U.S. patent application number 11/518609 was filed with the patent office on 2007-04-05 for secreted and transmembrane polypeptides and nucleic acids encoding the same.
This patent application is currently assigned to GENENTECH, INC.. Invention is credited to Avi Ashkenazi, David Bolstein, Luc Desnoyers, Dan L. Eaton, Napoleone Ferrara, Ellen Filvaroff, Sherman Fong, Wei-Qiang Gao, Hanspeter Gerber, Mary E. Gerritsen, Audrey Goddard, Paul J. Godowski, Christopher J. Grimaldi, Austin L. Gurney, Kenneth J. Hillan, Ivar J. Kljavin, Jennie P. Mather, James Pan, Nicholas F. Paoni, Margaret Ann Roy, Timothy A. Stewart, Daniel Tumas, P. Mickey Williams, William I. Wood.
Application Number | 20070077623 11/518609 |
Document ID | / |
Family ID | 39471780 |
Filed Date | 2007-04-05 |
United States Patent
Application |
20070077623 |
Kind Code |
A1 |
Ashkenazi; Avi ; et
al. |
April 5, 2007 |
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) ; Bolstein; David; (Belmont, CA) ;
Desnoyers; Luc; (San Francisco, CA) ; Eaton; Dan
L.; (San Rafael, CA) ; Ferrara; Napoleone;
(San Francisco, CA) ; Filvaroff; Ellen; (San
Francisco, CA) ; Fong; Sherman; (Alameda, CA)
; Gao; Wei-Qiang; (Palo Alto, CA) ; Gerber;
Hanspeter; (San Francisco, CA) ; Gerritsen; Mary
E.; (San Mateo, CA) ; Goddard; Audrey; (San
Francisco, CA) ; Godowski; Paul J.; (Hillsborough,
CA) ; Grimaldi; Christopher J.; (San Francisco,
CA) ; Gurney; Austin L.; (Belmont, CA) ;
Hillan; Kenneth J.; (San Francisco, CA) ; Kljavin;
Ivar J.; (Lafayette, CA) ; Mather; Jennie P.;
(Millbrae, CA) ; Pan; James; (Belmont, CA)
; Paoni; Nicholas F.; (Belmont, CA) ; Roy;
Margaret Ann; (San Francisco, CA) ; Stewart; Timothy
A.; (San Francisco, CA) ; Tumas; Daniel;
(Orinda, CA) ; Williams; P. Mickey; (Half Moon
Bay, CA) ; Wood; William I.; (Hillsborough,
CA) |
Correspondence
Address: |
HELLER EHRMAN LLP
275 MIDDLEFIELD ROAD
MENLO PARK
CA
94025-3506
US
|
Assignee: |
GENENTECH, INC.
South San Francisco
CA
|
Family ID: |
39471780 |
Appl. No.: |
11/518609 |
Filed: |
September 7, 2006 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10797366 |
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11518609 |
Sep 7, 2006 |
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09665350 |
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09665350 |
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Current U.S.
Class: |
435/69.1 ;
435/183; 435/320.1; 435/325; 530/350; 530/388.1; 536/23.5 |
Current CPC
Class: |
C07K 14/705 20130101;
C07K 14/47 20130101 |
Class at
Publication: |
435/069.1 ;
435/183; 435/320.1; 435/325; 530/350; 530/388.1; 536/023.5 |
International
Class: |
C12P 21/06 20060101
C12P021/06; C07H 21/04 20060101 C07H021/04; C12N 9/00 20060101
C12N009/00; C07K 14/705 20060101 C07K014/705; C07K 16/18 20060101
C07K016/18 |
Claims
1. Isolated nucleic acid having at least 80% sequence identity to a
nucleotide sequence that encodes a polypeptide comprising an amino
acid sequence selected from the group consisting of the amino acid
sequence shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4), FIG.
6 (SEQ ID NO: 12), FIG. 9 (SEQ ID NO: 18), FIG. 11 (SEQ ID NO:23),
FIG. 13 (SEQ ID NO:28), FIG. 15 (SEQ ID NO:34), FIG. 17 (SEQ ID
NO:39), FIG. 19 (SEQ ID NO:49), FIG. 22 (SEQ ID NO:59), FIG. 24
(SEQ ID NO:64), FIG. 26 (SEQ ID NO:69), FIG. 28 (SEQ ID NO:71),
FIG. 30 (SEQ ID NO:73), FIG. 32 (SEQ ID NO:84), FIG. 34 (SEQ ID
NO:91), FIG. 36 (SEQ ID NO:96), FIG. 38 (SEQ ID NO: 104), FIG. 40
(SEQ ID NO: 109), FIG. 42 (SEQ ID NO: 114), FIG. 44 (SEQ ID NO:
119), FIG. 46 (SEQ ID NO: 127), FIG. 48 (SEQ ID NO: 132), FIG. 50
(SEQ ID NO: 137), FIG. 52 (SEQ ID NO: 142), FIG. 54 (SEQ ID NO:
148), FIG. 56 (SEQ ID NO: 153), FIG. 58 (SEQ ID NO: 159), FIG. 60
(SEQ ID NO: 164), FIG. 62 (SEQ ID NO: 170), FIG. 64 (SEQ ID NO:
175), FIG. 66 (SEQ ID NO: 177), FIG. 68 (SEQ ID NO: 185), FIG. 70
(SEQ ID NO: 190), FIG. 72 (SEQ ID NO: 195), FIG. 74 (SEQ ID
NO:201), FIG. 76 (SEQ ID NO:207), FIG. 78 (SEQ ID NO:213), FIG. 80
(SEQ ID NO:221), FIG. 82 (SEQ ID NO:227), FIG. 84 (SEQ ID NO:236),
FIG. 86 (SEQ ID NO:245), FIG. 88 (SEQ ID NO:250), FIG. 90 (SEQ ID
NO:255), FIG. 92 (SEQ ID NO:257), FIG. 94 (SEQ ID NO:259), FIG. 96
(SEQ ID NO:261), FIG. 98 (SEQ ID NO:263), FIG. 100 (SEQ ID NO:285),
FIG. 102 (SEQ ID NO:290), FIG. 104 (SEQ ID NO:292), FIG. 106 (SEQ
ID NO:294), FIG. 108 (SEQ ID NO:310), FIG. 110 (SEQ ID NO:315),
FIG. 112 (SEQ ID NO:320), FIG. 114 (SEQ ID NO:325), FIG. 116 (SEQ
ID NO:332), FIG. 118 (SEQ ID NO:339), FIG. 120 (SEQ ID NO:341),
FIG. 122 (SEQ ID NO:377) and FIG. 124 (SEQ ID NO:423).
2. The nucleic acid of claim 1, wherein said nucleotide sequence
comprises a nucleotide sequence selected from the group consisting
of the sequence shown in FIG. 1 (SEQ ID NO:1), FIG. 3 (SEQ ID
NO:3), FIG. 5 (SEQ ID NO:11), FIG. 8 (SEQ ID NO:17), FIG. 10 (SEQ
ID NO:22), FIG. 12 (SEQ ID NO:27), FIG. 14 (SEQ ID NO:33), FIG. 16
(SEQ ID NO:38), FIG. 18 (SEQ ID NO:48), FIG. 21 (SEQ ID NO:58),
FIG. 23 (SEQ ID NO:63), FIG. 25 (SEQ ID NO:68), FIG. 27 (SEQ ID
NO:70), FIG. 29 (SEQ ID NO:72), FIG. 31 (SEQ ID NO:83), FIG. 33
(SEQ ID NO:90), FIG. 35 (SEQ ID NO:95), FIG. 37 (SEQ ID NO:103),
FIG. 39 (SEQ ID NO:108), FIG. 41 (SEQ ID NO:113), FIG. 43 (SEQ ID
NO:118), FIG. 45 (SEQ ID NO:126), FIG. 47 (SEQ ID NO:131), FIG. 49
(SEQ ID NO:136), FIG. 51 (SEQ ID NO:141), FIG. 53 (SEQ ID NO:147),
FIG. 55 (SEQ ID NO:152), FIG. 57 (SEQ ID NO:158), FIG. 59 (SEQ ID
NO:163), FIG. 61 (SEQ ID NO:169), FIG. 63 (SEQ ID NO:174), FIG. 65
(SEQ ID NO:176), FIG. 67 (SEQ ID NO:184), FIG. 69 (SEQ ID NO:189),
FIG. 71 (SEQ ID NO:194), FIG. 73 (SEQ ID NO:200), FIG. 75 (SEQ ID
NO:206), FIG. 77 (SEQ ID NO:212), FIG. 79 (SEQ ID NO:220), FIG. 81
(SEQ ID NO:226), FIG. 83 (SEQ ID NO:235), FIG. 85 (SEQ ID NO:244),
FIG. 87 (SEQ ID NO:249), FIG. 89 (SEQ ID NO:254), FIG. 91 (SEQ ID
NO:256), FIG. 93 (SEQ ID NO:258), FIG. 95 (SEQ ID NO:260), FIG. 97
(SEQ ID NO:262), FIG. 99 (SEQ ID NO:284), FIG. 101 (SEQ ID NO:289),
FIG. 103 (SEQ ID NO:291), FIG. 105 (SEQ ID NO:293), FIG. 107 (SEQ
ID NO:309), FIG. 109 (SEQ ID NO:314), FIG. 111 (SEQ ID NO:319),
FIG. 113 (SEQ ID NO:324), FIG. 115 (SEQ ID NO:331), FIG. 117 (SEQ
ID NO:338), FIG. 119 (SEQ ID NO:340), FIG. 121 (SEQ ID NO:376) and
FIG. 123 (SEQ ID NO:422), or the complement thereof.
3. The nucleic acid of claim 1, wherein said nucleotide sequence
comprises a nucleotide sequence selected from the group consisting
of the full-length coding sequence of the sequence shown in FIG. 1
(SEQ ID NO:1), FIG. 3 (SEQ ID NO:3), FIG. 5 (SEQ ID NO:11), FIG. 8
(SEQ ID NO:17), FIG. 10 (SEQ ID NO:22), FIG. 12 (SEQ ID NO:27),
FIG. 14 (SEQ ID NO:33), FIG. 16 (SEQ ID NO:38), FIG. 18 (SEQ ID
NO:48), FIG. 21 (SEQ ID NO:58), FIG. 23 (SEQ ID NO:63), FIG. 25
(SEQ ID NO:68), FIG. 27 (SEQ ID NO:70), FIG. 29 (SEQ ID NO:72),
FIG. 31 (SEQ ID NO:83), FIG. 33 (SEQ ID NO:90), FIG. 35 (SEQ ID
NO:95), FIG. 37 (SEQ ID NO:103), FIG. 39 (SEQ ID NO:108), FIG. 41
(SEQ ID NO:413), FIG. 43 (SEQ ID NO:118), FIG. 45 (SEQ ID NO:126),
FIG. 47 (SEQ ID NO:131), FIG. 49 (SEQ ID NO:136), FIG. 51 (SEQ ID
NO:141), FIG. 53 (SEQ ID NO:147), FIG. 55 (SEQ ID NO:152), FIG. 57
(SEQ ID NO:158), FIG. 59 (SEQ ID NO:163), FIG. 61 (SEQ ID NO:160),
FIG. 63 (SEQ ID NO:174), FIG. 65 (SEQ ID NO:176), FIG. 67 (SEQ ID
NO:184), FIG. 69 (SEQ ID NO:189), FIG. 71 (SEQ ID NO:194), FIG. 73
(SEQ ID NO:200), FIG. 75 (SEQ ID NO:206), FIG. 77 (SEQ ID NO:212),
FIG. 79 (SEQ ID NO:220), FIG. 81 (SEQ ID NO:226), FIG. 83 (SEQ ID
NO:235), FIG. 85 (SEQ ID NO:244), FIG. 87 (SEQ ID NO:249), FIG. 89
(SEQ ID NO:254), FIG. 91 (SEQ ID NO:256), FIG. 93 (SEQ ID NO:258),
FIG. 95 (SEQ ID NO:260), FIG. 97 (SEQ ID NO:262), FIG. 99 (SEQ ID
NO:284), FIG. 101 (SEQ ID NO:289), FIG. 103 (SEQ ID NO:291), FIG.
105 (SEQ ID NO:293), FIG. 107 (SEQ ID NO:309), FIG. 109 (SEQ ID
NO:314), FIG. 111 (SEQ ID NO:319), FIG. 113 (SEQ ID NO:324), FIG.
115 (SEQ ID NO:331), FIG. 117 (SEQ ID NO:338), FIG. 119 (SEQ ID
NO:340), FIG. 121 (SEQ ID NO:376) and FIG. 123 (SEQ ID NO:422), or
the complement thereof.
4. Isolated nucleic acid which comprises the full-length coding
sequence of the DNA deposited under accession number ATCC 209258,
ATCC 209256, ATCC 209264, ATCC 209250, ATCC 209375, ATCC 209378,
ATCC 209384, ATCC 209396, ATCC 209420, ATCC 209480, ATCC 209265,
ATCC 209257, ATCC 209262, ATCC 209253, ATCC 209402, ATCC 209401,
ATCC 209397, ATCC 209400, ATCC 209385, ATCC 209367, ATCC 209432,
ATCC 209263, ATCC 209251, ATCC 209255, ATCC 209252, ATCC 209373,
ATCC 209370, ATCC 209523, ATCC 209372, ATCC 209374, ATCC 209373,
ATCC 209382, ATCC 209383, ATCC 209403, ATCC 209398, ATCC 209399,
ATCC 209392, ATCC 209387, ATCC 209388, ATCC 209394, ATCC 209421,
ATCC 209393, ATCC 209418, ATCC 209485, ATCC 209483, ATCC 209482,
ATCC 209491, ATCC 209481, ATCC 209438, ATCC 209927, ATCC 209439,
ATCC 209489, ATCC 209433, ATCC 209488, ATCC 209434, ATCC 209395,
ATCC 209486, ATCC 209490, ATCC 209484, ATCC 209371 or ATCC
203553.
5. A vector comprising the nucleic acid of claim 1.
6. The vector of claim 5 operably linked to control sequences
recognized by a host cell transformed with the vector.
7. A host cell comprising the vector of claim 5.
8. The host cell of claim 7 wherein said cell is a CHO cell.
9. The host cell of claim 7 wherein said cell is an E. coli.
10. The host cell of claim 7 wherein said cell is a yeast cell.
11. A process for producing a PRO polypeptides comprising culturing
the host cell of claim 7 under conditions suitable for expression
of said PRO polypeptide and recovering said PRO polypeptide from
the cell culture.
12. Isolated native sequence PRO polypeptide having at least 80%
sequence identity to an amino acid sequence selected from the group
consisting of the amino acid sequence shown in FIG. 2 (SEQ ID
NO:2), FIG. 4 (SEQ ID NO:4), FIG. 6 (SEQ ID NO:12), FIG. 9 (SEQ ID
NO:18), FIG. 11 (SEQ ID NO:23), FIG. 13 (SEQ ID NO:28), FIG. 15
(SEQ ID NO:34), FIG. 17 (SEQ ID NO:39), FIG. 19 (SEQ ID NO:49),
FIG. 22 (SEQ ID NO:59), FIG. 24 (SEQ {D NO:64), FIG. 26 (SEQ ID
NO:69), FIG. 28 (SEQ ID NO:71), FIG. 30 (SEQ ID NO:73), FIG. 32
(SEQ ID NO:84), FIG. 34 (SEQ ID NO:91), FIG. 36 (SEQ ID NO:96),
FIG. 38 (SEQ ID NO:104), FIG. 40 (SEQ ID NO:109), FIG. 42 (SEQ ID
NO:114), FIG. 44 (SEQ ID NO:119), FIG. 46 (SEQ ID NO:127), FIG. 48
(SEQ ID NO:132), FIG. 50 (SEQ ID NO:137), FIG. 52 (SEQ ID NO:142),
FIG. 54 (SEQ ID NO:148), FIG. 56 (SEQ ID NO:153), FIG. 58 (SEQ ID
NO:159), FIG. 60 (SEQ ID NO:164), FIG. 62 (SEQ ID NO:170), FIG. 64
(SEQ ID NO:175), FIG. 66 (SEQ ID NO:177), FIG. 68 (SEQ ID NO:185),
FIG. 70 (SEQ IDNO:190), FIG. 72 (SEQ ID NO:195), FIG. 74 (SEQ ID
NO:201), FIG. 76 (SEQ ID NO:207), FIG. 78 (SEQ ID NO:213), FIG. 80
(SEQ ID NO:221), FIG. 82 (SEQ ID NO:227), FIG. 84 (SEQ ID NO:236),
FIG. 86 (SEQ ID NO:245), FIG. 88 (SEQ ID NO:250), FIG. 90 (SEQ ID
NO:255), FIG. 92 (SEQ ID NO:257), FIG. 94 (SEQ ID NO:259), FIG. 96
(SEQ ID NO:261), FIG. 98 (SEQ ID NO:263), FIG. 100 (SEQ ID NO:285),
FIG. 102 (SEQ ID NO:290), FIG. 104 (SEQ ID NO:292), FIG. 106 (SEQ
ID NO:294), FIG. 108 (SEQ ID NO:310), FIG. 110 (SEQ ID NO:315),
FIG. 112 (SEQ ID NO:320), FIG. 114 (SEQ ID NO:325), FIG. 116 (SEQ
ID NO:332), FIG. 118 (SEQ ID NO:339), FIG. 120 (SEQ ID NO:341),
FIG. 122 (SEQ ID NO:377) and FIG. 124 (SEQ ID NO:423).
13. Isolated PRO polypeptide having at least 80% sequence identity
to the amino acid sequence encoded by the nucleotide deposited
under accession number ATCC 209258, ATCC 209256, ATCC 209264, ATCC
209250, ATCC 209375, ATCC 209378, ATCC 209384, ATCC 209396, ATCC
209420, ATCC 209480, ATCC 209265, ATCC 209257, ATCC 209262, ATCC
209253, ATCC 209402, ATCC 209401, ATCC 209397, ATCC 209400, ATCC
209385, ATCC 209367, ATCC 209432, ATCC 209263, ATCC 209251, ATCC
209255, ATCC 209252, ATCC 209373, ATCC 209370, ATCC 209523, ATCC
209372, ATCC 209374, ATCC 209373, ATCC 209382, ATCC 209383, ATCC
209403, ATCC 209398, ATCC 209399, ATCC 209392, ATCC 209387, ATCC
209388, ATCC 209394, ATCC 209421, ATCC 209393, ATCC 209418, ATCC
209485, ATCC 209483, ATCC 209482, ATCC 209491, ATCC 209481, ATCC
209438, ATCC 209927, ATCC 209439, ATCC 209489, ATCC 209433, ATCC
209488, ATCC 209434, ATCC 209395, ATCC 209486, ATCC 209490, ATCC
209484, ATCC 209371 or ATCC 203553.
14. A chimeric molecule comprising a polypeptide according to claim
12 fused to a heterologous amino acid sequence.
15. The chimeric molecule of claim 14 wherein said heterologous
amino acid sequence is an epitope tag sequence.
16. The chimeric molecule of claim 14 wherein said heterologous,
amino acid sequence is a Fc region of an immunoglobulin.
17. An antibody which specifically binds to a PRO polypeptide
according to claim 12.
18. The antibody of claim 17 wherein said antibody is a monoclonal
antibody.
19. Isolated nucleic acid having at least 80% nucleic acid sequence
identity to: (a) a nucleotide sequence encoding the polypeptide
shown in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4), FIG. 6 (SEQ ID
NO:12), FIG. 9 (SEQ ID NO:18), FIG. 11 (SEQ ID NO:23), FIG. 13 (SEQ
ID NO:28), FIG. 15 (SEQ ID NO:34), FIG. 17 (SEQ ID NO:39), FIG. 19
(SEQ ID NO:49), FIG. 22 (SEQ ID NO:59), FIG. 24 (SEQ ID NO:64),
FIG. 26 (SEQ ID NO:69), FIG. 28 (SEQ ID NO:71), FIG. 30 (SEQ ID
NO:73), FIG. 32 (SEQ ID NO:84), FIG. 34 (SEQ ID NO:91), FIG. 36
(SEQ ID NO:96), FIG. 38 (SEQ ID NO:104), FIG. 40 (SEQ ID NO:109),
FIG. 42 (SEQ ID NO:114), FIG. 44 (SEQ ID NO:119), FIG. 46 (SEQ ID
NO:127), FIG. 48 (SEQ ID NO:132), FIG. 50 (SEQ ID NO:137), FIG. 52
(SEQ ID NO:142), FIG. 54 (SEQ ID NO:148), FIG. 56 (SEQ ID NO:153),
FIG. 58 (SEQ ID NO:159), FIG. 60 (SEQ ID NO:164), FIG. 62 (SEQ ID
NO:170), FIG. 64 (SEQ ID NO:175), FIG. 66 (SEQ ID NO:177), FIG. 68
(SEQ ID. NO:185), FIG. 70 (SEQ ID NO:190), FIG. 72 (SEQ ID NO:195),
FIG. 74 (SEQ ID NO:201), FIG. 76 (SEQ ID NO:207), FIG. 78 (SEQ ID
NO:213), FIG. 80 (SEQ ID NO:221), FIG. 82 (SEQ ID NO:227), FIG. 84
(SEQ ID NO:236), FIG. 86 (SEQ ID NO:245), FIG. 88 (SEQ ID NO:250),
FIG. 90 (SEQ ID NO:255), FIG. 92 (SEQ ID NO:257), FIG. 94 (SEQ ID
NO:259), FIG. 96 (SEQ ID NO:261), FIG. 98 (SEQ ID NO:263), FIG. 100
(SEQ ED NO:285), FIG. 102 (SEQ ID NO:290), FIG. 104 (SEQ ID
NO:292), FIG. 106 (SEQ ID NO:294), FIG. 108 (SEQ ID NO:310), FIG.
110 (SEQ ID NO:315), FIG. 112 (SEQ ID NO:320), FIG. 114 (SEQ ID
NO:325), FIG. 116 (SEQ ID NO:332), FIG. 118 (SEQ ID NO:339), FIG.
120 (SEQ ID NO:341), FIG. 122 (SEQ ID NO:377) or FIG. 124 (SEQ ID
NO:423), lacking its associated signal peptide; (b) a nucleotide
sequence encoding an extracellular domain of the polypeptide shown
in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4), FIG. 6 (SEQ ID
NO:12), FIG. 9 (SEQ ID NO:18), FIG. 11 (SEQ ID NO:23), FIG. 13 (SEQ
ID NO:28), FIG. 15 (SEQ ID NO:34), FIG. 17 (SEQ ID NO:39), FIG. 19
(SEQ ID NO:49), FIG. 22 (SEQ ID NO:59), FIG. 24 (SEQ ID NO:64),
FIG. 26 (SEQ ID NO:69), FIG. 28 (SEQ ID NO:71), FIG. 30 (SEQ ID
NO:73), FIG. 32 (SEQ ID NO:84), FIG. 34 (SEQ ID NO:91), FIG. 36
(SEQ ID NO:96), FIG. 38 (SEQ ID NO:104), FIG. 40 (SEQ ID NO:109),
FIG. 42 (SEQ ID NO:114), FIG. 44 (SEQ ID NO:119), FIG. 46 (SEQ ID
NO:127), FIG. 48 (SEQ ID NO:132), FIG. 50 (SEQ ID NO:137), FIG. 52
(SEQ ID NO:142), FIG. 54 (SEQ ID NO:148), FIG. 56 (SEQ ID NO:153),
FIG. 58 (SEQ ID NO:159), FIG. 60 (SEQ ID NO:164), FIG. 62 (SEQ ID
NO:170), FIG. 64 (SEQ ID NO:175), FIG. 66 (SEQ ID NO:177), FIG. 68
(SEQ ID NO:185), FIG. 70 (SEQ ID NO:190), FIG. 72 (SEQ ID NO:195),
FIG. 74 (SEQ ID NO:201), FIG. 76 (SEQ ID NO:207), FIG. 78 (SEQ ID
NO:213), FIG. 80 (SEQ ID NO:221), FIG. 82 (SEQ ID NO:227), FIG. 84
(SEQ ID NO:236), FIG. 86 (SEQ ID NO:245), FIG. 88 (SEQ ID NO:250),
FIG. 90 (SEQ ID NO:255), FIG. 92 (SEQ ID NO:257), FIG. 94 (SEQ ID
NO:259), FIG. 96 (SEQ ID NO:261), FIG. 98 (SEQ ID NO:263), FIG. 100
(SEQ ID NO:285), FIG. 102 (SEQ ID NO:290), FIG. 104 (SEQ ID
NO:292), FIG. 106 (SEQ ID NO:294), FIG. 108 (SEQ ID NO:310), Figure
re 110 (SEQ ID NO:315), FIG. 112 (SEQ ID NO:320), FIG. 114 (SEQ ID
NO:325), FIG. 116 (SEQ ID NO:332), FIG. 118 (SEQ ID NO:339), FIG.
120 (SEQ ID NO:341), FIG. 122 (SEQ ID NO:377) or FIG. 124 (SEQ ID
NO:423), with its associated signal peptide; or (c) a nucleotide
sequence encoding an extracellular domain of the polypeptide shown
in FIG. 2 (SEQ ID NO:2), FIG. 4 (SEQ ID NO:4), FIG. 6 (SEQ ID
NO:12), FIG. 9 (SEQ ID NO:18), FIG. 11 (SEQ ID NO:23), FIG. 13 (SEQ
ID NO:28), FIG. 15 (SEQ ID NO:34), FIG. 17 (SEQ ID NO:39), FIG. 19
(SEQ ID NO:49), FIG. 22 (SEQ ID NO:59), FIG. 24 (SEQ ID NO:64),
FIG. 26 (SEQ ID NO:69), FIG. 28 (SEQ ID NO:71), FIG. 30 (SEQ ID
NO:73), FIG. 32 (SEQ ID NO:84), FIG. 34 (SEQ ID NO:91), FIG. 36
(SEQ ID NO:96), FIG. 38 (SEQ ID NO:104), FIG. 40 (SEQ ID NO:109),
FIG. 4-2 (SEQ ID NO:114), FIG. 44 (SEQ ID NO:119), FIG. 46 (SEQ ID
NO:127), FIG. 48 (SEQ ID NO:132), FIG. 50 (SEQ ID NO:137), FIG. 52
(SEQ ID NO:142), FIG. 54 (SEQ ID NO:148), FIG. 56 (SEQ ID NO:153),
FIG. 58 (SEQ ID NO:159), FIG. 60 (SEQ ID NO:164), FIG. 62 (SEQ ID
NO:170), FIG. 64 (SEQ ID NO:175), FIG. 66 (SEQ ID NO:177), FIG. 68
(SEQ ID NO:185), FIG. 70 (SEQ ID NO:190), FIG. 72 (SEQ ID NO:195),
FIG. 74 (SEQ ID NO:201), FIG. 76 (SEQ ID NO:207), FIG. 78 (SEQ ID
NO:213), FIG. 80 (SEQ ID NO:221), FIG. 82 <SEQ ID NO:227), FIG.
84 (SEQ ID NO:236), FIG. 86 (SEQ ID NO:245), FIG. 88 (SEQ ID
NO:250), FIG. 90 (SEQ ID NO:255), FIG. 92 (SEQ ID NO:257), FIG. 94
(SEQ ID NO:259), FIG. 96 (SEQ ID NO:261), FIG. 98 (SEQ ID NO:263),
FIG. 100 (SEQ ID NO:285), FIG. 102 (SEQ ID NO:290), FIG. 104 (SEQ
ID NO:292), FIG. 106 (SEQ ID NO:294), FIG. 108 (SEQ ID NO:310),
FIG. 110 (SEQ ID NO:315), FIG. 112 (SEQ ID NO:320), FIG. 114 (SEQ
ID NO:325), FIG. 116 (SEQ ID NO:332), FIG. 118 (SEQ ID NO:339),
FIG. 120 (SEQ ID NO:341), FIG. 122 (SEQ ID NO:377) or FIG. 124 (SEQ
ID NO:423), lacking its associated signal peptide.
20. An isolated polypeptide having at least 80% amino acid sequence
identity to: (a) the polypeptide shown in FIG. 2 (SEQ ID NO:2),
FIG. 4 (SEQ ID NO:4), FIG. 6 (SEQ ID NO:12), FIG. 9 (SEQ ID NO:18),
FIG. 11 (SEQ ID NO:23), FIG. 13 (SEQ ID NO:28), FIG. 15 (SEQ ID
NO:34), FIG. 17 (SEQ ID NO:39), FIG. 19 (SEQ ID NO:49), FIG. 22
(SEQ ID NO:59), FIG. 24 (SEQ ID NO:64), FIG. 26 (SEQ ID NO:69),
FIG. 28 (SEQ ID NO:71), FIG. 30 (SEQ ID NO:73), FIG. 32 (SEQ ID
NO:84), FIG. 34 (SEQ ID NO:91), FIG. 36 (SEQ ID NO:96), FIG. 38
(SEQ ID NO:104), FIG. 40 (SEQ ID NO:109), FIG. 42 (SEQ ID NO:114),
FIG. 44 (SEQ ID NO:119), FIG. 46 (SEQ ID NO:127), FIG. 48 (SEQ ID
NO:132), FIG. 50 (SEQ ID NO:137), FIG. 52 (SEQ ID NO:142), FIG. 54
(SEQ ID NO:148), FIG. 56 (SEQ ID NO:153), FIG. 58 (SEQ ID NO:159),
FIG. 60 (SEQ ID NO:164), FIG. 62 (SEQ ID NO:170), FIG. 64 (SEQ ID
NO:175), FIG. 66 (SEQ ID NO:177), FIG. 68 (SEQ ID NO:185), FIG. 70
(SEQ ID NO:190), FIG. 72 (SEQ ID NO:195), FIG. 74 (SEQ ID NO:201),
FIG. 76 (SEQ ID NO:207), FIG. 78 (SEQ ID NO:213), FIG. 80 (SEQ ID
NO:221), FIG. 82 (SEQ ID NO:227), FIG. 84 (SEQ ID NO:236), FIG. 86
(SEQ ID NO:245), FIG. 88 (SEQ ID NO:250), FIG. 90 (SEQ ID NO:255),
FIG. 92 (SEQ ID NO:257), FIG. 94 SEQ ID NO:259), FIG. 96 (SEQ ID
NO:261), FIG. 98 (SEQ ID NO:263), FIG. 100 (SEQ ID NO:285), FIG.
102 (SEQ ID NO:290), FIG. 104 (SEQ ID NO:292), FIG. 106 (SEQ ID
NO:294), FIG. 108 (SEQ ID NO:310), FIG. 110 (SEQ ID NO:315), FIG.
112 (SEQ ID NO:320), FIG. 114 (SEQ ID NO:325), FIG. 116 (SEQ ID
NO:332), FIG. 118 (SEQ ID NO:339), FIG. 120 (SEQ ID NO:341), FIG.
122 (SEQ ID NO:377)or FIG. 124 (SEQ ID NO:423), lacking its
associated signal peptide; (b) an extracellular domain of the
polypeptide shown in FIG. 2 (SEQ ID NO:2 FIG. 4 (SEQ ID NO:4), FIG.
6 (SEQ ID NO:12), FIG. 9 (SEQ ID NO:18), FIG. 11 (SEQ ID NO:23),
FIG. 13 (SEQ ID NO:28), FIG. 15 (SEQ ID NO:34), FIG. 17 (SEQ ID
NO:39), FIG. 19 (SEQ ID NO:49), FIG. 22 (SEQ ID NO:59), FIG. 24
(SEQ ID NO:64), FIG. 26 (SEQ ID NO:69), FIG. 28 (SEQ ID NO:71),
FIG. 30 (SEQ ID NO:73), FIG. 32 (SEQ ID NO:84), FIG. 34 (SEQ ID
NO:91), FIG. 36 (SEQ ID NO:96), FIG. 38 (SEQ ID NO:104), FIG. 40
(SEQ ID NO:109), FIG. 42 (SEQ ID NO:114), FIG. 44 (SEQ ID NO:119),
FIG. 46 (SEQ ID NO:127), FIG. 48 (SEQ ID NO:132), FIG. 50 (SEQ ID
NO:137), FIG. 52 (SEQ ID NO:142), FIG. 54 (SEQ ID NO:148), FIG. 56
(SEQ ID NO:153), FIG. 58 (SEQ ID NO:159), FIG. 60 (SEQ ID NO:164),
FIG. 62 (SEQ ID NO:170), FIG. 64 (SEQ ID NO:175), FIG. 66 (SEQ ID
NO:177), FIG. 68 (SEQ ID NO:185), FIG. 70 (SEQ ID NO:190), FIG. 72
(SEQ ID NO:195), FIG. 74 (SEQ ID NO:201), FIG. 76 (SEQ ID NO:207),
FIG. 78 (SEQ ID NO:213), FIG. 80 (SEQ ID NO:221), FIG. 82 (SEQ ID
NO:227), FIG. 84 (SEQ ID NO:236), FIG. 86 (SEQ ID NO:245), FIG. 88
(SEQ ID NO:250), FIG. 90 (SEQ ID NO:255), FIG. 92 (SEQ ID NO:257),
FIG. 94 (SEQ ID NO:259), FIG. 96 (SEQ ID NO:261), FIG. 98 (SEQ ID
NO:263), FIG. 100 (SEQ ID NO:285), FIG. 102 (SEQ ID NO:290), FIG.
104 (SEQ ID NO:292), FIG. 106 (SEQ ID NO:294), FIG. 108 (SEQ ID
NO:310), FIG. 110 (SEQ ID NO:315), FIG. 112 (SEQ ID NO:320), FIG.
114 (SEQ ID NO:325), FIG. 116 (SEQ ID NO:332), FIG. 118 (SEQ ID
NO:339), FIG. 120 (SEQ ID NO:341), FIG. 122 (SEQ ID NO:377) or FIG.
124 (SEQ ID NO:423), with its associated signal peptide; or (c) an
extracellular domain of the polypeptide shown in FIG. 2 (SEQ ID
NO:2), FIG. 4 (SEQ ID NO:4), FIG. 6 (SEQ ID NO:12), FIG. 9 (SEQ ID
NO:18), FIG. 11 (SEQ ID NO:23), FIG. 13 (SEQ ID NO:28), FIG. 15
(SEQ ID NO:34), FIG. 17 (SEQ ID NO:39), FIG. 19 (SEQ ID NO:49),
FIG. 22 (SEQ ID NO:59), FIG. 24 (SEQ ID NO:64), FIG. 26 (SEQ ID
NO:69), FIG. 28 (SEQ ID NO:71), FIG. 30 (SEQ ID NO:73), FIG. 32
(SEQ ID NO:84), FIG. 34 (SEQ ID NO:91), FIG. 36 (SEQ ID NO:96),
FIG. 38 (SEQ ID NO:104), FIG. 40 (SEQ ID NO:109), FIG. 42 (SEQ ID
NO:114), FIG. 44 (SEQ ID NO:119), FIG. 46 (SEQ ID NO:127), FIG. 48
(SEQ ID NO:132), FIG. 50 (SEQ ID NO:137), FIG. 52 (SEQ ID NO:142),
FIG. 54 (SEQ ID NO:148), FIG. 56 (SEQ ID NO:153), FIG. 58 (SEQ ID
NO:159), FIG. 60 (SEQ IDNO:164), FIG. 62 (SEQ ID NO:170), FIG. 64
(SEQ ID NO:175), FIG. 66 (SEQ ID NO:177), FIG. 68 (SEQ ID NO:185),
FIG. 70 (SEQ ID NO:190), FIG. 72 (SEQ ID NO:195), FIG. 74 (SEQ ID
NO:201), FIG. 76 (SEQ ID NO:207), FIG. 78 (SEQ ID NO:213), FIG. 80
(SEQ ID NO:221), FIG. 82 (SEQ ID NO:227), FIG. 84 (SEQ ID NO:236),
FIG. 86 (SEQ ID NO:245), FIG. 88 (SEQ ID NO:250), FIG. 90 (SEQ ID
NO:255), FIG. 92 (SEQ ID NO:257), FIG. 94 (SEQ ID NO:259), FIG. 96
(SEQ ID NO:261), FIG. 98 (SEQ ID NO:263), FIG. 100 (SEQ ID NO:285),
FIG. 102 (SEQ ID NO:290), FIG. 104 (SEQ ID NO:292), FIG. 106 (SEQ
ID NO:294), FIG. 108 (SEQ ID NO:310), FIG. 110 (SEQ ID NO:315),
FIG. 112 (SEQ ID NO:320), FIG. 114 (SEQ ID NO:325), FIG. 116 (SEQ
ID NO:332), FIG. 118 (SEQ ID NO:339), FIG. 120 (SEQ ID NO:341),
FIG. 122 (SEQ ID NO:377) or FIG. 124 <SEQ ID NO:423), lacking
its associated signal peptide.
21. A method of detecting a PRO245 polypeptide in a sample
suspected of containing a PRO245 polypeptide, said method
comprising contacting said sample with a PRO] 868 polypeptide and
determining the formation of a PRO245/PRO 1868 polypeptide
conjugate in said sample, wherein the formation of said conjugate
is indicative of the presence of a PRO245 polypeptide in said
sample.
22. The method according to claim 21, wherein said sample comprises
cells suspected of expressing said PRO245 polypeptide.
23. The method according to claim 21, wherein said PRO1868
polypeptide is labeled with a detectable label.
24. The method according to claim 21, wherein said PRO 1868
polypeptide is attached to a solid support.
25. A method of detecting a PRO 1868 polypeptide in a sample
suspected of containing a PRO 1868 polypeptide, said method
comprising contacting said sample with a PRO245 polypeptide and
determining ihe formation of a PRO245/PRO 1868 polypeptide
conjugate in said sample, wherein the formation of said conjugate
is indicative of the presence of a PRO 1868 polypeptide in said
sample.
26. The method according to claim 25, wherein said sample comprises
cells suspected of expressing said PRO 1868 polypeptide.
27. The method according to claim 25, wherein said PRO245
polypeptide is labeled with a detectable label.
28. The method according to claim 25, wherein said PRO245
polypeptide is attached to a solid support.
29. A method of linking a bioactive molecule to a cell expressing a
PRO245 polypeptide, said method comprising contacting said cell
with a PRO 1868 polypeptide that is bound to said bioactive
molecule and allowing said PRO245 and PRO 1868 polypeptides to bind
to one another, thereby linking said bioactive molecules to said
cell.
30. The method according to claim 29, wherein said bioactive
molecule is a toxin, a radiolabel or an antibody.
31. The method according to claim 29, wherein said bioactive
molecule causes the death of said cell.
32. A method of linking a bioactive molecule to a cell expressing a
PRO1868 polypeptide, said method comprising contacting said cell
with a PRO245 polypeptide that is bound to said bioactive molecule
and allowing said PRO245 and PRO 1868 polypeptides to bind to one
another, thereby linking said bioactive molecules to said cell.
33. The method according to claim 32, wherein said bioactive
molecule is a toxin, a radiolabel or an antibody.
34. The method according to claim 32, wherein said bioactive
molecule causes the death of said cell.
35. A method of modulating at least one biological activity of a
cell expressing a PRO245 polypeptide, said method comprising
contacting said cell with a PRO 1868 polypeptide or an anti-PRO245
antibody, whereby said PRO 1868 polypeptide or said anti-PRO245
antibody binds to said PRO245 polypeptide, thereby modulating at
least one biological activity of said cell.
36. The method according to claim 35, wherein said cell is
killed.
37. A method of modulating at least one biological activity of a
cell expressing a PRO1868 polypeptide, said method comprising
contacting said cell with a PRO245 polypeptide or an anti-PRO1868
antibody, whereby said PRO245 polypeptide or said anti-PRO1868
antibody binds to said PRO1868 polypeptide, thereby modulating at
least one biological activity of said cell.
38. The method according to claim 37, wherein said cell is killed.
Description
RELATED APPLICATION
[0001] This is a continuation application claiming priority under
35 U.S.C. .sctn.120 to U.S. Ser. No. 10/797,366 filed Mar. 9, 2004,
which is a continuation of, and claims priority under 35 U.S.C.
.sctn.120 to U.S. Ser. No. 09/665,350 filed Sep. 18, 2000, now
abandoned, which is a continuation of, and claims priority under 35
U.S.C. .sctn.120 to, PCT Application number PCT/US00/04414 filed
Feb. 22, 2000, and where U.S. Ser. No. 09/665,350 is also a
continuation-in-part of, and claims priority under 35 U.S.C.
.sctn.120 to, PCT Application number PCT/US00/23328 filed Aug. 24,
2000, which is a continuation-in-part of, and claims priority under
35 U.S.C. .sctn.120 to, PCT Application number PCT/US00/20710 filed
Jul. 28, 2000, which claims priority under 35 U.S.C. .sctn.119 to
U.S. provisional application No. 60/146,222 filed Jul. 28, 1999,
and where PCT Application number PCT/US00/20710 is also a
continuation-in-part of, and claims priority under 35 U.S.C.
.sctn.120 to, PCT Application number PCT/US00/15264 filed Jun. 2,
2000, which is a continuation-in-part of, and claims priority under
35 U.S.C. .sctn.120 to, PCT Application number PCT/US00/14042
filed. May 22, 2000, which is a continuation-in-part of, and claims
priority under 35 U.S.C. .sctn.120 to, PCT Application number
PCT/US00/08439 filed Mar. 30, 2000, which is a continuation-in-part
of, and claims priority under 35 U.S.C. .sctn.120 to, PCT
Application number PCT/US00/07377 filed Mar. 20, 2000, which is a
continuation-in-part of, and claims priority under 35 U.S.C.
.sctn.120 to, PCT Application number PCT/US00/05841 filed Mar. 2,
2000, which is a continuation-in-part of, and claims priority under
35 U.S.C. .sctn.120 to, PCT Application number PCT/US00/05004 filed
Feb. 24, 2000, which is a continuation-in-part of, and claims
priority under 35 U.S.C. .sctn.120 to, PCT Application number
PCT/US00/04414 filed Feb. 22, 2000, which is a continuation-in-part
of, and claims priority under 35 U.S.C. .sctn.120 to, PCT
Application number PCT/US00/03565 filed Feb. 11, 2000, which is a
continuation-in-part of, and claims priority under 35 U.S.C.
.sctn.120 to, PCT Application number PCT/US00/00219 filed Jan. 5,
2000, which claims priority under 35 U.S.C. .sctn.119 to U.S.
provisional application No. 60/145,698 filed Jul. 26, 1999, and
where PCT Application number PCT/US00/00219 is also a
continuation-in-part of, and claims priority under 35 U.S.C.
.sctn.120 to, both PCT Application numbers: PCT/US99/30999 filed
Dec. 20, 1999 and PCT/US99/3091 filed Dec. 20, 1999, which claims
priority under 35 U.S.C. .sctn.119 to U.S. provisional application
No. 60/143,048 filed Jul. 7, 1999, and where PCT/US99/30999 and
PCT/US99/30911 are both continuations-in-part to, and claim
priority under 35 U.S.C. .sctn.120 to, PCT Application number
PCT/US99/30095 filed Dec. 16, 1999, which claims priority under 35
U.S.C. .sctn.119 to U.S. provisional application No. 60/113,296
filed Dec. 22, 1998, and where PCT Application number
PCT/US99/30095 is also a continuation-in-part of, and claims
priority under 35 U.S.C. .sctn.120 to, both PCT Application
numbers: PCT/US99/28565 and PCT/US99/28564 both filed Dec. 2, 1999,
which are also continuations-in-part of, and claim priority under
35 U.S.C. .sctn.120 to, PCT Application number PCT/US99/28301 filed
Dec. 1, 1999, which is a continuation-in-part of, and claims
priority under 35 U.S.C. .sctn.120 to, PCT Application number
PCT/US99/28313 filed Nov. 30, 1999, which is a continuation-in-part
of, and claims priority under 35 U.S.C. .sctn.120 to, PCT
Application number PCT/US99/28214 filed Nov. 29, 1999, which is a
continuation-in-part of, and claims priority under 35 U.S.C.
.sctn.120 to, PCT Application number PCT/US99/23089 filed Oct. 5,
1999, which claims priority under 35 U.S.C. .sctn.119 to U.S.
provisional application No. 60/104080 filed Oct. 13, 1998, and
where PCT Application number PCT/US99/23089 is also a
continuation-in-part of, and claims priority under 35 U.S.C.
.sctn.120 to, both PCT Application numbers: PCT/US99/21547and
PCT/US99/21090 both filed Sep. 15, 1999, which are also
continuations-in-part of, and claim priority under 35 U.S.C.
.sctn.120 to, PCT Application number PCT/US99/20944 filed Sep. 13,
1999, which is a continuation-in-part of, and claims priority-under
35 U.S.C. .sctn.120 to, PCT Application number PCT/US99/20594 filed
Sep. 8, 1999, which is a continuation-in-part of, and claims
priority under 35 U.S.C. .sctn.120 to, PCT Application number
PCT/US98/25108 filed Dec. 1, 1998, which is a continuation-in-part
of, and claims priority under 35 U.S.C. .sctn.120 to, PCT
Application number PCT/US98/19437 filed Sep. 17, 1998, which is a
continuation-in-part of, and claims priority under 35 U.S.C.
.sctn.120 to, PCT Application number PCT/US98/19330 filed Sep. 16,
1998, which is a continuation-in-part of, and claims priority under
35 U.S.C. .sctn.120 to, PCT Application number PCT/US98/19177 filed
Sep. 14, 1998, which is a continuation-in-part of, and claims
priority under 35 U.S.C. .sctn.120 to, PCT Application number
PCT/US98/18824 filed Sep. 10, 1998, which claims priority under 35
U.S.C. .sctn.119 to U.S. provisional application Nos. 60/059,115
filed Sep. 17, 1997, 60/059,184 filed Sep. 17, 1997, 60/059,122
filed Sep. 17, 1997, 60/059,117 filed Sep. 17, 1997, 60/059,113,
filed Sep. 17, 1997, 60/059,121 filed Sep. 17, 1997, 60/059,119
filed Sep. 17, 1997, 60/059,263 filed Sep. 18, 1997, 60/059,266
filed Sep. 18, 1997, 60/062,125 filed Oct. 15, 1997, 60/062,287
filed Oct. 17, 1997, 60/062,285 filed Oct. 17, 1997, 60/063,486
filed Oct. 21, 1997, 60/062,816 filed Oct. 24, 1997, 60/062,814
filed Oct. 24, 1997, 60/063,127 filed Oct. 24, 1997, 60/063,120
filed Oct. 24, 1997, 60/063,121 filed Oct. 24, 1997, 60/063,045
filed 10/24/1997, 60/063,128 filed Oct. 24, 1997, 60/063,329 filed
Oct. 27, 1997, 60/063,327 filed Oct. 27, 1997, 60/063,549 filed
10/28/1997, 60/063,541 filed Oct. 28, 1997, 60/063,550 filed Oct.
28, 1997, 60/063,542 filed Oct. 28, 1997, 60/063,544 filed Oct. 28,
1997, 60/063,564 filed Oct. 28, 1997, 60/063,734 filed Oct. 29,
1997, 60/063,738 filed Oct. 29, 1997, 60/063,704 filed Oct. 29,
1997, 60/063,435 filed Oct. 29, 1997, 60/064,215 filed 10/29/1997,
60/063,735 filed Oct. 29, 1997, 60/063,732 filed Oct. 29, 1997,
60/064,103 filed 10/31/1997, 60/063,870 filed Oct. 31, 1997,
60/064,248 filed Nov. 3, 1997, 60/064,809 filed Nov. 7, 1997,
60/065,186 filed Nov. 12, 1997, 60/065,846 filed Nov. 17, 1997,
60/065,693 filed Nov. 18, 1997, 60/066,120 filed Nov. 21, 1997,
60/066,364 filed 11/21/1997, 60/066,772 filed Nov. 24, 1997,
60/066,466 filed Nov. 24, 1997, 60/066,770 filed Nov. 24, 1997,
60/066,511 filed Nov. 24, 1997, 60/066,453 filed Nov. 24, 1997,
60/066,840 filed Nov. 25, 1997, 60/069,425 filed Dec. 12, 1997,
60/088,026 filed Jun. 4, 1998, 60/109,304 filed Nov. 20, 1998, the
entire disclosures of which are hereby incorporated by
reference.
FIELD OF THE INVENTION
[0002] The present invention relates generally to the
identification and isolation of novel DNA and to the recombinant
production of novel polypeptides.
BACKGROUND OF THE INVENTION
[0003] Extracellular proteins play important roles in, among other
things, the formation, differentiation and maintenance of
multicellular organisms. The fate of many individual cells, e.g.,
proliferation, migration, differentiation, or interaction with
other cells, is typically governed by information received from
other cells and/or the immediate environment. This information is
often transmitted by secreted polypeptides (for instance, mitogenic
factors, survival factors, cytotoxic factors, differentiation
factors, neuropeptides, and hormones) which are, in turn, received
and interpreted by diverse cell receptors or membrane-bound
proteins. These secreted polypeptides or signaling molecules
normally pass through the cellular secretory pathway to reach their
site of action in the extracellular environment.
[0004] Secreted proteins have various industrial applications,
including as pharmaceuticals, diagnostics, biosensors and
bioreactors. Most protein drugs available at present, such as
thrombolytic agents, interferons, interleukins, erythropoietins,
colony stimulating factors, and various other cytokines, are
secretory proteins. Their receptors, which are membrane proteins,
also have potential as therapeutic or diagnostic agents. Efforts
are being undertaken by both industry and academia to identify new,
native secreted proteins. Many efforts are focused on the screening
of mammalian recombinant DNA libraries to identify the coding
sequences for novel secreted proteins. Examples of screening
methods and techniques are described in the literature [see, for
example, Klein et al., Proc. Natl. Acad. Sci. 93:7108-7113 (1996);
U.S. Pat. No. 5,536,637)].
[0005] Membrane-bound proteins and receptors can play important
roles in, among other things, the formation, differentiation and
maintenance of multicellular organisms. The fate of many individual
cells, e.g., proliferation, migration, differentiation, or
interaction with other cells, is typically governed by information
received from other cells and/or the immediate environment. This
information is often transmitted by secreted polypeptides (for
instance, mitogenic factors, survival factors, cytotoxic factors,
differentiation factors, neuropeptides, and hormones) which are, in
turn, received and interpreted by diverse cell receptors or
membrane-bound proteins. Such membrane-bound proteins and cell
receptors include, but are not limited to, cytokine receptors,
receptor kinases, receptor phosphatases, receptors involved in
cell-cell interactions, and cellular adhesin molecules like
selectins and integrins. For instance, transduction of signals that
regulate cell growth and differentiation is regulated in part by
phosphorylation of various cellular proteins. Protein tyrosine
kinases, enzymes that catalyze that process, can also act as growth
factor receptors, Examples include fibroblast growth factor
receptor and nerve growth factor receptor.
[0006] Membrane-bound proteins and receptor molecules have various
industrial applications, including as pharmaceutical and diagnostic
agents. Receptor immunoadhesins, for instance, can be employed as
therapeutic agents to block receptor-ligand interactions. The
membrane-bound proteins can also be employed for screening of
potential peptide or small molecule inhibitors of the relevant
receptor/ligand interaction.
[0007] Efforts are being undertaken by both industry and academia
to identify new, native receptor or membrane-bound proteins. Many
efforts are focused on the screening of mammalian recombinant DNA
libraries to identify the coding sequences for novel receptor or
membrane-bound proteins.
[0008] 1. PRO211 and PRO217
[0009] Epidermal growth factor (EGF) is a conventional mitogenic
factor that stimulates the proliferation of various types of cells
including epithelial cells and fibroblasts. EGF binds to and
activates the EGF receptor (EGFR), which initiates intracellular
signaling and subsequent effects. The EGFR is expressed in neurons
of the cerebral cortex, cerebellum, and hippocampus in addition to
other regions of the central nervous system (CNS). In addition, EGF
is also expressed in various regions of the CNS. Therefore, EGF
acts not only on mitotic cells, but also on postmitotic neurons. In
fact, many studies have indicated that EGF has neurotrophic or
neuromodulatory effects on various types of neurons in the CNS. For
example, EGF acts directly on cultured cerebral cortical and
cerebellar neurons, enhancing neurite outgrowth and survival. On
the other hand, EGF also acts on other cell types, including septal
cholinergic and mesencephalic doparninergic neurons, indirectly
through glial cells. Evidence of the effects of EGF on neurons in
the CNS is accumulating, but the mechanisms of action remain
essentially unknown. EGF-induced signaling in mitotic cells is
better understood than in postrnitotic neurons. Studies of cloned
pheochromocytoma PC12 cells and cultured cerebral cortical neurons
have suggested that the EGF-induced neurotrophic actions are
mediated by sustained activation of the EGFR and mitogen-activated
protein kinase (MAPK) in response to EGF. The sustained
intracellular signaling correlates with the decreased rate of EGFR
down-regulation, which might determine the response of neuronal
cells to EGF. It is likely that EGF is a multi-potent growth factor
that acts upon various types of cells including mitotic cells and
postmitotic neurons.
[0010] EGF is produced by the salivary and Brunner's glands of the
gastrointestinal system, kidney, pancreas, thyroid gland, pituitary
gland, and the nervous system, and is found in body fluids such as
saliva, blood, cerebrospinal fluid (CSF), urine, amniotic fluid,
prostatic fluid, pancreatic juice, and breast milk, Plata-Salaman,
Peptides 12: 653-663 (1991).
[0011] EGF is mediated by its membrane specific receptor, which
contains an intrinsic tyrosine kinase. Stoscheck et al., J. Cell
Biochem. 31:135-152(1986). EGF is believed to function by binding
to the extracellular portion of its receptor which induces a
transmembrane signal that activates the intrinsic tyrosine
kinase.
[0012] Purification and sequence analysis of the EGF-like domain
has revealed the presence of six conserved cysteine residues which
cross-bind to create three peptide loops, Savage et al., J. Biol
Chem. 248: 7669-7672 (1979). It is now generally known that several
other peptides can react with the EGF receptor which share the same
generalized motif
X.sub.nCX.sub.7CX.sub.4/5CX.sub.10CXCX.sub.5GX.sub.2CX.sub.n, where
X represents any non-cysteine amino acid, and n is a variable
repeat number. Non isolated peptides having this motif include
TGF-.alpha., amphiregulin, schwannoma-derived growth factor (SDGF),
heparin-binding EGF-like growth factors and certain virally encoded
peptides (e.g., Vaccinia virus, Reisner, Nature 313: 801-803
(1985), Shope fibroma virus, Chang et al., Mol Cell Biol. 7:
535-540 (1987), Molluscum contagiosum, Porter and Archard, J Gen.
