrane
proteins 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 PRO704 polypeptides of the present invention for such
purposes.
[1465] PRO704 can be used in assays with the polypeptides to which
they have identity with to determine the x relative activities. The
results can be applied accordingly. PRO704 can be tagged or
measured for activity to measure endocytosis activity and thereby
used to screen for agents which effect endocytosis.
[1466] PRO706 polypeptides of the present invention which possess
biological activity related to that of the endogenous prostatic
acid phosphatase precursor 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 PRO706 polypeptides of the
present invention for such purposes.
[1467] PRO706 can be used in assays with human prostatic acid
phosphatase or human lysosomal acid phosphatase and its activity
thereon can be compared with that of human prostatic acid
phosphatase or human lysosomal acid phosphatase. The results can be
applied accordingly.
[1468] PRO707 polypeptides of the present invention which possess
biological activity related to that of cadherins 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 PRO707
polypeptides of the present invention for such purposes.
[1469] PRO707 can be used in assays to determine its activity in
relation to other cadherins, particularly cadherin FIB3. The
results can be applied accordingly.
[1470] PRO322 polypeptides of the present invention which possess
biological activity related to that of neuropsin 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 PRO322
polypeptides of the present invention for such purposes.
[1471] PRO322 can be used in assays to determine its activity
relative to neuropsin, trypsinogen, serine protease and neurosin,
and the results applied accordingly.
[1472] PRO526polypeptides of the present invention which possess
biological activity related to that of protein-protein binding
proteins 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 PRO526 polypeptides of the present invention for such
purposes.
[1473] Assays can be performed with growth factors and other
proteins which are known to form complexes to determine whether
PRO526 binds thereto and whether there is increased half-life due
to such binding. The results can be used accordingly.
[1474] PRO531 polypeptides of the present invention which possess
biological activity related to that of the protocadherins may be
employed both in iWvo for therapeutic purposes and in Witro. Those
of ordinary skill in the art will well know how to employ the
PRO531 polypeptides of the present invention for such purposes.
PRO531 can be used in assays against protocadherin 3 and other
protocadherins, to determine their relative activities. The results
can be applied accordingly.
[1475] PRO534 polypeptides of the present invention which possess
biological activity related to that of the protein disulfide
isomerase 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 PRO534 polypeptides of the present invention for such
purposes.
[1476] PRO534 can be used in assays with protein disulfide
isomerase to determine the relative activities. The results can be
applied accordingly.
[1477] PRO697 polypeptides of the present invention which possess
biological activity related to that of the sFRP family 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
PRO697 polypeptides of the present invention for such purposes.
[1478] PRO697 can be used in assays with sFRPs and SARPs to
determine the relative activities. The results can be applied
accordingly.
[1479] PRO731 polypeptides of the present invention which possess
biological activity related to that of any protocadherin 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
PRO731 polypeptides of the present invention for such purposes.
[1480] PRO731 can be used in assays with the polypeptides to which
they have identity with to determine the relative activities. The
results can be applied accordingly.
[1481] PRO768 polypeptides of the present invention which possess
biological activity related to that of integrins 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 PRO768
polypeptides of the present invention for such purposes.
[1482] PRO768 can be used in assays with the polypeptides to which
they have identity with to determine the relative activities. The
results can be applied accordingly.
[1483] PRO771 polypeptides of the present invention which possess
biological activity related to that of the testican protein may be
employed both in vivo for therapeutic purposes and in vitro. Those
of ordinary skdll in the art will well know how to employ the
PRO771 polypeptides of the present invention for such purposes.
[1484] PRO771 can be used in assays with the polypeptides to which
they have identity with to determine the relative activities. The
results can be applied accordingly.
[1485] PRO733 polypeptides of the present invention which possess
biological activity related to that of the proteins which bind the
TlIST2 receptor 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 PRO733 polypeptides of the present invention
for such purposes.
[1486] PRO733 can be used in assays with the polypeptides to which
they have identity with to determine the relative activities. The
results can be applied accordingly.
[1487] PRO162 polypeptides of the present invention which possess
biological activity related to that of the pancreatitis-associated
protein 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 PRO162 polypeptides of the present invention for such
purposes.
[1488] PRO162 can be used in assays with the polypeptides to which
they have identity with to determine the relative activities. The
results can be applied accordingly.
[1489] PRO788 polypeptides of the present invention which possess
biological activity related to that of the anti-neoplastic urinary
protein 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 PRO788 polypeptides of the present invention for such
purposes.
[1490] PRO788 can be used in assays with the polypeptides to which
they have identity with to determine the relative activities. The
results can be applied accordingly.
[1491] PRO1008 polypeptides of the present inventionwhich possess
biological activity related to that of dkk-1 imay 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 PRO1008
polypeptides of the present invention for such purposes.
[1492] PRO1008 can be used in assays with the polypeptides to which
they have identity with to determine the relative activities. The
results can be applied accordingly.
[1493] PRO1012 polypeptides of the present invention which possess
biological activity related to that of the protein disulfide
isomerase may be employed both in vivo and in vitro purposes. Those
of ordinary skill in the art will well know how to employ the
PRO1012 polypeptides of the present invention for such
purposes.
[1494] PRO1012 can be used in assays with the polypeptides to which
they have identity with to determine the relative activities. The
results can be applied accordingly.
[1495] PRO1014 polypeptides of the present invention which possess
biological activity related to that of reductase 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 PRO1014
polypeptides of the present invention for such purposes.
[1496] PRO1014 can be used in assays with the polypeptides to which
they have identity with to determine the relative activities.
Inhibitors of PRO1014 are particularly preferred. The results can
be applied accordingly.
[1497] PRO1017 polypeptides of the present invention which possess
biological activity related to that of sulfotransferase 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
PRO1017 polypeptides of the present invention for such
purposes.
[1498] PRO1017 can be used in assays with the polypeptides to which
they have identity with to determine the relative activities. The
results can be applied accordingly.
[1499] PRO474 polypeptides of the present invention which possess
biological activity related to that of dehydrogenase 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
PRO474 polypeptides of the present invention for such purposes.
[1500] PRO474 can be used in assays with the polypeptides to which
they have identity with to determine the relative activities. The
results can be applied accordingly.
[1501] PRO1031 polypeptides of the present invention which possess
biological activity related to that of IL,17 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 PRO1031
polypeptides of the present invention for such purposes.
[1502] PRO1031 can be used in assays with the polypeptides to which
they have identity with to determine the relative activities. The
results can be applied accordingly.
[1503] PRO938 polypeptides of the present invention which possess
biological activity related to that of protein disulfide isomerase
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
PRO938 polypeptides of the present invention for such purposes.
[1504] PRO1082 polypeptides of the present invention which possess
biological activity related to that of the LDL receptor may be
employed both in vivo for therapeutic purposes and in vitro. Those
of ordinary skiff in the art will well know how to employ the
PRO1082 polypeptides of the present invention for such
purposes.
[1505] PRO1082 can be used in assays with the polypeptides to which
they have identity with to determine the relative activities. The
results can be applied accordingly. PRO1082 can also be used in
assays to identify candidate agents which modulate the
receptors.
[1506] PRO1083 polypeptides of the present invention which possess
biological activity related to that of 7TM receptors 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
PRO1083 polypeptides of the present invention for such
purposes.
[1507] In particular PRO1083 can be used in assays to determine
candidate agents which control or modulate PRO1083, i.e., have an
effect on the receptor.
[1508] The VEGF-E molecules herein have a number of therapeutic
uses associated with survival, proliferation and/or differention of
cells. Such uses include the treatment of umbilical vein
endothelial cells, in view of the demonstrated ability of VEGF-E to
increase survival of human umbilical vein endothelial cells.
Treatment may be needed if the vein were subjected to traumata, or
situations wherein artificial means are employed to enhance the
survival of the umbilical vein, for example, where it is weak,
diseased, based on an artificial matrix, or in an artificial
environment. Other physiological conditions that could be improved
based on the selective mitogenic character of VEGF-E are also
included herein. Uses also include the treatment of fibroblasts and
myocytes, in view of the demonstrated ability of VEGF-E to induce
proliferation of fibroblasts and hypertrophy in myocytes. In
particular, VEGF-E can be used in wound healing, tissue growth and
muscle generation and regeneration.
[1509] For the indications referred to above, the VEGF-E molecule
will be formulated and dosed in a fashion consistent with good
medical practice taking into account the specific disorder to be
treated, the condition of the individual patient, the site of
delivery of the VEGF-E, the method of administration, and other
factors known to practitioners. Thus, for purposes herein, the
"therapeutically effective amount" of the VEGF-E is an amount that
is effective either to prevent, lessen the worsening of, alleviate,
or cure the treated condition, in particular that amount which is
sufficient to enhance the survival, proliferation and/or
differentiation of the treated cells in vivo.
[1510] VEGF-E amino acid variant sequences and derivatives that are
immunologically crossreactive with antibodies raised against native
VEGF are useful in immunoassays for VEGF-E as standards, or, when
labeled, as competitive reagents.
[1511] The VEGF-E is prepared for storage or administration by
mixing VEGF-E having the desired degree of purity with
physiologically acceptable carriers, excipients, or stabilizers.
Such materials are non-toxic to recipients at the dosages and
concentrations employed. If the VEGF-E is water soluble, it may be
formulated in a buffer such as phosphate or other organic acid salt
preferably at a pH of about 7 to 8. If the VEGF-E is only partially
soluble in water, it may be prepared as a microemulsion by
formulating it with a nonionic surfactant such as Tween, Pluronics,
or PEG, e.g., Tween 80, in an amount of 0.04-0.05% (w/v), to
increase its solubility.
[1512] Optionally other ingredients may be added such as
antioxidants, e.g., ascorbic acid; low molecular weight (less than
about ten residues) polypeptides, e.g., polyarginine or
tripeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids, such as glycine, glutamic acid, aspartic acid, or
arginine; monosaccharides, disaccharides, and other carbohydrates
including cellulose or its derivatives, glucose, mannose, or
dextrins; chelating agents such as EDTA; and sugar alcohols such as
mannitol or sorbitol.
[1513] The VEGF-E to be used for therapeutic administration must be
sterile. Sterility is readily accomplished by filtration through
sterile filtration membranes (e.g., 0.2 micron membranes). The
VEGF-E ordinarily will be stored in lyophilized form or as an
aqueous solution if it is highly stable to thermal and oxidative
denaturation. The pH of the VEGF-E preparations typically will be
about from 6 to 8, although higher or lower pH values may also be
appropriate in certain instances. It will be understood that use of
certain of the foregoing excipients, carriers, or stabilizers will
result in the formation of salts of the VEGF-E.
[1514] If the VEGF-E is to be used parenterally, therapeutic
compositions containing the VEGF-E 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.
[1515] Generally, where the disorder permits, one should formulate
and dose the VEGF-E for site-specific delivery. This is convenient
in the case of wounds and ulcers. Sustained release formulations
may also be prepared, and include the formation of microcapsular
particles and implantable articles. For preparing sustained-release
VEGF-E compositions, the VEGF-E is preferably incorporated into a
biodegradable matrix or microcapsule. A suitable material for this
purpose is a polylactide, although other polymers of
poly-(a-hydroxycarboxylic acids), such as
poly-D-(-)-3-hydroxybutyric acid (EP 133,988A), canbe used. Other
biodegradable polymers include poly(lactones), poly(acetals),
poly(orthoesters), or poly(orthocarbonates). The initial
consideration here must be that the carrier itself, or its
degradation products, is nontoxic in the target tissue and will not
further aggravate the condition. This can be determined by routine
screening in animal models of the target disorder or, if such
models are unavailable, in normal animals. Numerous scientific
publications document such animal models.
[1516] For examples of sustained release compositions, see U.S.
Pat. No. 3,773,919, EP 58,481A, U.S. Pat. No. 3,887,699, EP
158,277A, Canadian Patent No. 1176565, U. Sidman et al.,
Biopolymers 22, 547 [1983], and R. Langer et a., Chem. Tech. 12, 98
[1982].
[1517] When applied topically, the VEGF-E is suitably combined with
other ingredients, such as carriers and/or adjuvants. There are no
lunitations on the nature of such other ingredients, except that
they must be pharmaceutically acceptable and efficacious for their
intended administration, and cannot degrade the activity of the
active ingredients of the composition. Examples of suitable
vehicles include ointments, creams, gels, or suspensions, with or
without purified collagen. The compositions also may be impregnated
into transdermal patches, plasters, and bandages, preferably in
liquid or semi-liquid form.
[1518] For obtaining a gel formulation, the VEGF-E formulated in a
liquid composition may be mixed with an effective amount of a
water-soluble polysaccharide or synthetic polymer such as
polyethylene glycol to form a gel of the proper viscosity to be
applied topically. The polysaccharide that may be used includes,
for example, cellulose derivatives such as etherified cellulose
derivatives, including alkyl celluloses, hydroxyallyl celluloses,
and allylydroxyallyl celluloses, for example, methylcellulose,
hydroxyethyl cellulose, carboxymethyl cellulose, hydroxypropyl
methylcellulose, and hydroxypropyl cellulose; starch and
fractionated starch; agar; alginic acid and alginates; gumarabic;
pullullan; agarose; carrageenan; dextrans; dextdns; fructans;
inulin; mannans; xylans; arabinans; chitosans; glycogens; glucans;
and synthetic biopolymers; as well as gums such as xanthan gum;
guar gum; locust bean gum; gum arabic; tragacanth gum; and karaya
gum; and derivatives and mixtures thereof. The preferred gelling
agent herein is one that is inert to biological systems, nontoxic,
simple to prepare, and not too runny or viscous, and will not
destabilize the VEGF-E held within it.
[1519] Preferably the polysaccharide is an etherified cellulose
derivative, more preferably one that is well defind, purified, and
listed in USP, e.g., methylcellulose and the hydroxyalkyl cellulose
derivatives, such as hydroxypropyl cellulose, hydroxyethyl
cellulose, and hydroxypropyl methylcellulose. Most preferred herein
is methylcellulose.
[1520] The polyethylene glycol useful for gelling is typically a
mixture of low and high molecular weight polyethylene glycols to
obtain the proper viscosity. For example, a mixture of a
polyethylene glycol of molecular weight 400-600 with one of
molecular weight 1500 would be effective for this purpose when
mixed in the proper ratio to obtain a paste.
[1521] The term "water soluble" as applied to the polysaccharides
and polyethylene glycols is meant to include colloidal solutions
and dispersions. In general, the solubility of the cellulose
derivatives is determined by the degree of substitution of ether
groups, and the stabilizing derivatives useful herein should have a
sufficient quantity of such ether groups per anhydroglucose unit in
the cellulose chain to render the derivatives water soluble. A
degree of ether substitution of at least 0.35 ether groups per
anhydroglucose unit is generally sufficient. Additionally, the
cellulose derivatives may be in the form of alkali metal salts, for
example, the Li, Na, K, or Cs salts.
[1522] If methylcellulose is employed in the gel, preferably it
comprises about 2-5%, more preferably about 3%, of the gel and the
VEGF is present in an amount of about 300-1000 mg per ml of
gel.
[1523] The dosage to be employed is dependent upon the factors
described above. As a general proposition, the VEGF-E is formulated
and delivered to the target site or tissue at a dosage capable of
establishing in the tissue a VEGF-E level greater than about 0.1
ng/cc up to a maximum dose that is efficacious but not unduly
toxic. This intra-tissue concentration should be maintained if
possible by continuous infusion, sustained release, topical
application, or injection at empirically determined
frequencies.
[1524] It is within the scope hereof to combine the VEGF-E therapy
with other novel or conventional therapies (e.g., growth factors
such as VEGF, aFGF, bFGF, PDGF, IGF, NGF, anabolic steroids, EGF or
TGF-a) for enhancing the activity of any of the growth factors,
including VEGF-E, in promoting cell proliferation, survival,
differentiation and repair. It is not necessary that such
cotreatment drugs be included per se in the compositions of this
invention, although this will be convenient where such drugs are
proteinaceous. Such admixtures are suitably administered in the
same manner and for the same purposes as the VEGF-E used alone. The
useful molar ratio of VEGF-E to such secondary growth factors is
typically 1:0.1-10, with about equimolar amounts being
preferred.
[1525] The compounds of the present invention can be formulated
according to known methods to prepare pharmaceutically useful
compositions, whereby the PRO polypeptide hereof is combined in
admixture with a pharmaceutically acceptable carrier vehicle.
Suitable carrier vehicles and their formulation, inclusive of other
human proteins, e.g., human serum albumin, are described, for
example, in Remington's Pharnaceutical Sciences, 16th ed., 1980,
Mack Publishing Co., edited by Oslo et al. the disclosure of which
is hereby incorporated by reference. The VEGF-E herein may be
administered parenterally to subjects suffering from cardiovascular
diseases or conditions, or by other methods that ensure its
delivery to the bloodstream in an effective form.
[1526] Compositions particularly well suited for the clinical
administration of VEGF-E hereof employed in the practice of the
present invention include, for example, sterile aqueous solutions,
or sterile hydratable powders such as lyophilized protein. It is
generally desirable to include fther in the formulation an
appropriate amount of a pharmaceutically acceptable salt, generally
in an amount sufficient to render the formulation isotonic. A pH
regulator such as arginine base, and phosphoric acid, are also
typically included in sufficient quantities to maintain an
appropriate pH, generally from 5.5 to 7.5. Moreover, for
improvement of shelf-life or stability of aqueous formulations, it
may also be desirable to include further agents such as glycerol.
In this manner, variant t-PA formulations are rendered appropriate
for parenteral administration, and, in particular, intravenous
administration.
[1527] Dosages and desired drug concentrations of pharmaceutical
compositions of the present invention may vary depending on the
particular use envisioned. For example, in the treatment of deep
vein thrombosis or peripheral vascular disease, "bolus" doses, will
typically be preferred with subsequent administrations being given
to maintain an approximately constant blood level, preferably on
the order of about 3 ,g/ml.
[1528] However, for use in connection with emergency medical care
facilities where infuision capability is generally not available
and due to the generally critical nature of the underlying disease
(e.g., embolism, infarct), it will generally be desirable to
provide somewhat larger initial doses, such as an intravenous
bolus.
[1529] For the various therapeutic indications referred to for the
compounds hereof, the VEGF-E molecules will be formulated and dosed
in a fashion consistent with good medical practice taking into
account the specific disorder to be treated, the condition of the
individual patient, the site of delivery, the method of
administration and other factors known to practitioners in the
respective art. Thus, for purposes herein, the "therapeutically
effective amount" of the VEGF-E molecules hereof is an amount that
is effective either to prevent, lessen the worsening of, alleviate,
or cure the treated condition, in particular that amount which is
sufficient to enhance the survival, proliferation or
differentiation of targeted cells in vivo. In general a dosage is
employed capable of establishing in the tissue that is the target
for the therapeutic indication being treated a level of a VEGF-E
hereof greater than about 0.1 ng/cm.sup.3 up to a maximum dose that
is efficacious but not unduly toxic. It is contemplated that
intra-tissue administration may be the choice for certain of the
therapeutic indications for the compounds hereof.
[1530] The human Toll proteins of the present invention can also be
used in assays to identify other proteins or molecules involved in
Toll-mediated signal transduction. For example, PRO285 and PRO286
are useful in identifying the as of yet unknown natural ligands of
human Tolls, or other factors that participate (directly or
indirectly) in the activation of and/or signaling through a human
Toll receptor, such as potential Toll receptor associated kinases.
In addition, 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. Screening assays can be
designed to find lead compounds that mimic the biological activity
of a native Toll polypeptide or a ligand for a native Toll
polypeptide. 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.
[1531] In vitro assays employ a mixture of components including a
Toll receptor polypeptide, which may be part of fusion product with
another peptide or polypeptide, e.g., a tag for detecting or
anchoring, etc. The assay mixtures may further comprise (for
binding assays) a natural intra- or extracellular Toll binding
target (i.e. a Toll ligand, or another molecule known to activate
and/or signal through the Toll receptor). While native binding
targets may be used, it is frequently preferred to use portion of
such native binding targets (e.g. peptides), so long as the portion
provides binding affinity and avidity to the subject Toll protein
conveniently measurable in the assay. The assay mixture also
contains a candidate pharmacological agent. Candidate agents
encompass numerous chemical classes, through typically they are
organic compounds, preferably small organic compounds, and are
obtained from a wide variety of sources, including libraries of
synthetic or natural compounds. A variety of other reagents may
also be included in the mixture, such as, salts, buffers, neutral
proteins, e.g. albumin, detergents, protease inhibitors, nuclease
inhibitors, antimicrobial agents, etc.
[1532] In in vitro binding assays, the resultant mixture is
incubated under conditions whereby, but for the presence of the
candidate molecule, the Toll protein specifically binds the
cellular binding target, portion or analog, with a reference
binding affinity. The mixture components can be added in any order
that provides for the requisite bindings and incubations may be
performed at any temperature which facilitates optimal binding.
Incubation periods are likewise selected for optimal binding but
also minimized to facilitate rapid high-throughput screening.
[1533] After incubation, the agent-biased binding between the Toll
protein and one or more binding targets is detected by any
convenient technique. For cell-free binding type assays, a
separation step is often used to separate bound from unbound
components. Separationmay be effected by precipitation (e.g. TCA
precipitation, immunoprecipitation, etc.), immobilization (e.g on a
solid substrate), etc., followed by washing by, for example,
membrane filtration (e.g. Whatman's P-18 ion exchange paper,
Polyfiltronic's hydrophobic GFC membrane, etc.), gel chromatography
(e.g. gel filtration, affinity, etc.). For Toll-dependent
transcription assays, binding is detected by a change in the
expression of a Toll-dependent reporter.
[1534] Detection may be effected in any convenient way. For
cell-free binding assays, one of the components usually comprises
or is coupled to a label. The label may provide for direct
detection as radioactivity, luminescence, optical or electron
density, etc., or indirect detection, such as, an epitope tag, an
enzyme, etc. A variety of methods may be used to detect the label
depending on the nature of the label and other assay components,
e.g. through optical or electron density, radiative emissions,
nonradiative energy transfers, etc. or indirectly detected with
antibody conjugates, etc.
[1535] Nucleic acid encoding the Toll polypeptides disclosed herein
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 MnRNA.
Antisense RNAs and DNAs can be used as therapeutic agents for
blocking the expression of certain genes in vivo. It has already
been shown that short antisense oligonucleotides can be imported
into cells where they act as inhibitors, despite their low
intracellular concentrations caused by their restricted uptake by
the cell membrane. (Zamecnik et al., Proc. Natl. Acad. Sci. USA 83,
4143-4146 [1986]). The oligonucleotides can be modified to enhance
their uptake, e.g. by substituting their negatively charged
phosphodiester groups by uncharged groups.
[1536] 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 manmmalian cells in
vitro include the use of liposomes, electroporation,
microinjection, cell fusion, DEAE-dextran, the calcium phosphate
precipitation method, etc. The currently preferred in vivo gene
transfer techniques include transfection with viral (typically
retroviral) vectors and viral coat protein-liposome mediated
transfection (Dzau et al., Trends in Biotechnology 11, 205-210
[1993]). In some situations it is desirable to provide the nucleic
acid source with an agent that targets the target cells, such as an
antibody specific for a cell surface membrane protein or the target
cell, a ligand for a receptor on the target cell, etc. Where
liposomes are employed, proteins which bind to a cell surface
membrane protein associated with endocytosis may be used for
targeting and/or to facilitate uptake, e.g. capsid proteins or
fragments thereof tropic for a particular cell type, antibodies for
proteins which undergo internalization in cycling, proteins that
target intracellular localization and enhance intracellular
half-life. The technique of receptor-mediated endocytosis is
described, for example, by Wu et al., J. Biol. Chem. 262, 4429-4432
(1987); and Wagner et al., Proc. Natl. Acad. Sci. USA 87, 3410-3414
(1990). For review of the currently known gene marking and gene
therapy protocols see Anderson et al., Science 256, 808-813
(1992).
[1537] The various uses listed in connection with the Toll proteins
herein, are also available for agonists of the native Toll
receptors, which mimic at least one biological function of a native
Toll receptor.
[1538] Neurotrimin as well as other members of the IGLON subfamily
of the immnoglobulin superfamily have been identified to have
effect upon neural patterning, differentiation, maturation and
growth. As a result, PRO337 the human neurotrmin homolog
polypeptides would be expected to have utility in diseases which
are characterized by neural disfunction. For example, motoneuron
disorders such as amyotrophic lateral sclerosis (Lou Gehrig's
disease), Bell's palsy, and various conditions involving spinal
muscular atrophy, or paralysis. NGF variant formulations of the
invention can be used to treat human neurodegenerative disorders,
such as Alzheimer's disease, Parkinson's disease, epilepsy,
multiple sclerosis, Huntington's chorea, Down's Syndrome, nerve
deafness, and Meniere's disease. Moreover PRO337 polypeptide may
also be used as a cognitive enhancer, to enhance learning
particularly in dementia or trauma, such as those associated with
the above diseases.
[1539] Further, PRO337 may be employed to treat neuropathy, and
especially peripheral neuropathy. "Peripheral neuropathy" refers to
a disorder affecting the peripheral nervous system, most often
manifested as one or a combination of motor, sensory, sensorimotor,
or autonomic neural dysfunction. The wide variety of morphologies
exhibited by peripheral neuropathies can each be attributed
uniquely to an equally wide number of causes. For example,
peripheral neuropathies can be genetically acquired, can result
from a systemic disease, or can be induced by a toxic agent.
Examples include but are not limited to diabetic peripheral
neuropathy, distal sensorimotor neuropathy, or autonomic
neuropathies such as reduced motility of the gastrointestinal tract
or atony of the urinary bladder. Examples of neuropathies
associated with systemic disease include post-polio syndrome or
AIDS-associated neuropathy; examples of hereditary neuropathies
include Charcot-Marie-Tooth disease, Refsum's disease,
Abetalipoproteinemia, Tangier disease, Krabbe's disease,
Metachromatic leukodystrophy, Fabry's disease, and Dejerine-Sottas
syndrome; and examples of neuropathies caused by a toxic agent
include those caused by treatment with a chemotherapeutic agent
such as vincristine, cisplatin, methotrexate, or
3'-azido-3'-deoxythymidine. Correspondingly, neurotrimin
antagonists would be expected to have utility in diseases
characterized by excessive neuronal activity.
[1540] Endothelin is generated from inactive intermediates, the big
endothelins, by a unique processing event catalyzed by the zinc
metaloprotease, endothelin converting enzyme (ECE). ECE was
recently cloned, and its structure was shown to be a single pass
transmembrane protein with a short intracellular N-terminal and a
long extracellular C-termnnal that contains the catalytic domain
and numerous N-glycosylation sites. ECEs cleave the endothelin
propeptide between Trp73 and Val74 producing the active peptide,
ET, which appears to fraction as a local rather than a circulating
hormone (Rubanyi, G. M. & Polokoff, M. A., Pharmachological
Reviews 46: 325-415 (1994). Thus ECE activity is a potential site
of regulation of endothelin production and a possible target for
therapeutic intervention in the endothelin system. By blocking ECE
activity, it is possible stop the production of ET-1 by inhibiting
the conversion of the relatively inactive precursor, big ET-1, to
the physiologically active form.
[1541] ECE-2 is 64% identical to bovine ECE-2 at the amino acid
level. ECE-2 is closely related to ECE-1 (63% identical, 80%
conserved), neutral endopeptidase 24.11 and the Kell blood group
protein. Bovine ECE-2 is a type II membrane-bound metalloproteinase
localized in the trans-Golgi network where it acts as an
intracellular enzyme converting endogenous big endothelin-1 into
active endothelin (Emoto, N. and Yanangisawa, M., J. Biol. Chem.
270: 15262-15268 (1995). The bovine ECE-2 mRNA expression is
highest in parts of the brain, cerebral cortex, cerebellum and
adrenal medulla. It is expressed at lower levels in mymetrium,
testes, ovary, and endothelial cells. Bovine ECE-2 and ECE-1 both
are more active on ET-1 as a substrate compared to ET-2 or ET-3,
Emoto and Yanangisawa, supra.
[1542] Human ECE-2 is 736 amino acids in length with a 31 residue
amino-terminal tail, a 23 residue transmembrane helix and a 682
carboxy-terminal domain. It is 94% identical to bovine ECE-2 and
64% identical to human ECE-1. The predicted transmembrane domain is
highly conserved between the human and bovine ECE-2 proteins and
between human ECE-1 and human ECE-2, as are the putative N-linked
glycosylation sites, Cys residues conserved in the neutral
endopeptidase 24.11 and the Kell blood group protein family and the
putative zinc binding motif. The sequence suggests, that like other
members of the NEP-ECE-Kell family, human ECE-2 encodes a type II
transmembrane zinc-binding metalloproteinase, which, by
extrapolation from what is known about bovine ECE-2, is an
intracellular enzyme located within the secretory pathway which
processes endogenously produced big ET-1 while it is still in the
secretory vesicles. Emoto and Yanangisawa, supra.
[1543] The expression pattern of ECE-2 differs from that observed
for ECE-1. Northern blot analysis of mRNA levels indicated low
levels of expression of a 3.3 kb transcript in adult brain (highest
in the cerebellum, putamen, medulla and temporal lobe, and lower in
the cerebral cortex, occipital lobe and frontal lobe), spinal cord,
lung and pancreas and higher levels of a 4.5 kb transcript in fetal
brain and kidney. The two transcript sizes probably represent the
use of alternative polyadenylation sites as has been observed for
bovine ECE-2 (Emoto and Yanangisawa, supra) and ECE-1 (Xu et al.,
Cell 78: 473-485 (1994). PCR on cDNA libraries indicated low levels
of expression in fetal brain, fetal kidney, fetal small intestine
and adult testis. Fetal liver, fetal lung and adult pancreas were
all negative.
[1544] The endothelin (ET) family of peptides have potent vascular,
cardiac and renal actions which may be of pathophysiological
imnportanw in many human disease states. ET-1 is expressed as an
inactive 212 amino acid prepropeptide. The prepropeptide is first
cleaved at Arg52-Cys53 and Arg92-Ala93 and then the carboxy
terminal Lys91 and Arg92 are trimmed from the protein to generate
the propeptide big ET-1. ECEs then cleave the propeptide between
Trp73 and Val74, producing the active peptide, ET, which appears to
function as a local rather than a circulating hormone (Rubanyi and
Polokoff, Pharma. R. 46: 325-415 (1994).
[1545] Endothelins may play roles in the pathophysiology of a
number of disease states including: 1) cardiovascular diseases
(vasospasm, hypertension, myocardial ischemia; reperfusion injury
and acute myochardial infarction, stroke (cerebral ischemia),
congestive heart failure, shock, atherosclerosis, vascular
thickening); 2) kidney disease (acute and chronic renal failure,
glomerulonephritis, cirrhosis); 3) lung disease (bronchial asthma,
pulmonary hypertension);4) gastrointestinal disorders (gastric
ulcer, inflammatory bowel diseases); 5) reproductive disorders
(premature labor, dysmenorhea, preeclampsia) and 6) carcinogenesis.
Rubanyi & Polokoff, supra.
[1546] Diseases can be evaluated for the impact of ET upon them by
examining: 1) increased production of ETs; 2) increased reactivity
to ETs; and/or 3) efficacy of an ET receptor antagonist, antibody
or ECE inhibitor. Response to the previous criteria suggest that
ETs likely play roles in cerebral vasospasm following subarachnoid
hemorrhage, hypertension (fiiminantcomplications), acute renal
failure and congestive heart failure. While inlubitors of ET
production or activity have not been used in models of coronary
vasospasm, acute myocardial infarction, and atherosclerosis, they
do have elevated ET levels and increase reactivity to ETs. Shock
and pulmonary hypertension also exhibit elevated ET levels (Rubanyi
and Polokoff, supra). lnhibition of ECEs in these conditions may be
of therapeutic value.
[1547] The expression pattern of ECE-2 differs from that observed
for ECE-1. ECE-2 was observed at low levels in the adult brain,
lung and pancreas and higher levels in fetal brain and kidney by
Northern blot analysis (FIG. 8). PCR revealed low levels of
expression in additional tissues: fetal lung, fetal small intestine
and adult testis. Fetal liver was negative. A similar pattern was
reported for bovine ECE-2 (Emoto and Yanangisawa, supra). It is
expressed in brain tissues (cerebral cortex, cerebellum and adrenal
medulla), myometrium and testis, and in low levels in ovary and
very low levels in many other tissues. Bovine ECE-1 (Xu et al,
supra) is more widely and more abundantly expressed. It is observed
in vascular endothelial cells of most organs and in some
parenchymal cells. With the exception for brain, bovine ECE-2 MnRNA
was present at lower levels than ECE-1. Applicants believe ECE-2 to
be a particularly good target for the therapeutic intervention for
diseases such as cerebral vasospasm following subarachnoid
hemorrhage and stroke.
[1548] Uses of the herein disclosed molecules may also be based
upon the positive functional assay hits disclosed and described
below.
[1549] F. Anti-PRO Antibodies
[1550] The present invention further provides anti-PRO antibodies.
Exemplary antibodies include polyclonal, monoclonal, humanized,
bispecific, and heteroconjugate antibodies.
[1551] 1. Polvclonal Antibodies
[1552] 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 immunuization protocol
may be selected by one skilled in the art without undue
experimentation.
[1553] 2. Monoclonal Antibodies
[1554] 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.
[1555] 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]. Ihmorttliaed 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 conains 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.
[1556] 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, Virginia. 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].
[1557] 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).
[1558] After the desired hybridoma cells are identified, the clones
may be subdloned 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.
[1559] The monoclonal antibodies secreted by the subdlones 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.
[1560] 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 can be
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.
[1561] 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.
[1562] 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.
[1563] 3. Human and Humanized Antibodies
[1564] 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, nimmunoglobulin chains or fragments thereof (such
as Fv, Fab, Fab', F(ab').sub.2 or other antigen-binding
subsequences of antibodies) which contain mhniraal 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
irumunoglobulin 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)].
[1565] 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.
[1566] 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 368 856-859 (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).
[1567] 4. Bispecific Antibodies
[1568] 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.
[1569] 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).
[1570] 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 inrrnunoglobulin 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).
[1571] 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.
[1572] Bispecific antibodies can be prepared as fuill 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 aprocedure 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 CNB) 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.
[1573] 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.
[1574] Various technique for maldng 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 heterodiners. 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 (VH) connected to a light-chain
variable domain (VL) by a linker which is too short to allow
pairing between the two domains on the same chain. Accordingly, the
VH and VL 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).
[1575] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. Tutt et al.,
J. Immunol. 147:60 (1991).
[1576] 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-cefl 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 frther binds tissue factor
[1577] 5. Heteroconjugate Antibodies
[1578] Heteroconjugate antibodies are also withi 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 crosslinlung
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 methyl4mercaptobutyrizidate and those disclosed,
for example, in U.S. Pat. No. 4,676,980.
[1579] 6. Effector Function Engineering
[1580] 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 &is 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).
[1581] 7. Immunoconjugates
[1582] The invention also pertains to itmunoconjugates 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).
[1583] Chemotherapeutic agents useful in the generation of such
immunoconjugates have been described above. Enyyatically 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 amenicana 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.
[1584] Conjugates of the antibody and cytotoxic agent are made
using a variety of bifictional proteinoupling 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-14labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026.
[1585] 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).
[1586] 8. Immunoliposomes
[1587] 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.
[1588] Particularly useful liposomes can be generated by the
reverse-phase evaporation method with a lipid
compositioncomprisingphosphatidylcholine, cholesterol,
andPEG-derivatizedphosphatidylethanolarnine (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 canbe 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).
[1589] 9. Pharmaceutical Comiositions of Antibodies
[1590] 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.
[1591] 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 domaim 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.
[1592] 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.
[1593] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[1594] 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 y 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.
[1595] G. Uses for anti-PRO Antibodies
[1596] 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
irnmunoprecipitation 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).
[1597] 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.
[1598] The following examples are offered for illustrative purposes
only, and are not intended to limit the scope of the present
invention in any way.
[1599] All patent and literature references cited in the present
specification are hereby incorporated by reference in their
entirety.
EXAMPLES
[1600] 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
[1601] Extracellular Domain Homology Screening to Identify Novel
Polypeptides and cDNA Encoding Therefor
[1602] 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. LIESEQ.TM.,
Incyte Pharmaceuticals, Palo Alto, Calif.). The search was
performed using the computer program BLAST or BLAST2 (Altschul and
Gish, Methods in Enzymology 266: 460-480 (1996)) as a comparison of
the ECD protein sequences to a 6 frame translation of the EST
sequences. Those comparisons with a Blast score of 70 (or in some
cases 90) or greater that did not encode known proteins were
clustered and assembled into consensus DNA sequences with the
program "phrap" (Phil Green, University of Washington, Seattle,
Wash.).
[1603] Using this extracellular domain homology screen, consensus
DNA sequences were assembled relative to the other identified EST
sequences using phrap. In addition, the consensus DNA sequences
obtained were often (but not always) extended using repeated cycles
of BLAST and phrap to extend the consensus sequence as far as
possible using the sources of EST sequences discussed above.
[1604] Based upon the consensus sequences obtained as described
above, oligonucleotides were ten 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 1-1.5 kbp. In order to
screen several libraries for a full-length clone, DNA from the
libraries was screened by PCR amplification, as per Ausubel et al.,
Current Protocols in Molecular Biology, with the PCR primer pair. A
positive library was then used to isolate clones encoding the gene
of interest using the probe oligonucleotide and one of the primer
pairs.
[1605] The cDNA libraries used to isolate the cDNA clones were
constructed by standard methods using commercially available
reagents such as those from Invitrogen, San Diego, Calif. The cDNA
was primed with oligo dT containing a NotI site, linked with blunt
to SalI hemikinased adaptors, cleaved with NotI, sized
appropriately by gel electrophoresis, and cloned in a defined
orientation into a suitable cloning vector (such as pRKB or pRKD;
pRK5B is a precursor of pRK5D that does not contain the Sf11 site;
see, Holmes et al., Science, 253:1278-1280 (1991)) in the unique
XhoI and NotI sites.
Example 2
[1606] Isolation of cDNA Clones by Amylase Screening
[1607] 1. Preparation of Pligo dT Primed cDNA Library
[1608] mRNA was isolated from a human tissue of interest using
reagents and protocols from Invitrogen, San Diego, Calif. (Fast
Track 2). This RNA was used to generate an oligo dT primed cDNA
library in the vector pRK5D using reagents and protocols from Life
Technologies, Gaithersburg, Md. (Super Script Plasmid System). In
this procedure, the double stranded cDNA was sized to greater than
1000 bp and the Sall/NotI linkered cDNA was cloned into XhoVlNotI
cleaved vector. pRK5D is a cloning vector that has an sp6
transcription initiation site followed by an Sfil restriction
enzyme site preceding the XhoI/NotI cDNA cloning sites.
[1609] 2. Preparation of Random Primed cDNA Library
[1610] A secondary cDNA library was generated in order to
preferentially represent the 5' ends of the primary cDNA clones.
Sp6 RNA was generated from the primary library (described above),
and this RNA was used to generate a random primed cDNA library in
the vector pSST-AMY.0 using reagents and protocols from Life
Technologies (Super Script Plasmid System, referenced above). In
this procedure the double stranded cDNA was sized to 500-1000 bp,
Tinkered with blunt to Notd adaptors, cleaved with SflI, and cloned
into SfiVNotI cleaved vector. pSST-AMY.0 is a cloning vector that
has a yeast alcohol dehydrogenase promoter preceding the cDNA
cloning sites and the mouse amylase sequence (the mature sequence
without the secretion signal) followed by the yeast alcohol
dehydrogenase terminator, after the cloning sites. Thus, cDNAs
cloned into this vector that are fused in frame with amylase
sequence will lead to the secretion of amylase from appropriately
transfected yeast colonies.
[1611] 3. Transformation and Detection
[1612] DNA from the library described in paragraph 2 above was
chilled on ice to which was added electrocompetent DH10B bacteria
(Life Technologies, 20 ml). The bacteria and vector mixture was
then electroporated as recommended by the manufacturer.
Subsequently, SOC media (Life Technologies, 1 ml) was added and the
mixture was incubated at 37.degree. C. for 30 minutes. The
transformants were then plated onto 20 standard 150 mm LB plates
containing ampicillin and incubated for 16 hours (37.degree. C.).
Positive colonies were scraped off the plates and the DNA was
isolated from the bacterial pellet using standard protocols, e.g.
CsCl-gradient. The purified DNA was then carried on to the yeast
protocols below.
[1613] The yeast methods were divided into three categories: (1)
Transformation of yeast with the plasmid/cDNA combined vector; (2)
Detection and isolation of yeast clones secreting amylase; and (3)
PCR amplification of the insert directly from the yeast colony and
purification of the DNA for sequencing and flrther analysis.
[1614] The yeast strain used was HD56-5A (ATCC-90785). This strain
has the following genotype: MAT alpha, ura3-52, leu2-3, leu2-112,
his3-11, his3-15, MAL+, SUC+, GAL+. Preferably, yeast mutants can
be employed that have deficient post-translational pathways. Such
mutants may have translocation deficient alleles in sec71, sec72,
sec62, with truncated sec71 being most preferred. Alternatively,
antagonists (including antisense nucleotides and/or ligands) which
interfere with the normal operation of these genes, other proteins
implicated in this post translation pathway (e.g., SEC61p, SEC72p,
SEC62p, SEC63p, TDJ1p or SSA1p-4p) or the complex formation of
these proteins may also be preferably employed in combination with
the amylase-expressing yeast.
[1615] Transformation was performed based on the protocol outlined
by Gietz et al., Nucl. Acid. Res., 20:1425 (1992). Transformed
cells were then inoculated from agar into YEPD complex media broth
(100 ml) and grown overnight at 30.degree. C. The YEPD broth was
prepared as described in Kaiser et al., Methods in Yeast Genetics,
Cold Spring Harbor Press, Cold Spring Harbor, N.Y., p. 207 (1994).
The overnight culture was then diluted to about 2.times.10.sup.6
cells/ml (approx. OD.sub.600=0.1) into fresh YEPD broth (500 ml)
and regrown to 1.times.10.sup.7 cells/ml (approx.
OD.sub.600=0.4-0.5).
[1616] The cells were then harvested and prepared for
transformation by transfer into GS3 rotor bottles in a Sorval GS3
rotor at 5,000 rpm for 5 minutes, the supernatant discarded, and
then resuspended into sterile water, and centrifuged again in 50 ml
falcon tubes at 3,500 rpm in a Beckman GS-6KR centrifuge. The
supernatant was discarded and the cells were subsequently washed
with LiAc/TE (10 ml, 10 mM Tris-HCl, 1 mM EDTA pH 7.5, 100 mM
Li.sub.2OOCCH.sub.3), and resuspended into LiAc/TE (2.5 ml).
[1617] Transformnation took place by mixing the prepared cells (100
.mu.l) with freshly denatured single stranded salmon testes DNA
(Lofstrand Labs, Gaithersburg, Md.) and transforming DNA (1 .mu.g,
vol. <10 .mu.l) in microfuge tubes. The mixture was mixed
briefly by vortexing, then 40% PEG/TE (600 .mu.l, 40% polyethylene
glycol-4000, 10 mM Tris-HCl, 1 mM EDTA, 100 mM Li.sub.2OOCCH.sub.3,
pH 7.5) was added. This mixture was gently mixed and incubated at
30.degree. C. while agitating for 30 minutes. The cells were then
heat shocked at 42.degree. C. for 15 minutes, and the reaction
vessel centrifuged in a microfuge at 12,000 rpm for 5-10 seconds,
decanted and resuspended into TE (500 .mu.l, 10 mM Tris-HCl, 1 mM
EDTA pH 7.5) followed by recentrifligation. The cells were then
diluted into TE (1 ml) and aliquots (200 .mu.l) were spread onto
the selective media previously prepared in 150 mm growth plates
(VWR).
[1618] Alternatively, instead of multiple small reactions, the
transformation was performed using a single, large scale reaction,
wherein reagent amounts were scaled up accordingly.
[1619] The selective media used was a synthetic complete dextrose
agar lacking uracil (SCD-Ura) prepared as described in Kaiser et
al., Methods in Yeast Genetics, Cold Spring Harbor Press, Cold
Spring Harbor, N.Y., p. 208-210 (1994). Transformants were grown at
30.degree. C. for 2-3 days.
[1620] The detection of colonies secreting amylase was performed by
including red starch in the selective growth media. Starch was
coupled to the red dye (Reactive Red-120, Sigma) as per the
procedure described by Biely et al., Anal. Biochem., 172:176-179
(1988). The coupled starch was incorporated into the SCD-Ura agar
plates at a final concentration of 0.15% (w/v), and was buffered
with potassium phosphate to a pH of 7.0 (50-100 mM final
concentration).
[1621] The positive colonies were picked and streaked across fresh
selective media (onto 150 mm plates) in order to obtain well
isolated and identifiable single colonies. Well isolated single
colonies positive for amylase secretion were detected by direct
incorporation of red starch into buffered SCD-Ura agar. Positive
colonies were determined by their ability to break down starch
resulting in a clear halo around the positive colony visualized
directly.
[1622] 4. Isolation of DNA by PCR Amplification
[1623] When a positive colony was isolated, a portion of it was
picked by a toothpick and diluted into sterile water (30 .mu.l) in
a 96 well plate. At this time, the positive colonies were either
frozen and stored for subsequent analysis or immediately amplified.
An aliquot of cells (5 .mu.l) was used as a template for the PCR
reaction in a 25 .mu.l volume containing: 0.5 .mu.l Klentaq
(Clontech, Palo Alto, Calif.); 4.0 .mu.l 10 mM dNTP's (Perkin
Elmer-Cetus); 2.5 .mu.l Kentaq buffer (Clontech); 0.25 .mu.l
forward oligo 1; 0.25 .mu.l reverse oligo 2; 12.5 .mu.l distilled
water. The sequence of the forward oligonucleotide 1 was:
7 5'-TGTAAAACGACGGCCAGTTAAATAGACCTGCAATTATTAATCT-3' (SEQ ID NO:
324)
[1624] The sequence of reverse oligonucleotide 2 was:
8 5'-CAGGAAACAGCTATGACCACCTGCACACCTGCAAATCCATT-3' (SEQ ID NO:
325)
[1625] PCR was then performed as follows:
9 a. Denature 92.degree. C., 5 minutes b. 3 cycles of: Denature
92.degree. C., 30 seconds Anneal 59.degree. C., 30 seconds Extend
72.degree. C., 60 seconds c. 3 cycles of: Denature 92.degree. C.,
30 seconds Anneal 57.degree. C., 30 seconds Extend 72.degree. C.,
60 seconds d. 25 cycles of: Denature 92.degree. C., 30 seconds
Anneal 55.degree. C., 30 seconds Extend 72.degree. C., 60 seconds
e. Hold 4.degree. C.
[1626] The underlined regions of the oligonucleotides amnealed to
the ADH promoter region and the amylase region, respectively, and
amplified a 307 bp region from vector pSST-AMY.0 when no insert was
present. Typically, the first 18 nucleotides of the 5' end of these
oligonucleotides contained annealing sites for the sequencing
primers. Thus, the total product of the PCR reaction from an empty
vector was 343 bp. However, signal sequence-fused cDNA resulted in
considerably longer nucleotide sequences.
[1627] Following the PCR, an aliquot of the reaction (5 .mu.l) was
examined by agarose gel electrophoresis in a 1% agarose gel using a
Tris-Borate-EDTA (TBE) buffering system as described by Sambrook et
al., supra. Clones resulting in a single strong PCR product larger
than 400 bp were further analyzed by DNA sequencing after
purification with a 96 Qiaquick PCR clean-up column (Qiagen Inc.,
Chatsworth, Calif.).
Example 3
[1628] Isolation of cDNA Clones Encoding Human PRO213
[1629] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA28735. Based on
the DNA28735 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 PRO213.
[1630] A pair of PCR primers (forward and reverse) were
synthesized:
10 forward PCR primer 5'-TGGAGCAGCAATATGCCAGCC-3' (SEQ ID NO: 3)
reverse PCR primer 5'-TTTTCCACTCCTGTCGGGTTGG-3' (SEQ ID NO: 4)
[1631] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA28735 sequence which
had the following nucleotide sequence
[1632] hybridization probe
11 5'-GGTGACACTTGCCAGTCAGATGTGGATGAATGCAGTGCTAGGAGGG-3' (SEQ ID NO:
5)
[1633] 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 PRO213 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human fetal
lung tissue.
[1634] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO213 [herein designated as
UNQ187 (DNA30943-1163)] (SEQ ID NO:1) and the derived protein
sequence for PRO213.
[1635] The entire nucleotide sequence of UNQ187 (DNA30943-1163) is
shown in FIG. 1 (SEQ ID NO:1). Clone UNQ187 (DNA30943-1163)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 336-338 and ending at the
stop codon at nucleotide positions 1221-1223 (FIG. 1). The
predicted polypeptide precursor is 295 amino acids long (FIG. 2).
Clone UNQ187 (DNA30943-1163) has been deposited with ATCC.
[1636] Analysis of the amino acid sequence of the full-length
PRO213 polypeptide suggests that a portion of it possesses
significant homology to the human growth arrest-specific gene 6
protein. More specifically, an analysis of the Dayhoff database
(version 35.45 SwissProt 35) evidenced significant homology between
the PRO213 amino acid sequence and the following Dayhoff sequences,
HSMHC3W5A.sub.--6 and B48089.