Virol. 68: 673-682 (1987), and Myxoma virus, Upton et al, J. Virol.
61: 1271-1275 (1987), Prigent and Lemoine, Prog. Growth Factor Res.
4: 1-24 (1992).
[0013] EGF-like domains are not confined to growth factors but have
been observed in a variety of cell-surface and extracellular
proteins which have interesting properties in cell adhesion,
protein-protein interaction and development, Laurence and
Gusterson, Tumor Biol. 11: 229-261 (1990). These proteins include
blood coagulation factors (factors VI, IX, X, XII, protein C,
protein S, protein Z, tissue plasminogen activator, urokinase),
extracellular matrix components (laminin, cytotactin, entactin),
cell surface receptors (LDL receptor, thrombomodulin receptor) and
immunity-related proteins (complement Clr, uromodulin).
[0014] Even more interesting, the general structure pattern of
EGF-like precursors is preserved through lower organisms as well as
in mammalian cells. A number of genes with developmental
significance have been identified in invertebrates with EGF-like
repeats. For example, the notch gene of Drosophila encodes 36
tandemly arranged 40 amino acid repeats which show homology to EGF,
Wharton et al., Cell 43: 557-581 (1985). Hydropathy plots indicate
a putative membrane spanning domain, with the EGF-related sequences
being located on the extracellular side of the membrane. Other
homeotic genes with EGF-like repeats include Delta, 95F and 5ZD
which were identified using probes based on Notch, and the nematode
gene Lin-12 which encodes a putative receptor for a developmental
signal transmitted between two specified cells.
[0015] Specifically, EGF has been shown to have potential in the
preservation and maintenance of gastrointestinal mucosa and the
repair of acute and chronic mucosal lesions, Konturek et al., Eur.
J. Gastroenterol Hepatol. 7 (10), 933-37 (1995), including the
treatment of necrotizing enterocolitis, Zollinger-Ellison syndrome,
gastrointestinal ulceration gastrointestinal ulcerations and
congenital microvillus atrophy, Guglietta and Sullivan, Eur. J.
Gastroenterol Hepatol, 7(10), 945-50 (1995). Additionally, EGF has
been implicated in hair follicle differentiation; du Cros, J.
Invest. Dermatol. 101 (1 Suppl.), 106S-I 13S (1993), Hillier, Clin.
Endocrinol. 33(4),427-28 (1990); kidney function, Hamm et al.,
Semin. Nephrol. 13 (1): 109-15 (1993), Harris, Am. J Kidney Dis.
17(6): 627-30 (1991); tear fluid, van Setten et al., Int.
Ophthalmol 15(6); 359-62 (1991); vitamin K mediated blood
coagulation, Stenflo et al., Blood 18(7): 1637-51(1991). EGF is
also implicated various skin disease characterized by abnormal
keratinocyte differentiation, e.g., psoriasis, epithelial cancers
such as squamous cell carcinomas of the lung, epidermoid carcinoma
of the vulva and gliomas. King et al., Am. J. Med. Sci. 296:
154-158 (1988).
[0016] Of great interest is mounting evidence that genetic
alterations in growth factors signaling pathways are closely linked
to developmental abnormalities and to chronic diseases including
cancer. Aaronson, Science 254: 1146-1153 (1991). For example,
c-erb-2 (also known as HER-2). a proto-oncogene with close
structural similarity to EGF receptor protein, is overexpressed in
human breast cancer. King et al., Science 229: 974-976 (1985);
Gullick, Hormones and their actions, Cooke et al., eds, Amsterdam,
Elsevier, pp 349-360 (1986).
[0017] We herein describe the identification and characterization
of novel polypeptides having homology to EGF, wherein those
polypeptides are herein designated PRO211 and PRO217.
[0018] 2. PRO230
[0019] Nephritis is a condition characterized by inflammation of
the kidney affecting the structure and normal function of the
kidney. This condition can be chronic or acute and is generally
caused by infection, degenerative process or vascular disease. In
all cases, early detection is desirable so that the patient with
nephritis can begin treatment of the condition.
[0020] An approach to detecting nephritis is to determine the
antigens associated with nephritis and antibodies thereto. In
rabbit, a tubulointerstitial nephritis antigen (TIN-ag) has been
reported in Nelson, T. R., et al., J. Biol. Chem., 270(27):16265-70
(July 1995) (GENBANKAJ24270). This study reports that the rabbit
TIN-ag is a basement membrane glycoprotein having a predicted amino
acid sequence which has a carboxyl-terminal region exhibiting 30%
homology with human preprocathepsin B, a member of the cystein
proteinase family of proteins. It is also reported that the rabbit
TIN-ag has a domain in the amino-terminal region containing an
epidermal growth factor-like motif that shares homology with
laminin A and S chains, alpha 1 chain of type I collagen, von
Willebrand's factor and mucin, indicating structural and functional
similarities. Studies have also been conducted in mice. However, it
is desirable to identify tubulointerstitial nephritis antigens in
humans to aid in the development of early detection methods and
treatment of nephritis.
[0021] Proteins which have homology to tubulointerstitial nephritis
antigens are of particular interest to the medical and industrial
communities. Often, proteins having homology to each other have
similar function. It is also of interest when proteins having
homology do not have similar functions, indicating that certain
structural motifs identify information other than function, such as
locality of function. We herein describe the identification and
characterization of a novel polypeptide, designated herein as
PRO230, which has homology to tubulointerstitial nephritis
antigens.
[0022] 3. PRO232
[0023] Stem cells are undifferentiated cells capable of (a)
proliferation, (b) selfmaintenance, (c) the production of a large
number of differentiated functional progeny, (d) regeneration of
tissue after injury and/or (e) a flexibility in the use of these
options. Stem cells often express cell surface antigens which are
capable of serving as cell specific markers that can be exploited
to identify stem cells, thereby providing a means for identifying
and isolating specific stem cell populations.
[0024] Having possession of different stem cell populations will
allow for a number of important applications. For example,
possessing a specific stem cell population will allow for the
identification of growth factors and other proteins which are
involved in their proliferation and differentiation. In addition,
there may be as yet undiscovered proteins which are associated with
(1) the early steps of dedication of the stem cell to a particular
lineage, (2) prevention of such dedication, and (3) negative
control of stem cell proliferation, all of which may be identified
if one has possession of the stem cell population. Moreover, stem
cells are important and ideal targets for gene therapy where the
inserted genes promote the health of the individual into whom the
stem cells are transplanted. Finally, stem cells may play important
roles in transplantation of organs or tissues, for example liver
regeneration and skin grafting.
[0025] Given the importance of stem cells in various different
applications, efforts are currently being undertaken by both
industry and academia to identify new, native stem cell antigen
proteins so as to provide specific cell surface markers for
identifying stem cell populations as well as for providing insight
into the functional roles played by stem cell antigens in cell
proliferation and differentiation. We herein describe the
identification and characterization of novel polypeptides having
homology to a stem cell antigen, wherein those polypeptides are
herein designated as PRO232 polypeptides.
[0026] 4. PRO187
[0027] Growth factors are molecular signals or mediators that
enhance cell growth or proliferation, alone or in concert, by
binding to specific cell surface receptors. However, there are
other cellular reactions than only growth upon expression to growth
factors. As a result, growth factors are better characterized as
multifunctional and potent cellular regulators. Their biological
effects include proliferation, chemotaxis and stimulation of
extracellular matrix production. Growth factors can have both
stimulatory and inhibitory effects. For example, transforming
growth factor (TGF-.beta.) is highly pleiotropic and can stimulate
proliferation in some cells, especially connective tissue, while
being a potent inhibitor of proliferation in others, such as
lymphocytes and epithelial cells.
[0028] The physiological effect of growth stimulation or inhibition
by growth factors depends upon the state of development and
differentiation of the target tissue. The mechanism of local
cellular regulation by classical endocrine molecules involves
comprehends autocrine (same cell), juxtacrine (neighbor cell), and
paracrine (adjacent cells) pathways. Peptide growth factors are
elements of a complex biological language, providing the basis for
intercellular communication. They permit cells to convey
information between each other, mediate interaction between cells
and change gene expression. The effect of these multifunctional and
pluripotent flictors is dependent on the presence or absence of
other peptides.
[0029] FGF-8 is a member of the fibroblast growth factors (FGFs)
which are a family of heparin-binding, potent mitogens for both
normal diploid fibroblasts and established cell lines,
Gospodarowicz et al. (1984), Proc. Natt Acad Sci. USA 81:6963. The
FGF family comprises acidic FGF (FGF-1), basic FGF (FGF-2), INT-2
(FGF-3), K-FGF/HST (FGF4), FGF-5, FGF-6, KGF (FGF-7), AIGF (FGF-8)
among others. All FGFs have two conserved cysteine residues and
share 30-50% sequence homology at the amino acid level. These
factors are mitogenic for a wide variety of normal diploid
mesoderm-derived and neural crest-derived cells, including
granulosa cells, adrenal cortical cells, chondrocytes, myoblasts,
corneal and vascular endothelial cells (bovine or human), vascular
smooth muscle cells, lens, retina and prostatic epithelial cells,
oligodendrocytes, astrocytes, chrondocytes, myoblasts and
osteoblasts.
[0030] Fibroblast growth factors can also stimulate a large number
of cell types in a non-mitogenic manner. These activities include
promotion of cell migration into wound area (chemotaxis),
initiation of new blood vessel formulation (angiogenesis),
modulation of nerve regeneration and survival (neurotrophism),
modulation of endocrine functions, and stimulation or suppression
of specific cellular protein expression, extracellular matrix
production and cell survival. Baird & Bohlen, Handbook of Exp.
Pharmacol. 95(1): 369-418, Springer, (1990). These properties
provide a basis for using fibroblast growth factors in therapeutic
approaches to accelerate wound healing, nerve repair, collateral
blood vessel formation, and the like. For example, fibroblast
growth factors have been suggested to minimize myocardium damage in
heart disease and surgery (U.S. Pat. No. 4,378,347).
[0031] FGF-8, also known as androgen-induced growth factor (AIGF),
is a 215 amino acid protein which shares 30-40% sequence homology
with the other members of the FGF family. FGF-8 has been proposed
to be under androgenic regulation and induction in the mouse
mammary carcinoma cell line SCI Tanaka et al., Proc. Nad. Acad.
Sci. USA 89: 8928-8932 (1992); Sato et al., J. Steroid Biochem.
Molec. Biol. 47: 91-98 (1993). As a result, FGF-8 may have a local
role in the prostate, which is known to be an androgen-responsive
organ. FGF-8 can also be oncogenic, as it displays transforming
activity when transfected into NIA-373 fibroblasts. Kouhara et al.,
Oncogene 9 455-462 (1994). While FGF-8 has been detected in heart,
brain, lung, kidney, testis, prostate and ovary, expression was
also detected in the absence of exogenous androgens. Schmitt et
al., J. Steroid Biochem. Mot Biol. 57 (3-4): 173-78 (1996).
[0032] FGF-8 shares the property with several other FGFs of being
expressed at a variety of stages of murine embryogenesis, which
supports the theory that the various FGFs have multiple and perhaps
coordinated-roles in differentiation and embryogenesis. Moreover,
FGF-8 has also been identified as a protooncogene that cooperates
with Wnt-1 in the process of mammary tumorigenesis (Shackleford et
al, Proc. Nati. Acad. Sci. USA 90 740-744 (1993); Heikinheimo et
al, Mech. Dev. 48: 129-138 (1994)).
[0033] In contrast to the other FGFs, FGF-8 exists as three protein
isoforms, as a result of alternative splicing of the primary
transcript. Tanaka et al., supra. Normal adult expression of FGF-8
is weak and confined to gonadal tissue, however northern blot
analysis has indicated that FGF-8 mRNA is present from day 10
through day 12 or murine gestation, which suggests that FGF-8 is
important to normal development. Heikinheimo et al., Mech Dev.
48(2): 129-38 (1994). Further in situ hybridization assays between
day 8 and 16 of gestation indicated initial expression in the
surface ectoderm of the first bronchial arches, the frontonasal
process, the forebrain and the midbrain-hindbrain junction. At days
10-12, FGF-8 was expressed in the surface ectoderm of the forelimb
and hindlimb buds, the nasal its and nasopharynx, the infundibulum
and in the telencephalon, diencephalon and metencephalon.
Expression continues in the developing hindlimbs through day 13 of
gestation, but is undetectable thereafter. The results suggest that
FGF-8 has a unique temporal and spatial pattern in embryogenesis
and suggests a role for this growth factor in multiple regions of
ectodermal differentiation in the post-gastrulation embryo.
[0034] We herein describe the identification of novel poypeptides
having homology to FGF-8, wherein those polypeptides are heein
designated PRO187 polypeptides.
[0035] 5. PRO265
[0036] Protein-protein interactions include receptor and antigen
complexes and signaling mechanisms. As more is known about the
structural and functional mechanisms underlying protein-protein
interactions, protein-protein interactions can be more easily
manipulated to regulate the particular result of the
protein-protein interaction. Thus, the underlying mechanisms of
protein-protein interactions are of interest to the scientific and
medical community.
[0037] All proteins containing leucine-rich repeats are thought to
be involved in protein-protein interactions. Leucine-rich repeats
are short sequence motifs present in a number of proteins with
diverse functions and cellular locations. The crystal structure of
ribonuclease inhibitor protein has revealed that leucine-rich
repeats correspond to beta-alpha structural units. These units are
arranged so that they form a parallel beta-sheet with one surface
exposed to solvent, so that the protein acquires an unusual,
nonglubular shape. These two features have been indicated as
responsible for the protein-binding functions of proteins
containing leucine-rich repeats. See, Kobe and Deisenhofer, Trends
Biochem. Sci. 19(10):415-421 (October 1994).
[0038] A study has been reported on leucine-rich proteoglycans
which serve as tissue organizers, orienting and ordering collagen
fibrils during ontogeny and are involved in pathological processes
such as wound healing, tissue repair, and tumor stroma formation.
Iozzo, R. V., Crit. Rev. Biochem. Mol. Biol., 32(2): 141-174
(1997). Others studies implicating leucine rich proteins in wound
healing and tissue repair are De La Salle, C., et al., Vouv. Rev.
Fr. Hernatol. (Germany), 37(4):215-222 (1995), reporting mutations
in the leucine rich motif in a complex associated with the bleeding
disorder Bemard-Soulier syndrome and Chlemetson, K. J., Thromb.
Haemost. (Germany), 74(1): 111-116 (July 1995), reporting that
platelets have leucine rich repeats. Another protein of particular
interest which has been reported to have leucine-rich repeats is
the SLIT protein which has been reported to be useful in treating
neuro-degenerative diseases such as Alzheimer's disease, nerve
damage such as in Parkinson's disease, and for diagnosis of cancer,
see, Artavanistsakonas, S. and Rothberg, J. M., WO9210518-A1 by
Yale University. Other studies reporting on the biological
functions of proteins having leucine-rich repeats include: Tayar,
N., et al., Mol. Cell Endocrinol., (Ireland), 125(1-2):65-70
(December 1996) (gonadotropin receptor involvement); Miura, Y., et
al., Nippon Rinsho (Japan), 54(7): 1784-1789 (July 1996) (apoptosis
involvement); Harris, P. C., et al., J. Am. Soc. Nephrol., 6(4):
1125-1133 (October 1995) (kidney disease involvement); and
Ruoslahti, E. I., et al., W09110727-A by La Jolla Cancer Research
Foundation (decorin binding to transforming growth factor-.beta.
involvement for treatment for cancer, wound healing and scarring).
Also of particular interest is fibromodulin and its use to prevent
or reduce dermal scarring. A study of fibromodulin is found in U.S.
Pat. No. 5,654,270 to Ruoslahti, et al.
[0039] Efforts are therefore being undertaken by both industry and
academia to identify new proteins having leucine rich repeats to
better understand protein-protein interactions. Of particular
interest are those proteins having leucine rich repeats and
homology to known proteins having leucine rich repeats such as
fibromodulin, the SLIT protein and platelet glycoprotein V. Many
efforts are focused on the screening of mammalian recombinant DNA
libraries to identify the coding sequences for novel secreted and
membrane-bound proteins having leucine rich repeats. We herein
describe the identification and characterization of novel
polypeptides having homology to fibromodulin, herein designated as
PRO265 polypeptides.
[0040] 6. PRO219
[0041] Human matrilin-2 polypeptide is a member of the von
Willebrand factor type A-like module superfamily. von Willebrand
factor is a protein which plays an important role in the
maintenence of hemostasis. More specifically, von Willebrand factor
is a protein which is known to participate in platelet-vessel wall
interactions at the site of vascular injury via its ability to
interact and form a complex with Factor VIII. The absence of von
Willebrand factor in the blood causes an abnormality with the blood
platelets that prevents platelet adhesion to the vascular wall at
the site of the vascular injury. The result is the propensity for
brusing, nose bleeds, intestinal bleeding, and the like comprising
von Willebrand's disease.
[0042] Given the physiological importance of the blood clotting
factors, efforts are currently being undertaken by both industry
and academia to identify new, native proteins which may be involved
in the coagulation process. We herein describe the identification
of a novel full-length polypeptide which possesses homology to the
human matrilin-2 precursor polypeptide.
[0043] 7. PRO246
[0044] 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.
[0045] In light of the physiological importance of membrane-bound
proteins and specifically those which serve a cell surface receptor
for viruses, efforts are currently being undertaken by both
industry and academia to identify new, native membrane-bound
receptor proteins. Many of these efforts are focused on the
screening of mammalian recombinant DNA libraries to identify the
coding sequences for novel receptor proteins. We herein describe a
novel membrane-bound polypeptide (designated herein as PRO246)
having homology to the cell surface protein HCAR and to various
tumor antigens including A33 and carcinoembryonic antigen, wherein
this polypeptide may be a novel cell surface virus receptor or
tumor antigen.
[0046] 8. PRO228
[0047] There are a number of known seven transmembrane proteins and
within this family is a group which includes CD97 and EMRL CD97 is
a seven-span transmembrane receptor which has a cellular ligand,
CD55, DAF. Hamann, et al., J. Exp. Med. (U.S.), 184(3): 1189
(1996). Additionally, CD97 has been reported as being a
dedifferentiation marker in human thyroid carcinomas and as
associated with inflammation. Aust, et al., Cancer Res. (U.S.),
57(9): 1798 (1997); Gray, et al., J. Immunol. (U.S.), 157(12):5438
(1996). CD97 has also been reported as being related to the
secretin receptor superfamily, but unlike known members of that
family, CD97 and EMR1 have extended extracellular regions that
possess several EGF domains at the N-terminus. Hamann, et al.,
Genomics 32(1):144 (1996); Harmann, et al., J. Immunol. 155(4):1942
(1995). EMRI is further described in Lin, et al., Genomics, 41(3):
301 (1997) and Baud, et al., Genomics 26(2):334 (1995). While CD97
and EMRI appear to be related to the secretin receptors, a known
member of the secretin family of G protein-coupled receptors
includes the alpha-latroxin receptor, latrophilin, which has been
described as calcium independent and abundant among neuronal
tissues. Lelianova, et al., J. Biol. Chem. 272(34), 21504 (1997);
Davletov, et al., J. Biol. Chem. (U.S.), 271(38):23239(1996). Both
members of the secretin receptor superfamily and non-members which
are related to the secretin receptor superfamily, or CRF and
calcitonin receptors are of interest. In particular, new members of
these families, identified by their homology to known proteins, are
of interest.
[0048] Efforts are being undertaken by both industry and academia
to identify new membrane-bound receptor proteins, particularly
transmembrane proteins with EGF repeats and large N-terminuses
which may belong to the family of seven-transmembrane proteins of
which CD97 and EMR1 are members. We herein describe the
identification and charactization of novel polypeptides having
homology to CD97 and EMR1, designated herein as PRO228
polypeptides.
[0049] 9. PRO533
[0050] Growth factors are molecular signals or mediators that
enhance cell growth or proliferation, alone or in concert, by
binding to specific cell surface receptors. However, there are
other cellular reactions than only growth upon expression to growth
factors. As a result, growth factors are better characterized as
multifunctional and potent cellular regulators. Their biological
effects include proliferation, chemotaxis and stimulation of
extracellular matrix production. Growth factors can have both
stimulatory and inhibitory effects. For example, transforming
growth factors (TGF-.beta.) is highly pleiotropic and can stimulate
proliferation in some cells, especially connective tissues, while
being a potent inhibitor of proliferation in others, such as
lymphocytes and epithelial cells.
[0051] The physiological effect of growth stimulation or inhibition
by growth factors depends upon the state of development and
differentiation of the target tissue. The mechanism of local
cellular regulation by classical endocrine molecules comprehends
autocrine (same cell), juxtacrine (neighbor cell), and paracrine
(adjacent cell) pathways. Peptide growth factors are elements of a
complex biological language, providing the basis for intercellular
communication. They permit cells to convey information between each
other, mediate interaction between cells and change gene
expression. The effect of these multifunctional and pluripotent
factors is dependent on the presence or absence of other
peptides.
[0052] Fibroblast growth factors (FGFs) are a family of
heparin-binding, potent mitogens for both normal diploid
fibroblasts and established cell lines, Godpodarowicz, D. et al.
(1984), Proc. Natl. Acad. Sci. USA 81: 6983. The FGF family
comprises acidic FGF (FGF-1), basic FGF (FGF-2), INT-2 (FGF-3),
K-FGF/HST (FGF4), FGF-5, FGF-:6, KGF (FGF-7), AIGF (FGF-8) among
others. All FGFs have two conserved cysteine residues and share
30-50% sequence homology at the amino acid level. These factors are
mitogenic for a wide variety of normal diploid mesoderm-derived and
neural crest-derived cells, inducing granulosa cells, adrenal
cortical cells, chrondocytes, myoblasts, corneal and vascular
endothelial cells (bovine or human), vascular smooth muscle cells,
lens, retina and prostatic epithelial cells, oligodendrocytes,
astrocytes, chrondocytes, myoblasts and osteoblasts.
[0053] Fibroblast growth factors can also stimulate a large number
of cell types in a non-mitogenic manner. These activities include
promotion of cell migration into a wound area (chemotaxis),
initiation of new blood vessel formulation (angiogenesis),
modulation of nerve regeneration and survival (neurotrophism),
modulation of endocrine functions, and stimulation or suppression
of specific cellular protein expression, extracellular matrix
production and cell survival. Baird, A. & Bohlen, P., Handbook
of Exp. Phrmacol. 95(1): 369-418 (1990). These properties provide a
basis for using fibroblast growth factors in therapeutic approaches
to accelerate wound healing, nerve repair, collateral blood vessel
formation, and the like. For example, fibroblast growth factors,
have been, suggested to minimize myocardium damage in heart disease
and surgery (U.S. Pat. No. 4,378,437).
[0054] We herein describe the identification and characterization
of novel polypeptides having homology to FGF, herein designated
PRO533 polypeptides.
[0055] 10. PRO245
[0056] Some of the most important proteins involved in the above
described regulation and modulation of cellular processes are the
enzymes which regulate levels of protein phosphorylation in the
cell. For example, it is known that the transduction of signals
that regulate cell growth and differentiation is regulated at least
in part by phosphorylation and dephosphorylation of various
cellular proteins. The enzymes that catalyze these processes
include the protein kinases, which function to phosphorylate
various cellular proteins, and the protein phosphatases, which
function to remove phosphate residues from various cellular
proteins. The balance of the level of protein phosphorylation in
the cell is thus mediated by the relative activities of these two
types of enzymes.
[0057] Although many protein kinase enzymes have been identified,
the physiological role played by many of these catalytic proteins
has yet to be elucidated. It is well known, however, that a number
of the known protein kinases function to phosphorylate tyrosine
residues in proteins, thereby leading to a variety of different
effects. Perhaps most importantly, there has been a great deal of
interest in the protein tyrosine kinases since the discovery that
many oncogene products and growth factors possess intrinsic protein
tyrosine kinase activity. There is, therefore, a desire to identify
new members of the protein tyrosine kinase family.
[0058] Given the physiological importance of the protein kinases,
efforts are being undertaken by both industry and academia to
identify new, native kinase proteins. Many of these efforts are
focused on the screening of mammalian recombinant DNA libraries to
identify the coding sequences for novel kinase proteins. We herein
describe the identification and characterization of novel
polypeptides having homology to tyrosine kinase proteins,
designated herein as PRO245 polypeptides.
[0059] 11. PRO220, PRO221 and PRO227
[0060] Protein-protein interactions include receptor and antigen
complexes and signaling mechanisms. As more is known about the
structural and functional mechanisms underlying protein-protein
interactions, protein-protein interactions can be more easily
manipulated to regulate the particular result of the
protein-protein interaction. Thus, the underlying mechanisms of
protein-protein interactions are of interest to the scientific and
medical community.
[0061] All proteins containing leucine-rich repeats are thought to
be involved in protein-protein interactions. Leucine-rich repeats
are short sequence motifs present in a number of proteins with
diverse functions, and cellular locations. The crystal structure of
ribonuclease inhibitor protein has revealed that leucine-rich
repeats correspond to beta-alpha structural units. These units are
arranged so that they form a parallel beta-sheet with one surface
exposed to solvent, so that the protein acquires an unusual,
nonglubular shape. These two features have been indicated as
responsible for the protein-binding functions of proteins
containing leucine-rich repeats. See, Kobe and Deisenhofer, Trends
Biochem. Sci., 19(10):415-421 (October 1994).
[0062] A study has been reported on leucine-rich proteoglycans
which serve as tissue organizers, orienting and ordering collagen
fibrils during ontogeny and are involved in pathological processes
such as wound healing, tissue repair, and tumor stroma formation.
Iozzo, R. V., Crit. Rev. Biochem. Mol. Biol., 32(2):141-174 (1997).
Others studies implicating leucine rich proteins in wound healing
and tissue repair are De La Salle, C., et al., Vouv. Rev. Fr.
Hematol. (Germany), 37(4):215-222 (1995), reporting mutations in
the leucine rich motif in a complex associated with the bleeding
disorder Bemard-Soulier syndrome and Chlemetson, K. J., Thromb.
Haemost. (Germany), 74(1): 111-116 (July 1995), reporting that
platelets have leucine rich repeats. Another protein of particular
interest which has been reported to have leucine-rich repeats is
the SLIT protein which has been reported to be useful in treating
neuro-degenerative diseases such as Alzheimer's disease, nerve
damage such as in Parkinson's disease, and for diagnosis of cancer,
see, Artavanistsakonas, S. and Rothberg, J. M., W09210518-A1 by
Yale University. Other studies reporting on the biological
functions of proteins having leucine-rich repeats include: Tayar,
N., et al., Mol. Cell Endocrinol., (Ireland), 125(1-2):65-70
(December 1996) (gonadotropin receptor involvement); Miura, Y., et
al., Nippon Rinsho (Japan), 54(7):1784-1789 (July 1996) (apoptosis
involvement); Harris, P. C., et al., J. Am. Soc. Nephrol.,
6(4):1125-1133 (October 1995) (kidney disease involvement); and
Ruoslahti, E. I., et al., W09110727-A by La Jolla Cancer Research
Foundation (decorin binding to transforming growth factory
involvement for treatment for cancer, wound healing and
scarring).
[0063] Efforts are therefore being undertaken by both industry and
academia to identify new proteins having leucine rich repeats to
better understand protein-protein interactions. Of particular
interest are those proteins having leucine rich repeats and
homology to known proteins having leucine rich repeats such as the
SLIT protein and platelet glycoprotein V.
[0064] 12. PRO258
[0065] Immunoglobulins are antibody molecules, the proteins that
function both as receptors for antigen on the B-cell membrane and
as the secreted products of the plasma cell. Like all antibody
molecules, immunoglobulins perform two major functions: they bind
specifically to an antigen and they participate in a limited number
of biological effector functions. Therefore, new members of the Ig
superfamily are always of interest. Molecules which act as
receptors by various viruses and those which act to regulate immune
function are of particular interest. Also of particular interest
are those molecules which have homology to known Ig family members
which act as virus receptors or regulate immune function. Thus,
molecules having homology to poliovirus receptors, CRTAM and CD166
(a ligand for lymphocyte antigen CD6) are of particular
interest.
[0066] 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.
[0067] We herein describe the identification and characterization
of novel polypeptides having homology to CRTAM, designated herein
as PRO258 polypeptides.
[0068] 13. PRO266
[0069] Protein-protein interactions include receptor and antigen
complexes-and signaling mechanisms. As more is known about the
structural and functional mechanisms underlying protein-protein
interactions, protein-protein interactions can be more easily
manipulated to regulate the particular result of the
protein-protein interaction. Thus, the underlying mechanisms of
protein-protein interactions are of interest to the scientific and
medical community.
[0070] 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).
[0071] A study has been reported on leucine-rich proteoglycans
which serve as tissue organizers, orienting and ordering collagen
fibrils during ontogeny and are involved in pathological processes
such as wound healing, tissue repair, and tumor stroma formation.
Iozzo, R. V., Crit. Rev. Biochem. Mol. Biol., 32(2):141-174 (1997).
Others studies implicating leucine rich proteins in wound healing
and tissue repair are De La Salle, C., et al., Vouv. Rev. Fr.
Hematol. (Germany), 37(4):215-222 (1995), reporting mutations in
the leucine rich motif in a complex associated with the bleeding
disorder Bernard-Soulier syndrome and Chlemetson, K. J., Thromb.
Haemost. (Germany), 74(1):111-116 (July 1995), reporting that
platelets have leucine rich repeats. Another protein of particular
interest which has been reported to have leucine-rich repeats is
the SLIT protein which has been reported to be useful in treating
neuro-degenerative diseases such as Alzheimer's disease, nerve
damage such as in Parkinson's disease, and for diagnosis of cancer,
see, Artavanistsakonas, S. and Rothberg, J. M., W09210518-A1 by
Yale University. Other studies reporting on the biological
functions of proteins having leucine-rich repeats include: Tayar,
N., et al., Mol. Cell Endocrinol., (Ireland), 125(1-2):65-70
(December 1996) (gonadotropin receptor involvement); Miura, Y., et
al., Nippon Rinsho (Japan), 54(7): 1784-1789 (July 1996) (apoptosis
involvement); Harris, P. C., et al., J. Am. Soc. Nephrol.,
6(4):1125-1133 (October 1995) (kidney disease involvement); and
Ruoslahti, E. I., et al., W09110727-A by La Jolla Cancer Research
Foundation (decorin binding to transforming growth factors
involvement for treatment for cancer, wound healing and
scarring).
[0072] Efforts are therefore being undertaken by both industry and
academia to identify new proteins having leucine rich repeats to
better understand protein-protein interactions, neuronal
development and adhesin molecules. Of particular interest are those
proteins having leucine rich repeats and homology to known proteins
having leucine rich repeats such as the SLIT protein. We herein
describe novel polypeptides having homology to SLIT, designated
herein as PRO266 polypeptides.
[0073] 14. PRO269
[0074] Thrombomodulin binds to and regulates the activity of
thrombin. It is important in the control of blood coagulation.
Thrombomodulin functions as a natural anticoagulant by accelerating
the activation of protein C by thrombin. Soluble thrombomodulin may
have therapeutic use as an antithrombotic agent with reduced risk
for hemorrhage as compared with heparin. Thrombomodulin is a cell
surface trans-membrane glycoprotein, present on endothelial cells
and platelets. A smaller, functionally active form of
thrombomodulin circulates in the plasma and is also found in urine.
(In Haeberli, A., Human Protein Data, VCH Oub., N.Y., 1992).
Peptides having homology to thrombomodulin are particularly
desirable.
[0075] We herein describe the identification and characterization
of novel polypeptides having homology to thrombomodulin, designated
herein as PRO269 polypeptides.
[0076] 15. PRO287
[0077] Procollagen C-proteinase enhancer protein binds to and
enhances the activity of bone morphogenic protein
"BMP1"/procollagen C-proteinase (PCP). It plays a role in
extracellular matrix deposition. BMP1 proteins may be used to
induce bone and/or cartilage formation and in wound healing and
tissue repair. Therefore, procollagen C-proteinase enhancer
protein, BMP1 and proteins having homology thereto, are of interest
to the scientific and medical communities.
[0078] We herein describe the identification and characterization
of novel polypeptides having homology to procollagen C-proteinase
enhancer protein precursor and procollagen C-proteinase enhancer
protein, designated herein as PRO287 polypeptides.
[0079] 16. PRO214
[0080] Growth factors are molecular signals or mediators that
enhances cell growth or proliferation, alone or in concert, by
binding to specific cell surface receptors. However, there are
other cellular reactions than only growth upon expression to growth
factors. As a result, growth factors are better characterized as
multifunctional and potent cellular regulators. Their biological
effects include proliferation, chemotaxis and stimulation of
extracellular matrix production. Growth factors can have both
stimulatory and inhibitory effects. For example, transforming
growth factor .beta. (TGF-.beta.) is highly pleiotropic and can
stimulate proliferation in some cells, especially connective
tissue, while being a potent inhibitor of proliferation in others,
such as lymphocytes and epithelial cells.
[0081] The physiological effect of growth stimulation or inhibition
by growth factors depends upon the state of development and
differentiation of the target tissue. The mechanism of local
cellular regulation by classical endocrine molecules involves
comprehends autocrine (same cell), juxtacrine (neighbor cell), and
paracrine (adjacent cells) pathways. Peptide growth factors are
elements of a complex biological language, providing the basis for
intercellular communication. They permit cells to convey
information between each other, mediate interaction between cells
and change gene expression. The effect of these multifunctional and
pluripotent factors is dependent on the presence or absence of
other peptides.
[0082] Epidermal growth factor (EGF) is a conventional mitogenic
factor that stimulates the proliferation of various types of cells
including epithelial cells and fibroblasts. EGF binds to and
activates the EGF receptor (EGFR), which initiates intracellular
signaling and subsequent effects. The EGFR is expressed in neurons
of the cerebral cortex, cerebellum, and hippocampus in addition to
other regions of the central nervous system (CNS). In addition, EGF
is also expressed in various regions of the CNS. Therefore, EGF
acts not only on mitotic cells, but also on postmitotic neurons. In
fact, many studies have indicated that EGF has neurotrophic or
neuromodulatory effects on various types of neurons in the CNS. For
example, EGF acts directly on cultured cerebral cortical and
cerebellar neurons, enhancing neurite outgrowth and survival. On
the other hand, EGF also acts on other cell types, including septal
cholinergic and mesencephalic dopaminergic neurons, indirectly
through glial cells. Evidence of the effects of EGF on neurons in
the CNS is accumulating, but the mechanisms of action remain
essentially unknown. EGF-induced signaling in mitotic cells is
better understood than in postmitotic neurons. Studies of cloned
pheochromocytoma PC12 cells and cultured cerebral cortical neurons
have suggested that the EGF-induced neurotrophic actions are
mediated by sustained activation of the EGFR and mitogen-activated
protein kinase (MAPK) in response to EGF. The sustained
intracellular signaling correlates with the decreased rate of EGFR
down-regulation, which might determine the response of neuronal
cells to EGF. It is likely that EGF is a multi-potent growth factor
that acts upon various types of cells including mitotic cells and
postmitotic neurons.
[0083] EGF is produced by the salivary and Brunnees glands of the
gastrointestinal system, kidney, pancreas, thyroid gland, pituitary
gland, and the nervous system, and is found in body fluids such as
saliva, blood, cerebrospinal fluid (CSF), urine, amniotic fluid,
prostatic fluid, pancreatic juice, and breast milk, Plata-Salaman,
C R Peptides 12: 653-663 (1991).
[0084] EGF is mediated by its membrane specific receptor, which
contains an intrinsic tyrosine kinase. Stoscheck C M et al, J Cell
Biochem. 31: 135-152 (1986). EGF is believed to function by binding
to the extraceflular portion of its receptor which induces a
transmembrane signal that activates the intrinsic tyrosine
kinase.
[0085] Purification and sequence analysis of the EGF-like domain
has revealed the presence of six conserved cysteine residues which
cross-bind to create three peptide loops, Savage C R et al, J Biol.
Chem. 248: 7669-7672 (1979). It is now generally known that several
other peptides can react with the EGF receptor which share the same
generalized motif
X.sub.nCX.sub.7CX.sub.4/5CX.sub.10CXCX.sub.5GX.sub.2CX.sub.n, where
X represents any non-cysteine amino acid, and n is a variable
repeat number. Non isolated peptides having this motif include
TGF-a, amphiregulin, schwannoma-derived growth factor (SDGF),
heparin-binding EGF-like growth factors and certain virally encoded
peptides (e.g., Vaccinia virus, Reisner A H, Nature 313: 801-803
(1985), Shope fibroma virus, Chang W., et al., Mol Cell Biol. 7:
535-540 (1987), Molluscum contagiosurn, Porter C D & Archard L
C, J Gen. Virol. 68: 673-682(1987), and Myxoma virus, Upton C et
al., J Virol. 61: 1271-1275 (1987). Prigent S A & Lemoine N.
R., Prog Growth Factor Res. 4: 1-24 (1992).
[0086] EGF-like domains are not confined to growth factors but have
been observed in a variety of cell-surface and extracellular
protein s which have interesting properties in cell adhesion,
protein-protein interaction and development, Laurence D J R &
Gusterson B A, Tumor Biol. 11: 229-261 (1990). These proteins
include blood coagulation factors (factors VI, IX, X, XII, protein
C, protein S, protein Z, tissue plasminogen activator, urokinase),
extracellular matrix components (laminin, cytotactin, entactin),
cell surface receptors (LDL receptor, thrombomodulin receptor) and
immunity-related proteins (complement Clr, uromodulin).
[0087] Even more interesting, the general structure pattern of
EGF-like precursors is preserved through lower organisms as well as
in mammalian cells. A number of genes with developmental
significance have been identified in invertebrates with EGF-like
repeats. For example, the notch gene of Drosophila encodes 36
tandemly arranged 40 amino acid repeats which show homology to EGF,
Wharton W et al., Cell 43: 557-581 (1985). Hydropathy plots
indicate a putative membrane spanning domain, with the EGF-related
sequences being located on the extracellular side of the membrane.
Other homeotic genes with EGF-like repeats include Delta, 95F and
5ZD which were identified using probes based on Notch, and the
nematode gene Lin-12 which encodes a putative receptor for a
developmental signal transmitted between two specified cells.
[0088] Specifically, EGF has been shown to have potential in the
preservation and maintenance of gastrointestinal mucosa and the
repair of acute and chronic mucosal lesions, Konturek, P C et al.,
Eur. J. Gastroenterol Hepatol. 7(10), 933-37 (1995), including the
treatment of necrotizing enterocolitis, Zollinger-Ellison syndrome,
gastrointestinal ulceration gastrointestinal ulcerations and
congenital microvillus atrophy, A. Guglietta & P B Sullivan,
Eur. J Gastroenterol Hepatol, 7(10), 945-50 (1995). Additionally,
EGF has been implicated in hair follicle differentiation; C. L. du
Cros, J. Invest. Dermatol. 101(1 Suppl.), 106S-113 S (1993), S G
Hillier, Clin. Endocrinol. 33(4), 427-28 (1990); kidney function,
L. L. Hamm et al., Semin. Nephrol. 13(1): 109-15 (1993), R C
Harris, Am. J. Kidney Dis. 17(6): 627-30 (1991); tear fluid, G B
van Setten et al., Int. Ophthalmol 15(6); 359-62 (1991); vitamin K
mediated blood coagulation, J. Stenflo et al., Blood 78(7): 1637-51
(1991). EGF is also implicated various skin disease characterized
by abnormal keratinocyte differentiation, e.g., psoriasis,
epithelial cancers such as squamous cell carcinomas of the lung,
epidermoid carcinoma of the vulva and gliomas. King, L E et al.,
Am. J Med. Sci. 296:154-158(1988).
[0089] Of great interest is mounting evidence that genetic
alterations in growth factors signaling pathways are closely linked
to developmental abnormalities and to chronic diseases including
cancer. Aaronson S A, Science 254: 1146-1153 (1991). For example,
c-erb-2 (also known as HER-2), a proto-oncogene with close
structural similarity to EGF receptor protein, is overexpressed in
human breast cancer. King et al., Science 229: 974-976 (1985);
Gullick, W J, Hormones and their actions, Cooke B A et al., eds,
Amsterdam, Elsevier, pp 349-360 (1986).
[0090] 17. PRO317
[0091] The TGF-.beta. supergene family, or simply TGF-.beta.
superfamily, a group of secreted proteins, includes a large number
of related growth and differentiation factors expressed in
virtually all phyla. Superfamily members bind to specific cell
surface receptors that activate signal transduction mechanisms to
elicit their multifunctional cytokine effects. Kolodziejczyk and
Hall, Biochem. Cell. Biol. 74: 299-314 (1996); Attisano and Wrana,
Cytokine Growth Factor Rev., 7: 327-339 (1996); and Hill, Cellular
Signaling, 8: 533-544 (1996). Members of this family include five
distinct forms of TGF-.beta. (Sporn and Roberts, in Peptide Growth
Factors and Their Receptors, Sporn and Roberts, eds.
(Springer-Verlag: Berlin, 1990) pp. 419-472), as well as the
differentiation factors vgl (Weeks and Melton, Cell, 51: 861-867
(1987)) and DPP-C polypeptide (Padgett et al., Nature, 325: 81-84
(1987)), the hormones activin and inhibin (Mason et al., Nature
318: 659-663 (1985); Mason et al., Growth Factors, 1: 77-88
(1987)), the Mullerian-inhibiting substance (MIS) (Cate et al.,
Cell, 45: 685-698 (1986)), the bone morphogenetic proteins (BMPS)
(Wozney et al., Science, 242: 1528-1534 (1988); PCT WO 88/00205
published Jan. 14, 1988; U.S. Pat. No. 4,877,864 issued Oct. 31,
1989), the developmentally regulated proteins Vgr-1 (Lyons et al.,
Proc. Natl. Acad. Sci. USA. 86: 4554-4558 (1989)) and Vgr-2 (Jones
et al., Molec. Endocrinol., 6: 1961-1968 (1992)), the mouse growth
differentiation factor (GDF), such as GDF-3 and GDF-9 (Kingsley,
Genes Dev., 8: 133-146 (1994); McPherron and Lee, J. Biol. Chem.,
268:3444-3449(1993)), the mouse lefty/Stral (Meno et al., Nature
381: 151-155 (1996); Bouillet et al., Dev. Biol., 170:420-433
(1995)), glial cell line-derived neurotrophic factor (GDNF) (Lin et
al., Science 260: 1130-1132 (1993), neurturin (Kotzbaueretal.,
Nature 384:467-470 (1996)), and endometrial bleeding-associated
factor (EBAF) (Kothapalli et a., J. Clin. Invest., 99: 2342-2350
(1997)). The subset BMP-2A and BMP-2B is approximately 75%
homologous in sequence to DPP-C and may represent the mammalian
equivalent of that protein.
[0092] The proteins of the TGF-.beta. superfamily are
disulfide-linked homo- or heterodimers encoded by larger precursor
polypeptide chains containing a hydrophobic signal sequence, a long
and relatively poorly conserved N-terminal pro region of several
hundred amino acids, a cleavage site (usually polybasic), and a
shorter and more highly conserved C-terminal region. This
C-terminal region corresponds to the processed mature protein and
contains approximately 100 amino acids with a characteristic
cysteine motif, ie., the conservation of seven of the nine cysteine
residues of TGF-.beta. among all known family members. Although the
position of the cleavage site between the mature and pro regions
varies among the family members, the C-terminus of all of the
proteins is in the identical position, ending in the sequence
Cys-X-Cys-X, but differing in every case from the TGF-.beta.
consensus C-terminus of Cys-Lys-Cys-Ser. Sporn and Roberts, 1990,
supra.
[0093] There are at least five forms of TGF-.beta. currently
identified, TGF-.beta.1, TGF-.beta.2, TGF-.beta.3, TGF-.beta.4, and
TGF-.beta.5. The activated form of TGF-.beta.1 is a homodimer
formed by dimerization of the carboxy-terminal 112 amino acids of a
390 amino acid precursor. Recombinant TGF-.beta.1 has been cloned
(Derynck et al., Nature, 316:701-705 (1985)) and expressed in
Chinese hamster ovary cells (Gentry et al., Mol. Cell. Biol. 7:
3418-3427 (1987)). Additionally, recombinant human TGF-02 (deMartin
et al, EMBO J., 6: 3673 (1987)), as well as human and porcine
TGF-.beta.3 (DeryncketaL, EMBO J., 7: 3737-3743 (1988); ten Dijke
et al., Proc. Natl. Acad. Sci. USA, 85:4715 (1988)) have been
cloned. TGF-.beta.2 has a precursor form of 414 amino acids and is
also processed to a homodimer from the carboxy-terminal 112 amino
acids that shares approximately 70% homology with the active form
of TGF-.beta.1 (Marquardt et al., J. Biol. Chem. 262: 12127
(1987)). See also EP 200,341; 169,016; 268,561; and 267,463; U.S.
Pat. No. 4,774,322; Cheifetz et al., Cell 48: 409-415 (1987);
Jakowlew et al., Molecular Endocrin., 2: 747-755 (1988); Derynck et
al., J. Biol. Chem., 261: 4377-4379 (1986); Sharpies et al, DNA,
6:239-244 (1987); Derynck et al., Nucl. Acids. Res., 15: 3188-3189
(1987); Derynck et al., Nucl. Acids. Res., 15: 3187 (1987); Seyedin
et al., J. Biol. Chem., 261: 5693-5695 (1986); Madisen et al., DNA,
7: 1-8 (1988); and Hanks et al., Proc. Nati. Acad. Sci. (U.S.A.)
85: 79-82 (1988).
[0094] TGF-.beta.4 and TGF-.beta.5 were cloned from a chicken
chondrocyte cDNA library (Jakowlew et al., Molec. Endocrinol. 2:
1186-1195 (1988)) and from a frog oocyte cDNA library,
respectively.
[0095] The pro region of TGF-.beta. associates non-covalently with
the mature TGF-.beta. dimer (Wakefield et al., J. Biol. Chem. 263:
7646-7654 (1988); Wakefield et al., Growth Factors, 1: 203-218
(1989)), and the pro regions are found to be necessary for proper
folding and secretion of the active mature dimers of both
TGF-.beta. and activin (Gray and Mason, Science, 247: 1328-1330
(1990)). The association between the mature and pro regions of
TGF-.beta. masks the biological activity of the mature dimer,
resulting in formation of an inactive latent form. Latency is not a
constant of the TGF-.beta. superfamily, since the presence of the
pro region has no effect on activin or inhibin biological
activity.
[0096] A unifying feature of the biology of the proteins from the
TGF-.beta. superfamily is their ability to regulate developmental
processes. TGF-.beta. has been shown to have numerous regulatory
actions on a wide variety of both normal and neoplastic cells.
TGF-.beta. is multifunctional, as it can either stimulate or
inhibit cell proliferation, differentiation, and other critical
processes in cell function (Sporn and Roberts, supra).
[0097] One member of the TGF-.beta. superfamily, EBAF, is expressed
in endometrium only in the late secretory phase and during abnormal
endometrial bleeding. Kothapalli et al, J. Clin. Invest. 99:
2342-2350 (1997). Human endometrium is unique in that it is the
only tissue in the body that bleeds at regular intervals. In
addition, abnormal endometrial bleeding is one of the most common
manifestations of gynecological diseases, and is a prime indication
for hysterectomy. In situ hybridization showed that the mRNA of
EBAF was expressed in the stroma without any significant mRNA
expression in the endometrial glands or endothelial cells.
[0098] The predicted protein sequence of EBAF showed a strong
homology to the protein encoded by mouse lefty/stra3 of the
TGF-.beta. superfamily. A motif search revealed that the predicted
EBAF protein contains most of the cysteine residues which are
conserved among the TGF-.beta.-related proteins and which are
necessary for the formation of the cysteine knot structure. The
EBAF sequence contains an additional cysteine residue, 12 amino
acids upstream from the first conserved cysteine residue. The only
other family members known to contain an additional cysteine
residue are TGF-.beta.s, inhibins, and GDF-3. EBAF, similar to
LEFTY, GDF-3/Vgr2, and GDF-9, lacks the cysteine residue that is
known to form the intermolecular disulfide bond. Therefore, EBAF
appears to be an additional member of the TGF-.beta. superfamily
with an unpaired cysteine residue that may not exist as a dimer.
However, hydrophobic contacts between the two monomer subunits may
promote dimer formation. Fluorescence in situ hybridization showed
that the ebaf gene is located on human chromosome I at band
q42.1.
[0099] Additional members of the TGF-.beta. superfamily, such as
those related to EBAF, are being searched for by industry and
academics. We herein describe the identification and
characterization of novel polypeptides having homology to EBAF,
designated herein as PRO317 polypeptides.
[0100] 18. PRO301
[0101] The widespread occurrence of cancer has prompted the
devotion of considerable resources and discovering new treatments
of treatment. One particular method involves the creation of tumor
or cancer specific monoclonal antibodies (mAbs) which are specific
to tumor antigens. Such mAbs, which can distinguish between normal
and cancerous cells are useful in the diagnosis, prognosis and
treatment of the disease. Particular antigens are known to
be-associated with neoplastic diseases, such as colorectal
cancer.
[0102] One particular antigen, the A33 antigen is expressed in more
than 90% of primary or metastatic colon cancers as well as normal
colon epithelium. Since colon cancer is a widespread disease, early
diagnosis and treatment is an important medical goal. Diagnosis and
treatment of colon cancer can be implemented using monoclonal
antibodies (mAbs) specific therefore having fluorescent, nuclear
magnetic or radioactive tags. Radioactive gene, toxins and/or drug
tagged mAbs can be used for treatment in situ with minimal patient
description. mAbs can also be used to diagnose during the diagnosis
and treatment of colon cancers. For example, when the serum levels
of the A33 antigen are elevated in a patient, a drop of the levels
after surgery would indicate the tumor resection was successful. On
the other hand, a subsequent rise in serum A33 antigen levels after
surgery would indicate that metastases of the original tumor may
have formed or that new primary tumors may have appeared. Such
monoclonal antibodies can be used in lieu of, or in conjunction
with surgery and/or other chemotherapies. For example, U.S. Pat.
No. 4,579,827 and U.S. Ser. No. 424,991 (E.P. 199,141) are directed
to therapeutic administration of monoclonal antibodies, the latter
of which relates to the application of anti-A33 mAb.
[0103] Many cancers of epithelial origin have adenovinis receptors.
In fact, adenovirus-derived vectors have been proposed as a means
of inserting antisense nucleic acids into tumors (U.S. Pat. No.
5,518,885). Thus, the association of viral receptors with
neoplastic tumors is not unexpected.
[0104] We herein describe the identification and characterization
of novel polypeptides having homology to certain cancer-associated
antigens, designated herein as PRO301 polypeptides.
[0105] 19. PRO224
[0106] Cholesterol uptake can have serious implications on one's
health. Cholesterol uptake provides cells with most of the
cholesterol they require for membrane synthesis. If this uptake is
blocked, cholesterol accumulates in the blood and can contribute to
the formation of atherosclerotic plaques in blood vessel walls.
Most cholesterol is transported in the blood bound to protein in
the form of complexes known as low-density lipoproteins (LDLs).
LDLs are endocytosed into cells via LDL receptor proteins.
Therefore, LDL receptor proteins, and proteins having homology
thereto, are of interest to the scientific and medical communities.
Membrane-bound proteins and receptors can play an important role in
the formation, differentiation and maintenance of multicellular
organisms. The LDL receptors are an example of membrane-bound
proteins which are involved in the synthesis and formation of cell
membranes, wherein the health of an individual is affected directly
and indirectly by its function. Many membrane-bound proteins act as
receptors such as the LDL receptor. These receptors can function to
endocytose substrates or they can function as a receptor for a
channel. Other membrane-bound proteins function as signals or
antigens.
[0107] Membrane-bound proteins and receptor molecules have various
industrial applications, including as pharmaceutical and diagnostic
agents. The membrane-bound proteins can also be employed for
screening of potential peptide or small molecule regulators of the
relevant receptor/ligand interaction. In the case of the LDL
receptor, it is desirable to find molecules which enhance
endocytosis so as to lower blood cholesterol levels and plaque
formation. It is also desirable to identify molecules which inhibit
endocytosis so that these molecules can be avoided or regulated by
individuals having high blood cholesterol. Polypeptides which are
homologous to lipoprotein receptors but which do not function as
lipoprotein receptors are also of interest in the determination of
the function of the fragments which show homology.
[0108] The following studies report on previously known low density
lipoprotein receptors and related proteins including
apolipoproteins: Sawamura, et al., Nippon Chemiphar Co, Japan
patent application J09098787; Novak, S., et al., J. Biol. Chem.
271:(20)11732-6 (1996); Blaas, D., J. Virol., 69(11)7244-7
(November 1995); Scott, J., J. Inherit. Metab. Dis. (UK), 9/Supp.
1(3-16) (1986); Yamamoto, et al., Cell, 39:27-38(1984); Rebece, et
al., Neurobiol. Aging 15:5117 (1994); Novak, S., et al., J. Biol.
Chemistry 271:11732-11736 (1996); and Sestavel and Fruchart, Cell
Mol. Biol., 40(4):461-81 (June 1994). These publications and others
published prior to the filing of this application provide further
background to peptides already known in the art.
[0109] Efforts are being undertaken by both industry and academia
to identify new, native membrane-bound receptor proteins,
particularly those having homology to lipoprotein receptors. We
herein describe the identification and characterization of novel
polypeptides having homology to lipoprotein receptors, designated
herein as PRO224 polypeptides.
[0110] 20. PRO222
[0111] Complement is a group of proteins found in the blood that
are important in humoral immunity and inflammation. Complement
proteins are sequentially activated by antigen-antibody complexes
or by proteolytic enzymes. When activated, complement proteins kill
bacteria and other microorganisms, affect vascular permeability,
release histamine and attract white blood cells. Complement also
enhances phagocytosis when bound to target cells. In order to
prevent harm to autologous cells, the complement activation pathway
is tightly regulated.
[0112] Deficiencies in the regulation of complement activation or
in the complement proteins themselves may lead to immune-complex
diseases, such as systemic lupus erythematosus, and may result in
increased susceptibility to bacterial infection. In all cases,
early detection of complement deficiency is desirable so that the
patient can begin treatment. Thus, research efforts are currently
directed toward identification of soluble and membrane proteins
that regulate complement activation.
[0113] Proteins known to be important in regulating complement
activation in humans include Factor H and Complement receptor type
1 (CR1). Factor H is a 150 kD soluble serum protein that interacts
with complement protein C3b to accelerate the decay of C3
convertase and acts as a cofactor for Factor I-mediated cleavage of
complement protein C4b. Complement receptor type 1 is a 190-280 kD
membrane bound protein found in mast cells and most blood cells.
CR1 interacts with complement proteins C3b, C4b, and iC3b to
accelerate dissociation of C3 convertases, acts as a cofactor for
Factor I-mediated cleavage of C3b and C4b, and binds immune
complexes and promotes their dissolution and phagocytosis.
[0114] Proteins which have homology to complement proteins are of
particular interest to the medical and industrial communities.
Often, proteins having homology to each other have similar
function. It is also of interest when proteins having homology do
not have similar functions, indicating that certain structural
motifs identify information other than function, such as locality
of function.
[0115] Efforts are being undertaken by both industry and academia
to identify new, native secreted and membrane-bound proteins,
particularly those having homology to known proteins involved in
the complement pathway. Proteins involved in the complement pathway
were reviewed in Birmingham D J (1995), Critical Reviews in
Immunology, 15(2):133-154 and in Abbas A K, et al. (1994) Cellular
and Molecular Immunology, 2nd Ed. W. B. Saunders Company,
Philadelphia, pp 295-315.
[0116] We herein describe the identification and characterization
of novel polypeptides having homology to complement receptors,
designated herein as PRO222 polypeptides.
[0117] 21. PRO234
[0118] The successful function of many systems within multicellular
organisms is dependent on cell-cell interactions. Such interactions
are affected by the alignment of particular ligands with particular
receptors in a manner which allows for ligand-receptor binding and
thus a cell-cell adhesion. While protein-protein interactions in
cell recognition have been recognized for some time, only recently
has the role of carbohydrates in physiologically relevant
recognition been widely considered (see B. K. Brandley et al., J.
Leuk Biol. 40: 97 (1986) and N. Sharon et al., Science 246: 227
(1989). Oligosaccharides are well positioned to act as recognition
novel lectins due to their cell surface location and structural
diversity. Many oligosaccharide structures can be created through
the differential activities of a smaller number of
glycosyltransferases. The diverse structures of oligosaccharides
can be generated by transcription of relatively few gene products,
which suggests that the oligosaccharides are a plausible mechanism
by which is directed a wide range of cell-cell interactions.
Examples of differential expression of cell surface carbohydrates
and putative carbohydrate binding proteins (lectins) on interacting
cells have been described (J. Dodd & T. M. Jessel, J Neurosci.
5: 3278 (1985); L. J. Regan et al., Proc. Natl. Acad. Sci. USA 83:
2248-(1986); M. Constantine-Paton et al., Nature 324: 459 (1986);
and M. Tierneyer et al., J Biol. Chem. 263: 1671 (1989). One
interesting member of the lectin family are selectins.
[0119] The migration of leukocytes to sites of acute or chronic
inflammation involves adhesive interactions between these cells and
the endothelium. This specific adhesion is the initial event in the
cascade that is initiated by inflammatory insults, and it is,
therefore, of paramount importance to the regulated defense of the
organism.
[0120] The types of cell adhesion molecules that are involved in
the interaction between leukocytes and the endothelium during an
inflammatory response currently stands at four: (1) selectins; (2)
(carbohydrate and glycoprotein) ligands for selectins; (3)
integrins; and (4) integrin ligands, which are members of the
immunoglobulin gene superfamily.
[0121] The selectins are cell adhesion molecules that are unified
both structurally and functionally. Structurally, selectins are
characterized by the inclusion of a domain with homology to a
calcium-dependent lectin (C-lectins), an epidermal growth factor
(egf)-like domain and several complement binding-like domains,
Bevilacqua, M. P. et al., Science 243: 1160-1165 (1989); Johnston
et al., Cell 56: 1033-1044 (1989); Lasky et al, Cell 56: 1045-1055
(1989); Siegalman, M. et al., Science 243: 1165-1172 (1989);
Stoolman, L. M., Cell 56: 907-910 (1989). Functionally, selectins
share the common property of their ability to mediate cell binding
through interactions between their lectin domains and cell surface
carbohydrate ligands (Brandley, B, et al., Cell 63, 861-863 (1990);
Springer, T. and Lasky, L. A., Nature, 349 19-197 (1991);
Bevilacqua, M. P. and Nelson, R. M., J. Clin. Invest. 91 379-387
(1993) and Tedder et al., J. Exp. Med. 170: 123-133 (1989).
[0122] There are three members identified so far in the selectin
fwnily of cell adhesion molecules: L-selectin (also called
peripheral lymph node homing receptor (pnHR), LEC-CAM-1, LAM-1,
gp90.sup.MEL, gp100.sup.MEL, gp110.sup.MEL, MEL-14 antigen, Leu-8
antigen, TQ-1 antigen, DREG antigen), E-selectin (LEC-CAM-2,
LECAM-2, ELAM-1) and P-selectin (LEC-CAM-3, LECAM-3, GMP-140,
PADGEM).
[0123] The identification of the C-lectin domain has led to an
intense effort to define carbohydrate binding ligands; for proteins
containing such domains. E-selectin is believed to recognize the
carbohydrate sequence
NeuNAc.alpha.2-3Gal.beta.1-4(Fuc.alpha.l-3)GIcNAc (sialyl-Lewis x,
or sLe.sup.x) and related oligosaccharides, Berg et al., J. Mot
Chem. 265: 14869-14872 (199 1); Lowe et al., Cell 63: 475-484
(1990); Phillips et al., Science 250: 1130-1132 (1990); Tiemeyer et
al., Proc. Nat. Acad Sci. USA 88: 1138-1142 (1991).
[0124] L-selectin, which comprises a lectin domain, performs its
adhesive function by recognizing carbohydrate-containing ligands on
endothelial cells. L-selectin is expressed on the surface of
leukocytes, such as lymphocytes, neutrophils, monocytes and
eosinophils, and is involved with the trafficking of lymphocytes to
peripheral lymphoid tissues (Gallatin et al., Nature 303: 30-34
(1983)) and with acute neutrophil-medicated inflammatory responses
(Watson, S. R., Nature 349: 164-167 (1991)). The amino acid
sequence of L-selectin and the encoding nucleic acid sequence are,
for example, disclosed in U.S. Pat. No. 5,098,833 issued 24 Mar.
1992.
[0125] L-selectin (LECAM-1) is particularly interesting because of
its ability to block neutrophil influx (Watson et al., Nature 349:
164-167 (1991). It is expressed in chronic lymphocytic leukemia
cells which bind to HEV (Spertini et al., Nature 349: 691-694
(1991). It is also believed that REV structures at sites of chronic
inflammation are associated with the symptoms of diseases such as
rheumatoid arthritis, psoriasis and multiple sclerosis.
[0126] E-selectin (ELAM-1), is particularly interesting because of
its transient expression on endothelial cells in response to IL-1
or TNF. Bevilacqua et al., Science 243: 1160 (1989). The time
course of this induced expression (2-8 h) suggests a role for this
receptor in initial neutrophil induced extravasation in response to
infection and injury. It has further been reported that anti-ELAM-1
antibody blocks the influx of neutrophils in a primate asthma model
and thus is beneficial for preventing airway obstruction resulting
from the inflammatory response. Gundel et al., J. Clin. Invest. 88:
1407 (1991).
[0127] The adhesion of circulating neutrophils to stimulated
vascular endothelium is a primary event of the inflammatory
response. P-selectin has been reported to recognize the Lewis x
structure (Gal.beta.1-4(Fuc.alpha.l-3) GIcNAc), Larsen et al., Cell
63: 467-474(1990). Others report that an additional terminal linked
sialic acid is required for high affinity binding, Moore et al., J.
Cell. Biol 112: 491-499 (199 1). P-selectin has been shown to be
significant in acute lung injury. Anti-P-selectin antibody has been
shown to have strong protective effects in a rodent lung injury
model. M. S. Mulligan et al., J. Clin. Invest. 90: 1600 (1991).
[0128] We herein describe the identification and characterization
of novel polypeptides having homology to lectin proteins, herein
designated as PRO234 polypeptides.
[0129] 22. PRO231
[0130] Some of the most important proteins involved in the above
described regulation and modulation of cellular processes are the
enzymes which regulate levels of protein phosphorylation in the
cell. For example, it is known that the transduction of signals
that regulate cell growth and differentiation is regulated at least
in part by phosphorylation and dephosphorylation of various
cellular proteins. The enzymes that catalyze these processes
include the protein kinases, which function to phosphorylate
various cellular proteins, and the protein phosphatases, which
function to remove phosphate residues from various cellular
proteins. The balance of the level of protein phosphorylation in
the cell is thus mediated by the relative activities of these two
types of enzymes.
[0131] Protein phosphatases represent a growing family of enzymes
that are found in many diverse forms, including both membrane-bound
and soluble forms. While many protein phosphatases have been
described, the finictions of only a very few are beginning to be
understood (Tonks, Semin. Cell Biol. 4:373453 (1993) and Dixon,
Recent Prog. Horm. Res. 51:405414 (1996)). However, in general, it
appears that many of the protein phosphatases function to modulate
the positive or negative signals induced by various protein
kinases. Therefore, it is likely that protein phosphatases play
critical roles in numerous and diverse cellular processes.
[0132] Given the physiological importance of the protein
phosphatases, efforts are being undertaken by both industry and
academia to identify new, native phosphatase proteins. Many of
these efforts are focused on the screening of mammalian recombinant
DNA libraries to identify the coding sequences for novel
phosphatase proteins. Examples of screening methods and techniques
are described in the literature [see, for example, Klein et al.,
Proc. Natl.' Acad. Sci, 93:7108-7113 (1996); U.S. Pat. No.
5,536,637)].
[0133] We herein describe the identification and characterization
of novel polypeptides having homology to acid phosphatases,
designated herein as PRO231 polypeptides.
[0134] 23. PRO229
[0135] Scavenger receptors are known to protect IgG molecules from
catabolic degradation. Riechmann and Hollinger, Nature
Biotechnology 15:617 (1997). In particular, studies of the CH2 and
CH3 domains have shown that specific sequences of these domains are
important in determining the half-lives of antibodies. Ellerson, et
al., J. Immunol. 116: 510 (1976); Yasmeen, et al., J. Immunol. 116:
518 (1976; Pollock, et al., Eur. J. Immunol.. 20:2021 (1990).
Scavenger receptor proteins and antibodies thereto are further
reported in U.S. Pat. No. 5,510,466 to Krieger, et al. Due to the
ability of scavenger receptors to increase the half-life of
polypeptides and their involvement in immune function, molecules
having homology to scavenger receptors are of importance to the
scientific and medical community.
[0136] Efforts are being undertaken by both industry and academia
to identify new, native secreted and membrane-bound receptor
proteins, particularly those having homology to scavenger
receptors. Many efforts are focused on the screening of mammalian
recombinant DNA libraries to identify the coding sequences for
novel secreted and membrane-bound receptor proteins. Examples of
screening methods and techniques are described in the literature
[see, for example, Klein et al., Proc. Natl. Acad. Sci; 93:
7108-7113 (1996); U.S. Pat. No. 5,536,637)].
[0137] We herein describe the identification and characterization
of novel polypeptides having homology to scavenger receptors,
designated herein as PRO229 polypeptides.
[0138] 24. PRO238
[0139] Oxygen free radicals and antioxidants appear to play an
important role in the central nervous system after cerebral
ischemia and reperfusion. Moreover, cardiac injury, related to
ischaemia and reperfusion has been reported to be caused by the
action of free radicals. Additionally, studies have reported that
the redox state of the cell is a pivotal determinant of the fate of
the cells. Furthermore, reactive oxygen species have been reported
to be cytotoxic, causing inflammatory disease, including tissue
necrosis, organ failure, atherosclerosis, infertility, birth
defects, premature aging, mutations and malignancy. Thus, the
control of oxidation and reduction is important for a number of
reasons including for control and prevention of strokes, heart
attacks, oxidative stress and hypertension. In this regard,
reductases, and particularly, oxidoreductases, are of interest.
Publications further describing this subject matter include Kelsey,
et al., Br. J. Cancer 76(7):852-4 (1997); Friedrich and Weiss, J.
Theor. Biol., 187(4):52940 (1997) and Pieulle, et al., J.
Bacteriol., 179(18):5684-92 (1997).
[0140] Efforts are being undertaken by both industry and academia
to identify new, native secreted and membrane-bound receptor
proteins, particularly secreted proteins which have homology to
reductase. Many efforts are focused on the screening of mammalian
recombinant DNA libraries to identify the coding sequences for
novel secreted and membrane-bound receptor proteins. Examples of
screening methods and techniques are described in the literature
[see, for example, Klein et al., Prop. Natl. Acad. Sci.,
93:7108-7113 (1996); U.S. Pat. No. 5,536,637)].
[0141] We herein describe the identification and characterization
of novel polypeptides: having homology to reductase, designated
herein as PRO238 polypeptides.
[0142] 25. PRO233
[0143] Studies have reported that the redox state of the cell is an
important determinant of the fate of the cell. Furthermore,
reactive oxygen species have been reported to be cytotoxic, causing
inflammatory disease, including tissue necrosis, organ failure,
atherosclerosis, infertility, birth defects, premature aging,
mutations and malignancy. Thus, the control of oxidation and
reduction is important for a number of reasons, including the
control and prevention of strokes, heart attacks, oxidative stress
and hypertension. Oxygen free radicals and antioxidants appear to
play an important role in the central nervous system after cerebral
ischemia and reperfusion. Moreover, cardiac injury, related to
ischaemia and reperfusion has been reported to be caused by the
action of free radicals. In this regard, reductases, and
particularly, oxidoreductases, are of interest. In addition, the
transcription factors, NF-kappa B and AP-1, are known to be
regulated by redox state and to affect the expression of a large
variety of genes thought to be involved in the pathogenesis of
AIDS, cancer, atherosclerosis and diabetic complications.
Publications further describing this subject matter include Kelsey,
et al., Br. J. Cancer, 76(7):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 novel
polypeptides which have homology to reductase, designated herein as
PRO233 polypeptides.
[0144] 26. PRO223
[0145] The carboxypeptidase family of exopeptidases constitutes a
diverse group of enzymes that hydrolyze carboxyl-terminal amide
bonds in polypeptides, wherein a large number of mammalian tissues
produce these enzymes. Many of the carboxypeptidase enzymes that
have been identified to date exhibit rather strong cleavage
specificities for certain amino acids in polypeptides. For example,
carboxypeptidase enzymes have been identified which prefer lysine,
arginine, serine or amino acids with either aromatic or branched
aliphatic side chains as substrates at the carboxyl terminus of the
polypeptide.
[0146] With regard to the serine carboxypeptidases, such amino acid
specific enzymes have been identified from a variety of different
mammalian and non-mammalian organisms. The mammalian serine
carboxypeptidase enzymes play important roles in many different
biological processes including, for example, protein digestion,
activation, inactivation, or modulation of peptide, hormone
activity, and alteration of the physical properties of proteins and
enzymes.
[0147] In light of the physiological importance of the serine
carboxypeptidases, efforts are being undertaken by both industry
and academia to identify new, native secreted and membrane-bound
receptor proteins and specifically novel carboxypeptidases. Many of
these efforts are focused on the screening of mammalian recombinant
DNA libraries to identify the coding sequences for novel secreted
and membrane-bound receptor proteins. We describe herein novel
polypeptides having homology to one or more serine carboxypeptidase
polypeptides, designated herein as PRO223 polypeptides.
[0148] 27. PRO235
[0149] Plexin was first identified in Xenopus tadpole nervous
system as a membrane glycoprotein which was shown to mediate cell
adhesion via a homophilic binding mechanism in the presence of
calcium ions. Strong evolutionary conservation between Xenopus,
mouse and human homologs of plexin has been observed. [Kaneyama et
al., Biochem. And Biophys. Res. Comm. 226: 524-529 (1996)]. Given
the physiological importance of cell adhesion mechanisms in vivo,
efforts are currently being under taken to identify new, native
proteins which are involved in cell adhesion. We describe herein
the identification of a novel polypeptide which has homology to
plexin, designated herein as PRO235.
[0150] 28. PRO236 and PRO262
[0151] .beta.-galactosidase is a well known enzymatic protein which
functions to hydrolyze .beta.-galactoside molecules.
.beta.-galactosidase has been employed for a variety of different
applications, both in vitro and in vivo and has proven to be an
extremely useful research tool. As such, there is an interest in
obtaining novel polypeptides which exhibit homology to the
.beta.-galactosidase polypeptide.
[0152] Given the strong interest in obtaining novel polypeptides
having homology to .beta.-galactosidase, efforts are currently
being undertaken by both industry and academia to identify new,
native .beta.-galactosidase homolog proteins. Many of these efforts
are focused on the screening of mammalian recombinant DNA libraries
to identify the coding sequences for novel
.beta.-galactosidase-like proteins. Examples of screening methods
and techniques are described in the literature [see, for example,
Klein et al., Proc. Nad. Acad. Sci., 93:7108-7113 (1996); U.S. Pat.
No. 5,536,637)]. We herein describe novel poylpeptides having
siginificant homology to the .beta.-galactosidase enzyme,
designated herein as PRO236 and PRO262 polypeptides.
[0153] 29. PRO239
[0154] Densin is a glycoprotein which has been isolated from the
brain which has all the hallmarks of an adhesion molecule. It is
highly concentrated at synaptic sites in the brain and is expressed
prominently in dendritic processes in developing neurons. Densin
has been characterized as a member of the O-linked
sialoglycoproteins. Densin has relevance to medically important
processes such as regeneration. Given the physiological importance
of synaptic processes and cell adhesion mechanisms in vivo, efforts
are currently being under taken to identify new, native proteins
which are involved in synaptic machinery and cell adhesion. We
describe herein the identification of novel polypeptides which have
homology to densin, designated herein as PRO239 polypeptides.
[0155] 30. PRO257
[0156] Ebnerin is a cell surface protein associated with von Ebner
glands in mammals. Efforts are being undertaken by both industry
and academia to identify new, native cell surface receptor proteins
and specifically those which possess sequence homology to cell
surface proteins such as ebnerin. Many of these efforts are focused
on the screening of mammalian recombinant DNA libraries to identify
the coding sequences for novel receptor proteins. We herein
describe the identification of novel polypeptides having
significant homology to the von Ebner's gland-associated protein
ebnerin, designated herein as PRO257 polypeptides.
[0157] 31. PRO260
[0158] Fucosidases are enzymes that remove fucose residues from
fucose containing proteoglycans. In some pathological conditions,
such as cancer, rheumatoid arthritis, and diabetes, there is an
abnormal fucosylation of serum proteins. Therefore, facosidases,
and proteins having homology to facosidase, are of importance to
the study and abrogation of these conditions. In particular,
proteins having homology to the alpha-1-fucosidase precursor are of
interest. Fucosidases and fucosidase inhibitors are further
described in U.S. Pat. Nos. 5,637,490, 5,382,709, 5,240,707,
5,153,325, 5,100,797, 5,096,909 and 5,017,704. Studies are also
reported in Valk, et al., J. Virol., 71(9):6796 (1997), Aktogu, et
al., Monaldi. Arch. Chest Dis. (Italy), 52(2):118 (1997) and
Focarelli, et al., Biochem. Biophys. Res. Commun (U.S.), 234(1):54
(1997).
[0159] Efforts are being undertaken by both industry and academia
to identify new, native secreted and membrane-bound receptor
proteins. Of particular interest are proteins having homology to
the alpha-1-fucosidase precursor. Many efforts are focused on the
screening of mammalian recombinant DNA libraries to identify the
coding sequences for novel secreted and membrane-bound receptor
proteins. Examples of screening methods and techniques are
described in the literature [see, for example, Klein et al., Proc.
Natl. Acad. Sci., 93:7108-7113 (1996); U.S. Pat. No.
5,536,637)].
[0160] We herein describe the identification and characterization
of novel polypeptides having homology to fucosidases, designated
herein as PRO260 polypeptides.
[0161] 32. PRO263
[0162] CD44 is a cell surface adhesion molecule involved in
cell-cell and cell-matrix interactions. Hyaluronic acid, a
component of the extracellular matrix is a major ligand. Other
ligands include collagen, fibronectin, laminin, chrondroitin
sulfate, mucosal addressin, serglycin and osteoponin. CD44 is also
important in regulating cell traffic, lymph node homing,
transmission of growth signals, and presentation of chemokines and
growth factors to traveling cells. CD44 surface proteins are
associated with metastatic tumors and CD44 has been used as a
marker for HIV infection. Certain splice variants are associated
with metastasis and poor prognosis of cancer patients. Therefore,
molecules having homology with CD44 are of particular interest, as
their homology indicates that they may have functions related to
those functions of CD44. CD44 is further described in U.S. Pat.
Nos. 5,506,119, 5,504,194 and 5,108,904; Gerberick, et al.,
Toxicol. ARPI. Pharmacol., 146(1): 1(1997); Wittig, et al.,
Immunol. Letters (Netherlands), 57(1-3):217 (1997); and Oliveira
and Odell, Oral Oncol. (England), 33(4):260 (1997).
[0163] Efforts are being undertaken by both industry and academia
to identify new, native secreted and membrane-bound receptor
proteins, particularly transmembrane proteins with homology to CD44
antigen. Many efforts are focused on the screening of mammalian
recombinant DNA libraries to identify the coding sequences for
novel secreted and membrane-bound receptor proteins. Examples of
screening methods and techniques are described in the literature
[see, for example, Klein et al., Proc. Natl. Acad. Sci.
93:7108-7113 (1996); U.S. Pat. No. 5,536,637)].
[0164] We herein describe the identification and characterization
of novel polypeptides having homology to CD44 antigen, designated
herein as PRO263 polypeptides.
[0165] 33. PRO270
[0166] Thioredoxins effect reduction-oxidation (redox) state. Many
diseases are potentially related to redox state and reactive oxygen
species may play a role in many important biological processes. The
transcription factors, NF-kappa B and AP-1, are regulated by redox
state and are known to affect the expression of a large variety of
genes thought to be involved in the pathogenesis of AIDS, cancer,
atherosclerosis and diabetic complications. Such proteins may also
play a role in cellular antioxidant defense, and in pathological
conditions involving oxidative stress such as stroke and
inflammation in addition to having a role in apoptosis. Therefore,
thioredoxins, and proteins having homology thereto, are of interest
to the scientific and medical communities.
[0167] We herein describe the identification and characterization
of novel polypeptides having homology to thioredoxin,. designated
herein as PRO270 polypeptides.
[0168] 34. PRO271
[0169] The proteoglycan link protein is a protein which is
intimately associated with various extracellular matrix proteins
and more specifically with proteins such as collagen. For example,
one primary component of collagen is a large proteoglycan called
aggrecan. This molecule is retained by binding to the
glycosaminoglycan hyaluronan through the amino terminal G1 globular
domain of the core protein. This binding is stabilized by the
proteoglycan link protein which is a protein that is also
associated with other tissues containing hyaluronan binding
proteoglycans such as versican.
[0170] Link protein has been identified as a potential target for
autoimmune antibodies in individuals who suffer from juvenile
rheumatoid arthritis (see Guerassimov et al., J Rheumatology
24(5):959-964 (1997)). As such, there is strong interest in
identifying novel proteins having homology to link protein. We
herein describe the identification and characterization of novel
polypeptides having such homology, designated herein as PRO271
polypeptides.
[0171] 35. PRO272
[0172] Reticulocalbin is an endoplasmic reticular protein which may
be involved in protein transport and luminal protein processing.
Reticulocalbin resides in the lumen of the endopladsmic reticulum,
is known to bind calcium, and may be involved in a luminal
retention mechanism of the endoplasmic reticulum. It contains six
domains of the EF-hand motif associated with high affinity calcium
binding. We describe herein the identification and characterization
of a novel polypeptide which has homology to the reticulocalbin
protein, designated herein as PROM.
[0173] 36. PRO294
[0174] Collagen, a naturally occurring protein, finds wide
application in industry. Chemically hydrolyzed natural collagen can
be denatured and renatured by heating and cooling to produce
gelatin, which is used in photographic and medical, among other
applications. Collagen has important properties such as the ability
to form interchain aggregates having a conformation designated as a
triple helix. We herein describe the identification and
characterization of a novel polypeptide which has homology to
portions of the collagen molecule, designated herein as PRO294.
[0175] 37. PRO295
[0176] The integrins comprise a supergene family of cell-surface
glycoprotein receptors that promote cellular adhesion. Each cell
has numerous receptors that define its cell adhesive capabilities.
Integrins are involved in a wide variety of interaction between
cells and other cells or matrix components. The integrins are of
particular importance in regulating movement and function of immune
system cells. The platelet IIb/IIIA integrin complex is of
particular importance in regulating platelet aggregation. A member
of the integrin family, integrin .beta.-6, is expressed on
epithelial cells and modulates epithelial inflammation. Another
integrin, leucocyte-associated antigen-1 (LFA-1) is important in
the adhesion of lymphocytes during an immune response. The
integrins are expressed as heterodimers of non-covalently
associated alpha and beta subunits. Given the physiological
importance of cell adhesion mechanisms in vivo, efforts are
currently being under taken to identify new, native proteins which
are involved in cell adhesion. We describe herein the
identification and characterization of a novel polypeptide which
has homology to integrin, designated herein as PRO295.
[0177] 38. PRO293
[0178] Protein-protein interactions include receptor and antigen
complexes and signaling mechanisms. As more is known about the
structural and functional mechanisms underlying protein-protein
interactions, protein-protein interactions can be more easily
manipulated to regulate the particular result of the
protein-protein interaction. Thus, the underlying mechanisms of
protein-protein interactions are of interest to the scientific and
medical community.
[0179] All proteins containing leucine-rich repeats are thought to
be involved in protein-protein interactions. Leucine-rich repeats
are short sequence motifs present in a number of proteins with
diverse functions and cellular locations. The crystal structure of
ribonuclease inhibitor protein has revealed that leucine-rich
repeats correspond to beta-alpha structural units. These units are
arranged so that they form a parallel beta-sheet with one surface
exposed to solvent, so that the protein acquires an unusual,
nonglubular shape. These two features have been indicated as
responsible for the protein-binding functions of proteins
containing leucine-rich repeats. See, Kobe and Deisenhofer, Trends
Biochem. Sci. 19(10):415-421 (October 1994).
[0180] A study has been reported on leucine-rich proteoglycans
which serve as tissue organizers, orienting and ordering collagen
fibrils during ontogeny and are involved in pathological processes
such as wound healing, tissue repair, and tumor stroma formation.
Iozzo, R. V., Crit. Rev. Biochem. Mol. Biol., 32(2):141-174 (1997).
Others studies implicating leucine rich proteins in wound healing
and tissue repair are De La Salle, C., et al., Vouv. Rev. Fr.
Hematol. (Germany), 37(4):215-222 (1995), reporting mutations in
the leucine rich motif in a complex associated with the bleeding
disorder Bernard-Soulier syndrome and Chldmetson, K. J., Thromb.
Haemost. (Germany), 74(1): 111-116 (July 1995), reporting that
platelets have leucine rich repeats. Another protein of particular
interest which has been reported to have leucine-rich repeats is
the SLIT protein which has been reported to be useful in treating
neuro-degenerative diseases such as Alzheimer's disease, nerve
damage such as in Parkinson's disease, and for diagnosis of cancer,
see, Artavanistsakonas, S. and Rothberg, J. M., W09210518-A1 by
Yale University. Other studies reporting on the biological
functions of proteins having leucine-rich repeats include: Tayar,
N., et al., Mol. Cell Endocrinol., (Ireland), 125(1-2):65-70
(December 1996) (gonadotropin receptor involvement); Miura, Y., et
al., NipRqn Rinsho (Japan), 54(7):1784-1789 (July 1996) (apoptosis
involvement); Harris, P. C., et al., J. Am. Soc. Nephrol., 6(4):
1125-1133 (October 1995) (kidney disease involvement); and
Ruoslahti, E. I., et al., W09110727-A by La Jolla Cancer Research
Foundation (decorin binding to transforming growth factors
involvement for treatment for cancer, wound healing and
scarring).
[0181] Efforts are therefore being undertaken by both industry and
academia to identify new proteins having leucine rich repeats to
better understand protein-protein interactions. Of particular
interest are those proteins having leucine rich repeats and
homology to known neuronal leucine rich repeat proteins. Many
efforts are focused on the screening of mammalian recombinant DNA
libraries to identify the coding sequences for novel secreted and
membrane-bound proteins having leucine rich repeats. Examples of
screening methods and techniques are described in the literature
[see, for example, Klein et al., Proc. Nat. Acad. Sci.,
93:7108-7113 (1996); U.S. Pat. No. 5,536,637)].
[0182] We describe herein the identification and characterization
of a novel polypeptide which has homology to leucine rich repeat
proteins, designated herein as PRO293.
[0183] 39. PRO247
[0184] Protein-protein interactions include receptor and antigen
complexes and signaling mechanisms. As more is known about the
structural and functional mechanisms underlying protein-protein
interactions, protein-protein interactions can be more easily
manipulated to regulate the particular result of the
protein-protein interaction. Thus, the underlying mechanisms of
protein-protein interactions are of interest to the scientific and
medical community.
[0185] All proteins containing leucine-rich repeats are thought to
be involved in protein-protein interactions. Leucine-rich repeats
are short sequence motifs present in a number of proteins with
diverse functions and cellular locations. The crystal structure of
ribonuclease inhibitor protein has revealed that leucine-rich
repeats correspond to beta-alpha structural units. These units are
arranged so that they form a parallel beta-sheet with one surface
exposed to solvent, so that the protein acquires an unusual,
nonglubular shape. these two features have been indicated as
responsible for the protein-binding functions of proteins
containing leucine-rich repeats. See, Kobe and Deisenhofer, Trends
Biochem. Sci., 19(10):415-421 (October 1994).
[0186] A study has been reported on leucine-rich proteoglycans
which serve as tissue organizers, orienting and ordering collagen
fibrils during ontogeny and are involved in pathological processes
such as wound healing, tissue repair, and tumor stroma formation.
Iozzo, R. V., Crit. Rev. Biochem. Mol. Biol., 32(2):141-174 (1997).
Others studies implicating leucine rich proteins in wound healing
and tissue repair are De La Salle, C., et al., Vouv. Rev. Fr.
Hematol. (Germany), 37(4):215-222 (1995), reporting mutations in
the leucine rich motif in a complex associated with the bleeding
disorder Bernard-Soulier syndrome and Chlemetson, K. J., Thromb.
Haemost. (Germany), 74(1):111-116 (July 1995), reporting that
platelets have leucine rich repeats. Another protein of particular
interest which has been reported to have leucine-rich repeats is
the SLIT protein which has been reported to be useful in treating
neuro-degenerative diseases such as Alzheimer's disease, nerve
damage such as in Parkinson's disease, and for diagnosis of cancer,
see, Artavanistsakonas, S. and Rothberg, J. M., W09210518-A1 by
Yale University. Other studies reporting on the biological
functions of proteins having leucine-rich repeats include: Tayar,
N., et al., Mol. Cell Endocrinol., (Ireland), 125(1-2):65-70
(December 1996) (gonadotropin receptor involvement); Miura, Y., et
al., Nippon Rinsho (Japan), 54(7): 1784-1789 (July 1996) (apoptosis
involvement); Harris, P. C., et al., J. Am. Soc. Nephrol.,
6(4):1125-1133 (October 1995) (kidney disease involvement); and
Ruoslahti, E. I., et al., W09110727-A by La Jolla Cancer Research
Foundation (decorin binding to transforming growth factors
involvement for treatment for cancer, wound healing and
scarring).
[0187] Densin is a glycoprotein which has been isolated from the
brain which has all the hallmarks of an adhesion molecule. It is
highly concentrated at synaptic sites in the brain and is expressed
prominently in dendritic processes in developing neurons. Densin
has been characterized as a member of the O-linked
sialoglycoproteins. Densin has relevance to medically important
processes such as regeneration. Given the physiological importance
of synaptic processes and cell adhesion mechanisms in vivo, efforts
are currently being under taken to identify new, native proteins
which are involved in synaptic machinery and cell adhesion. Densin
is further described in Kennedy, M. B, Trends Neurosci. (England),
20(6):264 (1997) and Apperson, et al., J. Neurosci., 16(21):6839
(1996).
[0188] Efforts are therefore being undertaken by both industry and
academia to identify new proteins having leucine rich repeats to
better understand protein-protein interactions. Of particular
interest are those proteins having leucine rich repeats and
homology to known proteins having leucine rich repeats such as
KIAA0231 and densin. Many efforts are focused on the screening of
mammalian recombinant DNA libraries to identify the coding
sequences for novel secreted and membrane-bound proteins having
leucine rich repeats. Examples of screening methods and techniques
are described in the literature [see, for example, Klein et al.,
Proc. Natl. Acad. Sci. 93:7108-7113 (1996); U.S. Pat. No.
5,536,637)].
[0189] We describe herein the identification and characterization
of a novel polypeptide which has homology to leucine rich repeat
proteins, designated herein as PRO247.
[0190] 40. PRO302, PRO303, PRO304, PRO307 and PRO343
[0191] 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.
[0192] In light of the important physiological roles played by
protease enzymes, efforts are currently being undertaken by both
industry and academia to identify new, native protease homologs.
Many of these efforts are focused on the screening of mammalian
recombinant DNA libraries to identify the coding sequences for
novel secreted and membrane-bound receptor proteins. Examples of
screening methods and techniques are described in the literature
[see, for example, Klein et al., Proc. Natl. Acad. Sci.
93:7108-7113 (1996); U.S. Pat. No. 5,536,637)]. We herein describe
the identification of novel polypeptides having homology to various
protease enzymes, designated herein as PRO302, PRO303, PRO304,
PRO307 and PRO343 polypeptides.
[0193] 41. PRO328
[0194] The GLIP protein family has been characterized as comprising
zinc-finger proteins which play important roles in embryogenesis.
These proteins may function as transcriptional regulatory proteins
and are known to be amplified in a subset of human tumors. Glioma
pathogenesis protein is structurally related to a group of plant
pathogenesis-related proteins. It is highly expressed in
glioblastoma. See U.S. Pat. No. 5,582,981 (issued Dec. 10, 1996)
and U.S. Pat. No. 5,322,801 (issued June 21, 1996), Ellington, A.
D. et al., Nature 346:818 (1990), Grindley, J. C. et al., Dev.
Biol., 188(2):337 (1997), Marine, J. C. et al., Mech. Dev.,
63(2):211 (1997), The CRISP or cysteine rich secretory protein
family are a group of proteins which are also structurally related
to a group of plant pathogenesis proteins. [Schwidetzky, U.,
Biochem. J. 321:325 (1997), Pfisterer, P., Mol. Cell Biol.,
16(11):6160 (1996), Kratzschmar, J., Eur. J. Biochem., 236(3):827
(1996)]. We describe herein the identification of a novel
polypeptide which has homology to GLIP and CRISP, designated herein
as PRO328 polypeptides.
[0195] 42. PRO335, PRO331 and PRO326
[0196] Protein-protein interactions include receptor and antigen
complexes and signaling mechanisms. As more is known about the
structural and functional mechanisms underlying protein-protein
interactions, protein-protein interactions can be more easily
manipulated to regulate the particular result of the
protein-protein interaction. Thus, the underlying mechanisms of
protein-protein interactions are of interest to the scientific and
medical community.
[0197] 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 prote in has revealed that leucine-rich
repeats correspond to beta-alpha structural units. These units are
arranged so that they form a parallel beta-sheet with one surface
exposed to solvent, so that the protein acquires an unusual,
nonglubular shape. These two features have been indicated as
responsible for the protein-binding functions of proteins
containing leucine-rich repeats. See, Kobe and Deisenhofer, Trends
Biochem. Sci., 19(10):415-421 (October 1994).
[0198] A study has been reported on leucine-rich proteoglycans
which serve as tissue organizers, orienting and ordering collagen
fibrils during ontogeny and are involved in pathological processes
such as wound healing, tissue repair, and tumor stroma formation.
Iozzo, R. V., Crit. Rev. Biochem. Mol. Biol., 32(2):141-174 (1997).
Others studies implicating leucine rich proteins in wound healing
and tissue repair are De La Salle, C., et al., Vouv. Rev. Fr.
Hematol. (Germany), 37(4):215-222 (1995), reporting mutations in
the leucine rich motif in a complex associated with the bleeding
disorder Bernard-Soulier syndrome, Chlemetson, K. J., Thromb.
Haemost (Germany), 74(1):111-116 (July 1995), reporting that
platelets have leucine rich repeats and Ruoslahti, E. I., et al.,
W09110727-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 connective
tissue, skin and bone; in promoting body growth in humans and
animals; and in stimulating other growth-related processes. The
acid labile subunit of IGF (ALS) is also of interest in that it
increases the half-life of IGF and is part of the IGF complex in
vivo. Another protein which has been reported to have leucine-rich
repeats is the SLIT protein which has been reported to be useful in
treating neuro-degenerative diseases such as Alzheimer's disease,
nerve damage such as in Parkinson's disease, and for diagnosis of
cancer, see, Artavanistsakonas, S. and Rothberg, J. M.,
W09210518-A1 by Yale University. Of particular interest is LIG-1, a
membrane glycoprotein that is expressed specifically in glial cells
in the mouse brain, and has leucine rich repeats and
immunoglobulin-like domains. Suzuki, et al., J. Biol. Chem. (U.S.),
271(37):22522 (1996). Other studies reporting on the biological
functions of proteins having leucine rich repeats include: Tayar,
N., et al., Mol. Cell Endocrinol., (Ireland), 125(1-2):65-70
(December 1996) (gonadotropin receptor involvement); Miura, Y., et
al., Nippon Rinsho (Japan), 54(7): 1784-1789 (July 1996) (apoptosis
involvement); Harris, P. C., et al., J. Am. Soc. Nephrol.,
6(4):1125-1133 (October 1995) (kidney disease involvement).
[0199] Efforts are therefore being undertaken by both industry and
academia to identify new proteins having leucine rich repeats to
better understand protein-protein interactions. Of particular
interest are those proteins having leucine rich repeats and
homology to known proteins having leucine rich repeats such as
LIG-1, ALS and decorin. Many efforts are focused on the screening
of mammalian recombinant DNA libraries to identify the coding
sequences for novel secreted and membrane-bound proteins having
leucine rich repeats. Examples of screening methods and techniques
are described in the literature [see, for example, Klein et al.,
Proc. Natl. Acad. Sci., 93:7108-7113 (1996); U.S. Pat. No.
5,536,637)].
[0200] We describe herein the identification and characterization
of novel polypeptides which have homology to proteins of the
leucine rich repeat superfamily, designated herein as PRO335,
PRO331 and PRO326 polypeptides.
[0201] 43. PRO332
[0202] Secreted proteins comprising a repeat characterized by an
arrangement of conserved leucine residues (leucine-rich repeat
motif) have diverse biological roles. Certain proteoglycans, such
as biglycan, fibromodulin and decorin, are, for example,
characterized by the presence of a leucine-rich repeat of about 24
amino acids [Ruoslahti, Ann. Rev. Cell. Biol. 4 229-255 (1988);
Oldberg et al, EMBO J. 8, 12601-2604 (1989)]. In general,
proteoglycans are believed to play a role in regulating
extracellular matrix, cartilage or bone function. The proteoglycan
decorin binds to collagen type I and II and affects the rate of
fibril formation. Fibromodulin also binds collagen and delays
fibrilformation. Both fibromodulin and decorin inhibit the activity
of transforming growth factor beta (TGF-.beta.) (U.S. Pat. No.
5,583,103 issued Dec. 10, 1996). TGF-.beta. is known to play a key
role in the induction of extracellular matrix and has been
implicated in the development of fibrotic diseases, such as cancer
and glomerulonephritis. Accordingly, proteoglycans have been
proposed for the treatment of fibrotic cancer, based upon their
ability to inhibit TGF-.beta.'s growth stimulating activity on the
cancer cell. Proteoglycans have also been described as potentially
useful in the treatment of other proliferative pathologies,
including rheumatoid arthritis, arteriosclerosis, adult respiratory
distress syndrome, cirrhosis of the liver, fibrosis of the lungs,
post-myocardial infarction, cardiac fibrosis, post-angioplasty
restenosis, renal interstitial fibrosis and certain dermal fibrotic
conditions, such as keloids and scarring, which might result from
burn injuries, other invasive skin injuries, or cosmetic or
reconstructive surgery (U.S. Pat. No. 5,654,270, issued Aug. 5,
1997).
[0203] We describe herein the identification and characterization
of novel polypeptides which have homology to proteins of the
leucine rich repeat superfamily, designated herein as PRO332
polypeptides.
[0204] 44. PRO334
[0205] Microfibril bundles and proteins found in association with
these bundles, particularly attachment molecules, are of interest
in the field of dermatology, particularly in the study of skin
which has been damaged from aging, injuries or the sun. Fibrillin
microfibrils define the continuous elastic network of skin, and are
present in dermis as microfibril bundles devoid of measurable
elastin extending from the dermal-epithelial junction and as
components of the thick elastic fibres present in the deep
reticular dermis. Moreover, Marfan syndrome has been linked to
mutations which interfere with multimerization of fibrillin
monomers or other connective tissue elements.
[0206] Fibulin-1 is a modular glycoprotein with amino-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. Thus,
fibrillin, fibulin, and molecules related thereto are of interest,
particularly for the use of preventing skin from being damaged from
aging, injuries or the sun, or for restoring skin damaged from
same. Moreover, these molecules are generally of interest in the
study of connective tissue and attachment molecules and related
mechanisms. Fibrillin, fibulin and related molecules are further
described in Adams, et al., J. Mol. Biol., 272(2):226-36 (1997);
Kielty and Shuttleworth, Microsc. Res. Tech., 38(4):413-27 (1997);
and Child., J. Card. Sure., 12(2 Supp.):131-5 (1997).
[0207] Currently, efforts are being undertaken by both industry and
academia to identify new, native secreted and membrane-bound
receptor proteins, particularly secreted proteins which have
homology to fibulin and fibrillin. Many efforts are focused on the
screening of mammalian recombinant DNA libraries to identify the
coding sequences for novel secreted and membrane-bound receptor
proteins. Examples of screening methods and techniques are
described in the literature [see, for example, Klein et al., Proc.
Natl. Acad. Sci. 93:7108-7113 (1996); U.S. Pat. No.
5,536,637)].
[0208] We herein describe the identification and characterization
of novel polypeptides having homology to fibulin and fibrillin,
designated herein as PRO334 polypeptides.
[0209] 45. PRO346
[0210] The widespread occurrence of cancer has prompted the
devotion of considerable resources and discovering new treatments
of treatment. One particular method involves the creation of tumor
or cancer specific monoclonal antibodies (mAbs) which are specific
to tumor antigens. Such mAbs, which can distinguish between normal
and cancerous cells are useful in the diagnosis, prognosis and
treatment of the disease. Particular antigens are known to be
associated with neoplastic diseases, such as colorectal and breast
cancer. Since colon cancer is a widespread disease, early diagnosis
and treatment is an important medical goal. Diagnosis and treatment
of cancer can be implemented using monoclonal antibodies (mAbs)
specific therefore having fluorescent, nuclear magnetic or
radioactive tags. Radioactive genes, toxins and/or drug tagged mAbs
can be used for treatment in situ with minimal patient
description.
[0211] Carcinoembryonic antigen (CEA) is a glycoprotein found in
human colon cancer and the digestive organs of a 2-6 month human
embryos. CEA is a known human tumor marker and is widely used in
the diagnosis of neoplastic diseases, such as colon cancer. For
example, when the serum levels of CEA are elevated in a patient, a
drop of CEA levels after surgery would indicate the tumor resection
was successful. On the other hand, a subsequent rise in serum CEA
levels after surgery would indicate that metastases of the original
tumor may have formed or that new primary tumors may have appeared.
CEA may also be a target for mAb, antisense nucleotides.
[0212] 46. PRO268
[0213] Protein disulfide isomerase is an enzymatic protein which is
involved in the promotion of correct refolding of proteins through
the establishment of correct disulfide bond formation. Protein
disulfide isomerase was initially identified based upon its ability
to catalyze the renaturation of reduced denatured RNAse (Goldberger
et al., J. Biol. Chem. 239:1406-14 10.about.1964) and Epstein et
al., Cold Spring Harbor Symp. Quant. Biol. 28:439-''9 (1963)).
Protein disulfide isomerase has been shown to be a resident enzyme
of the endoplasmic reticulum which is retained in the endoplasmic
reticulum via a--KDEL or--HDEL amino acid sequence at its
C-terminus.
[0214] Given the importance of disulfide bond-forming enzymes and
their potential uses in a number of different applications, for
example in increasing the yield of correct refolding of
recombinantly produced proteins, efforts are currently being
undertaken by both industry and academia to identify new, native
proteins having homology to protein disulfide isomerase. Many of
these efforts are focused on the screening of mammalian recombinant
DNA libraries to identify the coding sequences for novel protein
disulfide isomerase homologs. We herein describe a novel
polypeptide having homology to protein disulfide isomerase,
designated herein as PRO268.
[0215] 47. PRO330
[0216] Prolyl 4-hydroxylase is an enzyme which functions to
post-translationally hydroxylate proline residues at the Y position
of the amino acid sequence Gly-X-Y, which is a repeating three
amino acid sequence found in both collagen and procollagen.
Hydroxylation of proline residues at the Y position of the Gly-X-Y
amino acid triplet to form 4-hydroxyproline residues at those
positions is required before newly synthesized collagen polypeptide
chains may fold into their proper three-dimensional triple-helical
conformation. If hydroxylation does not occur, synthesized collagen
polypeptides remain non-helical, are poorly secreted by cells and
cannot assemble into stable functional collagen fibrils. Vuorio et
al., Proc. Nati. Acad. Sc. USA 89:7467-7470 (1992). Prolyl
4-hydroxylase is comprised of at least two different polypeptide
subunits, alpha and beta.
[0217] Efforts are being undertaken by both industry and academia
to identify new, native secreted and membrane-bound receptor
proteins. Many efforts are focused on the screening of mammalian
recombinant DNA libraries to identify the coding sequences for
novel secreted and membrane-bound receptor proteins. Examples of
screening methods and techniques are described in the literature
[see, for example, Klein et al., Proc. Nati. Acad. Sci.,
93:7108-7113 (1996); U.S. Pat. No. 5,536,637)]. Based upon these
efforts, Applicants have herein identified and describe a novel
polypeptide having homology to the alpha subunit of prolyl
4-hydroxylase, designated herein as PRO330.
[0218] 48. PRO339 and PRO310
[0219] Fringe is a protein which specifically blocks
serrate-mediated activation of notch in the dorsal compartment of
the Drosophila wing imaginal disc. Fleming, et al., Development,
124(15):2973-81 (1997). Therefore, fringe is of interest for both
its role in development as well as its ability to regulate serrate,
particularly serrate's signaling abilities. Also of interest are
novel polypeptides which may have a role in development and/or the
regulation of serrate-like molecules. Of particular interest are
novel polypeptides: having homology to fringe as identified and
described herein, designated herein as PRO339 and PRO310
polypeptides.
[0220] 49. PRO244
[0221] Lectins are a class of proteins comprising a region that
binds carbohydrates specifically and non-covalently. Numerous
lectins have been identified in higher animals, both membrane-bound
and soluble, and have been implicated in a variety of
cell-recognition phenomena and tumor metastasis.
[0222] Most lectins can be classified as either C-type
(calcium-dependent) or S-type (thiol-dependent).
[0223] Lectins are thought to play a role in regulating cellular
events that are initiated at the level of the plasma membrane. For
example, plasma membrane associated molecules are involved in the
activation of various subsets of lymphoid cells, e.g.
T-lymphocytes, and it is known that cell surface molecules are
responsible for activation of these cells and consequently their
response during an immune reaction.
[0224] A particular group of cell adhesion molecules, selecting,
belong in the superfamily of C-type lectins. This group includes
L-selectin (peripheral lymph node homing receptor (pnHR),
LEC-CAM-1, LAM-1, gp90.sup.MEL, gp100.sup.MEL, gp110.sup.MEL,
MEL-14 antigen, Leu-8 antigen, TQ-1 antigen, DREG antigen),
E-selectin (LEC-CAM-2, LECAM-2, ELAM-1), and P-selectin (LEC-CAM-3,
LECAM-3, GMP-140, PADGEM). The structure of selectins consists of a
C-type lectin (carbohydrate binding) domain, an epidermal growth
factor-like (EGF-like) motif, and variable numbers of complement
regulatory (CR) motifs. Selectins are associated with leukocyte
adhesion, e.g. the attachment of neutrophils to venular endothelial
cells adjacent to inflammation (E-selectin), or with the
trafficking of lymphocytes from blood to secondary lymphoid organs,
e.g. lymph nodes and Peyer's patches (L-selectin).
[0225] Another exemplary lectin is the cell-associated macrophage
antigen, Mac-2 that is believed to be involved in cell adhesion and
immune responses. Macrophages also express a lectin that recognizes
Tn Ag, a human carcinoma-associated epitope.
[0226] Another C-type lectin is CD95 (Fas antigen/APO-1) that is an
important mediator of immunologically relevant regulated or
programmed cell death (apoptosis). "Apoptosis" is a non-necrotic
cell death that takes place in metazoan animal cells following
activation of an intrinsic cell suicide program. The cloning of Fas
antigen is described in PCT publication WO 91/10448, and European
patent application EP510691. The mature Fas molecule consists of
319 amino acids of which 157 are extracellular, 17 constitute the
transmembrane domain, and 145 are intracellular. Increased levels
of Fas expression at T cell surface have been associated with tumor
cells and HIV-infected cells. Ligation of CD95 triggers apoptosis
in the presence of interleukin-1 (IL-2). C-type lectins also
include receptors for oxidized low-density lipoprotein (LDL). This
suggests a possible role in the pathogenesis of
atherosclerosis.
[0227] We herein describe the identification and characterization
of novel polypeptides having homology to C-type lectins, designated
herein as PRO244 polypeptides.
SUMMARY OF THE INVENTION
[0228] 1. PRO211 and PRO217
[0229] Applicants have identified cDNA clones that encode novel
polypeptides having homology to EGF, designated in the present
application as "PRO211" and "PRO217" polypeptides.
[0230] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO211 or PRO217
polypeptide. In one aspect, the isolated nucleic acid comprises DNA
encoding EGF-like homologue PRO211 and PRO217 polypeptides of FIG.
2 (SEQ ID NO:2) and/or 4 (SEQ ID NO:4) indicated in FIG. 1 (SEQ ID
NO: 1) and/or FIG. 3 (SEQ ID NO:3), respectively, or is
complementary to such encoding nucleic acid sequence, and remains
stably bound to it under at least moderate, and optionally, under
high stringency conditions.
[0231] In another embodiment, the invention provides isolated
PRO211 and PRO217 EGF-like homologue PRO211 and PRO217
polypeptides. In particular, the invention provides isolated native
sequence PRO211 and PRO217 EGF-like homologue polypeptides, which
in one embodiment, includes an amino acid sequence comprising
residues: 1 to 353 of FIG. 2 (SEQ ID NO:2) or (2) 1 to 379 of FIG.
4 (SEQ ID NO: 4).
[0232] 2. PRO230
[0233] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as PRO230".
[0234] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO230 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO230 polypeptide having amino acid residues 1 through 467 of FIG.
6 (SEQ ID NO: 12), or is complementary to such encoding nucleic
acid sequence, and remains stably bound to it under at least
moderate, and optionally, under high stringency conditions.
[0235] In another embodiment, the invention provides isolated
PRO230 polypeptide. In particular, the invention provides isolated
native sequence PRO230 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 through 467
of FIG. 6 (SEQ ID NO: 12).
[0236] In another embodiment, the invention provides an expressed
sequence tag (EST) comprising the nucleotide sequence of SEQ ID NO:
13 (FIG. 7) which is herein designated as DNA20088.
[0237] 3. PRO232
[0238] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as "PRO232".
[0239] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO232 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO232 polypeptide having amino acid residues 1 to 114 of FIG. 9
(SEQ ID NO: 18), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0240] In another embodiment, the invention provides isolated
PRO232 polypeptide. In particular, the invention provides isolated
native sequence PRO232 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 114 of
FIG. 9 (SEQ ID NO: 18).
[0241] 4. PRO187
[0242] Applicants have identified a cDNA clone that encodes a novel
polypeptide,.designated in the present application as "PRO187".
[0243] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO187 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO187 polypeptide of FIG. 11 (SEQ ID NO:23), or is complementary
to such encoding nucleic acid sequence, and remains stably bound to
it under at least moderate, and optionally, under high stringency
conditions. In another aspect, the invention provides a nucleic
acid comprising the coding sequence of FIG. 10 (SEQ ID NO:22) or
its complement. In another aspect, the invention provides a nucleic
acid of the full length protein of clone DNA27864-1155, deposited
with the ATCC under accession number ATCC 209375, alternatively the
coding sequence of clone DNA27864-1155, deposited under accession
number ATCC 209375.
[0244] In yet another embodiment, the invention provides isolated
PRO187 polypeptide. In particular, the invention provides isolated
native sequence PRO187 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 205 of
FIG. 11 (SEQ ID NO:23). Alternatively, the invention provides a
polypeptide encoded by the nucleic acid deposited under accession
number ATCC 209375.
[0245] 5. PRO265
[0246] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as "PRO265".
[0247] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO265 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO265 polypeptide having amino acid residues 1 to 660 of FIG. 13
(SEQ ID NO:28), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0248] In another embodiment, the invention provides isolated
PRO265 polypeptide. In particular, the invention provides isolated
native sequence PRO265 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 660 of
FIG. 13 (SEQ ID NO:28). An additional embodiment of the present
invention is directed to an isolated extracellular domain of a
PRO265 polypeptide.