Example 4
[1637] Isolation of cDNA Clones Encoding Human PRO274
[1638] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA36469. Based on
the DNA36469 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 PRO274. ESTs proprietary to
Genentech were employed in the consensus assembly. The ESTs are
shown in FIGS. 5-7 and are herein designated DNA17873, DNA36157 and
DNA28929, respectively.
[1639] Pairs of PCR primers (forward and reverse) were
synthesized:
12 forward PCR primer 1 (36469.f1) 5'-CTGATCCGGTTCTTGGTGCCCCTG-3'
(SEQ ID NO: 11) forward PCR primer 2 (36469.f2)
5'-GCTCTGTCACTCACGCTC-3' (SEQ ID NO: 12) forward PCR primer 3
(36469.f3) 5'-TCATCTCTTCCCTCTCCC-3' (SEQ ID NO: 13) forward PCR
primer 4 (36469.f4) 5'-CCTTCCGCCACGGAGTTC-3' (SEQ ID NO: 14)
reverse PCR primer 1 (36469.r1) 5'-GGCAAAGTCCACTCCGATGATGTC-3' (SEQ
ID NO: 15) reverse PCR primer 2 (36469.r2)
5'-GCCTGCTGTGGTCACAGGTCTCCG-3' (SEQ ID NO: 16)
[1640] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA36469 sequence which
had the following nucleotide sequence
[1641] hybridization probe (36469.p1)
13 5'-TCGGGGAGCAGGCCTTGAACCGGGGCATTGCTGCTGTCAAGGAGG-3' (SEQ ID NO:
17)
[1642] In order to screen several libraries for a source of a
fAll-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 PRO274 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human fetal
liver tissue (LIB229).
[1643] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO274 [herein designated as
UNQ241 (DNA39987-1184)] (SEQ ID NO:1) and the derived protein
sequence for PRO274.
[1644] The entire nucleotide sequence of UNQ241 (DNA39987-1184) is
shown in FIG. 3 (SEQ ID NO:6). Clone UNQ241 (DNA39987-1184)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 83-85 and ending at the
stop codon at nucleotide positions 1559-1561 (FIG. 3). The
predicted polypeptide precursor is 492 amino acids long (FIG. 4),
has an estimated molecular weight of about 54,241 daltons and an
estimated pl of about 8.21. Clone UNQ241 (DNA39987-1184) has been
deposited with ATCC and is assigned ATCC deposit no. 209786.
[1645] Analysis of the amino acid sequence of the full-length
PRO274 polypeptide suggests that it possesses significant homology
to the Fn54 protein. More specifically, an analysis of the Dayhoff
database (version 35.45 SwissProt 35) evidenced significant
homology between the PRO274 amino acid sequence and the following
Dayhoff sequences, MMFN54S2.sub.--1, MMFN54S1.sub.--1,
CELF48C1.sub.--8, CEF38B7.sub.--6, PRP3_RAT, INL3_PIG,
MTCY07A7.sub.--13, YNAX_KLEAE, A47234 and HME2_MOUSE.
Example 5
[1646] Isolation of cDNA Clones Encoding Human PRO300
[1647] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA35930. Based on
the DNA35930 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 PRO300.
[1648] Forward and reverse PCR pruners were synthesized:
14 forward PCR primer 1 (35930.f1) 5'-GCCGCCTCATCTTCACGTTCTTCC-'
(SEQ ID NO: 20) forward PCR primer 2 (35930.f2)
5'-TCATCCAGCTGGTGCTGCTC-3' (SEQ ID NO: 21) forward PCR primer 3
(35930.f3) 5'-CTTCTTCCACTTCTGCCTGG-3' (SEQ ID NO: 22) forward PCR
primer 4 (35930.f4) 5'-CCTGGGCAAAAATGCAAC-3' (SEQ ID NO: 23)
reverse PCR primer 1 (35930.r1) 5'-CAGGAATGTAGAAGGCACCCACGG-3' (SEQ
ID NO: 24) reverse PCR primer 2 (35930.r2)
5'-TGGCACAGATCTTCACCCACACGG-3' (SEQ ID NO: 25)
[1649] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA35930 sequence which
had the following nucleotide sequence
[1650] hybridization probe (35930.p1)
15 5'-TGTCCATCATTATGCTGAGCCCGGGCGTGGAGAGTCAGCTCTACAAGCTG-3' (SEQ ID
NO: 26)
[1651] In order to screen several libraries for a source of a
fiull-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 PRO300 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human fetal
kidney tissue.
[1652] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO300 [herein designated as
UNQ263 (DNA40625-1189)] (SEQ ID NO:18) and the derived protein
sequence for PRO300.
[1653] The entire nucleotide sequence of UNQ263 (DNA40625-1189) is
shown in FIG. 8 (SEQ ID NO:18). Clone UNQ263 (DNA40625-1189)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 45-47 and ending at the
stop codon at nucleotide positions 1416-1418 (FIG. 8). The
predicted polypeptide precursor is 457 amino acids long (FIG. 9).
Clone UNQ263 (DNA40625-1189) has been deposited with ATCC and is
assigned ATCC deposit no. 209788.
[1654] Analysis of the amino acid sequence of the full-length
PRO300 polypeptide suggests that portions of it possess significant
homology to the Diff 33 protein. More specifically, an analysis of
the Dayhoff database (version 35.45 SwissProt 35) evidenced
significant homology between the PRO300 amino acid sequence and the
following Dayhoff sequence, HSU49188.sub.--1.
Example 6
[1655] Isolation of cDNA Clones Encoding Human PRO284
[1656] Two cDNA sequences were isolated in the amylase screen
described in Example 2 and those cDNA sequences are
hereindesignated DNA12982 (see FIG. 12; human placenta-derived) and
DNA15886 (see FIG. 13; human salivary gland-derived). The DNA12982
and DNA15886 sequences were then clustered and aligned, giving rise
to a consensus nucleotide sequence herein designated DNA18832.
[1657] Based on the DNA18832 consensus sequence, oligonucleotide
probes were generated and used to screen a human placenta library
(LIB89) prepared as described in paragraph 1 of Example 2 above.
The cloning vector was pRK5B (pRK5B is a precursor of pRKSD that
does not contain the Sfil site; see, Holmes et al., Science,
253:1278-1280 (1991)), and the cDNA size cut was less than 2800
bp.
[1658] PCR primers (forward and reverse) were synthesized:
16 forward PCR primer 1 (18832.est.f) 5'-TCGTACAGTTACGCTCTCCC-3'
(SEQ ID NO: 31) forward PCR primer 2 (18832.f)
5'-CTTGAGGAGCGTCAGAAGCG-3' (SEQ ID NO: 32) reverse PCR primer
(18832.r) 5'-ATAACGAATGAAGCCTCGTG-3' (SEQ ID NO: 33)
[1659] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the DNA18832 sequence which had the
following nucleotide sequence
[1660] hybridization probe (18832.p)
17 5'-GCTAATATCTGTAAGACGGCAGCTACAGCAGGCATCATTG-3' (SEQ ID NO:
34)
[1661] 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
PRO284 gene using the probe oligonucleotide and one of the PCR
primers.
[1662] A fill length clone was identified that contained a single
open reading frame with an apparent translational initiation site
at nucleotide positions 167-169 and ending at the stop codon found
at nucleotide positions 1022-1024 (FIG. 10; SEQ ID NO:27). The
predicted polypeptide precursor is 285 amino acids long, has a
calculated molecular weight of approximately 32,190 daltons and an
estimated pI of approximately 9.03. Analysis of the full-length
PRO284 sequence shown in FIG. 11 (SEQ ID NO:28) evidences the
presence of the following: a signal peptide from about amino acid 1
to about amino acid 24, transmembrane domains from about amino acid
76 to about amino acid 96 and from about amino acid 171 to about
amino acid 195 and a potential N-glycosylation site from about
amino acid 153 to about amino acid 156. Clone UNQ247
(DNA23318-1211) has been deposited with ATCC on Apr. 21, 1998 and
is assigned ATCC deposit no. 209787.
[1663] Analysis of the amino acid sequence of the full-length
PRO284 polypeptide suggests that it possesses no significant
sequence similarity to any known protein. However, an analysis of
the Dayhoff database (version 35.45 SwissProt 35) evidenced some
degree of homology between the PRO284 amino acid sequence and the
following Dayhoff sequences, JQO124, CELE04A4.sub.--5,
AB006451.sub.--1, AF030162.sub.--1, UM23_YEAST, S71194, NIA_CUCMA,
IM17_YEAST, I50479 and HUMZFHP.sub.--1.
Example 7
[1664] Isolation of cDNA Clones Encoding Human PRO296
[1665] A cDNA sequence isolated in the amylase screen as described
in Example 2 above was found, by BLAST and FastA sequence
alignment, to have sequence homology to a nucleotide sequence
encoding sarcoma-associatedprotein SAS. This cDNA sequence is
herein designated DNA23020 (see FIG. 16). The DNA23020 sequence was
then compared to a variety of expressed sequence tag (EST)
databases which included public EST databases (e.g., GenBank) and a
proprietary EST DNA database (LIFESEQ.TM., Incyte Pharmaceuticals,
Palo Alto, Calif.) to identify existing homologies. The homology
search was performed using the computer program BLAST or BLAST2
(Altshul et al., Methods in Enzymology 266:460480 (1996)). Those
comparisons resulting in a BLAST score of 70 (or in some cases 90)
or greater that did not encode known proteins were clustered and
assembled into a consensus DNA sequence with the program "phrap"
(Phil Green, University of Washington, Seattle, Wash.;
http://bozeman.mbt.washington.edu/phrap.docs/phrap.html). The
consensus sequence obtained therefrom is herein designated
DNA35858. Two proprietary Genentech ESTs were employed in the
assembly wherein those EST sequences are herein identified as
DNA21971 (FIG. 17; SEQ ID NO:38) and DNA29037 (FIG. 18; SEQ ID
NO:39).
[1666] Based on the DNA35858 consensus sequence, oligonucleotide
probes were generated and used to screen a human kidney library
(LIB228) library prepared as described in paragraph 1 of Example 2
above. The cloning vector was pRKSB (pRK5B is a precursor of pRKSD
that does not contain the Sfil site; see, Holmes et al., Science,
253:1278-1280 (1991)), and the cDNA size cut was less than 2800
bp.
[1667] PCR primers (forward and reverse) were synthesized:
18 forward PCR primer 1 (35858.f1) 5'-ACCCACGTCTGCGTTGCTGCC-3' (SEQ
ID NO:40) forward PCR nrimer 2 (35858.f2) 5'-GAGAATATGCTGGAGAGG-3'
(SEQ ID NO:41) reverse PCR primer (35858.r1)
5'-AGGAATGCACTAGGATTCGCGCGG-3' (SEQ ID NO:42)
[1668] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA35858 sequence which
had the following nucleotide sequence
[1669] hybridization probe (35858.p1)
19 5'-GGCCCCAAAGGCAAGGACAAAGCAGCTGTCAGGGAACCTCCGCCG-3' (SEQ ID
NO:43)
[1670] 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 PRO296 gene
using the probe oligonucleotide and one of the PCR priners.
[1671] A full length clone was identified that contained a single
open reading frame with an apparent translational initiation site
at nucleotide positions 174-176 and ending at the stop codon found
at nucleotide positions 786-788 (FIG. 14; SEQ ID NO:35). The
predicted polypeptide precursor is 204 amino acids long, has a
calculated molecular weight of approximately 22,147 daltons and an
estimated pI of approximately 8.37. Analysis of the full-length
PRO296 sequence shown in FIG. 15 (SEQ ID NO:36) evidences the
presence of the following: a signal peptide from about amino acid 1
to about amino acid 34 and transmembrane domains from about amino
acid 47 to about amino acid 63, from about amino acid 72 to about
amino acid 95 and from about amino acid 162 to about amino acid
182. Clone UNQ260 (DNA39979-1213) has been deposited with ATCC on
Apr. 21, 1998 and is assigned ATCC deposit no. 209789.
[1672] Analysis of the amino acid sequence of the full-length
PRO296 polypeptide suggests that it possesses significant sequence
similarity to the sarcoma-amplified SAS protein, thereby indicating
that PRO296 may be a novel SAS homolog. More specifically, an
analysis of the Dayhoff database (version 35.45 SwissProt 35)
evidenced significant homology between the PRO296 amino acid
sequence and the following Dayhoff sequences, 158391, GENI1061,
SSC2B04.sub.--1, HSU81031.sub.--2, CD63_RAT, CD63_MOUSE,
CD63_HUMAN, AF022813.sub.--1, CD63_RABIT and CO02_HUMAN.
Example 8
[1673] Isolation of cDNA Clones Encoding Human PRO329
[1674] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA35612. Based on
the DNA35612 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 PRO329.
[1675] PCR primers (forward and reverse) were synthesized:
20 forward PCR primer 1 (35612.f1) 5'-TGGGCTGTGTCCTCATGG-3' (SEQ ID
NO:46) forward PCR primer 2 (35612.f2) 5'-TTTCCAGCGCCAATTCTC-3'
(SEQ ID NO:47) reverse PCR primer 1 (35612.r1)
5'-AGTTCTTGGACTGTGATAGCCAC-3' (SEQ ID NO:48) reverse PCR primer 2
(35612.r2) 5'-AAACTTGGTTGTCCTCAGTGGCTG-3' (SEQ ID NO:49)
[1676] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA35612 sequence which
had the following nucleotide sequence
[1677] hybridization probe (35612.p1)
21 5'-GTGAGGGACCTGTCTGCACTGAGGAGAGCAGCTGCCACACGGAGG-3' (SEQ ID
NO:50)
[1678] 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
PRO329 gene using the probe oligonucleotide and one of the PCR
primers. RNA for construction of the cDNA libraries was isolated
from human fetal liver tissue (LIB6).
[1679] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO329 [herein designated as
UNQ291 (DNA40594-1233)] (SEQ ID NO:44) and the derived protein
sequence for PRO329.
[1680] The entire nucleotide sequence of UNQ291 (DNA40594-1233) is
shown in FIG. 19 (SEQ ID NO:44). Clone UNQ291 (DNA40594-1233)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 9-11 and ending at the stop
codon at nucleotide positions 1086-1088 (FIG. 19). The predicted
polypeptide precursor is 359 amino acids long (FIG. 20). The
full-length PRO329 protein shown in FIG. 20 has an estimated
molecular weight of about 38,899 daltons and apT of about 5.21.
Clone UNQ291 (DNA40594-1233) has been deposited with ATCC on Feb.
5, 1998 and is assigned ATCC deposit no. 209617.
[1681] Analysis of the amino acid sequence of the full-length
PRO329 polypeptide suggests that it possesses significant sequence
similarity to a high affinity immunoglobulin F.sub.c receptor
protein. More specifically, an analysis of the Dayhoff database
(version 35.45 SwissProt 35) evidenced significant homology between
the PRO329 amino acid sequence and the following Dayhoff sequences,
FCG1_HUMAN, FCG0_HUMAN, P_R1439, P_R22549, P_R91438, P_W00859,
P_R20811, P_R22550, HUMCD6406.sub.--1 and FCG1_MOUSE.
Example 9
[1682] Isolation of cDNA Clones Encoding Human PRO362
[1683] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA42257. Based on
the DNA42257 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 PRO362.
[1684] PCR primers (forward and reverse) were synthesized:
22 forward PCR primer 1 (42257.f1) 5'-TATCCCTCCAATTGAGCACCCTGG-3'
(SEQ ID NO:53) forward PCR primer 2 (42257.f2)
5'-GTCGGAAGACATCCCAACAAG-3' (SEQ ID NO:54) reverse PCR primer 1
(42257.r1) 5'-CTTCACAATGTCGCTGTGCTGCTC-3' (SEQ ID NO:55) reverse
PCR primer 2 (42257.r2) 5'-AGCCAAATCCAGCAGCTGGCTTAC-3' (SEQ ID
NO:56)
[1685] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA42257 sequence which
had the following nucleotide sequence
[1686] hybridization Probe (42257.p1)
23 5'-TGGATGACCGGAGCCACTACACGTGTGAAGTCACCTGGCAGACTCCTGAT-3' (SEQ ID
NO:57)
[1687] 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
PRO362 gene using the probe oligonucleotide and one of the PCR
primers. RNA for construction of the cDNA libraries was isolated
from human fetal brain tissue (LIB153).
[1688] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO362 [herein designated as
UNQ317 (DNA45416-125 1)] (SEQ ID NO:51) and the derived protein
sequence for PRO362.
[1689] The entire nucleotide sequence of UNQ317 (DNA45416-1251) is
shown in FIG. 21 (SEQ ID NO:51). Clone UNQ317 (DNA45416-1251)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 119-121 and ending at the
stop codon at nucleotide positions 1082-1084 (FIG. 21). The
predicted polypeptide precursor is 321 amino acids long (FIG. 22).
The full-length PRO362 protein shown in FIG. 2 has an estimated
molecular weight of about 35,544 daltons and a pi of about 8.51.
Analysis of the full-length PRO362 polypeptide as shown in FIG. 22
evidences the presence of a glycosaminoglycan attachment site at
about amino acid 149 to about amino acid 152 and a transmembrane
domain from about amino acid 276 to about amino acid 306. Clone
UNQ317 (DNA454161251) has been deposited with ATCC on Feb. 5, 1998
and is assigned ATCC deposit no. 209620.
[1690] Analysis of the amino acid sequence of the full-length
PRO362 polypeptide suggests that it possesses significant sequence
similarity to the A33 antigen protein and the HCAR protein. More
specifically, an analysis of the Dayhoff database (version 35.45
SwissProt 35) evidenced significant homology between the PRO362
amino acid sequence and the following Dayhoff sequences,
AB002341.sub.--1, HSU55258.sub.--1, HSC7NRCAM.sub.--1,
RNU81037.sub.--1, A33_HUMAN, P_W14158, NMNCAMRI.sub.--1,
HSTITINN2.sub.--1, S71824.sub.--1 and HSU63041.sub.--1.
Example 10
[1691] Isolation of cDNA Clones Encoding Human PRO363
[1692] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA42828. Based on
the DNA42828 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 PRO363.
[1693] A pair of PCR primers (forward and reverse) were
synthesized:
24 forward PCR primer (42828.f1) 5'-CCAGTGCACAGCAGGCAACGAAGC-3'
(SEQ ID NO:60) reverse PCR primer (42828.r1)
5'-ACTAGGCTGTATGCCTGGGTGGGC-3' (SEQ ID NO:61)
[1694] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA42828 sequence which
had the following nucleotide sequence
[1695] hybridization probe (42828.p1)
25 5'-GTATGTACAAAGCATCGGCATGGTTGCAGGAGCAGTGACAGGC-3' (SEQ ID
NO:62)
[1696] 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 PRO363 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human fetal
kidney tissue (LIB227).
[1697] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO363 [herein designated as
UNQ318 (DNA45419-1252)] (SEQ ID NO:58) and the derived protein
sequence for PRO363.
[1698] The entire nucleotide sequence of UNQ318 (DNA45419-1252) is
shown in FIG. 23 (SEQ ID NO:58). Clone UNQ318 (DNA45419-1252)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 190-192 and ending at the
stop codon at nucleotide positions 1309-1311 (FIG. 23). The
predicted polypeptide precursor is 373 amino acids long (FIG. 24).
The full-length PRO363 protein shown in FIG. 24 has an estimated
molecular weight of about 41,281 daltons and a pl of about 8.33. A
transmermbrane domain exists at amino acids 221 to 254 of the amino
acid sequence shown in FIG. 24 (SEQ a ID NO:59). The PRO363
polypeptide also possesses at least two myelin P0 protein domains
from about amino acids 15 to 56 and from about amino acids 87 to
116. Clone UNQ318 (DNA45419-1252) has been deposited with ATCC on
Feb. 5, 1998 and is assigned ATCC deposit no. 209616.
[1699] Analysis of the amino acid sequence of the full-length
PRO363 polypeptide suggests that it possesses significant sequence
similarity to the cell surface protein HCAR, thereby indicating
that PRO363 may be a novel HCAR homolog. More specifically, an
analysis of the Dayhoff database (version 35.45 SwissProt 35)
evidenced significant homology between the PRO363 amino acid
sequence and the following Dayhoff sequences, HS46KDA.sub.--1,
HSU90716.sub.--1, MMCARH.sub.--1, MMCARHOM.sub.--1,
MMU90715.sub.--1, A33_HUMAN, P_W14146, P_W14158, A42632 and
B42632.
Example 11
[1700] Isolation of cDNA Clones Encoding Human PRO868
[1701] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA38133. Based on
the DNA38133 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 PRO868.
[1702] A pair of PCR primers (forward and reverse) were
synthesized:
26 forward PCR primer (38133.f1) 5'-GTAGCAGTGCACATGGGGTGTTGG-3'
(SEQ ID NO:65) reverse PCR primer (38133.r1)
5'-ACCGCACATCCTCAGTCTGTGTCC-3' (SEQ ID NO:66)
[1703] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA38133 sequence which
had the following nucleotide sequence
[1704] hybridization probe (38133.p1)
27 5'-ACGATGATCGCGGGCTCCCTTCTCCTGCTTGGATTCCTTAGCACCACCAC-3' (SEQ ID
NO:67)
[1705] 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 PRO868 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human fetal
kidney tissue (LIB227).
[1706] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO868 [herein designated as
UNQ437 (DNA52594-1270)] (SEQ ID NO:63) and the derived protein
sequence for PRO868.
[1707] The entire nucleotide sequence of UNQ437 (DNA52594-1270) is
shown in FIG. 25 (SEQ ID NO:63). Clone UNQ437 (DNA52594-1270)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 325-327 and ending at the
stop codon at nucleotide positions 2290-2292 (FIG. 25). The
predicted polypeptide precursor is 655 amino acids long (FIG. 26).
The full-length PRO868 protein shown in FIG. 26 has an estimated
molecular weight of about 71,845 daltons and a pl of about 8.22.
Analysis of the full-length PRO868 polypeptide sequence
demonstrates the presence of conserved cysteine-containing domains
from about amino acid 66 to about amino acid 78 and from about
amino acid 123 to about amino acid 134 of the sequence shown in
FIG. 26 (SEQ ID NO:3), a TNFR death domain from about amino acid 85
to about amino acid 110, a FASA_mouse death domain block from about
amino acid 159 to about amino acid 175 and a transmembrane domain
from about amino acid 347 to about amino acid 375. Clone UNQ437
(DNA52594-1270) has been deposited with ATCC on Mar. 17, 1998 and
is assigned ATCC deposit no. 209679
[1708] Analysis of the amino acid sequence of the fiull4ength
PRO868 polypeptide suggests that it possesses significant sequence
similarity to the tumor necrosis factor receptor protein, thereby
indicating that PRO868 may be a novel member of the tumor necrosis
factor receptor family. More specifically, an analysis of the
Dayhoff database (version35.45 SwissProt 35) evidenced
significanthomology between the PRO868 amino acid sequence and the
following Dayhoff sequences, RNU94330.sub.--1, P_R99933, P_R99945,
P_R99950, HSU94332.sub.--1, CD40_HUMAN, S63368.sub.--1, TNR2_HUMAN,
MVU87844.sub.--1 AND CVU87837.sub.--1.
Example 12
[1709] Isolation of cDNA Clones Encodinz Human PRO382
[1710] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA30892. Based on
the DNA30892 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 PRO382.
[1711] A pair of PCR primers (forward and reverse) were
synthesized:
28 forward PCR primer 5'-TGACATCGCCCTTATGAAGCTGGC-3' (SEQ ID NO:
70) reverse PCR primer 5'-TACACGTCCCTGTGGTTGCA- GATC-3' (SEQ ID NO:
71)
[1712] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA30892 sequence which
had the following nucleotide sequence
[1713] hybridization probe
29 5'-CGTTCAATGCAGAAATGATCCAGCCTGTGTG (SEQ ID NO: 72)
CCTGCCCAACTCTGAAGAG-3'
[1714] 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 PRO382 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from humnan fetal
kidney tissue (LIB227).
[1715] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO382 [herein designated as
UNQ323 (DNA45234-1277)] (SEQ ID NO:68) and the derived protein
sequence for PRO382.
[1716] The entire nucleotide sequence of UNQ323 (DNA45234-1277) is
shown in FIG. 27 (SEQ ID NO:68). Clone UNQ323 (DNA45234-1277)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 126-128 and ending at the
stop codon at mncleotide positions 1485-1487 (FIG. 27). The
predicted polypeptide precursor is 453 amino acids long (FIG. 28).
The fiui-length PRO382 protein shown in FIG. 28 has an estimated
molecular weight of about 49,334 daltons and a pI of about 6.32.
Analysis of the native PRO382 amino acid sequence shown in FIG. 28
(SEQ ID NO:69) indicates the presence of a putative transmembrane
domain from about amino acid 240 to about amino acid 284, a
putative signal peptide at about amino acid 1 to about amino acid
20, a putative apple domain at about amino acid 386 to about amino
acid 419, a putative Kringle domain at about amino acid 394 to
about amino acid 406 and a histidine-containing protease active
site at about amino acid 253 to about amino acid 258. Clone UNQ323
(DNA45234-1277) has been deposited with ATCC on Mar. 5, 1998 and is
assigned ATCC deposit no. 209654.
[1717] Analysis of the amino acid sequence of the flll-length
PRO382 polypeptide suggests that it possess significant homology to
serine protease proteins, thereby indicating that PRO382 may be a
novel serine protease. Specifically, an analysis of the Dayhoff
database (version 35.45 SwissProt 35) evidenced significant
homology between the PRO382 amino acid sequence and the following
Dayhoff sequences, HSU75329.sub.--1, ENTK_MOUSE, HEPS_HUMAN,
AF030065.sub.--1, HEPS_RAT, PLMN_PIG, P_R89430, P_R89435,
PLMN_HORSE, PLMN_BOVIN and P_R83959.
Example 13
[1718] Isolation of cDNA Clones Encodine Human PRO545
[1719] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA44706. An EST
proprietary to Genentech was employed in the consensus assembly and
is herein designated DNA13217 (FIG. 31; SEQ ID NO:75). Based on the
DNA44706 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 PRO545.
[1720] Forward and reverse PCR primers were synthesized:
30 forward PCR primer 1 5'-GTCTCAGCACGTGTTCTGGTCTCAGGG-3' (SEQ ID
NO: 76) forward PCR primer 2 5'-CATGAGCATGTGCACGGC-3' (SEQ ID NO:
77) forward PCR primer 3 5'-TACCTGCACGATGGGCAC-3' (SEQ ID NO: 78)
forward PCR primer 4 5'-CACTGGGCACCTCCCTTC-3' (SEQ ID NO: 79)
reverse PCR primer 1 5'-CTCCAGGCTGGTCTCCAAGTCCTTCC-3' (SEQ ID NO:
80) reverse PCR primer 2 5'-TCCCTGTTGGACTCTGCAGCTTCC-3' (SEQ ID NO:
81) reverse PCR primer 3 5'-CTTCGCTGGGAAGAGTTTG-3' (SEQ ID NO:
82)
[1721] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA44706 sequence which
had the following nucleotide sequence
[1722] hybridization probe
31 5'-GTGCAACCAACAGATACAAACTCTTCCCAGC (SEQ ID NO: 83)
GAAGAAGCTGAAAAGCGTC-3'
[1723] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with one of the PCR primer pairs identified above. A
positive library was then used to isolate clones encoding the
PRO545 gene using the probe oligonucleotide and one of the PCR
primers. RNA for construction of the cDNA libraries was isolated
from human placenta tissue (LIB90).
[1724] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO545 [herein designated as
UNQ346 (DNA49624-1279)] (SEQ ID NO:73) and the derived protein
sequence for PRO545.
[1725] The entire nucleotide sequence of UNQ346 (DNA49624-1279) is
shown in FIG. 29 (SEQ ID NO:73). Clone UNQ346 (DNA49624-1279)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 311-313 and ending at the
stop codon at nucleotide positions 2516-2518 (FIG. 29). The
predicted polypeptide precursor is 735 amino acids long (FIG. 30).
The full-length PRO545 protein shown in FIG. 30 has an estimated
molecular weight of about 80,177 daltons and a pi of about 7.08.
Important regions of the PRO545 amino acid sequence include the
signal peptide, corresponding to amino acids 1-28, five potential
N-glycosylation sites, from about amino acid 111-114, amino acids
146-149, amino acids 348-351, amino acids 449452, and amino acids
648-651, and a neutral zinc metallopeptidase, zinc-binding region
signature sequence, from about amino acids 344-353. Clone UNQ346
(DNA496241279) has been deposited with ATCC and is assigned ATCC
deposit no. 209655.
Example 14
[1726] Isolation of cDNA Clones Encoding Human PRO617
[1727] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA42798. Based on
the DNA42798 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 PRO617.
[1728] A pair of PCR primers (forward and reverse) were
synthesized:
32 forward PCR primer 5'-ACGGGCACACTGGATCCCAAATG-3' (SEQ ID NO: 86)
reverse PCR primer 5'-GGTAGAGATGTAGAAGGGCAAGCAAG (SEQ ID NO: 87)
ACC-3'
[1729] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA42798 sequence which
had the following nucleotide sequence
[1730] hybridization probe
33 5'-GCTCCCTACCCGTGCAGGTTTCTTCATTTGT (SEQ ID NO: 88)
TCCTTTAACCAGTATGCCG-3'
[1731] 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 PRO617 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human fetal
kidney tissue (LIB227).
[1732] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO617 [herein designated as
UNQ353 (DNA48309-1280)] (SEQ ID NO:1) and the derived protein
sequence for PRO617.
[1733] The entire nucleotide sequence of UNQ353 (DNA48309-1280) is
shown in FIG. 32 (SEQ ID NO:84). Clone UNQ353 (DNA48309-1280)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 723-725 and ending at the
stop codon at nucleotide positions 924-926 (FIG. 32). The predicted
polypeptide precursor is 67 amino acids long FIG. 33). The
fiul-length PRO617 protein shown in FIG. 33 has an estimated
molecular weight of about 6,981 daltons and a pI of about 7.47.
Analysis of the PRO617 amino acid sequence also evidences the
existence of a putative signal peptide from about amino acid 15 to
about amino acid 27 and a putative protein kinase C phosphorylation
site from about amino acid 41 to about amino acid 43. Clone UNQ353
(DNA48309-1280) has been deposited on Mar. 5, 1998 with ATCC and is
assigned ATCC deposit no. 209656.
[1734] Analysis of the amino acid sequence of the full-length
PRO617 polypeptide suggests that it possesses significant homology
to the CD24 protein, thereby indicating that PRO617 may be a novel
CD24 homolog. More specifically, an analysis of the Dayhoff
database (version 35.45 SwissProt 35) evidenced significant
homology between the PRO617 amino acid sequence and the following
Dayhoff sequences, CD24_HUMAN, CD24_MOUSE, S15785, CD24_RAT, VGE
BPG4, MSE5_HUMAN, HSMHC3W36A.sub.--2, MLU15184.sub.--8, P R85075,
SEPL_HUMAN and MTCY63.sub.--13.
Example 15
[1735] Isolation of cDNA Clones Encoding Human PRO700
[1736] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA30837. Based on
the DNA30837 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 PRO700.
[1737] Forward and reverse PCR primers were synthesized:
34 forward PCR primer 1 5'-ATGTTCTTCGCGCCCTGGTG-3' (SEQ ID NO: 91)
forward PCR primer 2 5'-CCAAGCCAACACACTCTACAG-3' (SEQ ID NO: 92)
reverse PCR primer 1 5'-AAGTGGTCGCCTTGTGCAACGTGC-3' (SEQ ID NO: 93)
reverse PCR primer 2 5'-GGTCAAAGGGGATATATCGCCAC-3' (SEQ ID NO:
94)
[1738] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA30837 sequence which
had the following nucleotide sequence
[1739] hybridization probe
35 5'-GCATGGAAGATGCCAAAGTCTATGTGGCTAA (SEQ ID NO: 95)
AGTGGACTGCACGGCCCA-3'
[1740] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with one of the PCR primer pairs identified above. A
positive library was then used to isolate clones encoding the
PRO700 gene using the probe oligonucleotide and one of the PCR
primers. RNA for construction of the cDNA libraries was isolated
from human fetal kidney tissue (LIB227).
[1741] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO700 [herein designated as
UNQ364 (DNA46776-1284)] (SEQ ID NO:89) and the derived protein
sequence for PRO700.
[1742] The entire nucleotide sequence of UNQ364 (DNA46776-1284) is
shown in FIG. 34 (SEQ ID NO:89). Clone UNQ364 (DNA46776-1284)
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 1329-1331 (FIG. 34). The
predicted polypeptide precursor is 432 amino acids long (FIG. 35).
The full-length PRO700 protein shown in FIG. 35 has an estimated
molecular weight of about 47,629 daltons and a pI of about 5.90.
Important regions of the amino acid sequence of PRO700 include the
signal peptide, corresponding to amino acids from about 1 to 33,
regions homologous to disulfide isomerase, corresponding to amino
acids from about 82-99, 210-255, and 345-360, a tyrosine kinase
phosphorylation site, corresponding to amino acids from about
143-151, and an endoplasmic reticulum targeting sequence,
corresponding to amino acids from about 429-432. Clone UNQ364
(DNA46776-1284) has been deposited with ATCC and is assigned ATCC
Deposit No. 209721.
Example 16
[1743] Isolation of cDNA Clones Encoding Human PRO702
[1744] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA36623. Based on
the DNA36623 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
ftfl-length coding sequence for PRO702.
[1745] A pair of PCR primers (forward and reverse) were
synthesized:
36 forward PCR primer (36623.f1) 5'-CGCTGACTATGTTGCCAAGAGTG- G-3'
(SEQ ID NO: 98) reverse PCR primer (36623.r1)
5'-GATGATGGAGGCTCCATACCTCAG-3' (SEQ ID NO: 99)
[1746] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA36623 sequence which
had the following nucleotide sequence hybridization probe
(36623.p1)
37 5'-GTGTTCATTGGCGTGAATGACCTTGAAAGGG (SEQ ID NO: 100)
ACAGTACATGTTCAC-3'
[1747] 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 PRO702 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human fetal
liver tissue (LIB229).
[1748] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO702 [herein designated as
UNQ366 (DNA50980-1286)] (SEQ ID NO:96) and the derived protein
sequence for PRO702.
[1749] The entire nucleotide sequence of UNQ366 (DNA50980-1286) is
shown in FIG. 36 (SEQ ID NO:96). Clone UNQ366 (DNA50980-1286)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 22-24 and ending at the
stop codon at nucleotide positions 853-855 (FIG. 36). The predicted
polypeptide precursor is 277 amino acids long (FIG. 37). The
full-length PRO702 protein shown in FIG. 37 has an estimated
molecular weight of about 30,645 daltons and a pl of about 7.47.
Analysis of the fblliength native PRO702 amino acid sequence
evidences the presence of a putative signal peptide from about
amino acid 1 to about amino acid 25, potential N-glycosylation
sites from about amino acid 230 to about amino acid 233 and from
about amino acid 258 to about amino acid 261 and a C-type lectin
domain signature sequence from about amino acid 248 to about amino
acid 270. Clone UNQ366 (DNA50980-1286) has been deposited with ATCC
on Mar. 31, 1998 and is assigned ATCC deposit no. 209717.
[1750] Analysis of the amino acid sequence of the full-length
PRO702 polypeptide suggests that it possesses significant sequence
similarity to the conglutinin protein, thereby indicating that
PRO702 may be a novel conglutinin homolog. More specifically, an
analysis of the Dayhoff database (version 35.45 SwissProt 35)
evidenced significant homology between the PRO702 amino acid
sequence and the following Dayhoff sequences, S32436, P_R75642,
P_W18780, P_W18781, A53330, AC002528.sub.--1, HSPPA2IC0.sub.--1,
CA21_HUMAN, CA14_HUMAN and A61262.
Example 17
[1751] Isolation of cDNA Clones Encoding Human PRO703
[1752] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example I above, wherein the
consensus sequence obtained is herein designated DNA43047. Based on
the DNA43047 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 PRO703.
[1753] Forward and reverse PCR primers were synthesized:
38 forward PCR primer 5'-GAGAGCCATGGGGCTCCACCTG-3' (SEQ ID NO: 103)
reverse PCR primer 1 5'-GGAGAATGTGGCCACAAC-3' (SEQ ID NO: 104)
reverse PCR primer 2 5'-GCCCTGGCACAGTGACTCCATAGACG-3' (SEQ ID NO:
105) reverse PCR primer 3 5'-ATCCACTTCAGCGGACAC-3' (SEQ ID NO:
106)
[1754] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA40654 sequence which
had the following nucleotide sequence
[1755] hybridization probe
39 5'-CCAGTGCCAGGATACCTCTCTTCCCCCCAGA (SEQ ID NO: 107)
GCATAACAGACACG-3'
[1756] In order to screen several libraries for a source of a
fufll-length clone, DNA from the libraries was screened by PCR
amplification with one of the PCR primer pairs identified above. A
positive library was then used to isolate clones encoding the
PRO703 gene using the probe oligonucleotide and one of the PCR
primers. RNA for construction of the cDNA libraries was isolated
from human fetal kidney tissue (LIB227).
[1757] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO703 [herein designated as
UNQ367 (DNA50913-1287)] (SEQ ID NO:101) and the derived protein
sequence for PRO703.
[1758] The entire nucleotide sequence of UNQ367 (DNA50913-1287) is
shown in FIG. 38 (SEQ ID NO:101). Clone UNQ367 (DNA50913-1287)
contains a single open reading frame withan apparenttranslational
initiation site at nucleotide positions 115-117 and ending at the
stop codon at nucleotide positions 2305-2307 (FIG. 38). The
predicted polypeptide precursor is 730 amino acids long (FIG. 39).
The full-length PRO703 protein shown in FIG. 39 has an estimated
molecular weight of about 78,644 daltons, and a pl of about: 7.65.
Important regions of the PRO703 amino acid sequence include the
signal peptide, a cAMP- and cGMPependent protein kinase
phosphorylation site, a CUB domain protein motif, N-glycosylation
sites and a putative AMP-binding domain signature. Clone UNQ367
(DNA50913-1287) has been deposited with ATCC and is assigned ATCC
deposit no. 209716.
Example 18
[1759] Isolation of cDNA Clones Encoding Human PRO705
[1760] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA43437. Based on
the DNA43437 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 PRO705.
[1761] A pair of PCR primers (forward and reverse) were
synthesized:
40 forward PCR primer 5'-AAGCGTGACAGCGGGCACGTC-3' (SEQ ID NO: 11O)
reverse PCR primer 5'-TGCACAGTCTCTGCAGTGCCCAGG-3' (SEQ ID NO:
111)
[1762] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA43437 sequence which
had the following nucleotide sequence
[1763] hybridization Drobe (43437.p1)
41 5'-GAATGCTGGAACGGOCACAGCAAAGCCAGAT (SEQ ID NO: 112)
ACTTGCCTG-3'
[1764] 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 PRO705 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human fetal
kidney tissue (LIB227).
[1765] DNA sequencing of the clones isolated as described above
gave the filI-length DNA sequence for PRO705 [herein designated as
UNQ369 (DNA50914-1289)] (SEQ ID NO:108) and the derived protein
sequence for PRO705.
[1766] The entire nucleotide sequence of UNQ369 (DNA50914-1289) is
shown in FIG. 40 (SEQ ID NO:108). Clone UNQ369 (DNA50914-1289)
contains a single open reading frame with anapparent translational
initiation site at nucleotide positions 566-568 and ending at the
stop codon at nucleotide positions 2231-2233 (FIG. 40). The
predicted polypeptide precursor is 555 amino acids long FIG. 41).
The full-length PRO705 protein shown in FIG. 41 has an estimated
molecular weight of about 62,736 daltons and a pl of about 5.36.
Analysis of the full-length PRO705 sequence as shown in FIG. 41
evidences the presence of the following: a signal peptide from
about amino acid 1 to about amino acid 23, a eukaryotic DNA
topoisomerase I active site from about amino acid 418 to about
amino acid 436, and various regions that show homology to various
glypican proteins from about amino acid 237 to about amino acid
279, about amino acid 421 to about amino acid 458, about amino acid
53 to about amino acid 74, about amino acid 466 to about amino acid
504, about amino acid 308 to about amino acid 355, about amino acid
104 to about amino acid 156 and about amino acid 379 to about amino
acid 410. Clone UNQ369 (DNA50914-1289) has been deposited with ATCC
on Mar. 31, 1998 and is assigned ATCC deposit no. 209722.
[1767] Analysis of the amino acid sequence of the fll-length PRO705
polypeptide suggests that it possesses significant sequence
similanty to the K-glypican protein, thereby indicating that PRO705
may be a novel glypican protein family member. More specifically,
an analysis of the Dayhoff database (version 35.45 SwissProt 35)
evidenced significant homology between the PRO705 amino acid
sequence and the following Dayhoff sequences, GPCK_MOUSE,
GLYP_CHICK, GLYP_RAT, GLYP_HUMAN, GPC2_RAT, GPC5_HUMAN, GPC3_HUMAN,
GPC3_RAT, P_R30168 and CEC03H12.sub.--2.
Example 19
[1768] Isolation of cDNA Clones Encoding Human PRO708
[1769] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA34024. Based on
the DNA34024 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 PRO708.
[1770] A pair of PCR primers (forward and reverse) were
synthesized:
42 forward PCR primer 5'-CCCAACCCAACTGTTTACCTCTGG-3' (SEQ ID NO:
115) reverse PCR primer 5'-CTCTCTGAGTGTACATCTGTGTGG-3' (SEQ ID NO:
116)
[1771] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA34024 sequence which
had the following nucleotide sequence
[1772] hybridization probe
43 5'-GCCACCCTACCTCAGAAACTGAAGGAGGTTG (SEQ ID NO: 117)
GNTATTCAACGCATATGGTCGG-3'
[1773] 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 PRO708 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human bone
marrow tissue (LIB255).
[1774] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO708 [herein designated as
UNQ372 (DNA48296-1292)] (SEQ ID NO:113) and the derived protein
sequence for PRO708.
[1775] The entire nucleotide sequence of UNQ372 (DNA48296-1292) is
shown in FIGS. 42A-B (SEQ ID NO:113). Clone UNQ372 (DNA48296-1292)
contains a singleopenreading frame with anapparenttranslational
initiation site at nucleotide positions 891-893 and ending at the
stop codon at nucleotide positions 2436-2438 (FIGS. 42A-B). The
predicted polypeptide precursor is 515 amino acids long (FIG. 43).
The full-length PRO708 protein shown in FIG. 43 has an estimated
molecular weight of about 56,885 daltons and a pI of about 6.49.
Analysis of the PRO708 amino acid sequence shown in FIG. 43 (SEQ ID
NO:114) evidences the existence of a putative signal peptide at
about amino acid 1 to about amino acid 37, putative sulfatase
signature sequences at about amino acid 120 to about amino acid 132
and about amino acid 168 to about amino acid 177, a putative
tyrosine kinase phosphorylation site from about amino acid 163 to
about amino acid 169 and potential N-glycosylation sites from about
amino acid 157 to about amino acid 160, about amino acid 306 to
about amino acid 309 and about amino acid 318 to about amino acid
321. Clone UNQ372 (DNA48296-1292) has been deposited with ATCC on
Mar. 11, 1998 and is assigned ATCC deposit no. 209668.
[1776] Analysis of the amino acid sequence of the flil-length
PRO708 polypeptide suggests that it possesses significant homology
to the aryl sulfatase proteins, thereby indicating that PRO708 may
be a novel aryl sulfatase homolog. More specifically, an analysis
of the Dayhoff database (version 35.45 SwissProt 35) evidenced
significant homology between the PRO708 amino acid sequence and the
following Dayhoff sequences, ARSB_HUMAN, CELC54D2.sub.--2, G02857,
STS_HUMAN, I37186, I37187, GEN12648, CELD1014.sub.--7, GA6S_HUMAN
and SPHM_HUMAN.
Example 20
[1777] Isolation of cDNA Clones Encoding Human PRO320
[1778] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA28739. Based on
the DNA28739 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 PRO320.
[1779] A pair of PCR primers (forward and reverse) were
synthesized:
44 forward PCR primer 5'-CCTCAGTGGCCACATGCTCATG-3' (SEQ ID NO: 120)
reverse PCR primer 5'-GGCTGCACGTATGGCTATCCATAG-3' (SEQ ID NO:
121)
[1780] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA28739 sequence which
had the following nucleotide sequence
[1781] hybridization probe
45 5'-GATAAACTGTCAGTACAGCTGTGAAGACACA (SEQ ID NO: 122)
GAAGAAGGGCCACAGTGCC-3'
[1782] In order to screen several libraries for a source of a
fmll-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 PRO320 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human fetal
lung tissue (LIB25).
[1783] DNA sequencing of the clones isolated as described above
gave the fullength DNA sequence for PRO320 [herein designated as
UNQ281 (DNA32284-1307)] (SEQ ID NO:118) and the derived protein
sequence for PRO320.
[1784] The entire nucleotide sequence of UNQ281 (DNA32284-1307) is
shown in FIG. 44 (SEQ ID NO:118). Clone UNQ281 (DNA32284-1307)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 135-137 and ending at the
stop codon at nucleotide positions 1149-1151 (FIG. 44). The
predicted polypeptide precursor is 338 amino acids long (FIG. 45).
The full-length PRO320 protein shown in FIG. 45 has an estimated
molecular weight of about 37,143 daltons and a pl of about 8.92.
Important regions of the PRO320 amino acid sequence include the
signal peptide, corresponding to amino acids 1-21, an EGF-like
domain cysteine pattern signature, corresponding to amino acids
80-91, and three calcium-binding EGF-like domains, corresponding to
amino acids 103-124, 230-151 and 185-206, respectively. Clone
UNQ281 (DNA32284-1307) has been deposited with ATCC and is assigned
ATCC deposit no. 209670.
Example 21
[1785] Isolation of cDNA Clones Encoding Human PRO324
[1786] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA34347. Based on
the DNA34347 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 PRO324.
[1787] PCR primers (forward and reverse) were synthesized:
46 forward PCR primer 1 5'-GCAATGAACTGGGAGCTGC-3' (SEQ ID NO: 125)
forward PCR primer 2 5'-CTGTGAATAGCATCCTGGG-3' (SEQ ID NO: 126)
forward PCR primer 3 5'-CTTTTCAAGCCACTGGAGGG-3' (SEQ ID NO: 127)
reverse PCR primer 1 5'-CTGTAGACATCCAAGCTGGTATCC-3' (SEQ ID NO:
128) reverse PCR primer 2 5'-AAGAGTCTGCATCCACACCAC- TC-3' (SEQ ID
NO: 129)
[1788] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA34347 sequence which
had the following nucleotide sequence
[1789] hybridization probe
47 5'-ACCTGACGCTACTATGGGCCGAGTGGCAGGG (SEQ ID NO: 130)
ACGACGCCCAGAATG-3'
[1790] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with one of the PCR primer pairs identified above. A
positive library was then used to isolate clones encoding the
PRO324 gene using the probe oligonucleotide and one of the PCR
primers. RNA for construction of the cDNA libraries was isolated
from human fetal liver tissue (LIB6).
[1791] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO324 [herein designated as
UNQ285 (DNA36343-13 10)] (SEQ ID NO:123) and the derivedprotein
sequence for PRO324.
[1792] The entire nucleotide sequence of UNQ285 (DNA36343-1310) is
shown in FIG. 46 (SEQ ID NO:123). Clone UNQ285 (DNA36343-1310)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 144146 and ending at the
stop codon at nucleotide positions 1011-1013 (FIG. 46). The
predicted polypeptide precursor is 289 amino acids long (FIG. 47).
The fiul-length PRO324 protein shown in FIG. 47 has an estimated
molecular weight of about 32,268 daltons and a pi of about 9.21.
Analysis of the PRO324 polypeptide sequence shown in FIG. 47 (SEQ
ID NO:124) evidence the presence of the following: a signal peptide
from about amino acid 1 to about amino acid 31, a transmembrane
domain from about amino acid 136 to about amino acid 157, tyrosine
kinase phosphorylation sites from about amino acid 106 or about
amino acid 107 to about amino acid 113 and regions that are
homologous to short-chain alcohol dehydrogenase regions from about
amino acid 80 to about amino acid 90, from about amino acid 131 to
about amino acid 168, from about amino acid 1 to about amino acid
13 and from about amino acid 176 to about amino acid 185. Clone
UNQ285 (DNA36343-1310) has been deposited with ATCC on Mar.30, 1998
and is assigned ATCC deposit no. 209718.
[1793] Analysis of the amino acid sequence of the full-length
PRO324 polypeptide suggests that it possesses significant sequence
similarity to oxidoreductases, thereby indicating that PRO324 may
be a novel oxidoreductase homolog. More specifically, an analysis
of the Dayhoff database (version 35.45 SwissProt 35) evidenced
significant homology between the PRO324 amino acid sequence and the
following Dayhoff sequences, B61209, A69965, YQJQ_BACSU, D69930,
S76124, FABG_ECOLI, C70023, S77280, FABG_VIBHA and
MTV013.sub.--6.
Example 22
[1794] Isolation of cDNA Clones Encoding Human PRO351
[1795] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA35950. Based on
the DNA35950 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-lengh coding sequence for PRO351.
[1796] Forward and reverse PCR primers were synthesized:
48 forward PCR primer 5'-CCTGTGCTGTGCCTCGAGCCTGAC-3' (SEQ ID NO:
133) reverse PCR primer 5'-GTGGGCAGCAGTTAGCACCGCCTC-3' (SEQ ID NO:
134)
[1797] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA35950 sequence which
had the following nucleotide sequence
[1798] hybridization probe
49 5'-GGCTGGCATCATCAGCTTTGCATCAAGCTGT (SEQ ID NO: 135)
GCCCAGGAGGACGC-3'
[1799] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with one of the PCR primer pairs identified above. A
positive library was then used to isolate clones encoding the
PRO351 gene using the probe oligonucleotide and one of the PCR
primers. RNA for construction of the cDNA libraries was isolated
from human fetal liver tissue (ILB230).