[0249] 6. PRO219
[0250] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as "PRO219".
[0251] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO219 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO219 polypeptide having amino acid residues 1 to 915 of FIG. 15
(SEQ ID NO:34), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0252] In another embodiment, the invention provides isolated
PRO219 polypeptide. In particular, the invention provides isolated
native sequence PRO219 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 915 of
FIG. 15 (SEQ ID NO:34).
[0253] 7. PRO246
[0254] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as "PRO246".
[0255] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO246 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO246 polypeptide having amino acid residues 1 to 390 of FIG. 17
(SEQ ID NO:39), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0256] In another embodiment, the invention provides isolated
PRO246 polypeptide. In particular, the invention provides isolated
native sequence PRO246 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 390 of
FIG. 17 (SEQ ID NO:39). An additional embodiment of the present
invention is directed to an isolated extracellular domain of a
PRO246 polypeptide.
[0257] 8. PRO228
[0258] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to CD97, EMR1 and latrophilin, wherein
the polypeptide is designated in the present application as
"PRO228".
[0259] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO228 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO228 polypeptide having amino acid residues 1 to 690 of FIG. 19
(SEQ ID NO:49), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0260] In another embodiment, the invention provides isolated
PRO228 polypeptide. In particular, the invention provides isolated
native sequence PRO228 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 690 of
FIG. 19 (SEQ ID NO:49). An additional embodiment of the present
invention is directed to an isolated extracellular domain of a
PRO228 polypeptide.
[0261] In another embodiment, the invention provides an expressed
sequence tag (EST) comprising the nucleotide sequence of SEQ ID
NO:50, designated herein as DNA21951.
[0262] 9. PRO533
[0263] Applicants have identified a cDNA clone (DNA49435-1219) that
encodes a novel polypeptide, designated in the present application
as PRO533.
[0264] In one embodiment, the invention provides an isolated
nucleic acid molecule having at least about 80% sequence identity
to (a) a DNA molecule encoding a PRO533 polypeptide comprising the
sequence of amino acids 23 to 216 of FIG. 22 (SEQ ID NO:59), or (b)
the complement of the DNA molecule of (a). The sequence identity
preferably is about 85%, more preferably about 90%, most preferably
about 95%. In one aspect, the isolated nucleic acid has at least
about 80%, preferably at least about 85%, more preferably at least
about 90%, and most preferably at least about 95% sequence identity
with a polypeptide having amino acid residues 23 to 216 of FIG. 22
(SEQ ID NO:59). Preferably, the highest degree of sequence identity
occurs within the secreted portion (amino acids 23 to 216 of FIG.
22, SEQ ID NO:59). In a further embodiment, the isolated nucleic
acid molecule comprises DNA encoding a PRO533 polypeptide having
amino acid residues 1 to 216 of FIG. 22 (SEQ ID NO:59), or is
complementary to such encoding nucleic acid sequence, and remains
stably bound to it under at least moderate, and optionally, under
high stringency conditions. In another aspect, the invention
provides a nucleic acid of the full length protein of clone
DNA49435-1219, deposited with the ATCC under accession number ATCC
209480.
[0265] In yet another embodiment, the invention provides isolated
PRO533 polypeptide. In particular, the invention provides isolated
native sequence PRO533 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 23 to 216 of
FIG. 22 (SEQ ID NO:59). Native PRO533 polypeptides with or without
the native signal sequence (amino acids 1 to 22 in FIG. 22 (SEQ ID
NO:59)), and with or without the initiating methionine are
specifically included. Alternatively, the invention provides a
PRO533 polypeptide encoded by the nucleic acid deposited under
accession number ATCC 209480.
[0266] 10. PRO245
[0267] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as "PRO245".
[0268] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO245 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO245 polypeptide having amino acid residues 1 to 312 of FIG. 24
(SEQ ID NO:64), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0269] In another embodiment, the invention provides isolated
PRO245 polypeptide. In particular, the invention provides isolated
native sequence PRO245 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 312 of
FIG. 24 (SEQ ID NO:64).
[0270] 11. PRO220, PRO221 and PRO227
[0271] Applicants have identified cDNA clones that each encode
novel polypeptides, all having leucine rich repeats. These
polypeptides are designated in the present application as PRO220,
PRO221 and PRO227.
[0272] In one embodiment, the invention provides isolated nucleic
acid molecules comprising DNA respectively encoding PRO220, PRO221
and PRO227, respectively. In one aspect, provided herein is an
isolated nucleic acid comprises DNA encoding the PRO220 polypeptide
having amino acid residues 1 through 708 of FIG. 26 (SEQ ID NO:69),
or is complementary to such encoding nucleic acid sequence, and
remains stably bound to it under at least moderate, and optionally,
under high stringency conditions. Also provided herein is an
isolated nucleic acid comprises DNA encoding the PRO221 polypeptide
having amino acid residues 1 through 259 of FIG. 28 (SEQ ID NO
:71), or is complementary to such encoding nucleic acid sequence,
and remains stably bound to it under at least moderate, and
optionally, under high stringency conditions. Moreover, also
provided herein is an isolated nucleic acid comprises DNA encoding
the PRO227 polypeptide having amino acid residues 1 through 620 of
FIG. 30 (SEQ ID NO:73), or is complementary to such encoding
nucleic acid sequence, and remains stably bound to it under at
least moderate, and optionally, under high stringency
conditions.
[0273] In another embodiment, the invention provides isolated
PRO220, PRO221 and PRO227 polypeptides. In particular, provided
herein is the isolated native sequence for the PRO220 polypeptide,
which in one embodiment, includes an amino acid sequence comprising
residues 1 to 708 of FIG. 26 (SEQ ID NO:69). Additionally provided
herein is the isolated native sequence for the PRO221 polypeptide,
which in one embodiment, includes an amino acid sequence comprising
residues 1 to 259 of FIG. 28 (SEQ ID NO:71). Moreover, provided
herein is the isolated native sequence for the PRO227 polypeptide,
which in one embodiment, includes an amino acid sequence comprising
residues 1 to 620 of FIG. 30 (SEQ ID NO:73).
[0274] 12. PRO258
[0275] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to CRTAM and poliovirus receptor
precursors, wherein the polypeptide is designated in the present
application as "PRO258".
[0276] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO258 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO258 polypeptide having amino acid residues 1 to 398 of FIG. 32
(SEQ ID NO:84), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0277] In another embodiment, the invention provides isolated
PRO258 polypeptide. In particular, the invention provides isolated
native sequence PRO258 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 398 of
FIG. 32 (SEQ ID NO:84). An additional embodiment of the present
invention is directed to an isolated extracellular domain of a
PRO258 polypeptide.
[0278] 13. PRO266
[0279] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as "PRO266".
[0280] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO266 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO266 polypeptide having amino acid residues 1 to 696 of FIG. 34
(SEQ ID NO:91), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0281] In another embodiment, the invention provides isolated
PRO266 polypeptide. In particular, the invention provides isolated
native sequence PRO266 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 696 of
FIG. 34 (SEQ ID NO:91).
[0282] 14. PRO269
[0283] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as PRO269.
[0284] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO269 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO269 polypeptide having amino acid residues 1 to 490 of FIG. 36
(SEQ ID NO:96), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0285] In another embodiment, the invention provides isolated
PRO269 polypeptide. In particular, the invention provides isolated
native sequence PRO269 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 490 of
FIG. 36 (SEQ ID NO:96). An additional embodiment of the present
invention is directed to an isolated extracellular domain of a
PRO269 polypeptide.
[0286] 15. PRO287
[0287] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as "PRO287".
[0288] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO287 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO287 polypeptide having amino acid residues 1 to 415 of FIG. 38
(SEQ ID NO: 104), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0289] In another embodiment, the invention provides isolated
PRO287 polypeptide. In particular, the invention provides isolated
native sequence PRO287 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 415 of
FIG. 38 (SEQ ID NO: 104).
[0290] 16. PRO214
[0291] Applicants have identified a cDNA clone that encodes a novel
polypeptide, designated in the present application as "PRO214".
[0292] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO214 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO214 polypeptide of FIG. 40 (SEQ ID NO: 109), or is complementary
to such encoding nucleic acid sequence, and remains stably bound to
it under at least moderate, and optionally, under high stringency
conditions. In another aspect, the invention provides a nucleic
acid comprising the coding sequence of FIG. 39 (SEQ ID NO: 108) or
its complement. In another aspect, the invention provides a nucleic
acid of the full length protein of clone DNA32286-1191, deposited
with ATCC under accession number ATCC 209385.
[0293] In yet another embodiment, the invention provides isolated
PRO214 polypeptide. In particular, the invention provides isolated
native sequence PRO214 polypeptide, which in one embodiment,
includes an amino acid sequence comprising the residues of FIG. 40
(SEQ ID NO: 109). Alternatively, the invention provides a
polypeptide encoded by the nucleic acid deposited under accession
number ATCC 209385.
[0294] 17. PRO317
[0295] Applicants have identified a cDNA clone that encodes a novel
polypeptide, designated in the present application as "PRO317".
[0296] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding PRO317 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA (SEQ ID
NO:113) encoding PRO317 polypeptide having amino acid residues 1 to
366 of FIG. 42, or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0297] In another embodiment, the invention provides isolated
PRO317 polypeptide. In particular, the invention provides isolated
native-sequence PRO317 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 366 of
FIG. 42 (SEQ ID NO: 114).
[0298] In yet another embodiment, the invention supplies a method
of detecting the presence of PRO317 in a sample, the method
comprising:
[0299] a) contacting a detectable anti-PRO317 antibody with a
sample suspected of containing PRO317; and
[0300] b) detecting binding of the antibody to the sample; wherein
the sample is selected from the group consisting of a body fluid, a
tissue sample, a cell extract, and a cell culture medium.
[0301] In a still further embodiment a method is provided for
determining the presence of PRO317 mRNA in a sample, the method
comprising:
[0302] a) contacting a sample suspected of containing PRO317 mRNA
with a detectable nucleic acid probe that hybridizes under moderate
to stringent conditions to PRO317 mRNA; and
[0303] b) detecting hybridization of the probe to the sample.
[0304] Preferably, in this method the sample is a tissue sample and
the detecting step is by in situ hybridization, or the sample is a
cell extract and detection is by Northern analysis.
[0305] Further, the invention provides a method for treating a
PRO317-associated disorder comprising administering to a mammal an
effective amount of the PRO317 polypeptide or a composition thereof
containing a carrier, or with an effective amount of a PRO317
agonist or PRO317 antagonist, such as an antibody which binds
specifically to PRO317.
[0306] 18. PRO301
[0307] Applicants have identified A cDNA clone (DNA40628-1216) that
encodes a novel polypeptide, designated in the present application
as "PRO301".
[0308] In one embodiment, the invention provides an isolated
nucleic acid molecule having at least about 80% sequence identity
to (a) a DNA molecule encoding a PRO301 polypeptide comprising the
sequence of amino acids 28 to 258 of FIG. 44 (SEQ ID NO: 119), or
(b) the complement of the DNA molecule of (a). The sequence
identity preferably is about 85%, more preferably about 90%, most
preferably about 95%. In one aspect, the isolated nucleic acid has
at least about 80%, preferably at least about 85%, more preferably
at least about 90%, and most preferably at least about 95% sequence
identity with a polypeptide having amino acid residues 28 to 258 of
FIG. 44 (SEQ ID NO: 119). Preferably, the highest degree of
sequence identity occurs within the extracellular domains (amino
acids 28 to 258 of FIG. 44, SEQ ID NO: 119). In a further
embodiment, the isolated nucleic acid molecule comprises DNA
encoding a PRO301 polypeptide having amino acid residues 28 to 299
of FIG. 44 (SEQ ID NO: 119), or is complementary to such encoding
nucleic acid sequence, and remains stably bound to it under at
least moderate, and optionally, under high stringency conditions.
In another aspect, the invention provides a nucleic acid of the
full length protein of clone DNA40628-1216, deposited with the ATCC
under accession number ATCC 209432, alternatively the coding
sequence of clone DNA40628-1216, deposited under accession number
ATCC 209432.
[0309] In yet another embodiment, the invention provides isolated
PRO301 polypeptide. In particular, the invention provides isolated
native sequence PRO301 polypeptide, which in one embodiment,
includes an amino acid sequence comprising the extracellular domain
residues 28 to 258 of FIG. 44 (SEQ ID NO: 119). Native PRO301
polypeptides with or without the native signal sequence (amino
acids 1 to 27 in FIG. 44 (SEQ ID NO: 119), and with or without the
initiating methionine are specifically included. Additionally, the
sequences of the invention may also comprise the transmembrane
domain (residues 236 to about 258 in FIG. 44; SEQ ID NO: 119)
and/or the intracellular domain (about residue 259 to 299 in FIG.
44; SEQ ID NO: 119). Alternatively, the invention provides a PRO301
polypeptide encoded by the nucleic acid deposited under accession
number ATCC 209432.
[0310] 19. PRO224
[0311] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as "PRO224".
[0312] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO224 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO224 polypeptide having amino acid residues 1 to 282 of FIG. 46
(SEQ ID NO: 127), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0313] In another embodiment, the invention provides isolated
PRO224 polypeptide. In particular, the invention provides isolated
native sequence PRO224 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 282 of
FIG. 46 (SEQ ID NO: 127).
[0314] 20. PRO222
[0315] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as "PRO222".
[0316] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO222 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO222 polypeptide having amino acid residues 1 to 490 of FIG. 48
(SEQ ID NO: 132), or is complementary to such encoding nucleic;
acid sequence, and remains stably bound to it under at least
moderate, and optionally, under high stringency conditions.
[0317] In another embodiment, the invention provides isolated
PRO222 polypeptide. In particular, the invention provides isolated
native sequence PRO222 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 490 of
FIG. 48 (SEQ ID NO:132).
[0318] 21. PRO234
[0319] Applicants have identified a cDNA clone that encodes a novel
lectin polypeptide molecule, designated in the present application
as "PRO234".
[0320] In one embodiment, the invention provides an isolated
nucleic acid encoding a novel lectin comprising DNA encoding a
PRO234 polypeptide. In one aspect, the isolated nucleic acid
comprises the DNA encoding. PRO234 polypeptides having amino acid
residues 1 to 382 of FIG. 50 (SEQ ID NO: 137), or is complementary
to such encoding nucleic acid sequence, and remains stably bound to
it under at least moderate, and optionally, under high stringency
conditions. In another aspect, the invention provides an isolated
nucleic acid molecule comprising the nucleotide sequence of FIG. 49
(SEQ ID NO: 136).
[0321] In another embodiment, the invention provides isolated novel
PRO234 polypeptides. In particular, the invention provides isolated
native sequence PRO234 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 382 of
FIG. 50 (SEQ ID NO: 137).
[0322] In yet another embodiment, the invention provides
oligonucleotide probes useful for isolating genomic and cDNA
nucleotide sequences.
[0323] 22. PRO231
[0324] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to a putative acid phosphatase, wherein
the polypeptide is designated in the present application as
"PRO231".
[0325] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO231 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO231 polypeptide having amino acid residues 1 to 428 of FIG. 52
(SEQ ID NO: 142), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0326] In another embodiment, the invention provides isolated
PRO231 polypeptide. In particular, the invention provides isolated
native sequence PRO231 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 428 of
FIG. 52 (SEQ ID NO: 142).
[0327] 23. PRO229
[0328] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to scavenger receptors wherein the
polypeptide is designated in the present application as
"PRO229".
[0329] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO229 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO229 polypeptide having amino acid residues 1 to 347 of FIG. 54
(SEQ ID NO: 148), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0330] In another embodiment, the invention provides isolated
PRO229 polypeptide. In particular, the invention provides isolated
native sequence PRO229 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 347 of
FIG. 54 (SEQ ID NO: 148).
[0331] 24. PRO238
[0332] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to reductase, wherein the polypeptide
is designated in the present application as "PRO238".
[0333] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO238 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO238 polypeptide having amino acid residues 1 to 310 of FIG. 56
(SEQ ID NO: 153), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0334] In another embodiment, the invention provides isolated
PRO238 polypeptide. In particular, the invention provides isolated
native sequence PRO238 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 310 of
FIG. 56 (SEQ ID NO: 153).
[0335] 25. PRO233
[0336] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as "PRO233".
[0337] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO233 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO233 polypeptide having amino acid residues 1 to 300 of FIG. 58
(SEQ ID NO: 159), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0338] In another embodiment, the invention provides isolated
PRO233 polypeptide. In particular, the invention provides isolated
native sequence PRO233 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 300 of
FIG. 58 (SEQ ID NO: 159).
[0339] 26. PRO223
[0340] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to serine carboxypeptidase
polypeptides, wherein the polypeptide is designated in the present
application as "PRO223".
[0341] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO223 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO223 polypeptide having amino acid residues 1 to 476 of FIG. 60
(SEQ ID NO: 164), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0342] In another embodiment, the invention provides isolated
PRO223 polypeptide. In particular, the invention provides isolated
native sequence PRO223 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 476 of
FIG. 60 (SEQ ID NO: 164).
[0343] 27. PRO235
[0344] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as "PRO235".
[0345] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO235 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO235 polypeptide having amino acid residues 1 to 552 of FIG. 62
(SEQ ID NO: 170), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0346] In another embodiment, the invention provides isolated
PRO235 polypeptide. In particular, the invention provides isolated
native sequence PRO235 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 552 of
FIG. 62 (SEQ ID NO: 170).
[0347] 28. PRO236 and PRO262
[0348] Applicants have identified cDNA clones that encode novel
polypeptides having homology to galactosidase, wherein those
polypeptides are designated in the present application as "PRO236"
and "PRO262".
[0349] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO236 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO236 polypeptide having amino acid residues 1 to 636 of FIG. 64
(SEQ ID NO: 175), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0350] In another embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO262 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO262 polypeptide having amino acid residues 1 to 654 of FIG. 66
(SEQ ID NO: 177), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0351] In another embodiment, the invention provides isolated
PRO236 polypeptide. In particular, the invention provides isolated
native sequence PRO236 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 636 of
FIG. 64 (SEQ ID NO: 175).
[0352] In another embodiment, the invention provides isolated
PRO262 polypeptide. In particular, the invention provides isolated
native sequence PRO262 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 654 of
FIG. 66 (SEQ ID NO: 177).
[0353] 29. PRO239
[0354] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as "PRO239".
[0355] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO239 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO239 polypeptide having amino acid residues 1 to 501 of FIG. 68
(SEQ ID NO: 185), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0356] In another embodiment, the invention provides isolated
PRO239 polypeptide. In particular, the invention provides isolated
native sequence PRO239 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 501 of
FIG. 68 (SEQ ID NO: 185).
[0357] 30. PRO257
[0358] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as "PRO257".
[0359] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO257 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO257 polypeptide having-amino acid residues 1 to 607 of FIG. 70
(SEQ ID NO: 190), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0360] In another embodiment, the invention provides isolated
PRO257 polypeptide. In particular, the invention provides isolated
native sequence PRO257 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 607 of
FIG. 70 (SEQ ID NO: 190). An additional embodiment of the present
invention is directed to an isolated extracellular domain of a
PRO257 polypeptide.
[0361] 31. PRO260
[0362] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as "PRO260".
[0363] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO260 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO260 polypeptide having amino acid residues 1 to 467 of FIG. 72
(SEQ ID NO: 195), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0364] In another embodiment, the invention provides isolated
PRO260 polypeptide. In particular, the invention provides isolated
native sequence PRO260 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 467 of
FIG. 72 (SEQ ID NO: 195).
[0365] 32. PRO263
[0366] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to CD44 antigen, wherein the
polypeptide is designated in the present application as
"PRO263".
[0367] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO263 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO263 polypeptide having amino acid residues 1 to 322 of FIG. 74
(SEQ ID NO:201), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0368] In another embodiment, the invention provides isolated
PRO263 polypeptide. In particular, the invention provides isolated
native sequence PRO263 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 322 of
FIG. 74 (SEQ ID NO:201). An additional embodiment of the present
invention is directed to an isolated extracellular domain of a
PRO263 polypeptide.
[0369] 33. PRO270
[0370] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as "PRO270".
[0371] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO270 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA which
includes the sequence encoding the PRO270 polypeptide having amino
acid residues 1 to 296 of FIG. 76 (SEQ ID NO:207), or is
complementary to such encoding nucleic acid sequence, and remains
stably bound to it under at least moderate, and optionally, under
high stringency conditions.
[0372] In another embodiment, the invention provides isolated
PRO270 polypeptide. In particular, the invention provides isolated
native sequence PRO270 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 296 of
FIG. 76 (SEQ ID NO:207).
[0373] 34. PRO271
[0374] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to the proteoglycan link protein,
wherein the polypeptide is designated in the present application as
"PRO271".
[0375] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO271 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO271 polypeptide having amino acid residues 1 to 360 of FIG. 78
(SEQ ID NO:213), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0376] In another embodiment, the invention provides isolated
PRO271 polypeptide. In particular, the invention provides isolated
native sequence PRO271 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 360 of
FIG. 78 (SEQ ID NO:213).
[0377] 35. PRO272
[0378] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as "PRO272".
[0379] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO272 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO272 polypeptide having amino acid residues 1 to 328 of FIG. 80
(SEQ ID NO:221), or is complementary to such encoding nucleic; acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0380] In another embodiment, the invention provides isolated
PRO272 polypeptide. In particular, the invention provides isolated
native sequence PRO272 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 328 of
FIG. 80 (SEQ ID NO:211).
[0381] 36. PRO294
[0382] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as "PRO294".
[0383] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO294 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO294 polypeptide having amino acid residues 1 to 550 of FIG. 82
(SEQ ID NO:227), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0384] In another embodiment, the invention provides isolated
PRO294 polypeptide. In particular, the invention provides isolated
native sequence PRO294 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 550 of
FIG. 82 (SEQ ID NO:227).
[0385] 37. PRO295
[0386] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as "PRO295".
[0387] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO295 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO295 polypeptide having amino acid residues 1 to 350 of FIG. 84
(SEQ 113 NO:236), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0388] In another embodiment, the invention provides isolated
PRO295 polypeptide. In particular, the invention provides isolated
native sequence PRO295 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 350 of
FIG. 84 (SEQ ID NO:236).
[0389] 38. PRO293
[0390] Applicants have identified a cDNA clone that encodes a novel
human neuronal leucine rich repeat polypeptide, wherein the
polypeptide is designated in the present application as
"PRO293".
[0391] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO293 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO293 polypeptide having amino acid residues 1 to 713 of FIG. 86
(SEQ ID NO:245), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0392] In another embodiment, the invention provides isolated
PRO293 polypeptide. In particular, the invention provides isolated
native sequence PRO293 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 713 of
FIG. 86 (SEQ ID NO:245). An additional embodiment of the present
invention is directed to an isolated extracellular domain of a
PRO293 polypeptide.
[0393] 39. PRO247
[0394] Applicants have identified a cDNA clone that encodes a novel
polypeptide having leucine rich repeats wherein the polypeptide is
designated in the present application as "PRO247".
[0395] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO247 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO247 polypeptide having amino acid residues 1 to 546 of FIG. 88
(SEQ ID NO:250), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0396] In another embodiment, the invention provides isolated
PRO247 polypeptide. In particular, the invention provides isolated
native sequence PRO247 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 546 of
FIG. 88 (SEQ ID NO:250). An additional embodiment of the present
invention is directed to an isolated extracellular domain of a
PRO247 polypeptide.
[0397] 40. PRO302, PRO303, PRO304, PRO307 and PRO343
[0398] Applicants have identified cDNA clones that encode novel
polypeptides having homology to various proteases, wherein those
polypeptide are designated in the present application as "PRO302",
"PRO303", "PRO304", "PRO307" and "PRO343" polypeptides.
[0399] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO302 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO302 polypeptide having amino acid residues 1 to 452 of FIG. 90
(SEQ ID NO:255), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0400] In another embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO303 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO303 polypeptide having amino acid residues 1 to 314 of FIG. 92
(SEQ ID NO:257), or is complementary to such encoding nucleic acid
sequence; and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0401] In yet another embodiment, the invention provides an
isolated nucleic acid molecule comprising DNA encoding a PRO304
polypeptide. In one aspect, the isolated nucleic acid comprises DNA
encoding the PRO304 polypeptide having amino acid residues 1 to 556
of FIG. 94 (SEQ ID NO:259), or is complementary to such encoding
nucleic acid sequence, and remains stably bound to it under at
least moderate, and optionally, under high stringency
conditions.
[0402] In another embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO307 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO307 polypeptide having amino acid residues 1 to 383 of FIG. 96
(SEQ ID NO:261), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0403] In another embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO343 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO343 polypeptide having amino acid residues 1 to 317 of FIG. 98
(SEQ ID NO:263), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0404] In another embodiment, the invention provides isolated
PRO302 polypeptide. In particular, the invention provides isolated
native sequence PRO302 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 452 of
FIG. 90 (SEQ ID NO:255).
[0405] In another embodiment, the invention provides isolated
PRO303 polypeptide. In particular, the invention provides isolated
native sequence PRO303 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 314 of
FIG. 92 (SEQ ID NO.257).
[0406] In another embodiment, the invention provides isolated
PRO304 polypeptide. In particular, the invention provides isolated
native sequence PRO304 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 556 of
FIG. 94 (SEQ ID NO:259).
[0407] In another embodiment, the invention provides isolated
PRO307 polypeptide. In particular, the invention provides isolated
native sequence PRO307 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 383 of
FIG. 96 (SEQ ID NO:261).
[0408] In another embodiment, the invention provides isolated
PRO343 polypeptide. In particular, the invention provides isolated
native sequence PRO343 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 317 of
FIG. 98 (SEQ ID NO:263).
[0409] 41. PRO328
[0410] Applicants have identified a cDNA clone that encodes a novel
polypeptide, wherein the polypeptide is designated in the present
application as "PRO328".
[0411] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO328 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO328 polypeptide having amino acid residues 1 to 463 of FIG. 100
(SEQ ID NO:285), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0412] In another embodiment, the invention provides isolated
PRO328 polypeptide. In particular, the invention provides isolated
native sequence PRO328 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 463 of
FIG. 100 (SEQ ID NO:285). An additional embodiment of the present
invention is directed to an isolated extracellular domain of a
PRO306 polypeptide.
[0413] 42. PRO335, PRO331 and PRO326
[0414] Applicants have identified three cDNA clones that
respectively encode three novel polypeptides, each having leucine
rich repeats and homology to LIG-1 and ALS. These polypeptides are
designated in the present application as PRO335, PRO331 and PRO326,
respectively.
[0415] In one embodiment, the invention provides three isolated
nucleic acid molecules comprising DNA respectively encoding PRO335,
PRO331 and PRO326, respectively. In one aspect, herein is provided
an isolated nucleic acid comprising DNA encoding the PRO335
polypeptide having amino acid residues 1 through 1059 of FIG. 102
(SEQ ID NO:290), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions. Also provided
herein is an isolated nucleic acid comprises DNA encoding the
PRO331 polypeptide having amino acid residues 1 through 640 of FIG.
104 (SEQ ID NO:292), or is complementary to such encoding nucleic
acid sequence, and remains stably bound to it under at least
moderate, and optionally, under high stringency conditions.
Additionally provided herein is an isolated nucleic acid comprises
DNA encoding the PRO326 polypeptide having amino acid residues 1
through 1119 of FIG. 106 (SEQ ID NO:294), or is complementary to
such encoding nucleic acid sequence, and remains stably bound to it
under at least moderate, and optionally, under high stringency
conditions.
[0416] In another embodiment, the invention provides isolated
PRO335, PRO331 and PRO326 polypeptides or extracellular domains
thereof. In particular, the invention provides isolated native
sequence for the PRO335 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 through 1059
of FIG. 102 (SEQ ID NO:290). Also provided herein is the isolated
native sequence for the PRO331 polypeptide, which in one
embodiment, includes an amino acid sequence comprising residues 1
through 640 of FIG. 104 (SEQ ID NO:292). Also provided herein is
the isolated native sequence for the PRO326 polypeptide, which in
one embodiment, includes an amino acid sequence comprising residues
1 through 1119 of FIG. 106 (SEQ ID NO:294).
[0417] 43. PRO332
[0418] Applicants have identified a cDNA clone (DNA40982-1235) that
encodes a novel polypeptide, designated in the present application
as "PRO332."
[0419] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA having at least about 80%
sequence identity to (a) a DNA molecule encoding a PRO358
polypeptide comprising the sequence of amino acids 49 to 642 of
FIG. 108 (SEQ ID NO: 310), or (b) the complement of the DNA
molecule of (a). The sequence identity preferably is about 85%,
more preferably about 90%, most preferably about 95%. In one
aspect, the isolated nucleic acid has at least about 80%,
preferably at least about 85%, more preferably at least about 90%,
and most preferably at least about 95% sequence identity with a
polypeptide having amino acid residues 1 to 642 of FIG. 108 (SEQ ID
NO:310). Preferably, the highest degree of sequence identity occurs
within the leucine-rich repeat domains (amino acids 116 to 624 of
FIG. 108, SEQ ID NO:310). In a further embodiment, the isolated
nucleic acid molecule comprises DNA encoding a PRO332 polypeptide
having amino acid residues 49 to 642 of FIG. 108 (SEQ ID NO:310),
or is complementary to such encoding nucleic acid sequence, and
remains stably bound to it under at least moderate, and optionally,
under high stringency conditions.
[0420] In another embodiment, the invention provides isolated
PRO332 polypeptides. In particular, the invention provides isolated
native sequence PRO332 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 49 to 624 of
FIG. 108 (SEQ ID NO:310). Native PRO332 polypeptides with or
without the native signal sequence (amino acids 1 to 48 in FIG.
108, SEQ ID NO:310), and with or without the initiating methionine
are specifically included.
[0421] 44. PRO334
[0422] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to fibulin and fibrillin, wherein the
polypeptide is designated in the present application as
"PRO334".
[0423] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO334 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO334 polypeptide having amino acid residues 1 to 509 of FIG. 110
(SEQ ID NO:315), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0424] In another embodiment, the invention provides isolated
PRO334 polypeptide. In particular, the invention provides isolated
native sequence PRO334 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 509 of
FIG. 110 (SEQ ID NO:315).
[0425] 45. PRO346
[0426] Applicants have identified a cDNA clone (DNA44167-1243) that
encodes a novel polypeptide, designated in the present application
as "PRO346."
[0427] In one embodiment, the invention provides an isolated
nucleic acid molecule having at least about 80% sequence identity
to (a) a DNA molecule encoding a PRO346 polypeptide comprising the
sequence of amino acids 19 to 339 of FIG. 112 (SEQ ID NO: 320), or
(b) the complement of the DNA molecule of (a). The sequence
identity preferably is about 85%, more preferably about 90%, most
preferably about 95%. In one aspect, the isolated nucleic acid has
at least about 80%, preferably at least about 85%, more preferably
at least about 90%, and most preferably at least about 95% sequence
identity with a polypeptide having amino acid residues 19 to 339 of
FIG. 112 (SEQ ID NO:320). Preferably, the highest degree of
sequence identity occurs within the extracellular domains (amino
acids 19 to 339 of FIG. 112, SEQ ID NO:320). In alternative
embodiments, the polypeptide by which the homology is measured
comprises the residues 1-339, 19-360 or 19-450 of FIG. 112, SEQ ID
NO:320). In a further embodiment, the isolated nucleic acid
molecule comprises DNA encoding a PRO346 polypeptide having amino
acid residues 19 to 339 of FIG. 112 (SEQ ID NO:320), alternatively
residues 1-339, 19-360 or 19-450 of FIG. 112 (SEQ ID NO:320) or is
complementary to such encoding nucleic acid sequence, and remains
stably bound to it under at least moderate, and optionally, under
high stringency conditions. In another aspect, the invention
provides a nucleic acid of the full length protein of clone
DNA44167-1243, deposited with the ATCC under accession number ATCC
209434, alternatively the coding sequence of clone DNA44167-1243,
deposited under accession number ATCC 209434.
[0428] In yet another embodiment, the invention provides isolated
PRO346 polypeptide. In particular, the invention provides isolated
native sequence PRO346 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 19 to 339 of
FIG. 112 (SEQ ID NO:320). Native PRO346 polypeptides with or
without the native signal sequence (residues 1 to 18 in FIG. 112
(SEQ ID NO:320), with or without the initiating methionine, with or
without the transmembrane domain (residues 340 to 360) and with or
without the intracellular domain (residues 361 to 450) are
specifically included. Alternatively, the invention provides a
PRO346 polypeptide encoded by the nucleic acid deposited under
accession number ATCC 209434.
[0429] 46. PRO268
[0430] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to protein disulfide isomerase,.wherein
the polypeptide is designated in the present application as
"PRO268".
[0431] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO268 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO268 polypeptide having amino acid residues 1 to 280 of FIG. 114
(SEQ ID NO:325), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0432] In another embodiment, the invention provides isolated
PRO268 polypeptide. In particular, the invention provides isolated
native sequence PRO268 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 280 of
FIG. 114 (SEQ ID NO:325). An additional embodiment of the present
invention is directed to an isolated extracellular domain of a
PRO268 polypeptide.
[0433] 47. PRO330
[0434] Applicants have identified a cDNA clone that encodes a novel
polypeptide having homology to the alpha subunit of prolyl
4-hydroxylase, wherein the polypeptide is designated in the present
application as "PRO330".
[0435] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding a PRO330 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding the
PRO330 polypeptide having amino acid residues 1 to 533 of FIG. 116
(SEQ ID NO:332), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0436] In another embodiment, the invention provides isolated
PRO330 polypeptide. In particular, the invention provides isolated
native sequence PRO330 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 533 of
FIG. 116 (SEQ ID NO:332).
[0437] 48. PRO339 and PRO310
[0438] Applicants have identified two cDNA clones wherein each
clone encodes a novel polypeptide having homology to fringe,
wherein the polypeptides are designated in the present application
as "PRO339" and "PRO310".
[0439] In one embodiment, the invention provides isolated nucleic
acid molecules comprising DNA encoding a PRO339 and/or a PRO310
polypeptide. In one aspect, the isolated nucleic acid comprises DNA
encoding the PRO339 polypeptide having amino acid residues 1 to 772
of FIG. 118 (SEQ ID NO:339), or is complementary to such encoding
nucleic acid sequence, and remains stably bound to it under at
least moderate, and optionally, under high stringency conditions.
In another aspect, the isolated nucleic acid comprises DNA encoding
the PRO310 polypeptide having amino acid residues 1 to 318 of FIG.
120 (SEQ ID NO:341), or is complementary to such encoding nucleic
acid sequence, and remains stably bound to it under at least
moderate, and optionally, under high stringency conditions.
[0440] In another embodiment, the invention provides isolated
PRO339 as well as isolated PRO310 polypeptides. In particular, the
invention provides isolated native sequence PRO339 polypeptide,
which in one embodiment, includes an amino acid sequence comprising
residues 1 to 772 of FIG. 118 (SEQ ID NO:339). The invention
further provides isolated native sequence PRO310 polypeptide, which
in one embodiment, includes an amino acid sequence comprising
residues 1 to 318 of FIG. 120 (SEQ ID NO:341).
[0441] 49. PRO244
[0442] Applicants have identified a cDNA clone that encodes a novel
polypeptide, designated in the present application as "PRO244".
[0443] In one embodiment, the invention provides an isolated
nucleic acid molecule comprising DNA encoding PRO244 polypeptide.
In one aspect, the isolated nucleic acid comprises DNA encoding
PRO244 polypeptide having amino acid residues 1 to 219 of FIG. 122
(SEQ ID NO:377), or is complementary to such encoding nucleic acid
sequence, and remains stably bound to it under at least moderate,
and optionally, under high stringency conditions.
[0444] In another embodiment, the invention provides isolated
PRO244 polypeptide. In particular, the invention provides isolated
native sequence PRO244 polypeptide, which in one embodiment,
includes an amino acid sequence comprising residues 1 to 219 of
FIG. 122 (SEQ ID NO:377).
[0445] 50. Additional Embodiments
[0446] 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.
[0447] 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.
[0448] 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.
[0449] In yet other embodiments, the invention provides
oligonucleotide probes useful for isolating genomic and cDNA
nucleotide sequences, wherein those probes may be derived from any
of the above or below described nucleotide sequences.
[0450] In other embodiments, the invention provides an isolated
nucleic acid molecule comprising a nucleotide sequence that encodes
a PRO polypeptide.
[0451] In one aspect, the isolated nucleic acid molecule comprises
a nucleotide sequence having at least about 80% sequence identity,
preferably at least about 81% sequence identity, more preferably at
least about 82% sequence identity, yet more preferably at least
about 83% sequence identity, yet more preferably at least about 84%
sequence identity, yet more preferably at least about 85% sequence
identity, yet more preferably at least about 86% sequence identity,
yet more preferably at least about 87% sequence identity, yet more
preferably at least about 88% sequence identity, yet more
preferably at least about 89% sequence identity, yet more
preferably at least about 90% sequence identity, yet more
preferably at least about 91% sequence identity, yet more
preferably at least about 92% sequence identity, yet more
preferably at least about 93% sequence identity, yet more
preferably at least about 94% sequence identity, yet more
preferably at least about 95% sequence identity, yet more
preferably at least about 96% sequence identity, yet more
preferably at least about 97% sequence identity, yet more
preferably at least about 98% sequence identity and yet more
preferably at least about 99% sequence identity to (a) a DNA
molecule encoding a PRO polypeptide having a full-length amino acid
sequence as disclosed herein, an amino acid sequence lacking the
signal peptide as disclosed herein or an extracellular domain of a
transmembrane protein, with or without the signal peptide, as
disclosed herein, or (b) the complement of the DNA molecule of
(a).
[0452] In other aspects, the isolated nucleic acid molecule
comprises a nucleotide sequence having at least about 80% sequence
identity, preferably at least about 81% sequence identity, more
preferably at least about 82% sequence identity, yet more
preferably at least about 83% sequence identity, yet more
preferably at least about 84% sequence identity, yet more
preferably at least about 85% sequence identity, yet more
preferably at least about 86% sequence identity, yet more
preferably at least about 87% sequence identity, yet more
preferably at least about 88% sequence identity, yet more
preferably at least about 89% sequence identity, yet more
preferably at least about 90% sequence identity, yet more
preferably at least about 91% sequence identity, yet more
preferably at least about 92% sequence identity, yet more
preferably at least about 93% sequence identity, yet more
preferably at least about 94% sequence identity, yet more
preferably at least about 95% sequence identity, yet more
preferably at least about 96% sequence identity, yet more
preferably at least about 97% sequence identity, yet more
preferably at least about 98% sequence identity and yet more
preferably at least about 99% sequence identity to (a) a DNA
molecule comprising the coding sequence of a full-length PRO
polypeptide cDNA as disclosed herein, the coding sequence of a PRO
polypeptide lacking the signal peptide as disclosed herein or the
coding sequence of an extracellular domain of a transmembrane PRO
polypeptide, with or without the signal peptide, as disclosed
herein, or (b) the complement of the DNA molecule of (a).
[0453] 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).
[0454] Another aspect the invention provides an isolated nucleic
acid molecule comprising a nucleotide sequence encoding a PRO
polypeptide which is either transmembrane domain-deleted or
transmembrane domain-inactivated, or is complementary to such
encoding nucleotide sequence, wherein the transmembrane domain(s)
of such polypeptide are disclosed herein. Therefore, soluble
extracellular domains of the herein described PRO polypeptides are
contemplated.
[0455] Another embodiment is directed to fragments of a PRO
polypeptide coding sequence, or the complement thereof, that may
find use as, for example, hybridization probes or for encoding
fragments of a PRO polypeptide that may optionally encode a
polypeptide comprising a binding site for an anti-PRO antibody.
Such nucleic acid fragments are usually at least about 20
nucleotides in length, preferably at least about 30 nucleotides in
length, 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-encoding nucleotide sequence with other known
nucleotide sequences using any of a number of well known sequence
alignment programs and determining which PRO polypeptide-encoding
nucleotide sequence fragment(s) are novel. All of such PRO
polypeptide-encoding nucleotide sequences are contemplated herein.
Also contemplated are the PRO polypeptide fragments encoded by
these nucleotide molecule fragments, preferably those PRO
polypeptide fragments that comprise a binding site for an anti-PRO
antibody.
[0456] In another embodiment, the invention provides isolated PRO
polypeptide encoded by any of the isolated nucleic acid sequences
hereinabove identified.
[0457] In a certain aspect, the invention concerns an isolated PRO
polypeptide, comprising an amino acid sequence having at least
about 80% sequence identity, preferably at least about 81% sequence
identity, more preferably at least about 82% sequence identity, yet
more preferably at least about 83% sequence identity, yet more
preferably at least about 84% sequence identity, yet more
preferably at least about 85% sequence identity, yet more
preferably at least about 86% sequence identity, yet more
preferably at least about 87% sequence identity, yet more
preferably at least about 88% sequence identity, yet more
preferably at least about 89% sequence identity, yet more
preferably at least about 90% sequence identity, yet more
preferably at least about 91% sequence identity, yet more
preferably at least about 92% sequence identity, yet more
preferably at least about 93% sequence identity, yet more
preferably at least about 94% sequence identity, yet more
preferably at least about 95% sequence identity, yet more
preferably at least about 96% sequence identity, yet more
preferably at least about 97% sequence identity, yet more
preferably at least about 98% sequence identity and yet more
preferably at least about 99% sequence identity to a PRO
polypeptide having a full-length amino acid sequence as disclosed
herein, an amino acid sequence lacking the signal peptide as
disclosed herein or an extracellular domain of a transmembrane
protein, with or without the signal peptide, as disclosed
herein.
[0458] In a further aspect, the invention concerns an isolated PRO
polypeptide comprising an amino acid sequence having at least about
80% sequence identity, preferably at least about 81% sequence
identity, more preferably at least about 82% sequence identity, yet
more preferably at least about 83% sequence identity, yet more
preferably at least about 84% sequence identity, yet more
preferably at least about 85% sequence identity, yet more
preferably at least about 86% sequence identity, yet more
preferably at least about 87% sequence identity, yet more
preferably at least about 88% sequence identity, yet more
preferably at least about 89% sequence identity, yet more
preferably at least about 90% sequence identity, yet more
preferably at least about 91% sequence identity, yet more
preferably at least about 92% sequence identity, yet more
preferably at least about 93% sequence identity, yet more
preferably at least about 94% sequence identity, yet more
preferably at least about 95% sequence identity, yet more
preferably at least about 96% sequence identity, yet more
preferably at least about 97% sequence identity, yet more
preferably at least about 98% sequence identity and yet more
preferably at least about 99% sequence identity to an amino acid
sequence encoded by any of the human protein cDNAs deposited with
the ATCC as disclosed herein.
[0459] In a further aspect, the invention concerns an isolated PRO
polypeptide comprising an amino acid sequence scoring at least
about 80% positives, preferably at least about 81% positives, more
preferably at least about 82% positives, yet more preferably at
least about 83% positives, yet more preferably at least about 84%
positives, yet more preferably at least about 85% positives, yet
more preferably at least about 86% positives, yet more preferably
at least about 87% positives, yet more preferably at least about
88% positives, yet more preferably at least about 89% positives,
yet more preferably at least about 90% positives, yet more
preferably at least about 91% positives, yet more preferably at
least about 92% positives, yet more preferably at least about 93%
positives, yet more preferably at least about 94% positives, yet
more preferably at least about 95% positives, yet more preferably
at least about 96% positives, yet more preferably at least about
97% positives, yet more preferably at least about 98% positives and
yet more preferably at least about 99% positives when compared with
the amino acid sequence of a PRO polypeptide having a full-length
amino acid sequence as disclosed herein, an amino acid sequence
lacking the signal peptide as disclosed herein or an extracellular
domain of a transmernbrane protein, with or without the signal
peptide, as disclosed herein.
[0460] 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.
[0461] 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.
[0462] 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.
[0463] 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.
[0464] In a still fitrther 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.
[0465] 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
[0466] FIG. 1 shows a nucleotide sequence (SEQ ID NO: 1) of a
native sequence PRO211 cDNA, wherein SEQ ID NO: 1 is a clone
designated herein as "DNA32292-1131".
[0467] 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.
[0468] FIG. 3 shows a nucleotide sequence (SEQ ID NO:3) of a native
sequence PRO217 cDNA, wherein SEQ ID NO:3 is a clone designated
herein as "DNA33094-1131".
[0469] FIG. 4 shows the amino acid sequence (SEQ ID NO:4) derived
from the coding sequence of SEQ ID NO:3 shown in FIG. 3.
[0470] FIG. 5 shows a nucleotide sequence (SEQ ID NO: 11) of a
native sequence PRO230 cDNA, wherein SEQ ID NO: 11 is a clone
designated herein as "DNA33223-1136".
[0471] FIG. 6 shows the amino acid sequence (SEQ ID NO: 12) derived
from the coding sequence of SEQ ID NO: 11 shown in FIG. 5.
[0472] FIG. 7 shows a nucleotide sequence designated herein as
DNA20088 (SEQ ID NO: 13).
[0473] FIG. 8 shows a nucleotide sequence (SEQ ID NO: 17) of a
native sequence PRO232 cDNA, wherein SEQ ID NO: 17 is a clone
designated herein as "DNA34435-1140".
[0474] FIG. 9 shows the amino acid sequence (SEQ ID NO: 18) derived
from the coding sequence of SEQ ID NO: 17 shown in FIG. 8.
[0475] FIG. 10 shows a nucleotide sequence (SEQ ID NO:22) of a
native sequence PRO187 cDNA, wherein SEQ ID NO:22 is a clone
designated herein as "DNA27864-1155".
[0476] FIG. 11 shows the amino acid sequence (SEQ ID NO:23) derived
from the coding sequence of SEQ ID NO:22 shown in FIG. 10.
[0477] FIG. 12 shows a nucleotide sequence (SEQ ID NO:27) of a
native sequence PRO265 cDNA, wherein SEQ ID NO:27 is a clone
designated herein as "DNA36350-1158".
[0478] FIG. 13 shows the amino acid sequence (SEQ ID NO:28) derived
from the coding sequence of SEQ ID NO:27 shown in FIG. 12.
[0479] FIG. 14 shows a nucleotide sequence (SEQ ID NO:33) of a
native sequence PRO219 cDNA, wherein SEQ ID NO:33 is a clone
designated herein as "DNA32290-1164".
[0480] FIG. 15 shows the amino acid sequence (SEQ ID NO:34) derived
from the coding sequence of SEQ ID NO:33 shown in FIG. 14.
[0481] FIG. 16 shows a nucleotide sequence (SEQ ID NO:38) of a
native sequence PRO246 cDNA, wherein SEQ ID NO:38 is a clone
designated herein as "DNA35639-1172".
[0482] FIG. 17 shows the amino acid sequence (SEQ ID NO:39) derived
from the coding sequence of SEQ ID NO:38 shown in FIG. 16.
[0483] FIG. 18 shows a nucleotide sequence (SEQ ID NO:48) of a
native sequence PRO228 cDNA, wherein SEQ ID NO:48 is a clone
designated herein as "DNA33092-1202".
[0484] FIG. 19 shows the amino acid sequence (SEQ ID NO:49) derived
from the coding sequence of SEQ ID NO:48 shown in FIG. 18.
[0485] FIG. 20 shows a nucleotide sequence designated herein as
DNA21951 (SEQ ID NO:50).
[0486] FIG. 21 shows a nucleotide sequence (SEQ ID NO:58) of a
native sequence PRO533 cDNA, wherein SEQ ID NO:58 is a clone
designated herein as "DNA49435-1219".
[0487] FIG. 22 shows the amino acid sequence (SEQ ID NO:59) derived
from the coding sequence of SEQ ID NO:58 shown in FIG. 21.
[0488] FIG. 23 shows a nucleotide sequence (SEQ ID NO:63) of a
native sequence PRO245 cDNA, wherein SEQ ID NO:63 is a clone
designated herein as "DNA35638-1141".
[0489] FIG. 24 shows the amino acid sequence (SEQ ID NO:64) derived
from the coding sequence of SEQ ID NO:63 shown in FIG. 23.
[0490] FIG. 25 shows a nucleotide sequence (SEQ ID NO:68) of a
native sequence PRO220 cDNA, wherein SEQ ID NO:68 is a clone
designated herein as "DNA32298-1132".
[0491] FIG. 26 shows the amino acid sequence (SEQ ID NO:69) derived
from the coding sequence of SEQ ID NO:68 shown in FIG. 25.
[0492] FIG. 27 shows a nucleotide sequence (SEQ ID NO:70) of a
native sequence PRO221 cDNA, wherein SEQ ID NO:70 is a clone
designated herein as "DNA33089-1132".
[0493] FIG. 28 shows the amino acid sequence (SEQ ID NO:71) derived
from the coding sequence of SEQ ID NO:70 shown in FIG. 27.
[0494] FIG. 29 shows a nucleotide sequence (SEQ ID NO:72) of a
native sequence PRO227 cDNA, wherein SEQ ID NO:72 is a clone
designated herein as "DNA33786-1132".
[0495] FIG. 30 shows the amino acid sequence (SEQ ID NO:73) derived
from the coding sequence of SEQ ID NO:72 shown in FIG. 29.
[0496] FIG. 31 shows a nucleotide sequence (SEQ ID NO:83) of a
native sequence PRO258 cDNA, wherein SEQ ID NO:83 is a clone
designated herein as "DNA35918-1174".
[0497] FIG. 32 shows the amino acid sequence (SEQ ID NO:84) derived
from the coding sequence of SEQ ID NO:83 shown in FIG. 31.
[0498] FIG. 33 shows a nucleotide sequence (SEQ ID NO:90) of a
native sequence PRO266 cDNA, wherein SEQ ID NO:90 is a clone
designated herein as "DNA37150-1178".
[0499] FIG. 34 shows the amino acid sequence (SEQ ID NO:91) derived
from the coding sequence of SEQ ID NO:90 shown in FIG. 33.
[0500] FIG. 35 shows a nucleotide sequence (SEQ ID NO:95) of a
native sequence PRO269 cDNA, wherein SEQ ID NO:95 is a clone
designated herein as "DNA38260-1180".
[0501] FIG. 36 shows the amino acid sequence (SEQ ID NO:96) derived
from the coding sequence of SEQ ID NO:95 shown in FIG. 35.
[0502] FIG. 37 shows a nucleotide sequence (SEQ ID NO: 103) of a
native sequence PRO287 cDNA, wherein SEQ ID NO: 103 is a clone
designated herein as "DNA39969-1185".
[0503] FIG. 38 shows the amino acid sequence (SEQ ID NO: 104)
derived from the coding sequence of SEQ ID NO: 103 shown in FIG.
37.
[0504] FIG. 39 shows a nucleotide sequence (SEQ ID NO: 108) of a
native sequence PRO214 cDNA, wherein SEQ ID NO: 108 is a clone
designated herein as "DNA32286-1191.
[0505] FIG. 40 shows the amino acid sequence (SEQ ID NO: 109)
derived from the coding sequence of SEQ ID NO: 108 shown in FIG.
39.
[0506] FIG. 41 shows a nucleotide sequence (SEQ ID NO: 113) of a
native sequence PRO317 cDNA, wherein SEQ ID NO: 113 is a clone
designated herein as "DNA33461-1199".
[0507] FIG. 42 shows the amino acid sequence (SEQ ID NO: 114)
derived from the coding sequence of SEQ ID NO: 113 shown in FIG.
41.
[0508] FIG. 43 shows a nucleotide sequence (SEQ ID NO: 118) of a
native sequence PRO301 cDNA, wherein SEQ ID NO: 118 is a clone
designated herein as "DNA40628-1216".
[0509] FIG. 44 shows the amino acid sequence (SEQ ID NO: 119)
derived from the coding sequence of SEQ ID NO: 118 shown in FIG.
43.
[0510] FIG. 45 shows a nucleotide sequence (SEQ ID NO: 126) of a
native sequence PRO224 cDNA, wherein SEQ ID NO: 126 is a clone
designated herein as "DNA33221-1133".
[0511] FIG. 46 shows the amino acid sequence (SEQ ID NO: 127)
derived from the coding sequence of SEQ ID NO: 126 shown in FIG.
45.
[0512] FIG. 47 shows a nucleotide sequence (SEQ ID NO: 131) of a
native sequence PRO222 cDNA, wherein SEQ ID NO: 131 is a clone
designated herein as "DNA33107-1135".
[0513] FIG. 48 shows the amino acid sequence (SEQ ID NO: 132)
derived from the coding sequence of SEQ ID NO: 131 shown in FIG.
47.
[0514] FIG. 49 shows a nucleotide sequence (SEQ ID NO:136) of a
native sequence PRO234 cDNA, wherein SEQ ID NO: 136 is a clone
designated herein as "DNA35557-1137.
[0515] FIG. 50 shows the amino acid sequence (SEQ ID NO: 137)
derived from the coding sequence of SEQ ID NO:136 shown in FIG.
49.
[0516] FIG. 51 shows a nucleotide sequence (SEQ ID NO: 141) of a
native sequence PRO231 cDNA, wherein SEQ ID NO: 141 is a clone
designated herein as "DNA34434-1139".
[0517] FIG. 52 shows the amino acid sequence (SEQ ID NO: 142)
derived from the coding sequence of SEQ ID NO: 141 shown in FIG.
51.
[0518] FIG. 53 shows a nucleotide sequence (SEQ ID NO: 147) of a
native sequence PRO229 cDNA, wherein SEQ ID NO: 147 is a clone
designated herein as "DNA33100-1159".
[0519] FIG. 54 shows the amino acid sequence (SEQ ID NO: 148)
derived from the coding sequence of SEQ ID NO: 147 shown in FIG.
53.
[0520] FIG. 55 shows a nucleotide sequence (SEQ ID NO: 152) of a
native sequence PRO238 cDNA, wherein SEQ ID NO: 152 is a clone
designated herein as "DNA35600-1162".
[0521] FIG. 56 shows the amino acid sequence (SEQ ID NO: 153)
derived from the coding sequence of SEQ ID NO: 152 shown in FIG.
55.
[0522] FIG. 57 shows a nucleotide sequence (SEQ ID NO: 158) of a
native sequence PRO233 cDNA, wherein SEQ ID NO: 158 is a clone
designated herein as "DNA34436-1238".
[0523] FIG. 58 shows the amino acid sequence (SEQ ID NO: 159)
derived from the coding sequence of SEQ ID NO: 158 shown in FIG.
57.
[0524] FIG. 59 shows a nucleotide sequence (SEQ ID NO: 163) of a
native sequence PRO223 cDNA, wherein SEQ ID NO: 163 is a clone
designated herein as "DNA33206-1165".
[0525] FIG. 60 shows the amino acid sequence (SEQ 1ID NO: 164)
derived from the coding sequence of SEQ ID NO: 163 shown in FIG.
59.
[0526] FIG. 61 shows a nucleotide sequence (SEQ ID NO: 169) of a
native sequence PRO235 cDNA, wherein SEQ ID NO: 169 is a clone
designated herein as "DNA35558-1167".
[0527] FIG. 62 shows the amino acid sequence (SEQ ID NO: 170)
derived from the coding sequence of SEQ ID NO: 169 shown in FIG.
61.
[0528] FIG. 63 shows a nucleotide sequence (SEQ ID NO: 174) of a
native sequence PRO236 cDNA, wherein SEQ ID NO: 174 is a clone
designated herein as "DNA35599-1168".
[0529] FIG. 64 shows the amino acid sequence (SEQ ID NO: 175)
derived from the coding sequence of SEQ ID NO: 174 shown in FIG.
63.
[0530] FIG. 65 shows a nucleotide sequence (SEQ ID NO: 176) of a
native sequence PRO262 cDNA, wherein SEQ ID NO: 176 is a clone
designated herein as "DNA36992-1168".
[0531] FIG. 66 shows the amino acid sequence (SEQ ID NO: 177)
derived from the coding sequence of SEQ ID NO: 176 shown in FIG.
65.
[0532] FIG. 67 shows a nucleotide sequence (SEQ ID NO: 184) of a
native sequence PRO239 cDNA, wherein SEQ ID NO: 184 is a clone
designated herein as "DNA34407-1169".
[0533] FIG. 68 shows the amino acid sequence (SEQ ID NO: 185)
derived from the coding sequence of SEQ ID NO: 184 shown in FIG.
67.
[0534] FIG. 69 shows a nucleotide sequence (SEQ ID NO: 189) of a
native sequence PRO257 cDNA, wherein SEQ ID NO: 189 is a clone
designated herein as "DNA35841-1173".
[0535] 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.
[0536] FIG. 71 shows a nucleotide sequence (SEQ ID NO: 194) of a
native sequence PRO260 cDNA, wherein SEQ ID NO: 194 is a clone
designated herein as "DNA33470-1175".
[0537] FIG. 72 shows the amino acid sequence (SEQ ID NO: 195)
derived from the coding sequence of SEQ ID NO: 194 shown in FIG.
71.
[0538] FIG. 73 shows a nucleotide sequence (SEQ ID NO:200) of a
native sequence PRO263 cDNA, wherein SEQ ID NO:200 is a clone
designated herein as "DNA34431-1177".
[0539] FIG. 74 shows the amino acid sequence (SEQ ID NO:201)
derived from the coding sequence of SEQ ID NO:200 shown in FIG.
73.
[0540] FIG. 75 shows a nucleotide sequence (SEQ ID NO:206) of a
native sequence PRO270 cDNA, wherein SEQ ID NO:206 is a clone
designated herein as "DNA39510-118 V.
[0541] FIG. 76 shows the amino acid sequence (SEQ ID NO:207)
derived from the coding sequence of SEQ ID NO:206 shown in FIG.
75.
[0542] FIG. 77 shows a nucleotide sequence (SEQ ID NO:212) of a
native sequence PRO271 cDNA, wherein SEQ ID NO:212 is a clone
designated herein as "DNA39423-1182".
[0543] FIG. 78 shows the amino acid sequence (SEQ ID NO:213)
derived from the coding sequence, of SEQ ID NO:212 shown in FIG.
77.
[0544] FIG. 79 shows a nucleotide sequence (SEQ ID NO:220) of a
native sequence PRO272 cDNA, wherein SEQ ID NO:220 is a clone
designated herein as "DNA40620-1183.".
[0545] FIG. 80 shows the amino acid sequence (SEQ ID NO:221)
derived from the coding sequence of SEQ ID NO:220 shown in FIG.
79.
[0546] FIG. 81 shows a nucleotide sequence (SEQ ID NO:226) of a
native sequence PRO294 cDNA, wherein SEQ ID NO:226 is a clone
designated herein as "DNA40604-1187".
[0547] FIG. 82 shows the amino acid sequence (SEQ ID NO:227)
derived from the coding sequence of SEQ ID NO:226 shown in FIG.
81.
[0548] FIG. 83 shows a nucleotide sequence (SEQ ID NO:235) of a
native sequence PRO295 cDNA, wherein SEQ ID NO:235 is a clone
designated herein as "DNA38268-1188".
[0549] FIG. 84 shows the amino acid sequence (SEQ ID NO:236)
derived from the coding sequence of SEQ ID NO:235 shown in FIG.
83.
[0550] FIG. 85 shows a nucleotide sequence (SEQ ID NO:244) of a
native sequence PRO293 cDNA, wherein SEQ ID NO:244 is a clone
designated herein as "DNA37151-1193".
[0551] FIG. 86 shows the amino acid sequence (SEQ ID NO:245)
derived from the coding sequence of SEQ ID NO:244 shown in FIG.
85.
[0552] FIG. 87 shows a nucleotide sequence (SEQ ID NO:249) of a
native sequence PRO247 cDNA, wherein SEQ ID NO:249 is a clone
designated herein as, "DNA35673-1201".
[0553] FIG. 88 shows the amino acid sequence (SEQ ID NO:250)
derived from the coding sequence of SEQ ID NO:249 shown in FIG.
87.
[0554] FIG. 89 shows a nucleotide sequence (SEQ ID NO:254) of a
native sequence PRO302 cDNA, wherein SEQ ID NO:254 is a clone
designated herein as "DNA40370-1217".
[0555] FIG. 90 shows the amino acid sequence (SEQ ID NO:255)
derived from the coding sequence of SEQ ID NO:254 shown in FIG.
89.
[0556] FIG. 91 shows a nucleotide sequence (SEQ ID NO:256) of a
native sequence PRO303 cDNA, wherein SEQ ID NO:256 is a clone
designated herein as "DNA42551-1217".
[0557] FIG. 92 shows the amino acid sequence (SEQ ID NO:257)
derived from the coding sequence of SEQ ID NO:256 shown in FIG.
91.
[0558] FIG. 93 shows a nucleotide sequence (SEQ ID NO:258) of a
native sequence PRO304 cDNA, wherein SEQ ID NO:258 is a clone
designated herein as "DNA39520-1217".
[0559] FIG. 94 shows the amino acid sequence (SEQ ID NO:259)
derived from the coding sequence of SEQ ID NO:258 shown in FIG.
93.
[0560] FIG. 95 shows a nucleotide sequence (SEQ ID NO:260) of a
native sequence PRO307 cDNA, wherein SEQ ID NO:260 is a clone
designated herein as "DNA41225-1217".
[0561] FIG. 96 shows the amino acid sequence (SEQ ID NO:261)
derived from the coding sequence of SEQ ID NO:260 shown in FIG.
95.
[0562] FIG. 97 shows a nucleotide sequence (SEQ ID NO:262) of a
native sequence PRO343 cDNA, wherein SEQ ID NO:262 is a clone
designated herein as "DNA43318-1217".
[0563] FIG. 98 shows the amino acid sequence (SEQ ID NO:263)
derived from the coding sequence of SEQ ID NO:262 shown in FIG.
97.
[0564] FIG. 99 shows a nucleotide sequence (SEQ ID NO:284) of a
native sequence PRO328 cDNA, wherein SEQ ID NO:284 is a clone
designated herein as "DNA40587-1231".
[0565] FIG. 100 shows the amino acid sequence (SEQ ID NO:285)
derived from the coding sequence of SEQ ID NO:284 shown in FIG.
99.
[0566] FIG. 101 shows a nucleotide sequence (SEQ ID NO:289) of a
native sequence PRO335 cDNA, wherein SEQ ID NO:289 is a clone
designated herein as "DNA41388-1234".
[0567] FIG. 102 shows the amino acid sequence (SEQ ID NO:290)
derived from the coding sequence of SEQ ID NO:289 shown in FIG.
101.
[0568] FIG. 103 shows a nucleotide sequence (SEQ ID NO:291) of a
native sequence PRO331 cDNA, wherein SEQ ID NO:291 is a clone
designated herein as "DNA40981-1234".
[0569] FIG. 104 shows the amino acid sequence (SEQ ID NO:292)
derived from the coding sequence of SEQ ID NO:291 shown in FIG.
103.
[0570] FIG. 105 shows a nucleotide sequence (SEQ ID NO:293) of a
native sequence PRO326 cDNA, wherein SEQ ID NO:293 is a clone
designated herein as "DNA37140-1234".
[0571] FIG. 106 shows the amino acid sequence (SEQ ID NO:294)
derived from the coding sequence of SEQ ID NO:293 shown in FIG.
105.
[0572] FIG. 107 shows a nucleotide sequence (SEQ ID NO:309) of a
native sequence PRO332 cDNA, wherein SEQ ID NO:309 is a clone
designated herein as "DNA40982-1235".
[0573] FIG. 108 shows the amino acid sequence (SEQ ID NO:310)
derived from the coding sequence of SEQ ID NO:309 shown in FIG.
107.
[0574] FIG. 109 shows a nucleotide sequence (SEQ ID NO:314) of a
native sequence PRO334 cDNA, wherein SEQ ID NO:314 is a clone
designated herein as "DNA41379-1236".
[0575] FIG. 110 shows the amino acid sequence (SEQ ID NO:315)
derived from the coding sequence of SEQ ID NO:314 shown in FIG.
109.
[0576] FIG. 111 shows a nucleotide sequence (SEQ ID NO:319) of a
native sequence PRO346 cDNA, wherein SEQ ID NO:319 is a clone
designated herein as "DNA44167-1243".
[0577] FIG. 112 shows the amino acid sequence (SEQ ID NO:320)
derived from the coding sequence of SEQ ID NO:319 shown in FIG.
111.
[0578] FIG. 113 shows a nucleotide sequence (SEQ ID NO:324) of a
native sequence PRO268 cDNA, wherein SEQ ID NO:324 is a clone
designated herein as "DNA39427-1179".
[0579] FIG. 114 shows the amino acid sequence (SEQ ID NO:325)
derived from the coding sequence of SEQ ID NO:324 shown in FIG.
113.
[0580] FIG. 115 shows a nucleotide sequence (SEQ ID NO:331) of a
native sequence PRO330 cDNA, wherein SEQ ID NO:331 is a clone
designated herein as "DNA40603-1232".
[0581] FIG. 116 shows the amino acid sequence (SEQ ID NO:332)
derived from the coding sequence of SEQ ID NO:331 shown in FIG.
115.
[0582] FIG. 117 shows a nucleotide sequence (SEQ ID NO:338) of a
native sequence PRO339 cDNA, wherein SEQ ID NO:338. is a clone
designated herein as "DNA43466-1225".
[0583] FIG. 118 shows the amino acid sequence (SEQ ID NO:339)
derived from the coding sequence of SEQ ID NO:338 shown in FIG.
117.
[0584] FIG. 119 shows a nucleotide sequence (SEQ ID NO:340) of a
native sequence PRO310 cDNA, wherein SEQ ID NO:340 is a clone
designated herein as "DNA43046-1225".
[0585] FIG. 120 shows the amino acid sequence (SEQ ID NO:341)
derived from the coding sequence of SEQ ID NO:340 shown in FIG.
119.
[0586] FIG. 121 shows a nucleotide sequence (SEQ ID NO:376) of a
native sequence PRO244 cDNA, wherein SEQ ID NO:376 is a clone
designated herein as "DNA35668-1171".
[0587] FIG. 122 shows the amino acid sequence (SEQ ID NO:377)
derived from the coding sequence of SEQ ID NO:376 shown in FIG.
121.
[0588] FIG. 123 shows a nucleotide sequence (SEQ ID NO:422) of a
native sequence PRO 1868 cDNA, wherein SEQ ID NO:422 is a clone
designated herein as "DNA77624-2515".
[0589] FIG. 124 shows the amino acid sequence (SEQ ID NO:423)
derived from the coding sequence of SEQ ID NO:422 shown in FIG.
123.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
I. Definitions
[0590] The terms "PRO polypeptide" and "PRO" as used herein and
when immediately followed by a numerical designation refer to
various polypeptides, wherein the complete designation (i.e.,
PRO/number) refers to specific polypeptide sequences as described
herein. The terms "PRO/number polypeptide" and "PRO/number, wherein
the term "number" is provided as an actual numerical designation as
used herein encompass native sequence polypeptides and polypeptide
variants (which are further defined herein). The PRO polypeptides
described herein may be isolated from a variety of sources, such as
from human tissue types or from another source, or prepared by
recombinant or synthetic methods.
[0591] 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.
[0592] 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 contemplated by the present
invention.
[0593] 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.
[0594] "PRO polypeptide variant" means an active PRO polypeptide as
defined above or below having at least about 80% amino acid
sequence identity with a full-length native sequence PRO
polypeptide sequence as disclosed herein, a PRO polypeptide
sequence lacking the signal peptide as disclosed herein, an
extracellular domain of a PRO polypeptide, with or without the
signal peptide, as disclosed herein or any other fragment of a
full-length PRO polypeptide sequence as disclosed herein. Such PRO
polypeptide variants include, for instance, PRO polypeptides
wherein one or more amino acid residues are added, or deleted, at
the N-- or C-terminus of the full-length native amino acid
sequence. Ordinarily, a PRO polypeptide variant will have at least
about 80% amino acid sequence identity, preferably at least about
81% amino acid sequence identity, more preferably at least about
82% amino acid sequence identity, more preferably at least about
83% amino acid sequence identity, more preferably at least about
84% amino acid sequence identity, more preferably at least about
85% amino acid sequence identity, more preferably at least about
86% amino acid sequence identity, more preferably at least about
87% amino acid sequence identity, more preferably at least about
88% amino acid sequence identity, more preferably at least about
89% amino acid sequence identity, more preferably at least about
90% amino acid sequence identity, more preferably at least about
91% amino acid sequence identity, more preferably at least about
92% amino acid sequence identity, more preferably at least about
93% amino acid sequence identity, more preferably at least about
94% amino acid sequence identity, more preferably at least about
95% amino acid sequence identity, more preferably at least about
96% amino acid sequence identity, more preferably at least about
97% amino acid sequence identity, more preferably at least about
98% amino acid sequence identity and most preferably at least about
99% amino acid sequence identity with a full-length native sequence
PRO polypeptide sequence as disclosed herein, a PRO polypeptide
sequence lacking the signal peptide as disclosed herein, an
extracellular domain of a PRO polypeptide, with or without the
signal peptide, as disclosed herein or any other specifically
defined fiagment of a full-length PRO polypeptide sequence as
disclosed herein. Ordinarily, PRO variant polypeptides are at least
about 10 amino acids in length, often at least about 20 amino acids
in length, more often at least about 30 amino acids in length, more
often at least about 40 amino acids in length, more often at least
about 50 amino acids in length, more often at least about 60 amino
acids in length, more often at least about 70 amino acids in
length, more often at least about 80 amino acids in length, more
often at least about 90 amino acids in length, more often at least
about 100 amino acids in length, more often at least about 150
amino acids in length, more often at least about 200 amino acids in
length, more often at least about 300 amino acids in length, or
more.
[0595] "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.
[0596] In situations where ALIGN-2 is employed for amino acid
sequence comparisons, the % amino acid sequence identity of a given
amino acid sequence A to, with, or against a given amino acid
sequence B (which can alternatively be phrased as a given amino
acid sequence A that has or comprises a certain % amino acid
sequence identity to, with, or against a given amino acid sequence
B) is calculated as follows: 100 times the fraction X/Y where X is
the number of amino acid residues scored as identical matches by
the sequence alignment program ALIGN-2 in that program's alignment
of A and B, and where Y is the total number of amino acid residues
in B. It will be appreciated that where the length of amino acid
sequence A is not equal to the length of amino acid sequence B, the
% amino acid sequence identity of A to B will not equal the % amino
acid sequence identity of B to A. As examples of % amino acid
sequence identity calculations using this method, Tables 2 and 3
demonstrate how to calculate the % amino acid sequence identity of
the amino acid sequence designated "Comparison Protein" to the
amino acid sequence designated "PRO", wherein "PRO" represents the
amino acid sequence of a hypothetical PRO polypeptide of interest,
"Comparison Protein" represents the amino acid sequence of a
polypeptide against which the "PRO" polypeptide of interest is
being compared, and "X, "Y" and "Z" each represent different
hypothetical amino acid residues.
[0597] Unless specifically stated otherwise, all % amino acid
sequence identity values used herein are obtained as described in
the immediately preceding paragraph using the ALIGN-2 computer
program. However, % amino acid sequence identity values may also be
obtained as described below by using the WU-BLAST-2 computer
program (Altschul et al., Methods in Enzymology 266:460480 (1996)).
Most of the W-BLAST-2 search parameters are set to the default
values. Those not set to default values, ie., 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 W-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 W-BLAST-2 by (b) the total
number of amino acid residues of the PRO polypeptide of interest.
For example, in the statement "a polypeptide comprising an the
amino acid sequence A which has or having at least 80% amino acid
sequence identity to the amino acid sequence B", the amino acid
sequence A is the comparison amino acid sequence of interest and
the amino acid sequence B is the amino acid sequence of the PRO
polypeptide of interest.
[0598] Percent amino acid sequence identity may also be determined
using the sequence comparison program NCBI-BLAST2 (Altschul et al.,
Nucleic Acids Res. 25:3389-3402 (1997)). 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.
[0599] In situations where NCBI-BLAST2 is employed for amino acid
sequence comparisons, the % amino acid sequence identity of a given
amino acid sequence A to, with, or against a given amino acid
sequence B (which can alternatively be phrased as a given amino
acid sequence A that has or comprises a certain % amino acid
sequence identity to, with, or against a given amino acid sequence
B) is calculated as follows: 100 times the fraction X/Y 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.
[0600] "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.
[0601] 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.
[0602] "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.
[0603] In situations where ALIGN-2 is employed for nucleic acid
sequence comparisons, the % nucleic acid sequence identity of a
given nucleic acid sequence C to, with, or against a given nucleic
acid sequence D (which can alternatively be phrased as a given
nucleic acid sequence C that has or comprises a certain % nucleic
acid sequence identity to, with, or against a given nucleic acid
sequence D) is calculated as follows: 100 times the fraction W/Z
where W is the number of nucleotides scored as identical matches by
the sequence alignment program ALIGN-2 in that program's alignment
of C and D, and where Z is the total number of nucleotides in D. It
will be appreciated that where the length of nucleic acid sequence
C is not equal to the length of nucleic acid sequence D, the %
nucleic acid sequence identity of C to D will not equal the %
nucleic acid sequence identity of D to C. As examples of % nucleic
acid sequence identity calculations, Tables 4 and 5, demonstrate
how to calculate the % nucleic acid sequence identity of the
nucleic acid sequence designated "Comparison DNA" to the nucleic
acid sequence designated "PRO-DNA", wherein "PRO-DNA" represents a
hypothetical PRO-encoding nucleic acid sequence of interest,
"Comparison DNA" represents the nucleotide sequence of a nucleic
acid molecule against which the "PRO-DNA" nucleic acid molecule of
interest is being compared, and "N", "L" and "V" each represent
different hypothetical nucleotides.
[0604] 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 (ie., 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.
[0605] 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)).
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.
[0606] 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
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.
[0607] 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.
[0608] 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 (ie., 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.
[0609] 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.
[0610] 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 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.
[0611] "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.
[0612] 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.
[0613] The term "control sequences" refers to DNA sequences
necessary for the expression of an operably linked coding sequence
in a particular host organism. The control sequences that are
suitable for prokaryotes, for example, include a promoter,
optionally an operator sequence, and a ribosome binding site.
Eukaryotic cells are known to utilize promoters, polyadenylation
signals, and enhancers.
[0614] 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.
[0615] 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.
[0616] "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).
[0617] "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 alburnin/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
chloridelsodium 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.
[0618] "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.
[0619] 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).
[0620] As used herein, the term "immunoadhesin" designates
antibody-like molecules which combine the binding specificity of a
heterologous protein (an "adhesin") with the effector functions of
immunoglobulin constant domains. Structurally, the immunoadhesins
comprise a fusion of an amino acid sequence with the desired
binding specificity which is other than the antigen recognition and
binding site of an antibody (i.e., is "heterologous"), and an
immunoglobulin constant domain sequence. The adhesin part of an
immunoadhesin molecule typically is a contiguous amino acid
sequence comprising at least the binding site of a receptor or a
ligand. The immunoglobulin constant domain sequence in the
immunoadhesin may be obtained from any immunoglobulin, such as
IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and
IgA-2), IgE, IgD or IgM.
[0621] "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.
[0622] 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.
[0623] "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.
[0624] "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.
[0625] "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.
[0626] Administration "in combination with" one or more further
therapeutic agents includes simultaneous (concurrent) and
consecutive administration in any order.
[0627] "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.quadrature., polyethylene glycol (PEG), and
PLURONICS.quadrature..
[0628] "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.
[0629] 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.
[0630] "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.
[0631] 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.
[0632] 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.
[0633] Depending on the amino acid sequence of the constant domain
of their heavy chains, immunoglobulins can be assigned to different
classes. There are five major classes of immunoglobulins: IgA, IgD,
IgE, IgG, and IgM, and several of these may be further divided into
subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and
IgA2.
[0634] "Single-chain Fv" or "sFv" antibody fragments comprise the
V.sub.H and V.sub.L domains of antibody, wherein these domains are
present in a single polypeptide chain. Preferably, the Fv
polypeptide further comprises a polypeptide linker between the
V.sub.H and V.sub.L domains which enables the sFv to form the
desired structure for antigen binding. For a review of sFv, see
Pluckthunin The Pharmacology of Monoclonal Antibodies, vol. 113,
Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315
(1994).
[0635] 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. Nad.
Acad. Sci. USA 90:6444-6448 (1993).
[0636] 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.
[0637] 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.
[0638] 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.
[0639] 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.
[0640] A "small molecule" is defined herein to have a molecular
weight below about 500 Daltons.
[0641] "PRO317-associated disorder refers to a pathological
condition or disease wherein PRO317 is over- or underexpressed.
Such disorders include diseases of the female genital tract or of
the endometrium of a mammal, including hyperplasia, endometritis,
endometriosis, wherein the patient is at risk for infertility due
to endometrial factor, endometrioma, and endometrial cancer,
especially those diseases involving abnormal bleeding such as a
gynecological disease. They also include diseases involving
TABLE-US-00001 TABLE 2 PRO XXXXXXXXXXXXXXX (Length = 15 amino
acids) Comparison XXXXXYYYYYYY (Length = 12 amino acids) Protein %
amino acid sequence identity = (the number of identically matching
amino acid residues between the two polypeptide sequences as
determined by ALIGN-2) divided by (the total number of amino acid
residues of the PRO polypeptide) = 5 divided by 15 = 33.3%
[0642] TABLE-US-00002 TABLE 3 PRO XXXXXXXXXX (Length = 10 amino
acids) Comparison XXXXXYYYYYYZZYZ (Length = 15 amino acids) Protein
% amino acid sequence identity = (the number of identically
matching amino acid residues between the two polypeptide sequences
as determined by ALIGN-2) divided by (the total number of amino
acid residues of the PRO polypeptide) = 5 divided by 10 = 50%
[0643] TABLE-US-00003 TABLE 4 PRO-DNA NNNNNNNNNNNNNN (Length = 14
nucleotides) Comparison NNNNNNLLLLLLLLLL (Length = 16 nucleotides)
DNA % nucleic acid sequence identity = (the number of identically
matching nucleotides between the two nucleic acid sequences as
determined by ALIGN-2) divided by (the total number of nucleotides
of the PRO-DNA nucleic acid sequence) 6 divided by 14 = 42.9%
[0644] TABLE-US-00004 TABLE 5 PRO-DNA NNNNNNNNNNNN (Length = 12
nucleotides) Comparison DNA NNNNLLLVV (Length = 9 nucleotides) %
nucleic acid sequence identity = (the number of identically
matching nucleotides between the two nucleic acid sequences as
determined by ALIGN-2) divided by (the total number of nucleotides
of the PRO-DNA nucleic acid sequence) = 4 divided by 12 = 33.3%
II. Compositions and Methods of the Invention
[0645] A. Full-Length PRO Polypeptides
[0646] 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.
[0647] As disclosed in the Examples below, various cDNA clones have
been deposited with the ATCC. The actual nucleotide sequences of
those clones can readily be determined by the skilled artisan by
sequencing of the deposited clone using routine methods in the art.
The predicted amino acid sequence can be determined from the
nucleotide sequence using routine skill. For the PRO polypeptides
and encoding nucleic acids described herein, Applicants have
identified what is believed to be the reading frame best
identifiable with the sequence information available at the
time.
[0648] 1. Full-Length PR0211 and PR0217 Polypeptides
[0649] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PR0211 and PR0217. In particular, Applicants
have identified and isolated cDNA encoding PR0211 and PR0217
polypeptides, as disclosed in further detail in the Examples below.
Using BLAST (FastA format) sequence alignment computer programs,
Applicants found that cDNA sequences encoding full-length native
sequence PR0211 and PR0217 have homologies to known proteins having
EGF-like domains. Specifically, the cDNA sequence DNA32292-1131
(FIG. 1, SEQ ID NO: 1) has certain identify and a Blast score of
209 with PAC6_RAT and certain identify and a Blast score of 206
with Fibulin-1, isoform c precursor. The cDNA sequence
DNA33094-1131 (FIG. 3, SEQ ID NO:3) has 36% identity and a Blast
score of 336 with eastern newt tenascin, and 37% identity and a
Blast score of 331 with human tenascin-X precursor. Accordingly, it
is presently believed that PRO211 and PRO217 polypeptides disclosed
in the present application are newly identified members of the
EGF-like family and possesses properties typical of the EGF-like
protein family.
[0650] 2. Full-Length PR0230 Polypeptides
[0651] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO230. In particular, Applicants have
identified and isolated cDNA encoding a PR0230 polypeptide, as
disclosed in further detail in the Examples below. Using known
programs such as BLAST and FastA sequence alignment computer
programs, Applicants found that a cDNA sequence encoding
full-length native sequence PR0230 has 48% amino acid identity with
the rabbit tubulointerstitial nephritis antigen precursor.
Accordingly, it is presently believed that PR0230 polypeptide
disclosed in the present application is a newly identified member
of the tubulointerstitial nephritis antigen family and possesses
the ability to be recognized by human autoantibodies in certain
forms of tubulointerstitial nephritis.
[0652] 3. Full-Length PR0232 Polypeptides
[0653] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO232. In particular, Applicants have
identified and isolated cDNA encoding a PR0232 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that a
portion of the full-length native sequence PRO232 (shown in FIG. 9
and SEQ ID NO:18) has 35% sequence identity with a stem cell
surface antigen from Gallus gallus. Accordingly, it is presently
believed that the PR0232 polypeptide disclosed in the present
application may be a newly identified stem cell antigen.
[0654] 4. Full-Length PRO187 Polypeptides
[0655] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO187. In particular, Applicants have
identified and isolated cDNA encoding a PRO187 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that a
full-length native sequence PRO187 (shown in FIG. 15) has 74% amino
acid sequence identity and BLAST score of 310 with various
androgen-induced growth factors and FGF-8. Accordingly, it is
presently believed that PRO187 polypeptide disclosed in the present
application is a newly-identified member of the FGF-8 protein
family and may possess identify activity or property typical of the
FGF-8-like protein family.
[0656] 5. Full-Length PR0265 Polypeptides
[0657] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides; referred to in the
present application as PRO265. In particular, Applicants have
identified and isolated cDNA encoding a PR0265 polypeptide, as
disclosed in further detail in the Examples below. Using programs
such as BLAST and FastA sequence alignment computer programs,
Applicants found that various portions of the PR0265 polypeptide
have significant homology with the fibromodulin protein and
fibromodulin precursor protein. Applicants have also found that the
DNA encoding the PR0265 polypeptide has significant homology with
platelet glycoprotein V, a member of the leucine rich related
protein family-involved in skin and wound repair. Accordingly, it
is presently believed that PR0265 polypeptide disclosed in the
present application is a newly identified member of the leucine
rich repeat family and possesses protein protein binding
capabilities, as well as be involved in skin and wound repair as
typical of this family.
[0658] 6. Full-Length PR0219 Polypeptides
[0659] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PR0219. In particular, Applicants have
identified and isolated cDNA encoding a PR0219 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 PR0219 polypeptide have significant
homology with the mouse and human matrilin-2 precursor
polypeptides. Accordingly, it is presently believed that PR0219
polypeptide disclosed in the present application is related to the
matrilin-2 precursor polypeptide.
[0660] 7. Full-Length PR0246 Polypeptides
[0661] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PR0246. In particular, Applicants have
identified and isolated cDNA encoding a PR0246 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 PR0246 polypeptide has significant homology with the
human cell surface protein HCAR. Accordingly, it is presently
believed that PR0246 polypeptide disclosed in the present
application may be a newly identified membrane-bound virus receptor
or tumor cell-specific antigen.
[0662] 8. Full-Length PR0228 Polypeptides
[0663] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PR0228. In particular, Applicants have
identified and isolated cDNA encoding a PR0228 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 PR0228 polypeptide have significant
homology with the EMR1 protein. Applicants have also found that the
DNA encoding the PR0228 polypeptide has significant homology with
latrophilin, macrophage-restricted cell surface glycoprotein,
B0457.1 and leucocyte antigen CD97 precursor. Accordingly, it is
presently believed that PR0228 polypeptide disclosed in the present
application is a newly identified member of the seven transmembrane
superfamily and possesses characteristics and functional properties
typical of this family. In particular, it is believed that PR0228
is a new member of the subgroup within this family to which CD97
and EMRI belong.
[0664] 9. Full-Length PRO533 Polypeptides
[0665] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PR0533. In particular, Applicants have
identified and isolated cDNA encoding a PR0533 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST-2
and FastA sequence alignment computer programs, Applicants found
that a full-length native, sequence PR0533 (shown in FIG. 22 and
SEQ ID NO:59) has a Blast score of 509 and 53% amino acid sequence
identity with fibroblast growth factor (FGF). Accordingly, it is
presently believed that PR0533 disclosed in the present application
is a newly identified member of the fibroblast growth factor family
and may possess activity typical of such polypeptides.
[0666] 10. Full-Length PR0245 Polypeptides
[0667] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO245. In particular, Applicants have
identified and isolated cDNA encoding a PR0245 polypeptide, as
disclosed in farther detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that a
portion of the amino acid sequence of the PR0245 polypeptide has
60% amino acid identity with the human c-myb protein. Accordingly,
it is presently believed that the PR0245 polypeptide disclosed in
the present application may be a newly identified member of the
transmembrane protein tyrosine kinase family.
[0668] 11. Full-Length PR0220, PR0221 and PR0227 Polypeptides
[0669] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PR0220, PR0221 and PRO227. In particular,
Applicants have identified and isolated cDNAs encoding a PRO220,
PR0221 and PR0227 polypeptide, respectively, as disclosed in
further detail in the Examples below. Using BLAST and FastA
sequence alignment computer programs, PRO220 has amino acid
identity with the amino acid sequence of a leucine rich protein
wherein the identity is 87%. PR0220 additionally has amino acid
identity with the neuronal leucine rich protein wherein the
identity is 55%. The neuronal leucine rich protein is further
described in Taguchi, et al., Mol. Brain Res., 35:31-40 (1996).
[0670] PR0221 has amino acid identity with the SLIT protein
precursor, wherein different portions of these two proteins have
the respective percent identities of 39%, 38%, 34%, 31%, and
30%.
[0671] PR0227 has amino acid identity with the amino acid sequence
of platelet glycoprotein V precursor. The same results were
obtained for human glycoprotein V. Different portions of these two
proteins show the following percent identities of 30%, 28%, 28%,
31%, 35%, 39% and 27%.
[0672] Accordingly, it is presently believed that PR0220, PR0221
and PR0227 polypeptides disclosed in the present application are
newly identified members of the leucine rich repeat protein
superfamily and that each possesses protein-protein binding
capabilities typical of the leucine rich repeat protein
superfamily. It is also believed that they have capabilities
similar to those of SLIT, the leucine rich repeat protein and human
glycoprotein V.
[0673] 12. Full-Length PR0258 Polypeptides
[0674] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PR0258. In particular, Applicants have
identified and isolated cDNA encoding a PR0258 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 PR0258 polypeptide have significant
homology with the CRTAM and poliovirus receptors. Accordingly, it
is presently believed that PRO258 polypeptide disclosed in the
present application is a newly identified member of the Ig
superfamily and possesses virus receptor capabilities or regulates
immune function as typical of this family.
[0675] 13. Full-Length PR0266 Polypeptides
[0676] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PR0266. In particular, Applicants have
identified and isolated cDNA encoding a PR0266 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 PR0266 polypeptide have significant
homology with the SLIT protein from Drosophilia . Accordingly, it
is presently believed that PR0266 polypeptide disclosed in the
present application is a newly identified member of the leucine
rich repeat family and possesses ligand-ligand binding activity and
neuronal development typical of this family. SLIT has been shown to
be useful in the study and treatment of Alzheimer's disease, supra,
and thus, PR0266 may have involvement in the study and cure of this
disease.
[0677] 14. Full-Length PR0269 Polypeptides
[0678] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO269. In particular, Applicants have
identified and isolated cDNA encoding a PRO269 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST,
FastA and sequence alignment computer programs, Applicants found
that the amino acid sequence encoded by nucleotides 314 to 1783 of
the full-length native sequence PR0269 (shown in FIG. 35 and SEQ ID
NO:95) has significant homology to human urinary thrombomodulin and
various thrombomodulin analogues respectively, to which it was
aligned. Accordingly, it is presently believed that PR0269
polypeptide disclosed in the present application is a newly
identified member of the tbrombomodulin family.
[0679] 15. Full-Length PR0287 Polypeptides
[0680] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PR0287. In particular, Applicants have
identified and isolated cDNA encoding a PR0287 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 PR0287 polypeptide have significant
homology with the type 1 procollagen C-proteinase enhancer protein
precursor and type 1 procollagen C-proteinase enhancer protein.
Accordingly, it is presently believed that PR0287 polypeptide
disclosed in the present application is a newly identified member
of the C-proteinase enhancer protein family.
[0681] 16. Full-Length PR0214 Polypeptides
[0682] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PR0214. In particular, Applicants have
identified and isolated cDNA encoding a PRO214 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that a
full-length native sequence PRO214 polypeptide (shown in FIG. 40
and SEQ ID NO: 109) has 49% amino acid sequence identity with HT
protein, a known member of the EGF-family. The comparison resulted
in a BLAST score of 920, with 150 matching nucleotides.
Accordingly, it is presently believed that the PR0214 polypeptide
disclosed in the present application is a newly identified member
of the family comprising EGF domains and may possess activities or
properties typical of the EGF-domain containing family.
[0683] 17. Full-Length PR0317 Polypeptides
[0684] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides, referred to in the
present application as PR0317. In particular, cDNA encoding a
PR0317 polypeptide has been identified and isolated, as disclosed
in further detail in the Examples below. Using BLAST.TM. and
FastA.TM. sequence alignment computer programs, it was found that a
full-length native-sequence PR0317 (shown in FIG. 42 and SEQ ID NO:
114) has 92% amino acid sequence identity with EBAF-1. Further, it
is closely aligned with many other members of the
TGF-superfamily.
[0685] Accordingly, it is presently believed that PR0317 disclosed
in the present application is a newly identified member of the
TGF-superfamily and may possess properties that are therapeutically
useful in conditions of uterine bleeding, etc. Hence, PR0317 maybe
useful in diagnosing or treating abnormal bleeding involved in
gynecological diseases, for example, to avoid or lessen the need
for a hysterectomy. PR0317 may also be useful as an agent that
affects angiogenesis in general, so PR0317 may be useful in
anti-tumor indications, or conversely, in treating coronary
ischemic conditions.
[0686] Library sources reveal that ESTs used to obtain the
consensus DNA for generating PROM primers and probes were found in
normal tissues (uterus, prostate, colon, and pancreas), in several
tumors (colon, brain (twice), pancreas, and mullerian cell), and in
a heart with ischemia. PR0317 has shown up in several tissues as
well, but it does look to have a greater concentration in uterus.
Hence, PR0317 may have a broader use by the body than EBAF-1. It is
contemplated that, at least for some indications, PR0317 may have
opposite effects from EBAF-1.
[0687] 18. Full-Length PR0301 Polypeptides
[0688] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PR0301. In particular, Applicants have
identified and isolated cDNA encoding a PR0301 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that a
full-length native sequence PR0301 (shown in FIG. 44 and SEQ ID NO:
119) has a Blast score of 246 corresponding t o 30% amino acid
sequence identity with human A33 antigen precursor. Accordingly, it
is presently believed that PR0301 disclosed in the present
application is a newly identified member of the A33 antigen protein
family and may be expressed in human neoplastic diseases such as
colorectal cancer.
[0689] 19. Full-Length PR0224 Polypeptides
[0690] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PR0224. In particular, Applicants have
identified and isolated cDNA encoding a PR0224 polypeptide, as
disclosed in further detail in the Examples below. Using known
programs such as BLAST and FastA sequence alignment computer
programs, Applicants found that full-length native PR0224 (FIG. 46,
SEQ ID NO: 127) has amino acid identity with apolipoprotein E
receptor 2906 from homo sapiens. The alignments of different
portions of these two polypeptides show amino acid identities of
37%,36%, 30%,44%,44% and 28% respectively. Full-length native
PR0224 (FIG. 46, SEQ ID NO: 127) also has amino acid identity with
very low-density lipoprotein receptor precursor from gall. The
alignments of different portions of these two polypeptides show
amino acid identities of 38%,37%,42%,33%, and 37% respectively.
Additionally, full-length native PR0224 (FIG. 46, SEQ ID NO: 127)
has amino acid identity with the chicken oocyte receptor P95 from
Gallus gal lus. The alignments of different portions of these two
polypeptides show amino acid identities of 38%, 37%, 42%, 33%, and
37% respectively. Moreover, full-length native PR0224 (FIG. 46, SEQ
ID NO: 127) has amino acid identity with very low density
lipoprotein receptor short form precursor from humans. The
alignments of different portions of these two polypeptides show
amino acid identities of 32%, 38%, 34%, 45%, and 31%, respectively.
Accordingly, it is presently believed that PR0224 polypeptide
disclosed in the present application is a newly identified member
of the low density lipoprotein receptor family and possesses the
structural characteristics required to have the functional ability
to recognize and endocytose low density lipoproteins typical of the
low density lipoprotein receptor family. (The alignments described
above used the following scoring parameters: T=7, S+65, S2=36,
Matrix: BLOSUM62.)
[0691] 20. Full-Length PR0222 Polypeptides
[0692] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PR0222. In particular, Applicants have
identified and isolated cDNA encoding a PR0222 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that a
sequence encoding full-length native sequence PR0222 (shown in FIG.
48 and SEQ ID NO: 132) has 25-26% amino acid identity with mouse
complement factor h precursor, has 27-29% amino acid identity with
complement receptor, has 25-47% amino acid identity with mouse
complement C3b receptor type 2 long form precursor, has 40% amino
acid identity with human hypothetical protein kiaa0247.
Accordingly, it is presently believed that PRO222 polypeptide
disclosed in the present application is a newly identified member
of the complement receptor family and possesses activity typical of
the complement receptor family.
[0693] 21. Full-Length PR0234 Polypeptides
[0694] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PR0234. In particular, Applicants have
identified and isolated cDNA encoding a PR0234 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST
(FastA-format) sequence alignment computer programs, Applicants
found that a cDNA sequence encoding full-length native sequence
PR0234 has 31% identity and Blast score of 134 with E-selectin
precursor. Accordingly, it is presently believed that the PR0234
polypeptides disclosed in the present application are newly
identified members of the lectin/selectin family and possess
activity typical of the lectin/selectin family.
[0695] 22. Full-Length PR0231 Polypeptides
[0696] The present invention provides newly identified and isolated
nucleotide sequence encoding polypeptides referred to in the
present application as PR0231. In particular, Applicants have
identified and isolated cDNA encoding a PR0231 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
the full-length native sequence PR0231 polypeptide (shown in FIG.
52 and SEQ ID NO: 142) has 30% and 31% amino acid identity with
human and rat prostatic acid phosphatase precursor proteins,
respectively. Accordingly, it is presently believed that the PR0231
polypeptide disclosed in the present application may be a newly
identified member of the acid phosphatase protein family.
[0697] 23. Full-Length PR0229 Polypeptides
[0698] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides; referred to in the
present application as PR0229. In particular, Applicants have
identified and isolated cDNA encoding a PR0229 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 PR0229 polypeptide have significant
homology with antigen wc1.1, M130 antigen, T cell surface
glycoprotein CD6 and CD6. It also is related to Sp-alpha.
Accordingly, it is presently believed that PR0229 polypeptide
disclosed in the present application is a newly identified member
of the family containing scavenger receptor homology, a sequence
motif found in a number of proteins involved in immune function and
thus possesses immune function and /or segments which resist
degradation, typical of this family.
[0699] 24. Full-Length PR0238 Polypeptides
[0700] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PROM. In particular, Applicants have
identified and isolated cDNA encoding a PR0238 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 PR0238 polypeptide have significant
homology with reductases, including oxidoreductase and fatty
acyl-CoA reductase. Accordingly, it is presently believed that
PR0238 polypeptide disclosed in the present application is a newly
identified member of the reductase family and possesses reducing
activity typical of the reductase family.
[0701] 25. Full-Length PR0233 Polypeptides
[0702] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PR0233. In particular, Applicants have
identified and isolated cDNA encoding a PR0233 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 PR0233-polypeptide have significant
homology with the reductase protein. Applicants have also found
that the DNA encoding the PR0233 polypeptide has significant
homology with proteins from Caenorhabditis elegans. Accordingly, it
is presently believed that PR0233 polypeptide disclosed in the
present application is a newly identified member of the reductase
family and possesses the ability to effect the redox state of the
cell typical of the reductase family.
[0703] 26. Full-Length PR0223 Polypeptides
[0704] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PR0223. In particular, Applicants have
identified and isolated cDNA encoding a PR0223 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
the PR0223 polypeptide has significant homology with various serine
carboxypeptidase polypeptides. Accordingly, it is presently
believed that PR0223 polypeptide disclosed in the present
application is a newly identified serine carboxypeptidase.
[0705] 27. Full-Length PR0235 Polypeptides
[0706] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PR0235. In particular, Applicants have
identified and isolated cDNA encoding a PRO235 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
various portions of the PR0235 polypeptide have significant
homology with the various plexin proteins. Accordingly, it is
presently believed that PR0235 polypeptide disclosed in the present
application is a newly identified member of the plexin family and
possesses cell adhesion properties typical of the plexin
family.
[0707] 28. Full-Length PR0236 and PR0262 Polypeptides
[0708] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PR0236 and PRO262. In particular, Applicants
have identified and isolated cDNA encoding PR0236 and PR0262
polypeptides, as disclosed in further detail in the Examples below.
Using BLAST and FastA sequence alignment computer programs,
Applicants found that various portions of the PR0236 and PR0262
polypeptides have significant homology with various
.beta.-galactosidase and .beta.-galactosidase precursor
polypeptides. Accordingly, it is presently believed that the PR0236
and PR0262 polypeptides disclosed in the present application are
newly identified P-galactosidase homologs.
[0709] 29. Full-Length PR0239 Polypeptides
[0710] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PROM. In particular, Applicants have
identified and isolated cDNA encoding a PR0239 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 PR0239 polypeptide have significant
homology with densin proteins. Accordingly, it is presently
believed that PR0239 polypeptide disclosed in the present
application is a newly identified member of the densin family and
possesses cell adhesion and the ability to effect synaptic
processes as is typical of the densin family.
[0711] 30. Full-Length PR0257 Polypeptides
[0712] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO257. In particular, Applicants have
identified and isolated cDNA encoding a PR0257 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
various portions of the PRO257 polypeptide have significant
homology with the ebnerin precursor and ebnerin protein.
Accordingly, it is presently believed that PR0257 polypeptide
disclosed in the present application is a newly identified protein
member which is related to the ebnerin protein.
[0713] 31. Full-Length PR0260 Polypeptides
[0714] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PR0260. In particular, Applicants have
identified and isolated cDNA encoding a PR0260 polypeptide, as
disclosed in further detail in the Examples below. Using programs
such as BLAST and FastA sequence alignment computer programs,
Applicants found that various portions of the PR0260 polypeptide
have significant homology with the alpha-1-fucosidase precursor.
Accordingly, it is presently believed that PR0260 polypeptide
disclosed in the present application is a newly identified member
of the fucosidase family and possesses enzymatic activity related
to fucose residues typical of the fucosidase family.
[0715] 32. Full-Length PR0263 Polypeptides
[0716] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PROM. In particular, Applicants have
identified and isolated cDNA encoding a PR0263 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 PR0263 polypeptide have significant
homology with the CD44 antigen and related proteins. Accordingly,
it is presently believed that PRO263 polypeptide disclosed in the
present application is a newly identified member of the CD44
antigen family and possesses at least one of the properties
associated with these antigens, i.e., cancer and HIV marker,
cell-cell or cell-matrix interactions, regulating cell traffic,
lymph node homing, transmission of growth signals, and presentation
of chemokines and growth facors to traveling cells.
[0717] 33. Full-Length PR0270 Polypeptides
[0718] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PROM. In particular, Applicants have
identified and isolated cDNA encoding a PRO270 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST,
FastA and sequence alignment computer programs, Applicants found
that that various portions of the PR0270 polypeptide have
significant homology with various thioredoxin proteins.
Accordingly, it is presently believed that PR0270 polypeptide
disclosed in the present application is a newly identified member
of the thioredoxin family and possesses the ability to effect
reduction-oxidation (redox) state typical of the thioredoxin
family.
[0719] 34. Full-Length PR0271 Polypeptides
[0720] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PR0271. In particular, Applicants have
identified and isolated cDNA encoding a PR0271 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
the PRO271 polypeptide has significant homology with various link
proteins and precursors thereof. Accordingly, it is presently
believed that PR0271 polypeptide disclosed in the present
application is a newly identified link protein homolog.
[0721] 35. Full-Length PR0272 Polypeptides
[0722] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PROM. In particular, Applicants have
identified and isolated cDNA encoding a PR0272 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 PR0272 polypeptide have significant
homology with the human reticulocalbin protein and its precursors.
Applicants have also found that the DNA encoding the PR0272
polypeptide has significant homology with the mouse reticulocalbin
precursor protein. Accordingly, it is presently believed that
PR0272 polypeptide disclosed in the present application is a newly
identified member of the reticulocalbin family and possesses the
ability to bind calcium typical of the reticulocalbin family.
[0723] 36. Full-Length PR0294 Polypeptides
[0724] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PR0294. In particular, Applicants have
identified and isolated cDNA encoding a PR0294 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 PR0294 polypeptide have significant
homology with the various portions of a number of collagen
proteins. Accordingly, it is presently believed that PR0294
polypeptide disclosed in the present application is a newly
identified member of the collagen family.
[0725] 37. Full-Length PR0295 Polypeptides
[0726] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PR0295. In particular, Applicants have
identified and isolated cDNA encoding a PR0295 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 PR0295 polypeptide have significant
homology with integrin proteins. Accordingly, it is presently
believed that PR0295 polypeptide disclosed in the present
application is a newly identified member of the integrin family and
possesses cell adhesion typical of the integrin family.
[0727] 38. Full-Length PR0293 Polypeptides
[0728] The present invention provides newly-identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PR0293. In particular, Applicants have
identified and isolated cDNA encoding a PRO293 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs. Applicants found that
portions of the PR0293 polypeptide have significant homology with
the neuronal leucine rich repeat proteins 1 and 2, (NLRR-1 and
NLRR-2), particularly NLRR-2. Accordingly, it is presently believed
that PR0293 polypeptide disclosed in the present application is a
newly identified member of the neuronal leucine rich repeat protein
family and possesses ligand-ligand binding activity typical of the
NRLL protein family.
[0729] 39. Full-Length PR0247 Polypeptides
[0730] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PR0247. In particular, Applicants have
identified and isolated cDNA encoding a PR0247 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 PR0247 polypeptide have significant
homology with densin. Applicants have also found that the DNA
encoding the PRO247 polypeptide has significant homology with a
number of other proteins, including KIAA0231. Accordingly, it is
presently believed that PR0247 polypeptide disclosed in the present
application is a newly identified member of the leucine rich repeat
family and possesses ligand binding abilities typical of this
family.
[0731] 40. Full-Length PRO302, PR0303, PROM, PR0307 and PR0343
Polypeptides
[0732] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PR0302, PR0303, PR0304, PR0307 and PR0343.
In particular, Applicants have identified and isolated cDNA
encoding PR0302, PROM, PR0304, PR0307 and PR0343 polypeptides, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
various portions of the PR0302, PROM, PR0304, PR0307 and PROM
polypeptides have significant homology with various protease
proteins. Accordingly, it is presently believed that the PROM,
PR0303, PR0304, PR0307 and PR0343 polypeptides disclosed in the
present application are newly identified protease proteins.
[0733] 41. Full-Length PR0328 Polypeptides
[0734] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PR0328. In particular, Applicants have
identified and isolated cDNA encoding a PR0328 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 PR0328 polypeptide have significant
homology with the human glioblastoma protein ("GLIP"). Further,
Applicants found that various portions of the PR0328 polypeptide
have significant homology with the cysteine rich secretory protein
("CRISP") as identified by BLAST homology [ECCRISP3.sub.'1, S68683,
and CRS3_HUMAN]. Accordingly, it is presently believed that PR0328
polypeptide disclosed in the present application is a newly
identified member of the GLIP or CRISP families and possesses
transcriptional regulatory activity typical of the GLIP or CRISP
families.
[0735] 42. Full-Length PR0335, PR0331 and PR0326 Polypeptides
[0736] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PROM, PR0331 or PR0326. In particular,
Applicants have identified and isolated cDNA encoding a PROM,
PR0331 or PR0326 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 PROM,
PR0331 or PR0326 polypeptide have, significant homology with LIG-1,
ALS and in the case of PROM, additionally, decorin. Accordingly, it
is presently believed that the PROM, PR0331 and PR0326 polypeptides
disclosed in the present application are newly identified members
of the leucine rich repeat superfamily, and particularly, are
related to LIG-1 and possess the biological functions of this
family as discussed and referenced herein.
[0737] 43. Full-Length PR0332 Polypeptides
[0738] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PR0332. In particular, Applicants have
identified and isolated cDNA encoding PR0332 polypeptides, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that a
full-length native sequence PR0332 (shown in FIG. 108 and SEQ ID
NO:310) has about 30-40% amino acid sequence identity with a series
of known proteoglycan sequences, including, for example,
fibromodulin and fibromodulin precursor sequences of various
species (FMOD_BOVIN, FMOD_CHICK, FMOD_RAT, FMOD_MOUSE, FMOD_HUMAN,
P_R36773), osteomodulin sequences (AB000114.sub.--1,
AB007848.sub.--1), decorin sequences (CFU83141.sub.--1,
OCU03394.sub.--1, P_R42266, P_R42267, PR42260, P_R89439), keratan
sulfate proteoglycans (BTU48360.sub.--1, AF022890.sub.--1), corneal
proteoglycan (AF022256.sub.--1), and bone/cartilage proteoglycans
and proteoglycane precursors (PGS1_BOVIN, PGS2_MOUSE, PGS2_HUMAN).
Accordingly, it is presently believed that PR0332 disclosed in the
present application is a new proteoglycan-type molecule, and may
play a role in regulating extracellular matrix, cartilage, and/or
bone function.
[0739] 44. Full-Length PR0334 Polypeptides
[0740] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PR0334. In particular, Applicants have
identified and isolated cDNA encoding a PR0334 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 PR0334 polypeptide have significant
homology with fibulin and fibrillin. Accordingly, it is presently
believed that PR0334 polypeptide disclosed in the present
application is a newly identified member of the epidermal growth
factor family and possesses properties and activities typical of
this family.
[0741] 45. Full-Length PR0346 Polypeptides
[0742] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO346. In particular, Applicants have
identified and isolated cDNA encoding a PR0346 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that a
full-length native sequence PR0346 (shown in FIG. 112 and SEQ ID
NO:320) has 28% amino acid sequence identity with carcinoembryonic
antigen. Accordingly, it is presently believed that PR0346
disclosed in the present application is a newly identified member
of the carcinoembryonic protein family and may be expressed in
association with neoplastic tissue disorders.
[0743] 46. Full-Length PR0268 Polypeptides
[0744] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PRO268. In particular, Applicants have
identified and isolated cDNA encoding a PR0268 polypeptide, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that
portions of the PR0268 polypeptide have significant homology with
the various protein disulfide isomerase proteins. Accordingly, it
is presently believed that PR0268 polypeptide disclosed in the
present application is a homolog of the protein disulfide isomerase
p5 protein.
[0745] 47. Full-Length PR0330 Polypeptides
[0746] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PR0330. In particular, Applicants have
identified and isolated cDNA encoding a PR0330 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 PR0330 polypeptide have significant
homology. with the murine prolyl 4-hydroxylase alpha-II subunit
protein. Accordingly, it is presently believed that PR0330
polypeptide disclosed in the present application is a novel prolyl
4-hydroxylase subunit polypeptide.
[0747] 48. Full-Length PR0339 and PR0310 Polypeptides
[0748] The present invention provides newly identified and isolated
nucleotide sequences encoding polypeptides referred to in the
present application as PR0339 and PR0310. In particular, Applicants
have identified and isolated cDNA encoding a PR0339 polypeptide, as
disclosed in further detail in the Examples below. Applicants have
also identified and isolated cDNA encoding a PR03 10 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 PR0339 and PR0310 polypeptides have
significant homology with small secreted proteins from C. elegans
and are distantly related to fringe. PR0339 also shows homology to
collagen-like polymers. Sequences which were used to identify PROM,
designated herein as DNA405D and DNA42267, also show homology to
proteins from C. elegans. Accordingly, it is presently believed
that the PR0339 and PR0310 polypeptides disclosed in the present
application are newly identified member of the family of proteins
involved in development, and which may have regulatory abilities
similar to the capability of fringe to regulate serrate.