[1800] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO351 [herein designated as
UNQ308 (DNA40571-1315)] (SEQ ID NO:131) and the derived protein
sequence for PRO351.
[1801] The entire nucleotide sequence of UNQ308 (DNA40571-1315) is
shown in FIG. 48 (SEQ ID NO:131). Clone UNQ308 (DNA40571-1315)
contains two open reading frames with an apparent translational
initiation site at nucleotide positions 189-191 and a second open
reading frame beginning atnucleotide 470, with the two open reading
frames ending at the stop codons at nucleotide positions 363-365
and 2009-2011, respectively (FIG. 48). The predicted polypeptide
precursor is 571 amino acids long (FIG. 49). inportant regions of
the amino acid sequence of PRO351 include the signal peptide,
regions having sequence similarity to serine proteases of the
trypsin family, two N-glycosylation sites, and three Kringle
domains. Clone UNQ308 (DNA40571-1315) has been deposited with ATCC
and is assigned ATCC deposit no. 209784.
Example 23
[1802] Isolation of cDNA Clones Encoding Human PRO352
[1803] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA36950. Based on
the DNA36950 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 PRO352.
[1804] PCR primers (forward and reverse) were synthesized:
50 forward PCR primer 1 5'-CTGGCACAGCTCAACCTCATCTGG-3' (SEQ ID NO:
138) forward PCR primer 2 5'-GCTGTCTGTCTGTCTCATTG-3' (SEQ ID NO:
139) forward PCR primer 3 5'-GGACACAGTATACTGACCAC-3' (SEQ ID NO:
140) reverse PCR primer 1 5'-TGCGAACCAGGCAGCTGTAAGTGC-3' (SEQ ID
NO: 141) reverse PCR primer 2 5'-TGGAAGAAGAGGGTGGTGATG- TGG-3' (SEQ
ID NO: 142)
[1805] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA36950 sequence which
had the following nucleotide sequence
[1806] hybridization probe
51 5'-CAGCTGACAGACACCAAACAGCTGGTGCACA (SEQ ID NO: 143)
GTTTCACCGAAGGC-3'
[1807] 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 PRO352 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human fetal
kidney tissue (LB227).
[1808] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO352 [herein designated as
UNQ309 (DNA41386-1316)] (SEQ ID NO:136) and the derived protein
sequence for PRO352.
[1809] The entire nucleotide sequence of UNQ309 (DNA41386-1316) is
shown in FIG. 50 (SEQ ID NO:136). Clone UNQ309 (DNA41386-1316)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 152-154 and ending at the
stop codon at nucleotide positions 1100-1102 (FIG. 50). The
predicted polypeptide precursor is 316 amino acids long (FIG. 51).
The full-length PRO352 protein shown in FIG. 2 has an estimated pl
of about 4.62. Analysis of the full-length PRO352 sequence
evidences the presence of a signal peptide from about amino acid 1
to about amino acid 28, a transmembrane domain from about amino
acid 251 to about amino acid 270, potential N-glycosylation sites
from about amino acid 91 to about amino acid 94, about amino acid
104 to about amino acid 107, about amino acid 189 to about amino
acid 192 and about amino acid 215 to about amino acid 218 and a
region having homology to immunoglobulins and MHC from about amino
acid 217 to about amino acid 234. Clone UNQ309 (DNA41386 1316) has
been deposited with ATCC on Mar. 26, 1998 and is assigned ATCC
deposit no. 209703.
[1810] Analysis of the amino acid sequence of the full-length
PRO352 polypeptide suggests that it possesses significant sequence
similarity to the butyrophilin protein, thereby indicating that
PRO352 is a novel butyrophilin homolog. More specifically, an
analysis of the Dayhoff database (version 35.45 SwissProt 35)
evidenced significant homology between the PRO352 amino acid
sequence and the following Dayhoff sequences, BUTY_HUMAN,
HSB73.sub.--1, GGCD80.sub.--1, I46690, A33_HUMAN, P_R67988,
CD86_MOUSE, P_R71360, B39371andD50558.sub.--1.
Example 24
[1811] Isolation of cDNA Clones Encoding Human PRO381
[1812] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA39651. Based on
the DNA39651 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 PRO381.
[1813] A pair of PCR primers (forward and reverse) were
synthesized:
52 forward PCR primer 5'-CTTTCCTTGCTTCAGCAACATGAGGC-3' (SEQ ID NO:
146) reverse PCR primer 5'-GCCCAGAGCAGGAGGAATGATGAGC-3' (SEQ ID NO:
147)
[1814] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA39651 sequence which
had the following nucleotide sequence
[1815] hybridization probe
53 5'-GTGGAACGCGGTCTTGACTCTGTTCGTCACT (SEQ ID NO: 148)
TCTTTGATTGGGGCTTTG-3'
[1816] 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 PRO381 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human fetal
kidney tissue (LEB227).
[1817] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO381 [herein designated as
UNQ322 (DNA44194-1317)] (SEQ ID NO:144) and the derived protein
sequence for PRO381.
[1818] The (entire nucleotide sequence of UNQ322 (DNA441941317) is
shown in FIG. 52 (SEQ ID NO:144). Clone UNQ322 (DNA44194-1317)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 174-176 and ending at the
stop codon at nucleotide positions 807-809 (FIG. 52). The predicted
polypeptide precursor is 211 amino acids long (FIG. 53). The
full-length PRO381 protein shown in FIG. 53 has an estimated
molecular weight of about 24,172 daltons and a pI of about 5.99.
Analysis of the full-length PRO381 polypeptide shown in FIG. 53
(SEQ ID NO:145) evidences the presence of the following: a signal
peptide from about amino acid 1 to about amino acid 20, a potential
N-glycosylation site from about amino acid 176 to about amino acid
179, potential casein kinase II phosphorylation sites from about
amino acid 143 to about amino acid 146, from about amino acid 156
to about amino acid 159, from about amino acid 178 to about amino
acid 181, and from about amino acid 200 to about amino acid 203, an
endoplasmic reticulum targeting sequence from about amino acid 208
to about amino acid 211, FKBP-type peptidyl-prolyl cis-trans
isomerase sites from about amino acid 78 to about amino acid 114
and from about amino acid 118 to about amino acid 131, EF-hand
calcium binding domains from about amino acid 191 to about amino
acid 203, from about amino acid 184 to about amino acid 203 and
from about amino acid 140 to about amino acid 159, and an
S-100/ICaBP type calcium binding domain from about amino acid 183
to about amino acid 203. Clone UNQ322 (DNA44194-1317) has been
deposited with ATCC on Apr. 28, 1998 and is assigned ATCC deposit
no. 209808.
[1819] Analysis of the amino acid sequence of the full-length
PRO381 polypeptide suggests that it possesses significant sequence
similarity to FKBP immunophin proteins, thereby indicating that
PRO381 may be a novel FKBP immunophilin homolog. More specifically,
an analysis of the Dayhoff database (version 35.45 SwissProt 35)
evidenced significant homology between the PRO381 amino acid
sequence and the following Dayhoff sequences, AF040252.sub.--1,
I49669, P_R93551, S71238, CELC05C8.sub.--1, CEU27353.sub.--1,
MIP_TRYCR, CEZC455.sub.--3, FKB4_HUMAN and I40718.
Example 25
[1820] Isolation of cDNA Clones Encoding Human PRO386
[1821] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA40674. Two
proprietary Genentech EST sequences were employed in the consensus
sequence assembly, wherein those EST sequences are herein
designated DNA23350 (FIG. 56; SEQ ID NO:151) and DNA23536 (FIG. 57;
SEQ ID NO:152). Based on the DNA40674 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
PRO386.
[1822] A pair of PCR primers (forward and reverse) were
synthesized:
54 forward PCR primer 5'-ACGGAGCATGGAGGTCCACAGTAC-3' (SEQ ID NO:
153) reverse PCR primer 5'-GCACGTTTCTCAGCATCACCGAC-3' (SEQ ID NO:
154)
[1823] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA40674 sequence which
had the following nucleotide sequence
[1824] hybridization probe
55 5'-CGCCTGCCCTGCACCTTCAACTCCTGCTACA (SEQ ID NO: 155)
CAGTGAACCACAAACAGTT-3'
[1825] In order to screen several libraries fcr a source of a
fuil-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 PRO386 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human fetal
brain tissue (LIB153).
[1826] DNA sequencing of the clones isolated as described above
gave the fuill-length DNA sequence for PRO386 [herein designated as
UNQ326 (DNA45415-1318)] (SEQ ID NO:149) and the derived protein
sequence for PRO386.
[1827] The entire nucleotide sequence of UNQ326 (DNA45415-1318) is
shown in FIG. 54 (SEQ ID NO:149). Clone UNQ326 (DNA45415-1318)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 146-148 and ending at the
stop codon at nucleotide positions 791-793 (FIG. 54). The predicted
polypeptide precursor is 215 amino acids long (FIG. 55). The
full-length PRO386 protein shown in FIG. 55 has an estimated
molecular weight of about 24,326 daltons and a pI of about 6.32.
Analysis of the full-length PRO386 sequence shown in FIG. 55 (SEQ
ID NO:150) evidences the presence of the following: a signal
peptide from about amino acid 1 to about amino acid 20, a
trausmembrane domain from about amino acid 161 to about amino acid
179, an immunoglobulin-like fold from about amino acid 83 to about
amino acid 127 and potential N-glycosylation sites from about amino
acid 42 to about amino acid 45, from about amino acid 66 to about
amino acid 69 and from about amino acid 74 to about amino acid 77.
Clone UNQ326 (DNA45415-1318) has been deposited withATCC on Apr.
28, 1998 and is assigned ATCC deposit no. 209810.
[1828] Analysis of the amino acid sequence of the full-length
PRO386 polypeptide suggests that it possesses significant sequence
similarity to the sodium chamnel beta-2 subunit, thereby indicating
that PRO386 is a novel homolog thereof. More specifically, an
analysis of the Dayhoff database (version 35.45 SwissProt 35)
evidenced significant homology between the PRO386 amino acid
sequence and the following Dayhoff sequences, A57843, MYP0_HUMAN,
GEN14531, JC4024, HS46KDA.sub.--1, HSU90716.sub.--1,
D86996.sub.--2, MUSIGLVD.sub.--1, DMU42768.sub.--1 and S19247.
Example 26
[1829] Isolation of cDNA Clones Encoding Human PRO540
[1830] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA39631. Based on
the DNA39631 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 PRO540.
[1831] Forward and reverse PCR primers were synthesized:
56 forward PCR primer 5'-CTGGGGCTACACACGGGGTGAGG-3' (SEQ ID NO:
158) reverse PCR primer 5'-GGTGCCGCTGCAGAAAGTAGAGCG-3' (SEQ ID NO:
159)
[1832] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA40654 sequence which
had the following nucleotide sequence
[1833] hybridization probe
57 5'-GCCCCAAATGAAAACGGGCCCTACTTCCTGG (SEQ ID NO: 160)
CCCTCCGCGAGATG-3'
[1834] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with one of the PCR primner pairs identified above. A
positive library was then used to isolate clones encoding the
PRO540 gene using the probe oligonucleotide and one of the PCR
priners. RNA for construction of the cDNA libraries was isolated
from human fetal kidney tissue (LIB227).
[1835] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO540 [herein designated as
UNQ341 (DNA44189-1322)] (SEQ ID NO:156) and the derived protein
sequence for PRO540.
[1836] The entire nucleotide sequence of UNQ341 (DNA44189-1322) is
shown in FIG. 58 (SEQ ID NO:156). Clone UNQ341 (DNA44189-1322)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 21-23 and ending at the
stop codon at nucleotide positions 1257-1259 (FIG. 58). The
predicted polypeptide precursor is 412 amino acids long (FIG. 59).
The full-length PRO540 protein shown in FIG. 59 has an estimated
molecular weight of about 46,658 daltons and a pl of about 6.65.
Important regions of the amino acid sequence of PRO540 include the
signal peptide, potential N-glycosylation sites, a potential lipid
substrate binding site, a sequence typical of lipases and serine
proteins, and a beta-transducin family Trp-Asp repeat. Clone UNQ341
(DNA44189-1322) has been deposited with ATCC and is assigned ATCC
deposit no. 209699.
Example 27
[1837] Isolation of cDNA Clones Encoding Human PRO615
[1838] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA42240. Based on
the DNA42240 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 PRO615.
[1839] Forward and reverse PCR primers were synthesized:
58 forward PCR primer 5'-TGGTCTTCGCCTTGATCGTGTCT-3' (SEQ ID NO:
163) forward PCR primer 5'-GTGTACTGAGCGGCGGTTAG-3' (SEQ ID NO: 164)
reverse PCR primer 5'-CTGAAGGTGATGGCTGCCCTCAC-3' (SEQ ID NO: 165)
reverse PCR primer 5'-CCAGGAGGCTCATGGGAAAGTCC-3' (SEQ ID NO:
166)
[1840] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA42240 sequence which
had the following nucleotide sequence:
[1841] hybridization probe
59 5'-CCACGAGTCTAAGCAGATGTACTGCGTGTTC (SEQ ID NO: 167)
AACCGCAACGAGGATGCCT-3'
[1842] In order to screen several libraries for a source of a
fufll-length clone, DNA from the libraries was screened by PCR
amplification with one of the PCR primer pairs identified above. A
positive library was then used to isolate clones encoding the
PRO615 gene using the probe oligonucleotide and one of the PCR
primers. RNA for construction of the cDNA libraries was isolated
from human bone marrow tissue (LIB255).
[1843] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO615 [herein designated as
UNQ352 (DNA48304-1323)] (SEQ ID NO:161) andthe derived protein
sequence for PRO615.
[1844] The entire nucleotide sequence of UNQ352 (DNA48304-1323) is
shown in FIG. 60 (SEQ ID NO:161). Clone UNQ352 (DNA48304-1323)
contains a single open reading frame withan apparent translational
initiation site at nucleotide positions 51-53 and ending at the
stop codon at nucleotide positions 723-725 (FIG. 60). The predicted
polypeptide precursor is 224 amino acids long (FIG. 61). The
full-length PRO615 protein shown in FIG. 61 has an estimated
molecular weight of about 24,810 daltons and a pI of about 4.75.
Important regions of the amino acid sequence of PRO615 include a
type II transmembrane domain, corresponding to about amino acids
24-43, other transmembrane domains, corresponding to about amino
acids 74-90, 108-126, and 145-161, respectively, and a potential
N-glycosylation site, corresponding to about amino acids 97-100.
Clone UNQ352 (DNA48304-1323) has been deposited with ATCC and is
assigned ATCC deposit no. 209811.
Example 28
[1845] Isolation of cDNA Clones Encoding Human PRO618
[1846] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA30900. Based on
the DNA30900 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 PRO618.
[1847] Forward and reverse PCR primers were synthesized:
60 forward PCR primer 5'-TAACAGCTGCCCACTGCTTCCAGG-3' (SEQ ID NO:
171) reverse PCR primer 5'-TAATCCAGCAGTGCAGGCCGGG-3' (SEQ ID NO:
172)
[1848] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA30900 sequence which
had the following nucleotide sequence
[1849] hybridization probe
61 5'-ATGGCCTCCACGGTGCTGTGGACCGTGTTCC (SEQ ID NO: 173)
TGGGCAAGGTGTGGCAGAA-3'
[1850] Screening of the above described library gave rise to the
partial cDNA clone designated herein DNA35597 (SEQ ID NO:170).
Extension of this sequence using repeated cycles of BLAST and phrap
gave rise to a nucleotide sequence designated herein as DNA43335.
Primers based upon the DNA43335 consensus sequence were then
prepared as follows.
62 forward PCR primer 5'-TGCCTATGCACTGAGGAGGCAGAAG-3' (SEQ ID NO:
174) reverse PCR primer 5'-AGGCAGGGACACAGAGTCCATTCAC-3' (SEQ ID NO:
175)
[1851] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA43335 sequence which
had the following nucleotide sequence
[1852] hybridization probe
63 5'-AGTATGATTTGCCGTGCACCCAGGGCCAGTG (SEQ ID NO: 176)
GACGATCCAGAACAGGAGG-3'
[1853] In order to screen several libraries for a source of a
ftifl-length clone, DNA from the libraries was screened by PCR
amplification with one of the PCR primer pairs identified above. A
positive library was then used to isolate fill length clones
encoding the PRO618 gene using the second probe oligonucleotide and
one of the second set of PCR primers. RNA for construction of the
cDNA libraries was isolated from human fetal liver tissue
(LEB229).
[1854] DNA sequencing of the clones isolated as described above
gave the fiill-length DNA sequence for PRO618 [herein designated as
UNQ354 (DNA49152-1324)] (SEQ ID NO:168) and the derived protein
sequence for PRO618.
[1855] The entire nucleotide sequence of UNQ354 (DNA49152-1324) is
shown in FIG. 62 (SEQ ID NO:168). Clone UNQ354 (DNA49152-1324)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 73-75 and ending at the
stop codon at nucleotide positions 2479-2481 (FIG. 62). The
predicted polypeptide precursor is 802 amino acids long (FIG. 63).
The full-length PRO618 protein shown in FIG. 63 has an estimated
molecular weight of about 88,846 daltons and a pI of about 6.41.
Important regions of the amino acid sequence of PRO618 include type
II transmembrane domain, a sequence typical of a protease, trypsin
family, histidine active site, multiple N-glycosylation sites, two
sequences typical of a Kringle domain, two regions having sequence
similarity to Kallikrein light chain, and a region having sequence
similarity to lowensity lipoprotein receptor. Clone UNQ354
(DNA49152-1324) has been deposited with ATCC and is assigned ATCC
deposit no. 209813.
Example 29
[1856] Isolation of cDNA Clones Encoding Human PRO719
[1857] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA44851. Based on
the DNA44851 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
fiillength coding sequence for PRO719.
[1858] A pair of PCR primers (forward and reverse) were
synthesized:
64 forward PCR primer 5'-GTGAGCATGAGCGAGCCGTCCAC-3' (SEQ ID NO:
179) reverse PCR primer 5'-GCTATTACAACGGTTCTTGCGGCAGC-3' (SEQ ID
NO: 180)
[1859] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA44851 sequence which
had the following nucleotide sequence
[1860] hybridization probe
65 5'-TTGACTCTCTGGTGAATCAGGACAAGCCGAG (SEQ ID NO: 181)
TTTTGCCTTCCAG-3'
[1861] 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 PRO719 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human placenta
tissue (LIB90).
[1862] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO719 [herein designated as
UNQ387 (DNA49646-1327)] (SEQ ID NO:177) and the derived protein
sequence for PRO719.
[1863] The entire nucleotide sequence of UNQ387 (DNA49646-1327) is
shown in FIG. 65 (SEQ ID NO:177). Clone UNQ387 (DNA49646-1327)
contains a single open reading frame with anapparenttranslational
initiation site at nucleotide positions 223-225 and ending at the
stop codon at nucleotide positions 1285-1287 (FIG. 65). The
predicted polypeptide precursor is 354 amino acids long (FIG. 66).
The full-length PRO719 protein shown in FIG. 66 has an estimated
molecular weight of about 39,362 daltons and a pl of about 8.35.
Analysis of the full length PRO719 sequence evidences the presence
of a signal peptide from about amino acid 1 to about amino acid 16
as shown in FIG. 66 (SEQ ID NO:178), a lipase-associated
serine-containing active site at about amino acid 163 to about
amino acid 172, and two potential N-glycosylation sites from about
amino acid 80 to about amino acid 83 and about amino acid 136 to
about amino acid 139. Clone UNQ387 (DNA49646-1327) has been
deposited with ATCC on Mar. 26, 1998 and is assigned ATCC deposit
no. 209705.
[1864] Analysis of the amino acid sequence of the full-length
PRO719 polypeptide suggests that it possesses significant sequence
similarity to the lipoprotein lipase H protein, thereby indicating
that PRO719 may be a novel lipoprotein lipase homolog. More
specifically, an analysis of the Dayhoff database (version 35.45
SwissProt 35) evidenced significant homology between the PRO719
amino acid sequence and the following Dayhoff sequences,
LIPL_HUMAN, LIPH_HUMAN, D83548.sub.--1, A24059.sub.--1, P_R30740,
D88666.sub.--1, A43357, A46696, B43357 and A49488.
Example 30
[1865] Isolation of cDNA Clones Encodinz Human PRO724
[1866] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA35603. Based on
the DNA35603 consensus sequence, oligonucleotides were synthesized:
1) to identify by PCR a cDNA library that contained the sequence of
interest, and 2) for use as probes to isolate a clone of the
full-length coding sequence for PRO724.
[1867] Pairs of PCR primers (forward and reverse) were
synthesized:
66 forward PCR primer 1 5'-GGCTGTCACTGTGGAGACAC-3' (SEQ ID NO: 184)
forward PCR primer 2 5'-GCAAGGTCATTACAGCTG-3' (SEQ ID NO: 185)
reverse PCR primer 1 5'-AGAACATAGGAGCAGTCCCACTC-3' (SEQ ID NO: 186)
reverse PCR primer 2 5'-TGCCTGCTGCTGCACAATCTCAG-3' (SEQ ID NO:
187)
[1868] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA35603 sequence which
had the following nucleotide sequence
[1869] hybridization probe
67 5'-GGCTATTGCTTGCCTTGGGACAGACCCTGTG (SEQ ID NO: 188)
GCTTAGGCTCTGGC-3'
[1870] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pairs identified above. A
positive library was then used to isolate clones encoding the
PRO724 gene using the probe oligonucleotide and one of the PCR
primers. RNA for construction of the cDNA libraries was isolated
from human fetal lung tissue (LIB26).
[1871] DNA sequencing of the clones isolated as desenbed above gave
the full-length DNA sequence for PRO724 [herein designated as
UNQ389 (DNA49631-1328)] (SEQ ID NO:182) and the derived protein
sequence for PRO724.
[1872] The entire nucleotide sequence of UNQ389 (DNA49631-1328) is
shown in FIG. 67 (SEQ ID , NO:182). Clone LN Q389 (DNA49631 -1328)
contains a single open reading frame with an apparenttranslational
initiation site at nucleotide positions 546-548 and ending at the
stop codon at nucleotide positions 2685-2687 (FIG. 67). The
predicted polypeptide precursor is 713 amino acids long (FIG. 68).
The fllll-length PRO724 protein shown in FIG. 68 has an estimated
molecular weight of about 76,193 daltons and a pl of about 5.42.
Analysis of the full-length PRO724 amino acid sequence shown in
FIG. 68 (SEQ ID NO:183) evidences the presence of the following: a
signal peptide from about amino acid 1 to about amino acid 16, a
transmembrane domain from about amino acid 442 to about amino acid
462 and LDL receptor class A domain regions from about amino acid
152 to about amino acid 171, about amino acid 331 to about amino
acid 350, about amino acid 374 to about amino acid 393 and about
amino acid 411 to about amino acid 430. Clone UNQ389 (D)NA4963
1-1328) has been deposited with ATCC on Apr. 28, 1998 and is
assigned ATCC deposit no. 209806 Analysis of the amino acid
sequence of the ftll-length PRO724 polypeptide suggests that it
possesses significant sequence similarity to the human LDL receptor
protein, thereby indicating that PRO724 may be a novel LDL receptor
homolog. More specifically, an analysis of the Dayhoff database
(version 35.45 SwissProt 35) evidenced significant homology between
the PRO724 amino acid sequence and the following Dayhoff sequences,
P_R48547, MMAM2R.sub.--1, LRP2_RAT, P_R60517, P_R47861, P_R05533,
A44513.sub.--1, A30363, P_R74692 and LMLIPOPHO.sub.--1.
Example 31
[1873] Isolation of cDNA Clones Encoding Human PRO772
[1874] One cDNA sequence was isolated in the amylase screen
described in Example 2, wherein that cDNA sequence is herein
designated DNA43509 (see FIG. 71). Based on the DNA43509 sequence,
oligonucleotide probes were generated and used to screen a human
fetal lung library (LIB25) prepared as described in paragraph 1 of
Example 2 above. The cloning vector was pRKSB (pRK5B is a precursor
of pRK5D that does not contain the SfiI site; see, Holmes et al.,
Science, 253:1278-1280 (1991)), and the cDNA size cut was less than
2800 bp.
[1875] A pair of PCR primers (forward and reverse) were synthesized
based on the DNA43509 sequence:
68 forward PCR primer 5'-CGTTTTGCAGAACCTACTCAGGCAG-3' (SEQ ID NO:
192) reverse PCR primer 5'-CCTCCACCAACTGTCAATGTTGTGG-3' (SEQ ID NO:
193)
[1876] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA43509 sequence which
had the following nucleotide sequence
[1877] hybridization probe
69 5'-AAAGTGCTGCTGCTGGGTCTGCAGACGCGAT (SEQ ID NO: 194)
GGATAACGT-3'
[1878] Using the above described primers and library, a full length
clone was identified that contained a single open reading frame
with an apparent translational initiation site at nucleotide
positions 131-133 and ending at the stop codon found at nucleotide
positions 587-589 (FIG. 69; SEQ ID NO:189). The predicted
polypeptide precursor is 152 amino acids long, has a calculated
molecular weight of approximately 17,170 daltons and an estimated
pI of approximately 9.62. Analysis of the flll-length PRO772
sequence shown in FIG. 70 (SEQ ID NO:190) evidences the presence of
the following: a potential type II transmembrane domain from about
amino acid 26 to about amino acid 42, other potential transmembrane
domains from about amino acid 44 to about amino acid 65, from about
amino acid 81 to about amino acid 101 and from about amino acid 109
to about amino acid 129, leucine zipper pattern sequences from
about amino acid 78 to about amino acid 99 and from about amino
acid 85 to about amino acid 106. Clone UNQ410 (DNA49645-1347) has
been deposited with ATCC on Apr. 28, 1998 and is assigned ATCC
deposit no. 209809.
[1879] Analysis of the amino acid sequence of the full-length
PRO772 polypeptide suggests that it possesses significant sequence
similarity to the human A4 protein, thereby indicating that PRO772
may be a novel A4 protein homolog. More specifically, an analysis
of the Dayhoff database (version 35.45 SwissProt 35) evidenced
significant homology between the PRO772 amino acid sequence and the
following Dayhoff sequences, HSU93305.sub.--1, A4P_HUMAN,
CELB0454.sub.--2, VPU_JSRV, CELC12D12.sub.--2, OCCM_AGRT1,
LBPHIG1E.sub.--50, YIGK_ECOLI, S76245 and P_R50807.
Example 32
[1880] Isolation of cDNA Clones Encoding Human PRO852
[1881] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA34364. Based on
the DNA34364 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 PRO852.
[1882] PCR primers (forward and reverse) were synthesized:
70 forward PCR primer 1 5'-CGCAGAAGCTACAGATTCTCG-3' (SEQ ID NO:
197) forward PCR primer 2 5'-GGAAATTGGAGGCCAAAGC-3' (SEQ ID NO:
198) forward PGR primer 3 5'-GGATGTAGCCAGCAACTGTG.3' (SEQ ID NO:
199) forward PCR primer 4 5'-GCCTTGGCTCGTTCTCTTC-3' (SEQ ID NO:
200) forward PCR primer 5 5'-GGTCCTGTGCCTGGATGG-3' (SEQ ID NO: 201)
reverse PCR primer 1 5'-GACAAGACTACCTCCGTTGGTC-3' (SEQ ID NO: 202)
reverse PCR primer 2 5'-TGATGCACAGTTCAGCACCTGTTG-3' (SEQ ID NO:
203)
[1883] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA34364 sequence which
had the following nucleotide sequence
[1884] hybridization probe
71 5'-CGCTCCAAGGGCTTTGACGTCACAGTGAAGT (SEQ ID NO: 204)
ACACACAAGGAAGCTG-3'
[1885] 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 PROB52 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human fetal
kidney tissue (LIB228).
[1886] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO852 [herein designated as
UNQ418 (DNA45493-1349)] (SEQ ID NO:195) and the derived protein
sequence for PRO852.
[1887] The entire nucleotide sequence of UNQ418 (DNA45493-1349) is
shown in FIG. 72 (SEQ ID NO:195). Clone UNQ418 (DNA45493-1349)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 94-96 and ending at the
stop codon at nucleotide positions 16748-1650 (FIG. 72). The
predictedpolypeptide precursor is 518 amino acids long (FIG. 73).
The fuli-lengthPRO852 protein shown in FIG. 73 has an estimated
molecular weight of about 56,180 daltons and a pI of about 5.08.
Analysis of the full-length PRO852 sequence shown in FIG. 73 (SEQ
ID NO:196) evidences the presence of the following: a signal
peptide from about amino acid 1 to about amino acid 20, a
transmembrane domain from about amino acid 466 to about amino acid
494, potential N-glycosylation sites from about amino acid 170 to
about amino acid 173 and about amino acid 366 to about amino acid
369, leucine zipper sequence pattern blocks from about amino acid
10 to about amino acid 31 and from about amino acid 197 to about
amino acid 218 and blocks of amino acids having sequence homology
to eukaryotic and viral aspartyl proteases from about amino acid
109 to about amino acid 118, from about amino acid 252 to about
amino acid 261 and from about amino acid 298 to about amino acid
310. Clone UNQ418 (DNA45493-1349) has been deposited with ATCC on
Apr. 28, 1998 and is assigned ATCC deposit no. 209805.
[1888] Analysis of the amino acid sequence of the full-length
PRO852 polypeptide suggests that it possesses significant sequence
similarity to various protease proteins, thereby indicating that
PRO852 may be a novel protease protein or homolog thereof. More
specifically, an analysis of the Dayhoff database (version 35.45
SwissProt 35) evidenced significant homology between the PRO852
amino acid sequence and the following Dayhoff sequences,
PEPC_HUMAN, S66516, S66517, PEPE_CHICK, CATD_HUMAN, P_R74207,
CARP_YEAST, PEP2_RABIT, CATE_HUMAN and RENI_MOUSE.
Example 33
[1889] Isolation of cDNA Clones Encoding Human PRO853
[1890] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA43050. Based on
the DNA43050 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
ftill-length coding sequence for PRO853.
[1891] Forward and reverse PCR primers were synthesized:
72 forward PCR primer 5'-CTTCATGGCCTTGGACTTGGCCAG-3' (SEQ ID NO:
207) reverse PCR primer 5'-ACGCCAGTGGCCTCAAGCTGGTTG-3' (SEQ ID NO:
208)
[1892] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA43050 sequence which
had the following nucleotide sequence
[1893] hybridization probe
73 5'-CTTTCTGAGCTCTGAGCCACGGTTGGACATC (SEQ ID NO: 209)
CTCATCCACAATGC-3'
[1894] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with one of the PCR primer pairs identified above. A
positive library was then used to isolate clones encoding the
PRO853 gene using the probe oligonucleotide and one of the PCR
primers. RNA for construction of the cDNA libraries was isolated
from human fetal kidney tissue (LIB228).
[1895] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO853 [hereindesignatedas
UNQ419 (DNA48227-1350)] (SEQIID NO:205) and the derived protein
sequence for PRO853.
[1896] The entire nucleotide sequence of UNQ419 (DNA48227-1350) is
shown in FIG. 74 (SEQ ID NO:205). Clone UNQ419 (DNA48227-1350)
contains a single open reading frame with anappareat translational
initiation site at nucleotide positions 128-130 and ending at the
stop codon at nucleotide positions 1259-1261 (FIG. 74). The
predicted polypeptide precursor is 377 amino acids long (FIG. 75).
The full-length PRO853 protein shown in FIG. 75 has an estimated
molecular weight of about 40,849 daltons and a pI of about 7.98.
Important regions of the amino acid sequence of PRO853 include the
signal peptide, corresponding to amino acids from about 1 to about
16 of SEQ ID NO:206, the glycosaminoglycan attachment site,
corresponding to amino acids from about 46 to about 49 of SEQ ID
NO:206, and two sequences typical of the short-chain alcohol
dehydrogenase family, corresponding to amino acids from about 37 to
about 49 and about 114 to about 124 of SEQ ID NO:206, respectively.
Clone UNQ419 (DNA48227-1350) has been deposited with ATCC and is
assigned ATCC deposit no. 209812.
Example 34
[1897] Isolation of cDNA Clones Encoding Human PRO860
[1898] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA38137. Based on
the DNA38137 consensu 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 PRO860.
[1899] Forward and reverse PCR primers were synthesized:
74 forward PCR primer 5'-GAAGGGACCTACATGTGTGTGGCC-3' (SEQ ID NO:
212) reverse PCR primer 5'-ACTGACCTTCCAGCTGAGCCACAC-3' (SEQ ID NO:
213)
[1900] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA40654 sequence which
had the following nucleotide sequence
[1901] hybridization probe
75 5'-AGGACTACACGGCGAATGTGGAGCTTCTGGC (SEQ ID NO: 214)
TGTGCGAATTCAGCTGGAA-3'
[1902] In order to screen several libraries for a source of a
fiull-length clone, DNA from the libraries was screened by PCR
amplification with one of the PCR primer pairs identified above. A
positive library was then used to isolate clones encoding the
PRO860 gene using the probe oligonucleotide and one of the PCR
primers. RNA for construction of the cDNA libraries was isolated
from human fetal lung tissue (LIB26).
[1903] DNA sequencing of the clones isolated as described above
gave the fuillength DNA sequence for PRO860 [herein designated as
UNQ421 (DNA41404-1352)] (SEQ ID NO:210) and the derived protein
sequence for PRO860.
[1904] The entire nucleotide sequence of UNQ421 (DNA41404-1352) is
shown in FIG. 76 (SEQ ID NO:210). Clone UNQ421 (DNA41404-1352)
contains a sinWeopenreading frame with an apparenttranslational
initiation site at nucleotide positions 58-60 and ending at the
stop codon at nucleotide positions 3013-3015 (FIG. 76). The
predicted polypeptide precursor is 985 amino acids long (FIG. 77).
The fuillength PRO860 protein shown in FIG. 77 has an estimated
molecular weight of about 105,336 daltons and a pI of about 6.55.
Important regions of the amino acid sequence of PRO860 include the
transmembrane region corresponding to about amino acids 448467, the
extracellular domain, corresponding to amino acids about 1-447,
several N-glycosylation sites, numerous N-myristoylation sites and
a sequence typical of phosphotyrosine interaction domain proteins..
Clone UNQ421 (DNA41404-1352) has been deposited with ATCC and is
assigned ATCC deposit no. 209844.
Example 35
[1905] Isolation of cDNA Clones Encoding Human PRO846
[1906] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA39949. Based on
the DNA39949 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 PRO846.
[1907] Forward and reverse PCR primers were synthesized:
76 forward PCR primer 5'-CCCTGCAGTGCACCTACAGGGAAG-3' (SEQ ID NO:
217) reverse PCR primer 5'-CTGTCTTCCCCTGCTTGGCTGTGG-3' (SEQ ID NO:
218)
[1908] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA39949 sequence which
had the following nucleotide sequence
[1909] hybridization probe
77 5'-GGTGCAGGAAGGGTGGGATCCTCTTCTCTCG (SEQ ID NO: 219)
CTGCTCTGGCCACATC-3'
[1910] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with one of the PCR primer pairs identified above. A
positive library was then used to isolate clones encoding the
PRO846 gene using the probe oligonucleotide and one of the PCR
primers. RNA for construction of the cDNA libraries was isolated
from human fetal kidney tissue (LIB227).
[1911] DNA sequencing of the clones isolated as described above
gave the fiill-length DNA sequence for PRO846 [herein designated as
UNQ422 (DNA44196-1353)] (SEQ ID NO:215) and the derived protein
sequence for PRO846.
[1912] The entire nucleotide sequence of UNQ422 (DNA44196-1353) is
shown in FIG. 78 (SEQ ID NO:215). Clone UN422 (DNA44196-1353)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 25-27 and ending at the
stop codon at nucleotide positions 1021-1023 (FIG. 78). The
predicted polypeptide precursor is 332 amino acids long (FIG. 79).
The full-length PRO846 protem shown in FIG. 79 has an estimated
molecular weight of about 36,143 daltons and a pI of about 5.89.
Important regions of the amino acid sequence of PRO846 include the
signal peptide, the transmembrane domain, an N-glycosylation site,
a sequence typical of fibrinogen beta and gamma chains C-terminal
domain, and a sequence typical of Ig like V-type domain as shown in
FIG. 79. Clone UNQ422 (DNA44196-1353) has been deposited with ATCC
and is assigned ATCC deposit no. 209847.
Example 36
[1913] Isolation of cDNA Clones Encoding Human PRO862
[1914] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA47370. Based on
the DNA47370 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
fiill-length coding sequence for PRO862.
[1915] Forward and reverse PCR primers were synthesized:
78 forward PCR primer 5'-GGGATCATGTTGTTGGCCCTGGTC-3' (SEQ ID NO:
222) reverse PCR primer 5'-GCAAGGCAGACCCAGTCAGCCAG-3' (SEQ ID NO:
223)
[1916] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA47370 sequence which
had the following nucleotide sequence
[1917] hybridization probe
79 5'-CTGCCTGCTACCCTCCAAGTGAGGCCAAGCT (SEQ ID NO: 225)
CTACGGTCGTTGTG-3'
[1918] In order to screen several libraries for a source of a
fll-length clone, DNA from the libraries was screened by PCR
amplification with one of the PCR primer pairs identified above. A
positive library was then used to isolate clones encoding the
PRO862 gene using the probe oligonucleotide and one of the PCR
primers. RNA for construction of the cDNA libraries was isolated
from human pancreas tissue (LB355).
[1919] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO862 [herein designated as
UNQ424 (DNA52187-1354)] (SEQ ID NO:220) and the derived protein
sequence for PRO862.
[1920] The entire nucleotide sequence of UNQ424 (DNA52187-1354) is
shown in FIG. 80 (SEQ ID NO:220). Clone UNQ424 (DNA52187-1354)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 410412 and ending at the
stop codon at nucleotide positions 848-850 (FIG. 80). The predicted
polypeptide precursor is 146 amino acids long (FIG. 81). The
full-length PRO862 protein shown in FIG. 81 has an estimated
molecular weight of about 16,430 daltons and a pl of about 5.05.
Inportant regions of the amino acid sequence of PRO862 include the
signal peptide, an N-myristoylation site, and sequences having
similarity to region to Alpha-lactalbuminilysozyme C proteins as
shown in FIG. 81. Clone UNQ424 (DNA52187-1354) has been deposited
with the ATCC and is assigned ATCC deposit no. 209845.
Example 37
[1921] Isolation of cDNA Clones Encoding Human PRO864
[1922] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA40666. Based on
the DNA40666 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 PRO864.
[1923] Forward and reverse PCR primers were synthesized:
80 forward PCR primer 540 -GCTGCAGCTGCAAATTCCACTGG-340 (SEQ ID NO:
227) reverse PCR primer 540 -TGGTGGGAGACTGTTTAAATTATCGGCC-340 (SEQ
ID NO: 228)
[1924] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA40666 sequence which
had the following nucleotide sequence
[1925] hybridization probe
81 5'-TGCTTCGTCAAGTGCCGGCAGTGCCAGCGGC (SEQ ID NO: 229)
TCGTGGAGTT-3'
[1926] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with one of the PCR primer pairs identified above. A
positive library was then used to isolate clones encoding the
PRO864 gene using the probe oligonucleotide and one of the PCR
primers. RNA for construction of the cDNA libraries was isolated
from human fetal brain tissue (LIB153).
[1927] DNA sequencing of the clones isolated as described above
gave the fiill-length DNA sequence for PRO864 [herein designated as
UNQ426 (DNA48328-1355)] (SEQ ID NO:225) and the derived protein
sequence for PRO864.
[1928] The entire nucleotide sequence of UNQ426 (DNA48328-1355) is
shown in FIG. 82 (SEQ ID NO:225). Clone UNQ426 (DNA48328-1355)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 37-39 and ending at the
stop codon at nucleotide positions 1090-1092 (FIG. 82). The
predicted polypeptide precursor is 351 amino acids long (FIG. 83).
The full-length PRO864 protein shown in FIG. 83 has an estimated
molecular weight of about 39,052 and a pI of about 8.97. Important
regions of the amino acid sequence of PRO864 include the signal
peptide, two N-glycosylation sites, a Wnt-I family signature
sequence, and sequence regions homologous to Wnt-1 family proteins
as shown in FIG. 83. Clone UNQ426 (DNA48328-1355) has been
deposited with ATCC and is assigned ATCC deposit no. 209843.
Example 38
[1929] Isolation of cDNA Clones Encoding Human PRO792
[1930] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA38106. Based on
the DNA38106 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 PRO792.
[1931] A pair of PCR primers (forward and reverse) were
synthesized:
82 forward PCR primer 5'-GCGAGAACTGTGTCATGATGCTGC-3' (SEQ ID NO:
232) reverse PCR primer 5'-GTTTCTGAGACTCAGCAGCGGTGG-3' (SEQ ID NO:
233)
[1932] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA38106 sequence which
had the following nucleotide sequence
[1933] hybridization probe
83 5'-CACCGTGTGACAGCGAGAAGGACGGCTGGAT (SEQ ID NO: 234)
CTGTGAGAAAAGGCACAAC-3'
[1934] 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 PRO792 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human bone
marrow tissue (LIB255).
[1935] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO792 [herein designated as
UNQ431 (DNA56352-1358)] (SEQ ID NO:230) and the derived protein
sequence for PRO792.
[1936] The entire nucleotide sequence of UNQ431 (DNA56352-1358) is
shown in FIG. 84 (SEQ ID NO:230). Clone UNQ431 (DNA56352-1358)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 67-69 and ending at the
stop codon at nucleotide positions 946-948 (FIG. 84). The predicted
polypeptide precursor is 293 amino acids long (FIG. 85). The
flll-length PRO792 protein shown in FIG. 85 has an estimated
molecular weight of about 32,562 daltons and a pI of about 6.53.
Analysis of the full-length PRO792 sequence shown in FIG. 85 (SEQ
ID NO:231) evidences the presence of the following: a type II
transmembrane domain from about amino acid 31 to about amino acid
54, potential N-glycosylation sites from about amino acid 73 to
about amino acid 76 and from about amino acid 159 to about amino
acid 162, a leucine zipper amino acid sequence pattern from about
amino acid 102 to about amino acid 123, potential N-myristolation
sites from about amino acid 18 to about amino acid 23, from about
amino acid 133 to about amino acid 138 and from about amino acid
242 to about amino acid 247 and a C-type lectin domain signature
block from about amino acid 264 to about amno acid 287. Clone
UNQ431 (DNA56352-1358) has been deposited with ATCC on May 6, 1998
and is assigned ATCC deposit no. 209846.
[1937] Analysis of the amino acid sequence of the full-length
PRO792 polypeptide suggests that it possesses significant sequence
similarity to the CD23 protein, thereby indicating that PRO792 may
be a novel CD23 homolog. More specifically, an analysis of the
Dayhoff database (version 35.45 SwissProt 35) evidenced significant
homology between the PRO792 amino acid sequence and the following
Dayhoff sequences, S34198, A07100.sub.--1, A05303.sub.--1,
P_R41689, P_P82839, A10871.sub.--1, P_R12796, P_R47199, A46274 and
P_R32188.
Example 39
[1938] Isolation of cDNA Clones Encoding Human PRO866
[1939] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA44708. Based on
the DNA44708 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 PRO866.
[1940] PCR primers (forward and reverse) were synthesized:
84 forward PCR nrimer 1 5'-CAGCACTGCCAGGGGAAGAGGG-3' (SEQ ID NO:
237) forward PCR primer 2 5'-CAGGACTCGCTACGTCCG-3' (SEQ ID NO: 238)
forward PCR primer 3 5'-CAGCCCCTTCTCCTCCTTTCTCCC-3' (SEQ ID NO:
239) reverse PCR primer 1 5'-GCAGTTATCAGGGACGCACTCAGCC-3' (SEQ ID
NO: 240) reverse PCR primer 2 5'-CCAGCGAGAGGCAGATAG-3' (SEQ ID NO:
241) reverse PCR primer 3 5'-CGGTCACCGTGTCCTGCGGGATG-3' (SEQ ID NO:
242)
[1941] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA44708 sequence which
had the following nucleotide sequence
[1942] hybridization probe
85 5'-CAGCCCCTTCTCCTCCTTTCTCCCACGTCCT (SEQ ID NO: 243)
ATCTGCCTCTC-3'
[1943] In order to screen several libraries for a source of a
flll-length clone, DNA from the libraries was screened by PCR
amplification with one of the PCR primer pairs identified above. A
positive library was then used to isolate clones encoding the
PRO866 gene using the probe oligonucleotide and one of the PCR
primers. RNA for construction of the cDNA libraries was isolated
from human fetal kidney tissue (LIB228).
[1944] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO866 [herein designated as
UNQ435 (DNA53971-1359)] (SEQ ID NO:235) and the derived protein
sequence for PRO866.
[1945] The entire nucleotide sequence of UNQ435 (DNA53971-1359) is
shown in FIG. 86 (SEQ ID NO:235). Clone UNQ435 (DNA53971-1359)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 275-277 and ending at the
stop codon at nucleotide positions 1268-1270 (FIG. 86). The
predicted polypeptide precursor is 331 amino acids long (FIG. 87).
The full-length PRO866 protein shown in FIG. 87 has an estimated
molecular weight of about 35,844 daltons and a pf of about 5.45.
Analysis of the full-length PRO866 sequence shown in FIG. 87 (SEQ
ID NO:236) evidences the presence of the following: a signal
peptide from about amino acid 1 to about amino acid 26. Clone
UNQ435 (DNA53971-1359) has been deposited with ATCC on Apr. 7, 1998
and is assigned ATCC deposit no. 209750.
[1946] Analysis of the amino acid sequence of the full-length
PRO866 polypeptide suggests that it possesses significant sequence
similarity to the mindin/spondin family of proteins, thereby
indicating that PRO866 may be a novel niindin homolog. More
specifically, an analysis of the Dayhoff database (version 35.45
SwissProt 35) evidenced significant homology between the PRO866
amino acid sequence and the following Dayhoff sequences,
AB006085.sub.--1, AB006084.sub.--1, AB006086.sub.--1,
AF017267.sub.--1, CWU42213.sub.--1, AC004160.sub.--1,
CPMICRP.sub.--1, S49108, A48569 and I46687.
Example 40
[1947] Isolation of cDNA Clones Encoding Human PRO871
[1948] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA40324. Based on
the DNA40324 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 PRO871.
[1949] PCR primers (forward and reverse) were synthesized:
86 forward PCR primer 1 5'-TGCGGAGATCCTACTGGCACAGGG-3' (SEQ ID NO:
246) forward PCR primer 2 5'-CGAGTTAGTCAGAGCATG-3' (SEQ ID NO: 247)
forward PCR primer 3 5'-CAGATGGTGCTGTTGCCG-3' (SEQ ID NO: 248)
reverse PCR primer 1 5'-CAACTGGAACAGGAACTGAGATGTGG (SEQ ID NO: 249)
ATC-3' reverse PCR primer 2 5'-CTGGTTCAGCAGTGCAAGGGTCTG-3' (SEQ ID
NO: 250) reverse PCR primer 3 5'-CCTCTCCGATTAAAACGC-3' (SEQ ID NQ:
251)
[1950] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA40324 sequence which
had the following nucleotide sequence
[1951] hybridization probe
87 5'-GAGAGGACTGGTTGCCATGGCAAATGCTGGT (SEQ ID NO: 252)
TCTCATGATAATGG-3'
[1952] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with one of the PCR primer pairs identified above. A
positive library was then used to isolate clones encoding the
PRO871 gene using the probe oligonucleotide and one of the PCR
primers. RNA for construction of the cDNA libraries was isolated
from human fetal kidney tissue (LIB227).
[1953] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO871 [herein designated as
UNQ438 (DNA50919-1361)] (SEQ ID NO:244) and the derived protein
sequence for PRO871.
[1954] The entire nucleotide sequence of UNQ438 (DNA50919-1361) is
shown in FIG. 88 (SEQ ID NO:244). Clone UNQ438 (DNA50919-1361)
contains a single open reading frame with an apparenttranslational
initiation site at nucleotide positions 191-193 and ending at the
stop codon at nucleotide positions 1607-1609 (FIG. 88). The
predicted polypeptide precursor is 472 amino acids long (FIG. 89).
The full-length PRO871 protein shown in FIG. 89 has an estimated
molecular weight of about 53,847 daltons and a pI of about 5.75.
Analysis of the full-length PRO871 sequence shown in FIG. 89 (SEQ
ID NO:245) evidences the presence of the following: a signal
peptide from about amino acid 1 to about amino acid 21, potential
N-glycosylation sites from about amino acid 109 to about amino acid
112 and from about amino acid 201 to about amino acid 204, a
cyclophilin-type peptidy-prolyl cis-trans isomerase signature
sequence from about amino acid 49 to about amino acid 66 and
regions that are homologous to cyclophilin-type peptidy-prolyl
cis-trans isomerases from about amino acid 96 to about amino acid
140, from about amino acid 49 to about amino acid 89 and from about
amino acid 22 to about amino acid 51. Clone UNQ438 (DNA50919-1361)
has been deposited with ATCC on May 6, 1998 and is assigned ATCC
deposit no. 209848.