[0749] 49. Full Length PR0244 Polypeptides
[0750] The present invention provides newly identified and isolated
nucleotide sequences encoding C-type lectins referred to in the
present application as PRO244. In particular, applicants have
identified and isolated cDNA encoding PR0244 polypeptides, as
disclosed in further detail in the Examples below. Using BLAST and
FastA sequence alignment computer programs, Applicants found that a
full-length native sequence PRO244 (shown in FIG. 122 and SEQ ID
NO:377) has 43% amino acid sequence identity with the hepatic
lectin gallus gallus (LECH-CHICK), and 42% amino acid sequence
identity with an HIV gp120 binding C-type lectin (A46274).
Accordingly, it is presently believed that PR0244 disclosed in the
present application is a newly identified member of the C-lectin
superfamily and may play a role in immune function, apoptosis, or
in the pa thogenesis of atherosclerosis. In addition, PR0244 may be
useful in identifying tumor-associated epitopes.
[0751] B. PRO Polypeptide Variants
[0752] 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.
[0753] Variations in the native full-length sequence PRO or in
various domains of the PRO described herein, can be made, for
example, using any of the techniques and guidelines for
conservative and non-conservative mutations set forth, for
instance, in U.S. Pat. No. 5,364,934. Variations may be a
substitution, deletion or insertion of one or more codons encoding
the PRO that results in a change in the amino acid sequence of the
PRO as compared with the native sequence PRO. Optionally the
variation is by substitution of at least one amino acid with any
other amino acid in one or more of the domains of the PRO. Guidance
in determining which amino acid residue may be inserted,
substituted or deleted without adversely affecting the desired
activity may be found by comparing the sequence of the PRO with
that of homologous known protein molecules and minimizing the
number of amino acid sequence changes made in regions of high
homology. Amino acid substitutions can be the result of replacing
one amino acid with another amino acid having similar structural
and/or chemical properties, such as the replacement of a leucine
with a serine, i.e., conservative amino acid replacements.
Insertions or deletions may optionally be in the range of about 1
to 5 amino acids. The variation allowed may be determined by
systematically making insertions, deletions or substitutions of
amino acids in the sequence and testing the resulting variants for
activity exhibited by the full-length or mature native
sequence.
[0754] 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.
[0755] 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.
[0756] 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. TABLE-US-00005 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 glu glu
Cys (C) ser ser Gin (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; ala; leu norleucine
[0757] 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: [0758] (1) hydrophobic: norleucine, met,
ala, val, leu, ile; [0759] (2) neutral hydrophilic: cys, ser, thr;
[0760] (3) acidic: asp, glu; [0761] (4) basic: asn, gIn, his, lys,
arg; [0762] (5) residues that influence chain orientation: gly,
pro; and [0763] (6) aromatic: trp, tyr, phe.
[0764] 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.
[0765] 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.
[0766] Scanning amino acid analysis can also be employed to
identify one or more amino acids along a contiguous sequence. Among
the preferred scanning amino acids are relatively small, neutral
amino acids. Such amino acids include alanine, glycine, serine, and
cysteine. Alanine is typically a preferred scanning amino acid
among this group because it eliminates the side-chain beyond the
beta-carbon and is less likely to alter the main-chain conformation
of the variant [Cunningham and Wells, Science, 244: 1081 -1085
(1989)]. Alanine is also typically preferred because it is the most
common amino acid. Further, it is frequently found in both buried
and exposed positions [Creighton, The Proteins, (W.H. Freeman &
Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)], If alanine
substitution does not yield adequate amounts of variant, an
isoteric amino acid can be used.
[0767] C. Modifications of PRO
[0768] Covalent modifications of PRO are included within the scope
of this invention. One type of covalent modification includes
reacting targeted amino acid residues of a PRO polypeptide with an
organic derivatizing agent that is capable of reacting with
selected side chains or the N-- or C-terminal residues of the PRO.
Derivatization with bifunctional agents is useful, for instance,
for crosslinking PRO to a water-insoluble support matrix or surface
for use in the method for purifying anti-PRO antibodies, and
vice-versa. Commonly used crosslinking agents include, e.g.,
1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,
N-hydroxysuccinimide esters, for example, esters with
4-azidosalicylic acid, homobifunctional imidoesters, including
disuccinimidyl esters such as
3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides
such as bis-N-maleimido-1,8-octane and agents such as
mediyl-3-[(p-azidophenyl)dithio]propioiniidate.
[0769] 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.
[0770] 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.
[0771] 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.
[0772] 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 11 Sep. 1987,
and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306
(1981).
[0773] Removal of carbohydrate moieties present on the PRO
polypeptide may be accomplished chemically or enzymatically or by
mutational substitution of codons encoding for amino acid residues
that serve as targets for glycosylation. Chemical deglycosylation
techniques are known in the art and described, for instance, by
Hakimuddin, et al., Arch. Biochem. Biophys. 259:52 (1987) and by
Edge et al., Anal. Biochem., 118:131 (1981). Enzymatic cleavage of
carbohydrate moieties on polypeptides can be achieved by the use of
a variety of endo- and exo-glycosidases as described by Thotakura
et al., Meth. Enamol. 138:350 (1987).
[0774] 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 glyco, 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.
[0775] 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.
[0776] In one embodiment, such a chimeric molecule comprises a
fusion of the PRO with a tag polypeptide which provides an epitope
to which an anti-tag antibody can selectively bind. The epitope tag
is generally placed at the amino- or carboxyl-terminus of the PRO.
The presence of such epitope-tagged forms of the PRO can be
detected using an antibody against the tag polypeptide. Also,
provision of the epitope tag enables the PRO to be readily purified
by affinity purification using an anti-tag antibody or another type
of affinity matrix that binds to the epitope tag. Various tag
polypeptides and their respective antibodies are well known in the
art. Examples include poly-histidine (poly-his) or
poly-histidine-glycine (poly-his-gly) tags; the flu HA tag
polypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol.,
8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7
and 9E10 antibodies thereto [Evan et al., Molecular and Cellular
Biology, 5:3610-3616 (1985)]; and the Herpes Simplex virus
glycoprotein D (gD) tag and its antibody [Paborsky et al., Protein
Engineering 3(6):547-553 (1990)]. Other tag polypeptides include
the Flag-peptide [Hopp et al., BioTechnology 6:1204-1210 (1988)];
the KT3 epitope peptide [Martin et al., Science 255:192-194
(1992)]; an .alpha.-tubulin epitope peptide [Skinner et al., J.
Biol. Chem. 266:15163-15166 (1991)]; and the T7 gene 10 protein
peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA
87:6393-6397 (1990)].
[0777] In an alternative embodiment, the chimeric molecule may
comprise a fusion of the PRO with an immunoglobulin or a particular
region of an immunoglobulin. For a bivalent form of the chimeric
molecule (also referred to as an "immunoadhesin"), such a fusion
could be to the Fc region of an IgG molecule. The Ig fusions
preferably include the substitution of a soluble (transmembrane
domain deleted or inactivated) form of a PRO polypeptide in place
of at least one variable region within an Ig molecule. In a
particularly preferred embodiment, the immunoglobulin fusion
includes the hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3
regions of an IgG1 molecule. For the production of immunoglobulin
fusions see also U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.
[0778] D. Preparation of PRO
[0779] 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.
[0780] 1. Isolation of DNA Encoding PRO
[0781] 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).
[0782] 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)].
[0783] 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.
[0784] Sequences identified in such library screening methods can
be compared and aligned to other known sequences deposited and
available in public databases such as GenBank or other private
sequence databases. Sequence identity (at either the amino acid or
nucleotide level) within defined regions of the molecule or across
the full-length sequence can be determined using methods known in
the art and as described herein.
[0785] 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.
[0786] 2. Selection and Transformation of Host Cells
[0787] 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.
[0788] Methods of eukaryotic cell transfection and prokaryotic tell
transformation are known to the ordinarily skilled artisan, for
example, CaC1.sub.2, CaPO.sub.4, liposome-mediated and
electroporation. Depending on the host cell used, transformation is
performed using standard techniques appropriate to such cells. The
calcium.treatment employing calcium chloride, as described in
Sambrook et al., supra, or electroporation is generally used for
prokaryotes. Infection with Agrobacterium tumefaciens is used for
transformation of certain plant cells, as described by Shaw et al.,
Gene, 23:315 (1983) and WO 89/05859 published 29 Jun. 1989. For
mammalian cells without such cell walls, the calcium phosphate
precipitation method of Graham and van der Eb, Virology 52:456457
(1978) can be employed. General aspects of mammalian cell host
system transfections have been described in U.S. Pat. No.
4,399,216. Transformations into yeast are typically carried out
according to the method of Van Solingen et al., J. Bact., 130:946
(1977) and Hsiao et al., Proc. Nati. Acad. Sci. (USA), 16:3829
(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).
[0789] 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
(ATCC27,325)and K5 772(ATCC53,635). Other suitable prokaryotic host
cells include Enterobacteriaceae such as Escherichia, e.g., E.
coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g.,
Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and
Shigella, as well as Bacilli such as B. subtilis and B.
licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710
published 12 Apr. 1989), Pseudomonas such as P. aeruginosa, and
Streptomyces. These examples are illustrative rather than limiting.
Strain W3110 is one particularly preferred host or parent host
because it is a common host strain for recombinant DNA product
fermentations. Preferably, the host cell secretes minimal amounts
of proteolytic enzymes. For example, swain W3110 may be modified to
effect a genetic mutation in the genes encoding proteins endogenous
to the host, with examples of such hosts including E. coli W3110
strain I A2, which has the complete genotype tonA ; E. coli W3110
strain 9E4, which has the complete genotype tonA ptr3; E. coli
W3110 strain 27C7 (ATCC 55,244), which has the complete genotype
tonA ptr3; phoA E15 (argF-lac)169 degP ompTkan'; E. coli W3110
strain 37D6, which has the complete genotype tonA ptr3phoA E15
(argF-lac) 169 degP ompT rbs7 ilvG kan'; E. coli W3110 strain 40B4,
which is strain 37D6 with a non-kanamycin resistant degP deletion
mutation; and an E. coli strain having mutant periplasmic protease
disclosed in U.S. Pat. No. 4,946,783 issued 7 Aug. 1990.
Alternatively, in vitro methods of cloning, e.g., PCR or other
nucleic acid polymerase reactions, are suitable.
[0790] 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); Kluyveromyces hosts (U.S.
Pat. No. 4,943,529; Fleer et al., Bio/Technology 9:968-975 (1991))
such as, e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et
al., J. Bacteriol. 737 {1983]), K. fragilis (ATCC 12,424), K
bulgaricus (ATCC 16,045), K wickeramii (ATCC 24,178) K waltii (ATCC
56,500), K drosophilarum (ATCC 36,906; Van den Berg et al.,
Bio/Technology 8:135 (1990)), K thermotolerans, and K marxianus;
yarrowia (EP 402,226); Pichia pastoris (EP 183,070; Sreekrishna et
al., J. Basic Microbiol. 28:265-278 [1988]); Candida,- Trichoderma
reesia (EP 244,234); Neurospora crassa (Case et al., Proc. Natl.
Acad. Sci. USA, 76:5259-5263 [1979]); Schwanniomyces such as
Schwanniomyces occidentalis (EP 394,538 published 31 Oct. 1990);
and filamentous fungi such as, e.g., Neurospora, Penicillium,
Tolypocladium (WO 91/00357 published 10 Jan. 1991), and Aspergillus
hosts such as A. nidulans (Ballance et al., Biochem. Biophys. Res.
Commun., 112:284-289 [1983]; Tilburn et al., Gene, 26:205-221
[1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81: 1470-1474
[1984]) and A. niger (Kelly and Hynes, EMBO J. 4:475-479 [1985]).
Methylotropic yeasts are suitable herein and include, but are not
limited to, yeast capable of growth on methanol selected from the
genem consisting of Hansenula, Candida, Kloeckera, Pichia,
Saccharomyces, Torulopsis, and Rhodotorula. A list of specific
species that are exemplary of this class of yeasts may be found in
C. Anthony, The Biochemistry Methylotrophs 269 (1982).
[0791] Suitable host cells for the expression of glycosylated PRO
are derived from multicellular organisms. Examples of invertebrate
cells include insect cells such as Drosophila S2 and Spodoptera
Sf9, as well as plant cells. Examples of useful mammalian host cell
lines include Chinese hamster ovary (CHO) and COS cells. More
specific examples include monkey kidney CV1 line transformed by
SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or
293 cells subcloned for growth in suspension culture, Graham et
al., J. Gen Virol., 36:59 (1977)); Chinese hamster ovary
cells/-DHFR (CHO, Urlaub and Chasin, Prop. Natl. Acad. Sci. USA
77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.
23:243-251 (1980)); human lung cells (W138, ATCC CCL 75); human
liver cells (Hep G2, HB 8065); and mouse mammary tumor (MMT 060562,
ATCC CCL51). The selection of the appropriate host cell is deemed
to be within the skill in the art.
[0792] 3. Selection and Use of a Replicable Vector
[0793] The nucleic acid (e.g., cDNA or genomic DNA) encoding PRO
may be inserted into a replicable vector for cloning (amplification
of the DNA) or for expression. Various vectors are publicly
available. The vector may, for example, be in the form of a
plasmid, cosmid, viral particle, or phage. The appropriate nucleic
acid sequence may be inserted into the vector by a variety of
procedures. In general, DNA is inserted into an appropriate
restriction endonuclease site(s) using techniques known in the art.
Vector components generally include, but are not limited to, one or
more of a signal sequence, an origin of replication, one or more
marker genes, an enhancer element, a promoter, and a transcription
termination sequence. Construction of suitable vectors containing
one or more of these components employs standard ligation
techniques which are known to the skilled artisan.
[0794] 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 the
vector, or it may be a part of the PRO-encoding DNA that is
inserted into the vector. The signal sequence may be a prokaryotic
signal sequence selected, for example, from the group of the
alkaline phosphatase, penicillinase, 1pp, or heat-stable
enterotoxin II leaders. For yeast secretion the signal sequence may
be, e.g., the yeast invertase leader, alpha factor leader
(including Saccharomyces and Kluyveromyces .alpha.-factor leaders,
the latter described in U.S. Pat. No. 5,010,182), or acid
phosphatase leader, the C albicans glucoarnylase leader (EP 362,179
published 4 Apr. 1990), or the signal described in WO 90/13646
published 15 Nov. 1990. In mammalian cell expression, mammalian
signal sequences may be used to direct secretion of the protein,
such as signal sequences from secreted polypeptides of the same or
related species, as well as viral secretory leaders.
[0795] Both expression and cloning vectors contain a nucleic acid
sequence that enables the vector to replicate in one or more
selected host cells. Such sequences are well known for a variety of
bacteria, yeast, and viruses. The origin of replication from the
plasmid pBR322 is suitable for most Gram-negative bacteria, the
2.mu. plasmid origin is suitable for yeast, and various viral
origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for
cloning vectors in mammalian cells.
[0796] 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.
[0797] 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. Nad. Acad. Sci.
USA. 77:4216 (1980). A suitable selection gene for use in yeast is
the trp1 gene present in the yeast plasmid YRp7 [Stinchcomb et al.,
Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979);
Tschemper et al., Gene 10: 157 (1980)]. The trp1 gene provides a
selection marker for a mutant strain of yeast lacking the ability
to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1
[Jones, Genetics 85:12 (1977)].
[0798] Expression and cloning vectors usually contain a promoter
operably linked to the PRO-encoding nucleic acid sequence to direct
mRNA synthesis. Promoters recognized by a variety of potential host
cells are well known. Promoters suitable for use with prokaryotic
hosts include the P-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. Nati.
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.
[0799] 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. Ady. 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.
[0800] 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,
glyceraidehyde-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.
[0801] PRO transcription from vectors in mammalian host cells is
controlled, for example, by promoters obtained from the genomes of
viruses such as polyorna virus, fowlpox virus (UK 2,211,504
published 5 Jul. 1989), adenovirus (such as Adenovirus 2), bovine
papilloma virus, avian sarcoma virus, cytomegalovirus, a
retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from
heterologous mammalian promoters, e.g., the actin promoter or an
immunoglobulin promoter, and from heat-shock promoters, provided
such promoters are compatible with the host cell systems.
[0802] 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 16 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.
[0803] Expression vectors used in eukaryotic host cells (yeast,
fungi, insect, plant, animal, human, or nucleated cells from other
multicellular organisms) will also contain sequences necessary for
the termination of transcription and for stabilizing the mRNA. Such
sequences are commonly available from the 5' and, occasionally 3',
untranslated regions of eukaryotic or viral DNAs or cDNAs. These
regions contain nucleotide segments transcribed as polyadenylated
fragments in the untranslated portion of the mRNA encoding PRO.
[0804] 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 (198
1); Mantei et al., Nature 281:4046 (1979); EP 117,060; and EP
117,058.
[0805] 4. Detecting Gene Amplification/Expression
[0806] Gene amplification and/or expression may be measured in a
sample directly, for example, by conventional Southern blotting,
Northern blotting to quantitate the transcription of mRNA [Thomas,
Proc. NatI. 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.
[0807] Gene expression, alternatively, may be measured by
immunological methods, such as immunohistochemical staining of
cells or tissue sections and assay of cell culture or body fluids,
to quantitate directly the expression of gene product. Antibodies
useful for immunohistochemical staining and/or assay of sample
fluids may be either monoclonal or polyclonal, and may be prepared
in any mammal. Conveniently, the antibodies may be prepared against
a native sequence 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.
[0808] 5. Purification of Polypeptide
[0809] 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.
[0810] 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.
[0811] E. Uses for PRO
[0812] 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.
[0813] The full-length native sequence PRO gene, or portions
thereof, may be used as hybridization probes for a cDNA library to
isolate the full-length PRO cDNA or to isolate still other cDNAs
(for instance, those encoding naturally-occurring variants of PRO
or PRO from other species) which have a desired sequence identity
to the native PRO sequence disclosed herein. Optionally, the length
of the probes will be about 20 to about 50 bases. The hybridization
probes may be derived from at least partially novel regions of the
full length native nucleotide sequence wherein those regions may be
determined without undue experimentation or from genomic sequences
including promoters, enhancer elements and introns of native
sequence PRO. By way of example, a screening method will comprise
isolating the coding region of the PRO gene using the known DNA
sequence to synthesize a selected probe of about 40 bases.
Hybridization probes may be labeled by a variety of labels,
including radionucleotides such as .sup.32P .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.
[0814] Any EST sequences disclosed in the present application may
similarly be employed as probes, using the methods disclosed
herein.
[0815] 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.
(BioTechnigues 6:958, 1988).
[0816] 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.
[0817] 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.
[0818] Antisense or sense oligonucleotides may be introduced into a
cell containing the target nucleic acid sequence by any gene
transfer method, including, for example, CaP0.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 retrovinis M-MuL V, N2 (a
retrovirus derived from M-MuL V), or the double copy vectors
designated DCT5A, DCT5B and DCT5C (see WO 90/13641).
[0819] Sense or antisense oligonucleotides also may be introduced
into a cell containing the target nucleotide sequence by formation
of a conjugate with a ligand binding molecule, as described in WO
91/04753. Suitable ligand binding molecules include, but are not
limited to, cell surface receptors, growth factors, other
cytokines, or other ligands that bind to cell surface receptors.
Preferably, conjugation of the ligand binding molecule does not
substantially interfere with the ability of the ligand binding
molecule to bind to its corresponding molecule or receptor, or
block entry of the sense or antisense oligonucleotide or its
conjugated version into the cell.
[0820] Alternatively, a sense or an antisense oligonucleotide may
be introduced into a cell containing the target nucleic acid
sequence by formation of an oligonucleotide-lipid complex, as
described in WO 90/10448. The sense or antisense
oligonucleotide-lipid complex is preferably dissociated within the
cell by an endogenous lipase.
[0821] Antisense RNA or DNA molecules are generally at least about
5 bases in length, about 10 bases in length, about 15 bases in
length, about 20 bases in length, about 25 bases in length, about
30 bases in length, about 35 bases in length, about 40 bases in
length, about 45 bases in length, about 50 bases in length, about
55 bases in length, about 60 bases in length, about 65 bases in
length, about 70 bases in length, about 75 bases in length, about
80 bases in length, about 85 bases in length, about 90 bases in
length, about 95 bases in length, about 100 bases in length, or
more.
[0822] The probes may also be employed in PCR techniques to
generate a pool of sequences for identification of closely related
PRO coding sequences.
[0823] 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.
[0824] 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.
[0825] 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 mi accordance with
established techniques and the genomic sequences used to generate
transgenic animals that contain cells which express DNA encoding
PRO. Methods for generating transgenic animals, particularly
animals such as mice or rats, have become conventional in the art
and are described, for example, in U.S. Pat. Nos. 4,736,866 and
4,870,009. Typically, particular cells would be targeted for PRO
transgene incorporation with tissue-specific enhancers. Transgenic
animals that include a copy of a transgene encoding PRO introduced
into the germ line of the animal at an embryonic stage can be used
to examine the effect of increased expression of DNA encoding PRO.
Such animals can be used as tester animals for reagents thought to
confer protection from, for example, pathological conditions
associated with its overexpression. In accordance with this facet
of the invention, an animal is treated with the reagent and a
reduced incidence of the pathological condition, compared to
untreated animals bearing the transgene, would indicate a potential
therapeutic intervention for the pathological condition.
[0826] Alternatively, non-human homologues of PRO can be used to
construct a PRO "knock out" animal which has a defective or altered
gene encoding PRO as a result of homologous recombination between
the endogenous gene encoding PRO and altered genomic DNA encoding
PRO introduced into an embryonic stem cell of the animal. For
example, cDNA encoding PRO can be used to clone genomic DNA
encoding PRO in accordance with established techniques. A portion
of the genomic DNA encoding PRO can be deleted or replaced with
another gene, such as a gene encoding a selectable marker which can
be used to monitor integration. Typically, several kilobases of
unaltered flanking DNA (both at the 5' and 3' ends) are included in
the vector [see e.g., Thomas and Capecchi, Cell, 51:503 (1987) for
a description of homologous recombination vectors]. The vector is
introduced into an embryonic stem cell line (e.g., by
electroporation) and cells in which the introduced DNA has
homologously recombined with the endogepous 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.
[0827] 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. (Zarnecnik
et al, Proc. Nati. 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.
[0828] There are a variety of techniques available for introducing
nucleic acids into viable cells. The techniques vary depending upon
whether the nucleic acid is transferred into cultured cells in
vitro, or in vivo in the cells of the intended host. Techniques
suitable for the transfer of nucleic acid into mammalian cells in
vitro include the use of liposomes, electroporation,
microinjection, cell fusion, DEAE-dextran, the calcium phosphate
precipitation method, etc. The currently preferred in vivo gene
transfer techniques include transfection with viral (typically
retroviral) vectors and viral coat protein-liposome mediated
transfection (Dzau et al., Trends in Biotechnology 11, 205-210
[1993]). In some situations it is desirable to provide the nucleic
acid source with an agent that targets the target cells, such as an
antibody specific for a cell surface membrane protein or the target
cell, a ligand for a receptor on the target cell, etc. Where
liposomes are employed, proteins which bind to a cell surface
membrane protein associated with endocytosis may be used for
targeting and/or to facilitate uptake, e.g. capsid proteins or
fragments thereoftropic 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).
[0829] 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.
[0830] 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.
[0831] 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.
[0832] The PRO polypeptides described herein may also be employed
as therapeutic agents. The PRO polypeptides of the present
invention can be formulated according to known methods to prepare
pharmaceutically useful compositions, whereby the PRO product
hereof is combined in admixture with a pharmaceutically acceptable
carrier vehicle. Therapeutic formulations are prepared for storage
by mixing the active ingredient having the desired degree of purity
with optional physiologically acceptable carriers, excipients or
stabilizers (Remington's Pharmaceutical Sciences 16th edition,
Osol, A. Ed. (1980)), in the form of lyophilized formulations or
aqueous solutions. Acceptable carriers, excipients or stabilizers
are nontoxic to recipients at the dosages and concentrations
employed, and include buffers such as phosphate, citrate and other
organic acids; antioxidants including ascorbic acid; low molecular
weight (less than about 10 residues) polypeptides; proteins, such
as serum albumin, gelatin or immunoglobulins; hydrophilic polymers
such as polyvinylpyrrolidone, amino acids such as glycine,
glutamine, asparagine, arginine or lysine; monosaccharides,
disaccharides and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugar alcohols such as
mannitol or sorbitol; salt-forming counterions such as sodium;
and/or nonionic surfactants such as TWEEN.TM., PLURONICS.TM. or
PEG.
[0833] 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.
[0834] Therapeutic compositions herein generally are placed into a
container having a sterile access port, for example, an intravenous
solution bag or vial having a stopper pierceable by a hypodermic
injection needle.
[0835] 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.
[0836] 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.
[0837] 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.
[0838] Where sustained-release administration of a PRO polypeptide
is desired in a formulation with release characteristics suitable
for the treatment of any disease or disorder requiring
administration of the PRO polypeptide, microencapsulation of the
PRO polypeptide is contemplated. Microencapsulation of recombinant
proteins for sustained release has been successfully performed with
human growth hormone (rhGH), interferon- (rhIFN-), interleukin-2,
and MN rgp 120. Johnson et al., Nat. Med., 2:795-799 (1996);
Yasuda, Biomed. Ther., 27:1221-1223 (1993); Hora et al.,
Bio/Technology, 8:755-758,(1990); Cleland, "Design and Production
of Single Immunization Vaccines Using Polylactide Polyglycolide
Microsphere Systems," in Vaccine Design: The Subunit and Adjuvant
Approach, Powell and Newman, eds, (Plenum Press: New York, 1995),
pp. 439-462; WO 97/03692, WO 96/40072, WO 96/07399; and U.S. Pat.
No. 5,654,010.
[0839] 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.
[0840] 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.
[0841] 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.
[0842] 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.
[0843] 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.
[0844] 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., Prod. Natl. Acad. Sci.
USA 88:9578-9582 (1991)) as disclosed by Cbevray and Nathans, Proc.
Natl. Acad. Sci. USA, 89: 5789-5793 (199 1). 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 GAL4, and
another, in which candidate activating proteins are fused to the
activation domain. The expression of a GAL1-1acZ reporter gene
under control of a GAL4-activated promoter depends on
reconstitution of GAL4 activity via protein-protein interaction.
Colonies containing interacting polypeptides are detected with a
chromogenic substrate for .beta.-galactosidase. A complete kit
(MATCHMAKER.TM.) for identifying protein-protein interactions
between two specific proteins using the two-hybrid technique is
commercially available from Clontech. This system can also be
extended to map protein domains involved in specific protein
interactions as well as to pinpoint amino acid residues that are
crucial for these interactions.
[0845] 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.
[0846] 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.
[0847] 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.
[0848] 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.
[0849] More specific examples of potential antagonists include an
oligonucleotide that binds to the fitsions 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.
[0850] Another potential PRO polypeptide antagonist is an antisense
RNA or DNA construct prepared using antisense technology, where,
e.g., an antisense RNA or DNA molecule acts to block directly the
translation of mRNA by hybridizing to targeted mRNA and preventing
protein translation. Antisense technology can be used to control
gene expression through triple-helix formation or antisense DNA or
RNA, both of which methods are based on binding of a polynucleotide
to DNA or RNA. For example, the 5' coding portion of the
polynucleotide sequence, which encodes the mature PRO polypeptides
herein, is used to design an antisense RNA oligonucleotide of from
about 10 to 40 base pairs in length. A DNA oligonucleotide is
designed to be complementary to a region of the gene involved in
transcription (triple helix--see Lee et al., Nucl. Acids Res.
6:3073 (1979); Cooney et al., Science 241: 456 (1988); Dervan et
al., Science 251:1360 (1991)), thereby preventing transcription and
the production of the PRO polypeptide. The antisense RNA
oligonucleotide hybridizes to the mRNA in vivo and blocks
translation o fthe 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 vtvo 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.
[0851] Potential antagonists include small molecules that bind to
the active site, the receptorbinding 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.
[0852] Ribozymes are enzymatic RNA molecules capable ofcatalyzing
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).
[0853] 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 fin-ther
details see, e.g., PCT publication No. WO 97/33551, supra.
[0854] 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.
[0855] With regard to the PR0211 and PR0217 polypeptide,
therapeutic indications include disorders associated with the
preservation and maintenance of gastrointestinal mucosa and the
repair of acute and chronic mucosal lesions (e.g., enterocolitis,
Zollinger-Ellison syndrome, gastrointestinal ulceration and
congenital microvillus atrophy), skin diseases associated with
abnormal keratinocyte differentiation (e.g., psoriasis, epithelial
cancers such as lung squarnous cell carcinoma, epidermoid carcinoma
of the vulva and gliomas.
[0856] Since the PR0232 polypeptide and nucleic acid encoding it
possess sequence homology to a cell surface stem cell antigen and
its encoding nucleic acid, probes based upon the PR0232 nucleotide
sequence may be employed to identify other novel stem cell surface
antigen proteins. Soluble forms of the PR0232 polypeptide may be
employed as antagonists of membrane bound PR0232 activity both in
vitro and in vivo. PR0232 polypeptides may be employed in screening
assays designed to identify agonists or antagonists of the native
PR0232 polypeptide, wherein such assays may take the forin of any
conventional cell-type or biochemical binding assay. Moreover, the
PR0232 polypeptide may serve as a molecular marker for the tissues
in which the polypeptide is specifically expressed.
[0857] With regard to the PRO187 polypeptides disclosed herein,
FGF-8 has been implicated in cellular differentiation and
embryogenesis, including the patterning which appears during limb
formation. FGF-8 and the PRO187 molecules of the invention
therefore are likely to have potent effects on cell growth and
development. Diseases which relate to cellular growth and
differentiation are therefore suitable targets for therapeutics
based on functionality similar to FGF-8. For example, diseases
related to growth or survival of nerve cells including Parkinson's
disease, Alzheimees disease, ALS, neuropathies. Additionally,
disease related to uncontrolled cell growth, e.g., cancer, would
also be expected therapeutic targets.
[0858] With regard to the PR0265 polypeptides disclosed herein,
other methods for use with PR0265 are described in U.S. Pat. No.
5,654,270 to Ruoslahti et al. In particular, PR0265 can be used in
comparison with the fibromodulin disclosed therein to compare its
effects on reducing dermal scarring and other properties of the
fibromodulin described therein including where it is located and
with what it binds and does not.
[0859] The PR0219 polypeptides of the present invention which play
a regulatory role in the blood coagulation cascade may be employed
in vivo for therapeutic purposes as well as for in vitro purposes.
Those ofordinary skill in the art will well know how to employ
PR0219 polypeptides for such uses.
[0860] The PR0246 polypeptides of the present invention which serve
as cell surface receptors for one or more viruses will find other
uses. For example, extracellular domains derived from these PR0246
polypeptides may be employed therapeutically in vivo for lessening
the effects of viral infection. Those PR0246 polypeptides which
serves as tumor specific antigens may be exploited as therapeutic
targets for anti-tumor drugs, and the like. Those of ordinary skill
in the art will well know how to employ PR0246 polypeptides for
such uses.
[0861] Assays in which connective growth factor and other growth
factors are usually used should be performed with PR0261. An assay
to determine whether TGF beta induces PR0261, indicating a role in
cancer is performed as known in the art. Wound repair and tissue
growth assays are also performed with PR026 1. The results are
applied accordingly.
[0862] PR0228 polypeptides should be used in assays in which EMR1,
CD97 and latrophilin would be used in to determine their relative
activities. The results can be applied accordingly. For example, a
competitive binding assay with PR0228 and CD97 can be performed
with the ligand for CD97, CD55.
[0863] Native PRO533 is a 216 amino acid polypeptide of which
residues 1-22 are the signal sequence. Residues 3 to 216 have a
Blast score of 509, corresponding to 53% homology to fibroblast
growth factor. At the nucleotide level, DNA47412, the EST from
which PCR oligos were generated to isolate the full length
DNA49435-1219, has been observed to map to 11 p15. Sequence
homology to the 11 p15 locus would indicate that PR0533 may have
utility in the treatment of Usher Syndrome or Atrophia areata.
[0864] As mentioned previously, fibroblast growth factors can act
upon cells in both a mitogenic and non-mitogenic manner. These
factors are mitogenic for a wide variety of normal diploid
mesoderm-derived and neural crest-derived cells, inducing granulosa
cells, adrenal cortical cells, chrondrocytes, myoblasts, corneal
and vascular endothelial cells (bovine or human), vascular smooth
muscle cells, lens, retina and prostatic epithelia] cells,
oligodendrocytes, astrocytes, chrondocytes, myoblasts and
osteoblasts.
[0865] Non-mitogenic actions of fibroblast growth factors include
promotion of cell migration into a wound area (chemotaxis),
initiation of new blood vessel formulation (angiogenesis),
modulation of nerve regeneration and survival (neurotrophism),
modulation of endocrine functions, and stimulation or suppression
of specific cellular protein expression, extracellular matrix
production and cell survival. Baird, A. & Bohlen, P., Handbook
of Exp. Phrmacol 95(1): 369418 (1990). These properties provide a
basis for using fibroblast growth factors in therapeutic approaches
to accelerate wound healing, nerve repair, collateral blood vessel
formation, and the like. For example, fibroblast growth factors,
have been suggested to minimize myocardium damage in heart disease
and surgery (U.S. Pat. No. 4,378,437).
[0866] Since the PRO245 polypeptide and nucleic acid encoding it
possess sequence homology to a transmembrane protein tyrosine
kinase protein and its encoding nucleic acid, probes based upon the
PR0245 nucleotide sequence may be employed to identify other novel
transmembrane tyrosine kinase proteins. Soluble forms of the PR0245
polypeptide may be employed as antagonists of membrane bound PR0245
activity both in vitro and in vivo. PR0245 polypeptides may be
employed in screening assays designed to identify agonists or
antagonists of the native PRO245 polypeptide, wherein such assays
may take the fbrm ofany conventional cell-type or biochemical
binding assay. Moreover, the PRO245 polypeptide may serve as a
molecular marker for the tissues in which the polypeptide is
specifically expressed.
[0867] PR0220, PR0221 and PR0227 all have leucine rich repeats.
Additionally, PR0220 and PRO221 have homology to SLIT and leucine
rich repeat protein. Therefore, these proteins are useful in assays
described in the literature, supra wherein the SLIT and leucine
rich repeat protein are used. Regarding the SLIT protein, PR0227
can be used in an assay to determine the affect of PR0227 on
neurodegenerative disease. Additionally, PR0227 has homology to
human glycoprotein V. In the case of PR0227, this polypeptide is
used in an assay to determine its affect on bleeding, clotting,
tissue repair and scarring.
[0868] The PR0266 polypeptide can be used in assays to determine if
it has a role in neurodegenerative diseases or their reversal.
[0869] PR0269 polypeptides and portions thereof which effect the
activity of thrombin may also be useful for in vivo therapeutic
purposes, as well as for various in vitro applications. In
addition, PR0269 polypeptides and portions thereof may have
therapeutic use as an antithrombotic agent with reduced risk for
hemorrhage as compared with heparin. Peptides having homology to
thrombomodulin are particularly desirable.
[0870] PR0287 polypeptides and portions thereof which effect the
activity of bone morphogenic protein "BMP1"/procollagen
C-proteinase (PCP) may also be usefiil for in vivo therapeutic
purposes, as well as for various in vitro applications. In
addition, PR0287 polypeptides and portions thereof may have
therapeutic applications in wound healing and tissue repair.
Peptides having homology to procollagen C-proteinase enhancer
protein and its precursor may also be used to induce bone and/or
cartilage formation and are therefore of particular interest to the
scientific and medical communities.
[0871] Therapeutic indications for PRO214 polypeptides include
disorders associated with the preservation and maintenance of
gastrointestinal mucosa and the repair of acute and chronic mucosal
lesions (e.g., enterocolitis, Zollinger-Ellison syndrome,
gastrointestinal ulceration and congenital microvillus atrophy),
skin diseases associated with abnormal keratinocyte differentiation
(e.g., psoriasis, epithelial cancers such as lung squamous cell
carcinoma, epidermoid carcinoma of the vulva and gliomas.
[0872] Studies on the generation and analysis of mice deficient in
members of the TGF-superfamily are reported in Matzuk, Trends in
Endocrinol. and Metabol. 6:120-127 (1995).
[0873] The PRO317 polypeptide, as well as PR0317-specific
antibodies, inhibitors, agonists, receptors, or their analogs,
herein are useful in treating PR0317-associated disorders. Hence,
for example, they may be employed in modulating endometrial
bleeding angiogenesis, and may also have an effect on kidney
tissue. Endometrial bleeding can occur in gynecological diseases
such as endometrial cancer as abnormal bleeding. Thus, the
compositions herein may find use in diagnosing and treating
abnormal bleeding conditions in the endometrium, as by reducing or
eliminating the need for a hysterectomy. The molecules herein may
also find use in angiogenesis applications such as anti-tumor
indications for which the antibody against vascular endothelial
growth factor is used, or, conversely, ischemic indications for
which vascular endothelial growth factor is employed.
[0874] Bioactive compositions comprising PR0317 or agonists or
antagonists thereof may be administered in a suitable therapeutic
dose determined by any of several methodologies including clinical
studies on mammalian species to determine maximal tolerable dose
and on normal human subjects to determine safe dose. Additionally,
the bioactive agent may be complexed with a variety of well
established compounds or compositions which enhance stability or
pharmacological properties such as half-life. It is contemplated
that the therapeutic, bioactive composition may be delivered by
intravenous infusion into the bloodstream or any other effective
means which could be used for treating problems of the kidney,
uterus, endometrium, blood vessels, or related tissue, e.g., in the
heart or genital tract.
[0875] Dosages and administration of PR0317, PR0317 agonist, or
PR0317 antagonist in a pharmaceutical composition may be determined
by one of ordinary skill in the art of clinical pharmacology or
pharmacokinetics. See, for example, Mordenti and Rescigno,
Pharmaceutical Research, 9:17-25 (1992); Morenti et al.,
Pharmaceutical Research, 8:1351-1359 (1991); and Mordenti and
Chappell, "The use of interspecies scaling in toxicokinetics" in
Toxicokinetics and New Drug DevelgRment Yacobi et al (eds)
(Pergarnon Press: NY, 1989), pp. 42-96. An effective amount of
PRO317, PR0317 agonist, or PR0317 antagonist to be employed
therapeutically will depend, for example, upon the therapeutic
objectives, the route of administration, and the condition of the
mammal. Accordingly, it will be necessary for the therapist to
titer the dosage and modify the route of administration as required
to obtain the optimal therapeutic effect. A typical daily dosage
might range from about 10 ng/kg to up to 100 mg/kg of the marnmal's
body weight or more per day, preferably about 1 .mu.gkg/day to 10
mg/kg/day. Typically, the clinician will administer PR317, PR0317
agonist, or PR0317 antagonist, until a dosage is reached that
achieves the desired effect for treatment of the above mentioned
disorders.
[0876] PR0317 or an PR0317 agonist or PR0317 antagonist may be
administered alone or in combination with another to achieve the
desired pharmacological effect. PR0317 itself, or agonists or
antagonists of PR0317 can provide different effects when
administered therapeutically. Such compounds for treatment will be
formulated in a nontoxic, inert, pharmaceutically acceptable
aqueous carrier nriedium preferably at a pH of about 5 to 8, more
preferably 6 to 8, although the pH may vary according to the
characteristics of the PRO317, agonist, or antagonist being
formulated and the condition to be treated. Characteristics of the
treatment compounds include solubility of the molecule, half-life,
and antigenicity/immunogenicity; these and other characteristics
may aid in defining an effective carrier.
[0877] PR0317 or PR0317 agonists or PR0317 antagonists may be
delivered by known routes of administration including but not
limited to topical creams and gels; transmucosal spray and aerosol,
transdermal patch and bandage; injectable, intravenous, and lavage
formulations; and orally administered liquids and pills,
particularly formulated to resist stomach acid and enzymes. The
particular formulation, exact dosage, and route of administration
will be determined by the attending physician and will vary
according to each specific situation.
[0878] Such determinations of administration are made by
considering multiple variables such as the condition to be treated,
the type of mammal to be treated, the compound to be administered,
and the pharmacokinetic profile of the particular treatment
compound. Additional factors which may be taken into account
include disease state (e.g. severity) of the patient, age, weight,
gender, diet, time of administration, drug combination, reaction
sensitivities, and tolerance/response to therapy. Long-acting
treatment compound formulations (such as liposomally encapsulated
PR0317 or PEGylated PR0317 or PR0317 polymeric microspheres, such
as polylactic acid-based microspheres) might be administered every
3 to 4 days, every week, or once every two weeks depending on
half-life and clearance rate of the particular treatment
compound.
[0879] 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
ug/kg/day to 10 mg/kg/day, depending upon the route of
administration. Guidance as to particular dosages and methods of
delivery is provided in the literature; see, for example, U.S. Pat.
Nos. 4,657,760; 5,206,344; or 5,225,212. It is anticipated that
different formulations will be effective for different treatment
compounds and different disorders, that administration targeting
the uterus, for example, may necessitate delivery in a manner
different from that to another organ or tissue, such as cardiac
tissue.
[0880] Where sustained-release administration of PR0317 is desired
in a formulation with release characteristics suitable for the
treatment of any disease or disorder requiring administration of
PRO317, microencapsulation of PR0317 is contemplated.
Microencapsulation of recombinant proteins for sustained release
has been successfully performed with human growth hormone (rhGH),
interferon-(rhIFN-), interleukin-2, and MN rgp 120. Johnson et al.,
Nat. Med., 2: 795-799 (1996); Yasuda, Biomed. Tber. 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 Adauvant Approach, Powell and Newman, eds, (Plenum
Press: New York, 1995), pp. 439-462; WO 97/03692, WO 96/40072, WO
96/07399; and U.S. Pat. No. 5,654,010.
[0881] It is contemplated that conditions or diseases of the
uterus, endometrial tissue, or other genital tissues or cardiac
tissues may precipitate damage that is treatable with PR0317 or
PR0317 agonist where PR0317 expression is reduced in the diseased
state; or with antibodies to PR0317 or other PR0317 antagonists
where the expression of PR0317 is increased in the diseased state.
These conditions or diseases may be specifically diagnosed by the
probing tests discussed above for physiologic and pathologic
problems which affect the function of the organ.
[0882] The PR0317, PR0317 agonist, or PR0317 antagonist may be
administered to a mammal with another biologically active agent,
either separately or in the same formulation to treat a common
indication for which they are appropriate. For example, it is
contemplated that PR0317 can be administered together with EBAF-1
for those indications on which they demonstrate the same
qualitative biological effects. Alternatively, where they have
opposite effects, EBAF-1 may be administered together with an
antagonist to PR0317, such as an anti-PRO317 antibody. Further,
PR0317 may be administered together with VEGF for coronary ischemia
where such indication is warranted, or with an anti-VEGF for cancer
as warranted, or, conversely, an antagonist to PR0317 may be
administered with VEGF for coronary ischemia or with anti-VEGF to
treat cancer as warranted. These administrations would be in
effective amounts for treating such disorders.
[0883] Native PR0301 (SEQ ID NO:119) has a Blast score of 246 and
30% homology at residues 24 to 282 of FIG. 44 with A33_HUMAN, an
A33 antigen precursor. A33 antigen precursor, as explained in the
Background is a tumor-specific antigen, and as such, is a
recognized marker and therapeutic target for the diagnosis and
treatment of colon cancer. The expression of tumor-specific
antigens is often associated with the progression of neoplastic
tissue disorders. Native PR0301 (SEQ ID NO: 119) and A33_HUMAN also
show a Blast score of 245 and 30% homology at residues 21 to 282 of
FIG. 44 with A33_HUMAN, the variation dependent upon how spaces are
inserted into the compared sequences. Native PR0301 (SEQ ID NO:
119) also has a Blast score of 165 and 29% homology at residues 60
to 255 of FIG. 44 with HS46KDA.sub.--1, a human coxsackie and
adenovirus receptor protein, also known as cell surface protein
HCAR. This region of PR0301 also shows a similar Blast score and
homology with HSU90716.sub.--1. Expression of such proteins is
usually associated with viral infection and therapeutics for the
prevention of such infection may be accordingly conceived. As
mentioned in the Background, the expression of viral receptors is
often associated with neoplastic tumors.
[0884] Therapeutic uses for the PR0234 polypeptides of the
invention includes treatments associated with leukocyte homing or
the interaction between leukocytes; and the endotheliurn during an
inflammatory response. Examples include asthma, rheumatoid
arthritis, psoriasis and multiple sclerosis.
[0885] Since the PR0231 polypeptide and nucleic acid encoding it
possess sequence homology to a putative acid phosphatase and its
encoding nucleic acid, probes based upon the PRO231 nucleotide
sequence may be employed to identify other novel phosphatase
proteins. Soluble forms of the PR0231 polypeptide may be employed
as antagonists of membrane bound PR0231 activity both in vitro and
in vivo. PR0231 polypeptides may. be employed in screening assays
designed to identify agonists or antagonists of the native PR0231
polypeptide, wherein such assays may take the form of any
conventional cell-type or biochemical binding assay. Moreover, the
PR0231 polypeptide may serve as a molecular marker for the tissues
in which the polypeptide is specifically expressed.
[0886] PR0229 polypeptides can be fused with peptides of interest
to determine whether the fusion peptide has an increased half-life
over the peptide of interest. The PR0229 polypeptides can be used
accordingly to increase the half-life of polypeptides of interest.
Portions of PR0229 which cause the increase in half-life are an
embodiment of the invention herein.
[0887] PR0238 can be used in assays which measure its ability to
reduce substrates, including oxygen and Aceyl-CoA, and
particularly, measure PR0238's ability to produce oxygen free
radicals. This is done by using assays which have been previously
described. PR0238 can further be used to assay for candidates which
block, reduce or reverse its reducing abilities. This is done by
performing side by side assays where candidates are added in one
assay having PR0238 and a substrate to reduce, and not added in
another assay, being the same but for the lack of the presence of
the candidate.
[0888] PR0233 polypeptides and portions thereof which have homology
to reductase may also be useful for in vivo therapeutic purposes,
as well as for various other applications. The identification of
novel reductase proteins and related molecules may be relevant to a
number of human disorders such as inflammatory disease, organ
failure, atherosclerosis, cardiac injury, infertility, birth
defects, premature aging, AIDS, cancer, diabetic complications and
mutations in general. Given that oxygen free radicals and
antioxidants appear to play important roles in a number of disease
processes, the identification of new reductase proteins and
reductase-like molecules is ofspecial 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 PR0233.
[0889] The PR0223 polypeptides of the present invention which
exhibit serine carboxypeptidease activity may be employed in vivo
for therapeutic purposes as well as for in vitro purposes. Those of
ordinary skill in the art will well know how to employ PR0223
polypeptides; for such uses.
[0890] PR0235 polypeptides and portions thereof which may be
involved in cell adhesion are also usefid for in vivo therapeutic
purposes, as well as for various in vitro applications. In
addition, PR0235 polypeptides and portions thereof may have
therapeutic applications in disease states which involve cell
adhesion. Given the physiological importance of cell adhesion
mechanisms in vivo, efforts are currently being under taken to
identify new, native proteins which are involved in cell adhesion.
Therefore, peptides having homology to plexin are of particular
interest to the scientific and medical communities.
[0891] Because the PR0236 and PR0262 polypeptides disclosed herein
are homologous to various known galactosidase proteins, the PR0236
and PRO262 polypeptides disclosed herein will find use in
conjugates of monoclonal antibodies and the polypeptide for
specific killing of tumor cells by generation of active drug from a
galactosylated prodrug (e.g., the generation of 5-fluorouridine
from the prodrug .beta.-D-galactosyl-5-fluorouridine). The PR0236
and PR0262 polypeptides disclosed herein may also find various uses
both in vivo and in vitro, wherein those uses will be similar or
identical to uses for which .beta.-galactosidase proteins are now
employed. Those of ordinary skill in the art will well know how to
employ PRO236 and PRO262 polypeptides for such uses.
[0892] PR0239 polypeptides and portions thereof which have homology
to densin may also be useful for in vivo therapeutic purposes, as
well as for various in vitro applications. In addition, PRO239
polypeptides and portions thereof may have therapeutic applications
in disease states which involve synaptic mechanisms, regeneration
or cell adhesion. Given the physiological importance of synaptic
processes, regeneration and cell adhesion mechanisms in vivo,
efforts are currently being under taken to identify new, native
proteins which are involved in synaptic machinery and cell
adhesion. Therefore, peptides having homology to densin are of
particular interest to the scientific and medical communities.
[0893] The PRO260 polypeptides described herein can be used in
assays to determine their relation to fucosidase. In particular,
the PR0260 polypeptides can be used in assays in determining their
ability to remove fucose or other sugar residues from
proteoglycans. The PR0260 polypeptides can be assayed to determine
if they have any functional or locational similarities as
fucosidase. The PR0260 polypeptides can then be used to regulate
the systems in which they are integral.
[0894] PR0263 can be used in assays wherein CD44 antigen is
generally used to determine PR0263 activity relative to that of
CD44. The results can be used accordingly.
[0895] PRO270 polypeptides and portions thereof which effect
reduction-oxidation (redox) state may also be useful for in vivo
therapeutic purposes, as well as for various in vitro applications.
More specifically, PRO270 polypeptides may affect the expression of
a large variety of genes thought to be involved in the pathogenesis
of AIDS, cancer, atherosclerosis, diabetic complications and in
pathological conditions involving oxidative stress such as stroke
and inflammation. In addition, PR0270 polypeptides and portions
thereof may affect the expression of a genes which have a role in
apoptosis. Therefore, peptides having homology to thioredoxin are
particularly desirable to the scientific and medical
communities.
[0896] PR0272 polypeptides and portions thereof which possess the
ability to bind calcium may also have numerous in vivo therapeutic
uses, as well as various in vitro applications. Therefore, peptides
having homology to reticulocalbin are particularly desirable. Those
with ordinary skill in the art will know how to employ PR0272
polypeptides and portions thereof for such purposes.
[0897] PR0294 polypeptides and portions thereofwhich have homology
to collagen may also be useful for in vivo therapeutic purposes, as
well as for various other applications. The identification of novel
collagens; and collage-like molecules may have relevance to a
number of human disorders. Thus, the identification of new
collagens and collage-like molecules is of special importance in
that such proteins may serve as potential therapeutics for a
variety of different human disorders. Such polypeptides may also
play important roles in biotechnological and medical research as
well as various industrial applications. Given the large number of
uses for collagen, there is substantial interest in polypeptides
with homology to the collagen molecule.
[0898] PR0295 polypeptides and portions thereof which have homology
to integrin may also be useful for in vivo therapeutic purposes, as
well as for various other applications. The identification of novel
integrins and integrin-like molecules may have relevance to a
number ofhuman disorders such as modulating the binding or activity
ofeells of the immune system. Thus, the identification of new
integrins and integrin-like molecules is of special importance in
that such proteins may serve as potential therapeutics for a
variety of different human disorders. Such polypeptides may also
play important roles in biotechnological and medical research as
well.as various industrial applications. As a result, there is
particular scientific and medical interest in new molecules, such
as PR0295.
[0899] As the PR0293 polypeptide is clearly a leucine rich repeat
polypeptide homologue, the peptide can be used in all applications
that the known NLRR-1 and NLRR-2 polypeptides are used. The
activity can be compared between these peptides and thus applied
accordingly.
[0900] The PR0247 polypeptides described herein can be used in
assays in which densin is used to determine the activity of PR0247
relative to densin or these other proteins. The results can be used
accordingly in diagnostics and/or therapeutic applications with
PR0247.
[0901] PR0302, PR0303, PR0304, PR0307 and PR0343 polypeptides of
the present invention which possess protease activity may be
employed both in vivo for therapeutic purposes and in vitro. Those
of ordinary skill in the art will well know how to employ the
PR0302, PR0303, PR0304, PRO307 and PR0343 polypeptides of the
present invention for such purposes.
[0902] PR0328 polypeptides and portions thereof which have homology
to GLIP and CRISP may also be useful for in vivo therapeutic
purposes, as well as for various other applications. The
identification of novel GLIP and CRISP-like molecules may have
relevance to a number of human disorders which involve
transcriptional regulation or are over expressed in human tumors.
Thus, the identification of new GLIP and CRISP-like molecules is of
special importance in that such proteins may serve as potential
therapeutics for a variety ofdifferent 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 PR0328.
[0903] Uses for PRO335, PR0331 or PR0326 including uses in
competitive assays with LIG-1, ALS and decorin to determine their
relative activities. The results can be used accordingly. PR0335,
PR0331 or PR0326 can also be used in assays where LIG-1 would be
used to determine if the same effects are incurred.
[0904] PR0332 contains GAG repeat (GKEK) at amino acid positions
625-628 in FIG. 108 (SEQ ID NO:310). Slippage in such repeats can
be associated with human disease. Accordingly, PR0332 can use
useful for the treatment of such disease conditions by gene
therapy, i.e. by introduction of a gene containing the correct GKEK
sequence motif.
[0905] Other uses of PR0334 include use in assays in which
fibrillin or fibulin would be used to determine the relative
activity of PR0334 to fibrillin or fibulin. In particular, PR0334
can be used in assays which require the mechanisms imparted by
epidermal growth factor repeats.
[0906] Native PR0346 (SEQ ID NO:320) has a Blast score of 230,
corresponding to 27% homology between amino acid residues 21 to 343
with residues 35 to 1040 CGM6_HUMAN, a carcinoembryonic antigen
cgm6 precursor. This homology region includes nearly all but 2
N-terminal extracellular domain residues, including an
immunoglobulin superfamily homology at residues 148 to 339 of
PR0346 in addition to several transmembrane residues (340-343).
Carcinoembryonic antigen precursor, as explained in the Background
is a tumor-specific: antigen, and as such, is a recognized marker
and therapeutic target for the diagnosis and treatment of colon
cancer. The expression of tumor-specific antigens is often
associated with the progression of neoplastic tissue disorders.