[1955] Analysis of the amino acid sequence of the full-length
PRO871 polypeptide suggests that it possesses significant sequence
similarity to the cyclophilin family of proteins, thereby
indicating that PRO871 may be a novel cyclophilin protein family
member. More specifically, an analysis of the Dayhoff database
(version 35.45 SwissProt 35) evidenced significant homology between
the PRO871 amino acid sequence and the following Dayhoff sequences,
SPBC16H5.sub.--5, S64705, YAL5_SCHPO, CYP4_CAEEL, CELC34D4.sub.--7,
CYPA_CAEEL, HUMORF006.sub.--1, CYPI_MYCTU, AF043642.sub.--1 and
HSSRCYP.sub.--1.
Example 41
[1956] Isolation of cDNA Clones Encoding Human PRO873
[1957] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA39621. Based on
the DNA39621 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 PRO873.
[1958] A pair of PCR primers (forward and reverse) were
synthesized:
88 forward PCR primer 5'-AGGTGCCTGCAGGAGTCCTGGGG-3' (SEQ ID NO:
255) reverse PCR primer 5'-CCACCTCAGGAAGCCGAAGATGCC-3' (SEQ ID NO:
256)
[1959] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA39621 sequence which
had the following nucleotide sequence:
[1960] hybridization probe
89 5'-GAACGGTACAAGTGGCTGCGCTTCAGCGAGG (SEQ ID NO: 257)
ACTGTCTGTACCTG-3'
[1961] 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 PRO873 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human fetal
liver tissue (LEB229).
[1962] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO873 [herein designated as
UNQ440 (DNA44179-1362)] (SEQ ID NO:253) and the derived protein
sequence for PRO873.
[1963] The entire nucleotide sequence of UNQ440 (DNA44179-1362) is
shown in FIG. 90 (SEQ ID NO:253). Clone UNQ440 (DNA44179-1362)
contains a single open reading frame with an apparenttranslational
initiation site at nucleotide positions 139-141 and ending at the
stop codon at nucleotide positions 1774-1776 (FIG. 90). The
predicted polypeptide precursor is 545 amino acids long (FIG. 91).
The full-lengdi PRO873 protein shown in FIG. 91 has an estimated
molecular weight of about 58,934 daltons and a pI of about 9.45.
Analysis of the full-length PRO873 sequence shown in FIG. 91 (SEQ
ID NO:254) evidences the presence of the following features: a
signal peptide from about amino acid 1 to about amino acid 29; a
carboxylesterase type-B serine active site at about amino acid 312
to about amino acid 327; a carboxylesterase type-B signature 2
motif at about amino acid 218 to about amino acid 228; and three
potential N-glycosylation sites at about amino acid 318 to about
amino acid 321, about amino acid 380 to about amino acid 383, and
about amino acid 465 to about amino acid 468. Clone UNQ440
(DNA44179-1362) has been deposited with ATCC on May 6, 1998 and is
assigned ATCC deposit no. 209851.
[1964] Analysis of the amino acid sequence of the full-length
PRO873 polypeptide suggests that it possesses significant sequence
similarity to a human liver carboxylesterase, thereby indicating
that PRO873 may be a novel carboxylesterase. More specifically, an
analysis of the Dayhoff database (version 35.45 SwissProt 35)
evidenced significant homology between the PRO873 amino acid
sequence and the following Dayhoff sequences: ES10_RAT, GEN12405,
AB010633.sub.--1, EST4_RAT, A48809, SASB_ANAPL, RNU41662.sub.--1,
RNU22952.sub.--1, BAL_RAT, GEN13522.
Example 42
[1965] Isolation of cDNA Clones Encoding Human PRO940
[1966] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA47442. Based on
the DNA47442 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 PRO940.
[1967] A pair of PCR primers (forward and reverse) were
synthesized:
90 forward PCR primer 5'-CAAAGCCTGCGCCTGGTCTGTG-3' (SEQ ID NO: 260)
reverse PCR primer 5'-TTCTGGAGCCCAGAGGGTGCTGAG-3' (SEQ ID NO:
262)
[1968] Additionaly, a synthetic oligonucleotide hybridiztion probe
was constructed from the consensus DNA47442 sequence which had the
following nucleotide sequence
[1969] hybridization probe
91 5'-GGAGCTGCCACCCATTCAAATGGAGCACGAA (SEQ ID NO: 263)
GGAGAGTTCACCTG-3'
[1970] 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 PRO940 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human fetal
liver tissue (LIB229).
[1971] DNA sequencing of the clones isolated as described above
gave the fllll-length DNA sequence for PRO940 [herein designated as
UNQ477 (DNA54002-1367)] (SEQ ID NO:258) and the derived protein
sequence for PRO940.
[1972] The entire nucleotide sequence of UNQ477 (DNA54002-1367) is
shown in FIG. 92 (SEQ ID NO:258). Clone UNQ477 (DNA54002-1367)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 4648 and ending at the stop
codon at nucleotide positions 1678-1680 (FIG. 92). The predicted
polypeptide precursor is 544 amino acids long (FIG. 93). The
full-length PRO940 protein shown in FIG. 93 has an estimated
molecular weight of about 60,268 daltons and a pI of about 9.53.
Analysis of the full-length PRO940 sequence shown in FIG. 93 (SEQ
ID NO:259) evidences the presence of the following: a signal
peptide from about amino acid 1 to about amino acid 15, potential
N-glycosylation sites from about amino acid 100 to about amino acid
103, from about amino acid 297 to about amino acid 300 and from
about amino acid 306 to about amino acid 309 and an immunoglobulin
and major histocompatibility complex signature sequence block from
about amino acid 365 to about amino acid 371. Clone UNQ477
(DNA54002-1367) has been deposited with ATCC on Apr. 7, 1998 and is
assigned ATCC deposit no. 209754.
[1973] Analysis of the amino acid sequence of the full-length
PRO940 polypeptide suggests that it possesses significant sequence
similarity to CD33 and the OD binding protein-2. More specifically,
an analysis of the Dayhoff database (version 35.45 SwissProt 35)
evidenced significant homology between the PRO940 amino acid
sequence and the following Dayhoff sequences, CD33_HUMAN,
HSU71382.sub.--1, HSU71383.sub.--1, D86359.sub.--1, PGBM_HUMAN,
MAGS_MOUSE, D86983.sub.--1, C22B_HUMAN, P_W01002 and
HVU24116.sub.--1.
Example 43
[1974] Isolation of cDNA Clones Encoding Human PRO941
[1975] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA35941. An EST
sequence proprietary to Genentech was employed in the assembly and
is herein designated DNA6415 (FIG. 96; SEQ ID NO:265). Based on the
DNA35941 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 PRO941.
[1976] A pair of PCR primers (forward and reverse) were
synthesized:
92 forward PCR primer 5'-CTTGACTGTCTCTGAATCTGCACC-3' (SEQ ID NO:
266) reverse PCR primer 5'-AAGTGGTGGAAGCCTCCAGTGTGG-3' (SEQ ID NO:
267)
[1977] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA35941 sequence which
had the following nucleotide sequence
[1978] hybridization probe
93 5'-CCACTACGGTATTAGAGCAAAAGTTAAAAAC (SEQ ID NO: 268)
CATCATGGTTCCTGGAGCAGC-3'
[1979] 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 PRO941 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human fetal
kidney tissue (LIB227).
[1980] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO941 [herein designated as
UNQ478 (DNA53906-1368)] (SEQ ID NO:263) and the derived protein
sequence for PRO941.
[1981] The entire nucleotide sequence of UNQ478 (I)NA53906-1368) is
shown in FIG. 94 (SEQ ID NO:263). Clone UNQ478 (DNA53906-1368)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 37-39 and ending at the
stop codon at nucleotide positions 2353-2355 (FIG. 94). The
predicted polypeptide precursor is 772 amino acids long (FIG. 95).
The full-length PRO941 protein shown in FIG. 95 has an estimated
molecular weight of about 87,002 daltons and a pl of about 4.64.
Analysis of the full-length PRO941 sequence shown in FIG. 95 (SEQ
ID NO:264) evidences the presence of the following: a signal
peptide from about amino acid 1 to about amino acid 21, potential
N-glycosylation sites from about amino acid 57 to about amino acid
60, from about amino acid 74 to about amino acid 77, from about
amino acid 419 to about amino acid 422, from about amino acid 437
to about amino acid 440, from about amino acid 508 to about amino
acid 511, from about amino acid 515 to about amino acid 518, from
about amino acid 516 to about amino acid 519 and from about amino
acid 534 to about amino acid 537, and cadherin extracellular
repeated domain signature sequences from about amino acid 136 to
about amino acid 146 and from about amino acid 244 to about amino
acid 254. Clone UNQ478 (DNA53906-1368) has been deposited with ATCC
on Apr. 7, 1998 and is assigned ATCC deposit no. 209747.
[1982] Analysis of the amino acid sequence of the full-length
PRO941 polypeptide suggests that it possesses significant sequence
similarity to a cadherin protein, thereby indicating that PRO941
may be a novel cadherin protein family member. More specifically,
an analysis of the Dayhoff database (version 35.45 SwissProt 35)
evidenced significant homology between the PRO941 amino acid
sequence and the following Dayhoff sequences, I50180, CADA_CHICK,
I50178, GEN12782, CADC_HUMAN, P_W25637, A38992, P_R49731, D38992
and G02678.
Example 44
[1983] Isolation of cDNA Clones Encoding Human PRO944
[1984] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA47374. A
variety of proprietary Genentech EST sequences were employed in the
assembly and are shown in FIGS. 99-107. Based on the DNA47374
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 PRO944.
[1985] A pair of PCR primers (forward and reverse) were
synthesized:
94 forward PCR primer 5'-CGAGCGAGTCATGGCCAACGC-3' (SEQ ID NQ: 280)
reverse PCR primer 5'-GTGTCACACGTAGTCTTTCCCGCT-3' (SEQ ID NO:
281)
[1986] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA47374 sequence which
had the following nucleotide sequence
[1987] hybridization probe
95 5'-CTGCAGCTGTTGGGCTTCATTCTCGCCTTCC (SEQ ID NO: 282)
TGGGATGGATCG-3'
[1988] 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 PRO944 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human fetal
kidney tissue (LIB227).
[1989] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO944 [herein designated as
UNQ481 (DNA52185-1370)] (SEQ ID NO:269) and the derived protein
sequence for PRO944.
[1990] The entire nucleotide sequence of UNQ481 (DNA52185-1370) is
shown in FIG. 97 (SEQ ID NO:269). Clone UNQ481 (DNA52185-1370)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 219-221 and ending at the
stop codon at nucleotide positions 852-854 (FIG. 97). The predicted
polypeptide precursor is 211 amino acids long (FIG. 98). The
full-length PRO944 protein shown in FIG. 98 has an estimated
molecular weight of about 22,744 daltons and a pI of about 8.51.
Analysis of the full-length PRO944 sequence shown in FIG. 98 (SEQ
ID NO:270) evidences the presence of the following: a signal
peptide from about amino acid 1 to about amino acid 21,
transmembrane domains from about amino acid 82 to about amino acid
102, from about amino acid 118 to about amino acid 142 and from
about amino acid 161 to about amino acid 187, a potential
N-glycosylation site from about amino acid 72 to about amino acid
75, a sequence block having homology to PMP-22/EMP/MP2O family of
proteins from about amino acid 70 to about amino acid 111 and a
sequence block having homology to ABC-2 type transport system
integral membrane protein from about amino acid 119 to about amino
acid 133. Clone UNQ481 (DNA52185-1370) has been deposited with ATCC
on May 14, 1998 and is assigned ATCC deposit no. 209861.
[1991] Analysis of the amino acid sequence of the ftlll-lengit
PRO944 polypeptide suggests that it possesses significant sequence
similarity to the CPE-R protein, thereby indicating that PRO944 may
be a novel CPE-R homolog. More specifically, an analysis of the
Dayhoff database (version 35.45 SwissProt 35) evidenced significant
homology between the PRO944 amino acid sequence and the following
Dayhoff sequences, AB000713.sub.--1, AB000714.sub.--1,
AF035814.sub.--1, AF000959.sub.--1, HSU89916.sub.--1, EMP2_HUMAN,
JC5732, CELF53B3.sub.--6, PM22_MOUSE and CGU49797.sub.--1.
Example 45
[1992] Isolation of cDNA Clones Encoding Human PRO983
[1993] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA47473. Various
proprietary Genentech EST sequences were employed in the assembly,
wherein those EST sequences are shown in FIGS. 110-116. Based on
the DNA47473 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 PRO983.
[1994] A pair of PCR primers (forward and reverse) were
synthesized:
96 forward PCR primer 5'-GCACCACCGTAGGTACTTGTGTGAGGC-3' (SEQ ID NO:
292) reverse PCR primer 5'-AACCACCAGAGCCAAGAGCCGGG-3' (SEQ ID NO:
293)
[1995] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA47473 sequence which
had the following nucleotide sequence
[1996] hybridization probe
97 5'-CAGCGGAATCATCGATGCAGGGGCCTCAATT (SEQ ID NO: 294)
AATGTATCTGTGATGTTAC-3'
[1997] 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 PRO983 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human bone
marrow (LIB256).
[1998] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO983 [herein designated as
UNQ484 (DNA53977-1371)] (SEQ ID NO:283) and the derived protein
sequence for PRO983.
[1999] The entire nucleotide sequence of UNQ484 (DNA53977-1371) is
shown in FIG. 108 (SEQ ID NO:283). Clone UNQ484 (DNA53977-1371)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 234-236 and ending at the
stop codon at nucleotide positions 963-965 (FIG. 108). The
predicted polypeptide precursor is 243 amino acids long (FIG. 109).
The full-length PRO983 protein shown in FIG. 109 has an estimated
molecular weight of about 27,228 daltons and a pl of about 7.43.
Analysis of the full-length PRO983 sequence shown in FIG. 109 (SEQ
ID NO:284) evidences the presence of the following features: a
putative transmembrane domain from about amino acid 224 to about
amino acid 239; a potential N-glycosylation site from about amino
acid 68 to about amino acid 71; and three potential
N-myristoylation sites from about amino acid 59 to about amino acid
64, from about amino acid 64 to about amino acid 69, and from about
amino acid 235 to about amino acid 240. Clone UNQ484
(DNA53977-1371) has been deposited with ATCC on May 14, 1998 and is
assigned ATCC deposit no. 209862.
[2000] Analysis of the amino acid sequence of the full-length
PRO983 polypeptide suggests that it possesses significant sequence
similarity to the vesicle-associated protein, VAP-33, thereby
indicating that PRO983 may be a novel vesicle associated membrane
protein. More specifically, an analysis of the Dayhoff database
(version 35.45 SwissProt 35) evidenced significant homology between
the PRO983 amino acid sequence and the following Dayhoff sequences:
VP33_APLCA, CELF33D11.sub.--12, CELF42G2.sub.--2, S50623,
YDFC_SCHPO, CELF54H5.sub.--2, CELZC196.sub.--8, CEF57A10.sub.--3,
MSP3_GLORO, CEC15H11.sub.--1.
Example 46
[2001] Isolation of cDNA Clones Encoding Human PRO1057
[2002] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA49808. Based on
the DNA49808 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 PRO1057.
[2003] PCR primers (forward and reverse) were synthesized:
98 forward PCR primer 5'-GCATCTGCAGGAGAGAGCGAAGGG-3' (SEQ ID NO:
297) reverse PCR primer 5'-CATCGTTCCCGTGAATCCAGAGGC-3' (SEQ ID NO:
298)
[2004] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA49808 sequence which
had the following nucleotide sequence
[2005] hybridization probe
99 5'-GAAGGGAGGCCTTCCTTTCAGTGGACCCGGG (SEQ ID NO: 299)
TCAAGAATACCCAC-3'
[2006] 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 PRO1057 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human fetal
kidney tissue (LIB227).
[2007] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO1057 [herein designated as
UNQ522 (DNA57253-1382)] (SEQ ID NO:295) and the derived protein
sequence for PRO1057.
[2008] The entire nucleotide sequence of UNQ522 (DNA57253-1382) is
shown in FIG. 117 (SEQ ID NO:295). Clone UNQ522 (DNA57253-1382)
contains a single open reading frame withan apparent translational
initiation site at nucleotide positions 275-277 and ending at the
stop codon at nucleotide positions 1514-1516 (FIG. 117). The
predicted polypeptide precursor is 413 amino acids long (FIG. 118).
The full-length PRO1057 protein shown in FIG. 118 has an estimated
molecular weight of about 47,070 daltons and a pl of about 9.92.
Analysis of the full-length PRO1057 sequence shown in FIG. 118 (SEQ
ID NO:296) evidences the presence of the following: a signal
peptide from about amino acid 1 to about amino acid 16, potential
N-glycosylation sites from about amino acid 90 to about amino acid
93, from about amino acid 110 to about amino acid 113 and from
about amino acid 193 to about amino acid 196, a glycosaminoglycan
attachment site from about amino acid 236 to about amino acid 239
and a serine protease histidine-contaiing active site from about
amino acid 165 to about amino acid 170. Clone UNQ522
(DNA57253-1382) has been deposited with ATCC on May 14, 1998 and is
assigned ATCC deposit no. 209867.
[2009] Analysis of the amino acid sequence of the full-length
PRO1057 polypeptide suggests that it possesses significant sequence
similarity to various protease proteins, thereby indicating that
PRO1057 may be a novel protease. More specifically, an analysis of
the Dayhoff database (version 35.45 SwissProt 35) evidenced
significant homology between the PRO1057 amino acid sequence and
the following Dayhoff sequences, TRYE_DROER, P_R14159, A69660,
EBN1_EBV, S65494, GEN12688, A51084.sub.--1, P_R99571, A57514 and
AF003200.sub.--1.
Example 47
[2010] Isolation of cDNA Clones Encoding Human PRO1071
[2011] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA53035. Based on
the DNA53035 consensus sequence, it was determined that that
consensus sequence shared significant sequence identity with Incyte
EST clone no. 2872569, a clone that upon review appeared to encode
a full length protein. As such, Incyte EST clone no. 2872569 was
purchased and its insert was obtained and sequenced so as to
confirm the proper sequence. This sequence is herein designated
UNQ528 or DNA58847-1383.
[2012] DNA sequencing of the clone isolated as described above gave
the lull-length DNA sequence for PRO1071 [herein designated as
UNQ528 (DNA58847-1383)] (SEQ ID NO:300) and the derived protein
sequence for PRO1071.
[2013] The entire nucleotide sequence of UNQ528 (DNA58847-1383) is
shown in FIG. 119 (SEQ ID NO:300). Clone UNQ528 (DNA58848-1383)
contains a single openreading frame with an apparei translational
initiation site at nucleotide positions 133-135 and ending at the
stop codon at nucleotide positions 1708-1710 (FIG. 119). The
predicted polypeptide precursor is 525 amino acids long (FIG. 120).
The flull-length PRO1071 protein shown in FIG. 120 has an estimated
molecular weight of about 58,416 daltons and a pI of about 6.62.
Analysis of the fullength PRO1071 sequence shown in FIG. 120 (SEQ
ID NO:301) evidences the presence of the following: a signal
peptide from about amino acid 1 to about amino acid 25, a potential
N-glycosylation site from about amino acid 251 to about amino acid
254, a thrombospondin-1 homology block from about amino acid 385 to
about amino acid 399 and von Willibrands factor type C homology
blocks from about amino acid 385 to about amino acid 399, from
about amino acid 445 to about amino acid 459 and from about amino
acid 42 to about amino acid 56. Clone UNQ528 (DNA58847-1383) has
been deposited with ATCC on May 20, 1998 and is assigned ATCC
deposit no. 209879.
[2014] Analysis of the amino acid sequence of the full-length
PRO1071 polypeptide suggests that it possesses significant sequence
similarity to the thrombospondin protein, thereby indicating that
PRO1071 may be a novel thrombospondin homolog. More specifically,
an analysis of the Dayhoff database (version 35.45 SwissProt 35)
evidenced significant homology between the PRO1071 amino acid
sequence and the following Dayhoff sequences, AB002364.sub.--1,
D67076.sub.--1, BTPCINPGN.sub.--1, CET13H10.sub.--1,
CEF25H8.sub.--5, CEF53B6.sub.--2, CEC26C6.sub.--6, HSSEMG.sub.--1,
CET21B6.sub.--4 and BTY08561.sub.--1.
Example 48
Isolation of cDNA Clones Encoding Human PRO1072
[2015] A consensus sequence was obtained relative to a variety of
EST sequences as described in Examnple 1 above, wherein the
consensus sequence obtained is herein designated DNA53125. Based on
the DNA53125 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 PRO1072.
[2016] PCR primers (forward and reverse) were synthesized:
100 forward PCR primer 5'-CCAGGAAATGCTCCAGGAAGAGCC-3' (SEQ ID NO:
305) reverse PCR primer 5'-GCCCATGACACCAAATTGAAGAGTGG-3' (SEQ ID
NO: 306)
[2017] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA53125 sequence which
had the following nucleotide sequence
[2018] hybridization probe
101 5'-AACGCAGGGATCTTCCAGTGCCCTTACATGA (SEQ ID NO: 307)
AGACTGAAGATGGG-3'
[2019] 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 PRO1072 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human fetal
lung tissue (LIB26).
[2020] DNA sequencing of the clones isolated as described above
gave the fuil-length DNA sequence for PRO1072 [herein designated as
UNQ529 (DNA58747-1384)] (SEQ ID NO:302) and the derived protein
sequence for PRO1072.
[2021] The entire nucleotide sequence of UNQ529 (DNA58747-1384) is
shown in FIG. 121 (SEQ ID NO:302). Clone UNQ529 (DNA58747-1384)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 65-67 and ending at the
stop codon at nucleotide positions 1073-1075 (FIG. 121). The
predicted polypeptide precursor is 336 amino acids long (FIG. 122).
The full-length PRO1072 protein shown in FIG. 122 has an estimated
molecular weight of about 36,865 daltons and a pl of about 9.15.
Analysis of the full-length PRO1072 sequence shown in FIG. 122 (SEQ
ID NO:303) evidences the presence of the following: a signal
peptide from about amino acid 1 to about amino acid 21, short-chain
alcohol dehydrogenase protein homology blocks from about amino acid
134 to about amino acid 144, from about amino acid 44 to about
amino acid 56 and from about amino acid 239 to about amino acid 248
and potential N-glycosylation sites from about amino acid 212 to
about amino acid 215 and from about amino acid 239 to about amino
acid 242. Clone UNQ529 (DNA58747-1384) has been deposited with ATCC
on May 14, 1998 and is assigned ATCC deposit no. 209868.
[2022] Analysis of the amino acid sequence of the full-length
PRO1072 polypeptide suggests that it possesses significant sequence
similarity to the reductase family of proteins, thereby indicating
that PRO1072 may be a novel reductase. More specifically, an
analysis of the Dayhoff database (version 35.45 SwissProt 35)
evidenced significant homology between the PRO1072 amino acid
sequence and the following Dayhoff sequences,
P_W03198,P_W15759,P_R60800, MTV037.sub.--3,
CEC15H11.sub.--6,ATAC00234314- , MTV022.sub.--13, SCU43704.sub.--1,
OXIR_STRAT AND CELC01G8.sub.--3.
Example 49
[2023] Isolation of cDNA Clones Encoding Human PRO1075
[2024] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA34363. Based on
the DNA34363 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
fAll-length coding sequence for PRO1075.
[2025] PCR primers (forward and reverse) were synthesized:
102 forward PCR primer 5'-TGAGAGGCCTCTCTGGAAGTTG-3' (SEQ ID NQ:
312) forward PCR primer 5'-GTCAGCGATCAGTGAAAC-3' (SEQ ID NO: 313)
forward PCR primer 5'-CCAGAATGAAGTAGCTCGGC-3' (SEQ ID NO: 314)
forward PCR primer 5'-CCGACTCAAAATGCATTGTC-3' (SEQ ID NO: 315)
forward PCR primer 5'-CATTTGGCAGGAATTGTCC-3' (SEQ ID NO: 316)
forward PCR primer 5'-GGTGCTATAGGCCAAGGGG-3' (SEQ ID NO: 317)
reverse PCR primer 5'-CTGTATCTCTGGGCTATGTCAGAG-3' (SEQ ID NO: 318)
reverse PCR primer 5'-CTACATATAATGGCACATGTCAGCC-3' (SEQ ID NO:
319)
[2026] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA34363 sequence which
had the following nucleotide sequence
[2027] hybridization probe
103 5'-CGTCTTCCTATCCTTACCCGACCTCAGATGC (SEQ ID NO: 320)
TCCCTTCTGCTCCTG-3'
[2028] 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 primner pair identified above. A
positive library was then used to isolate clones encoding the
PRO1075 gene using the probe oligonucleotide and one of the PCR
primers. RNA for construction of the cDNA libraries was isolated
from human skin tumor tissue (L[1324).
[2029] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO1075 [herein designated as
UNQ532 (DNA57689-1385)] (SEQ ID NO:308) and the derived protein
sequence for PRO1075.
[2030] The entire nucleotide sequence of UNQ532 (D)NA57689-1385) is
shown in FIG. 124 (SEQ ID NO:308). Clone UNQ532 (DNA57689-1385)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 137-139 and ending at the
stop codon at nucleotide positions 1355-1357 (FIG. 124). The
predicted polypeptide precursor is 406 amino acids long (FIG. 125).
The full-length PRO1075 protein shown in FIG. 125 has an estimated
molecular weight of about 46,927 daltons and a pl of about 5.21.
Analysis of the full-length PRO1075 sequence shown in FIG. 125 (SEQ
ID NO:309) evidences the presence of the following: a signal
peptide from about amino acid 1 to about amino acid 29, an
endoplasmic reticulum targeting sequence from about amino acid 403
to about amino acid 406, a tyrosine kinase phosphorylation site
from about amino acid 203 to about amino acid 211 and a sequence
block having homology to the thioredoxin family of proteins from
about amino acid 50 to about amino acid 66. Clone UNQ532
(DNA57689-1385) has been deposited with ATCC on May 14, 1998 and is
assigned ATCC deposit no. 209869.
[2031] Analysis of the amino acid sequence of the full-length
PRO1075 polypeptide suggests that it possesses significant sequence
similarity to protein disulfide isomerase, thereby indicating that
PRO1075 may be a novel protein disulfide isomerase. More
specifically, an analysis of the Dayhoff database (version 35.45
SwissProt 35) evidenced significant homology between the PRO1075
amino acid sequence and the following Dayhoff sequences,
CELC30H7.sub.--2, CELC06A6.sub.--3, CELF42G8.sub.--3, S57942,
ER72_CAEEL, CELC07A12.sub.--3, CEH06O01.sub.--4 and P_R51696.
Example 50
[2032] Isolation of cDNA Clones Encoding Human PRO181
[2033] A cDNA sequence isolated in the amylase screen described in
Example 2 above was found, by BLAST and FastA sequence alignment,
to have sequence homology to a nucleotide sequence encoding the
cornichon protein. This cDNA sequence is herein designated DNA13242
(FIG. 130; SEQ ID NO:323). Based on the sequence homology,
oligonucleotide probes were generated from the sequence of the DNA
13242 molecule and used to screen a human placenta (LIB89) library
prepared as described in paragraph 1 of Example 2 above. The
cloning vector was pRK5B (pRK5B is a precursor of pRK5D that does
not contain the Sf1I site; see, Holmes et al., Science,
253:1278-1280 (1991)), and the cDNA size cut was less than 2800
bp.
[2034] The oligonucleotide probes employed included:
104 forward PCR primer 5'-GTGCAGCAGAGTGGCTTACA-3' (SEQ ID NO: 326)
reverse PCR primer 5'-ACTGGACCAATTCTTCTGTG-3' (SEQ ID NO: 327)
hybridization probe 5'-GATATTCTAGCATATTGTCAGAAGGAAGGAT (SEQ ID NO:
328) GGTGCAAATTAGCT-3'
[2035] A full length clone was identified that contained a single
open reading frame with an apparent translational initiation site
at nucleotide positions 14-16 and ending at the stop codon found
atnucleotide positions 446448 (FIG. 128; SEQ ID NO:321). The
predicted polypeptide precursor is 144 amino acids long, has a
calculated molecular weight of approximately 16,699 daltons and an
estimated pI of approximately 5.6. Analysis of the fulT-length
PRO181 sequence shown in FIG. 129 (SEQ ID NO:322) evidences the
presence of the following: a signal peptide from about amino acid 1
to about amino acid 20, a putative type II transmembrane domain
from about amino acid 11 to about amino acid 31 and other
transmembrane domains from about amino acid 57 to about amino acid
77 and from about amino acid 123 to about amino acid 143. Clone
UNQ155 (DNA23330-1390) has been deposited with ATCC on Apr. 14,
1998 and is assigned ATCC deposit no. 209775.
[2036] Analysis of the amino acid sequence of the full-length
PRO181 polypeptide suggests that it possesses significant sequence
similarity to the cornichon protein, thereby indicating that PRO181
may be a novel cornichon homolog. More specifically, an analysis of
the Dayhoff database (version 35.45 SwissProt 35) evidenced
significanthomology betweenthe PRO181 amino acidsequence andthe
following Dayhoff sequences, AF022811.sub.--1, CET09E8.sub.--3,
S64058, YGF4_YEAST, YB60_YEAST, EBU89455.sub.--1, SIU36383.sub.--3
and PH1371.
Example 51
[2037] Isolation of cDNA Clones Encoding Human PRO195
[2038] A cDNA sequence was isolated in the amylase screen described
in Example 2 above and is herein designated DNA13199 (FIG. 134; SEQ
ID NO:332). The DNA13199 sequence was then compared to a variety of
expressed sequence tag (EST) databases which included public EST
databases (e.g., GenBank) to identify existing homologies. The
homology search was performed using the computer program BLAST or
BLAST2 (Altshul et al., Methods in Enzymology 266:460480 (1996)).
Those comparisons resulting in a BLAST score of 70 (or in some
cases 90) or greater that did not encode known proteins were
clustered and assembled into consensus DNA sequences with the
program "phrap" (Phil Green, University of Washington, Seattle,
Wash.; http://bozeman.mbt.washington.edu/phrap.docs/- phrap.html).
The consensus sequence obtained therefrom is herein designated as
DNA22778.
[2039] Based on the DNA22778 sequence, oligonucleotide probes were
generated and used to screen a human placenta library (LIB89)
prepared as described in paragraph 1 of Example 2 above. The
cloning vector was pRK5B (pRKSB is a precursor of pRK5D that does
not contain the SfRI site; see, Holmes et al., Science,
253:1278-1280 (1991)), and the cDNA size cut was less than 2800
bp.
[2040] PCR primers (forward and reverse) were synthesized:
105 forward PCR primer 5'-ACAAGCTGAGCTGCTGTGACAG-3' (SEQ ID NO:
333) reverse PCR primer 5'-TGATTCTGGCAACCAAGATGGC-3' (SEQ ID NO:
334)
[2041] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the DNA22778 sequence which had the
following nucleotide sequence
[2042] hybridization probe
106 5'-ATGGCCTTGGCCGGAGGTTCGGGGACCGCTT (SEQ ID NO: 335)
CGGCTGAAG-3'
[2043] 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 PRO195 gene
using the probe oligonucleotide and one of the PCR primers.
[2044] A full length clone was identified that contained a single
open reading frame with an apparent translational initiation site
at nucleotide positions 70-72 and ending atthe stop codon found at
nucleotide positions 1039-1041 (FIG. 132; SEQ ID NO:330). The
predicted polypeptide precursor is 323 amino acids long, has a
calculated molecular weight of approximately 36,223 daltons and an
estimated pl of approximately 5.06. Analysis of the fuil-length
PRO195 sequence shown in FIG. 132 (SEQ ID NO:330) evidences the
presence of the following: a signal peptide from about amino acid 1
to about amino acid 31, a transmembrane domain from about amino
acid 241 to about amino acid 260 and a potential N-glycosylation
site from about amino acid 90 to about amino acid 93. Clone UNQ169
(DNA26847-1395) has been deposited with ATCC on Apr. 14, 1998 and
is assigned ATCC deposit no. 209772.
[2045] Analysis of the amino acid sequence of the full-length
PRO195 polypeptide suggests that it possesses no significant
sequence similarity to any known protein. However, an analysis of
the Dayhoff database (version 35.45 SwissProt 35) evidenced some
degree of homology between the PRO195 amino acid sequence and the
following Dayhoff sequences, P_P191380, AF035118.sub.--1,
HUMTROPCS.sub.--1, NUOD_SALTY and E70002.
Example 52
[2046] Isolation of cDNA Clones Encoding Human PRO865
[2047] A cDNA sequence isolated in the amylase screen described in
Example 2 above was herein designated DNA37642 (FIG. 137, SEQ ID
NO:338). The DNA37642 sequence was then compared to a variety of
expressed sequence tag (EST) databases which included public EST
databases (e.g., GenBank) and a proprietary EST DNA database
(LIFESEQ.TM., Incyte Pharmaceuticals, Palo Alto, Calif.) to
identify homologies therebetween. The homology search was performed
using the computer program BLAST or BLAST2 (Altshul et al., Methods
in Enzymology 266:460-480 (1996)). Those comparisons resulting in a
BLAST score of 70 (or in some cases 90) or greater that did not
encode known proteins were clustered and assembled into consensus
DNA sequences with the program "phrap" (Phil Green, University of
Washington, Seattle, Wash.;
http://bozeman.mbt.washington.edu/phrap.docs/- phrap.html). The
consensus sequence obtained is herein designated DNA48615.
[2048] Based on the DNA48615 consensus sequence, probes were
generated and used to screen a human fetal kidney (LIB227) library
prepared as described in paragraph 1 of Example 2 above. The
cloning vector was pRK5B (pRK5B is a precursor of pRK5D that does
not contain the Sfil site; see, Holmes et al., Science,
253:1278-1280 (1991)), and the cDNA size cut was less than 2800
bp.
[2049] PCR primers (forward and reverse) were synthesized:
107 forward PCR primer 1 5'-AAGCTGCCGGAGCTGCAATG-3' (SEQ ID NO:
339) forward PCR primer 2 5'-TTGCTTCTTAATCCTGAGCGC-3' (SEQ ID NO:
340) forward PCR primer 3 5'-AAAGGAGGACTTTCGACTGC-3' (SEQ ID NO:
341) reverse PCR primer 1 5'-AGAGATTCATCCACTGCTCCAAGTCG-3' (SEQ ID
NO: 342) reverse PCR primer 2 5'-TGTCCAGAAACAGGCACATATCAGC-3' (SEQ
ID NO: 343)
[2050] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA48615 sequence which
had the following nucleotide sequence
[2051] hybridization probe
108 5'-AGACAGCGGCACAGAGGTGCTTCTGCCAGGT (SEQ ID NO: 344)
TAGTGGTTACTTGGATGAT-3'
[2052] 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
PRO865 gene using the probe oligonucleotide and one of the PCR
primers.
[2053] A full length clone was identified that contained a single
open reading frame with an apparent translational initiation site
at nucleotide positions 173-175 and ending at the stop codon found
at nucleotide positions 1577-1579 (FIG. 135; SEQ ID NO:336). The
predicted polypeptide precursor is 468 amino acids long, has a
calculated molecular weight of approximately 54,393 daltons and an
estimated pI of approximately 5.63. Analysis of the full-length
PRO865 sequence shown in FIG. 136 (SEQ ID NO:337) evidences the
presence of the following: a signal peptide from about amino acid 1
to about amino acid 23, potential N-glycosylation sites from about
amino acid 280 to about amino acid 283 and from about amino acid
384 to about amino acid 387, a potential amidation site from about
amino acid 94 to about amino acid 97, glycosaminoglycan attachment
sites from about amino acid 20 to about amino acid 23 and from
about amino acid 223 to about amino acid 226, an aminotransferase
class-V pyridoxyl-phosphate amino acid sequence block from about
amino acid 216 to about amino acid 222 and an amino acid sequence
block similar to that found in the interleukin-7 protein from about
amino acid 338 to about amino acid 343. Clone UNQ434
(DNA53974-1401) has been deposited with ATCC on Apr. 14, 1998 and
is assigned ATCC deposit no. 209774.
[2054] Analysis of the amino acid sequence of the full-length
PRO865 polypeptide suggests that it possesses no significant
sequence similarity to any known protein. However, an analysis of
the Dayhoff database (version 35.45 SwissProt 35) evidenced some
degree of homology between the PRO865 amino acid sequence and the
following Dayhoff sequences, YMN0_YEAST, ATFCA4.sub.--43, S44168,
P_W14549 and RABTCRG4.sub.--1.
Example 53
[2055] Isolation of cDNA Clones Encoding Human PRO827
[2056] A cDNA sequence isolated in the amylase screen described in
Example 2 above was found, by BLAST and FastA sequence alignment,
to have sequence homology to nucleotide sequences encoding various
integrin proteins. This cDNA sequence is herein designated DNA47751
(see FIG. 140; SEQ ID NO:347). Based on the sequence homology,
probes were generated from the sequence of the DNA47751 molecule
and used to screen a human fetal pigment epithelium library
(LIB113) prepared as described in paragraph 1 of Example 2 above.
The cloning vector was pRK5B (pRKSB is a precursor of pRK5D that
does not contain the SfiI site; see, Holmes et al., Science
253:1278-1280 (1991)), and the cDNA size cut was less than 2800
bp.
[2057] PCR primers (forward and reverse) were synthesized:
109 forward PCR primer 5'-AGGGACAGAGGCCAGAGGACTTC-3' (SEQ ID NO:
348) reverse PCR primer 5'-CAGGTGCATATTCACAGCAGGATG-3' (SEQ ID NO:
349)
[2058] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA47751 sequence which
had the following nucleotide sequence
[2059] hybridization probe
110 5'-GGAACTCCCCTTCGTCACTCACCTGTTCTTG (SEQ ID NO: 350)
CCCCTGGTGTTCCT-3'
[2060] In order to screen several libraries for a source of a
fullength 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 PRO827 gene
using the probe oligonucleotide and one of the PCR primers.
[2061] A full length clone was identified that contained a single
open reading frame with an apparent translational initiation site
at nucleotide positions 134-136 and ending at the stop codon found
at nucleofide positions 506-508 (FIG. 138; SEQ ID NO:345). The
predicted polypeptide precursor is 124 amino acids long, has a
calculated molecular weight of approximately 13,352 daltons and an
estimated pI of approximately 5.99. Analysis of the full-length
PRO827 sequence shown in FIG. 139 (SEQ ID NO:346) evidences the
presence of the following: a signal peptide from about amino acid 1
to about amino acid 22, a cell attachment sequence from about amino
acid 70 to about amino acid 72, a potential N-glycosylation site
from about amino acid 98 to about amino acid 101 and an integrin
alpha chain protein homology sequence from about amino acid 67 to
about amino acid 81. Clone UNQ468 (DNA57039-1402) has been
deposited with ATCC on Apr. 14, 1998 and is assigned ATCC deposit
no. 209777.
[2062] Analysis of the amino acid sequence of the full-length
PRO827 polypeptide suggests that it possesses significant sequence
similarity to the VLA-2 integrin protein and various other integrin
proteins, thereby indicating that PRO827 may be a novel integrin or
splice variant thereof. More specifically, an analysis of the
Dayhoff database (version 35.45 SwissProt 35) evidenced significant
homology between the PRO240 amino acid sequence and the following
Dayhoff sequences, S44142, ITA2_HUMAN, ITA1_RAT, ITA1_HUMAN,
ITA4_HUMAN, ITA9_HUMAN, AF032108.sub.--1, ITAM_MOUSE, ITA8_CHICK
and ITA6_CHICK.
Example 54
[2063] Isolation of cDNA Clones EncodinL Human PRO1114
[2064] A cDNA sequence isolated in the amylase screen described in
Example 2 was found, by the WU-BLAST2 sequence alignment computer
program, to have certain sequence identity to other known
interferon receptors. This cDNA sequence is herein designated
DNA48466 (FIG. 143; SEQ ID NO:352). Based on the sequence identity,
probes were generated from the sequence of the DNA48466 molecule
and used to screen a human breast carconoma library (LIB135)
prepared as described in paragraph 1 of Example 2 above. The
cloning vector was pRKSB (pRK5B is a precursor of pRKSD that does
not contain the SfiI site; see, Holmes et al., Science,
253:1278-1280 (1991)), and the cDNA size cut was less than 2800
bp.
111 forward PCR primer 5'-AGGCTTCGCTGCGACTAGACCTC-3' (SEQ ID NO:
354) reverse PCR primer 5'-CCAGGTCGGGTAAGGATGGTTGAG-3' (SEQ ID NO:
355) hybridization probe 5'-TTTCTACGCATTGATTCCATGTTTGCTCACA (SEQ ID
NO: 356) GATGAAGTGGCCATTCTGC-3'
[2065] A full length clone was identified that contained a single
open reading frame with an apparent translational initiation site
at nucleotide positions 250-252, and a stop signal at nucleotide
positions 1183-1185 (FIG. 141, SEQ ID NO:351). The predicted
polypeptide precursor is 311 amino acids long, has a calculated
molecular weight of approximately 35,076 daltons and an estimated
pl of approximately 5.04. Analysis of the fiull-length PRO1114
interferon receptor sequence shown in FIG. 142 (SEQ ID NO:352)
evidences the presence of the following: a signal peptide from
about amino acid 1 to about amino acid 29, a transmembrane domain
from about amino acid 230 to about amino acid 255, potential
N-glycosylation sites from about amino acid 40 to about amino acid
43 and from about amino acid 134 to about amino acid 137, an amino
acid sequence block having homology to tissue factor proteins from
about amino acid 92 to about amino acid 119 and an amino acid
sequence block having homology to integrin alpha chain proteins
from about amino acid 232 to about amino acid 262. Clone UNQ557
(DNA57033-1403) has been deposited with ATCC on May 27, 1998 and is
assigned ATCC deposit no. 209905.
[2066] 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. 142 (SEQ ID NO:352), evidenced
significant homology between the PROL 114 interferon receptor amino
acid sequence and the following Dayhoff sequences: G01418,
INR1_MOUSE, P_R71035, INGS_HUMAN, A26595.sub.--1, A26593.sub.--1,
I56215 and TF_HUMAN.
Example 55
[2067] Isolation of cDNA Clones Encoding Human PRO237
[2068] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA30905. Based on
the DNA30905 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 PRO237.
[2069] PCR primers (forward and reverse) were synthesized:
112 forward PCR primer 5'-TCTGCTGAGGTGCAGCTCATrCAC-3' (SEQ ID NO:
359) reverse PCR primer 5'-GAGGCTCTGGAAGATCTGAGATGG-3' (SEQ ID NO:
360)
[2070] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA30905 sequence which
had the following nucleotide sequence
[2071] hybridization probe
113 5'-GCCTCTTTGTCAACGTTGCCAGTACCTCTAA (SEQ ID NO: 361)
CCCATTCCTCAGTCGCCTC-3'
[2072] 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 PRO237 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human fetal
brain tissue (LIB153).
[2073] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO237 herein designated as
UNQ211 (DNA34353-1428)] (SEQ ID NO:357) and the derived protein
sequence for PRO237.
[2074] The entire nucleotide sequence of UNQ211 (DNA34353-1428) is
shown in FIG. 144 (SEQ ID NO:357). Clone UNQ211 (DNA34353-1428)
contains a single open reading framewith an apparent translational
initiation site at nucleotide positions 586-588 and ending at the
stop codon at nucleotide positions 1570-1572 (FIG. 144). The
predicted polypeptide precursor is 328 amino acids long (FIG. 145).
The full-length PRO237 protein shown in FIG. 145 has an estimated
molecular weight of about 36,238 daltons and a pl of about 9.90.
Analysis of the full-length PRO237 sequence shown in FIG. 145 (SEQ
ID NO:358) evidences the presence of the following: a signal
peptide from about amino acid 1 to about amino acid 23, a
transmembrane domain from about amino acid 177 to about amino acid
199, potential N-glycosylation sites from about amino acid 118 to
about amino acid 121, from about amino acid 170 to about amino acid
173 and from about amino acid 260 to about amino acid 263 and
eukaryotic-type carbonic anhydrase sequence homology blocks from
about amino acid 222 to about amino acid 270, from about amino acid
128 to about amino acid 164 and from about amino acid 45 to about
amino acid 92. Clone UNQ211 (DNA34353-1428) has been deposited with
ATCC on May 12, 1998 and is assigned ATCC deposit no. 209855.
[2075] Analysis of the amino acid sequence of the full-length
PRO237 polypeptide suggests that it possesses significant sequence
similarity to the carbonic anhydrase protein. More specifically, an
analysis of the Dayhoff database (version35.45 SwissProt35)
evidenced significanthomology betweenthe PRO237 amino acid sequence
and the following Dayhoff sequences, AF050106.sub.--1,
OACALP.sub.--1, CELD1022.sub.--8, CAH2_HUMAN, 1CAC, CAH5_HUMAN,
CAHP_HUMAN, CAH3_HUMAN, CAH1_HUMAN and 2CAB.
Example 56
[2076] Isolation of cDNA Clones Encoding Human PRO541
[2077] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA42259. Based on
the DNA42259 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 PRO541.
[2078] PCR primers (forward and reverse) were synthesized:
114 forward PCR primer 5'-GGACAGAATTTGGGAGCACACTGG-3' (SEQ ID NO:
364) forward PCR primer 5'-CCAAGAGTATACTGTCCTCG-3' (SEQ ID NO: 365)
reverse PCR primer 5'-AGCACAGATTTTCTCTACAGCCCCC-3' (SEQ ID NO: 366)
reverse PCR primer 5'-AACCACTCCAGCATGTACTGCTGC-3' (SEQ ID NO:
367)
[2079] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA42259 sequence which
had the following nucleotide sequence
[2080] hybridization probe
115 5'-CCATTCAGGTGTTCTGGCCCTGTATGTACAC (SEQ ID NO: 368)
ATTATACACAGGTCGTGTG-3'
[2081] In order to screen several libraries for a source of a
fiill-length clone, DNA from the libraries was screened by PCR
amplification with one of the PCR primer pairs identified above. A
positive library was then used to isolate clones encoding the
PRO541 gene using the probe oligonucleotide and one of the PCR
primers. RNA for construction of the cDNA libraries was isolated
froin human fetal kidney tissue (LIB227).
[2082] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO541 [herein designated as
UNQ342 (DNA45417-1432)] (SEQ ID NO:362) and the derived protein
sequence for PRO541.
[2083] The entire nucleotide sequence of UNQ342 (DNA45417-1432) is
shown in FIG. 146 (SEQ ID NO:362). Clone UNQ342 (DNA45417-1432)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 469-471 and ending at the
stop codon at nucleotide positions 1969-1971 (FIG. 146). The
predicted polypeptide precursor is 500 amino acids long (FIG. 147).
The fuill-length PRO541 protein shown in FIG. 147 has an estimated
molecular weight of about 56,888 daltons and a pl of about 8.53.
Analysis of the full-length PRO541 sequence shown in FIG. 147 (SEQ
ID NO:363) evidences the presence of the following: a signal
peptide from about amino acid 1 to about amino acid 20, amino acid
sequence blocks having homology to extracellular proteins
SCP/Tpx-1/Ag5/PR-1/Sc7 from about amino acid 165 to about amino
acid 186, from about amino acid 196 to about amino acid 218, from
about amino acid 134 to about amino acid 146, from about amino acid
96 to about amino acid 108 and from about amino acid 58 to about
amino acid 77 and a potential N-glycosylation site from about amino
acid 28 to about amino acid 31. Clone UNQ342 (DNA45417-1432) has
been deposited with ATCC on May27, 1998 and is assigned ATCC
deposit no. 209910.
[2084] Analysis of the amino acid sequence of the full-length
PRO541 polypeptide suggests that it possesses significant sequence
similarity to a trypsin inhibitor protein, thereby indicating that
PRO541 may be a novel trypsin inhiubitor. More specifically, an
analysis of the Dayhoff database (version 35.45 SwissProt 35)
evidenced significant homology between the PRO541 amino acid
sequence and the following Dayhoff sequences, D45027.sub.--1,
AB009609.sub.--1, JC5308, CRS3_HORSE, TPX1_HUMAN, HELO_HELHO,
GEN14351, A28112.sub.--1, CET05A10.sub.--4 and P_W11485.
Example 57
[2085] Isolation of cDNA Clones Encoding Human PRO273
[2086] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA36465. Based on
the DNA36465 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 PRO273.
[2087] A pair of PCR primers (forward and reverse) were
synthesized:
116 forward PCR primer 5'-CAGCGCCCTCCCCATGTCCCTG-3' (SEQ ID NO:
371) reverse PCR primer 5'-TCCCAACTGGTTTGGAGTTTTCCC-3' (SEQ ID NO:
372)
[2088] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA36465 sequence which
had the following nucleotide sequence
[2089] hybridization probe
117 5'-CTCCGGTCAGCATGAGGCTCCTGGCGGCCGC (SEQ ID NO: 373)
TGCTCCTGCTGCTG-3'
[2090] 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 PRO273 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human fetal
kidney tissue.
[2091] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO273 [herein designated as
UNQ240 (DNA39523-1192)] (SEQ ID NO:369) and the derived protein
sequence for PRO273.
[2092] The entire nucleotide sequence of UNQ240 (DNA39523-1192) is
shown in FIG. 148 (SEQ ID NO:369). Clone UNQ240 (DNA39523-1192)
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 500-502 (FIG. 148). The
predicted polypeptide precursor is 111 amino acids long (FIG. 149).
Clone UNQ240 (DNA39523-1192) has been deposited with the ATCC. It
is understood that the deposited clone contains the actual sequence
and that the sequences provided herein are merely representative
based on current sequencing techniques. Moreover, given the
sequences provided herein and knowledge of the universal genetic
code, the corresponding nucleotides for any given amino acid can be
routinely identified and vice versa.
[2093] Analysis of the amino acid sequence of the full-length
PRO273 polypeptide suggests that portions of it possess sequence
identity with human macrophage inflammatory protein-2,
cytokine-induced neutrophil chemoattractant 2, and neutrophil
chemotactic factor 2-beta, thereby indicating that PRO273 is a
novel chemokine.
[2094] As discussed further below, the cDNA was subcloned into a
baculovirus vector and expressed in insect cells as a C-terminally
tagged IgG fusion protein. N-terminal sequencing of the resultant
protein identified the signal sequence cleavage site, yielding a
mature polypeptide of 77 amino acids. The mature sequence, showing
31-40% identity to other human CXC chemokines, includes the four
canonical cysteine residues but lacks the ELR motif. Northern
analysis demonstrates expression at least in the small intestine,
colon, spleen, lymph node and kidney. By in situ hybridization,
also described in detail below, mRNA is localized to the lamina
propria of intestinal vrlli and to renal tubules.
Example 58
[2095] Isolation of cDNA Clones Encoding Human PRO701
[2096] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example I above, wherein the
consensus sequence obtained is herein designated DNA39848. Based on
the DNA39848 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 PRO701.
[2097] A pair of PCR primers (forward and reverse) were
synthesized:
118 forward PCR primer 5'-GGCAAGCTACGGAAACGTCATCGTG-3' (SEQ ID NO:
376) reverse PCR primer 5'-AACCCCCGAGCCAAAAGATGGTCAC-3' (SEQ ID NO:
377)
[2098] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA39848 sequence which
had the following nucleotide sequence:
[2099] hybridization probe
119 5'-GTACCGGTGACCAGGCAGCAAAAGGCAACTATGGGCTCCTGGATCAG-3' (SEQ ID
NO: 378).
[2100] 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 PRO701 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human fetal
kidney tissue (L11B227).
[2101] DNA sequencing of the clones isolated as described above
gave the fillf-length DNA sequence for PRO701 [herein designated as
UNQ365 (DNA44205-1285)] (SEQ ID NO:374) and the derived protein
sequence for PRO701.
[2102] The entire nucleotide sequence of UNQ365 (DNA44205-1285) is
shown in FIG. 150 (SEQ ID NO:374). Clone UNQ365 (DNA44205-1285)
contains a single open reading frame with an apparenttranslational
initiation site at nucleotide positions 50-52 and ending at the
stop codon at nucleotide positions 2498-3000 (FIG. 150). The
predicted polypeptide precursor is 816 amino acids long (FIG. 151).
The full-length PRO701 protein shown in FIG. 151 has an estimated
molecular weight of about 91,794 daltons, a pl of about 5.88 and
NX(SIT) being 4. Clone UNQ365 (DNA44205-1285) has been deposited
with the ATCC on Mar. 31, 1998. It is understood that the clone was
the correct and actual sequence, wherein the sequences provided
herein are representative based on sequencing techniques.
[2103] Still regarding the amino acid sequence shown in FIG. 151,
there is a potential signal peptide cleavage site at about amino
acid 25. There are potential N-glycosylation sites at about amino
acid positions 83, 511, 716 and 803. The carboxylesterases type-B
signature 2 sequence is at about residues 125 to 135. Regions
homologous with carboxylesterase type-B are also at about residues
54-74, 197-212 and 221-261. A potential trausmembrane region
corresponds approximately to amino acids 671 through about 700. The
corresponding nucleic acids can be routinely determined from the
sequences provided herein.
[2104] Analysis of the amino acid sequence of the full-length
PRO701 polypeptide suggests that it possess significant homology to
the neuroligins from rattus norvegicus indicating that PRO701 may
be a novel human neuroligin.
Example 59
[2105] Isolation of cDNA Clones Encoding Human PRO704
[2106] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA43033. Based on
the DNA43033 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 PRO704.
[2107] A pair of PCR primers (forward and reverse) were
synthesized:
120 forward PCR primer 5'-CCTTGGGTCGTGGCAGCAGTGG-3' (SEQ ID
NO:381); reverse PCR primer 5'-CACTCTCCAGGCTGCATGCTCAGG-- 3' (SEQ
ID NQ:382).
[2108] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the DNA43033 consensus sequence which
had the following nucleotide sequence:
[2109] hybridization probe
121 5'-GTCAAACGTTCGAGTACTTGAAACGGGAGCACTCGCTGTCGAAGC-3' (SEQ ID NO:
383).
[2110] 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 PRO704 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human fetal
kidney tissue (LIB227).
[2111] DNA sequencing of the clones isolated as described above
gave the futl-length DNA sequence for PRO704 [herein designated as
UNQ368 (DNA50911-1288)] (SEQ ID NO:379) and the derived protein
sequence for PRO704.
[2112] The entire nucleotide sequence of UNQ368 (DNA50911-1288) is
shown in FIG. 152 (SEQ ID NO:379). Clone UNQ368 (DNA50911-1288)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 8-10 and ending at the stop
codon at nucleotide positions 1052-1054 (FIG. 152). The predicted
polypeptide precursor is 348 amino acids long (FIG. 153). The
full-length PRO704 protein shown in FIG. 153 has an estimated
molecular weight of about 39,711 and a pl of about 8.7. Clone
UNQ368 (DNA50911-1288) has been deposited with the ATCC on Mar. 31,
1998. Regarding the sequence, it is understood that the deposited
clone contains the correct sequence, and the sequences provided
herein are based on known sequencing techniques.
[2113] Analysis of the amino acid sequence of the full-length
PRO704 polypeptide suggests that portions of it possess significant
homology to the vesicular integral membrane protein 36, thereby
indicating that PRO704 may be a novel vesicular integral membrane
protein.
[2114] Still analyzing the amino acid sequence of SEQ ID NO:380,
the putative signal peptide is at about amino acids 1-39 of SEQ ID
NO:380. The transmembrane domain is at amino acids 310-335 of SEQ
ID NO:380. A potential N-glycosylation site is at about amino acids
180-183 of SEQ ID NO:380. The corresponding nucleotides can be
routinely determined given the sequences provided herein.
Example 60
[2115] Isolation of cDNA Clones Encoding Human PRO706
[2116] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA40669. Based on
the DNA40669 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 PRO706.
[2117] A pair of PCR primers (forward and reverse) were
synthesized:
122 forward PCR primer 5'-CCAAGCAGCTTAGAGCTCCAGACC-3' (SEQ ID NO:
386) reverse PCR primer 5'-TTCCCTATGCTCTGTATTGGCATGG-3' (SEQ ID NO:
387)
[2118] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA40669 sequence which
had the following nucleotide sequence
[2119] hybridization probe
123 5'-GCCACTTCTGCCACAATGTCAGCTTTCCCTGTACCAGAAATGGCTGTGTT-3' (SEQ
ID NO: 388)
[2120] 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 PRO706 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human fetal
brain tissue (LIB153).
[2121] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO706 [herein designated as
UNQ370 (DNA48329-1290)] (SEQ ID NO:384) and the derived protein
sequence for PRO706. It is understood that the deposited clone
contains the actual sequence, and that the sequences provided
herein are representative based on current sequencing
techniques.
[2122] The entire nucleotide sequence of UNQ370 (DNA48329-1290) is
shown in FIG. 154 (SEQ ID NO:384). CloneUNQ370 (DNA48329-1290)
contains asingleopenreadingframewithan apparenttranslational
initiation site at nucleotide positions 279-281 and ending at the
stop codon at nucleotide positions 1719-1721 (FIG. 154). The
predicted polypeptide precursor is 480 amino acids long (FIG. 155).
The full-length PRO706 protein shown in FIG. 155 has an estimated
molecular weight of about 55,239 daltons and a pI of about 9.30.
Clone UNQ370 (DNA48329-1290) has been deposited with the ATCC on
Apr. 21, 1998.
[2123] Still regarding the amino acid sequence shown in FIG. 155,
there is a potential signal peptide cleavage site at about amino
acid 19. There are potential N-glycosylation sites at about amino
acid positions 305 and 354. There is a potential tyrosine kinase
phosphorylation site at about amino acid position 333. A region
homologous with histidine acid phosphatase is at about residues
87-102. The corresponding nucleic acid regions can be routinely
determined given the provided sequences, i.e., the codons can be
determined from the specifically named amino acids given.
[2124] Analysis of the amino acid sequence of the full-length
PRO706 polypeptide suggests that portions of it possess significant
homology to the human prostatic acid phosphatase precursor thereby
indicating that PRO706 may be a novel human prostatic acid
phosphatase.
Example 61
[2125] Isolation of cDNA Clones Encoding Human PRO707
[2126] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA42775. Based on
DNA42775, 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 PRO707.
[2127] A pair of PCR primers (forward and reverse) were
synthesized:
124 forward PCR primer 5'-TCCGTCTCTGTGAACCGCCCCAC-3' (SEQ ID NO:
391); reverse PCR primer 5'-CTCGGGCGCATTGTCGTTCTGGTC-3' (SEQ ID NO:
392).
[2128] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the DNA42775 sequence which had the
following nucleotide sequence:
[2129] hybridization probe
125 5'-CCGACTGTGAAAGAGAACGCCCCAGATCCACTTATTCCCC-3' (SEQ ID NO:
393).
[2130] 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 PRO707 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human fetal
kidney tissue (LIB227).
[2131] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO707 [herein designated as
UNQ371 (DNA48306-1291)] (SEQ ID NO:389) and the derived protein
sequence for PRO707.
[2132] The entire nucleotide sequence of UNQ371 (DNA48306-1291) is
shown in FIG. 156 (SEQ ID NO:389). Clone UNQ371 (DNA48306-1291)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 371-373 and ending at the
stop codon at nucleotide positions 3119-3121 of SEQ ID NO:389. The
predicted polypeptide precursor is 916 amino acids long (FIG. 157).
The full-length PRO707 protein shown in FIG. 157 has an estimated
molecular weight of about 100,204 daltons and a pl of about 4.92.
Clone UNQ371 (DNA483061291) has been deposited with ATCC on May 27,
1998. It is understood that the clone UNQ371 which is deposited is
that which encodes PRO707, and that the sequences herein are merely
representations based on known sequencing techniques which may be
subject to minor errors.
[2133] Regarding analysis of the amino acid sequence, the signal
sequence appears to be at about 1 through 30 of SEQ ID NO:390.
Cadherins extracellular repeated domain signature sequence is at
about amino acids 121-131, 230-240, 335-345, 440-450, and 550-560
of SEQ ID NO:390. Tyrosine kinase phosphorylation sites are at
about amino acids 124-132 and 580-586 of SEQ ID NO:390. A potential
transmembrane domain is at about amino acids 682-715.+-.5. The
nucleic acid positions can be derived by referring to the
corresponding codon for the named amino acid.
[2134] Analysis of the anmno acid sequence of the fAI-length PRO707
polypeptide suggests that portions of it possess significant
homology to the cadherin FIB3 protein, expressed in human
fibroblasts, thereby indicating that PRO707 may be a novel
cadherin.
Example 62
[2135] Isolation of cDNA Clones Encoding Human PRO322
[2136] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA48336. Based on
the DNA48336 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 PRO322.
[2137] A pair of PCR primers (forward and reverse) were
synthesized:
126 forward PCR primer 5'-CAGCCTACAGAATAAAGATGGCCC-3' (SEQ ID NO:
396) reverse PCR primer 5'-GGTGCAATGATCTGCCAGGCTGAT-3' (SEQ ID NO:
397)
[2138] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the DNA48336 consensus sequence which
had the following nucleotide sequence:
[2139] hybridization probe
127 5'-AGAAATACCTGTGGTTCAGTCCATCCCAAACCCCTGCTACAACAGCAG-3' (SEQ ID
NO: 398).
[2140] 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 PRO322 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human fetal
kidney tissue (LIB227).
[2141] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO322 [herein designated as
UNQ283 (DNA48336-1309)] (SEQ ID NO:394) and the derived protein
sequence for PRO322. It is understood that UNQ283 (DNA48336-1309)
in fact encodes PRO322, and that SEQ ID NO:394 is a representation
of the sequence based on sequencing techniques known in the
art.
[2142] The entire nucleotide sequence of UNQ283 (DNA48336-1309) is
shown in FIG. 158 (SEQ ID NO:394). Clone UNQ283 (DNA48336-1309)
contains a single openreading frame with an apparent translational
initiation site at nucleotide positions 166-168 and ending at the
stop codon at nucleotide positions 946-948 (FIG. 158). The
predicted polypeptide precursor is 260 amino acids long (FIG. 159).
The full-length PRO322 protein shown in FIG. 159 has an estimated
molecular weight of about 28,028 daltons and a pI of about 7.87.
Clone UNQ283 (DNA48336-1309) has been deposited with ATCC and is
assigned ATCC deposit no. 209669.
[2143] Regarding the amino acid sequence of FIG. 159, a potential
N-glycosylation site is at amino acid 110 of SEQ ID NO:395. The
serine proteases, trypsin family and histidine active site is
identified at amino acids 69 through 74 of SEQ ID NO:395 and the
consensus sequence is identified at amino acids 207 through 217 of
SEQ ID NO:395. The kringle domain proteins motif is identified at
amino acids 205 through 217 of SEQ ID NO:395. The putative signal
peptide is encoded at about amino acids 1-23.
[2144] Analysis of the amino acid sequence of the full-length
PRO322 polypeptide suggests that portions of it possess significant
homology to neuropsin and other serine proteases, thereby
indicating that PRO322 is a novel serine protease related to
neuropsin.
Example 63
[2145] Isolation of cDNA Clones Encoding Human PRO526
[2146] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA39626. Based on
the DNA39626 consensus sequence, oligonucleotides were synthesized:
1) to identify by PCR a cDNA library that contained the sequence of
interest, and 2) for use as probes to isolate a clone of the
full-length coding sequence for PRO526.
[2147] A pair of PCR primers (forward and reverse) were
synthesized:
128 forward PCR primer 5'-TGGCTGCCCTGCAGTACCTCTACC-3' (SEQ ID NO:
401); reverse PCR primer 5'-CCCTGCAGGTCATTGGCAGCTAGG-3' (SEQ ID NO:
402).
[2148] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the DNA39626 consensus sequence which
had the following nucleotide sequence:
[2149] hybridization probe
129 5'-AGGCACTGCCTGATGACACCTTCCGCGACCTGGGCAACCTCACAC-3' (SEQ ID NO:
403).
[2150] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones encoding the PRO526 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human fetal
liver tissue (LIB228).
[2151] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO526 [herein designated as
UNQ330 (DNA44184-1319)] (SEQ ID NO:399) and the derived protein
sequence for PRO526.
[2152] The entire nucleotide sequence of UNQ330 (DNA44184-1319) is
shown in FIG. 160 (SEQ ID NO:399). Clone UNQ330 (DNA44184-1319)
contains a single open reading frame with an apparenttranslational
initiation site at nucleotide positions 514-516 and ending at the
stop codon at nucleotide positions 1933-1935 (FIG. 160). The
predicted polypeptide precursor is 473 amino acids long (FIG. 161).
The fiill-length PRO526 protein shown in FIG. 161 has an estimated
molecular weight of about 50,708 daltons and a pl of about 9.28.
Clone UNQ330 (DNA44184-1319) has been deposited with the ATCC on
Mar. 26, 1998. It is understood that the clone contains the actual
sequence, whereas the sequences presented herein are representative
based on current sequencing techniques.
[2153] Analysis of the amino acid sequence of the flll-length
PRO526 polypeptide suggests that portions of it possess significant
homology to the leucine repeat rich proteins including ALS, SLIT,
carboxypeptidase and platelet glycoprotein V thereby indicating
that PRO526 is a novel protein which is involved in protein-protein
interactions.
[2154] Still analyzing SEQ ID NO:400, the signal peptide sequence
is at about amino acids 1-26. A leucine zipper pattern is at about
amino acids 135-156. A glycosaminoglycan attachment is at about
amino acids 436-439. N-glycosylation sites are at about amino acids
82-85, 179-182, 237-240 and 423-426. A von Willebrand factor (VWF)
type C domain(s) is found at about amino acids 411425. The skilled
artisan can understand which nucleotides correspond to these amino
acids based on the sequences provided herein.
Example 64
[2155] Isolation of cDNA Clones Encoding Human PRO531
[2156] An ECD database was searched and an expressed sequence tag
(EST) from LIFESEQ.TM., Incyte Pharmaceuticals, Palo Alto, Calif.
was identified which showed homology to protocadherin 3. Based on
this sequence, a search was performed using the computer program
BLAST or BLAST2 (Altshul et al., Methods in Enzymology 266:460-480
(1996)) as a comparison of the ECD protein sequences to a 6 frame
translation of the EST sequence. Those comparisons resulting in a
BLAST score of 70 (or in some cases 90) or greater that did not
encode known proteins were clustered and assembled into consensus
DNA sequences with the program "phrap" (Phil Green, University of
Washington, Seattle, Wash.).
[2157] A consensus DNA sequence was assembled relative to other EST
sequences using phrap. Based on the consensus sequence obtained,
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
PRO531.
[2158] A pair of PCR primers (forward and reverse) were
synthesized:
130 forward PCR primer 5'-CTGAGAACGCGCCTGAAACTGTG-3' (SEQ ID NO:
406); reverse PCR primer 5'-AGCGTTGTCATTGACATCGGCG-3' (SEQ ID NO:
407).
[2159] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA sequence which had the
following nucleotide sequence:
[2160] hybridization probe
131 5'-TTAGTTGCTCCATTCAGGAGGATCTACCCTTCCTCCTGAAATCCGCGGAA-3' (SEQ
ID NO: 408).
[2161] In order to screen several libraries for a source of a
full-length clone, DNA from tie libraries was screened by PCR
amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones encoding the PRO531 gene
using the probe oligonucleotide and one of the PCR priIners. RNA
for construction of the cDNA hbraries was isolated from human fetal
brain tissue (LIB153). 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 SaU 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;
pRKSB is a precursor of pRK5D that does not contain the SfiI site;
see, Holmes et al., Science, 253:1278-1280 (1991)) in the unique
Xhol and Notl sites.
[2162] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO531 [herein designated as
UNQ332 (DNA48314-1320)] (SEQ ID NO:404) and the derived protein
sequence for PRO531.
[2163] The entire representative nucleotide sequence of UNQ332
(DNA48314-1320) is shown in FIG. 162 (SEQ ID NO:404). It is
understood that the actual sequence is that within the clone
deposited with the ATCC as DNA48314-1320. Clone UNQ332
(DNA48314-1320) contains a single open reading frame with an
apparent translational initiation site at nucleotide positions
171-173 and ending at the stop codon at nucleotide positions
2565-2567 (FIG. 162). The predicted polypeptide precursor is 789
amino acids long (FIG. 163). The full-length PRO531 protein shown
in FIG. 163 has an estimated molecular weight of about 87,552
daltons and a pI of about 4.84. Clone UNQ332 (DNA48314-1320) has
been deposited with the ATCC on Mar. 26, 1998.
[2164] Analysis of the amino acid sequence of the fMIl-length
PRO531 polypeptide suggests that portions of it possess significant
homology to protocadherin 3. Moreover, PRO531 is found in the
brain, like other protocadherins, thereby indicating that PRO531 is
a novel member of the cadherin superfamily.
[2165] Still analyzing the amino acid sequence of SEQ ID NO:405,
the cadherin extracellular repeated domain signature is found at
about amino acids 122-132, 231-241, 336-346, 439-449 and 549-559 of
SEQ ID NO:405. An ATP/GTP-binding site motif A (P-loop) is found at
about amino acids 285-292 of SEQ ID NO:405. N-glycosylation sites
are found at least at about amino acids 567-570, 786790, 418-421
and 336-339 of SEQ ID NO:405. The signal peptide is at about amino
acids 1-26, and the transmembrane domain is at about amino acids
685-712 of SEQ ID NO:405.
Example 65
[2166] Isolation of cDNA Clones Encoding Human PRO534
[2167] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA43038. Based on
the 43048 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
fifll-length coding sequence for PRO534.
[2168] A pair of PCR primers (forward and reverse) were
synthesized:
132 forward PCR primer 5'-CACAGAGCCAGAAGTGGCGGAATC-3' (SEQ ID NO:
411); reverse PCR primer 5'-CCACATGTTCCTGCTCTTGTCCTGG-3' (SEQ ID
NO: 412).
[2169] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA43038 sequence which
had the following nucleotide sequence:
[2170] hybridization probe
133 5'-CGGTAGTGACTGTACTCTAGTCCTGTTTTACACCCCGTGGTGCCG-3' (SEQ ID NO:
413).
[2171] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
ampliication with the PCR primer pair identified above. A positive
library was then used to isolate clones encoding the PRO534 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human fetal
lung tissue (LIB26).
[2172] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO534 [herein designated as
UNQ335 (DNA48333-1321)] (SEQ ID NO:409) and the derived protein
sequence for PRO534.
[2173] The entire nucleotide sequence of UNQ335 (DNA48333-1321) is
shown in FIG. 164 (SEQ ID NO:409). Clone UNQ335 (DNA48333-1321)
contains a single open reading frame with an apparenttranslational
initiation site at nucleotide positions 87-89 and ending at the
stop codon at nucleotide positions 1167-1169 (FIG. 164). The
predicted polypeptide precursor is 360 amino acids long (FIG. 165).
The full-length PRO534 protein shown in FIG. 165 has an estimated
molecular weight of about 39,885 daltons and a pl of about 4.79.
Clone UNQ335 (DNA48333-1321) has been deposited with ATCC on Mar.
26, 1998. It is understood that the deposited clone contains the
actual sequence, and that the sequences provided herein are
representative based on current sequencing techniques.
[2174] Analysis of the amino acid sequence of the full-length
PRO534 polypeptide suggests that portions of it possess significant
sequence identity with the protein disulfide isomerase, thereby
indicating that PRO534 may be a novel disulfide isomerase.
[2175] Still analyzing the amino acid sequence of PRO534, the
signal peptides is at about amino acids 1-25 of SEQ ID NO:410. The
transmembrane domain is at about amino acids 321-340 of SEQ ID
NO:410. The disulfide isomerase corresponding region is at amino
acids 212-302 of SEQ ID NO:410. The thioredoxin domain is at amino
acids 211-227 of SEQ ID NO:410. N-glycosylation sites are at:
165-168, 181-184, 187-190, 194-197, 206-209, 278-281, and 293-296
of SEQ ID NO:410. The corresponding nucleotides can routinely be
determined from the sequences provided herein. PRO534 has a
transmembrane domain rather than an ER retention peptide like other
protein disulfide isomerases. Additionally, PRO534 may have an
intron at the 5 prime end.
Example 66
[2176] Isolation of cDNA Clones Encoding Human PRO697
[2177] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA43052. 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 for PRO697.
[2178] A pair of PCR primers (forward and reverse) were
synthesized:
134 forward PCR primer 5'-CCTGGCTCGCTGCTGCTGCTC-3' (SEQ ID NQ:
416); reverse PCR primer 5'-CCTCACAGGTGCACTGCAAGCTGTC-3' (SEQ ID
NO: 417).
[2179] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the DNA43052 consensus sequence which
had the following nucleotide sequence:
[2180] hybridization probe
135 5'-CTCTTCCTCTTTGGCCAGCCCGACTTCTCCTACAAGCGCAGAATTGC-3' (SEQ ID
NO: 418).
[2181] 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 PRO697 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human fetal
kidney tissue (LIB227).
[2182] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO697 [herein designated as
UNQ361 (DNA50920-1325)] (SEQ ID NO:414) and the derived protein
sequence for PRO697.
[2183] The entire nucleotide sequence of UNQ361 (DNA50920-1325) is
shown in FIG. 166 (SEQ ID NO:414). Clone UNQ361 (DNA50920-1325)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 44-46 and ending at the
stop codon at nucleotide positions 929-931 (FIG. 166). The
predicted polypeptide precursor is 295 amino acids long (FIG. 167).
The full-length PRO697 protein shown in FIG. 167 has an estimated
molecular weight of about 33,518 daltons and a p1 of about 7.74.
Clone UNQ361 (DNA50920-1325) was deposited with the ATCC on Mar.
26, 1998. It is understood that the deposited clone contains the
actual sequence, and that the sequences provided herein are
representative based on current sequencing techniques.
[2184] Analysis of the amino acid sequence of the full-length
PRO697 polypeptide suggests that portions of it possess significant
sequence identity with sFRPs, thereby indicating that PRO697 may be
a novel sFRP family member.
[2185] Still analyzing the amino acid sequence of PRO697, the
signal peptides is at about amino acids 1-20 of SEQ ID NO:415. The
cystein rich domain, having identity with the frizzled N-terminus,
is at about amino acids 6153 of SEQ ID NO:415. The corresponding
nucleotides can routinely be determined from the sequences provided
herein.
Example 67
[2186] Isolation of cDNA Clones Encoding Human PRO717
[2187] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA42829. Based on
the DNA42829 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 PRO717.
[2188] A pair of PCR primers (forward and reverse) were
synthesized:
136 forward PCR primer 5'-AGCTTCTCAGCCCTCCTGGAGCAG-3' (SEQ ID NO:
421); reverse PCR primer 5'-CGGGTCAATAAACCTGGACGCTTGG-3' (SEQ ID
NO: 422).
[2189] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the DNA42829 consensus sequence which
had the following nucleotide sequence:
[2190] hybridization probe
137 5'-TATGTGGACCGGACCAAGCACTTCACTGAGGCCACCAAGATTG-3' (SEQ ID NO:
423).
[2191] 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 primner pair identified above. A
positive library was then used to isolate clones encoding the
PRO717 gene using the probe oligonucleotide and one of the PCR
primers. RNA for construction of the cDNA libraries was isolated
from human fetal liver tissue (LIB229).
[2192] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO717 [herein designated as
UNQ385 (DNA50988-1326)] (SEQ ID NO:419) and the derived protein
sequence for PRO717.
[2193] The entire nucleotide sequence of UNQ385 (DNA50988-1326) is
shown in FIG. 168 (SEQ ID NO:419). Clone UNQ385 (DNA50988-1326)
contains a singleopenreadingframewithanapparenttranslational
initiation site at nucleotide positions 17-19 and ending at the
stop codon at nucleotide positions 1697-1699 (FIG. 168). The
predicted polypeptide precursor is 560 amino acids long (FIG. 169).
The full-length PRO717 protein shown in FIG. 169 has an estimated
molecular weight of about 58,427 daltons and a pl of about 6.86.
Clone UNQ385 (DNA50988-1326) has been deposited with the ATCC on
Apr. 28, 1998. Regarding the sequence, it is understood that the
deposited clone contains the correct sequence, and the sequences
provided herein are based on known sequencing techniques.
[2194] Analysis of the amino acid sequence of the fullength PRO717
polypeptide suggests that PRO717 may be a novel 12 tranamembrane
receptor. The reverse complement strand of DNA50988 has a stretch
that matches identically with human regulatory myosm light
strand.
[2195] Still analyzing the amino acid sequence of SEQ ID NO:420,
transmembrane domains are at about amino acids 30-50, 61-79,
98-112, 126-146, 169-182, 201-215, 248-268, 280-300, 318-337,
341-357, 375-387, and 420441 of SEQ ID NO:420. N-glycosylation
sites are at about amino acids 40-43 and 4346 of SEQ ID NO:420. A
glycosaminoglyean attachment site is at about amino acids 468471 of
SEQ ID NO:420. The corresponding nucleotides can be routinely
determined given the sequences provided herein.
Example 68
[2196] Isolation of cDNA Clones Encoding Human PRO731
[2197] A database was used to search expressed sequence tag (EST)
databases. The EST database used herein was the proprietary EST DNA
database LIFESEQT, of Incyte Pharmaceuticals, Palo Alto, Calif.
Incyte clone 2581326 was herein identified and termed DNA42801.
Based on the DNA42801 sequence, oligonucleotides were synthesized:
1) to identify by PCR a cDNA library that contained the sequence of
interest, and 2) for use as probes to isolate a clone of the
full-length coding sequence for PRO731.
[2198] A pair of PCR primers (forward and reverse) were
synthesized:
138 forward PCR primer 5'-GTAAGCACATGCCTCCAGAGGTGC-3' (SEQ ID NO:
426); reverse PCR primer 5'-GTGACGTGGATGCTTGGGATGTTG-3' (SEQ ID NO:
427).
[2199] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the DNA42801 sequence which had the
following nucleotide sequence:
[2200] hybridization probe
139 5'-TGGACACCTTCAGTATTGATGCCAAGACAGGCCAGGTCATTCTGCGTCGA-3' (SEQ
ID NO:428).
[2201] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones encoding the PRO731 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human bone
marrow tissue (LIB255). The cDNA libraries used to isolate the cDNA
clones were constructed by standard methods using commercially
available reagents such as those from Invitrogen, San Diego, Calif.
The cDNA was primed with oligo dT containing a NotI site, linked
with blunt to Sall 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 SfEl site;
see, Holmes et al., Science, 253:1278-1280 (1991)) in the unique
XhoI and NotI sites.
[2202] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO731 [herein designated as
UNQ395 (DNA48331-1329)] (SEQ ID NO:424) and the derived protein
sequence for PRO731.
[2203] The entire nucleotide sequence of UNQ395 (DNA48331-1329) is
shown in FIG. 170 (SEQ ID NO:424). Clone UNQ395 (DNA48331-1329)
contains a singleopenreading frame with anapparenttranslational
initiation site at nucleotide positions 329-331 and ending at the
stop codon at nucleotide positions 3881-3883 (FIG. 170). The
predicted polypeptide precursor is 1184 amino acids long (FIG.
171). The fiill-length PRO731 protein shown in FIG. 171 has an
estitated molecular weight of about 129,022 daltons and a pI of
about 5.2. Clone UNQ395 (DNA48331-1329) was deposited with the ATCC
on Mar. 31, 1998. Regarding the sequence, it is understood that the
deposited clone contains the correct sequence, and the sequences
provided herein are based on known sequencing techniques.
[2204] Analysis of the amino acid sequence of the full-length
PRO731 polypeptide suggests that portions of it possess significant
identity and similarity to members of the protocadherin family,
thereby indicating that PRO731 may be a novel protocadherin.
[2205] Still analyzing the amino acid sequence of SEQ ID NO:425,
the putative signal peptide is at about amino acids 1-13 of SEQ ID
NO:425. The transmembrane domain is at amino acids 719-739 of SEQ
ID NO:425. The N-glycosylation of SEQ ID NO:425 are as follows:
415418, 582-586, 659-662, 662-665, and 857-860. The cadherin
extracellular repeated domain signatures are at about amino acids
(of SEQ ID NO:425): 123-133, 232-242, 340-350, 448458, and 553-563.
The corresponding nucleotides can be routinely determined given the
sequences provided herein.
Example 69
[2206] Isolation of cDNA Clones Encoding Human PRO218
[2207] A consensus sequence was obtained relative to a variety of
EST sequences as descnred in Example 1 above, wherein the consensus
sequence obtained is herein designated DNA17411. Two proprietary
Genentech EST sequences were employed in the consensus assembly and
are shown in FIGS. 174 and 175. Based on the DNA17411 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 PRO218.
[2208] A pair of PCR primers (forward and reverse) were
synthesized:
140 forward PCR primer 5'-AAGTGGAGCCGGAGCCTTCC-3' (SEQ ID NO:433);
reverse PCR primer 5'-TCGTTGTTTATGCAGTAGTCGG-3' (SEQ ID
NO:434).
[2209] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA17411 sequence which
had the following nucleotide sequence:
[2210] hybridization probe
141 5'-ATTGTTTAAAGACTATGAGATACGTCAGTATGTTGTACAGG-3' (SEQ ID
NO:435).
[2211] 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 PRO218 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human fetal
kidney tissue (LIB28).
[2212] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO218 [herein designated as
UNQ192 ()NA30867-1335)] (SEQ ID NO:429) andthe derivedprotein
sequence for PRO218.
[2213] The entire nucleotide sequence of UNQ192 (DNA30867-1335) is
shown in FIG. 172 (SEQ ID NO:429). Clone UNQ192 (DNA30867-1335)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 150-152 and ending at the
stop codon at nucleotide positions 1515-1517 (FIG. 172). The
predicted polypeptide precursor is 455 amino acids long (FIG. 173).
The full-length PRO218 protein shown in FIG. 173 has an estimated
molecular weight of about 52,917 daltons and a pl of about 9.5.
Clone UNQ192 (DNA30867-1335) has been deposited with the ATCC on
Apr.28, 1998. Regarding the sequence, it is understood that the
deposited clone contains the correct sequence, and the sequences
provided herein are based on known sequencing techniques.
[2214] Analysis of the amino acid sequence of the full-length
PRO218 polypeptide suggests that PRO218 may be a novel
transmembrane protein.
[2215] Still analyzing the amino acid sequence of SEQ ID NO:430,
the putative signal peptide is at about amino acids 1 through 23 of
SEQ ID NO:430. Transmembrane domains are potentially at about amino
acids 37-55, 81-102, 150-168, 288-311, 338-356, 375-398, and 425444
of SEQ ID NO:430. N-glycosylation sites are at about amino acids
67, 180, and 243 of SEQ ID NO:430. Eukaryotic cobalami-binding
protein is at about amino acids 151-160 of SEQ ID NO:430. The
corresponding nucleotides canbe routinely determined given the
sequences provided herein.
Example 70
[2216] Isolation of cDNA Clones Encoding Human PRO768
[2217] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA43448. Based on
the DNA43448 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 PRO768.
[2218] A pair of PCR primers (forward and reverse) were
synthesized:
142 forward PCR primer 5'-GGCTGACACCGCAGTGCTCTTCAG-3' (SEQ ID
NO:438); reverse PCR primer 5'-GCTGCTGGGGACTGCAATGTA- GCTG-3' (SEQ
ID NO:439).
[2219] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the DNA43448 consensus sequence which
had the following nucleotide sequence:
[2220] hybridization probe
143 5'-CATCCTCCATGTCTCCCATGAGGTCTCTATTGCTCCACGAAGCATC-3' (SEQ ID
NQ:440).
[2221] In order to screen several libraries for a source of a
fiill-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 PRO768 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human bone
marrow tissue (LIB255).
[2222] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO768 [herein designated as
UNQ406 (DNA55737-1345)] (SEQ ID NO:436) and the derived protein
sequence for PRO768.
[2223] The entire nucleotide sequence of UNQ406 (DNA55737-1345) is
shown in FIG. 176 (SEQ ID NO:436). Clone UNQ406 (DNA55737-1345)
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 3443-3445 (FIG. 176). The
predicted polypeptide precursor is 1141 amino acids long (FIG.
177). The fiull-length PRO768 protein shown in FIG. 177 has an
estimated molecular weight of about 124,671 daltons and a pI of
about 5.82. Clone UNQ406 (DNA55737-1345) has been deposited with
the ATCC on Apr. 6, 1998. Regarding the sequence, it is understood
that the deposited clone contains the correct sequence, and the
sequences provided herein are based on known sequencing
techniques.
[2224] Analysis of the amino acid sequence of the full-length
PRO768 polypeptide suggests that portions of it possess significant
sequence identity and similarity with integrin 7.
[2225] Still analyzing the amino acid sequence of SEQ ID NO:437,
the putative signal peptide is at about amino acids 1-33 of SEQ ID
NO:437. The transmembrane domain is at amino acids 1039-1064 of SEQ
ID NO:437. N-glycosylation sites are at amino acids: 86-89,
746-749, 949-952, 985-988 and 1005-1008 of SEQ ID NO:437. Integrin
alpha chain protein domains are identified at about amino acids:
1064-1071, 384-409, 1041-1071, 317-346, 443-465, 385-407, 215-224,
634-647, 85-99, 322-346, 470-479,442-466, 379-408 and 1031-1047 of
SEQ ID NO:437. The corresponding nucleotides can be routinely
determined given the sequences provided herein.
Example 71
[2226] Isolation of cDNA Clones Encoding Human PRO771
[2227] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example I above, wherein the
consensus sequence obtained is herein designated DNA43330. Based on
the DNA43330 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 PRO771.
[2228] A pair of PCR primers (forward and reverse) were
synthesized:
144 forward PCR primer 5'-CAGCAATATTCAGAAGCGGCAAGGG-3' (SEQ ID
NO:443); reverse PCR primer 5'-CATCATGGTCATCACCACCATCATC- ATC-3'
(SEQ ID NO:444).
[2229] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the DNA43330 consensus sequence which
had the following nucleotide sequence:
[2230] hybridization probe
145 5'-GGTTACTACAAGCCAACACAATGTCATGGCAGTGTTGGACAGTGCTGG-3' (SEQ ID
NO:445).
[2231] 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 PRO771 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human fetal
kidney tissue (LEB28).
[2232] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO771 [herein designated as
UNQ409 (DNA49829-1346)] (SEQ ID NO:441) and the derived protein
sequence for PRO771.
[2233] The entire nucleotide sequence of UNQ409 (DNA49829-1346) is
shown in FIG. 178 (SEQ ID NO:441). Clone UNQ409 (D3NA49829-1346)
contains a single open reading frame with an apparenttranslational
initiation site at nucleotide positions 134-136 and ending at the
stop codon at nucleotide positions 1442-1444 (FIG. 178). The
predicted polypeptide precursor is 436 amino acids long (FIG. 179).
The full-length PRO771 protein shown in FIG. 179 has an estimated
molecular weight of about 49,429 daltons and a pl of about 4.8.
Clone UNQ409 (DNA49829-1346) has been deposited with the ATCC on
Apr. 7, 1998. Regarding the sequence, it is understood that the
deposited clone contains the correct sequence, and the sequences
provided herein are based on known sequencing techniques.
[2234] Analysis of the amino acid sequence of the fufll-length
PRO771 polypeptide suggests that portions of it possess significant
homology to the testican protein, thereby indicating that PRO771
may be a novel testican homologue.
[2235] Still analyzing the amino acid sequence of SEQ ID NO:442,
the putative signal peptide, leucine zipper pattern,
N-myristoylation sites, and thyroglobulin type-1 repeats are also
shown in FIG. 179. The corresponding nucleotides can be routinely
determined given the sequences provided herein.
Example 72
[2236] Isolation of cDNA Clones Encoding Human PRO733
[2237] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA45600. Based on
the DNA45600 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 PRO733.
[2238] A pair of PCR primers (forward and reverse) were
synthesized:
146 forward PCR primer 5'-CCCAGCAGGGATGGGCGACAAGA-3' (SEQ ID
NO:448); reverse PCR primer 5'-GTCTTCCAGTTTCATATCCAATA-3- ' (SEQ ID
NO:449).
[2239] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the DNA45600 consensus sequence which
had the following nucleotide sequence:
[2240] hybridization probe
147 5'-CCAGAAGGAGCACGGGGAAGGGCAGCCAGATCTTGTCGCCCAT-3' (SEQ ID
NO:450).
[2241] In order to screen several libraries for a source of a
fiill-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 PRO733 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human bone
marrow tissue (LIB255).
[2242] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO733 [herein designated as
UNQ411 (DNA52196-1348)] (SEQ ID NO:446) and the derived protein
sequence for PRO733.
[2243] The entire nucleotide sequence of UNQ411 (DNA52196-1348) is
shown in FIG. 180 (SEQ ID NO:446). Clone UNQ411 (DNA52196-1348)
contains a single open reading frame with an apparenttranslational
initiation site at nucleotide positions 106-108 and ending at the
stop codon at nucleotide positions 793-795 (FIG. 180). The
predicted polypeptide precursor is 229 amino acids long (FIG. 181).
The full-length PRO733 protein shown in FIG. 181 has an estimated
molecular weight of about 26,017 daltons and a pI of about 4.73.
Clone UNQ411 (DNA52196-1348) has been deposited with the ATCC on
Apr. 7, 1998. Regarding the sequence, it is understood that the
deposited clone contains the correct sequence, and the sequences
provided herein are based on known sequencing techniques.
[2244] Analysis of the amino acid sequence of the full-length
PRO733 polypeptide suggests that portions of it possess significant
sequence identity and similarity to the T1/ST2 receptor binding
protein precursor and therefore may have a similar function in cell
signaling. If it is a cytokine, it may be useful in the treatment
of inflammation and cancer.
[2245] Still analyzing the amino acid sequence of SEQ ID NO:447,
the putative signal peptide, transmembrane domain, N-myristoylation
site, and tyrosine kinase site are also shown in FIG. 181. The
corresponding nucleotides can be routinely determined given the
sequences provided herein.
Example 73
[2246] Isolation of cDNA Clones Encoding Human PRO162
[2247] An expressed sequence tag (EST) DNA database
(Merck/Washington University) was searched and an EST AA397543 was
identified which showed homology to human pancreatitis-associated
protein. The EST AA397543 cole was purchased and its insert
obtained and sequenced and the sequence obtained is shown in FIG.
182 (SEQ ID NO:451).
[2248] The entire nucleotide sequence ofPRO162 is shown in FIG. 182
(SEQ ID NO:451). DNA sequencing of the clone gave the flll-length
DNA sequence for PRO162 [herein designated as UNQ429
(DNA56965-1356)] (SEQ ID NO:451) and the derived protein sequence
for PRO162. Clone UNQ429 (DNA56965-1356) contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 86-88 and ending at the stop codon at
nucleotide positions 611-613 (FIG. 182). The predicted polypeptide
precursor is 175 amino acids long (FIG. 183). The full-length
PRO162 protein shown in FIG. 183 has an estimated molecular weight
of about 19,330 daltons and a pI of about 7.25. Clone UNQ429
(DNA56965-1356) has been deposited with the ATCC. Regarding the
sequence, it is understood that the deposited clone contains the
correct sequence, and the sequences provided herein are based on
known sequencing techniques.
[2249] Analysis of the amino acid sequence of the full-length
PRO162 polypeptide suggests that portions of itpossess
significanthomology to the humanpancreatitis-associatedprotein,
thereby indicating that PRO162 may be a novel
pancreatitis-associated protein.
[2250] Still analyzing the amino acid sequence of SEQ ID NO:452,
the putative signal peptide is at about amino acids 1-26 of SEQ ID
NO:452. A C-type lectin domain signature is at about amino acids
146-171 of SEQ ID NO:452. The corresponding nucleotides can be
routinely determined given the sequences provided herein.
Example 74
[2251] Isolation of cDNA Clones Encoding Human PRO788
[2252] A consensus DNA sequence (designated herein as DNA49308) was
assembled relative to other EST sequences using phrap as described
in Example 1 above. Based upon an observed homology between the
DNA49308 consensus sequence and the Incyte EST cloOne no. 2777282,
the Incyte EST clone no. 2777282 was purchased and its insert
obtained and sequenced, which gave the full-length DNA sequence for
PRO788 [herein designated as UNQ430 (DNA56405-1357)] (SEQ ID
NO:453) and the derived protein sequence for PRO788.
[2253] Clone UNQ430 (DNA56405-1357) contains a single open reading
frame with an apparent translational initiation site at nucleotide
positions 84-86 and ending at the stop codon at nucleotide
positions 459461 (FIG. 184). The predicted polypeptide precursor is
125 amino acids long (FIG. 185). The full-length PRO788 protein
shown in FIG. 185 has an estimated molecular weight of about 13,115
daltons and a pI of about 5.90. Clone UNQ430 DNA56405-1357) has
been deposited with the ATCC. Regarding the sequence, it is
understood that the deposited clone contains the correct sequence,
and the sequences provided herein are based on known sequencing
techniques.
[2254] Still analyzing FIG. 185, a signal peptide is shown at about
amino acids 1-17 of SEQ ID NO:454. An N-glycosylation site is at
about amino acids 4649 of SEQ ID NO:454.
Example 75
[2255] Isolation of cDNA Clones Encoding Human PRO1008
[2256] 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 as DNA49804. An EST
proprietary to Genentech was employed in the consensus assembly and
is herein designated as DNA16508 (FIG. 188; SEQ ID NO:457). Based
upon an observed homology between the DNA49804 sequence and Merck
EST clone no. AA143670, the Merck EST clone no. AA143670 was
purchased and its insert obtained and sequenced. That sequence is
shown herein in FIG. 186 (SEQ ID NO:455).
[2257] Sequencing gave the fall length sequence for PRO1008 [herein
designated as UNQ4Y2 (DNA57530-1375)] (SEQ ID NO:455) and the
derived protein sequence for PRO1008 were identified.
[2258] The entire nucleotide sequence of UN0492 (DNA57530-1375) is
shown in FIG. 186 (SEQ ID NO:455). Clone UNQ492 (DNA57530-1375)
contains asingle openreadingframewithanapparenttranslational
initiation site at nucleotide positions 138-140 and ending at the
stop codon at nucleotide positions 936-938 (FIG. 186). The
predicted polypeptide precursor is 266 amino acids long (FIG. 187).
T[he full-length PRO1008 protein shown in FIG. 187 has an estimated
molecular weight of about 28,672 daltons and a pI of about 8.85.
Clone UNQ492 (DNA57530-1375) has been deposited with the ATCC on
May 20, 1998. Regarding the sequence, it is understood that the
deposited clone contains the correct sequence, and the sequences
provided herein are based on known sequencing techniques.
[2259] Analysis of the amino acid sequence of the full-length
PRO1008 polypeptide suggests that portions of it possess
significant sequence identity and/or similarity with mdkk-1,
thereby indicating that PRO1008 may be a novel member of this
family and have head inducing activity.
[2260] Still analyzing the amino acid sequence of SEQ ID NO:456,
the putative signal peptide is at about amino acids 1-23 of SEQ ID
NO:456. The N-glycosylation site is at about amino acids 256-259 of
SEQ ID NO:456, and the fungal zn2):cys(6) binuclear cluster domain
is at about amino acids 110-126 of SEQ ID NO:456. The corresponding
nucleotides can of all the amino acids can be routinely determined
given the sequences provided herein.
Example 76
[2261] Isolation of cDNA Clones Encoding Human PRO1012
[2262] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described in Example 1 above, wherein the
consensus sequence is herein designated DNA49313. Based on the
DNA49313 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 PRO1012.
[2263] A pair of PCR primers (forward and reverse) were
synthesized:
148 forward PCR primer 5'-ACTCCCCAGGCTGTTCACACTGCC-3' (SEQ ID
NO:460); reverse PCR primer 5'-GATCAGCCAGCCAATACCAGCAGC-- 3' (SEQ
ID NO:461).
[2264] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the DNA49313 consensus sequence which
had the following nucleotide sequence:
[2265] hybridization probe
149 5'-GTGGTGATGATAGAATGCTTTGCCGAATGAAAGGAGTCAACAGCTATCCC-3' (SEQ
ID NO:462).
[2266] 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 PRO1012 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human fetal
kidney tissue (LIB227).
[2267] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO1012 [herein designated as
UNQ495 (DNA56439-1376)] (SEQ ID NO:458) and the derived protein
sequence for PRO1012.
[2268] The entire nucleotide sequence of UNQ495 (DNA56439-1376) is
shown in FIG. 189 (SEQ ID NO:458). Clone UNQ495 (DNA56439-1376)
contains a single open reading framewith anapparenttranslational
initiation site at nucleotide positions 404406 and ending at the
stop codon at nucleotide positions 2645-2647 (FIG. 189). The
predicted polypeptide precursor is 747 amino acids long (FIG. 190).
The full-length PRO1012 protein shown in FIG. 190 has an estimated
molecular weight of about 86,127 daltons and a pl of about 7.46.
Clone UNQ495 (DNA56439-1376) has been deposited with ATCC on May
14, 1998. Regarding the sequence, it is understood that the
deposited clone contains the correct sequence, and the sequences
provided herein are based on known sequencing techniques.
[2269] Analysis of the amino acid sequence of the full-length
PRO1012 polypeptide suggests that portions of it possess sequence
identity with disulfide isomerase thereby indicating that PRO1012
may be a novel disulfide isomerase related protein.
[2270] Still analyzing the amino acid sequence of SEQ ID NO:459,
the cytochrome C family heie-binding site signature is at about
amino acids 158-163 of SEQ ID NO:459. The Nt-DNAJ domain signature
is at about amino acids 77-96 of SEQ ID NO:459. An N-glycosylation
site is at about amino acids 484-487 of SEQ ID NO:459. The ER
targeting sequence is at about amino acids 744-747 of SEQ ID
NO:459. It is understood that the polypeptide and nucleic acids
disclosed can be routinely formed with or without, these portions
as desired, in alternative embodiments. For example, it may be
desirable to produce PRO1012 without the ER targeting sequence. The
corresponding nucleotides can be routinely determined given the
sequences provided herein.
Example 77
[2271] Isolation of cDNA Clones Encoding Human PRO1014
[2272] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described in Example 1 abobe, wherein the
consensus sequence obtained is herein designated DNA49811 . Based
upon an observed homology between the DNA49811 sequence and Incyte
EST clone no. 2612207, Incyte EST clone no. 2612207 was purchased
and its insert was obtained and sequenced, wherein the sequence
obtained is shown in FIG. 191 (SEQ OD NO:463).
[2273] DNA sequencing gave the full-length DNA sequence for PRO1014
[herein designated as UNQ497 (DNA56409-1377)] (SEQ ID NO:463) and
the derived protein sequence for PRO1014.
[2274] The entire nucleotide sequence of UNQ497 (DNA56409-1377) is
shown in FIG. 191 (SEQ ID NO:463). Clone UNQ497 (ONA56409-1377)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 66-68 and ending at the
stop codon at nucleotide positions 966-968 (FIG. 191). The
predicted polypeptide precursor is 300 amino acids long (FIG. 192).