Native PR0346 (SEQ ID NO:320) and P_W06874, a human
carcinoembryonic antigen CEA-d have a Blast score of 224 and
homology of 28% between residues 2 to 343 and 67 to 342,
respectively. This homology includes the entire extracellular
domain residues of native PR0346, minus the initiator methionine
(residues 2 to 18) as well as several transmembrane residues
(340-343).
[0907] PRO268 polypeptides which have protein disulfide isornerase
activity will be usefid for many applications where protein
disulfide isomerase activity is desirable including, for example,
for use in promoting proper disulfide bond formation in
recombinantly produced proteins so as to increase the yield of
correctly folded protein. Those of ordinary skill in the art will
readily know how to employ such PR0268 polypeptides for such
purposes.
[0908] PR0330 polypeptides of the present invention which possess
biological activity related to that of the prolyl 4-hydroxylase
alpha subunit protein may be employed both in vivo for therapeutic
purposes and in vitro. Those of ordinary skill in the art will well
know how to employ the PR0330 polypeptides of the present invention
fbr such purposes.
[0909] Uses of the herein disclosed molecules may also be based
upon the positive functional assay hits disclosed and described
below.
[0910] F. Anti-PRO Antibodies
[0911] The present invention further provides anti-PRO antibodies.
Exemplary antibodies include polyclonal, monoclonal, humanized,
bispecific, and heteroconjugate antibodies.
[0912] 1. Polyclonal Antibodies
[0913] The anti-PRO antibodies may comprise polyclonal antibodies.
Methods of preparing polyclonal antibodies are known to the skilled
artisan. Polyclonal antibodies can be raised in a mammal, for
example, by one or more injections of an immunizing agent and, if
desired, an adjuvant. Typically, the immunizing agent and/or
adjuvant will be injected in the mammal by multiple subcutaneous or
intraperitoneal injections. The immunizing agent may include the
PRO polypeptide or a fusion protein thereof. It may be useful to
conjugate the immunizing agent to a protein known to be immunogenic
in the mammal being immunized. Examples of such immunogenic
proteins include but are not limited to keyhole limpet hemocyanin,
serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.
Examples of adjuvants which may be employed include Freund's
complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A,
synthetic trehalose dicorynomycolate). The immunization protocol
may be selected by one skilled in the art without undue
experimentation.
[0914] 2. Monoclonal Antibodies
[0915] 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.
[0916] The immunizing agent will typically include the PRO
polypeptide or a fusion protein thereof. Generally, either
peripheral blood lymphocytes ("PBLs") are used if cells of human
origin are desired, or spleen cells or lymph node cells are used if
non-human mammalian sources are desired. The lymphocytes are then
fused with an immortalized cell line using a suitable fusing agent,
such as polyethylene glycol, to form a hybridoma. cell [Goding,
Monoclonal Antibodies: Principles and Practice, Academic Press,
(1986) pp. 59-103]. Immortalized cell lines are usually transformed
mammalian cells, particularly myeloma cells of rodent, bovine and
human origin. Usually, rat or mouse myeloma cell lines are
employed. The hybridonia cells may be cultured in a suitable
culture medium that preferably contains one or more substances that
inhibit the growth or survival of the unfused, immortalized cells.
For example, if the parental cells lack the enzyme hypoxanthine
guanine phosphoribosyl transferase (HGPRT or HPRT), the culture
medium for the hybridomas typically will include hypoxanthine,
aminopterin, and thymidine ("HAT medium"), which substances prevent
the growth of HGPRT-deficient cells.
[0917] Preferred immortalized cell lines are those that fuse
efficiently, support stable high level expression of antibody by
the selected antibody-producing cells, and are sensitive to a
medium such as HAT medium. More preferred immortalized cell lines
are murine myeloma lines, which can be obtained, for instance, from
the Salk Institute Cell Distribution Center, San Diego, Calif. and
the American Type Culture Collection, Manassas, Va. Human myeloma
and mouse-human heteromyeloma cell lines also have been described
for the production of human monoclonal antibodies [Kozbor, J.
Immunol. 133:3001 (1984); Brodeur et al, Monoclonal Antibgdy
Production Technigues and Applications, Marcel Dekker, Inc., New
York, (1987) pp. 51-63].
[0918] 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).
[0919] After the desired hybridoma cells are identified, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods [Goding, supra]. Suitable culture media for this
purpose include, for example, Dulbecco's Modified Eagle's Medium
and RPMI-1640 medium. Alternatively, the hybridoma cells maybe
grown in vivo as ascites in a mammal.
[0920] The monoclonal antibodies secreted by the subclones may be
isolated or purified from the culture medium or ascites fluid by
conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0921] 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.
Patent 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-mmunoglobulin 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.
[0922] 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.
[0923] 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.
[0924] 3. Human and Humanized Antibodies
[0925] 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 immunoglobulns,
immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab',
F(ab').sub.2 or other antigen-binding subsequences of antibodies)
which contain mi nimal sequence derived from non-human
immunoglobulin. Humanized antibodies include human immunoglobulins
(recipient antibody) in which residues from a complementary
determining region (CDR) of the recipient are replaced by residues
from a CDR of a non-human species (donor antibody) such as mouse,
rat or rabbit having the desired specificity, affinity and
capacity. In some instances, Fv framework residues of the human
immunoglobulin are replaced by corresponding non-human residues.
Humanized antibodies may also comprise residues which are found
neither in the recipient antibody nor in the imported CDR or
framework sequences. In general, the humanized antibody will
comprise substantially all of at least one, and typically two,
variable domains, in which all or substantially all of the CDR
regions correspond to those of a non-human immunoglobulin and all
or substantially all of the FR regions are those of a human
immunoglobulin consensus sequence. The humanized antibody optimally
also will comprise at least a portion of an immunoglobulin constant
region (Fc), typically that of a human immunoglobulin [Jones et
al., Nature, 321:522-525 (1986); Riechmarm et al., Nature,
332:323-329(1988); and Presta, Curr. Op. Struct. Biol., 2:593-596
(1992)].
[0926] 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); Riechmarm 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.
[0927] 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).
[0928] 4. Bispecific Antibodies
[0929] 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.
[0930] 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 13 May
1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).
[0931] Antibody variable domains with the desired binding
specificities (antibody-antigen combining sites) can be fused to
immunoglobulin constant domain sequences. The fusion preferably is
with an immunoglobulin heavy-chain constant domain, comprising at
least part of the hinge, CH2, and CH3 regions. It is preferred to
have the first heavy-chain constant region (CH1) containing the
site necessary for light-chain binding present in at least one of
the fusions. DNAs encoding the immunoglobulin heavy-chain fusions
and, if desired, the immunoglobulin light chain, are inserted into
separate expression vectors, and are co-transfected into a suitable
host organism. For further details of generating bispecific
antibodies see, for example, Suresh et al., Methods in Enzymology
121:210 (1986).
[0932] 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.
[0933] Bispecific antibodies can be prepared as full length
antibodies or antibody fragments (e.g. F(ab').sub.2 bispecific
antibodies). Techniques for generating bispecific antibodies from
antibody fragments have been described in the literature. For
example, bispecific antibodies can be prepared can be prepared
using chemical linkage. Brennan et al, Science 229:81 (1985)
describe a procedure wherein intact antibodies are proteolytically
cleaved to generate F(ab').sub.2 fragments. These fragments are
reduced in the presence of the dithiol complexing agent sodium
arsenite to stabilize vicinal dithiols and prevent intermolecular
disulfide formation. The Fab' fragments generated are then
converted to thionitrobenzoate (TNB) derivatives. One of the
Fab'-TNB derivatives is then reconverted to the Fab'-thiol by
reduction with mercaptoethylamine and is mixed with an equimolar
amount of the other Fab'-TNB derivative to forrn the bispecific
antibody. The bispecific antibodies produced can be used as agents
for the selective immobilization of enzymes.
[0934] 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.
[0935] Various technique for making and isolating bispecific
antibody fragments directly from recombinant cell culture have also
been described. For example, bispecific antibodies have been
produced using leucine zippers. Kostelny et al, J. Immunol. 148(5):
1547-1553 (1992). The leucine zipper peptides from the Fos and Jun
proteins were linked to the Fab' portions of two different
antibodies by gene fusion. The antibody homodimers were reduced at
the hinge region to form monomers and then re-oxidized to form the
antibody heterodimers. This method can also be utilized for the
production of antibody homodimers. The "diabody" technology
described by Hollinger et al., Proc. Nad. Acad. Sci. USA
90:6444-6448 (1993) has provided an alternative mechanism for
making bispecific antibody fragments. The fragments comprise a
heavy-chain variable domain (V.sub.H) connected to a light-chain
variable domain (V.sub.L)by a linker which is too short to allow
pairing between the two domains on the. same chain. Accordingly,
the V.sub.H and V.sub.L domains of one fragment are forced to pair
with the complementary V.sub.L and V.sub.H domains of another
fragment, thereby forming two antigen-binding sites. Another
strategy for making bispecific antibody fragments by the use of
single-chain Fv (sFv) dimers has also been reported. See, Gruber et
al., J. Immunol. 152:5368 (1994).
[0936] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. Tutt et al, J.
Immunol. 147:60 (1991).
[0937] Exemplary bispecific antibodies may bind to two different
epitopes on a given PRO polypeptide herein. Alternatively, an
anti-PRO polypeptide arm may be combined with an arm Which binds to
a triggering molecule on a leukocyte such as a T-cell receptor
molecule (e.g. CD2, CD3, CD28, or B7), or Fc receptors for IgG
(Fc.gamma.R), such as Fc.gamma.RI (CD64), Fc.gamma.RII (CD32) and
Fc.gamma.RIII (CD16) so as to focus cellular defense mechanisms to
the cell expressing the particular PRO polypeptide. Bispecific
antibodies may also be used to localize cytotoxic agents to cells
which express a particular PRO polypeptide. These antibodies
possess a PRO-binding arm and an arm which binds a cytotoxic agent
or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA.
Another bispecific antibody of interest binds the PRO polypeptide
and further binds tissue factor (TF).
[0938] 5. Heteroconjugate Antibodies
[0939] Heteroconjugate antibodies are also within the scope of the
present invention. Heteroconjugate antibodies are composed of two
covalently joined antibodies. Such antibodies have, for example,
been proposed to target immune system cells to unwanted cells [U.S.
Pat. No. 4,676,980], and for treatment of HIV infection [WO
91/00360; WO 92/200373; EP 03089]. It is contemplated that the
antibodies may be prepared in vitro using known methods in
synthetic protein chemistry, including those involving crosslinking
agents. For example, immunotoxins may be constructed using a
disulfide exchange reaction or by forming a thioether bond.
Examples of suitable reagents for this purpose include
iminothiolate and methyl-4-mercaptobutyfimidate and those
disclosed, for example, in U.S. Pat. No. 4,676,980.
[0940] 6. Effector Function Engineering
[0941] It may be desirable to modify the antibody of the invention
with respect to effector function, so as to enhance, e.g., the
effectiveness of the antibody in treating cancer. For example,
cysteine residue(s) may be introduced into the Fc region, thereby
allowing interchain disulfide bond formation in this region. The
homodimeric antibody thus generated may have improved
internalization capability and/or increased complement-mediated
cell killing and antibody-dependent cellular cytotoxicity (ADCC).
See Caron et al., J. Exy 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).
[0942] 7. Immunoconjugates
[0943] The invention also pertains to immunoconjugates comprising
an antibody conjugated to a cytotoxic agent such as a
chemotherapeutic agent, toxin (e.g., an enymatically active toxin
of bacterial, fungal, plant, or animal origin, or fragments
thereof), or a radioactive isotope (i.e., a radioconjugate).
[0944] Chemotherapeutic agents useful in the generation of such
immunoconjugates have been described above. Enymatically active
toxins and fragments thereof that can be used include diphtheria A
chain, nonbinding active fi-agments of diphtheria toxin, exotoxin A
chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain,
modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin
proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S),
momordica charantia inhibitor, curcin, crotin, sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin, and the tricothecenes. A variety of
radionuclides are available for the production of radioconjugated
antibodies. Examples include .sup.212Bi, .sup.131I, .sup.131In,
.sup.90Y, and .sup.186Re. Conjugates of the antibody and cytotoxic
agent are made using a variety of bifunctional protein-coupling
agents such as N-succinimidyl-3-2-pyridyidithiol) propionate
(SPDP), iminothiolane (IT), bifunctional derivatives of imidoestets
(such as dimethyl adipimidate HCL), active esters (such as
disuccinimidyl suberate), aldehydes (such as glutareldehyde),
bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine),
bis-diazonium derivatives (such as
bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as
tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such
as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin
immunotoxin can be prepared as described in Vitetta et al.,
Science, 238: 1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyidiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating; agent for conjugation of
radionucleotide to the antibody. See W094/11026.
[0945] 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).
[0946] 8. Immunoliposomes
[0947] 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.
[0948] Particularly useful liposomes can be generated by the
reverse-phase evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol, and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter. Fab' fragments of the antibody of the present invention
can be conjugated to the liposomes as described in Martin et al.,
J. Biol. Chem., 257: 286-288 (1982) via a disulfide-interchange
reaction. A chemotherapeutic agent (such as Doxorubicin) is
optionally contained within the liposome. See Gabizon et al., J.
National Cancer Inst., 81 (19): 1484 (1989).
[0949] 9. Pharmaceutical Compositions of Antibodies
[0950] Antibodies specifically binding a PRO polypeptide identified
herein, as well as other molecules identified by the screening
assays disclosed bereinbefore, can be administered for the
treatment of various disorders in the form of pharmaceutical
compositions.
[0951] If the PRO polypeptide is intracellular and whole antibodies
are used as inhibitors, internalizing antibodies are preferred.
However, lipofections or liposomes can also be used to deliver the
antibody, or an antibody fragment, into cells. Where antibody
fragments are used, the smallest inhibitory fragment that
specifically binds to the binding domain of the target protein is
preferred. For example, based upon the variable-region sequences of
an antibody, peptide molecules can be designed that retain the
ability to bind the target protein sequence. Such peptides can be
synthesized chemically and/or produced by recombinant DNA
technology. See, e.g., Marasco et al, Proc. Nad. 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.
[0952] 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-(methylmediacylate)
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.
[0953] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0954] 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.quadrature.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 sulthydryl residues, lyophilizing from acidic solutions,
controlling moisture content, using appropriate additives, and
developing specific polymer matrix compositions.
[0955] G. Uses for Anti-PRO Antibodies
[0956] The anti-PRO antibodies of the invention have various
utilities. For example, anti-PRO antibodies may be used in
diagnostic assays for PRO, e.g., detecting its expression in
specific cells, tissues, or serum. Various diagnostic assay
techniques known in the art may be used, such as competitive
binding assays, direct or indirect sandwich assays and
immunoprecipitation assays conducted in either heterogeneous or
homogeneous phases [Zola, Monoclonal Antibodies: A Manual of
Techniques CRC Press, Inc. (1987) pp. 147-158]. The antibodies used
in the diagnostic assays can be labeled with a detectable moiety.
The detectable moiety should be capable of producing, either
directly or indirectly, a detectable signal. For example, the
detectable moiety may be a radioisotope, such as .sup.3H, .sup.14C,
.sup.32p, .sup.35S, or .sup.125I, a fluorescent or chemiluminescent
compound, such as fluorescein isothiocyanate, rhodamine, or
luciferin, or an enzyme, such as alkaline phosphatase,
beta-galactosidase or horseradish peroxidase. Any method known in
the art for conjugating the antibody to the detectable moiety may
be employed, including those methods described by Hunter et al.,
Nature. 144:945 (1962); David et al., Biochemistry 13:1014 (1974);
Pain et al., J. Immunol. Meth., 40:219 (1981); and Nygren, J.
Histochem. and Cytochem. 30:407 (1982).
[0957] 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.
[0958] The following examples are offered for illustrative purposes
only, and are not intended to limit the scope of the present
invention in any way.
[0959] All patent and literature references cited in the present
specification are hereby incorporated by reference in their
entirety.
EXAMPLES
[0960] Commercially available reagents referred to in the examples
were used according to manufacturer's instructions unless otherwise
indicated. The source of those cells identified in the following
examples, and throughout the specification, by ATCC accession
numbers is the American Type Culture Collection, Manassas, Va.
Example 1
Extracellular Domain Homology Screening to Identify Novel
Polyeptides and cDNA Encoding Therefor
[0961] The extracellular domain (ECD) sequences (including the
secretion signal sequence, if any) from about 950 known secreted
proteins from the Swiss-Prot public database were used to search
EST databases. The EST databases included public databases (e.g.,
Dayhoff, GenBank), and proprietary databases (e.g., LIFESEQ.TM.,
Incyte Pharmaceuticals, Palo Alto, Calif.). The search was
performed using the computer program BLAST or BLAST2 (Altschul, and
Gish, Methods in Enzymology 266: 460-80 (1996); 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.).
[0962] Using this extracellular domain homology screen, consensus
DNA sequences were assembled relative to the other identified EST
sequences. In addition, the consensus DNA sequences obtained were
often (but not always) extended using repeated cycles of BLAST and
phrap to extend the consensus sequence as far as possible using the
sources of EST sequences discussed above.
[0963] Based upon the consensus sequences obtained as described
above, oligonucleotides were then synthesized and used to identify
by PCR a cDNA library that contained the sequence of interest and
for use as probes to isolate a clone of the full-length coding
sequence for a PRO polypeptide. Forward (.f) and reverse (.r) PCR
primers generally range from 20 to 30 nucleotides and are often
designed to give a PCR product of about 100-1000 bp in length. The
probe (.p) sequences are typically 40-55 bp in length. In some
cases, additional oligonucleotides are synthesized when the
consensus sequence is greater than about 1-1.5 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.
[0964] The cDNA libraries us ed 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 Sfil site;
see, Holmes et al., Science, 253:1278-1280 (1991)) in the unique
Xhol and Notl sites.
Example 2
Isolation of cDNA Clones Encoding PRO211 and PRO217
[0965] Consensus DNA sequences were assembled as described in
Example 1 above and were designated as DNA28730 and DNA28760,
respectively. Based on these consensus sequences, oligonucleotides
were synthesized and used to identify by PCR a cDNA library that
contained the sequences of interest and for use as probes to
isolate a clone of the full-length coding sequence for the PR0211
and PR0217 polypeptides. The libraries used to isolate
DNA32292-1131 and DNA33094-1131 were fetal lung libraries.
[0966] cDNA clones were sequenced in their entirety. The entire
nucleotide sequences of PR0211 (DNA32292-1131) and PR0217 (UNQ 191)
are shown in FIG. 1 (SEQ ID NO: 1) and FIG. 3 (SEQ ID NO:3),
respectively. The predicted polypeptides are 353 and 379 amino acid
in length, respectively, with respective molecular weights of
approximately 38,190 and 41,520 daltons.
[0967] The oligonucleotide sequences used in the above procedures
were the following: TABLE-US-00006 28730.p (OLI516)
5'-AGGGAGCACGGACAGTGTGCAGATGTGGACG (SEQ ID NO:5) AGTGCTCACTAGCA-3'
28730.f (OLI517) 5'-AGAGTGTATCTCTGGCTACGC-3' (SEQ ID NO:6) 28730.r
(OLI518) (SEQ ID NO:7) 5'-TAAGTCCGGCACATTACAGGTC-3' 28760.p
(OLI617) 5'-CCCACGATGTATGAATGGTGGACTTTGTGACTC (SEQ ID NO:8)
CTGGTTTCTGCATC-3' 28760.f (OLI618) 5'-AAAGACGCATCTGCGAGTGTCC-3'
(SEQ ID NO:9) 28760.r (OLI619) 5'-TGCTGATTTCACACTGCTCTCCC-3' (SEQ
ID NO:10)
Example 3
Isolation of cDNA Clones Encoding Human PR0230
[0968] A consensus DNA sequence was assembled relative to the other
identified EST sequences as described in Example 1 above, wherein
the consensus sequence is designated herein as DNA30857. An EST
proprietary to Genentech was employed in the consensus assembly.
The EST is designated as DNA20088 and has the nucleotide sequence
shown in FIG. 7 (SEQ ID NO: 13).
[0969] Based on the DNA30857 consensus sequence, oligonucleotides
were synthesized to identify by PCR a cDNA library that contained
the sequence of interest and for use as probes to isolate a clone
of the full-length coding sequence for PRO230.
[0970] A pair of PCR primers (forward and reverse) were
synthesized: TABLE-US-00007 forward PCR primer
5'-TTCGAGGCCTCTGAGAAGTGGCCC-3' (SEQ ID NO:14) reverse PCR primer
5'-GGCGGTATCTCTCTGGCCTCCC-3' (SEQ ID NO:15)
Additionally, a. synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA30857 sequence which had the
following nucleotide sequence
[0971] Hybridization probe TABLE-US-00008
5'-TTCTCCACAGCAGCTGTGGCATCCGATCGTGTC (SEQ ID NO:16)
TCAATCCATTCTCTGGG-3'
[0972] In order to screen. several libraries for a source ofa
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 PR0230 gene
using the probe oligonucleotide and one of the PCR primers.
[0973] RNA for construction of the cDNA libraries was isolated from
human fetal lung tissue. DNA sequencing of the clones isolated as
described above gave the full-length DNA sequence for PR0230
(herein designated as DNA33223-1136 and the derived protein
sequence for PRO230.
[0974] The entire nucleotide sequence of DNA33223-1136 is shown in
FIG. 5 (SEQ ID NO:11). Clone DNA33223-1136 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 100-103 and ending at the stop codon at
nucleotide positions 1501-1503 (FIG. 5; SEQ ID NO:11). The
predicted polypeptide precursor is 467 amino acids long (FIG.
6).
Example 4
Isolation of cDNA Clones Encoding Human PR0232
[0975] A consensus DNA sequence was assembled relative to the other
identified EST sequences as described in Example 1 above, wherein
the consensus sequence is designated herein as DNA30935. Based on
the DNA30935 consensus sequence, oligonucleotides were synthesized
to identify by PCR a cDNA library that contained the sequence of
interest and for use as probes to isolate a clone of the
full-length coding sequence for PRO232.
[0976] A pair of PCR primers (forward and reverse) were
synthesized: TABLE-US-00009 forward PCR primer
5'-TGCTGTGCTACTCCTGCAAAGCCC-3' (SEQ ID NO:19) reverse PCR primer
5'-TGCACAAGTCGGTGTCACAGCACG-3' (SEQ ID NO:20)
Additionally, a synthetic oligonucleotide hybridization pr6be was
constructed from the consensus DNA30935 sequence which had the
following nucleotide sequence
[0977] hybridization probe TABLE-US-00010 (SEQ ID NO:21)
5'-AGCAACGAGGACTGCCTGCAGGTGGAGAACTGCACCCAGCTGGG-3'
[0978] 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 PRO232 gene
using the probe oligonucleotide and one of the PCR primers.
[0979] RNA for construction of the cDNA libraries was isolated from
human fetal kidney tissue.
[0980] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PR0232 [herein designated as
DNA34435-1140] and the derived protein sequence for PRO232.
[0981] The entire nucleotide sequence of DNA34435-1140 is shown in
FIG. 8 (SEQ ID NO:17). Clone DNA34435-1140 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 17-19 and ending at the stop codon at
nucleotide positions 359-361 (FIG. 8; SEQ ID NO: 17). The predicted
polypeptide precursor is 114 amino acids long (FIG. 9). Clone
DNA34435-1140 has been deposited with ATCC on Sep. 16, 1997 and is
assigned ATCC deposit no. ATCC 209250.
[0982] Analysis of the amino acid sequence of the full-length
PRO232 suggests that it possesses 35% sequence identity with a stem
cell surface antigen from Gallus gallus.
Example 5
Isolation of cDNA Clones Encoding PRO 187
[0983] A proprietary expressed sequence tag (EST) DNA database
(LIFESEQ.TM., Incyte Pharmaceuticals, Palo Alto, Calif.) was
searched and an EST (#843193) was identified which showed homology
to fibroblast growth factor (FIG. 8) also known as androgen-induced
growth factor. mRNA was isolated from human fetal lung tissue using
reagents and protocols from Invitrogen, San Diego, Calif. (Fast
Track 2). The cDNA libraries used to isolate the cDNA clones were
constructed by standard methods using commercially available
reagents (e.g., Invitrogen, San Diego, Calif., Life Technologies,
Gaithersburg, Md.). The cDNA was primed with oligo dT containing a
NotI site, linked with blunt to SalI hemikinased adaptors, cleaved
with NotI, sized appropriately by gel electrophoresis, and cloned
in a defined orientation into the cloning vector pRK5D using
reagents and protocols from Life Technologies, Gaithersburg, Md.
(Super Script Plasmid System). The double-stranded cDNA was sized
to greater than 1000 bp and the SalI/NotI linkered cDNA was cloned
into Xhol/Notl cleaved vector. pRK5D is a cloning vector that has
an sp6 transcription initiation site followed by an SfiI
restriction enzyme site -preceding the XhoI/NotI cDNA cloning
sites.
[0984] Several libraries from various tissue sources were screened
by PCR amplification with the following oligonucleotide probes:
TABLE-US-00011 IN843193.f (OLI315)
5'-CAGTACGTGAGGGACCAGGGCGCCATGA-3' (SEO ID NO:24) IN843193.r
(OLI317) 5'-CCGGTGACCTGCACGTGCTTGCCA-3' (SEO ID NO:25)
[0985] A positive library was then used to isolate clones encoding
the PRO187 gene using one of the above oligonucleotides and the
following oligonucleotide probe: TABLE-US-00012 IN843193.p (OLI316)
(SEQ ID NO:26) 5'-GCGGATCTGCCGCCTGCTCANCTGGTCGGTCATGGCGCCCT-3'
[0986] A cDNA clone was sequenced in entirety. The entire
nucleotide sequence of PRO 187 (DNA27864-1155) is shown in FIG. 10
(SEQ ID NO:22). Clone DNA278647-1155 contains a single open reading
frame with an apparent translational initiation site at nucleotide
position 1 (FIG. 10; SEQ ID NO:22). The predicted polypeptide
precursor is 205 amino acids long. Clone DNA27864-1155 has been
deposited with the ATCC (designation: DNA27864-1155) and is
assigned ATCC deposit no. ATCC 209375.
[0987] Based on a BLAST and FastA sequence alignment analysis
(using the ALIGN computer program) of the full-length sequence, the
PRO 187 polypeptide shows 74% amino acid sequence identity (Blast
score 310) to human fibroblast growth factor-8 (androgen-induced
growth factor).
Example 6
Isolation of cDNA Clones Encoding PR0265
[0988] A consensus DNA sequence was assembled relative to other EST
sequences as described in Example 1 above using phrap. This
consensus sequence is herein designated DNA33679. Based on the
DNA33679 consensus sequence, oligonucleotides were synthesized: 1)
to identify by PCR a cDNA library that contained the sequence of
interest, and 2) for use as probes to isolate a clone of the
full-length coding sequence for PR0265.
[0989] PCR primers (two forward and one reverse) were synthesized:
TABLE-US-00013 forward PCR primer A: 5'-CGGTCTACCTGTATGGCAACC-3';
(SEQ ID NO:29) forward PCR primer B: 5'-GCAGGACAACCAGATAAACCAC-3';
(SEQ ID NO:30) reverse PCR primer 5'-ACGCAGAMGAGAAGGCTGTC-3' (SEQ
ID NO:31)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA33679 sequence which had the
following nucleotide sequence
[0990] hybridization probe TABLE-US-00014
5'-TTCACGGGCTGCTCTTGCCCAGCTCTTGAAGCT (SEQ ID NO:32)
TGAAGAGCTGCAC-3'
[0991] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with PCR primer pairs identified above. A positive
library was then used to isolate clones encoding the PR0265 gene
using the probe oligonucleotide and one of the PCR primers.
[0992] RNA for construction of the cDNA libraries was isolated from
human a fetal brain library.
[0993] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO265 [herein designated as
DNA36350-1158] (SEQ ID NO:27) and the derived protein sequence for
PR0265.
[0994] The entire nucleotide sequence of DNA36350-1158 is shown in
FIG. 12 (SEQ ID NO:27). Clone DNA36350-1158 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 352-354 and ending at the stop codon at
positions 2332-2334 (FIG. 12). The predicted polypeptide precursor
is 660 amino acids long (FIG. 13). Clone DNA36350-1158 has been
deposited with ATCC and is assigned ATCC deposit no. ATCC
209378.
[0995] Analysis of the amino acid sequence of the full-length
PRO265 polypeptide suggests that portions of it possess significant
homology to the fibromodulin and the fibromodulin precursor,
thereby indicating that PR0265 may be a novel member of the leucine
rich repeat family, particularly related to fibromodulin.
Example 7
Isolation of cDNA Clones Encoding Human PR0219
[0996] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described in Example 1 above. This
consensus sequence is herein designated DNA28729. Based on the
DNA28729 consensus sequence, oligonucleotides were synthesized: 1)
to identify by PCR a cDNA library that contained the sequence of
interest, and 2) for use as probes to isolate a clone of the
full-length coding sequence for PRO219.
[0997] A pair of PCR primers (forward and reverse) were
synthesized: TABLE-US-00015 forward PCR primer
5'-GTGACCCTGGTTGTGAATACTCC-3' (SEQ ID NO:35) reverse PCR primer
5'-ACAGCCATGGTCTATAGCTTGG-3' (SEQ ID NO:36)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA28729 sequence which had the
following nucleotide sequence
[0998] hybridization pro TABLE-US-00016
5'-GCCTGTCAGTGTCCTGAGGGACACGTGCTCCGC (SEQ ID NO:37)
AGCGATGGGAAG-3'
[0999] In order to screen several libraries for a source ofa
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 PR0219 gene
using the probe oligonucleotide and one of the PCR primers.
[1000] RNA for construction of the cDNA libraries was isolated from
human fetal kidney tissue.
[1001] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PR0219 [herein designated as
DNA32290-1164] (SEQ ID NO:33) and the derived protein sequence for
PRO219.
[1002] The entire nucleotide sequence of DNA32290-1164 is shown in
FIG. 14 (SEQ ID NO:33). Clone DNA32290-1164 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 204-206 and ending at the stop codon at
nucleotide positions 2949-2951 (FIG. 14). The predicted polypeptide
precursor is 915 amino acids long (FIG. 15). Clone DNA32290-1164
has been deposited with ATCC and is assigned ATCC deposit no. ATCC
209384.
[1003] Analysis of the amino acid sequence of the full-length
PR0219 polypeptide suggests that portions of it possess significant
homology to the mouse and human matrilin-2 precursor
polypeptides.
Example 8
Isolation of cDNA Clones Encoding Human PR0246
[1004] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described in Example 1 above. This
consensus sequence is herein designated DNA30955. Based on the
DNA30955 consensus sequence, oligonucleotides were synthesized: 1)
to identify by PCR a cDNA library that contained the sequence of
interest, and 2) for use as probes to isolate a clone of the
full-length coding sequence for PR0246.
[1005] A pair of PCR primers (forward and reverse) were
synthesized: TABLE-US-00017 forward PCR primer
5'-AGGGTCTCCAGGAGAAAGACTC-3' (SEQ ID NO:40) reverse PCR primer
5'-ATTGTGGGCCTTGCAGACATAGAC-3' (SEQ ID NO:41)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA30955 sequence which had the
following nucleotide sequence
[1006] hybridization probe TABLE-US-00018
5'-GGCCACAGCATCAAAACCTTAGAACTCAATGTA (SEQ ID NO:42)
CTGGTTCCTCCAGCTCC-3'
[1007] 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 PR0246 gene
using the probe oligonucleotide and one of the PCR primers.
[1008] RNA for construction of the cDNA libraries was isolated from
human fetal liver tissue. DNA sequencing of the clones isolated as
described above gave the full-length DNA sequence for PR0246
[herein designated as DNA35639-1172] (SEQ ID NO:38) and the derived
protein sequence for PRO246.
[1009] The entire nucleotide sequence of DNA35639-1172 is shown in
FIG. 16 (SEQ ID NO:38). Clone DNA35639-1172 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 126-128 and ending at the stop codon at
nucleotide positions 1296-1298 (FIG. 16). The predicted polypeptide
precursor, is 390 amino acids long (FIG. 17). Clone DNA35639-1172
has been deposited with ATCC and is assigned ATCC deposit no. ATCC
209396.
[1010] Analysis of the amino acid sequence of the full-length
PR0246 polypeptide suggests that it possess significant homology to
the human cell surface protein HCAR, thereby indicating that PR0246
may be a novel cell surface virus receptor.
Example 9
Isolation of cDNA Clones Encoding Human PR0228
[1011] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described in Example 1 above. This
consensus sequence is herein designated DNA28758. An EST
proprietary to Genentech was employed in the consensus assembly.
This EST is shown in FIG. 20 (SEQ ID NO:50) and is herein
designated as DNA21951.
[1012] Based on the DNA28758 consensus sequence, oligonucleotides
were synthesized: 1) to identify by PCR a cDNA library that
contained the sequence of interest, and 2) for use as probes to
isolate a clone of the full-length coding sequence for PR0228.
[1013] PCR primers (forward and reverse) were synthesized:
TABLE-US-00019 forward PCR primer 5'-GGTAATGAGCTCCATTACAG-3' (SEQ
ID NO:51) forward PCR primer 5'-GGAGTAGAAAGCGCATGG-3' (SEQ ID
NO:52) forward PCR primer 5'-CACCTGATACCATGAATGGCAG-3' (SEQ ID
NO:53) reverse PCR primer 5'-CGAGCTCGAATTAATTCG-3' (SEQ ID NO:54)
reverse PCR primer 5'-GGATCTCCTGAGCTCAGG-3' (SEQ ID NO:55) reverse
PCR primer 5'-CCTAGTTGAGTGATCCTTGTAAG-3' (SEQ ID NO:56)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA28758 sequence which had the
following nucleotide sequence
[1014] hybridization probe TABLE-US-00020
5'-ATGAGACCCACACCTCATGCCGCTGTAATCACC (SEQ ID NO:57)
TGACACATTTTGCAATT-3'
[1015] 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
PR0228 gene using the probe oligonucleotide and one of the PCR
primers.
[1016] RNA for construction of the cDNA libraries was isolated from
human fetal kidney tissue.
[1017] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PR0228 [herein designated as
DNA33092-1202] (SEQ ID NO:48) and the derived protein sequence for
PR0228.
[1018] The entire nucleotide sequence of DNA33092-1202 is shown in
FIG. 18 (SEQ ID NO:48). Clone DNA33092-1202 contains a single open
reading fi-ame with an apparent translational initiation site at
nucleotide positions 24-26 of SEQ ID NO:48 and ending at the stop
codon after nucleotide position 2093 of SEQ ID NO:48. The predicted
polypeptide precursor is 690 amino acids long (FIG. 19). Clone
DNA33092-1202 has been deposited with ATCC and is assigned ATCC
deposit no. ATCC 209420.
[1019] Analysis of the amino acid sequence of the full-length
PR0228 polypeptide suggests that portions of it possess significant
homology to the secretin-related proteins CD97 and EMR1 as well as
the secretin member, latrophilin, thereby indicating that PR0228
may be a new member of the secretin related proteins.
Example 10
Isolation of cDNA Clones Encodiniz Human PR0533
[1020] The EST sequence accession number AF007268, a murine
fibroblast growth factor (FGF-15) was used to search various public
EST databases (e.g., GenBank, Dayhoff, etc.) The search was
performed using the computer program BLAST or BLAST2 as a
comparison of the ECD protein sequences to a 6 frame translation of
the EST sequences. The search resulted in a hit with GenBank EST
AA220994, which has been identified as Strategene NT2 neuronal
precursor 937230.
[1021] Based on the Genbank EST AA220994 sequence, oligonucleotides
were synthesized: 1) to identify by PCR a cDNA library that
contained the sequence of interest, and 2) for use as probes to
isolate a clone of the full-length coding sequence. Forward and
reverse PCR primers may range from 20 to 30, nucleotides (typically
about 24), and are designed to give a PCR product of 100-1000 bp in
length. The probe sequences are typically 40-55 bp (typically about
50) in length. In order to screen several libraries for a source of
a full-length clone, DNA from the libraries was screened by PCR
amplification, as per Ausubel et al., Current Protocols in
Molecular Biology, with the PCR primer pair. A positive library was
then used to isolate clones encoding the gene of interest using the
probe oligonucleotide and one of the PCR primers.
[1022] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pair identified below. A positive
library was then used to isolate clones encoding the PR0533 gene
using the probe oligonucleotide and one of the PCR primers.
[1023] RNA for construction of the cDNA libraries was isolated from
human fetal retina. The cDNA libraries used to isolated the cDNA
clones were constructed by standard methods using commercially
available reagents (e.g., Invitrogen, San Diego, Calif.; Clontech,
etc.) The cDNA was primed with oligo dT containing a NotI site,
linked with blunt to SalI hemikinased adaptors, cleaved with NotI,
sized appropriately by gel electrophoresis, and cloned in a defined
orientation into a suitable cloning vector (such as pRKB or pRKD;
pRK5B is a precursor of pRK5D that does not contain the SfiI site;
see, Holmes et al., Science, 253:1278-1280 (199 1)) in the unique
XhoI and NotI sites.
[1024] A cDNA clone was sequenced in its entirety. The full length
nucleotide sequence of PRO533 is shown in FIG. 21 (SEQ ID NO:58).
Clone DNA49435-1219 contains a single open reading frame with an
apparent translational initiation site at nucleotide positions
459-461 (FIG. 21; SEQ ID NO:58). The predicted polypeptide
precursor is 216 amino acids long. Clone DNA47412-1219 has been
deposited with ATCC and is assigned ATCC deposit no. ATCC
209480.
[1025] Based on a BLAST-2 and FastA sequence alignment analysis of
the full-length sequence, PR0533 shows amino acid sequence identity
to fibroblast growth factor (53%). The oligonucleotide sequences
used in the above procedure were the following: TABLE-US-00021 FGF
15.forward: (SEQ ID NO:60) 5'-ATCCGCCCAGATGGCTACAATGTGTA-3'; FGF
15.probe: (SEQ ID NO:61)
5'-GCCTCCCGGTCTCCCTGAGCAGTGCCAAACAGCGGCAGTGTA-3'; FGF 15.reverse:
(SEQ ID NO:62) 5'-CCAGTCCGGTGACAAGCCCAAA-3'.
Example 11
Isolation of cDNA Clones Encoding Human PRO245
[1026] A consensus DNA sequence was assembled relative to the other
identified EST sequences as described in Example 1 above, wherein
the consensus sequence is designated herein as DNA30954.
[1027] Based on the DNA30954 consensus sequence, oligonucleotides
were synthesized to identify by PCR a cDNA library that contained
the sequence of interest and for use as probes to isolate a clone
of the full-length coding sequence for PR0245.
[1028] A pair of PCR primers (forward and reverse.) were
synthesized: TABLE-US-00022 forward PCR primer
5'-ATCGTTGTGAAGTTAGTGCCCC-3' (SEQ ID NO:65) reverse PCR primer
5'-ACCTGCGATATCCAACAGAATTG-3' (SEQ ID NO:66)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA30954 sequence which had the
following nucleotide sequence
[1029] hybridization pro TABLE-US-00023
5'-GGAAGAGGATACAGTCACTCTGGAAGTATTAGT (SEQ ID NO:67)
GGCTCCAGCAGTTCC-3'
[1030] 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 PR0245 gene
using the probe oligonucleotide and one of the PCR primers.
[1031] RNA for construction of the cDNA libraries was isolated from
human fetal liver tissue. DNAsequencing of the clones isolated as
described above gave the full-length DNA sequence for PR0245
[herein designated as DNA35638-1141] and the derived protein
sequence for PRO245.
[1032] The entire nucleotide sequence of DNA35638-1141 is shown in
FIG. 23 (SEQ ID NO:63). Clone DNA35638-1141 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 89-91 and ending at the stop codon at
nucleotide positions 1025-1027 (FIG. 23; SEQ ID NO:63). The
predicted polypeptide precursor is 312 amino acids long (FIG. 24).
Clone DNA35638-1141 has been deposited with ATCC on Sep. 16, 1997
and is assigned ATCC deposit no. ATCC 209265.
[1033] Analysis of the amino acid sequence of the full-length
PR0245 suggests that a portion of it possesses 60% amino acid
identity with the human c-myb protein and, therefore, may be a new
member of the transmembrane protein receptor tyrosine kinase
family.
Example 12
Isolation of cDNA Clones Encoding Human PR0220, PR0221 and
PR0227
[1034] (a) PR0220
[1035] A consensus DNA sequence was assembled relative to d ie
other identified EST sequences as described in Example 1 above,
wherein the consensus sequence is designated herein as DNA28749.
Based on the DNA28749 consensus sequence, oligonucleotides were
synthesized to identify by PCR a cDNA library that contained the
sequence of interest and for use as probes to isolate a clone of
the full-length coding sequence for PRO220.
[1036] A pair of PCR primers (forward and reverse) were
synthesized: TABLE-US-00024 forward PCR primer
5'-TCACCTGGAGCCTTTATTGGCC-3' (SEQ ID NO:74) reverse PCR primer
5'-ATACCAGCTATAACCAGGCTGCG-3' (SEQ ID NO:75)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA28749 sequence which had the
following nucleotide sequence:
[1037] hybridization probe TABLE-US-00025
5'-CAACAGTAAGTGGTTTGATGCTCTTCCAAATCT (SEQ ID NO:76)
AGAGATTCTGATGATTGGG-3'.
[1038] 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 PR0220 gene
using the probe oligonucleotide and one of the PCR primers.
[1039] RNA for construction of the cDNA libraries was isolated from
human fetal lung tissue. DNA sequencing of the clones isolated as
described above gave the full-length DNA sequence for PR0220
[herein designated as DNA32298-1132 and the derived protein
sequence for PR0220.
[1040] The entire nucleotide sequence of DNA32298-1132 is shown in
FIG. 25 (SEQ ID NO:68). Clone DNA32298-1132 contains, a single open
reading frame with an apparent translational initiation site at
nucleofide positions 480-482 and ending at the stop codon at
nucleotide positions 2604-2606 (FIG. 25). The predicted polypeptide
precursor is 708 amino acids long (FIG. 26). Clone DNA32298-1132
has been deposited with ATCC and is assigned ATCC deposit no. ATCC
209257.
[1041] Analysis of the amino acid sequence of the full-length
PR0220 shows it has homology to member of the leucine rich repeat
protein superfamily, including the leucine rich repeat protein and
the neuronal leucine-rich repeat protein 1.
[1042] (b) PR0221
[1043] A consensus DNA sequence was assembled relative to the other
identified EST sequences as described in Example 1 above, wherein
the consensus sequence is designated herein as DNA28756. Based on
the DNA28756 consensus sequence, oligonucleotides were synthesized
to identify by PCR a cDNA library that contained the sequence of
interest and for use as probes to isolate a clone of the
full-length coding sequence for PR022 1.
[1044] A pair of PCR primers (forward and reverse) were
synthesized: TABLE-US-00026 forward PCR primer
5'-CCATGTGTCTCCTCCTACAAAG-3' (SEQ ID NO:77) reverse PCR primer
5'-GGGAATAGATGTGATCTGATFGG-3' (SEQ ID NO:78)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA28756 sequence which had the
following nucleotide sequence:
[1045] hybridization probe TABLE-US-00027
5'-CACCTGTAGCAATGCAAATCTCAAGGAAATACC (SEQ ID NO:79)
TAGAGATCTTCCTCCTG-3'
[1046] 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 PR0221 gene
using the probe oligonucleotide and one of the PCR primers.
[1047] RNA for construction of the cDNA libraries was isolated from
human fetal lung tissue. DNA sequencing of the clones isolated as
described above gave the full-length DNA sequence for PR0221
[herein designated as DNA33089-1132 and the derived protein
sequence for PR0221.
[1048] The entire nucleotide sequence of DNA33089-1132 is shown in
FIG. 27 (SEQ ID NO:70). Clone DNA33089-1132 con tains a single open
reading fi-ame with an apparent translational initiation site at
nucleotide positions 179-181 and ending at the stop codon at
nucleotide positions 956-958 (FIG. 27). The predicted polypeptide
precursor is 259 amino acids long (FIG. 28). PR0221 is believed to
have a transmembrane region at amino acids 206-225. Clone
DNA33089-1132 has been deposited with ATCC and is assigned ATCC
deposit no. ATCC 209262.
[1049] Analysis of the amino acid sequence of the full-length
PR0221 shows it has homology to member of the leucine rich repeat
protein superfamily, including the SLIT protein.
[1050] (c) PR0227
[1051] A consensus DNA sequence was assembled relative to the other
identified EST sequences as described in Example 1 above, wherein
the consensus sequence is designated herein as DNA28740. Based on
the DNA28740 consensus sequence, oligonucleotides were synthesized
to identify by PCR a cDNA library that contained the sequence of
interest and for use as probes to isolate a clone of the
full-length coding sequence for PR0227.
[1052] A pair of PCR primers (forward and reverse) were
synthesized: TABLE-US-00028 forward PCR primer
5'-AGCAACCGCCTGAAGCTCATCC-3' (SEQ ID NO:80) reverse PCR primer
5'-AAGGCGCGGTGAAAGATGTAGACG-3' (SEQ ID NO:81)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA28740 sequence which had the
following nucleotide sequence:
[1053] hybridization probe TABLE-US-00029
5'GACTACATGTTTCAGGACCTGTACAACCTCAAGT (SEQ ID NO:82)
CACTGGAGGTTGGCGA-3'.
[1054] 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 PR0227 gene
using the probe oligonucleotide and one of the PCR primers.
[1055] RNA for construction of the cDNA libraries was isolated from
human fetal lung tissue. DNA sequencing of the clones isolated as
described above gave the full-length DNA sequence for PR0227
[herein designated as DNA33786-1132 and the derived protein
sequence for PR0227.
[1056] The entire nucleotide sequence of DNA33786-1132 is shown in
FIG. 29 (SEQ ID NO:72). Clone DNA33786-1132 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 33-35 and ending at the stop codon at
nucleotide positions 1893-1895 (FIG. 29). The predicted polypeptide
precursor is 620 amino acids long (FIG. 30). PR0227 is believed to
have a transmembrane region. Clone DNA33786-1132 has been deposited
with ATCC and is assigned ATCC deposit no. ATCC 209253.
[1057] Analysis of the amino acid sequence of the full-length
PR0221 shows it has homology to member of the leucine rich repeat
protein superfamily, including the platelet glycoprotein V
precursor and the human glycoprotein V.
Example 13
Isolation of cDNA Clones Encoding Human PRO258
[1058] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described in Example 1 above. This
consensus sequence is herein designated DNA28746.
[1059] Based on the DNA28746 consensus sequence, oligonucleotides
were synthesized: 1) to identify by PCR a cDNA library that
contained the sequence of interest, and 2) for use as probes to
isolate a clone of the full-length coding sequence for PRO258.
[1060] PCR primers (forward and reverse) were synthesized:
TABLE-US-00030 forward PCR primer 5'-GCTAGGAATTCCACAGAAGCCC-3' (SEQ
ID NO:85) reverse PCR primer 5'-AACCTGGAATGTCACCGAGCTG-3' (SEQ ID
NO:86) reverse PCR primer 5'-CCTAGCACAGTGACGAGGGACTTGGC-3' (SEQ ID
NO:87)
Additionally, synthetic oligonucleotide hybridization probes were
constructed from the consensus DNA28740 sequence which had the
following nucleotide sequence:
[1061] hybridization probe TABLE-US-00031
5'-AAGACACAGCCACCCTAAACTGTCAGTCTTCTG (SEQ ID NO:88)
GGAGCAAGCCTGCAGCC-3' 5'-GCCCTGGCAGACGAGGGCGAGTACACCTGCTCA (SEQ ID
NO:89) ATCTTCACTATGCCTGT-3'
[1062] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones encoding the PRO258 gene
using the probe oligonucleotide and one of the PCR primers.
[1063] RNA for construction of the cDNA libraries was isolated from
human fetal lung tissue. DNA sequencing of the clones isolated as
described above gave the full-length DNA sequence for PRO258
[herein designated as DNA35918-1174] (SEQ ID NO:83) and the derived
protein sequence for PRO258.
[1064] The entire nucleotide sequence of DNA35918-1174 is shown in
FIG. 31 (SEQ ID NO:83). Clone DNA35918-1174 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 147-149 of SEQ ID NO:83 and ending at the stop
codon after nucleotide position 1340 of SEQ ID NO:83 (FIG. 31). The
predicted polypeptide precursor is 398 amino acids long (FIG. 32).
Clone DNA35918-1174 has been deposited with ATCC and is assigned
ATCC deposit no. ATCC 209402.
[1065] Analysis of the amino acid sequence of the full-length
PRO258 polypeptide suggests that portions of it possess significant
homology to the CRTAM and the poliovirus receptor and have an Ig
domain, thereby indicating that PRO258 is a new member of the Ig
superfamily.
Example 14
Isolation of cDNA Clones Encoding Human PRO266
[1066] An expressed sequence tag database was searched for ESTs
having homology to SLIT, resulting in the identification of a
single EST sequence designated herein as T73996. Based on the
T73996 EST sequence, oligonucleotides were synthesized: 1) to
identify by PCR a cDNA library that contained the sequence of
interest, and 2) for use as probes to isolate a clone of the
full-length coding sequence for PRO266.
[1067] A pair of PCR primers (forward and reverse) were
synthesized: TABLE-US-00032 forward PCR primer
5'-GTTGGATCTGGGCAACAATAAC-3' (SEQ ID NO:92) reverse PCR primer
5'-ATTGTTGTGCAGGCTGAGTTTAAG-3' (SEQ ID NO:93)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed which had the following nucleotide sequence
[1068] hybridization probe TABLE-US-00033
5'-GGTGGCTATACATGGATAGCAATTACCTGGACA (SEQ ID NO:94)
CGCTGTCCCGGG-3'
[1069] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones encoding the PRO266 gene
using the probe oligonucleotide and one of the PCR primers.
[1070] RNA for construction of the cDNA libraries was isolated from
human fetal brain tissue. DNA sequencing of the clones isolated as
described above gave the full-length DNA sequence for PRO266
[herein designated as DNA37150-1178] (SEQ ID NO:90) and the derived
protein sequence for PRO266.
[1071] The entire nucleotide sequence of DNA37150-1178 is shown in
FIG. 33 (SEQ ID NO:90). Clone DNA37150-1178 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 167-169 and ending at the stop codon after
nucleotide position 2254 of SEQ ID NO:90. The predicted polypeptide
precursor is 696 amino acids long (FIG. 34). Clone DNA37150-1178
has been deposited with ATCC and is assigned ATCC deposit no. ATCC
209401.
[1072] Analysis of the amino acid sequence of the full-length
PRO266 polypeptide suggests that portions of it possess significant
homology to the SLIT protein, thereby indicating that PRO266 may be
a novel leucine rich repeat protein.
Example 15
Isolation of cDNA Clones Encoding Human PR0269
[1073] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described in Example 1 above. This
consensus sequence is herein designated DNA35705. Based on the
DNA35705 consensus sequence, oligonucleotides were synthesized: 1)
to identify by PCR a cDNA library that contained the sequence of
interest, and 2) for use as probes to isolate a clone of the
full-length coding sequence for PRO269.
[1074] Forward and reverse PCR primers were synthesized:
TABLE-US-00034 forward PCR primer (.f1)
5'-TGGAAGGAGATGCGATGCCACCTG-3' (SEQ ID NO:97) forward PCR primer
(.f2) 5'-TGACCAGTGGGGAAGOACAG-3' (SEQ ID NO:98) forward PCR primer
(.f3) 5'-ACAGAGCAGAGGGTGCCTTG-3' (SEQ ID NO:99) reverse PCR primer
(.r1) 5'-TCAGGGACAAGTGGTGTCTCTCCC-3' (SEQ ID NO:100) reverse PCR
primer (.r2) 5-TCAGGGAAGGAGTGTGCAGTTCTG-3' (SEQ ID NO:101)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA35705 sequence which had the
following nuclootide sequence:
[1075] hybridization probe TABLE-US-00035
5'-ACAGCTCCCGATCTCAGTTACTTGCATCGCG (SEQ ID NO:102)
GACGAAATCGGCGCTCGCT-3'
[1076] 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
PR0269 gene using the probe oligonucleotide and one of the PCR
primers.
[1077] RNA for construction of the cDNA libraries was isolated
fi-om human fetal kidney tissue.
[1078] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO269 [herein designated as
DNA38260-1180] (SEQ ID NO:95) and the derived protein sequence for
PR0269.
[1079] The entire nucleotide sequence of DNA38260-1180 is shown in
FIG. 35 (SEQ ID NO:95). Clone DNA38260-1180 contains a single open
reading fi-ame with an apparent translational initiation site at
nucleotide positions 314-316 and ending at the stop codon at
nucleotide positions 1784-1786 (FIG. 35; SEQ ID NO:95). The
predicted polypeptide precursor is 490 amino acids long (FIG. 36).
Clone DNA38260-1180 has been deposited with ATCC and is assigned
ATCC deposit no. ATCC 209397.
[1080] Analysis of the amino acid sequence of the full-length
PR0269 suggests that portions of it possess significant homology to
the human thrombomodulin proteins, thereby indicating that PR0269
may possess one or more thrombomodulin-like domains.
Example 16
Isolation of cDNA Clones Encoding Human PR0287
[1081] A consensus DNA sequence encoding PR0287 was assembled
relative to the other identified EST sequences as described in
Example 1 above, wherein the consensus sequence is designated
herein as DNA28728. Based on the DNA28728 consensus sequence,
oligonucleotides were synthesized to identify by PCR a cDNA library
that contained the sequence of interest and for use as probes to
isolate a clone of the fidl-length coding sequence for PR0287.
[1082] A pair of PCR primers (forward and reverse) were
synthesized: TABLE-US-00036 forward PCR primer
5'-CCGATTCATAGACCTCGAGAGT-3' (SEQ ID NO:105) reverse PCR primer
5'-GTCAAGGAGTCCTCCACAATAC-3' (SEQ ID NO:106)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA28728 sequence which had the
following nucleotide sequence
[1083] hybridization probe TABLE-US-00037
5'-GTGTACAATGGCCATGCCAATGGCCAGCGCA (SEQ ID NO:107)
TTGGCCGCTTCTGT-3'
[1084] 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 PR0287 gene
using the probe oligonucleotide and one of the PCR primers.