The full-length PRO1014 protein shown in FIG. 192 has an estimated
molecular weight of about 33,655 daltons and a pI of about 9.31.
Clone UNO497 (DNA56409-1377) has been deposited with the ATCC on
May 20, 1998. Regarding the sequence, it is understood that the
deposited clone contains the correct sequence, and the sequences
provided herein are based on known sequencing techniques.
[2275] Analysis of the amino acid sequence of the full-length
PRO1014 polypeptide suggests that portions of it possess sequence
identity with reductase, thereby indicating that PRO1014 may be a
novel member of the reductase family.
[2276] Stil analyzing the amino acid sequence of SEQ ID NO:464, the
putative signal peptide is at about amino acids 1-19 of SEQ ID
NO:464. The cAMP and cGMP dependent protein kinase phosphorylation
sites are at about amino acids 30-33 and 58-61 of SEQ ID NO:464.
Short chain alcohol dehydrogenase family proteins are at about
amino acids 165-202, 37-49, 112-122 and 210-219 of SEQ ID NO:464.
The corresponding nucleotides of these domains and any other amino
acids provided herein can be routinely determined given the
sequences provided herein.
Example 78
[2277] Isolation of cDNA Clones Encoding Human PRO1017
[2278] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described in Example 1 above, wherein that
consensus DNA sequence is herein designated DNA53235. Based upon an
observed homology between the DNA53235 consensus sequence and the
Merck EST clone no. AA243086, the Merck EST clone no. AA243086 was
purchased and its insert obtained and sequenced, wherein the
sequence obtained is shown in FIG. 193 (SEQ ID NO:465). DNA
sequencing gave the full-length DNA sequence for PRO1017 [herein
designated as UNQ500 (DNA56112-1379)] (SEQ ID NO:465) and the
derived protein sequence for PRO1017.
[2279] The entire nucleotide sequence of UNQ500 (DNA56112-1379) is
shown in FIG. 193 (SEQ ID NO:465). Clone UNQ500 (DNA56112-1379)
contains a single open reading frame with an apparenttranslational
initiation site at nucleotide positions 128-130 and ending at the
stop codon at nucleotide positions 1370-1372 (FIG. 193). The
predicted polypeptide precursor is 414 amino acids long (FIG. 194).
The full-length PRO1017 protein shown in FIG. 194 has an estimated
molecular weight of about 48,414 daltons and a pI of about 9.54.
Clone UNQ500 DNA56112-1379) has been deposited with the ATCC.
Regarding the sequence, it is understood that the deposited clone
contains the correct sequence, and the sequences provided herein
are based on known sequencing techniques.
[2280] Analysis of the amino acid sequence of the full-length
PRO1017 polypeptide suggests that portions of it possess sequence
identity with HNK-1 sulfotransferase, thereby indicating that
PRO1017 may be a novel sulfotransferase.
[2281] Still analyzing the amino acid sequence of SEQ ID NO:466,
the putative signal peptide is at about amino acids 1-31 of SEQ ID
NO:466. N-glycosylation sites are at about amino acids 134-137,
209-212, 280-283 and 370-273 of SEQ ID NO:466. The TNFRINGFR family
cystein-rich region protein is at about amino acids 329-332 of SEQ
ID NO:466. The corresponding nucleotides can be routinely
determined given the sequences provided herein. The protein can be
secreted.
Example 79
[2282] Isolation of cDNA Clones Encoding Human PRO474
[2283] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described in Example 1 above, wherein the
consensus sequence obtained is herein designated DNA49818. Based
upon an observed homology between the DNA49818 consensus sequence
and the Merck EST clone no. H77889, the Merck EST clone no. H77889
was purchased and its insert obtained and sequenced, wherein the
sequence obtained is herein shown in FIG. 195 (SEQ ID NO:467). DNA
sequencing gave the full-length DNA sequence for PRO474 [herein
designated as UNQ502 (DNA56045-1380)] (SEQ ID NO:467) and the
derived protein sequence for PRO474.
[2284] The entire nucleotide sequence of UNQ502 (DNA56045-1380) is
shown in FIG. 195 (SEQ ID NO:467). Clone UNQ502 (DNA56045-1380)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 106-108 and ending at the
stop codon at nucleotide positions 916-918 (FIG. 195). The
predicted polypeptide precursor is 270 amino acids long (FIG. 196).
The full-length PRO474 protein shown in FIG. 196 has an estimated
molecular weight of about 28,317 daltons and a pI of about 6.0.
Clone UNQ502 (DNA56045-1380) has been deposited with the ATCC.
Regarding the sequence, it is understood that the deposited clone
contains the correct sequence, and the sequences provided herein
are based on known sequencing techniques.
[2285] Still analyzing the amino acid sequence of SEQ ID NO:468, an
N-glycosylation site is at about amino acids 138-141 of SEQ ID
NO:468. Short-chain alcohol dehydrogenase family proteins are at
about amino acids 10-22, 81-91, 134-171 and 176-185 of SEQ ID
NO:468. The corresponding nucleotides cmn be routinely determined
given the sequences provided herein.
Example 80
[2286] Isolation of cDNA Clones Encoding Human PRO1031
[2287] An initial consensus DNA sequence was assembled relative to
other EST sequences using phrap as described in Example 1 above,
wherein the consensus sequence obtained is herein designated as
DNA47332. Based upon an observed homology between the DNA47332
sequence and the Merck EST clone no. W74558, Merck EST clone no.
W74558 was purchased and its insert obtained and sequenced, wherein
the sequence obtained is shown in FIG. 197 (SEQ ID NO:469). DNA
sequencing gave the full-length DNA sequence for PRO1031 [herein
designated as UNQ516 (DNA59294-1381)] (SEQ ID NO:469) and the
derived protein sequence for PRO1031.
[2288] The entire nucleotide sequence of UNQ516 (DNA59294-1381) is
shown in FIG. 197 (SEQ ID NO:469). Clone UNQ516 (DNA592941381)
contains a single open reading frame with an apparenttrnnslational
initiation site at nucleotide positions 42-44 and ending at the
stop codon at nucleotide positions 582-584 (FIG. 197). The
predicted polypeptide precursor is 180 amino acids long (FIG. 198).
The full-length PRO1031 protein shown in FIG. 198 has an estimated
molecular weight of about 20,437 daltons and a pI of about 9.58.
Clone UNQ516 (DNA59294-1381) has been deposited with the ATCC.
Regarding the sequence, it is understood that the deposited clone
contains the correct sequence, and the sequences provided herein
are based on known sequencing techniques.
[2289] Analysis of the amino acid sequence of the full-length
PRO1031 polypeptide suggests that it is a novel cytokine.
[2290] Still analyzing the amino acid sequence of SEQ ID NO:470,
the putative signal peptide is at about amino acids 1-20 of SEQ ID
NO:470. An N-glycosylation site is at about amino acids 75-78 of
SEQ ID NO:470. A region having sequence identity with IL-17 is at
about amino acids 96180. The corresponding nucleotides can be
routinely determined given the sequences provided herein.
Example 81
[2291] Isolation of cDNA Clones Encoding Human PRO938
[2292] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described in Example 1 above, wherein that
consensus sequence is herein designated DNA49798. Based on the
DNA49798 DNA 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 PRO938.
[2293] A pair of PCR primers (forward and reverse) were
synthesized:
150 forward PCR primer 5'-GTCCAGCCCATGACCGCCTCCAAC-3' (SEQ ID
NO:473) reverse PCR primer 5'-CTCTCCTCATCCACACCAGCAGCC-3- ' (SEQ ID
NO:474)
[2294] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA49798 sequence which
had the following nucleotide sequence:
[2295] hybridization probe
151 hybridization probe 5'-GTGGATGCTGAAATTTTACGCCCCATGGTGT-
CCATCCTGCCAGC-3' (SEQ ID NO:475)
[2296] In order to screen several libraries for a source of a
fiillength 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 PRO938 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human fetal
kidney tissue (LIB227).
[2297] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO938 [herein designated as
UNQ475 (DNA56433-1406)] (SEQ ID NO:471) andthe derived protein
sequence for PRO938.
[2298] The entire nucleotide sequence of UNQ475 (DNA56433-1406) is
shown in FIG. 199 (SEQ ID NO:471). Clone UNQ475 (DNA56433-1406)
contains a single open reading frame with an apparenttranslational
initiation site at nucleotide positions 134-136 and ending at the
stop codon at nucleotide positions 1181-1183 (FIG. 199). The
predicted polypeptide precursor is 349 amino acids long (FIG. 200).
T]he fUll-length PRO938 protein shown in FIG. 200 has an estimated
molecular weight of about 38,952 daltons and a pI of about 4.34.
Analysis of the full-length PRO938 sequence shown in FIG. 200 (SEQ
ID NO:472) evidences the presence of the following features: a
signal peptide from amino 1 to about amino acid 22, a transmembrane
domain from about amino acid 191 to about amino acid 211, a
potential N-glycosylation site from about amino acid 46 to about
amino acid 49, a region homologous to disulfide isomerase from
about amino acid 56 to about amino acid 72, and a region having
sequence identity with flavodoxin proteins from about amino acid
173 to about amino acid 187.
[2299] Clone UNQ475 (DNA56433-1406) has been deposited with ATCC on
May 12, 1998, and is assigned ATCC Accession No. 209857.
[2300] Analysis of the amino acid sequence of the flll-length
PRO938 polypeptide suggests that it possesses significant sequence
similarity to protein disulfide isomerase, thereby indicating that
PRO938 may be a novel protein disulfide isomerase. An analysis of
the Dayhoff database (version 35.45 SwissProt 35) evidenced
significant homology between the PRO938 amino acid sequence and the
following Dayhoff sequences, P_W03626, P_W03627, P_R70491,
GARP_PLAFF, XLU85970.sub.--1, ACADISPROA.sub.--1, IE68_HSVSA,
KSU52064.sub.--1, U93872.sub.--83, P_R97866.
Example 82
[2301] Isolation of cDNA Clones Encoding Human PRO1082
[2302] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described in Example 1 above, wheein the
consensus sequence is herein designated DNA38097. 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 for PRO1082.
[2303] A set of PCR primers (two forward and one reverse) were
synthesized:
152 forward primer 1 5'-GTCCACAGACAGTCATCTCAGGAGCAG-3' (SEQ ID
NO:478); forward primer 2 5'-ACAAGTGTCTTCCCAACCTG-3' (SEQ ID
NO:479); reverse primer 1 5'-ATCCTCCCAGAGCCATGGTA- CCTC-3' (SEQ ID
NO:480).
[2304] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the DNA38097 consensus sequence which
had the following nucleotide sequence:
[2305] hybridization probe
153 5'-CCAAGGATAGCTGTTGTTTCAGAGAAAGGATCGTGTGCTGCATCTCCTCCT-3' (SEQ
ID NO:481).
[2306] 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 primers identified above. A positive
library was then used to isolate clones encoding the PRO1082 gene
using the probe oligonucleotide and one of the PCR primers. RNA for
construction of the cDNA libraries was isolated from human fetal
kidney tissue (LIB227).
[2307] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO1082 [herein designated as
UNQ539 (DNA53912-1457)] (SEQ ID NO:476) and the derived protein
sequence for PRO1082.
[2308] The entire nucleotide sequence of UNQ539 (DNA53912-1457) is
shown in FIG. 201 (SEQ ID NO:476). Clone UNQ539 (DNA53912-1457)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 160-162 and ending at the
stop codon at nucleotide positions 763-765 (FIG. 201). The
predicted polypeptide precursor is 201 amino acids long (FIG. 202).
The fll-length PRO1082 protein shown in FIG. 202 has an estimated
molecular weight of about 22,563 daltons and a pI of about 4.87.
Clone UNQ539 (DNA53912-1457) has been deposited with the ATCC.
Regarding the sequence, it is understood that the deposited clone
contains the correct sequence, and the sequences provided herein
are based on known sequencing techniques.
[2309] Still analyzing the amino acid sequence of SEQ ID NO:477,
the transmembrane domain is at about amino acids 45-65 of SEQ ID
NO:477. A cAMP- and cGMP-dependent protein linase phosphorylation
site is at about amino acids 197-200 of SEQ ID NO:477.
N-myristoylation sites are at about amino acids 3540 and 151-156 of
SEQ ID NO:477. The regions which share sequence identity with the
LDL receptor are at about amino acids 34-67 and 70-200 of SEQ ID
NO:477. The corresponding nucleotides of these amino acid regions
and others can be routinely determined given the sequences provided
herein.
Example 83
[2310] Isolation of cDNA Clones Encoding Human PRO1083
[2311] A cDNA sequence was identified using the amylase screening
technique described in Example 2 above, wherein that cDNA sequence
is designated herein as DNA24256 (FIG. 205; SEQ ID NO:484). That
cDNA sequence was then compared and aligned with other known EST
sequencees as described in Example 1 above to obtain a consensus
DNA sequence which is designated herein as DNA43422. Based on ttle
DNA 43422 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 PRO1083.
[2312] A pair of PCR primers (forward and reverse) were
synthesized:
154 forward PCR primer 5'-GGCATTGGAGCAGTGCTGGGTG-3' (SEQ ID
NO:485); reverse PCR primer 5'-TGGAGGCCTAGATGCGGCTGGACG-- 3' (SEQ
ID NO:486).
[2313] 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 PRO1083 gene
using the reverse PCR primer. RNA for construction of the cDNA
libraries was isolated from human fetal kidney tissue (LIB227).
[2314] DNA sequencing of the clones isolated as described above
gave the full-iength DNA sequence for PRO1083 [herein designated as
UNQ540 (DNA50921-1458)] (SEQ ID NO:482) and the derived protein
sequence for PRO1083.
[2315] The entire nucleotide sequence of UNQ540 (DNA50921-1458) is
shown in FIG. 203 (SEQ ID NO:482). Clone UNQ540 (DNA50921-1458)
contains a single open reading frame with an apparent translational
initiation site at nucleotide positions 214-216 and ending at the
stop codon at nucleotide positions 2293-2295 (FIG. 203). The
predicted polypeptide precursor is 693 amino acids long (FIG. 204).
The full-length PRO1083 protein shown in FIG. 204 has an estimated
molecular weight of about 77,738 daltons and a pI of about 8.87.
Clone UNQ540 (DNA50921-1458) has been deposited with the ATCC.
Regarding the sequence, it is understood that the deposited clone
contains the correct sequence, and the sequences provided herein
are based on known sequencing techniques.
[2316] Still analyzing the amino acid sequence of SEQ ID NO:483,
the putative signal peptide is at about amino acids 1-25 of SEQ ID
NO:483. The transmembrane domains are at about amino acids 382-398,
402420, 445-468, 473491, 519-537, 568-590 and 634-657 of SEQ ID
NO:483. A microbodies C-terminal targeting signal is at about amino
acids 691-693 of SEQ ID NO:483. cAMP- and cGMPdependent protein
kinase phosphorylation sites are at about amino acids 198-201 and
370-373 of SEQ ID NO:483. N-glycosylation sites are at about amino
acids 39-42, 148-151, 171-174,234-237,303-306,324-227 and 341-344
of SEQ ID NO:483. A G-protein coupled receptor family domain is at
about amino acids 475-504 of SEQ ID NO:483. The corresponding
nucleotides can be routinely determined given the sequences
provided herein.
Example 84
[2317] Isolation of cDNA Clones Encoding Human PRO200
[2318] Probes based on an expressed sequence tag (EST) identified
from the Incyte Pharmaceuticals database due to homology with VEGF
were used to screen a cDNA library derived from the human glioma
cell line G61. In particular, Incyte Clone "INC1302516" was used to
generate the following four probes:
155 ACTTCTCAGTGTCCATAAGGG (SEQ ID NO:489);
GAACTAAAGAGAACCGATACCATTTTCTGGCCAGGTTGTC (SEQ ID NO:490);
CACCACAGCGTTTAACCAGG (SEQ ID NO:491); and ACAACAGOCACAGTTCCCAC (SEQ
ID NO:492).
[2319] Nine positives were identified and characterized. Three
clones contained the full coding region and were identical in
sequence. Partial clones were also identified from a fetal lung
library and were identical with the glioma-derived sequence with
the exception of one nucleotide change which did not alter the
encoded amino acid.
Example 85
[2320] Expression Constructs for PRO200
[2321] For mammalian protein expression, the entire open reading
frame (ORF) was cloned into a CMV-based expression vector. An
epitope-tag (FLAG, Kodak) and Histidine-tag (His8) were inserted
between the ORF and stop codon. VEGF-E-His8 and VEGF-E-FLAG were
transfected into human embryonic kidney 293 cells by SuperFect
(Qiagen) and pulse-labeled for 3 hours with [.sup.35S]methionine
and [.sup.35C]cysteine. Both epitope-tagged proteins co-migrate
when 20 microliters of 15-fold concentrated serum-free conditioned
medium were electrophoresed on a polyacrylamide gel (Novex) in
sodium dodecyl sulfate sample buffer (SDS-PAGE). The VEGF-E-IgG
expression plasmid was constructed by cloning the ORF in front of
the human Fc (IgG) sequence.
[2322] The VEGF-E-IgG plasmid was co-transfected with Baculogold
Baculovirus DNA (Pharmingen) using Lipofectin (GibcoBRL) into
10.sup.5 Sf9 cells grown in Hink's TNM-FH medium (JRH Biosciences)
supplemented with 10% fetal bovine serum. 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 at an approximate multiplicity of infection (MOI) of 10.
Cells were incubated for 3 days, then supernatant harvested, and
expression of the recombinant plasmid determined by binding of 1 ml
of supernatant to 30 Izl of Protein-A Sepharose CL-4B beads
(Pharmacia) followed by subsequent SDS-PAGE analysis. The first
amplification supernatant was used to infect a 500 ml spinner
culture of Sf9 cells grown in ESF-921 medium (Expression Systems
LLC) at an approximate MOI of 0.1 Cells were treated as above,
except harvested supernatant was sterile filtered. Specific protein
was purified by binding to Protein-A Sepharose 4 Fast Flow
(Pharmacia) column.
Example 86
[2323] Northern Blot Analyses for PRO200
[2324] Blots of human poly(A) +RNA from multiple adult and fetal
tissues and tumor cell lines were obtained from Clontech (Palo
Alto, Calif.). Hybridization was carried out using .sup.32P-labeled
probes containg the entire coding region and washed in
0.1.times.SSC, 0.1% SDS at 63.degree. C.
[2325] VEGF-E mRNA was detectable in fetal lung, kidney, brain,
liver and adult heart, placenta, liver, skeletal muscle, kidney,
and pancreas. VEGF-E mRNA was also found in A549 lung
adenocarcinoma and HeLa cervical adenocarcinoma cell lines.
Example 87
[2326] In Situ Hybridization of Human Fetal Tissue Sections for
PRO200
[2327] Formalin-fixed, paraffin-embeddedhuman fetal brain, liver,
lower limb, small intestine, thyroid, lymph node, thymus, stomach,
trachea, skin, spleen, spinal cord, adrenal, placenta, cord, and
adult liver, pancreas, lung, spleen, lymph node, adrenal, heart,
aorta, and skin were sectioned, deparaffinized, deproteinated in
proteinase K (20 .mu.g/ml) for 15 minutes at 37.degree. C., and
further processed for in situ hybridization as described by Lu L H
and Gillett N A (Cell Vision 1:169-176, 1994). A
[.alpha..sup.33-P]UTP-Iabeled antisense riboprobe was generated
from a PCR product of 980 bp (primers GGCGGAATCCAACCTGAGTAG and
GCGGCTATCCTCCTGTGCTC, SEQ ID NOS: 493 and 494, respectively). The
slides were dipped in Kodak NTB2 nuclear track emulsion and exposed
for 4 weeks.
[2328] VEGF-E mRNA expression included localization at the growth
plate region and embracing fetal myocytes.
Example 88
[2329] Myocyte Hypertrophy Assay for PRO200
[2330] Myocytes from neonatal Harlan Sprague Dawley rat heart
ventricle (23 days gestation) were plated in duplicate at 75000
cellslml in a 96-well plate. Cells were treated for 48h with 2000,
200, 20, or 2 ng/ml VEGF-E-IgG. Myocytes were stained with crystal
violet to visualize morphology and scored on a scale of 3 to 7, 3
being nonstimulated and 7 being full-blown hypertrophy.
[2331] 2000 ng/ml and 200 ng/ml VEGF-E caused hypertrophy, scored
as a 5.
Example 89
[2332] Cell Proliferation Assay for PRO200
[2333] Mouse embryonic fibroblast C3HI0T1/2 cells (ATCC) were grown
in 50:50 Ham's F-12: low glucose DMEM medium containg 10% fetal
calf serum (FCS). Cells were plated in duplicate in a 24-well plate
at 1000, 2000, and 4000 cells/well. After 48 hours, cells were
switched to medium containing 2% FCS and were incubated for 72
hours with 200, 800, or 2000 ng/ml VEGF-E or no growth factor
added.
[2334] Approximately 1.5 fold greater number of cells were measured
in the presence of 200 ng/ml VEGF-E as in its absence, at all three
cell densities.
Example 90
[2335] Endothelial Cell Survival Assay for PRO200
[2336] Human umbilical vein endothelial cells (HUVEC, Cell Systems)
were maintained in Complete Media (Cell Systems) and plated in
triplicate in serum-free medium (Basic Media from Cell Systems
containing 0.1% BSA) at 20,000 cellslwell of a 48-well plate. Cells
were incubated for 5 days with 200 or 400 ng/ml VEGF-E-IgG, 100
ng/ml VEGF, 20 ng/ml basic FGF, or no addition.
[2337] Survival was 2-3 times greater with VEGF-E as compared to
lack of growth factor addition. VEGF and basic FGF were included as
positive controls.
Example 91
[2338] Isolation of cDNA Clones Encoding Human PRO285
[2339] A proprietary expressed sequence tag (EST) DNA database
(LIFESEQ.TM., Incyte Pharmaceuticals, Palo Alto, Calif.) was
searched and an EST (#2243209) was identified which showed homology
to the Drosophila Toll protein.
[2340] Based on the EST, a pair of PCR primers (forward and
reverse):
156 TAAAGACCCAGCTGTGACCG (SEQ ID NO:499) ATCCATGAGCCTCTGATGGG (SEQ
ID NO: 500), and a probe:
ATTTATGTCTCGAGGAAAGGGACTGGTTACCAGGGCAGCCAGTTC (SEQ ID NO: 501)
[2341] were synthesized.
[2342] mRNA for construction of the cDNA libraries was isolated
from human placenta tissue. The cDNA libraries used to isolate the
cDNA clones were constructed by standard methods using commercially
available reagents such as those from Invitrogen, San Diego, Calif.
(Fast Track 2). The cDNA was primed with oligo dT containing a NotI
site, linked with blunt to Sall hemnikinased adaptors, cleaved with
Notl, sized appropriately by gel electrophoresis, and cloned in a
defined orientation into the cloning vector pCR2.1 (nvitrogen,
Inc.) 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 cDNA was
cloned into BamHl/Notl cleaved vector. pCR2.1 is a commercially
available plasmid, designed for easy cloning of PCR fragments, that
carries AmpR and KanR genes for selection, and LacZ gene for
blue-white selection.
[2343] 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 PRO285 gene
using the probe oligonucleotide and one of the PCR primers.
[2344] A cDNA clone was sequenced in entirety. The entire
nucleotide sequence of DNA40021-1154 (encoding PRO285) is shown in
FIG. 208 (SEQ ID NO:495). Clone DNA40021-1154 contains a single
open reading frame with an apparent translational initiation site
at nucleotide positions 61-63 (FIG. 208). The predicted polypeptide
precursor is 1049 amino acids long, including a putative signal
peptide at amino acid positions 1-29, a putative transmembrane
domain between amino acid positions 837-860, and a leucine zipper
pattern at amino acid positions 132-153 and 704-725, respectively.
It is noted that the indicated boundaries are approximate, and the
actual limits of the indicated regions might differ by a few amino
acids. Clone DNA40021-1154 has been deposited with ATCC
(designation: DNA40021-1154) and is assigned ATCC deposit no.
209389.
[2345] Based on a BLAST and FastA sequence alignment analysis
(using the ALIGN computer program) of the full-length sequence is a
human analogue of the Drosophila Toll protein, and is homologous to
the following human Toll proteins: Toll1 (DNAX# HSU88540-1, which
is identical with the random sequenced full-length cDNA
#HUMRSC786-1); Toll2 (DNAX# HSU88878-1); To113 (DNAX# HSU88879-1);
and Tol14 (DNAX# HSU88880-1).
Example 92
[2346] Isolation of cDNA Clones Encoding Human PRO286
[2347] A proprietary expressed sequence tag (IST) DNA database
(LIFESEQ.TM., Incyte Pharmaceuticals, Palo Alto, Calif.) was
searched and an EST (#694401) was identified which showed homology
to the Drosophila Toll protein.
[2348] Based on the EST, a pair of PCR primers (forward and
reverse):
157 GCCGAGACAAAAACGTTCTCC (SEQ ID NO:502) CATCCATGTTCTCATCCATTAGCC
(SEQ ID NO: 503), and a probe:
TCGACAACCTCATGCAGAGCATCAACCAAAGCAAGAAAAACAGTATT (SEQ ID NO:
504)
[2349] were synthesized.
[2350] mRNA for construction of the cDNA libraries was isolated
from human placenta tissue. This RNA was used to generate an oligo
dT primed cDNA library in the vector pRK5D using reagents and
protocols from Life Technologies, Gaithersburg, Md. (Super Script
Plasmid System). pRK5D is a cloning vector that has an sp6
transcription initiation site followed by an Sffi restriction
enzyme site preceding the XhoI/Notl cDNA cloning sites. The cDNA
was primed with oligo dT containing a NotI site, linked with blunt
to SalI hemikinased adaptors, cleaved with NotI, sized to greater
than 1000 bp appropriately by gel electrophoresis, and cloned in a
defined orientation into XhoI/NotI-cleaved pRK5D.
[2351] 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 PRO286 gene
using the probe oligonucleotide identified above and one of the PCR
primers.
[2352] A cDNA clone was sequenced in entirety. The entire
nucleotide sequence of DNA42663-1154 (encoding PRO286) is shown in
FIG. 210 (SEQ ID NO:497). Clone DNA42663-1154 contains a single
open reading frame with an apparent translational initiation site
at nucleotide positions 57-59 (FIG. 211). The predicted polypeptide
precursor is 1041 amino acids long, including a putative signal
peptide at amino acid positions 1-26, a potential transmembrane
domain at amino acid positions 826-848, and leucine zipper patterns
at amino acids 130-151, 206-227, 662-684, 669-690 and 693-614,
respectively. It is noted that the indicated boundaries are
approximate, and the actual limits of the indicated regions might
differ by a few amino acids. Clone DNA42663-1154 has been deposited
with ATCC (designation: DNA42663-1154) and is assigned ATCC deposit
no. 209386.
[2353] Based on a BLAST and FastA sequence alignment analysis
(using the ALIGN computer program) of the fllll-length sequence of
PRO286, it is a human analogue of the Drosophila Toll protein, and
is homologous to the following human Toll proteins: Tolli (DNAX#
HSU88540-1, which is identical with the random sequenced
full-length cDNA #HUMRSC78&1); Toll2 (DNAX# HSU88878-1); To113
(DNAX# HSU88879-1); and Toll4 (DNAX# HSU88880-1).
Example 93
[2354] NF-.kappa.B Assay for PRO285 and PRO286
[2355] As the Toll proteins signal through the NF-.kappa.B pathway,
their biological activityr can be tested in an NF-.kappa.B assay.
In this asswy Jurkat cells are transiently transfected using
Lipofectamine reagent (Gibco BRL) according to the manufacturer's
instructions. 1 .mu.g pB2XLuc plasmid, containing
NF-.kappa.B-driven luciferase gene, is contransfected with 1 .mu.g
pSR.alpha.N expression vector with or without the insert encoding
PRO285 or PRO286. For a positive control, cells are treated with
PMA (phorbol myristyl acetate; 20 ng/ml) and PHA (phytohaemaglutin
2 .mu.g/ml) for three to four hours. Cells are lysed 2 or 3 days
later for measurement of luciferase activity using reagents from
Promega.
Example 94
[2356] Isolation of cDNA Clones Encoding Human PRO213-1, PRO1330
and PRO1449
[2357] 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 DNA28735. Based on the
DNA28735 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
ftfflength coding, sequence for PRO213-1, PRO1330 and/or PRO1449. A
pair of PCR primers (forward and reverse) were synthesized:
158 forward PCR primer 5'-TGGAGCAGCAATATGCCAGCC-3' (SEQ ID NO:511)
reverse PCR primer 5'-TTTTCCACTCCTGTCGGGTTGG-3' (SEQ ID NO:512)
[2358] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA28735 sequence which
had the following nucleotide sequence:
[2359] hybridization probe
159 5'-GGTGACACTTGCCAGTCAGATGTGGATGAATGCAGTGCTAGGAGGG-3' (SEQ ID
NO: 513)
[2360] 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 PRO213-1,
PRO1330 and/or PRO1449 gene using the probe oligonucleotide and one
of the PCR priners. RNA for construction of the cDNA libraries was
isolated from human fetal lung tissue.
[2361] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence encoding PRO213-1, PRO1330 and/or
PRO1449 [DNA30943-1-1163-1 (SEQ ID NO:505), DNA64907-1163-1 (SEQ ID
NO:507) and DNA64908-1163-1 (SEQ ID NO:509), respectively].
[2362] The entire nucleotide sequences corresponding to
DNA30943-1-1163-1 (SEQ ID NO:505), DNA64907-1163-1 (SEQ ID NO:507)
and DNA64908-1163-1 (SEQ ID NO:509), respectively. DNA30943-1163,
DNA64907-1163-1 and DNA64908-1163-1 contain a single open reading
frame with an apparent translational initiation site at nucleotide
positions 336-338, 488490 and 326-328, respectively, and ending at
the stop codon at nucleotide positions 1221-1223, 1307-1309 and
1145-1147, respectively (FIGS. 212, 214 and 216). The predicted
polypeptide precursor is 295, 273 and 273 amino acids long,
respectively (FIGS. 213, 215 and 217). DNA30943-1-1163-1,
DNA64907-1163-1 and DNA64908-1163-1 have been deposited with ATCC
and are assigned ATCC deposit no. 209791, 203242 and 203243,
respectively.
[2363] Analysis of the amino acid sequence of the full-length
PRO213-1 polypeptide suggests that a portion of it possess
significant homology to the human growth arrest-specific gene 6
protein. More specifically, an analysis of the Dayhoff database
(version 35.45 SwissProt 35) evidenced significant homology between
the PRO213 amino acid sequence and the following Dayhoff sequences,
HSMHC3W5A.sub.--6 and B48089.
[2364] Additional analysis of the amino acid sequence of the
full-length PRO1330 and PRO1449 polypeptide indicates significant
identity with notch4. More specifically, an analysis of the Dayhoff
database (version 35.130 SwissProt 35) evidenced significant
identity between PRO1330 andthe following Dayhoff sequences, D86566
1 and NEL HUMAN.
Example 95
[2365] Isolation of cDNA Clones Encoding Human PRO298
[2366] A cDNA isolated in the amylase screen described in Example 2
above is herein designated DNA26832 (FIG. 220; SEQ ID NO:516). The
sequence of DNA26832 was then used to search expressed sequence tag
(EST) databases. The EST databases included public EST databases
(e.g., GenBank) and a proprietary EST database (LIEESEQ.TM., Incyte
Pharmaceuticals, Palo Alto, Calif.). The search was performed using
the computer program BLAST or BLAST2 (Altshul et al., Methods in
Enzvmology 266: 469480 [1996]). Those comparisons resulting in a
BLAST score of 70 (or in some cases 90) or greater that did not
encode proteins were clustered and assembled into consensus DNA
sequences with the program "phrap" (Phil Green, University of
Washington, Seattle, Wash.;
http:/Ibozeman.mbt.washington.edu/phrap.do- cs/phrap.html).
[2367] A consensus DNA sequence was assembled relative to other EST
sequences using phrap. A consensus sequence was determined, which
was then 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. The extended assembly sequence
was designated DNA35861.Based on the DNA35861 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 of
PRO298. Forward and reverse 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 sequence is typically 40-55 bp in
length. In some cases, additional oligonucleotides are synthesized
when the consensus sequence is greater than about 1-1.5 kbp. In
order to screen several libraries for a full-length clone, DNA from
the libraries was screened by PCR amplification, as per Ausubel et
al., Current Protocols in Molecular Biology, with the PCR primer
pair. A positive library was used to isolate clones encoding the
gene of interest using the probe oligonucleotide and one of the
primer pairs.
[2368] PCR primers (forward and reverse) and a hybridization probe
were synthesized:
160 forward PCR primer 1 CAACGTGATTTCAAAGCTGGGCTC (SEQ ID NO: 517)
forward PCR primer 2 GCCTCGTATCAAGAATTTCC (SEQ ID NO: 518) forward
PCR primer 3 AGTGGAAGTCGACCTCCC (SEQ ID NO: 519) reverse PCR primer
1 CTCACCTGAAATCTCTCATAGCCC (SEQ ID NO: 520) hybridization probe 1
CGCAAAACCCATTTTGGGAGCAGGAATTCCAATCATGTCTGTGA- TGGTGG (SEQ ID NO:
521)
[2369] (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 PRO298 gene
using the probe oligonucleotide and one of the PCR primers.
[2370] RNA for construction of the cDNA libraries was isolated from
human fetal lung tissue (LIB25). The cDNA libraries used to
isolated 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 Notd
site, linked with blunt to SalI hemikinased adaptors, cleaved with
Notl, 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 Notl sites.
[2371] DNA sequencing of the clones isolated as descn1bed above
gave the full-length DNA sequence for PRO298 (herein designated
UNQ261 [DNA39975-1210]) (SEQ ID NO:514), and the derived protein
sequence for PRO298 (SEQ ID NO:515).
[2372] The entire nucleotide sequence of UNQ261 (DNA39975-1210) is
shown in FIG. 218 (SEQ ID NO:514). Clone DNA39975-1210 contains a
single open reading frame with an apparent translational initiation
site at nucleotide positions 375-377. The predicted polypeptide
precursor is 364 amino acids long. The protein contains four
putative transmembrane domains between amino acid positions 36-55
(type 11 TM), 65-84, 188-208, and 229-245, respectively. A putative
N-linked glycosylation site starts at amino acid position 253. In
addition, the following features have been identified in the
protein sequence: cAMP- and cGMPdependent protein kinase
phosphorylation site, starting at position 8; N-myristoylation
sites starting a position 173 and 262, respectively; and a ZP
domain between amino acid positions 45-60. Clone DNA39975-1210 has
been deposited with ATCC (Apr. 21, 1998) and is assigned ATCC
deposit no. 209783.
Example 96
[2373] Isolation of cDNA Clones Encoding Human PRO337
[2374] A cDNA sequence identified in the amylase screen described
in Example 2 above is herein designated DNA42301 (FIG. 223, SEQ ID
NO:524). The DNA42301 sequence was then compared to other EST
sequences using phrap as described in Example 1 above and a
consensus sequence designated herein as DNA28761 was identified.
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. In order to screen
several libraries for a source of a fiill-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 PRO337 gene using the probe oligonucleotide and
one of the PCR primers. RNA for construction of the cDNA libraries
was isolated from human fetal brain.
[2375] A cDNA clone was sequenced in its entirety. The full length
nucleotide sequence of DNA43316-1237 is shown in FIG. 221 (SEQ ID
NO:522). Clone DNA43316-1237 contains a single open reading frame
with an apparent translational initiation site at nucleotide
positions 134-136 (FIG. 221; SEQ ID NO:522). The predicted
polypeptide precursor is 344 amino acids long. Clone DNA43316-1237
has been deposited with ATCC and is assigned ATCC deposit no.
209487
[2376] Based on a BLAST-2 and FastA sequence alignment analysis of
the full-length sequence, PRO337 shows amino acid sequence identity
to rat neurotrimin (97%).
Example 97
[2377] Isolation of cDNA Clones Encoding Human PRO403
[2378] Introduction:
[2379] Human thromnbopoietin (THPO) is a glycosylated hormone of
352 amino acids consisting of two domains. The N-terminal domain,
sharing 50% similarity to erythropoietin, is responsible for the
biological activity. The C-terminal region is required for
secretion. The gene for thrombopoietin (TBPO) maps to human
chromosome 3q27-q28 where the six exons of this gene span 7
kilobase base pairs of genomic DNA (Chang et al., Genomics 26:
636-7 (1995); Foster et al., Proc. Natl. Acad. Sci. USA 91: 13023-7
(1994); Gurney et al., Blood 85: 981-988 (1995). In order to
determine whether there were any genes encoding TBPO homologues
located in close proximity to THPO, genomic DNA fragments from this
region were identified and sequenced. Three P1 clones and one PAC
clones (Genome Systems Inc., St. Louis, Mo.; cat. Nos. P1-2535 and
PAC-6539) encompassing the THPO locus were isolated and a 140 kb
region was sequenced using the ordered shotgun strategy (Chen et
al., Genomics 17: 651-656 (1993)), coupled with a PCR-based gap
filling approach. Analysis reveals that the region is gene-rich
with four additional genes located very close to THPO: tumor
necrosis factor-receptor type I associated protein 2 (TRAP2) and
elongation initiation factor gamma (eIF40, chloride channel 2
(CLCN2) and RNA polymerase II subunit HRPB17. While no THPO homolog
was found in the region, four novel genes have been predicted by
computer-assisted gene detection (GRAIL)(Xu et al., Gen. Engin. 16:
241-253 (1994), the presence of CpG islands (Cross, S. and Bird,
A., Curr. Opin. Genet. & Devel. 5: 109-314 (1995), and homology
to known genes (as detected by WU-BLAST2.0)(Altschul and Gish,
Methods Enzymol. 266: 460-480 (1996)
(http://blast.wustl.edulblast/README.html).
[2380] Procedures:
[2381] P1 and PAC clones:
[2382] The initial human P1 clone was isolated from a genomic PI
library (Genome Systems Inc., St. Louis, Mo.; cat. no.: P1-2535)
screened with PCR primers designed from the ThPO genomic sequence
(A.L. Gurney, et al., Blood 85: 981-88 (1995). PCR primers were
designed from the end sequences derived from this P1 clone were
then used to screen P1 and PAC libraries (Genome Systems, Cat.
Nos.: P1-2535 & PAC-6539) to identify overlapping clones (PAC1,
p1.t, and P1.u). The 3'-end sequence from PAC.z was used to define
the primers used for the screening of a human BAC library (Genome
Systems Inc., St. Louis, Mo.; Cat. No.: BDTW-4533A).
[2383] Ordered Shotgun Strategy:
[2384] The Ordered Shotgun Strategy (OSS) (Chenet al., Genomics 17:
651-656 (1993)) involves the mapping and sequencing of large
genomic DNA clones with ahierarchical approach. The PI or PAC clone
was sonicated and the fragments subcloned into lambda vector
(.lambda.Bluestar) (Novagen, Inc., Madison, Wis.; cat. no.
69242-3). The lambda subclone inserts were isolated by long-range
PCR (Barnes, W. Proc. Natl. Acad. Sci. USA 91: 2216-2220 (1994) and
the ends sequenced. The lambda-end sequences were overlapped to
create a partial map of the original clone. Those lambda clones
with overlapping end-sequences were identified, the insets
subeloned into a plasmid vector (pUC18 or pUC19, Hoefer Pharnacia
Biotech, Inc., San Francisco, Calif., Cat. Nos. 27-4949-01 and
274951-01) and the ends of the plasmid subclones were sequenced and
assembled to generate a contiguous sequence. This directed
sequencing strategy minimizes the redundancy required while
allowing one to scan for and concentrate on interesting
regions.
[2385] In order to define better the THPO locus and to search for
other genes related to the hematopoietin family, five genomic
clones were isolated from this region by PCR screening of human PI
and PAC libraries (Genome System, Inc., Cat. Nos.: P1-2535 and
PAC-6539).
[2386] The sizes of the genomic fragments are as follows: Pl.t is
40 kb; Pl.g is 70 kb; Pl.u is 70 kb; PAC.z is 200 kb; and BAC.1 is
80 kb. Approximately 75% (140 kb) of the 190 kb genomic DNA region
was sequenced by the Ordered Shotgun Strategy (OSS) (Chen et al.,
Geiomics 17: 651-56 (1993), and assembled into contigs using
AutoAssembler.TM. (Applied Biosystems, Perkin Elmer, Foster City,
Calif., cat. no. 903227). The preliminary order of these contigs
was determined by manual analysis. There were 47 contigs the 140 kb
region. A PCR-based approach to ordering the contigs and filling in
the gaps was employed. The following summarizes the number and
sizes of the gaps. The 50 kb of sequence unique to BAC.1 was
sequenced by a total shotgun approach with a ten-fold
redundancy.
161 Size of gap number <50 bp 13 50-150 bp 7 150-300 bp 7
300-1000 bp 10 1000-5000 bp 7 >5000 bp 2 ((15,000 bp)
[2387] DNA sequencing:
[2388] ABI DYE-primnerTM chemistry (PE AppliedBiosystems, Foster
City, Calif.; Cat. No.: 402112) was used to end-sequence the lambda
and plasmid subclones. ABI DYE-terminater.TM. chemistry (PE Applied
Biosystems, Foster City, Calif., Cat. No: 403044) was used to
sequence the PCR products with their respective PCR primers. The
sequences were collected with an ABI377 instrument. For PCR
products larger than lkb, walking primers were used. The sequences
of contigs generated by the OSS strategy in AutoAssemblerTM (PE
Applied Biosystems, Foster City, Calif.; Cat. No: 903227) and the
gap-filling sequencing trace files were imported into
Sequencher.TM. (Gene Codes Corp., Ann Arbor, Mich.) for overlapping
and editing. The sequences generated by the total shotgun strategy
were assembled using Phred and Phrap and edited using Consed
(http://chimera.biotech.washington.edu/uwgclprojects.htm) and GFP
(Genome Reconstruction Manager for Phrap), version 1.2
(http://stork.cellb.bcm.tm- c.edu/gfp/).
[2389] PCR-Based Gap filling Strategy:
[2390] Primers were designed based on the 5'- and 3'-end sequenced
of each contig, avoiding repetitive and low quality sequence
regions. All primers were designed to be 19-24-mers with 50-70% G/C
content. Oligos were synthesized and gel-purified by standard
methods.
[2391] Since the orientation and order of the contigs were unknown,
permutations of the primers were used in the amplification
reactions. Two PCR kits were used: first, XL PCR kit (Perkin Elmer,
Norwalk, Conn.; Cat. No.: N8080205), with extension times of
approximately 10 minutes; and second, the Taq polymerase PCR kit
(Qiagen Inc., Valencia, Calif.; Cat. No.: 201223) was used under
high strngency conditions if smeared or multiple products were
observed with the XL PCR kit. The main PCR product from each
successful reaction was extracted from a 0.9% low melting agarose
gel and purified with the Geneclean DNA Purification kit prior to
sequencing.
[2392] Analysis:
[2393] The identification and characterization of coding regions
was carried out as follows: First, repetitive sequences were masked
using RepeatMasker (A.F.A. Smit & P. Green,
http://ftp.genome.washington.edu/RM- /RM_details.html) which
screens DNA sequences in FastA format against a library of
repetitive elements and returns a masked query sequence. Repeats
not masked were identified by comparing the sequence to the GenBank
database using WUBLAST2.0 [Altschul, S & Gish, W., Methods
Enzymol. 266: 460-480 (1996);
http://blast.wustl.edu/blast/README.html] and were masked
manually.
[2394] Next, known genes were revealed by comparing the genomic
regions against Genentech's protein database using the WUBLAST2.0
algorithm and then annotated by aligning the genomic and cDNA
sequences for each gene, respectively, using a Needleman-Wunch
(Needleman and Wunsch, J. Mol. Biol. 48: 443453 (1970) algorithm to
find regions of local identity between sequences. The strategy
results in detection of all exons of the five known genes in the
region, THPO, TRAP2, eIF4g, CLCN2 and hRPB17 (see below).
162 Known genes Map position eukaryotic translation initiation
factor 4 gamma 3q27-qter thrombopoietin 3q26-q27 chloride channel 2
3q26-qter TNF receptor associated protein 2 not previously mapped
RNA polymerase II subunit hRPB17 not previously mapped
[2395] Finally, novel transcription units were predicted using a
number of approaches. CpG islands (S. Cross & Bird, A., Curr.
Opin. Genet. Dev. 5: 109-314 (1995) islands were used to define
promoter regions and were identified as clusters of sites cleaved
by enzymes recognizing GC-rich, 6 or 8-mer palindromic sequences
(NotI, NarI, BssHII, XhoI. CpG islands are usually associated with
promoter regions of genes. WUBLAST2.0 analysis of short genomic
regions (10-20 kb) versus GenBank revealed matches to ESTs. The
individual EST sequences (or where possible, their sequence
chromatogram files) were retrieved and assembled with Sequencer to
provide a theoretical cDNA sequence (DNA36443). GRAML2 (ApoCom
Inc., Knoxville, Tenn., command line version for the DEC alpha) was
used to predict a novel exon. The five known genes in the region
served as internal controls for the success of the GRAIL
algorithm.
[2396] Isolation:
[2397] A partial endothelin converting enzyme-2 (ECE-2) cDNA clone
was isolated by first splicing in silico the ECE-2 exons predicted
in the genomic sequence to generate a putative sequence (DNA36443).
An oligonucleotide probe: GAAGCAGTGCAGCCAGCAGTAGAGAGGCACCTGCTAAGA)
(SEQ ID NO:530) was designed and used to screen a human fetal small
intestine library (LIB110) and internal PCR primers (36443fl)
(ECE2.f:ACGCAGCTGGAGCTGGTCTTAGCA) (SEQ ID NO:531) and (36443rl)
(ECE2.r) (GGTACTGGACCCCTAGGGCCACAA) (SEQ ID NO:532) were used to
confirm clones hybridizing to the probe prior to sequencing. One
positive clone was obtained, however this cDNA (DNA49830)
represented a partially spliced transcript containing appropriately
spliced exons 1 through 6, followed by intron 6 sequence. The oligo
dT primer annealed to a polyA-stretch within an Alu element present
in intron 6. An additional ECE-2 cDNA fragment (DNA49831) was
obtained by PCR from a human fetal kidney library (LIB227) with
primers designed from the presumed cDNA sequence [36443f3:
CCTCCCAGCCGAGACCAGTGG (SEQ ID NO:533) and 36443r2:
GGTCCTATAAGGGCCAAGACC (SEQ ID NO:534)]. This PCR product extended
from exon 13 into the 3' untranslated region in exon 18.
[2398] A full length endothelin converting enzyme 2 (ECE-2) cDNA
clone (DNA55800-1263) was isolated from an oligo-dT-primed human
fetal brain library. RNA from human fetal brain tissue (20 weeks
gestation, #283005)(SRC175) was isolated by guanidine thiocyanate
and 5 Ig used to generate double stranded cDNA which was cloned
into the vector pRK5E. The 3'-primer
(pGACTAGTTCTAGATCGCGAGCGGCCGCCCTTTTTTTTTTTTTTT) (SEQ ID NO:535) and
the 5 -linker (pCGGACGCGTGGGTCGA) (SEQ ID NO:536) were designed to
introduce XhoI and NotI restriction sites. The library was screened
with PCR primers [36443pcrf1: CGGCCGTGATGGCTGGTGACG (SEQ ID NO:537)
and 36443r3: GGCAGACTCCTTCCTATGGG (SEQ ID NO:538)] designed from
the partial human ECE-2 cDNA sequences (DNA49830 and DNA49831). PCR
products were cloned into the vector pCR2.1-TOPO (Invitrogen Corp.,
Carlsbad, Calif., Cat. No. K4500-01) and sequenced with
DYEterminator chemnistry as described above.
Example 98
[2399] Northern Blot and in situ RNA Hvbridization Analysis for
PRO403
[2400] Expression of PRO403 rnRNA in human tissues was examined by
Northern blot analysis. Human polyA+RNA blots derived from human
fetal and aduit tissues (Clontech, Palo Alto, Calif.; Cat. Nos.
7760-1, 7756-1 and 7755-1) were hybridized to a
[32P-ec]dATP-labelled cDNA fragments from probe based on the fill
length PRO403 cDNA. Blots were incubated with the probes in
hybridization buffer (5.times.SSPE; 2X Denhardt's solution; 100
mg/mL denatured sheared salmon sperm DNA; 50% formamide; 2% SDS)
for 18 hours at 42.degree. C., washed to high stringency
(0.1.times.SSC, 0.1% SDS, 50.degree. C.) and autoradiographed. The
blots were developed after overnight exposure by phosphorimager
analysis (Fuji).
[2401] PRO403 mRNA transcripts were detected. Analysis of the
expression pattern showed the strongest signal of the expected 3.3
kb transcript in adult brain (highest in the cerebellum, putamen,
medulla, and temporal lobe, and lower in the cerebral cortex,
occipital lobe and frontal lobe), spinal cord, lung and pancreas
and higher levels of a 4.5 kb transcript in fetal brain and
kidney.
Example 99
[2402] Use of PRO Polypeptide-Encoding Nucleic Acid as
Hybridization Probes
[2403] The following method describes use of a nucleotide sequence
encoding a PRO polypeptide as a hybridization probe.
[2404] 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
IDNAs (such as those encoding naturally-occurring variants of the
PRO polypeptide) in human tissue cDNA libraries or human tissue
genomic libraries.
[2405] 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, 2x 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.
[2406] 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 100
[2407] Expression of PRO Polypeptides in E. coli
[2408] This example illustrates preparation of an unglycosylated
form of a desired PRO polypeptide by recombinant expression in E.
coli.
[2409] 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 enterolinase cleavage site), the specific PRO
polypeptide coding region, lambda transcriptional terminator, and
an argu gene.
[2410] 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 confimed by restriction analysis and DNA
sequencing.
[2411] 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.
[2412] After culturing the cells for several more hours, the cells
can be harvested by centriflgation. 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.
[2413] PRO181, PRO195, PRO200, PRO237, PRO273, PRO540, PRO322,
PRO1017, PRO938, PRO162, PRO1114, PRO827 and PRO1008 were expressed
in E. coli in a poly-His tagged form, using the following
procedure. The DNA encoding the PRO polypeptide 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-amnplified, poly-His tagged sequences
were then ligated into an expression vector, which was used to
transform an E. coli 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).sub.2SO.sub.4, 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.
[2414] E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets)
was resuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris,
pH 8 buffer. Solid sodium sulfite and sodium tetrathionate is added
to make final concentrations of 0.1M and 0.02 M, respectively, and
the solution 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 S ml Qiagen
Ni-NTA metal chelate column equilibrated in the metal chelate
column buffer. The column was washed with additional buffer
containg 50 ir im idazole (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.
[2415] 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
Ri/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 concentrations of acetonitrile since those
species are the most compact with their hydrophobic interiors
shielded from interaction with the reversed phase resin. Aggregated
species are usually eluted at higher acetonitrile concentrations.
In addition to resolving misfolded forms of proteins from the
desired form, the reversed phase step also removes endotoxin from
the samples.
[2416] Fractions containing the desired folded PRO proteins 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.
[2417] Many of the PRO polypeptides described herein were
successfully expressed as described above.
Example 101
[2418] Expression of PRO Polypeptides in Mammalian Cells
[2419] This example illustrates preparation of a glycosylated form
of a desired PRO polypeptide by recombinant expression in mammalian
cells.
[2420] The vector, pRK5 (see EP 307,247, published Mar. 15, 1989),
is employed as the expression vector. Optionally, the PRO
polypeptide ncoding 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.
[2421] 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 rnure 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 at25.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.
[2422] 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.
[2423] In an alternative technique, PRO polypeptide may be
inroduced 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 centrifigation and
washed with PBS. The DNA-dextan 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 spimner 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.
[2424] 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 contaiing the expressed PRO polypeptide can then be
concentrated and purified by any selected method.
[2425] Epitope-tagged PRO polypeptide may also be expressed in host
CHO cells. The PRO polypeptide may be subeloned 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
thenbe 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.
[2426] 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 IgGl constant region sequence containing
the hinge, CH2 and CH2 domains and/or is a poly-His tagged
form.
[2427] Following PCR amplification, the respective DNAs were
subeloned 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.
[2428] 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.
[2429] 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 mnL spinners were seeded with
3.times.105 cells/mnL. 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 actualy 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.
[2430] 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 contaning 0.3 M NaCl
and 5 mM imidazole at a flow rate of 45 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 inmdazole. 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.
[2431] 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.
[2432] Many of the PRO polypeptides described herein were
successfully expressed as described above.
Example 102
[2433] Expression of PRO Polvvettides in Yeast
[2434] The following method describes recombinant expression of a
desired PRO polypeptide in yeast.
[2435] 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 ADH21GAPDH promoter, the
yeast alpha-factor secretory signal/leader sequence, and linker
sequences (if needed) for expression of the PRO polypeptide.
[2436] Yeast cells, such as yeast strain AB110, can thenbe
transformed with the expressionplasmids 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.
[2437] Recombinant PRO polypeptide can subsequently be isolated and
purified by removing the yeast cells from the fermentation medium
by centriflgation and then concentrating the medium using selected
cartridge filters. The concentrate containing the PRO polypeptide
may further be purified using selected column chromatography
resins.
[2438] Many of the PRO polypeptides described herein were
successfilly expressed as described above.
Example 103
[2439] Expression of PRO Polypeptides in Baculovirus-Infected
Insect Cells
[2440] The following method describes recombinant expression of PRO
polypeptides in Baculovirus-infected insect cells.
[2441] 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 extracelular
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 subdloned into the expression vector.
[2442] 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 45 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).
[2443] 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 Aw 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 Ano 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.
[2444] 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.
[2445] PRO195, PRO526, PRO540, PRO846, PRO362, PRO363, PRO700,
PRO707, PRO322, PRO719, PRO1083, PRO868, PRO866, PRO768, PRO788,
PRO938, PRO827 and PRO1031 were successfiully expressed in
baculovirus infected Sf9 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 IgGl constant region sequence
contaning the hinge, CH2 and CH3 domains and/or in poly-His tagged
forms.
[2446] For expression in baculovirus infected Sf9 cells, following
PCR amplification, the respective coding sequences were subdloned
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 (Pharmiingen) 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 Rink'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 inHink's TNM-FH medium supplementedwith 10% FBS atan
approximate multiplicity of infection (MOI) 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 CL4B beads (Pharmacia) for IgG tagged proteins
followed by SDS-PAGE analysis comparing to a known concentration of
protein standard by Coomassie blue staining.
[2447] 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.
[2448] 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, imtdazole 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.
[2449] 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.
[2450] PRO181, PRO195, PRO200, PRO320, PRO237, PRO273, PRO285,
PRO337, PRO526, PRO540, PRO846, PRO362, PRO363, PRO617, PRO322,
PRO1083, PRO868, 768, PRO792, PRO788, PRO162, PRO1114, PRO827,
PRO1075 and PRO1031 were successfiully expressed in baculovirus
infected Hi5 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.
[2451] For expression in baculovirus-infected His insect cells, the
PRO polypeptide-encoding DNA may be amplified with suitable
systems, such as Pfu (Stratagene), or fused upstream (5'-of) 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 subeloned into the expression vector. For example,
derivatives of pVL1393 can include the Fc region of human IgG
(pb.PH.1gG) or an 8 histidine (pb.PH.His) tag downstream (3'of) the
NAME sequence. Preferably, the vector construct is sequenced for
confrination.
[2452] Hi5 cells are grown to a confluency of 50% under the
conditions of, 27.degree. C., no CO2, NO pen/strep. For each 150 mm
plate, 30 ug of pIE based vector containing PRO polypeptide is
mixed with 1 ml Ex-Cell medium (Media: Ex-Cell 401+{fraction
(1/100)} L-Glu JRH Biosciences #14401-78P (note: this media is
light sensitive)), and in a separate tube, 100 ul of CellFectin
(CellFECTIN (GibcoBRL #10362-010) (vortexed to. mix)) is mixed with
1 ml of Ex-Cell medium. The two solutions are combined and allowed
to incubate at room temperature for 15 minutes. 8 nl of Ex-Cell
media is added to the 2ml of DNA/CellFECTIN mix and this is layered
on Hi5 cells that have been washed once with Ex-Cell media. The
plate is then incubated in darkness for 1 hour at room temperature.
The DNA/CeUFECTlN mix is then aspirated, and the cells are washed
once with Ex-Cell to remove excess CellFECTIN. 30 ml of fresh
Ex-Cell media is added and the cells are incubated for 3 days at
28.degree. C. The supernatant is harvested and the expression of
the PRO polypeptide in the baculovirus expression vector can be
determined by batch binding of 1 ml of supernatent to 25 mL of
Ni-NTA beads (QIAGEN) for histidine tagged proteins or Protein-A
Sepharose CL4B beads (Pharmacia) for IgG tagged proteins followed
by SDS-PAGE analysis comparing to a known concentration of protein
standard by Coomassie blue staining.
[2453] The conditioned media from the transfected cells (0.5 to 3
L) is harvested by centrifugation to remove the cells and filtered
through 0.22 micron filters. For the poly-His tagged constructs,
the protein comprising the PRO polypeptide is purified using a
Ni-NTA column (Qiagen). Before purification, imldazole is added to
the conditioned media to a concentration of 5 mM. The conditioned
media is pumped onto a 6 ml Ni-NTA column equilibrated in 20 mM
Hepes, pH 7.4, buffer contaiing 0.3 M NaCl and S mM imidazole at a
flow rate of 4-5 ml/min. at 4.degree. C. After loading, the column
is washed with additional equilibration buffer and the protein
eluted with equilibration buffer containing 0.25 M imidazole. The
highly purified protein is subsequently deslated into a storage
buffer containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol, pH 6.8,
with a 25 ml G25 Superfme (Pharmacia) column and stored at
-80.degree. C.
[2454] Immunoadhesin (Fc containing) constructs of proteins are
purified from the conditioned media as follows. The conditioned
media is pumped onto a 5 ml Protein A column (Pharmacia) which had
been equilibrated in 20 mM Na phosphate buffer, pH 6.8. After
loading, the column is washed extensively with equilibration buffer
before elution with 100 mM citric acid, pH 3.5. The eluted protein
is immediately neutralized by collecting 1 ml fractions into tubes
containing 275 mL of 1 M Tris buffer, pH 9. The highly purified
protein is subsequently desalted into storage buffer as described
above for the poly-His tagged proteins. The homogeneity of PRO
polypeptide can be assessed by SDS polyacrylamide gels and by
N-terminal amino acid sequencing by Edman degradation and other
analytical procedures as desired or necessary.
[2455] Many of the PRO polypeptides described herein were
successfully expressed as described above.
Example 104
[2456] Preparation of Antibodies that Bind to PRO Polypeptides
[2457] This example illustrates preparation of monoclonal
antibodies which can specifically bind to a PRO polypeptide.
[2458] Techniques for producing the monoclonal antibodies are known
in the art and are described, for instance, in Goding, surra.
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 experinentation.
[2459] 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 inMPL-TDM
adjuvant (Ribi hinmunochemical Research, Hamilton, Mont.) and
injected into the aniimal'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.
[2460] 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 P3X63AgU.1, available
from ATCC, No. CRL 1597. The fusions generate hybridoma cells which
can then be plated in 96 well tissue culture plates containing HAT
(hypoxanthine, aminopterin, and thynidine) medium to inhibit
proliferation of non-fused cells, myeloma hybrids, and spleen cell
hybrids.
[2461] 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.
[2462] 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 105
[2463] Chimeric PRO Polypeptides
[2464] 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 (Olvitrogen, San Diego Cafif.) between the
purification domain and the PRO polypeptide sequence may be useful
to facilitate expression of DNA encoding the PRO polypeptide.
Example 106
[2465] Purification of PRO Polypeptides Using Specific
Antibodies
[2466] 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.
[2467] Polyclonal immunoglobulins are prepared fiom 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.
[2468] 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.
[2469] 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 107
[2470] Drug Screening
[2471] 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 cellor target receptors caused by the agent being
tested.
[2472] 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.
[2473] 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 84103564,
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.
[2474] 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 108
[2475] Rational Drug Design
[2476] 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)).
[2477] In one approach, the three imensional 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, usefuil 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).
[2478] 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.
[2479] 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 109
[2480] Ability of PRO Polypeptides to Inhibit Vascular Endothelial
Growth Factor (VEGF) Stimulated Proliferation of Endothelial Cell
Growth (Assay 9)
[2481] 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.
[2482] Specifically, bovine adrenal cortical capilary endothelial
cells (ACE) (from primary culture, maxim 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. penicfflin/streptomycin/fungizone. Control
wells included the following: (1) no ACE cells added; (2) ACE cells
alone; (3) ACE cells plus 5 ng/ml IFGF; (4) ACE cells plus 3 ng/ml
VEGF; (5) ACE cells plus 3 ngiml 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
37OC/5% C0.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. IM 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 nm.
[2483] 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 rm)
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
ng/ml 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).
[2484] The following polypeptides tested positive in this assay:
PRO200, PRO322 and PRO320.
Example 10
[2485] Retinal Neuron Survival (Assay 52)
[2486] 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.
[2487] 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 DMEMlF12 supplemented with N2 and
with or without the specific test PRO polypeptide. Cells for all
experiments are grown at 370C 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
deterination 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.
[2488] 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.
[2489] The following PRO polypeptides tested positive in this assay
using polypeptide concentrations within the range of 0.01% to 1.0%
in the assay: PRO200, PRO322, PRO540, PRO846 and PRO617.
Example 111
[2490] Rod Photoreceptor Survival (Assay 56)
[2491] 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 inmammals 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+,
M.sup.2+-free PBS. The retinas are incubated at 37.degree. C. for
7-10 minutes after which the trypsin is inactivated by adding I ml
soybean trypsin inhibitor. The cells are plated at 100,000 cells
per well in 96 well plates in DMEM/FI2 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.
[2492] The following polypeptides tested positive in this assay:
PRO200, PRO322, PRO540, PRO846 and PRO617.
Example 112
[2493] Ability of PRO Polypeltides to Stimulate the Release of
Proteoglycans from Cartilage (Assay 97)
[2494] The ability of various PRO polypeptides to stimulate the
release of proteoglycans from cartilage tissue was tested as
follows.
[2495] 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 100U/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
polypetides were then added at 1% either alone or in combination
with 18 ng/ml interleukin-la, a known stimulator of proteoglycan
release from cartilage tissue. The supernatant was then harvested
and assayed for the amount of proteoglycans using the
1,9dimethyl-methylene blue (DM13) 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, osteoardritis or rheumatoid
arthritis.
[2496] When various PRO polypeptides were tested in the above
assay, the polypeptides demonstrated armarked ability to stimulate
release of proteoglycans from cartilage tissue both basally and
after stimulation with interleulin-la 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-relatedjoint problems, articular cartilage defects,
osteoarthritis or rheumatoid arthritis. The polypeptides testing
positive in this assay are: PRO200.
Example 113
[2497] In Vitro Antiproliferative Assav (Assay 161)
[2498] 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]).
[2499] 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 96-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.
[2500] 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.
[2501] 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:
[2502] NSCL=non-small cell lung carcinoma
[2503] CNS=central nervous system
163TABLE 7 Test compound Tumor Cell Line Type Cell Line Designation
PRO181 Leukemia RPMI-8226 PRO181 NSCL NCI-H226; NCI-H522 PRO181
Melanoma MALME-3M; SK-MEL-5 PRO181 Ovarian OVCAR-4 PRO181 Breast
NCI/ADR-RES PRO181 Leukemia MOLT-4 PRO181 NSCL NCI-H226* PRO181 CNS
SNB-19 PRO181 Ovarian OVCAR-3; OVCAR-8 PRO181 Renal A498 PRO181
Breast MDA-MB-231/ATCC; MDA-N PRO181 Melanoma LOX IMVI PRO181
Leukemia CCRF-CEM; RPMI-8226* PRO181 NSCL HOP-62 PRO181 Leukemia
HL-60 (TB) PRO237 Leukemia K-562 PRO237 NSCL NCI-H322M PRO237 Colon
HCC-2998; HCT-15 PRO237 Colon KM12 PRO237 Prostate DU-145 PRO237
Breast MDA-N PRO526 NSCL HOP-62; NCI-H322M PRO526 Colon HCT-116
PRO526 Melanoma LOX IMVI; SK-MEL-2 PRO526 Ovarian OVCAR-3 PRO526
Prostate PC-3 PRO526 NSCL NCI-H226 PRO526 CNS SF-539 PRO526 Renal
CAKI-1; RXF 393 PRO362 NSCL NCI-H322M PRO362 Colon HCT-116 PRO362
CNS SF-295 PRO362 Melanoma LOX IMVI PRO362 Leukemia MOLT-4;
RPMI-8226; SR PRO362 Colon COLO 205 PRO362 Breast HS 578T; MDA-N
PRO362 Prostate PC-3 PRO362 Leukemia HL-60 (TB); K-562 PRO362 NSCL
EKVX; NCI-H23 PRO362 Colon HCC-2998 PRO362 CNS U251 PRO362 Melanoma
UACC-257; UACC-62 PRO362 Ovarian OVCAR-8 PRO362 Breast T-47D PRO362
NSCL NCI-H522 PRO362 Renal RXF 393; UO-31 PRO362 Breast MDA-MB-435
PRO362 NSCL HOP-62; NCI-H522 PRO362 Colon KM12 PRO362 Melanoma
MALME-3M; SK-MEL-2 PRO362 Melanoma SK-MEL-28; SK-MEL-5 PRO362
Ovarian OVCAR-3; OVCAR-4 PRO362 Breast MCF7 PRO866 Leukemia HL-60
(TB); MOLT-4; SR PRO866 NSCL HOP-62 PRO866 NSCL HOP-92 PRO866 Colon
KM12 PRO866 CNS SF-295 PRO866 Ovarian IGROV1 PRO866 Breast
MDA-MB-435 PRO866 Melanoma LOX IMVI PRO320 Leukemia CCRF-CEM;
RPMI-8226 PRO320 NSCL HOP62; NCI H322M PRO320 Colon HCT-116 PRO320
Renal SN12C PRO320 Breast MDA-N PRO320 Ovarian OVCAR-3 PRO320
Melanoma MALME-3M *cytotoxic
[2504] The results of these assays demonstrate that the positive
testing PRO polypeptides are useful for inhibiting neoplastic
growth in a number 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 114
[2505] Gene Amirlification in Tumors
[2506] 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.
[2507] 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 (forexample, ABI Prizm7700 Sequence
Detection System.TM. (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.
[2508] The results of the TaqMan.TM. are reported in delta
(.DELTA.) 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-encodig 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:
164 PRO853 (DNA48227-1350) 48227am.f1 5'-GGCACTTCATGGTCCTTGAAAA-3'
(SEQ ID NO: 539) 48227.tm.p1 5'-CGGATGTGTGTGAGGCCATGCC-3' (SEQ ID
NO: 540) 48227.tm.r1 5'-GAAAGTAACCACGGAGGTCAAGAT-3' (SEQ ID NO:
541) PRO1017 (DNA56112-1379): 56112.tm.f1
5'-CCTCCTCCGAGACTGAAAGCT-3' (SEQ ID NO: 542) 56112.tm.p1
5'-TCGCGTTGCTTTTTCTCGCGTG-3' (SEQ ID NO: 543) 56112.tm.r1
5'-GCGTGCGTCAGGTTCCA-3' (SEQ ID NO: 544) PRO213-1
(DNA30943-1163-1): 30943.tm.f3: 5'-CGTTCGTGCAGCGTGTGTA-3' (SEQ ID
NO: 545) 30943.tm.p3: 5'-CTTCCTCACCACCTGCGACGGG-3' (SEQ ID NO: 546)
30943.tm.r3: 5'-GGTAGGCGGTCCTATAGATGGTT-3' (SEQ ID NO: 547)
30943.tm.f1: 5'-AGATGTGGATGAATGCAGTGCTA-3' (SEQ ID NO: 548)
30943.tm.p1: 5'-ATCAACACCGCCGGCATTACTGCG-3' (SEQ ID NO: 549)
30943.tm.r1: 5'-ACAGAGTGTACCGTCTGCAGACA-3' (SEQ ID NO: 550)
30943.3trn-5: 5'-AGCCTCCTGGTGCACTCCT-3' (SEQ ID NO: 551)
30943.3trn-probe: 5'-CGACTCCCTGAGCGAGCAGATT (SEQ ID NO: 552) TCC-3'
5'-GCTGGGCAGTCACGAGTCTT-3' (SEQ ID NO: 553) PRO237 (DNA34353-1428):
34353.tm.f: 5'-AATCCTCCATCTCAGATCTTCCAG-3' (SEQ ID NO: 554)
34353.tm.p: 5'-CCTCAGCGGTAACAGCCGGCC-3' (SEQ ID NO: 555)
34353.tm.r: 5'-TGGGCCAAGGGCTGC-3' (SEQ ID NO: 556) PRO324
(DNA36343-1310): 36343.tmf1: 5'-TGGTGGATAACCAACAAGATGG-3' (SEQ ID
NO: 557) 36343.tmp1: 5'-GAGTCTGCATCCACACCACTCTTAAAGTTC (SEQ ID NO:
558) TCAA-3' 36343.tmr1: 5'-CAGGTGCTCTTTTCAGTCATGTTT-3' (SEQ ID NO:
559) PRO351 (DNA40571-1315): 40571.tm.f1:
5'-TGGCCATTCTCAGGACAAGAG-3' (SEQ ID NO: 560) 40571.tm.p1:
5'-CAGTAATGCCATTTGCCTGCCTGCAT-3' (SEQ ID NO: 561) 40571.tm.r1:
5'-TGCCTGGAATCACATGACA-3' (SEQ ID NO: 562) PRO362 (DNA45416-1251):
45416.tm.f1: 5'-TGTGGCACAGACCCAATCCT-3' (SEQ ID NO: 563)
45416.tm.p1: 5'-GACCCTGAAGGCCTCCGGCCT-3' (SEQ ID NO: 564)
45416.tm.r1: 5'-GAGAGAGGGAAGGCAGCTATGTC-3' (SEQ ID NO: 565) PRO615
(DNA48304-1323): 48304.tm.f1: 5'-CAGCCCCTCTCTTTCACCTGT-3' (SEQ ID
NO: 566) 48304.tm.p1: 5'-CCATCCTGTGCAGCTGACACACAGC-3' (SEQ ID NO:
567) 48304.tm.r1: 5'-GC CAGGCTATGA GGCTCCTT-3' (SEQ ID NO: 568)
PRO531 (DNA48314-1320): 48314.tm.f1: 5'-TTCAAGTTCCTGAAGCCGATTAT-3'
(SEQ ID NO: 569) 48814.tm.p1: 5'-CCAACTTCCCTCCCCAGTGCCCT-3' (SEQ ID
NO: 570) 48814.tm.r1: 5'-TTGGGGAAGGTAGAATTTCCTTGTAT-3' (SEQ ID NO:
571) PRO618 (DNA49152-1324): 49152.tm.f1:
5'-CCCTTCTGCCTCCCAATTCT-3' (SEQ ID NO: 572) 49152.tm.p1:
5'-TCTCCTCCGTCCCCTTCCTCCACT-3' (SEQ ID NO: 573) 49152.tm.r1:
5'-TGAGCCACTGCCTTGCATTA-3' (SEQ ID NO: 574) PRO772 (DNA49645-1347):
49645.tm.f2: 5'-TCTGCAGACGCGATGGATAA-3' (SEQ ID NO: 575)
49645.tm.p2: 5'-CCGAAAATAAAACATCGCCCCTTCTGC-3' (SEQ ID NO: 576)
49645.tm.r2: 5'-CACGTGGCCTTTCACACTGA-3' (SEQ ID NO: 577)
49645.tm.f1: 5'-ACTTGTGACAGCAGTATGCTGT- CTT-3' (SEQ ID NO: 578)
49645.tm.p1: 5'-AAGCTTCTGTTCAATCCCAGCGGTCC-3' (SEQ ID NO: 579)
49645.tm.r1: 5'-ATGCACAGGCTTTTTCTGGTAA-3' (SEQ ID NO: 580) PRO703
(DNA50913-1287): 50913.tm.f1: 5'-GCAGGAAACCTTCGAATCTGAG-3' (SEQ ID
NO: 581) 50913.tm.p1: 5'-ACACCTGAGGCACCTGAGAGAGGAAC (SEQ ID NO:
582) TCT-3' 50913.tm.r1: 5'-GACAGCCCAGTACACCTGCAA-3' (SEQ ID NO:
583) PRO792 (DNA56352-1358): 56352.tm.f1:
5'-GACGGCTGGATCTGTGAGAAA-3' (SEQ ID NO: 584) 56352.tm.p1:
5'-CACAACTGCTGACCCCGCCCA-3' (SEQ ID NO: 585) 56352.tm.r1:
5'-CCAGGATACGACATGCTGCAA-3' (SEQ ID NO: 586) PRO474
(DNA56045-1380): 56045.tm.f1: 5'-AAACTCCAACCTGTATCAGATGCA-3' (SEQ
ID NO: 587) 56045.tm.p1: 5'-CCCCCAAGCCCTTAGACTCTAAGCCC-3' (SEQ ID
NO: 588) 56045.tm.r1: 5'-GACCCGGCACCTTGCTAAC-3' (SEQ ID NO: 589)
PRO274 (DNA39987-1184): 39987.tm.f: 5'-GGACGGTCAGTCAGGATGACA-3'
(SEQ ID NO: 590) 39987.tm.p: 5'-TTCGGCATCATCTCTT2CCCTCTCCC-3' (SEQ
ID NO: 591) 39987.tm.r: 5'-ACAAAAAAAAGGGAACAAAATACGA-3' (SEQ ID NO:
592) PRO381 (DNA44194-1317) 44194.tm.f:
5'-CTTTGAATAGAAGACTTCTGGACAA (SEQ ID NO: 593) TTT-3' 44194.tm.p:
5'-TTGCAACTGGGAATATACCACGACATG (SEQ ID NO: 594) AGA-3' 44194.tm.r:
5'-TAGGGTGCTAATTTGTGCTATAACCT-3' (SEQ ID NO: 595) 44194.tm.f2:
5'-GGCTCTGAGTCTCTGCTTGA-3' (SEQ ID NO: 596) 44194.tm.p2:
5'-TCCAACAACCATTTTCCTCTGGTCC-3' (SEQ ID NO: 597) 44194.tm.r2:
5'-AAGCAGTAGCCATTAACAAGTCA-3' (SEQ ID NO: 598) PRO717
(DNA50988-1326): 50988.tm.f3: 5'-CAAGCGTCCAGGTTTATGA-3' (SEQ ID NO:
599) 50988.tm.r3: 5'-GACTACAAGGCGCTCAGCTA-3' (SEQ ID NO: 600)
50988.tm.p3: 5'-CCGGCTGGGTCTCACTCCTCC-3' (SEQ ID NO: 601) PRO1330
and PRO1449 (DNA64907-1163 and DNA64908-1163. respectively):
30943.tm.f3: 5'-CGTTCGTGCAGCGTGTGTA-3' (SEQ ID NO: 602)
30943.tm.p3: 5'-CTTCCTCACCACCTGCGACG GG-3' (SEQ ID NO: 603)
30943.tm.r3: 5'-GGTAGGCGGTCCTATAGATGGTT-3' (SEQ ID NO: 604)
30943.tm.f1: 5'-AGATG TGGATGAATG CAGTGCTA-3' (SEQ ID NO: 605)
30943.tm.p1: 5'-ATCAACACCGCCGGCAGTTACTGG-3' (SEQ ID NO: 606)
30943.tm.r1: 5'-ACAGAGTGTACCGTCTGCAGACA-3' (SEQ ID NO: 607)
30943.3trn-5: 5'-AGCCTCCTGGTGCACTCCT-3' (SEQ ID NO: 608)
30943.3trn-probe: 5'-CGACTCCCTGAGCGAGCAGATTTCC-3' (SEQ ID NO: 609)
30943.3trn-3: 5'-GCTGGGGGCAGTCACGAGTCTT-3- ' (SEQ ID NO: 610)
[2509] 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.
[2510] 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.
[2511] 5' Nuclease assay data are initially expressed as Ct, or the
threshold cycle. This is defined as the cycle at which the reporter
signal accumulates above the background level of fluorescence. The
ACt values are used as quantitative measurement of the relative
number of starting copies of a particular target sequence in a
nucleic acid sample when comparing cancer DNA results to normal
human DNA results.
[2512] 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.
165TABLE 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)
[CT10] 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
[2513] DNA Preparation:
[2514] 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.
[2515] Cell Culture Lysis:
[2516] Cells were washed and trypsinized at a concentration of
7.5.times.10.sup.8 per tip and pelleted by centrifiging 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 clituted
into 6.25 ml cold ddH.sub.2O to a fina 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 mg/ml) to a final concentration
of 200 .mu.g/ml.
[2517] Buffer C1 (10 ml, 4.degree. C.) and ddH20 (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 4PC for 15 minutes. The supernatant was discarded and the
nuclei were suspended with a vortex into 2 ml Buffer Cl (at 4PC)
and 6 ml ddH20, 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. Quiagen protease (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.).
[2518] Solid Human Tumor Sample Preparation and Lysis:
[2519] 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 fmal 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
Jtumor 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.20, followed by G2 buffer
(50 ml). If tissue was still present on the generator tip, the
apparatus was disassembled and cleaned.
[2520] 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 centrigation 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.).
[2521] Human Blood Preparation and Lysis:
[2522] 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,vg/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 Cl
buffer (4.degree. C.) and 6 ml ddH20 (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.).
[2523] Purification of Cleared Lysates:
[2524] (1) Isolation of genomic DNA:
[2525] Genomic DNA was equilibrated (1 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 silanied, autoclaved 30 ml Corex tubes with 15
m1l QF buffer (50.degree. C.). Isopropanol (10.5 ml) was added to
each sample, the tubes covered with parafin and mixed by repeated
inversion until the DNA precipitated. Samples were pelleted by
cenrifugation 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,C) was added. Samples were
pelleted. again by centrifigation on the SS-34 rotor at 10,000 rpm
for 10 minutes at 49C. 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.
[2526] 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.
[2527] (2) Quantitation of Zenomic DNA and Preparation for Gene
Amplification Assay:
[2528] 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.
[2529] 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.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.
[2530] 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 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.
[2531] Gene Amplification Assay:
[2532] 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.
166TABLE 9 .DELTA.Ct values in lung and colon primary tumor and
cell line models PRO1330 Tumor or PRO PRO PRO PRO PRO PRO PRO PRO
PRO PRO PRO PRO PRO PRO PRO PRO PRO and Cell Line 213-1 237 324 351
362 615 531 853 1017 618 772 703 792 474 274 381 717 PRO1449 LT-1
1.60 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 1.60 LT-1a
1.24 1.04 -- -- -- -- 1.70 -- 1.785 -- 1.33 1.22 1.16 1.94 -- -- --
1.24 1.62 LT2 -- -- -- -- 1.39 -- -- -- -- -- -- -- -- -- -- -- --
-- LT3 1.51 1.74 -- -- -- 1.31 1.95 -- 2.38 1.03 1.11 1.77 1.10
2.55 -- -- -- 1.51 1.55 1.24 1.52 1.44 LT4 2.26 -- -- -- 1.00 --
1.46 -- -- -- -- -- -- -- 1.24 -- -- 2.26 LT6 1.56 1.16 -- -- --
1.00 2.07 -- 2.80 -- 1.07 1.15 1.81 2.10 -- -- -- 1.56 2.28 LT7
2.45 1.44 -- -- -- 1.09 -- -- 1.12 -- -- 1.44 -- 1.06 -- -- -- 2.45
1.03 LT9 1.24 -- -- 1.19 -- 1.04 1.10 -- 2.74 1.39 1.62 -- 1.99
2.56 -- -- -- 1.24 1.14 1.11 2.59 LT10 -- 1.20 -- 1.06 1.69 1.18
1.96 -- 3.52 1.29 1.46 1.48 2.00 2.63 -- -- -- -- 1.11 1.16 1.29
2.85 LT11 2.26 -- 1.34 1.02 -- 1.46 1.79 1.03 1.54 1.84 1.45 1.90
1.20 1.36 -- -- -- 2.26 2.85 1.72 2.94 1.83 5.21 2.85 2.25 1.27
1.41 2.25 1.79 1.25 1.79 1.06 LT12 1.86 -- 1.92 -- -- 2.08 1.86
1.18 1.77 -- -- 1.38 -- 1.64 -- -- -- 1.86 4.32 1.87 3.02 1.62 5.01
4.32 2.59 1.41 1.82 2.59 1.55 1.50 1.55 1.25 LT13 1.98 1.05 -- 1.23
-- 1.39 2.53 1.33 1.55 -- 1.18 1.33 1.33 1.03 -- -- 7.03 1.98 2.52
1.09 2.06 2.14 1.20 1.00 2.52 2.38 1.03 1.31 2.03 4.54 2.38 1.14
1.65 LT15 1.40 -- -- 1.14 -- 1.67 2.56 1.28 2.23 -- 1.47 1.45 1.04
1.35 -- -- 2.71 1.40 1.58 1.47 2.95 2.01 1.44 1.86 1.58 2.69 1.09
1.31 2.50 4.97 2.69 1.05 1.52 LT16 1.22 1.22 1.63 1.09 -- 1.32 --
1.33 2.98 -- -- 1.07 -- 4.23 1.00 -- 5.48 1.22 2.77 1.38 1.77 1.52
2.77 1.75 1.17 1.75 LT17 4.58 1.07 1.75 1.46 -- 1.66 1.12 1.21 2.90
1.04 1.42 1.24 1.35 1.40 -- -- -- 4.58 3.73 1.59 1.53 1.62 1.61
1.115 5.45 3.73 5.55 1.21 5.55 1.50 1.13 LT18 -- -- -- 1.07 -- --
-- -- 3.28 -- -- -- -- 5.31 1.61 -- -- -- 1.68 LT19 1.03 -- 1.90
1.33 -- 1.59 2.08 -- 2.54 -- 1.60 1.38 1.62 1.59 -- -- -- 1.03 1.22
1.50 2.95 2.98 1.19 1.84 1.22 1.26 1.03 1.21 4.84 1.26 1.48 LT21
1.86 -- 1.15 1.27 -- 1.19 -- -- 3.14 -- -- 1.22 -- 5.15 -- -- --
1.86 1.83 1.09 1.83 3.21 1.06 3.21 LT22 1.61 -- -- -- -- -- -- --
-- -- -- -- -- -- -- -- -- 1.61 CT2 1.61 -- -- -- -- 1.36 2.21 2.4
3.72 -- -- 2.10 1.46 2.67 -- -- -- 1.61 2.11 1.25 2.55 2.55 1.65
2.11 1.90 1.48 CT3 -- -- -- -- -- 1.12 1.50 1.52 3.91 -- -- 1.62 --
2.41 -- -- -- -- 1.58 1.02 CT8 2.80 -- -- -- -- -- 1.15 1.55 2.66
-- -- 1.06 -- 2.34 -- -- -- 2.80 1.34 CT10 2.39 -- -- -- -- 1.55
1.75 1.97 3.57 -- -- 1.96 -- 2.23 -- -- -- 2.39 1.47 1.78 1.21 CT12
3.45 -- -- -- -- 1.08 1.93 1.36 3.50 -- -- 1.57 -- 2.46 -- -- --
3.45 1.30 1.08 CT14 3.79 -- -- -- -- 1.76 1.47 1.75 3.88 -- -- 1.19
-- 2.83 -- -- -- 3.79 1.02 1.11 1.86 CT15 3.66 -- -- -- -- 1.23
2.44 1.75 3.62 -- -- 1.70 -- 2.89 -- -- 2.61 3.66 1.33 CT16 2.66 --
-- -- -- 1.29 1.95 1.11 3.12 -- -- 1.51 -- 2.60 -- -- 2.21 2.66
CT17 3.63 -- -- -- -- 1.44 2.19 1.11 3.34 -- -- 1.31 -- 2.33 -- --
3.31 3.63 CT1 -- -- -- -- -- -- -- 1.09 -- -- -- 1.08 -- 1.00 -- --
-- -- CT4 1.18 -- -- -- -- 1.17 -- 1.16 1.11 -- -- 1.63 -- 1.13 --
-- -- 1.18 1.07 CT5 1.25 -- -- -- -- 1.12 1.59 1.95 2.21 -- -- 1.50
-- 1.84 -- -- -- 1.25 1.16 1.35 2.05 2.11 CT6 1.27 -- -- -- -- --
-- -- 1.12 -- -- 1.38 -- 1.24 -- -- -- 1.27 1.36 CT7 -- -- -- -- --
-- -- 1.14 -- -- -- 1.50 -- -- -- -- -- -- CT9 -- -- -- -- -- --
1.28 -- 1.29 -- -- -- -- -- -- -- -- -- CT11 -- -- -- -- -- 1.74
1.49 1.88 1.48 -- -- 1.99 -- 2.11 -- -- -- -- 1.17 2.13 CT18 -- --
-- -- -- 1.36 -- -- -- -- -- 1.15 -- 9.66 -- -- -- -- Calu-1 1.35
-- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 1.77 1.35 2.95 2.95
H441 2.00 -- -- -- -- -- -- 1.71 -- -- -- -- -- -- -- -- 2.57 2.00
H522 -- -- -- -- -- -- -- 1.03 -- -- -- -- -- -- -- -- 3.78 -- H810
2.76 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 1.84 2.76 HT29
1.31 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 1.71 1.31 SW403
2.08 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 2.09 2.08 LS174T
1.61 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 2.90 1.61 HCT15
1.22 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 1.46 1.22 HCC2998
1.73 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 1.20 1.73
HF-000643 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 4.83 -- --
HF-000840 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 1.08 -- --
HF-000811 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 2.09 -- --
3.15 HF-001294 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 1.14 --
-- 1.08 HF-001296 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 3.18
-- -- 3.53 HF-001291 -- -- -- -- -- -- -- -- -- -- -- -- -- -- --
1.17 -- -- A549 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --
1.66 -- H460 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 2.50
-- SKMES1 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 2.15 --
SW620 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 2.36 --
Colo320 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 1.99 --
2.73 HCT116 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 1.90 --
SKCO1 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 3.13 --
Colo205 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 1.48 --
KM12 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- 1.67 --
[2533] Summary
[2534] 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 115
[2535] Induction of c-fos in Endothelial Cells (Assay 34)
[2536] 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.
[2537] 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.4cells/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% (wlv) 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.
[2538] 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 weU 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.
[2539] 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 maling 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.
[2540] 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.
[2541] The following PRO polypeptides tested positive in this
assay: PRO938, PRO200, PRO865, PRO788 and PRO1013.
Example 116
[2542] Proliferation of Rat Utricular Supporting Cells (Assay
54)
[2543] 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 thle regeneration of auditory hair cells and treating
hearing loss in mammals. The assay is performed as follows. Rat
UEC4 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 serum-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.
[2544] The following polypeptide tested positive in this assay:
PRO337, PRO363 and PRO1012.
Example 117
[2545] Detection of PRO Polypeptides That Affect Glucose or FFA
Uptake by Primary Rat Adipocytes (Assay 94)
[2546] 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.
[2547] In a 96 well format, PRO polypeptides to be assayed are
added to priary 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 HFA 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.
[2548] The following PRO polypeptides tested positive as
stimulators of glucose and/or FFA uptake in this assay: PRO181,
PRO200, PRO337, PRO362, PRO363, PRO731, PRO534, PRO1114 and
PRO1075.
[2549] The following PRO polypeptides tested positive as
imhibitorss of glucose and/or FFA uptake in this assay: PRO195,
PRO322, PRO862, PRO868, PRO865 and PRO162.
Example 118
[2550] Detection of Polypeptides That Affect Glucose and/or FFA
Uptake in Skeletal Muscle (Assay 106)
[2551] 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.
[2552] 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 PFA 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.
[2553] The following PRO polypeptides tested positive as either
stimulatorrs or inhibitors of glucose and/or FFA uptake in this
assay: PRO181, PRO200, PRO1083, PRO865, PRO162, PRO1008 and
PRO1330.
Example 119
[2554] Stimulation of Heart Neonatal Hypertrophy (Assay 1)
[2555] 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.
[2556] 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
previouslycoatedwith 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.
[2557] The following polypeptides tested positive in this assay:
PRO195, PRO200, PRO526 and PRO792.
Example 120
[2558] Enhancement of Heart Neonatal Hypertrophy Induced by F2a
(Assay 37)
[2559] 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.
[2560] 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-wellplates previously coated with DMEM/F12+4% FCS. Test samples
containing the test PRO polypeptide (20 yl/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.sup.-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.
[2561] 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 transferiin, 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 {fraction (1/10)} (0.4%) and
{fraction (1/100)} (0.04%), but not at {fraction (1/1000)}
(0.004%). Therefore the test sample buffer containing mannitol is
not run.
[2562] The following PRO polypeptides tested positive in this
assay: PRO195.
Example 121
[2563] Guinea Pig Vascular Leak (Assays 32 and 51)
[2564] 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.
[2565] 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
slin 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 blueolored
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.
[2566] The following PRO polypeptides tested positive in this
assay: PRO200.
Example 122
[2567] Skin Vascular Permeability Assay (Assay 64)
[2568] 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 inflammnatory cell
infiltration into the skin. Sites with visible inflammatory cell
inflammation are scored as positive. nflammatory cells may be
neutrophilic, eosinophilic, monocytic or lymphocytic. At least a
minimal perivascular infiltrate at the injection site is scored as
positve, no infiltrate at the site of injection is scored as
negative.
[2569] The following polypeptide tested positive in this assay:
PRO200, PRO362 and PRO1031.
Example 123
[2570] Induction of c-fos in Cortical Neurons (Assay 83)
[2571] 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.
[2572] 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 tie
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. MnRNA 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.
[2573] The following PRO polypeptides tested positive in this
assay: PRO200.
Example 124
[2574] Mouse Kidney Mesangial Cell Proliferation Assay (Assay
92)
[2575] 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 ml 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, 20pl
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.
[2576] The following polypeptide tested positive in this assay:
PRO200, PRO363, PRO731, PRO534, PRO866 and PRO1031.
Example 125
[2577] Pericvte c-Fos Induction (Assay 93)
[2578] 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 pericyteassociated 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
gl of PRO polypeptide test samples and controls (positive
control=DME+5% serum +/-PDGF at 500 ng/ml; negative control=protein
32). Replicates are averaged and SDICV 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+1X pen strep+1X fungizone. Assay Media=low
glucose DMEM +5% FBS.
[2579] The following polypeptides tested positive in this assay:
PRO200.
Example 126
[2580] Chondrocyte Re-differentiation Assay (Assay 110)
[2581] 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 metacarpophalangealjoints 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% PBS 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 100w1 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 .mu.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.
[2582] The following polypeptide testedpositive in this assay:
PRO200, PRO285, PRO337, PRO526, PRO362, PRO363, PRO531, PRO1083,
PRO862, PRO733, PRO1OI7, PRO792, PRO788, PRO1008, PRO1075, PRO725
and PRO1031.
Example 127
[2583] Fetal Hemoglobin Induction in an Erythroblastic Cell Line
(Assay 107)
[2584] 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.
[2585] The following polypeptides tested positive in this assay:
PRO237, PRO381, PRO362, PRO724, PRO866, PRO1114, PRO725 and
PRO1071.
Example 128
[2586] Induction of Pancreatic (3-Cell Precursor Proliferation
(Assay 117)
[2587] This assay shows that certain polypeptides of the invention
act to induce an increase in the number of pancreatic .beta.-cell
precursor cells and, therefore, are useful for treating various
insulin deficient states in mammals, including diabetes mellitus.
The assay is performed as follows. The assay uses a primary culture
of mouse fetal pancreatic cells and the primary readout is an
alteration in the expression of markers that represent either
.beta.-cell precursors or mature .beta.-cells. Marker expression is
measured by real time quantitative PCR (RTQ-PCR); wherein the
marker being evaluated is a transcription factor called Pdxl.
[2588] The pancreata are dissected from E14 embryos (CD1 mice). The
pancreata are then digested with collagenase/dispase in F12DMEM at
37.degree. C. for 40 to 60 minutes (collagenase/dispase, 1.37
mg/ml, Boehringer Mannheim, #1097113). The digestion is then
neutralized with an equal volume of 5% BSA and the cells are washed
once with RPAI1640. At day 1, the cells are seeded into 12-well
tissue culture plates (pre-coated with lamin, 20 .mu.g/ml in PBS,
Boehringer Manmheim, #124317). Cells from pancreata from 1-2
embryos are distributel per well. The culture medium for this
primary cuture is 14F/1640. At day 2, the media is removed and the
attached cells washed with RPMI/1640. Two mls of minimal media are
added in addition to the protein to be tested. At day 4, the media
is removed and RNA prepared from the cells and marker expression
analyzed by real time quantitative RT-PCR. A protein is considered
to be active in the assay if it increases the expression of the
relevant P-ceU marker as compared to untreated controls.
[2589] 14F/1640 is RPMI1640 (Gibco) plus the following:
[2590] group A 1:1000
[2591] group B 1:1000
[2592] recombinant human insulin 10 .mu.g/ml
[2593] Aprotinin (50 .mu.g/ml) 1:2000 (Boehringer manheim
#981532)
[2594] Bovine pituitary extract (BPE) 60 .mu.g/ml
[2595] Gentamycin 100 ng/ml
[2596] Group A: (in 10 ml PBS)
[2597] Transferrin, 100 mg (Sigma T2252)
[2598] Epidermal Growth Factor, 100 .mu.g (BRL 100004)
[2599] Triiodothyronine, 10 .mu.l of 5.times.10.sup.4 M (Sigma
T5516)
[2600] Ethanolamine, 100 .mu.l of 10.sup.-1 M (Sigma E0135)
[2601] Phosphoethalamine, 100 .mu.l of 10.sup.-1 M (Sigma
P0503)
[2602] Selenium, 4 .mu.l of 10.sup.-1 M (Aesar #12574)
[2603] Group C: (in 10 ml 100% ethanol)
[2604] Hydrocortisone, 2 .mu.l of 5.times.10.sup.-3 M (Sigma
#H0135)
[2605] Progesterone, 100 .mu.l of 1.times.10.sup.-3 M (Sigma
#P6149)
[2606] Forskolin, 500 .mu.l of 20 mM (Calbiochem #344270)
[2607] Minimal media:
[2608] RPMI 1640 plus transferrin (10 .mu.g/ml), insulin (1
.mu.g/ml), gentamycin (100 ng/ml), aprotinin (50 .mu.g/ml) and BPE
(15 .mu.g/ml).
[2609] Defined media:
[2610] RPMI 1640 plus transferrin (10 .mu.g/ml), insulin (1
.mu.g/ml), gentamycin (100 ng/ml) and aprotinin (50 .mu.g/ml).
[2611] The following polypeptides tested positive in this assay:
PRO237 and PRO731.
Example 129
[2612] Stimulatory Activity in Mixed Lymphocyte Reaction (MLR)
Assay (Assay 24)
[2613] 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.
[2614] 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.
[2615] 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/mi of assay media (RPMI 10% fetal bovine
serum, 1% peniclllinistreptomycin, 1% glutamtne, 1% HEPES, 1%
nonessential amino acids, 1% pyruvate). The stimulator PBMCs are
prepared by irradiating the cells (about 3000 Rads).
[2616] The assay is prepared by plating in triplicate wells a
mixture of:
[2617] 100:1 of test sample diluted to 1% or to 0.1%,
[2618] 50:1 of irradiated stimulator cells, and
[2619] 50:1 of responder PBMC cells.
[2620] 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.
[2621] 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% enicillin/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),
centriging 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.
[2622] 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.
[2623] The following PRO polypeptides tested positive in this
assay: PRO273, PRO526, PRO381, PRO719, PRO866 and PRO1031.
Example 130
[2624] Inhibitory Activity in Mixed Lymphocyte Reaction (MLR) Assay
(Assay 67)
[2625] 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.
[2626] 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.
[2627] More specifically, in one assay variant, peripheral blood
mononuclear cells (PBMC) are isolated from mannalian 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% penicillinistreptomycin, 1% glutamine, 1% HEPES, 1%
non-essential amino acids, 1% pyruvate). The stimulator PBMCs are
prepared by irradiating the cells (about 3000 Rads).
[2628] The assay is prepared by plating in triplicate wells a
mixture of:
[2629] 100:1 of test sample diluted to 1% or to 0.1%,
[2630] 50:1 of irradiated stimulator cells, and
[2631] 50:1 of responder PBMC cells.
[2632] 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.
[2633] In another variant of this assay, PBMCs are isolated from
the spleens of Balbic mice and C57B6 mice. The cells are teased
from freshly harvested spleens in assay media (RPMI; 10% fetal
bovine serum, 1% penicillinistreptomycin, 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 resuspencing
the cells to 1.times.10.sup.7 cells/ml of assay media. The assay is
then conducted as described above.
[2634] 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.
[2635] The following polypeptide tested positive in this assay:
PRO273, PRO526, PRO381, PRO701, PRO363, PRO531, PRO1083, PRO865,
PRO788 and PRO1114.
Example 131
[2636] Fibroblast (BHK-21) Proliferation (Assay 98)
[2637] 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
ceilslwell 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 lcggml 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 405nm. A positive in the assay is acid
phosphatase activity which is at least 50% above the negative
control.
[2638] The following PRO polypeptide tested positive in this assay:
PRO273 and PRO731.
Example 132
[2639] Induction of Endothelial Cell Apoptosis (ELISA) (Assay
109)
[2640] 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.
pen/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.
[2641] 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
.mu.l of a 3.times. stock of staurosporine were used. The cells
were incubated for 24 to 35 hours prior to ELISA.
[2642] 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 mn, 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.
[2643] The following PRO polypeptides tested positive in this
assay: PRO846.
Example 133
[2644] Induction of Endothelial Cell Apontosis (Assay 73)
[2645] The ability of PRO polypeptides to induce apoptosis in
endothelial cells was tested in human venous umbilical vein
endothelhal 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.
[2646] 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.
[2647] 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.35Streptavidin 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.
[2648] The following PRO polypeptides tested positive in this
assay: PRO719.
Example 134
[2649] Human Venous Endothelial Cell Calcium Flux Assay (Assay
68)
[2650] 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.
[2651] 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/ml 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 3x 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.
[2652] The fluorescence change from baseline to the maximum rise of
the curve (.DELTA. change) was calculated, and replicates averaged.
The rate of fluorescence increase was monitored, and only those
samples which had a .DELTA. change greater than 1000 and a rise
within 60 seconds, were considered positive.
[2653] The following PRO polypeptides tested positive in the
present assay: PRO771.
Example 135
[2654] Induction of c-fos in Endothelial Cells (Assay 34)
[2655] 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 cr 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.
[2656] 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 BEPES, 140 mM NaCl, 4% (wlv) 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.
[2657] 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.
[2658] 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.
[2659] 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.
[2660] The following PRO polypeptides tested positive in this
assay: PRO474.
Example 136
[2661] Induction of Pancreatic 6-Cell Precursor Differentiation
(Assay 89)
[2662] This assay shows that certain polypeptides of the invention
act to induce differentiation of pancreatic .beta.-cell precursor
cells into mature pancreatic .beta.-cells and, therefore, are
useful for treating various insulin deficient states in mammals,
including diabetes mellitus. The assay is performed as follows. The
assay uses a primary culture of mouse fetal pancreatic cells and
the primary readout is an alteration in the expression of markers
that represent either .beta.-cell precursors or mature
.beta.-cells. Marker expression is measured by real time
quantitative PCR (RTQ-PCR); wherein the marker being evaluated is
insulin.
[2663] The pancreata are dissected from E14 embryos (CD1 mice). The
pancreata are then digested with collagenase/dispase in F12/DMEM at
37.degree. C. for 40 to 60 minutes (collagenase/dispase, 1.37
mg/ml, Boehringer Mannheim, #1097113). The digestion is then
neutralized with an equal volume of 5% BSA and the cells are washed
once with RPMI1640. At day 1, the cells are seeded into 12-well
tissue culture plates (pre-coated with laminin, 20 .mu.g/ml in PBS,
Boehiinger Mannheim, #124317). Cells from pancreata from 1-2
embryos are distributed per well. The culture medium for this
primary cuture is 14F/1640. At day 2, the media is removed and the
attached cells washed with RPMI/1640. Two mls of minimal media are
added in addition to the protein to be tested. At day 4, the media
is removed and RNA prepared from the cells and marker expression
analyzed by real time quantitative RT-PCR. A protein is considered
to be active in the assay if it increases the expression of the
relevant .beta.-cell marker as compared to untreated controls.
14F/1640 is RPMI1640 (Gibco) plus the following:
[2664] group A 1:1000
[2665] group B 1:1000
[2666] recombinant human insulin 10 .mu.g/ml
[2667] A protinin (50 .mu.g/ml) 1:2000 (Boehringer manheim
#981532)
[2668] Bovine pituitary extract (3PE) 60 .mu.g/ml
[2669] Gentamycin 100 ng/ml
[2670] Group A: (in 10 ml PBS)
[2671] Transferrin, 100 mg (Sigma T2252)
[2672] Epidermal Growth Factor, 100 .mu.g (BRL 100004)
[2673] Triiodothyronine, 10 .mu.l of 5.times.10.sup.-6 M (Sigma
T5516)
[2674] Ethanolamine, 100 .mu.l of 10.sup.-1 M (Sigma E0135)
[2675] Phosphoethalamine, 100 .mu.l of 10.sup.-1 M (Sigma
P0503)
[2676] Selenium, 4 .mu.l of 10.sup.-1 M (Aesar #12574)
[2677] Group C: (in 10 ml 100% ethanol)
[2678] Hydrocortisone, 2 .mu.l of 5.times.10.sup.-3 M (Sigma
#H0135)
[2679] Progesterone, 100 .mu.l of 1.times.10.sup.-3 M (Sigma
#P6149)
[2680] Forskolin, 500 .mu.l of 20 mM (Calbiochem #344270)
[2681] Minimal media:
[2682] RPMI 1640 plus transferrin (10 .mu.g/ml), insulin (1 Ig/ml),
gentamycin (100 ng/ml), aprotimn (50 .mu.g/ml) and BPE (15
.mu.g/ml).
[2683] Defined media:
[2684] RPMI 1640 plus transferrin (10 .mu.g/ml), insulin (1
.mu.g/ml), gentamycin (100 ng/ml) and aprotinin (50 .mu.g/ml).
[2685] The following polypeptides were positive in this assay:
PRO788 and PRO162.
Example 137
[2686] Stimulation of Endothelial Cell Proliferation (Assay 8)
[2687] This assay is designed to determine whether PRO polypeptides
of the present invention show the ability to stimulate adrenal
cortical capillary endothelial cell (ACE) growth. 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.
[2688] Bovine adrenal cortical capillary endothelial (ACE) cells
(from primary culture, maximum of 12-14 passages) were plated in
96-well plates at 500 cels/well per 100 microliter. Assay media
included low glucose DMEM, 10% calf serun, 2 mM glutamine, and
1.times. peniclllin/streptomycin/fimgizone. Control wells included
the following: (1) no ACE cells added; (2) ACE cells alone; (3) ACE
cells plus VEGF (5 ng/ml); and (4) ACE cells plus FGF (5ng/ml). The
control or test sample, (in 100 microliter volumes), was 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. After the incubation, the media in the wells was aspirated, and
the cells were washed 1X 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 nm.
[2689] The activity of a PRO polypeptide was calculated as the fold
increase in proliferation (as determined by the acid phosphatase
activity, OD 405 nm) relative to (1) cell only background, and (2)
relative to maximum stimulation by VEGF. VEGF (at 3-10 ng/ml) and
FGF (at 1-5 ng/ml) were employed as an activity reference for
maximum stimulation. Results of the assay were considered
"positive" if the observed stimulation was .gtoreq.50% increase
over background. VEGF (5 ng/ml) control at 1% dilution gave 1.24
fold stimulation; FGF (5 ng/ml) control at 1% dilution gave 1.46
fold stimulation.
[2690] The following PRO polypeptides tested positive in this
assay: PRO1075.
Example 138
[2691] Mouse Mesengial Cell Inhibition Assay (Assay 114)
[2692] This assay is designed to determine whether PRO polypeptides
of the present invention show the ability to inhibit the
proliferation of mouse mesengial cells in culture. PRO polypeptides
testing positive in this assay would be expected to be useful for
the therapeutic treatment of such diseases or conditions where
inhibition of mesengial cell proliferation would be beneficial such
as, for example, cystic renal dysplasia, polycystic kidney disease,
or other kidney disease assoiciated with abnormal mesengial cell
proliferation, renal tumors, and the like.
[2693] On day 1, mouse mesengial cells are plated on a 96 well
plate in growth medium (a 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 then are allowed to grow
overnght. On day 2, the PRO polypeptide is diluted at 2 different
concentrations (1%, 0.1%) in serum-free medium and is added to the
cells. The negative control is growth medium without added PRO
polypeptide. After the cells are allowed to incubate for 48 hours,
20 .mu.l of the Cell Titer 96 Aqueous one solution reagent
(Promega) is added to each well and the colormetric reaction is
allowed to proceed for 2 hours. The absorbance (OD) is then
measured at 490 nm. A positive in the assay is an absorbance
reading which is at least 10% above the negative control.
[2694] The following PRO polypeptides tested positive in this
assay: PRO200 and PRO697.
Example 139
[2695] Chondrocyte Proliferation Assay (Assay 111)
[2696] 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.
[2697] Porcine chondrocytes are isolated by overnight collagenase
digestion of articular carilage 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 Ig/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
37PC. The fluorescence is then measured in each well (Ex:530 um;
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.
[2698] The following PRO polypeptides tested positive in this
assay: PRO181, PRO200 and PRO322.
Example 140
[2699] Rat DRG Neuronal Survival Inhibition Assay
[2700] This assay is designed to determine whether PRO polypeptides
of the present invention show the ability to inhibit the survival
of neural cells in culture. Polypeptides testing positive in this
assay are expected to be useful for the therapeutic treatment of
neuropathic conditions which are associated with undesirable neural
cell proliferation including, for example, neuroblastomas, gilomas,
giloblastomas, and the like.
[2701] A heterogeneous population of neural cells freshly isolated
from E14 rat embryo dorsal root ganglia are diluted in complete
medium and are plated at 5,000 cells/well on polyurethane
pretreated plates containing 504 .mu.l F12 complete media. Test PRO
polypeptides (50 .mu.l, one concentration) with 504 .mu.l
additional assay media are then added to test for survival
inhibition activity. Negative controls are treated with 100 .mu.l
of complete medium alone. After 3 days incubation, the cells are
stained with CMFDA and fixed after 1 hour with 4% paraformaldehyde.
Cells are then quantified by NIH image analysis. A positive in the
assay is cell numbers in the treated well(s) being less than 0.5 of
the untreated control well(s).
[2702] The following PRO polypeptides tested positive in this
assay: PRO195 and PRO701.
Example 141
[2703] Tissue Expression Distribution
[2704] 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 useftl 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.
167 Tissues With Tissues Lacking DNA Molecule Significant
Expression Significant Expression DNA40954-1233 liver, lung brain
DNA41404-1352 lung, kidney liver, retina, pancreas DNA44179-1362
liver lung, brain DNA45234-1277 kidney liver, placenta, brain
DNA45415-1318 thyroid, brain, kidney liver, bone marrow
DNA45417-1432 thyroid, brain, kidney, bone liver marrow
DNA45493-1349 liver, kidney brain DNA48306-1291 brain, kidney
pancreas, liver DNA48328-1355 thyroid, brain, liver, kidney bone
marrow DNA48329-1290 brain, bone marrow, kidney liver, thyroid
DNA49624-1279 placenta liver, lung, kidney, brain DNA50911-1288
brain placenta DNA50914-1289 brain, kidney, liver placenta
DNA53906-1368 lung, kidney brain DNA53912-1457 lung, liver, kidney,
pancreas brain DNA53977-1371 lung, liver, kidney, bone brain,
pancreas marrow DNA54002-1367 bone marrow, liver, kidney lung,
thyroid, brain DNA55737-1345 bone marrow, kidney liver, brain
DNA57039-1402 pigment epithelium lung, brain, liver, kidney
DNA57253-1382 lung, brain, liver, kidney placenta DNA58747-1384
lung, brain, kidney, liver pancreas, thyroid DNA23318-1211 spleen,
brain, heart, cartilage colon tumor, prostate DNA39975-1210 brain,
colon tumor, heart THP-1 macrophages DNA39979-1213 dendrocytes,
cartilage, heart spleen, substantia nigra, uterus, prostate
DNA41386-1316 HUVEC, cartilage, substantia nigra, dendrocytes colon
tumor, uterus DNA50919-1361 HUVEC, brain, spleen, prostate,
cartilage, colon tumor heart, uterus DNA52185-1370 dendrocytes
substantia nigra, hippocampus, uterus DNA42663-1154 uterus, spleen,
bone marrow cartilage, HUVEC, colon tumor DNA50980-1286 placenta,
adrenal gland, bone marrow, uterus, prostate cartilage
Example 142
[2705] In situ Hybridization
[2706] In situ hybridization is a powerfull 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.
[2707] In situ hybridization was performed following an optimized
version of the protocol by Lu and Gillett, Cell Vision 1:169-176
(1994), using PCR-generated .sup.33P-labeled riboprobes. Briefly,
formalin-fixed, paraffin-embedded human tissues were sectioned,
deparaffinized, deproteinated in proteinase K (20 glml) for 15
minutes at 37.degree. C., and further processed for in situ
hybridization as described by Lu and Gillett, supra. A
[.sup.33-P]UTP-labeled antisense riboprobe was generated from a PCR
product and hybridized at 55.degree. C. overnight. The slides were
dipped in Kodak NTB2 nuclear track emulsion and exposed for 4
weeks.
[2708] .sup.33P-Riboprobe synthesis
[2709] 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:
[2710] 2.0 .mu.l 5.times. transcription buffer
[2711] 1.0 .mu.l DTT (100 mM)
[2712] 2.0 .mu.l NTP mix (2.5 mM: 10.mu.; each of 10 mM GTP, CTP
& ATP+10 .mu.l H.sub.2O)
[2713] 1.0 .mu.l UTP (50 .mu.M)
[2714] 1.0 .mu.l Rnasin
[2715] 1.0 .mu.l DNA template (1 .mu.g)
[2716] 1.0 .mu.l H.sub.2O
[2717] 1.0 .mu.l RNA polymerase (for PCR products T3=AS, T7=S,
usually)
[2718] 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 filal product was pipetted on DE81 paper and counted
in 6 ml of Biofluor II.
[2719] The probe was run on a TBE/urea gel. 1-3 .mu.l of the probe
or 5 41 of RNA Mrk mI 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.
[2720] 33P-Hybridization
[2721] A. Pretreatment of Frozen Sections
[2722] 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.20). After deproteination in 0.5 .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.
[2723] B. Pretreatment of Paraffin-embedded Sections
[2724] 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 Al of 10 mg/ml in 250 ml RNase-free RNase buffer; 37.degree.
C., 15 minutes)--human embryo, or 8.times.proteinase K (100 ,ul 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.
[2725] C. Prehybridization
[2726] 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.
[2727] D. Hybridization 1.0.times.10.sup.6 cpm probe and 1.0 .mu.l
tRNA (50 mg/ml stock) per slide were heated at 95C 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.
[2728] E. Washes
[2729] 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).
[2730] F. Oligonucleotides
[2731] In situ analysis was performed on a variety of DNA sequences
disclosed herein. The oligonucleotides employed for these analyses
were derived from the nucleotide sequences disclosed herein and
generally range from about 40 to 55 nucleotides in length.
[2732] G. Results
[2733] In situ analysis was performed on a variety of DNA sequences
disclosed herein. The results from these analyses are as
follows.
[2734] (1) DNA29101-1122 (PRO200)
[2735] Fetal: Lower limb expression in developing lower limb bones
at the edge of the cartlagenous anlage (i.e. around the outside
edge); in developing tendons, in vascular smooth muscle and in
cells embracing developing skeletal muscle myocytes and myotubes.
Expression also observed at the epiphyseal growth plate. Lymph node
expression in marginal sinus of developing lymaph nodes. Thymus
expression in the subeapsular region of the thymic cortex, possibly
representing either the subeapsular epithelial cells or the
proliferating, double negative, thymocytes that are found in this
region. Spleen is negative. Trachea expression in smooth muscle.
Brain (cerebral cortex) focal expression in cortical neurones.
Spinal cord negative. Small intestine expression in smooth muscle.
Thyroid - generalized expression over thyroid epithelium. Adrenal
is negative. Liver expression in ductal plate cells. Stomach
expression in mural smooth muscle. Fetal skin expression in basal
layer of squamous epithelium. Placenta expression in interstitial
cells in trophoblastic villi. Cord expression in wall of arteries
and vein.
[2736] Comments: Expression pattern suggests that PRO200 may be
involved in cell differentiation/proliferation.
[2737] High expression was observed at the following additional
sites: Chimp ovary--granulosa cells of maturing follicles, lower
intensity signal observed over thecal cells. Chimp
parathyroid--high expression over chief cells. Human fetal
testis--moderate expression over stromal cells surrounding
developing tubules. Human fetal lung--high expression over
chondrocytes in developing bronchial tree, and low level expression
over branching bronchial epithelium. Specific expression was not
observed over the renal cell, gastric and colonic carcinomas. 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. 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.
Acetorninophen induced liver injury and hepatic cirrhosis.
[2738] (2) DNA30867-1335 (PRO218)
[2739] Low level expi ession over numerous epithelia including
fetal small intestine, fetal thyroid, chimp gastric epithelium.
Expression also seen over malignant cells in a renal cell
carcinoma. Expression in fetal brain, over cortex. The distribution
does not suggest an obvious function. 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. Adult human tissues
examined: kidney (normal and end-stage), bladder, adrenal, spleen,
lymph node, pancreas, lung, skin, eye (inc. retina), colon,
bladder, liver (normal, cirrhotic, acute failure), heart, clear
cell carcinoma of kidney, gastric adenocarcinoma, colorectal
carcinoma. Non-humanprimate tissues examined: Chimp tissues:
salivary gland, stomach, thyroid, parathyroid, tongue, thymus,
ovary, lymph node, peripheral nerve. Rhesus Monkey tissues:
cerebral cortex, hippocampus, cerebellum, penis.
[2740] (3) DNA40021-1154 (PRO285)
[2741] Low levels of expression observed in the placenta and over
hematopoietic cells in the mouse fetal liver. No expression was
detected in either human fetal, adult or chimp lymph node and no
expression was detected in human fetal or human adult spleen. Fetal
tissues examined (E12-El6 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. Adult tissues examined: liver, kidney, adrenal, myocardium,
aorta, spleen, lymph node, pancreas, lung, skin, cerebral cortex
(rm), hippocampus (rm), cerebellum (rm), brain infarct (human),
cerebritis (human), penis, eye, bladder, stomach, gastric
carcinoma, colon, colonic carcinoma, thyroid (chimp), parathyroid
(chimp) ovary (chimp) and chondrosarcoma. Acetominophen induced
liver injury and hepatic cirrhosis.
[2742] (4) DNA39523-1192 (PRO273)
[2743] Expression over epithelium of mouse embryo skin as wel as
over basal epithelium and dermis of human fetal skin. Basal
epithelial pegs of the squamous mucosa of the chimp tongue are also
positive. Expression over a subset of cells in developing glomeruli
of fetal kidney, adult renal tubules, and over "thyroidized"
epithelium in end-stage renal disease, low expression in a renal
cell carcinoma, probably over the epithelial cells. Low level
expression over stromal cells in fetal lung. Expression over
stromal cells in the apical portion of gastric glands. High
expression in the lamina propria of the fetal small intestinal
villi, normal colonic mucosa and over stromal cells in a colonic
carcinoma. Strong expression over benign connective tissue cells in
the hylanized stroma of a sarcoma. Expression over stromal cells in
the placental villi and the splenic red pulp. In the brain,
expression over cortical neurones. Connective tissue surrounding
developing bones and over nerve sheath cells in the fetus. 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. Adult
tissues examined: liver, kidney, adrenal, myocardium, aorta,
spleen, lymph node, pancreas, lung, skin, cerebral cortex (rm),
hippocampus(rm), eye, stomach, gastric carcinoma, colon, colonic
carcinoma, thyroid (chimp), parathyroid (chimp) ovary (chimp) and
chondrosarcoma. Acetominophen induced liver injury and hepatic
cirrhosis.
[2744] Expression was present in many cells in the outer layers (I
and II) of the monkey cerebral cortex. A small subset of cells in
the deeper cortical layers also expressed mRNA for this chemokine
homolog. Scattered cells within the molecular layers of the
hippocampus and bordering the inner edge of the dentate gyrus
contained chemokine homolog mRNA. No expression was detected within
the cerebellar cortex. Chemokine homolog expression is not observed
in infarcted brain, where cell death has occurred in the regions
where the chemokine homolog normally is expressed. This probe could
possibly serve as a marker of a subset of neurons of outer layers
of the cerebral cortex and could possibly reveal neuronal migration
disorders. Abnormal neuronal migration is a possible cause of some
seizure disorders and schizophrenia. In order to gain a better
appreciation of the distribution of this mRNA we will test whether
the probe will cross-hybridize with mouse brain tissue.
[2745] Also shows intriguing and specific patterns of hybridization
within postnatal day (P) 10 and adult mouse brains. In one sagittal
section of P1O mouse brain, strong signal was observed scattered
within the molecular layer of the hippocampus and inner edges of
the dentate gyrus. Cells in the presubiculum were moderately
labeled; the signal extended in a strong band through outer layers
of the retrosplenial cortes to the occipital cortex, where the
signal dinnshed to background levels. A small set of positive
neurons were detected in deeper regions of P10 motor cortex;
neurons in outer layers of P10 cortex did not exhibit signal above
background levels. Moderate hybridization signal was also detected
in the inferior colliculus. Chemokine homolog signal in the adult
mouse brain was evaluated in three coronal sections at different
levels. Strong signal was detected in the septum and in scattered
neurons in the pontine nuclei and motor root of the trigeminal
nerve; moderate signal was seen in the molecular layers of the
hippocampus and outer layers of the retrosplenial cortex.
[2746] (5) DNA39979-1213 (PRO296)
[2747] Widespread expression in fetal in adult tissues. Expressed
in a variety of fetal and adult epithelia, skeletal and cardiac
muscle, developing (including retina) and adult CNS, thymic
epithelium, placental viri, hepatocytes in cirrhotic and
acetaminophen induced toxicity. Highly expressed in hypertrophic
chondrocytes in developing skeletal system.The overall expression
pattern, while not completely ovelapping (not expressed in
glomeruli, more widely expressed in CNS), is not disimilar to VEGF.
A possible role in angiogenesis should therefore be considered.
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. Adult
human tissues examined: kidney (normal and end-stage), adrenal,
spleen, lymph node, pancreas, lung, eye (inc. retina), bladder,
liver (normal, cirrhotic, acute failure). Non-human primate tissues
examined: Chimp tissues: adrenal. Rhesus Monkey tissues: cerebral
cortex, hippocampus, cerebellum.
[2748] (6) DNA52594-1270 (PRO868)
[2749] Expression over neuronal cells in fetal dorsal root ganglia,
spinal cord, developing enteric neurons, cortical neurons. Low
level expression also seen in placental trophoblast. In adult
tissues the only site where notable expression was observed was the
normal adult prostate; as such it may represent a possible prostate
cell surface receptor target antigen. Studies to fer characterize
the expression in adult tissues seem warranted. Low level
expression also observed in a liposarcoma. Fetal tissues examined
(E2-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. Adult human tissues
examined: liver, kidney, adrenal, myocardium, aorta, spleen, lung,
skin, chondrosarcoma, eye, stomach, gastric carcinoma, colon,
colonic carcinoma, renal cell carcinoma, prostate, bladder mucosa
and gaU bladder. Acetominophen induced liver injury and hepatic
cirrhosis. Rhesus tissues examined: cerebral cortex (rm),
hippocampus(rm), cerebellum. Chimp tissues examined: thyroid,
parathyroid, ovary, nerve, tongue, thymus, adrenal, gastric mucosa
and salivary gland. WIG-1(WISP-1), WIG-2 (WISP-2) and WIG-5
(WISP-3) expression in human breast carcinoma and normal breast
tissue, Wig-2 in lung carcinoma, and Wig-5 in colon carcinoma.
[2750] (7) DNA64907-1163 (PRO1330)
[2751] In human fetal tissues there was strong specific expression
over artrerial, venous, capillary and sinusoidal endothelium in all
tissues examined, except for fetal brain. En normal adult tissues
expression was low to absent, but when present appeared expression
was confined to the vasculature. Highest expression in adult
tissues was observed regionally in vessels running within the white
matter of rhesus brain - the significance of this pattern is
unclear. Elevated expression observed in vasculature of many
inflamed and diseased tissues, including tumor vasculature. In some
of these tissues it was unclear if expression was soley confmed to
vascular endothelium. In the 15 lung tumors examined no expression
was seen over the malignant epithelium, however, vascular
expression was observed in many of the tumors and adjacent lung
tissue. Moderate, apparently non-specific background, was seen with
this probe over hyalinised collagen and sites of tissue necrosis.
In the abscence of a sense control, however, it is not possible to
be absolutely certain that all of this signal is non-specific. Some
signal, also thought to be non-specific, was seen over the chimp
gastric mucosa, transitional cell epithelium of human adult bladder
and fetal retina.
[2752] (8) DNA49624-1279 (PRO545)
[2753] Expression of the ADAM family molecule, ADAM 12
(DNA49624-1279) observed in normal human lung, lung tumor, normal
colon and colon carcinoma.
[2754] (9) DNA59294-1381 (PRO1031)
[2755] The expression of this IL17 homologue was evaluated in a
panel consisting of normal adult and fetal tissues and tissues with
inflammation, predominantly chronic lymphocytic inflammation. This
panel is designed to specifically evaluate the expression pattern
in immune mediated inflammatory disease of novel proteins that
modulate T lymphocyte function (stimulatory or inhibitory). This
protein when expressed as an Ig-fusion protein was
immunostimulatory in a dose dependent fashion in the human mixed
lymphocyte reaction (MLR); it caused a 285% and 147% increase above
the baseline stimulation index when utilized at two different
concentrations (1.0% and 0.1% of a 560 nM stock). Summary:
expression was restricted to muscle, certain types of smooth muscle
in the adult and in skeletal and smooth muscle in the human fetus.
Expression in adult human was in smooth muscle of tubular organs
evaluated including colon and gall bladder. There no expression in
the smooth muscle of vessels or bronchi. No adult human skeletal
muscle was evaluated. In fetal tissues there was moderate to high
diffuse expression in skeletal muscle the axial skeleton and limbs.
There was weak expression in the smooth muscle of the intestinal
wall but no expression in cardiac muscle. Adult human tissues with
expression: Colon. there was low level diffuse expression in the
smooth muscle (tunica muscularis) in 5 specimens with chronic
inflammatory bowel disease. GaU bladder: there was weak to low
level expression in the smooth muscle of the gall bladder. Fetal
human tissues with expression: there was moderate diffuse
expression in skeletal muscle and weak tolow expression in smooth
muscle; there was no expression in fetal heart or any other fetal
organ including liver, spleen, CNS, kidney, gut, lung. Human
tissues with no expression: lung with chronic granulomatous
inflammation and chronic bronchitis (5 patients), peripheral nerve,
prostate, heart, placenta, liver (disease multiblock), brain
(cerbrum and cerebellum), tonsil (reactive hyperplasia), peripheral
lymph node, thymus.
[2756] (10) DNA45416-1251 (PRO362)
[2757] The expression of this novel protein was evaluated in a
variety of human and non-human primate tissues and was found to be
highly restricted. Expression was present only in alveolar
macrophages in the lung and in Kupffer cells of the hepatic
sinusoids. Expression in these cells was significantly increased
when these distinct cell populations were activated. Though these
two subpopulations of tissue macrophages are located in different
organs, they have similar biological functions. Both types of these
phagocytes act as biological filters to remove material from the
blood stream or airways including pathogens, senescent cells and
proteins and both are capable of secreting a wide variery of
important prointlammatory cytokes. In inflamed lung (7 patient
samples) expression was prominent in reactive alveolar macrophage
cell populations defined as large, pale often vacuolated cells
present singly or in aggregates within alveoli and was weak to
negative in normal, non-reactive macrophages (single scattered
cells of normal size). Expression in alveolar macrophages was
increased during inflammation when these cells were both increased
in numbers and size (activated). Despite the presence of histocytes
in areas of interstial inflamantion and peribronchial lymphoid
hyperplasia in these tissues, expression was restricted to alveolar
macrophages. Many of the inflamed lungs also had some degree of
suppurative inflammation; expression was not present in
neutrophilic granulocytes. In liver, there was strong expression in
reactive/activated Kupffer cells in livers with acute centrilobular
necrosis (acetominophen toxicity) or fairly marked periportal
inflammntiont. However there was weak or no expression in Kupffer
cells in normal liver or in liver with only mild inflammation or
mild to moderate lobular hyperplasia/hypertrophy. Thus, as in the
lung, there was increased expression in acivated/reative cells.
There was no expression of this molecule in histiocytes/macropahges
present in inflamed bowel, hyperplastic/reactive tonsil or normal
lymph node. The lack of expression in these tissues which all
contained histiocytic inflammation or resident macrophage
populations strongly supports restricted expression to the unique
macrophage subset populations defined as alveolar macrophage and
hepatic Kupffer cells. Spleen or bone marrow was not available for
evaluation. HFuman tissues evaluated which had no detectable
expression included: Inflammatory bowel disease (7 patient samples
with moderate to severe disease), tonsil with reactive hyperplasia,
peripheral lymphnode, psoriatic skin (2 patient samples with mild
to moderate disease), heart, peripheral nerve. Chimp tissues
evaluated which had no detectable expression included: tongue,
stomach, thymus.
[2758] (11) DNA52196-1348 (PRO733)
[2759] Generalized low level signal in many tissues and in many
cell types. While endothelial cell expression was observed it wias
not a prominent feature in either fetal, normal or diseased
tissues. Human tissues: moderate expression over fetal liver
(mainly hepatocytes), lung, skin, adrenal and heart. Fetal spleen,
small intestine, brain and eye are negative. Adult normal kidney,
bladder epithelium, lung, adrenal, pancreas, skin - all negative.
Expression in adult human liver (normal and diseased), renal
tubules in end-stage renal disease, adipose tissue, sarcoma, colon,
renal cell carcinoma, hepatocellular carcinoma, squamous cell
carcinoma. Non human primate tissues: chimp salivary gland,
vessels, stomach, tongue, peripheral nerve, thymus, lymph node,
thyroid and parathyroid. Rhesus spinal cord negative, cortical and
hippocampal neurones positive.
Example 143
[2760] Isolation of cDNA Clones Encoding a Human PRO4993
[2761] 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 DNA85042. In some cases,
the DNA85042 consensus sequence derives from an intermediate
consensus DNA sequence which was extended using repeated cycles of
BLAST and phrap to extend that intermediate consensus sequence as
far as possible using the sources of EST sequences discussed above.
Based on the DNA85042 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 flitlength coding sequence for PRO4993.
[2762] PCR primers (forward and reverse) were synthesized:
168 forward PCR primer 5'-AGATGTGAAGGTGCAGGTGTGCCG-3' (SEQ ID NO:
619) reverse PCR primer 5'-GAACATCAGCGCTCCCGGTAATTCC-3' (SEQ ID NO:
620)
[2763] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA8S042 sequence which
had the following nucleotide sequence
[2764] hybridization probe
169 5'-CCAGCCTTTGAATGGTACAAAGGAGAGAAGAAGCTCTTCAATGGCC-3' (SEQ ID
NO: 621)
[2765] RNA for construction of the cDNA libraries was isolated from
human fetal brain tissue.
[2766] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for a full-length PRO4993
polypeptide (designated herein as DNA94832-2659 [FIG. 229, SEQ ID
NO:611]) and the derived protein sequence for that PRO4993
polypeptide.
[2767] The full length clone identified above contained a single
open reading frame with an apparent translational initiation site
at nucleotide positions 305-307 and a stop signal at nucleotide
positions 1361-1363 (FIG. 229, SEQ ID NO:61 1). The predicted
polypeptide precursor is 352 amino acids long, has a calculated
molecular weight of approximately 38,429 daltons and an estimated
pl of approximately 6.84. Analysis of the fiull-length PRO4993
sequence shown in FIG. 230 (SEQ ID NO:612) evidences the presence
of a variety of important polypeptide domains as shown in FIG. 230,
wherein the locations given for those important polypeptide domains
are approximate as described above. Clone DNA94832-2659 has been
deposited with ATCC on Jun. 15, 1999 and is assigned ATCC deposit
no. 240-PTA.
[2768] An analysis of the Dayhoff database (version 35.45 SwissProt
35), using the ALIGN-2 sequence alignment analysis of the
full-length sequence shown in FIG. 230 (SEQ ID NO:612), evidenced
sequence identity between the PRO4993 amino acid sequence and the
following Dayhoff sequences: P_W05152; LAMP_HUMAN; PW05157;
P_W05155; I56551; OPCM_RAT; AMAL_DROME; DMU78177.sub.--1; I37246;
and NCA1_HUMAN.
Example 144
[2769] Isolation of cDNA Clones Encodine Human PRO1559, PRO725 and
PRO739
[2770] A consensus sequence was obtained relative to a variety of
EST sequences as described in Example 1 above. Based upon an
observed homology between this consensus sequence and an EST
sequence contained within incyte EST clone No. 4242090, Incyte EST
clone No. 4242090 was purchased and its insert was obtained and
sequenced. It was discovered that the insert sequence encoded a
full-length protein designated herein as PRO1559 (FIG. 232; SEQ ID
NO:614). The DNA sequence of the insert (DNA68886) is shown in FIG.
231 (SEQ ID NO:613).
[2771] A cDNA sequence isolated in the amylase screen described in
Example 2 above is herein designated DNA43301. The DNA43301
sequence was then compared to a variety of expressed sequence tag
(EST) databases which included public EST databases (e.g., GenBank)
and a proprietary EST DNA database (LIFESBQ.TM., Incyte
Pharmaceuticals, Palo Alto, Calif.) to identify existing
homologies. The homology search was performed using the computer
program BLAST or BLAST2 (Altshul et al., Methods in Enzymology
266:460-480 (1996)). Those comparisons resulting in a BLAST score
of 70 (or in some cases 90) or greater that did not encode known
proteins were clustered and assembled into consensus DNA sequences
with the program "phrap" (Phil Green, University of Washington,
Seattle, Wash.). The consensus sequence obtained therefrom is
herein designated DNA45458. Based on the DNA45458 consensus
sequence, oligonucleotide probes were generated and used to screen
a human fetal brain (LIB153) library prepared as described in
paragraph 1 of Example 2 above. The cloning vector was pRK5B (pRK5B
is a precursor of pRK5D that does not contain the Sfll site; see,
Holmes et al., Science, 253:1278-1280 (1991)), and the cDNA size
cut was less than 2800 bp.
[2772] PCR primers (forward and reverse) were synthesized:
170 forward PCR primer (45458.f1) 5'-CCAAACTCACCCAGTGAGTGTG- AGC-3'
(SEQ ID NQ: 619) reverse PCR primer (45458.r1)
5'-TGGGAAATCAGGAATGGTGTTCTCC-3' (SEQ ID NO: 620)
[2773] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA45458 sequence which
had the following nucleotide sequence
[2774] hybridization probe (45458.p1)
171 5'-CTTGTTTTCACCATTGGGCTAACTTTGCTGCTAGGAGTTCAAGCCATGCC-3' (SEQ
ID NO: 621)
[2775] 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 PRO725 gene
using the probe oligonucleotide and one of the PCR primers.
[2776] A full length clone was identified that contained a single
open reading frame with an apparent translational initiation site
at nucleotide positions 161-163 and ending at the stop codon found
at nucleotide tiS positions 455-457 (FIG. 233; SEQ ID NO:615). The
predicted polypeptide precursor is 98 amino acids long, has a
calculated molecular weight of approximately 11,081 daltons and an
estimated pI of approximately 6.68. Analysis of the full-length
PRO725 sequence shown in FIG. 234 (SEQ ID NO:616) evidences the
presence of the following: a signal peptide from about amino acid 1
to about amino acid 20, a potential N-glycosylation site from about
amino acid 72 to about amino acid 75 and a tyrosine kinase
phosphorylation site from about amino acid 63 to about amino acid
70. Clone DNA52758-1399 has been deposited with ATCC on Apr. 14,
1998 and is assigned ATCC deposit no. 209773.
[2777] Analysis of the amino acid sequence of the full-length
PRO725 polypeptide suggests that it possesses no significant
sequence similarity to any known protein. However, an analysis of
the Dayhoff database (version 35.45 SwissProt 35) evidenced some
degree of homology between the PRO725 amino acid sequence and the
following Dayhoff sequences, POL_BLVAU, PSSP_RAT, CELC36C5.sub.--7,
AF019234.sub.--1, I48862, P_R12498, P_P10125, P_R26861, A64527 and
P_W20495.
[2778] DNA52756, as shown in FIG. 235 (SEQ ID NO:617) and which
encodes native PRO739 polypeptide (FIG. 236; SEQ ID NO:618) was
obtained from GenBank.
Example 145
[2779] Identification of Recentor/Ligand Interactions
[2780] 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
preparalon 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.
[2781] 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 fiuction 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. s FTC 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.
[2782] 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.
[2783] Using these assays, the following receptor/ligand
interactions havebeenherein identified: PRO337 binds to PRO4993,
PRO1559 binds to PRO725, PRO1559 binds to PRO700 and PRO1559 binds
to PRO739.
[2784] Deposit of Material
[2785] The following materials have been deposited with the
American Type Culture Collection, 12301 Parklawn Drive, Rockville,
Md., USA (ATCC):
172 Material ATCC Dep. No. Deposit Date DNA39987-1184 ATCC 209786
April 21, 1998 DNA40625-1189 ATCC 209788 April 21, 1998
DNA23318-1211 ATCC 209787 April 21, 1998 DNA39979-1213 ATCC 209789
April 21, 1998 DNA40594-1233 ATCC 209617 February 5, 1998
DNA45416-1251 ATOC 209620 February 5, 1998 DNA45419-1252 ATCC
209616 February 5, 1998 DNA52594-1270 ATCC 209679 March 17, 1998
DNA45234-1277 ATCC 209654 March 5, 1998 DNA49624-1279 ATCC 209655
March 5, 1998 DNA48309-1280 ATCC 209656 March 5, 1998 DNA46776-1284
ATCC 209721 March 31, 1998 DNA50980-1286 ATCC 209717 March 31, 1998
DNA50913-1287 ATCC 209716 March 31, 1998 DNA50914-1289 ATCC 209722
March 31, 1998 DNA48296-1292 ATCC 209668 March 11, 1998
DNA32284-1307 ATCC 209670 March 11, 1998 DNA36343-1310 ATCC 209718
March 31, 1998 DNA40571-1315 ATCC 209784 April 21, 1998
DNA41386-1316 ATCC 209703 March 26, 1998 DNA44194-1317 ATCC 209808
April 28, 1998 DNA45415-1318 ATCC 209810 April 28, 1998
DNA44189-1322 ATCC 209699 March 26, 1998 DNA48304-1323 ATCC 209811
April 28, 1998 DNA49152-1324 ATCC 209813 April 28, 1998
DNA49646-1327 ATCC 209705 March 26, 1998 DNA49631-1328 ATCC 209806
April 28, 1998 DNA49645-1347 ATCC 209809 April 28, 1998
DNA45493-1349 ATCC 209805 April 28, 1998 DNA48227-1350 ATCC 209812
April 28, 1998 DNA41404-1352 ATCC 209844 May 6, 1998 DNA44196-1353
ATCC 209847 May 6, 1998 DNA52187-1354 ATCC 209845 May 6, 1998
DNA48328-1355 ATCC 209843 May 6, 1998 DNA56352-1358 ATCC 209846 May
6, 1998 DNA53971-1359 ATCC 209750 April 7, 1998 DNA50919-1361 ATCC
209848 May 6, 1998 DNA44179-1362 ATCC 209851 May 6, 1998
DNA54002-1367 ATCC 209754 April 7, 1998 DNA53906-1368 ATCC 209747
April 7, 1998 DNA52185-1370 ATCC 209861 May 14, 1998 DNA53977-1371
ATCC 209862 May 14, 1998 DNA57253-1382 ATCC 209867 May 14, 1998
DNA58847-1383 ATCC 209879 May 20, 1998 DNA58747-1384 ATCC 209868
May 14, 1998 DNA57689-1385 ATCC 209869 May 14, 1998 DNA23330-1390
ATCC 209775 April 14, 1998 DNA26847-1395 ATCC 209772 April 14, 1998
DNA53974-1401 ATCC 209774 April 14, 1998 DNA57039-1402 ATCC 209777
April 14, 1998 DNA57033-1403 ATCC 209905 May 27, 1998 DNA34353-1428
ATCC 209855 May 12, 1998 DNA45417-1432 ATCC 209910 May 27, 1998
DNA39523-1192 ATCC 209424 October 31, 1997 DNA44205-1285 ATCC
209720 March 31, 1998 DNA50911-1288 ATCC 209714 March 31,1998
DNA48329-1290 ATCC 209785 April 21, 1998 DNA48306-1291 ATCC 209911
May 27, 1998 DNA48336-1309 ATCC 209669 March 11, 1998 DNA44184-1319
ATCC 209704 March 26, 1998 DNA48314-1320 ATCC 209702 March 26, 1998
DNA48333-1321 ATCC 209701 March 26, 1998 DNA50920-1325 ATCC 209700
March 26, 1998 DNA50988-1326 ATCC 209814 April 28, 1998
DNA48331-1329 ATCC 209715 March 31, 1998 DNA30867-1335 ATCC 209807
April 28, 1998 DNA55737-1345 ATCC 209753 April 7, 1998
DNA49829-1346 ATCC 209749 April 7, 1998 DNA52196-1348 ATCC 209748
April 7, 1998 DNA56965-1356 ATCC 209842 May 6, 1998 DNA56405-1357
ATCC 209849 May 6, 1998 DNA57530-1375 ATCC 209880 May 20, 1998
DNA56439-1376 ATCC 209864 May 14, 1998 DNA56409-1377 ATCC 209882
May 20, 1998 DNA56112-1379 ATCC 209883 May 20, 1998 DNA56045-1380
ATCC 209865 May 14, 1998 DNA59294-1381 ATCC 209866 May 14, 1998
DNA56433-1406 ATCC 209857 May 12, 1998 DNA53912-1457 ATCC 209870
May 14, 1998 DNA50921-1458 ATCC 209859 May 12, 1998 DNA29101-1122
ATCC 209653 March 5, 1998 DNA40021-1154 ATCC 209389 October 17,
1997 DNA42663-1154 ATCC 209386 October 17, 1997 DNA30943-1-1163-1
ATCC 209791 April 21, 1998 DNA64907-1163-1 ATCC 203242 September 9,
1998 DNA64908-1163-1 ATCC 203243 September 9, 1998 DNA39975-1210
ATCC 209783 April 21, 1998 DNA43316-1237 ATCC 209487 November 21,
1997 DNA55800-1263 ATCC 209680 March 17, 1998 DNA94832-2659 240-PTA
June 15, 1999 DNA52758-1399 ATCC 209773 April 14, 1998
[2786] 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
Commnissioner's rules pursuant thereto (including 37 CFR .sctn.
1.14 with particular reference to 886 OG 638).
[2787] 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.
[2788] 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
fiustrations that it represents. Indeed, various modifications of
the invention in addition to those shown and described herem will
become apparent to those skilled in the art from the foregoing
description and fall within the scope of the appended claims.
APPENDIX A
[2789] I hereby claim the benefit under Title 35, United States
Code, .sctn.119(e) of any United States provisional applications
listed below:
173 1. 60/062250 filed October 17, 1997 2. 60/064249 filed November
3, 1997 3. 60/065311 filed November 13, 1997 4. 60/066364 filed
November 21, 1997 5. 60/077450 filed March 10, 1998 6. 60/077632
filed March 11, 1998 7. 60/077641 filed March 11, 1998 8. 60/077649
filed March 11, 1998 9. 60/077791 filed March 12, 1998 10.
60/078004 filed March 13, 1998 11. 60/078886 filed March 20, 1998
12. 60/078936 filed March 20, 1998 13. 60/078910 filed March 20,
1998 14. 60/078939 filed March 20, 1998 15. 60/079294 filed March
25, 1998 16. 60/079656 filed March 26, 1998 17. 60/079664 filed
March 27, 1998 18. 60/079689 filed March 27, 1998 19. 60/079663
filed March 27, 1998 20. 60/079728 filed March 27, 1998 21.
60/079786 filed March 27, 1998 22. 60/079920 filed March 30, 1998
23. 60/079923 filed March 30, 1998 24. 60/080105 filed March 31,
1998 25. 60/080107 filed March 31, 1998 26. 60/080165 filed March
31, 1998 27. 60/080194 filed March 31, 1998 28. 60/080327 filed
April 1, 1998 29. 60/080328 filed April 1, 1998 30. 60/080333 filed
April 1, 1998 31. 60/080334 filed April 1, 1998 32. 60/081070 filed
April 8, 1998 33. 60/081049 filed April 8, 1998 34. 60/081071 filed
April 8, 1998 35. 60/081195 filed April 9, 1998 36. 60/081203 filed
April 9, 1998 37. 60/081229 filed April 9, 1998 38. 60/081955 filed
April 15, 1998 39. 60/081817 filed April 15, 1998 40. 60/081819
filed April 15, 1998 41. 60/081952 filed April 15, 1998 42.
60/081838 filed April 15, 1998 43. 60/082568 filed April 21, 1998
44. 60/082569 filed April 21, 1998 45. 60/082704 filed April 22,
1998 46. 60/082804 filed April 22, 1998 47. 60/082700 filed April
22, 1998 48. 60/082797 filed April 22, 1998 49. 60/082796 filed
April 23, 1998 50. 60/083336 filed April 27, 1998 51. 60/083322
filed April 28, 1998 52. 60/083392 filed April 29, 1998 53.
60/083495 filed April 29, 1998 54. 60/083496 filed April 29, 1998
55. 60/083499 filed April 29, 1998 56. 60/083545 filed April 29,
1998 57. 60/083554 filed April 29, 1998 58. 60/083558 filed April
29, 1998 59. 60/083559 filed April 29, 1998 60. 60/083500 filed
April 29, 1998 61. 60/083742 filed April 30, 1998 62. 60/084366
filed May 5, 1998 63. 60/084414 filed May 6, 1998 64. 60/084441
filed May 6, 1998 65. 60/084637 filed May 7, 1998 66. 60/084639
filed May 7, 1998 67. 60/084640 filed May 7, 1998 68. 60/084598
filed May 7, 1998 69. 60/084600 filed May 7, 1998 70. 60/084627
filed May 7, 1998 71. 60/084643 filed May 7, 1998 72. 60/085339
filed May 13, 1998 73. 60/085338 filed May 13, 1998 74. 60/085323
filed May 13, 1998 75. 60/085582 filed May 15, 1998 76. 60/085700
filed May 15, 1998 77. 60/085689 filed May 15, 1998 78. 60/085579
filed May 15, 1998 79. 60/085580 filed May 15, 1998 80. 60/085573
filed May 15, 1998 81. 60/085704 filed May 15, 1998 82. 60/085697
filed May 15, 1998 83. 60/086023 filed May 18, 1998 84. 60/086430
filed May 22, 1998 85. 60/086392 filed May 22, 1998 86. 60/086486
filed May 22, 1998 87. 60/086414 filed May 22, 1998 88. 60/087208
filed May 28, 1998 89. 60/087106 filed May 28, 1998 90. 60/087098
filed May 28, 1998 91. 60/091010 filed June 26, 1998 92. 60/090863
filed June 26, 1998 93. 60/091359 filed July 1, 1998 94. 60/094651
filed July 30, 1998 95. 60/100038 filed September 11, 1998 96.
60/109304 filed November 20, 1998 97. 60/113296 filed December 22,
1998 98. 60/113621 filed December 23, 1998 99. 60/123957 filed
March 12, 1999 100. 60/126773 filed March 29, 1999 101. 60/130232
filed April 21, 1999 102. 60/131022 filed April 26, 1999 103.
60/131445 filed April 28, 1999 104. 60/134287 filed May 14, 1999
105. 60/139557 filed June 16, 1999 106. 60/141037 filed June 23,
1999 107. 60/142680 filed July 7, 1999 108. 60/145698 filed July
26, 1999 109. 60/146222 filed July 28, 1999 110. 60/162506 filed
October 29, 1999
APPENDIX B
[2790] I hereby claim the benefit under Title 35, United States
Code, .sctn.120 of any United States and PCT applications listed
below:
174 US APPLICATIONS 1. 09/040220 filed March 17, 1998 2. 09/105413
filed June 26, 1998 3. 09/168978 filed October 7, 1998 4. 09/184216
filed November 2, 1998 5. 09/187368 filed November 6, 1998 6.
09/202054 filed December 7, 1998 7. 09/218517 filed December 22,
1998 8. 09/254465 filed March 5, 1999 9. 09/265686 filed March 10,
1999 10. 09/267213 filed March 12, 1999 11. 09/284291 filed April
12, 1999 12. 09/311832 filed May 14, 1999 13. 09/380137 filed
August 25, 1999 14. 09/380138 filed August 25, 1999 15. 09/380142
filed August 25, 1999 16. 09/709238 filed November 8, 2000 17.
09/723749 filed November 27, 2000 18. 09/747259 filed December 20,
2000 19. 09/816744 filed March 22, 2001 20. 09/816920 filed March
22, 2001 21. 09/854280 filed May 10, 2001 22. 09/854208 filed May
10, 2001 23. 09/872035 filed June 1, 2001 24. 09/874503 filed June
5, 2001 25. 09/882636 filed June 14, 2001 26. 09/886342 filed June
19, 2001 PCT APPLICATIONS 1. PCT/US98/21141 filed October 7, 1998
2. PCT/US98/24855 filed November 20, 1998 3. PCT/US99/00106 filed
January 5, 1999 4. PCT/US99/05028 filed March 8, 1999 5.
PCT/US99/05190 filed March 10, 1999 6. PCT/US99/10733 filed May 14,
1999 7. PCT/US99/12252 filed June 2, 1999 8. PCT/US99/28313 filed
November 30, 1999 9. PCT/US99/28551 filed December 2, 1999 10.
PCT/US99/28565 filed December 2, 1999 11. PCT/US99/30095 filed
December 16, 1999 12. PCT/US99/31243 filed December 30, 1999 13.
PCT/US99/31274 filed December 30, 1999 14. PCT/US00/00219 filed
January 5, 2000 15. PCT/US00/00277 filed January 6, 2000 16.
PCT/US00/00376 filed January 6, 2000 17. PCT/US00/03565 filed
February 11, 2000 18. PCT/US00/04341 filed February 18, 2000 19.
PCT/US00/05841 filed March 2, 2000 20. PCT/US00/07532 filed March
21, 2000 21. PCT/US00/05004 filed February 24, 2000 22.
PCT/US00/06319 filed March 10, 2000 23. PCT/US00/08439 filed March
30, 2000 24. PCT/US00/13705 filed May 17, 2000 25. PCT/US00/14042
filed May 22, 2000 26. PCT/US00/14941 filed May 30, 2000 27.
PCT/US00/15264 filed June 2, 2000 28. PCT/US00/20710 filed July 28,
2000 29. PCT/US00/23328 filed August 24, 2000 30. PCT/US00/32678
filed December 1, 2000 31. PCT/US00/34956 filed December 20, 2000
32. PCT/US01/06520 filed February 28, 2001 33. PCT/US01/09552 filed
March 22, 2001 34. PCT/US01/17092 filed May 25, 2001 35.
PCT/US01/17800 filed June 1, 2001 36. PCT/US01/19692 filed June 20,
2001 37. PCT/US01/21066 filed June 29, 2001 38. PCT/US01/21735
filed July 9, 2001
[2791]
Sequence CWU 0
0
* * * * *
References