[1085] RNA for construction of the cDNA libraries was isolated from
human fetal kidney tissue.
[1086] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PR0287 [herein designated as
DNA3 9969-1185, SEQ ID NO: 103] and the derived protein sequence
for PRO287.
[1087] The entire nucleotide sequence of DNA39969-1185 is shown in
FIG. 37 (SEQ ID NO:103). Clone DNA39969-1185 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 307-309 and ending at the stop codon at
nucleotide positions 1552-1554 (FIG. 37; SEQ ID NO: 103). The
predicted polypeptide precursor is 415 amino acids long (FIG. 38).
Clone DNA39969-1185 has been deposited with ATCC and is assigned
ATCC deposit no. ATCC 209400.
[1088] Analysis of the amino acid sequence of the full-length
PR0287 suggests that it may possess one or more procollagen
C-proteinase enhancer protein precursor or procollagen C-proteinase
enhancer protein-like domains. Based on a BLAST and FastA sequence
alignment analysis of the full-length sequence, PRO287 shows
nucleic acid sequence identity to procollagen C-proteinase enhancer
protein precursor and procollagen C-proteinase enhancer protein (47
and 54%, respectively).
Example 17
Isolation of cDNA Clones Encoding Human PR0214
[1089] A consensus DNA sequence was assembled using phrap as
described in Example 1 above. This consensus DNA sequence is
designated herein as DNA28744. Based on this consensus sequence,
oligonucleotides were synthesized: 1) to identify by PCR a cDNA
library that contained the sequence of interest, and 2) for use as
probes to isolate a clone of the full-length coding sequence.
[1090] In order to screen several libraries for a source ofa
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pair identified below. A positive
library was then used to isolate clones encoding the PR0214 gene
using the probe oligonucleotide and one of the PCR primers.
[1091] RNA for construction of the cDNA libraries was isolated from
human fetal lung tissue.
[1092] A cDNA clone was sequenced in its entirety. The full length
nucleotide sequence of DNA32286-1191 is shown in FIG. 39 (SEQ ID
NO: 108). DNA32286-1191 contains a single open reading frame with
an apparent translational initiation site at nucleotide position
103 (FIG. 39; SEQ ID NO: 108). The predicted polypeptide precursor
is 420 amino acids long (SEQ ID NO: 109).
[1093] Based on a BLAST and FastA sequence alignment analysis of
the full-length sequence, PRO214 polypeptide shows amino acid
sequence identity to HT protein and/or Fibulin (49% and 38%,
respectively).
[1094] The oligonucleotide sequences used in the above procedure
were the following: TABLE-US-00038 28744.p (OLI555)
5'-CCTGGCTATCAGCAGGTGGGCTCCAAGTGTC (SEQ ID NO:110)
TCGATGTGGATGAGTGTGA-3' 28744.f (OLI556)
5'-ATTCTGCGTGAACACTGAGGGC-3' (SEQ ID NO:111) 28744.r (OLI557)
5'-ATCTGCTTGTAGCCCTCGGGAC-3' (SEQ ID NO:112)
Example 18
Isolation of cDNA Clones Encoding Human PR0317
[1095] A consensus DNA sequence was assembled using phrap as
described in Example 1 above, wherein the consensus sequence is
herein designated as DNA28722. Based on this consensus sequence,
oligonucleotides were synthesized: 1) to identify by PCR a cDNA
library that contained the sequence of interest, and 2) for use as
probes to isolate a clone of the full-length coding sequence. The
forward and reverse PCR primers, respectively, synthesized for this
purpose were: TABLE-US-00039 5'-AGGACTGCCATAACTTGCCTG (OL1489) (SEQ
ID NO:115) and 5'-ATAGGAGTTGAAGCAGCGCTGC (SEQ ID NO:116)
(OLI490).
[1096] The probe synthesized for this purpose was: TABLE-US-00040
5'-TGTGTGGACATAGACGAGTGCCGCTACCGCT (SEQ ID NO:117) ACTGCCAGCACCGC
(OLI488)
[1097] mRNA for construction of the cDNA libraries was isolated
from human fetal kidney tissue.
[1098] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification, as per Ausubel et al., Current Protocols in
Molecular Biology (1989), with the PCR primer pair identified
above. A positive library was then used to isolate clones
containing the PRO317 gene using the probe oligonucleotide
identified above and one of the PCR primers.
[1099] A cDNA clone was sequenced in its entirety. The entire
nucleotide sequence of DNA33461-1199 (encoding PR0317) is shown in
FIG. 41 (SEQ ID NO: 113). Clone DNA33461-1 199 contains a single
open reading fi-ame with an apparent translational initiation site
at nucleotide positions 68-70 (FIG. 41; SEQ ID NO: 113). The
predicted polypeptide precursor is 366 amino acids long. The
predicted signal seq uence is amino acids 1-18 of FIG. 42 (SEQ ID
NO: 114). There is one predicted N-linked glycosylation site at
amino acid residue 160. Clone DNA33461-1199 has been deposited with
ATCC and is assigned ATCC deposit no. ATCC 209367.
[1100] Based on BLAST.TM. and FastA.TM. sequence alignment analysis
(using the ALIGN.TM. computer program) of the full-length
PR0317sequence, PR0317 shows the most amino acid sequence identity
to EBAF-1 (92%). The results also demonstrate a significant
homology between human PR0317 and mouse LEFTY protein. The
C-terminal end of the PR0317 protein contains many conserved
sequences consistent with the pattern expected of a member of the
TGF-superfamily.
[1101] In situ expression analysis in human tissues performed as
described below evidences that there is distinctly strong.
expression of the PR0317 polypeptide in pancreatic tissue.
Example 19
Isolation of cDNA clones Encoding Human PR0301
[1102] A consensus DNA sequence designated herein as DNA35936 was
assembled using phrap as described in Example 1 above. Based on
this consensus sequence, oligonucleotides were synthesized: 1) to
identify by PCR a cDNA library that contained the sequence of
interest, and 2) for use as probes to isolate a clone of the
full-length coding sequence.
[1103] 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 below. A positive
library was then used to isolate clones encoding the PR0301 gene
using the probe oligonucleotide and one of the PCR primers.
[1104] RNA for construction of the cDNA libraries was isolated from
human fetal kidney.
[1105] A cDNA clone was sequenced in its entirety. The full length
nucleotide sequence of native sequence PR0301 is shown in FIG. 43
(SEQ ID NO: 118). Clone DNA40628-1216 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 52-54 (FIG. 43; SEQ ID NO: 118). The predicted
polypeptide precursor is 299 amino acids long with a predicted
molecular weight of 32,583 daltons and pI of 8.29. Clone
DNA40628-1216 has been deposited with ATCC and is assigned ATCC
deposit No. ATCC 209432.
[1106] Based on a BLAST and FastA sequence alignment analysis of
the full-length sequence, PR0301 shows amino acid sequence identity
to A33 antigen precursor (30%) and coxsackie and adenovirus
receptor protein (29%).
[1107] The oligonucleotide sequences used in the above procedure
were the following: TABLE-US-00041 OLI2162 (3593611)
5'-TCGCGGAGCTGTGTTCTGTTTCCC-3- (SEQ ID NO:120) OLI2163 (35936.p1)
5'-TGATCGCGATGGGGACAAAGGCGCAAGCTCG (SEQ ID NO:121)
AGAGGAAACTGTTGTGCCT-3' OLI2164 (35936.f2)
5'-ACACCTGGTTCAAAGATGGG-3' (SEQ ID NO:122) OLI2165 (35936.r1)
5'-TAGGAAGAGTTGCTGAAGGCACGG-3' (SEQ ID NO:123) OLI2166 (35936.0)
5'-TTGCCTTACTCAGGTGCTAC-3' (SEQ ID NO:124) OLI2167 (35936.r2)
5'-ACTCAGCAGTGGTAGGAAAG-3' (SEQ ID NO:125)
Example 20
Isolation of cDNA Clones Encoding Human PR0224
[1108] A consensus DNA sequence assembled relative to the other
identified EST sequences as described in Example 1, wherein the
consensus sequence is designated herein as DNA30845. Based on the
DNA30845 consensus sequence, oligonucleotides were synthesized to
identify by PCR a cDNA library that contained the sequence of
interest and for use as probes to isolate a clone of the
full-length coding sequence for PR0224.
[1109] A pair of PCR primers (forward and reverse) were
synthesized: TABLE-US-00042 forward PCR primer
5'-AAGTTCCAGTGCCGCACCAGTGGC-3' (SEQ ID NO-128) reverse PCR primer
5'-TTGGTTCCACAGCCGAGCTCGTCG-3' (SEQ ID NO:129)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA30845 sequence which had the
following nucleotide sequence
[1110] hybridization probe TABLE-US-00043
5'-GAGGAGGAGTGCAGGATTGAGCCATGTACCC (SEQ ID NO:130)
AGAAAGGGCAATGCCCACC-3'
[1111] 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 PR0224 gene
using the probe oligonucleotide and one of the PCR primers.
[1112] RNA for construction of the cDNA libraries was isolated from
human fetal liver tissue.
[1113] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO224 [herein designated as
DNA33221-1133] and the derived protein sequence for PR0224.
[1114] The entire nucleotide sequence of DNA33221-1133 is shown in
FIG. 45 (SEQ ID NO:126). Clone DNA33221-1133 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 33-35 and ending at the stop codon at
nucleotide positions 879-899 (FIG. 45; SEQ ID NO: 126). The start
of a transmembrane region begins at nucleotide position 777. The
predicted polypeptide precursor is 282 amino acids long (FIG. 46).
Clone DNA33221-1133 has been deposited with ATCC and is assigned
ATCC deposit no. ATCC 209263.
[1115] Analysis of the amino acid sequence of the full-length
PR0224 suggests that it has homology to very low-density
lipoprotein receptors, apolipoprotein E receptor and chicken oocyte
receptors P95. Based on a BLAST and FastA sequence alignment
analysis of the full-length sequence, PR0224 has amino acid
identity to portions of the se proteins in the range from 28% to
45%, and overall identity with these proteins in the range from 33%
to 39%.
Example 21
Isolation of cDNA Clones Encodinit Human PR0222
[1116] A consensus DNA sequence was assembled relative to the other
identified EST sequences as described in Example 1 above, wherein
the consensus sequence is designated herein as DNA28771. Based on
the DNA28771 consensus sequence, oligonucleotides were synthesized
to identify by PCR a cDNA library that contained the sequence of
interest and for use as probes to isolate a clone of the
full-length coding sequence for PR0222.
[1117] A pair of PCR primers (forward and reverse) were
synthesized: TABLE-US-00044 forward PCR primer
5-ATCTCCTATCGCTGCTTTCCCGG-3' (SEQ ID NO:133) reverse PRC primer
5'-AGCCAGGATCGCAGTAAAACTCC-3' (SEQ ID NO:134)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA28771 sequence which had the
following nucleotide sequence:
[1118] hybridization probe TABLE-US-00045
5'-ATTTAAACTTGATGGGTCTGCGTATCTTGAG (SEQ ID NO:135)
TGCTTACAAAACCTTATCT-3'
[1119] In order to screen several libraries fora 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 PR0222 gene
using the probe oligonucleotide and one of the PCR primers.
[1120] RNA for construction of the cDNA libraries was isolated from
human-fetal kidney tissue.
[1121] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PR0222 [herein designated as
DNA33107-11351 and the derived protein sequence for PR0222.
[1122] The entire nucleotide sequence of DNA33107-1135 is shown in
FIG. 47 (SEQ ID NO: 131). Clone DNA33107-1135 contains a single
open reading frame with an apparent translational initiation site
at nucleotide positions 159-161 and ending at the stop codon at
nucleotide positions 1629-1631 (FIG. 47; SEQ ID NO: 131). The
predicted polypeptide precursor is 490 amino acids long (FIG. 48).
Clone DNA33107-1135 has been deposited with ATCC and is assigned
ATCC deposit no. ATCC 20925 1.
[1123] Based on a BLAST and FastA sequence alignment analysis of
the full-length sequence, PRO222 shows amino acid sequence identity
to mouse complement factor h precursor (25-26%), complement
receptor (27-29%), mouse complement C3b receptor type 2 long form
precursor (25-47%) and human hypothetical protein kiaaO247
(40%).
Example 22
Isolation of cDNA clones Encoding PRO234
[1124] A consensus DNA sequence was assembled (DNA30926) using
phrap as described in Example 1 above. Based on this consensus
sequence, oligonucleotides were synthesized: 1) to identify by PCR
a cDNA library that contained the sequence of interest, and 2) for
use as probes to isolate a clone of the full-length coding
sequence.
[1125] RNA for the construction of the cDNA libraries was isolated
using standard isolation protocols, e.g., Ausubel et al., Current
Protocols in Molecular Biology, from tissue or cell line sources or
it was purchased from commercial sources (e.g., Clontech). The cDNA
libraries used to isolate the cDNA clones were. constructed by
standard methods (e.g., Ausubel et al.) using commercially
available reagents (e.g., Invitrogen). This library was derived
from 22 week old fetal brain tissue.
[1126] A cDNA clone was sequenced in its entirety. The entire
nucleotide sequence of PR0234 is shown in FIG. 49 (SEQ ID NO:136).
The predicted polypeptide precursor is 382 amino acids long and has
a cal culated molecular weight of approximately 43.1 kDa.
[1127] The oligonucleotide sequences used in the above procedure
were the following: 30926.p TABLE-US-00046 (OL1826) (SEQ ID
NO:138): 5-GTTTCATTGAAAACCTCTTGCCATCTGATGGTGACTTCTGGATTGGGC TCA-3'
30926.f (OL1827) (SEQ ID NOI 39): 5'-AAGCCAAAGAAGCCTGCAGGAGGG-3'
30926.r (OL1828) (SEQ ID NO:140):
5'-CAGTCCAAGCATAAAGGTCCTGGC-3'
Example 23
Isolation of cDNA Clones Encoding Human PR0231
[1128] A consensus DNA sequence was assembled relative to the other
identified EST sequences as described in Example 1 above, wherein
the consensus sequence was designated herein as DNA30933. Based on
the DNA30933 consensus sequence, oligonucleotides were synthesized
to identify by PCR a cDNA library that contained the sequence of
interest and for use as probes to isolate a clone of the
full-length coding sequence for PR0231.
[1129] Three PCR primers (two forward and one reverse) were
synthesized: TABLE-US-00047 forward PCR primer 1
5'-CCAACTACCAAAGCTGCTGGAOCC-3' (SEQ ID NO:143) forward PCR primer 2
5'-GCAGCTCTATTACCACGGGAAGGA-3' (SEQ ID NO:144) reverse PCR primer
5'-TCCTTCCCGTGGTAATAGAGCTGC-3' (SEQ ID NO:145)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA30933 sequence which had the
following nucleotide sequence
[1130] hybridization probe TABLE-US-00048
5'-GGCAGAGAACCAGAGGCCGGAGGAGACTGCC (SEQ ID NO:146)
TCTTTACAGCCAGG-3'
[1131] 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
PR0231 gene using the probe oligonucleotide and one of the PCR
primers.
[1132] RNA for construction of the cDNA libraries was isolated from
human fetal liver tissue.
[1133] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO231 [herein designated as
DNA34434-1139] and the derived protein sequence for PR023 1.
[1134] The entire nucleotide sequence of DNA34434-1139 is shown in
FIG. 51 (SEQ ID NO: 141). Clone DNA34434-1139 contains a single
open reading frame with an apparent translational initiation site
at nucleotide positions 173-175 and ending at the stop codon at
nucleotide positions 1457-1459 (FIG. 51; SEQ ID NO: 141). The
predicted polypeptide precursor is 428 amino acids long (FIG. 52).
Clone DNA34434-1139 has been deposited with ATCC on Sep. 16, 1997
and is assigned ATCC deposit no. ATCC 209252.
[1135] Analysis of the amino acid sequence of the full-length
PR0231 suggests that it possesses 30% and 31% amino acid identity
with the human and rat prostatic acid phosphatase precursor
proteins, respectively.
Example 24
Isolation of cDNA Clones Encoding Human PRO229
[1136] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described in Example 1 above. This
consensus sequence is herein designated DNA28762. Based on the
DNA28762 consensus sequence, oligonucleotides were synthesized: 1)
to identify by PCR a cDNA library that contained the sequence of
interest, and 2) for use as probes to isolate a clone of the
full-length coding sequence for PR0229.
[1137] A pair of PCR primers (forward and reverse) were
synthesized: TABLE-US-00049 forward PCR primer
5'-TTCAGCTCATCACCTTCACCTGCC-3' (SEQ ID NO:149) reverse PCR primer
5-GGCTCATACAAAATACCACTAGGG-3' (SEQ ID NO:150)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA28762 sequence which had the
following nucleotide sequence
[1138] hybridization probe TABLE-US-00050
5'-GGGCCTCCACCGCTGTGAAGGGCGGGTGGAG (SEQ ID NO:151)
GTGGAACAGAAAGGCCAGT-3'
[1139] 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 PR0229 gene
using the probe oligonucleotide and one of the PCR primers.
[1140] RNA for construction of the cDNA libraries was isolated from
human fetal liver tissue.
[1141] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO229 [herei n designated as
DNA33100-1159] (SEQ ID NO: 147) and the derived protein sequence
for PR0229.
[1142] The entire nucleotide sequence of DNA33100-1159 is shown in
FIG. 53 (SEQ ID NO: 147). Clone DNA33100-1159 contains a single
open reading frame with an apparent translational initiation site
at nucleotide positions 98-100 and ending at the stop codon at
nucleotide positions 1139-1141 (FIG. 53). The predicted polypeptide
precursor is 347 amino acids long (FIG. 54). Clone DNA33100-1159
has been deposited with ATCC and is assigned ATCC deposit no.ATCC
209377
[1143] Analysis of the amino acid sequence of the full-length
PR0229 polypeptide suggests that portions of it possess significant
homology to antigen wc1.1, M1 30 antigen and CD6.
Example 25
Isolation of cDNA Clones Encoding Human PRO238
[1144] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described above in Example 1. This
consensus sequence is herein designated DNA30908. Based on the
DNA30908 consensus sequence, oligonucleotides were synthesized: 1)
to identify by PCR a cDNA library that contained the sequence of
interest, and 2) for use as probes to isolate a clone of the
full-length coding sequence for PRO238.
[1145] PCR primers (forward and reverse) were synthesized:
TABLE-US-00051 forward PCR primer 1 5'-GGTGCTAAACTGGTGCTCTGTGGC-3'
(SEQ ID NO:154) forward PCR primer 2 5'-CAGGGCAAGATGAGCATTCC-3'
(SEQ ID NO:155) reverse PCR primer 5'-TCATACTGTTCCATCTCGGCACGC-3'
(SEQ ID NO:156)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA30908 sequence which had the
following nucleotide sequence
[1146] hybridization probe TABLE-US-00052
51-AATGGTGGGGCCCTAGAAGAGCTCATCAGAG (SEQ ID NO:157)
AACTCACCGCTTCTCATGC-3'
[1147] In order to screen several libraries for a source ofa
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pair identified above. A positive
librwy was then used to isolate clones encoding the PR0238 gene
using the probe oligonucleotide and one of the PCR primers.
[1148] RNA for construction of the cDNA libraries was isolated from
human fetal liver tissue.
[1149] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PR0238 and the derived
protein sequence for PRO238.
[1150] The entire nucleotide sequence of DNA35600-1162 is shown in
FIG. 55 (SEQ ID NO: 152). Clone DNA35600-1162 contains a single
open reading frame with an apparent translational initiation site
at nucleotide positions 134-136 and ending prior to the stop codon
at nucleotide positions 1064-1066 (FIG. 55). The predicted
polypeptide precursor is 310 amino acids long (FIG. 56). Clone
DNA35600-1162 has been deposited with ATCC and is assigned ATCC
deposit no. ATCC 209370.
[1151] Analysis of the amino acid sequence of the full-length
PR0238 polypeptide suggests that portions of it possess significant
homology to reductase, particularly oxidoreductase, thereby
indicating that PR0238 may be a novel reductase.
Example 26
Isolation of cDNA Clones Encoding Human PR0233
[1152] The extracellular domain (ECD) sequences (including the
secretion signal, if any) of from about 950 known secreted proteins
from the Swiss-Prot public protein database were used to search
expressed sequence tag (EST) databases. The EST databases included
public EST databases (e.g., GenBank) and a proprietary EST DNA
database (LIFESEQ.TM., Incyte Pharmaceuticals, Palo Alto, Calif.).
The search was performed using the computer program BLAST or BLAST2
(Altshul et al., Methods in Enzymology 266:460-480 (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.
[1153] An expressed sequence tag (EST) was identified by the EST
database search and a consensus DNA sequence was assembled relative
to other EST sequences using phrap. This consensus sequence is
herein design and DNA30945. Based on the DNA30945 consensus
sequence, oligonucleotides were synthesized: 1) to identify by PCR
a cDNA library that contained the sequence of interest, and 2) for
use as probes to isolate a clone of the full-length coding sequence
for PR0233.
[1154] Forward and reverse PCR primers were synthesized:
TABLE-US-00053 forward PCR primer 5-GGTGAAGGCAGAAATTGGAGATG-3' (SEQ
ID NO:160) reverse PCR primer 5'-ATCCCATGCATCAGCCTGACC-3' (SEQ ID
NO:161)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA30945 sequence which had the
following nucleotide sequence
[1155] hybridization probe TABLE-US-00054
5'-GCTGGTGTAGTCTATACATCAGATTTGTTTG (SEQ ID NO:162)
CTACACAAGATCCTCAG-3'
[1156] In order to screen several libraries for a source ofa
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 PR0233 gene
using the probe oligonucleotide.
[1157] RNA for construction of the cDNA libraries was isolated from
human fetal brain tissue.
[1158] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PR0233 [herein designated as
DNA34436-12381 (SEQ ID NO: 158) and the derived protein sequence
for PR0233.
[1159] The entire nucleotide sequence of DNA34436-1238 is shown in
FIG. 57 (SEQ ID NO: 158). Clone DNA34436-1238 contains a single
open reading frame with an apparent translational initiation site
at nucleotide positions 101-103 and ending at the stop codon at
nucleotide positions 1001-1003 (FIG. 57). The predicted polypeptide
precursor is 300 amino acids long (FIG. 58). The full-length PR0233
protein shown in FIG. 58 has an estimated molecular weight of about
32,964 daltons; and a pI of about 9.52. Clone DNA34436-1238 has
been deposited with ATCC and is assigned ATCC deposit no. ATCC
209523.
[1160] Analysis of the amino acid sequence of the full-length
PR0233 polypeptide suggests that portions of it possess significant
homology to reductase proteins, thereby indicating that PR0233 may
be a novel reductase.
Example 27
Isolation of cDNA Clones Encoding Human PR0223
[1161] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described in Example 1 above. This
consensus sequence is herein designated DNA30836. Based on the
DNA30836 consensus sequence, oligonucleotides were synthesized: 1)
to identify by PCR a cDNA library that contained the sequence of
interest, and 2) for use as probes to isolate a clone of the
full-length coding sequence for PRO223.
[1162] PCR primer pairs (one forward and two reverse) were
synthesized: TABLE-US-00055 forward PCR primer
5'-TTCCATGCCACCTAAGGGAGACTC-3' (SEQ ID NO:165) reverse PCR primer 1
5'-TGGATGAGGTGTGCAATGGCTGGC-3' (SEQ ID NO:166) reverse PCR primer 2
5'-AGCTCTCAGAGGCTGGTCATAGGG-3' (SEQ ID NO:167)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA30836 sequence which had the
following nucleotide sequence:
[1163] hybridization probe TABLE-US-00056
5'-GTCGGCCCTTTCCCAGGACTGAACATGAAGA (SEQ ID NO:168)
GTTATGCCGGCTTCCTCAC-3'
[1164] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones encoding the PRO223 gene
using the probe oligonucleotide and one of the PCR primers.
[1165] RNA for construction of the cDNA libraries was isolated from
human fetal liver tissue.
[1166] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO223 [herein designated as
DNA33206-1165] (SEQ ID NO:163) and the derived protein sequence for
PRO223.
[1167] The entire nucleotide sequence of DNA33206-1165 is shown in
FIG. 59 (SEQ ID NO:163). Clone DNA33206-1165 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 97-99 and ending at the stop codon at
nucleotide positions 1525-1527 (FIG. 59). The predicted polypeptide
precursor is 476 amino acids long (FIG. 60). Clone DNA33206-1165
has been deposited with ATCC and is assigned ATCC deposit no. ATCC
209372.
[1168] Analysis of the amino acid sequence of the full-length
PRO223 polypeptide suggests that it possesses significant homology
to various serine carboxypeptidase proteins, thereby indicating
that PRO223 may be a novel serine carboxypeptidase.
Example 28
Isolation of cDNA Clones Encoding Human PRO235
[1169] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described in Example 1 above. This
consensus sequence is herein designated "DNA30927". Based on the
DNA30927 consensus sequence, oligonucleotides were synthesized: 1)
to identify by PCR a cDNA library that contained the sequence of
interest, and 2) for use as probes to isolate a clone of the
full-length coding sequence for PROM.
[1170] A pair of PCR primers (forward and reverse) were
synthesized: TABLE-US-00057 forward PCR primer
5'-TGGAATACCGCCTCCTGCAG-3' (SEQ ID NO:171) reverse PCR primer
5'-CTTCTGCCCTTTGGAGAAGATGGC-3' (SEQ ID NO:172)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA30927 sequence which had the
following nucleotide sequence.
[1171] hybridization probe TABLE-US-00058 (SEQ ID NO:173)
5'-GGACTCACTGGCCCAGGCCTTCAATATCACCAGCCAGGACGAT-3'
[1172] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones encoding the PRO235 gene
using the probe oligonucleotide and one of the PCR primers.
[1173] RNA for construction of the cDNA libraries was isolated from
human fetal liver tissue.
[1174] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO235 [herein designated as
DNA35558-1167] (SEQ ID NO:169) and the derived protein sequence for
PROM.
[1175] The entire nucleotide sequence of DNA35558-1167 is shown in
FIG. 61 (SEQ ID NO:169). Clone DNA35558-1167 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 667-669 and ending at the stop codon at
nucleotide positions 2323-2325 (FIG. 61). The predicted polypeptide
precursor is 552 amino acids long (FIG. 62). Clone DNA35558-1167
has been deposited with ATCC and is assigned ATCC deposit no.
209374.
[1176] Analysis of the amino acid sequence of the full-length
PRO235 polypeptide suggests that portions of it possess significant
homology to the human, mouse and Xenopus plexin protein, thereby
indicating that PRO235 may be a novel plexin protein.
Example 29
Isolation of cDNA Clones Encoding Human PRO236 and Human PRO262
[1177] Consensus DNA sequences were assembled relative to other EST
sequences using phrap as described in Example 1 above. These
consensus sequences are herein designated DNA30901 and DNA30847.
Based on the DNA30901 and DNA30847 consensus sequences,
oligonucleotides were synthesized: 1) to identify by PCR a cDNA
library that contained the sequence of interest, and 2) for use as
probes to isolate a clone of the full-length coding sequence for
PRO236 and PRO262, respectively.
[1178] Based upon the DNA30901 consensus sequence, a pair of PCR
primers (forward and reverse) were synthesized: TABLE-US-00059
forward PCR primer 5'-TGGCTACTCCAAGACCCTGGCATG-3' (SEQ ID NO:178)
reverse PCR primer 5'-TGGACAAATCCCCTTGCTCAGCCC-3' (SEQ ID
NO:179)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA30901 sequence which had the
following nucleotide sequence
[1179] hybridization probe TABLE-US-00060
5'-GGGCTTCACCGAAGCAGTGGACCTTTATTTT (SEQ ID NO:180)
GACCACCTGATGTCCAGGG-3'
[1180] Based upon the DNA30847 consensus sequence, a pair of PCR
primers (forward and reverse) were synthesized: TABLE-US-00061
forward PCR primer 5'-CCAGCTATGACTATGATGCACC-3' (SEQ ID NO:181)
reverse PCR primer 5'-TGGCACCCAGAATGGTGTTGGCTC-3' (SEQ ID
NO:182)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA30847 sequence which had the
following nucleotide sequence
[1181] hybridization probe TABLE-US-00062
5'-CGAGATGTCATCAGCAAGTTCCAGGAAGTTC (SEQ ID NO:183)
CTTTTGGGACCTTTACCTCC-3'
[1182] In order to screen several libraries for a source of
full-length clones, DNA from the libraries was screened by PCR
amplification with the PCR primer pairs identified above. Positive
libraries were then used to isolate clones encoding the PRO236 and
PRO262 genes using the probe oligonucleotides and one of the PCR
primers.
[1183] RNA for construction of the cDNA libraries was isolated from
human fetal lung tissue for PRO236 and human fetal liver tissue for
PRO262.
[1184] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO236 [herein designated as
DNA35599-1168] (SEQ ID NO:174), the derived protein sequence for
PRO236, the full-length DNA sequence for PRO262 {herein designated
as DNA36992-1168] (SEQ ID NO:176) and the derived protein sequence
for PRO262.
[1185] The entire nucleotide sequence of DNA35599-1168 is shown in
FIG. 63 (SEQ ID NO:174). Clone DNA35509-1168 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 69-71 and ending at the stop codon at
nucleotide positions 1977-1979 (FIG. 63). The predicted polypeptide
precursor is 636 amino acids long (FIG. 64). Clone DNA35599-1168
has been deposited with ATCC and is assigned ATCC deposit no. ATCC
209373.
[1186] The entire nucleotide sequence of DNA36992-1168 is shown in
FIG. 65 (SEQ ID NO:176). Clone DNA36992-1168 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 240-242 and ending at the stop codon at
nucleotide positions 2202-2204 (FIG. 65). The predicted polypeptide
precursor is 654 amino acids long (FIG. 66). Clone DNA36992-1168
has been deposited with ATCC and is assigned ATCC deposit no. ATCC
209382.
[1187] Analysis of the amino acid sequence of the full-length
PRO236 and PRO262 polypeptides suggests that portions of those
polypeptides possess significant homology to Beta-galactosidase
proteins derived from various sources, thereby indicating that
PRO236 and PRO262 may be novel Beta-galactosidase homologs.
Example 30
Isolation of cDNA Clones Encoding Human PRO239
[1188] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described in Example 1 above. This
consensus sequence is herein designated DNA30909. Based on the
DNA30909 consensus sequence, oligonucleotides were synthesized: 1)
to identify by PCR a cDNA library that contained the sequence of
interest, and 2) for use as probes to isolate a clone of the
full-length coding sequence for PRO239.
[1189] A pair of PCR primers (forward and reverse) were
synthesized: TABLE-US-00063 forward PCR primer
5'-CCTCCCTCTATTACCCATGTC-3' (SEQ ID NO:186) reverse PCR primer
5'-GACCAACTTTCTCTGGGAGTGAGG-3' (SEQ ID NO:187)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA30909 sequence which had the
following nucleotide sequence [1190] hybridization probe [1191]
(SEQ ID NO: 188)
[1192] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones encoding the PRO239 gene
using the probe oligonucleotide and one of the PCR primers.
[1193] RNA for construction of the cDNA libraries was isolated from
human fetal lung tissue.
[1194] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO239 [herein designated as
DNA34407-1169] (SEQ ID NO:184) and the derived. protein sequence
for PROM.
[1195] The entire nucleotide sequence of DNA34407-1169 is shown in
FIG. 67 (SEQ ID NO:184). Clone DNA34407-1169 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 72-74 and ending at the stop codon at
nucleotide positions 1575-1577 (FIG. 67). The predicted polypeptide
precursor is 501 amino acids long (FIG. 68). Clone DNA34407-1169
has been deposited with ATCC and is assigned ATCC deposit no.ATCC
209383.
[1196] Analysis of the amino acid sequence of the full-length
PRO239.polypeptide suggests that portions of it possess significant
homology to the densin protein, thereby indicating that PRO239 may
be a novel molecule in the densin family.
Example 31
Isolation of cDNA Clones Encoding Human PRO257
[1197] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described in Example 1 above. This
consensus sequence is herein designated DNA28731. Based on the
DNA28731 consensus sequence, oligonucleotides were synthesized: 1)
to identify by PCR a cDNA library that contained the sequence of
interest, and 2) for use as probes to isolate a clone of the
full-length coding sequence for PRO257.
[1198] A pair of PCR primers (forward and reverse) were
synthesized: TABLE-US-00064 forward PCR primer
5'-TCTCTATTCCAAACTGTGGCG-3' (SEQ ID NO:191) reverse PCR primer
5'-TTTGATGACGATTCGAAGGTGG-3' (SEQ ID NO:192)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA28731 sequence which had the
following nucleotide sequence
[1199] hybridization probe TABLE-US-00065
5'-GGAAGGATCCTTCACCAGCCCCAATTACCCA (SEQ ID NO:193)
AAGCCGCATCCTGAGC-3'
[1200] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones encoding the PRO257 gene
using the probe oligonucleotide and one of the PCR primers.
[1201] RNA for construction of the cDNA libraries was isolated from
human fetal kidney tissue.
[1202] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO257 [herein designated as
DAN 841 -1173 (SEQ ID NO- 189) and the derived protein sequence for
PRO257.
[1203] The entire nucleotide sequence of DNA35841-1173 is shown in
FIG. 69 (SEQ ID NO:189). Clone DNA35841-1173 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 964-966 and ending at the stop codon at
nucleotide positions 2785-2787 (FIG. 69). The predicted polypeptide
precursor is 607 amino acids long (FIG. 70). Clone DNA35841-1173
has been deposited with ATCC and is assigned ATCC deposit no. ATCC
209403.
[1204] Analysis of the amino acid sequence of the full-length
PRO257 polypeptide suggests that portions of it possess significant
homology to the ebnerin protein, thereby indicating that PRO257 may
be a novel protein member related to the ebnerin protein.
Example 32
Isolation of cDNA Clones Encoding Human PRO260
[1205] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described in Example 1 above. This
consensus sequence is herein designated DNA30834. Based on the
DNA30834 consensus sequence, oligonucleotides were synthesized: 1)
to identify by PCR a cDNA library that contained the sequence of
interest, and 2) for use as probes to isolate a clone of the
full-length coding sequence for PRO260.
[1206] PCR primers (forward and two reverse) were synthesized:
TABLE-US-00066 forward PCR primer: 5'-TGGTTTGACCAGGCCAAGTTCGG-3';
(SEQ ID NO:196) reverse PCR primer A:
5'-GGATTCATCCTCAAGGAAGAGCGG-3'; (SEQ ID NO:197) and reverse PCR
primer B: 5'AACTTGCAGCATCAGCCACTCTGC-3' (SEQ ID NO:198)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA30834 sequence which had the
following nucleotide sequence:
[1207] hybridization probe: TABLE-US-00067
5'-TTCCGTGCCCAGCTTCGGTAGCGAGTGGTTC (SEQ ID NO:199)
TGGTGGTATTGGCA-3'
[1208] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones encoding the PRO260 gene
using the probe oligonucleotide and one of the PCR primers.
[1209] RNA for construction of the cDNA libraries was isolated from
human fetal kidney tissue.
[1210] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO260 [herein designated as
DNA33470-11751 (SEQ ID NO:194) and the derived protein sequence for
PRO260.
[1211] The entire nucleotide sequence of DNA33470-1175 is shown in
FIG. 71 (SEQ ID NO:194). Clone DNA33470-1175 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 67-69 and ending at the stop codon 1468-1470
(see FIG. 71). The predicted polypeptide precursor is 467 amino
acids long (FIG. 72). Clone DNA33470-1175 has been deposited with
ATCC and is assigned ATCC deposit no. ATCC 209398.
[1212] Analysis of the amino acid sequence of the full-length
PRO260 polypeptide suggests that portions of it possess significant
homology to the alpha-l-fucosidase precursor, thereby indicating
that PRO260 may be a novel fucosidase.
Example 33
Isolation of cDNA Clones Encoding Human PRO263
[1213] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described in Example 1 above. This
consensus sequence is herein designated DNA30914. Based on the
DNA30914 consensus sequence, oligonucleotides were synthesized: 1)
to identify by PCR a cDNA library that contained the sequence of
interest, and 2) for use as probes to isolate a clone of the
full-length coding sequence for PRO263.
[1214] PCR primers (tow forward and one reverse) were synthesized:
TABLE-US-00068 forward PCR primer 1:
5'-GAGCTTTCCATCCAGGTGTCATGC-3'; (SEQ ID NO:202) forward PCR primer
2: 5'-GTCAGTGACAGTACCTACTCGG-3'; (SEQ ID NO:203) reverse PCR
primer: 5'-TGGAGCAGGAGGAGTAGTAGTAGG-3' (SEQ ID NO:204)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA30914 sequence which had the
following nucleotide sequence:
[1215] hybridization probe: TABLE-US-00069
5'-AGGAGGCCTGTAGGCTGCTGGGACTAAGTTT (SEQ ID NO:205)
GGCCGGAAGGACCAAGTT-3'
[1216] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones encoding the PRO263 gene
using the probe oligonucleotide and one of the PCR primers.
[1217] RNA for construction of the cDNA libraries was isolated from
human fetal liver tissue.
[1218] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO263 [herein designated as
DNA34431 -1177] (SEQ ID NO:200) and the derived protein sequence
for PRO263.
[1219] The entire nucleotide sequence of DNA34431 -1177 is shown in
FIG. 73 (SEQ ID NO:200). Clone DNA34431 -1177 contains a single
open reading frame with an apparent translational initiation site
at nucleotide positions 160-162 of SEQ ID NO:200 and ending at the
stop codon after the nucleotide at position 1126-1128 of SEQ ID
NO:200 (FIG. 73). The predicted polypeptide precursor is 322 amino
acids long (FIG. 74). Clone DNA34431-1177 has been deposited with
ATCC and is assigned ATCC deposit no. ATCC 209399.
[1220] Analysis of the amino acid sequence of the full-length
PRO263 polypeptide suggests that portions of it possess significant
homology to CD44 antigen, thereby indicating that PRO263 may be a
novel cell surface adhesion molecule.
Example 34
Isolation of cDNA Clones Encoding Human PRO270
[1221] A consensus DNA sequence was assembled relative to the other
identified EST sequences as described in Example 1 above, wherein
the consensus sequence was designated herein as DNA35712. Based on
the DNA35712 consensus sequence, oligonucleotides were synthesized:
1) to identify by PCR a cDNA library that contained the sequence of
interest, and 2) for use as probes to isolate a clone of the
full-length coding sequence for PROM.
[1222] Forward and reverse PCR primers were synthesized:
TABLE-US-00070 forward PCR primer (.f1)
5'-GCTTGGATATTCGCATGGGCCTAC-3' (SEQ ID NO:208) forward PCR primer
(.f2) 5'-TGGAGACAATATCCCTGAGG-3' (SEQ ID NO:209) reverse PCR primer
(.r1) 5'-AACAGTTGGCCACAGCATGGCAGG-3' (SEQ ID NO:210)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA35712 sequence which had the
following nucleotide sequence
[1223] hybridization probe TABLE-US-00071
5'-CCATTGATGAGGAACTAGAACGGGACAAGAG (SEQ ID NO:211)
GGTCACTTGGATTGTGGAG-3'
[1224] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by P CR
amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones encoding the PRO270 gene
using the probe oligonucleotide and one of the PCR primers.
[1225] RNA for construction of the cDNA libraries was isolated from
human fetal lung tissue.
[1226] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO270 [herein designated as
DNA39510-1181] (SEQ ID NO:206) and the derived protein sequence for
PROM.
[1227] The entire nucleotide sequence of DNA39510-1181 is shown in
FIG. 75 (SEQ ID NO:206). Clone DNA39510-1181 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 3-5 and ending at the stop codon at nucleotide
positions 891-893 (FIG. 75; SEQ ID NO:206). The predicted
polypeptide precursor is 296 amino acids long (FIG. 76). Clone
DNA39510-1181 has been deposited with ATCC and is assigned ATCC
deposit no, ATCC 209392.
[1228] Analysis of the amino acid sequence of the full-length
PRO270 suggests that portions of it possess significant homology to
the thioredoxin-protein, thereby indicating that the PRO270 protein
may be a novel member of the thioredoxin family.
Example 35
Isolation of cDNA Clones Encoding Human PRO271
[1229] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described in Example 1 above. This
consensus sequence is herein designated DNA35737. Based on the
DNA35737 consensus sequence, oligonucleotides were synthesized: 1)
to identify by PCR a cDNA library that contained the sequence of
interest, and 2) for use as probes to isolate a clone of the
full-length coding sequence for PRO271.
[1230] Forward and reverse PCR primers were synthesized:
TABLE-US-00072 forward PCR primer 1 5'-TGCTTCGCTACTGCCCTC-3' (SEQ
ID NO:214) forward PCR primer 2 5'-TTCCCTTGTGGGTTGGAG-3' (SEQ ID
NO:215) forward PCR primer 3 5'-AGGGCTGGAAGCCAGTTC-3' (SEQ ID
NO:216) reverse PCR primer 1 5'-AGCCAGTGAGGAAATGCG-3' (SEQ ID
NO:217) reverse PCR primer 2 5'-TGTCCAAAGTACACACACCTGAGG-3' (SEQ ID
NO:218)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA35737 sequence which had the
following nucleotide sequence
[1231] hybridization probe TABLE-US-00073
5'-GATGCCACGATCGCCAAGGTGGGACAGCTCT (SEQ ID NO:219)
TTGCCGCCTGGAAG-3'
[1232] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones encoding the PRO271 gene
using the probe oligonucleotide and one of the PCR primers.
[1233] RNA for construction of the cDNA libraries was isolated from
human fetal brain tissue.
[1234] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO271 [herein designated as
DNA39423-1182] (SEQ ID NO:212) and the derived protein sequence for
PRO271.
[1235] The entire nucleotide sequence of DNA39423-1182 is shown in
FIG. 77 (SEQ ID NO:212). Clone DNA39423-1182 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 101 -103 and ending at the stop codon at
nucleotide positions 1181 -1183 (FIG. 77). The predicted
polypeptide precursor is 360 amino acids long (FIG. 78). Clone
DNA39423-1182 has been deposited with ATCC and is assigned ATCC
deposit no. ATCC 209387.
[1236] Analysis of the amino acid sequence of the full-length
PRO271 polypeptide suggests that it possess significant homology to
the proteoglycan link protein, thereby indicating that PRO271 may
be a link protein homolog.
Example 36
Isolation of cDNA Clones Encoding Human PRO272
[1237] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described in Example 1 above. This
consensus sequence is herein designated DNA36460. Based on the
DNA36460 consensus sequence, oligonucleotides were synthesized: 1)
to identify by PCR a cDNA library that contained the sequence of
interest, and 2) for use as probes to isolate a clone of the
full-length coding sequence for PROM.
[1238] Forward and reverse PCR primers were synthesized:
TABLE-US-00074 forward PCR primer (.f1) 5'-CGCAGGCCCTCATGGCCAGG-3'
(SEQ ID NO:222) forward PCR primer (.f2) 5'-GAAATCCTGGGTAATTGG-3'
(SEQ ID NO:223) reverse PCR primer 5'-GTGCGCGGTGCTCACAGCTCATC-3'
(SEQ ID NO:224)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA36460 sequence which had the
following nucleotide sequence
[1239] hybridization probe TABLE-US-00075
5'-CCCCCCTGAGCGACGCTCCCCCATGATGACG (SEQ ID NO:225)
CCCACGGGAACTTC-3'
[1240] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pairs identified above. A
positive library was then used to isolate clones encoding the
PRO272 gene using the probe oligonucleotide and one of the PCR
primers.
[1241] RNA for construction of the cDNA libraries was isolated from
human fetal lung tissue.
[1242] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO272 [herein designated as
DNA40620-1183] (SEQ ID NO:220) and the derived protein sequence for
PROM.
[1243] The entire nucleotide sequence of DNA40620-1183 is shown in
FIG. 79 (SEQ ID NO:220). Clone DNA40620-1183 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 35-37 and ending at the stop codon at
nucleotide positions 1019-1021 (FIG. 79). The predicted polypeptide
precursor is 328 amino acids long (FIG. 80). Clone DNA40620-1183
has been deposited with ATCC and is assigned ATCC deposit no. ATCC
209388.
[1244] Analysis of the amino acid sequence of the full-length
PRO272 polypeptide suggests that portions of it possess significant
homology to the human and mouse reticulocalbin proteins,
respectively, thereby indicating that PRO272 may be a novel
reticulocalbin protein.
Example 37
Isolation of cDNA Clones Encoding Human PRO294
[1245] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described in Example 1 above. This
consensus sequence is herein designated DNA35731. Based on the
DNA35731 consensus sequence, oligonucleotides were synthesized: 1)
to identify by PCR a cDNA library that contained the sequence of
interest, and 2) for use as probes to isolate a clone of the
full-length coding sequence for PRO294.
[1246] Forward and reverse PCR primers were synthesized:
TABLE-US-00076 forward PCR primer (.f1) 5'-TGGTCTCGCACACCGATC-3'
(SEQ ID NO:228) forward PCR primer (.f2) 5'-CTGCTGTCCACAGGGGAG-3'
(SEQ ID NO:229) forward PCR primer (.f3) 5'-CCTTGAAGCATACTGCTC-3'
(SEQ ID NO:230) forward PCR primer (.f4) 5'-GAGATAGCAATTTCCGCC-3'
(SEQ ID NO:231) reverse PCR primer (.r1) 5'-TTCCTCAAGAGGGCAGCC-3'
(SEQ ID NO:232) reverse PCR primer (.r2)
5'-CTTGGCACCAATGTCCGAGATTTC-3' (SEQ ID NO:233)
[1247] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA35731 sequence which
had the following nucleotide sequence TABLE-US-00077 hybridization
probe 5'-GCTCTGAGGAAGGTGACGCGCGGGGCCTCCG (SEQ ID NO:234)
AACCCTTGGCCTTG-3'
[1248] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pairs identified above. A
positive library was then used to isolate clones encoding the
PRO294 gene using the probe oligonucleotide and one of the PCR
primers.
[1249] RNA for construction of the cDNA libraries was isolated from
human fetal brain tissue.
[1250] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO294 [herein designated as
DNA40604-1187] (SEQ ID NO:226) and the derived protein sequence for
PRO294.
[1251] The entire nucleotide sequence of DNA40604-1187 is shown in
FIG. 81 (SEQ ID NO:226). Clone DNA40604-1187 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 396-398 and ending at the stop codon at
nucleotide positions 2046-2048 (FIG. 81). The predicted polypeptide
precursor is 550 amino acids long (FIG. 82). Clone DNA40604-1187
has been deposited with ATCC and is assigned ATCC deposit no.
209394.
[1252] Analysis of the amino acid sequence of the full-length
PRO294 polypeptide suggests that portions of it possess significant
homology to portions of various collagen proteins, thereby
indicating that PRO294 may be collagen-like molecule.
Example 38
Isolation of cDNA Clones Encoding Human PRO295
[1253] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described in Example 1 above. This
consensus sequence is herein designated DNA35814. Based on the
DNA35814 consensus sequence, oligonucleotides were synthesized: 1)
to identify by PCR a cDNA library that contained the sequence of
interest, and 2) for use as probes to isolate a clone of the
full-length coding sequence for PRO295.
[1254] Forward and reverse PCR primers were synthesized:
TABLE-US-00078 forward PCR primer (.f1)
5'-GCAGAGCGGAGATGCAGCGGCTTG-3' (SEQ ID NO:238) forward PCR primer
(.f2) 5'-CCCAGCATGTACTGCCAG-3' (SEQ ID NO:239) forward PCR primer
(.f3) 5'-TTGGCAGCTTCATGGAGG-3' (SEQ ID NO:240) forward PCR primer
(.f4) 5'-CCTGGGCAAAAATGCAAC-3' (SEQ ID NO:241) reverse PCR primer
(.r1) 5'-CTCCAGCTCCTGGCGCACCTCCTC-3' (SEQ ID NO:242)
[1255] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA35814 sequence which
had the following nucleotide sequence TABLE-US-00079 hybridization
probe 5'-GGCTCTCAGCTACCGCGCAGGAGCGAGGCCA (SEQ ID NO:243)
CCCTCAATGAGATG-3'
[1256] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pairs identified above. A
positive library was then used to isolate clones encoding the
PRO295 gene using the probe oligonucleotide and one of the PCR
primers.
[1257] RNA for construction of the cDNA libraries was isolated from
human fetal lung tissue.
[1258] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO295 [herein designated as
DNA38268-1188] (SEQ ID NO:235) and the derived protein sequence for
PRO295.
[1259] The entire nucleotide sequence of DNA38268-1188 is shown in
FIG. 83 (SEQ ID NO:235). Clone DNA38268-1188 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 153-155 and ending at the stop codon at
nucleotide positions 1202-1204 (FIG. 83). The predicted polypeptide
precursor is 350 amino acids long (FIG. 84). Clone DNA38268-1188
has been deposited with ATCC and is assigned ATCC deposit no.
209421.
[1260] Analysis of the amino acid sequence of the full-length
PRO295 polypeptide suggests that portions of it possess significant
homology to the integrin proteins, thereby indicating that PRO295
may be a novel integrin.
Example 39
Isolation of cDNA Clones Encoding Human PRO293
[1261] The extracellular domain (ECD) sequences (including the
secretion signal, if any) of from about 950 known secreted proteins
from the Swiss-Prot public protein database were used to search
expressed sequence tag (EST) databases. The EST databases included
public EST databases (e.g., GenBank) and a proprietary EST DNA
database (LIFESEQ.TM., Incyte Pharmaceuticals, Palo Alto, Calif.).
The search was performed using the computer program BLAST or BLAST2
(Altshul et al., Methods in Enzymology 266:460-480 (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.
[1262] Based on an expression tag sequence designated herein as
T08294 identified in the above analysis, oligonucleotides were
synthesized: 1) to identify by PCR a cDNA library that contained
the sequence of interest, and 2) for use as probes to isolate a
clone of the full-length coding sequence for PRO293.
[1263] A pair of PCR primers (forward and reverse) were
synthesized: TABLE-US-00080 forward PCR primer
5'-AACAAGGTAAGATGCCATCCTG-3' (SEQ ID NO:246) reverse PCR primer
5'-AAACTTGTCGATGGAGACCAGCTC-3' (SEQ ID NO:247)
[1264] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the expression sequence tag which had
the following nucleotide sequence TABLE-US-00081 hybridization
probe 5'-AGGGGCTGCAAAGCCTGGAGAGCCTCTCCTT (SEQ ID NO:248)
CTATGACAACCAGC-3'
[1265] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones encoding the PRO293 gene
using the probe oligonucleotide and one of the PCR primers.
[1266] RNA for construction of the cDNA libraries was isolated from
human fetal brain tissue.
[1267] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO293 [herein designated as
DNA37151-1193] (SEQ ID NO:244) and the derived protein sequence for
PRO293.
[1268] The entire nucleotide sequence of DNA37151-1193 is shown in
FIG. 85 (SEQ ID NO:244). Clone DNA37151-1193 contains a single open
reading frame with an apparent translational initiation site a at
nucleotide positions 881-883 and ending at the stop codon after
nucleotide position 3019 of SEQ ID NO:244, FIG. 85). The predicted
polypeptide precursor is 713 amino acids long (FIG. 86). Clone
DNA37151-1193 has been deposited with ATCC and is assigned ATCC
deposit no. ATCC 209393.
[1269] Analysis of the amino acid sequence of the full-length
PRO293 polypeptide suggests that portions of it possess significant
homology to the NLRR proteins, thereby indicating that PRO293 may
be a novel NLRR protein.
Example 40
Isolation of cDNA Clones Encoding Human PRO247
[1270] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described in Example 1 above. This
consensus sequence is herein designated DNA33480. Based on the
DNA33480 consensus sequence, oligonucleotides were synthesized: 1)
to identify by PCR a cDNA library that contained the sequence of
interest, and 2) for use as probes to isolate a clone of the
full-length coding sequence for PRO247.
[1271] A pair of PCR primers (forward and reverse) were
synthesized: TABLE-US-00082 forward PCR primer
5'-CAACAATGAGGGCACCAAGC-3' (SEQ ID NO:251) reverse PCR primer
5-GATGGCTAGGTTCTGGAGGTTCTG-3' (SEQ ID NO:252)
[1272] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the DNA33480 expression sequence tag
which had the following nucleotide sequence hybridization probe
TABLE-US-00083 5'-CAACCTGCAGGAGATTGACCTCAAGGACAAC (SEQ ID NO:253)
AACCTCAAGACCATCG-3'
[1273] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones encoding the PRO247 gene
using the probe oligonucleotide and one of the PCR primers.
[1274] RNA for construction of the cDNA libraries was isolated from
human fetal brain tissue.
[1275] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO247 [herein designated as
DNA35673-1201]- (SEQ ID NO:249) and the derived protein sequence
for PRO247.
[1276] The entire nucleotide sequence of DNA35673-1201 is shown in
FIG. 89 (SEQ ID NO:249). Clone DNA35673-1201 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 80-82 of SEQ ID NO:249 and ending at the stop
codon after nucleotide position 1717 of SEQ ID NO:249 (FIG. 89).
The predicted polypeptide precursor is 546 amino acids long (FIG.
88). Clone DNA35673-1201 has been deposited with ATCC and is
assigned ATCC deposit no. 209418.
[1277] Analysis of the amino acid sequence of the full-length
PRO247 polypeptide suggests that portions of it possess significant
homology to the densin molecule and KIAA0231, thereby indicating
that PRO247 may be a novel leucine rich repeat protein.
Example 41
Isolation of cDNA Clones Encoding Human PRO302, PRO303, PRO304,
PRO307 and PRO343
[1278] Consensus DNA sequences were assembled relative to other EST
sequences using phrap as described in Example 1 above. These
consensus sequences are herein designated DNA35953, DNA35955,
DNA35958, DNA37160 and DNA30895. Based on the DNA35953 consensus
sequence, oligonucleotides were synthesized: 1) to identify by PCR
a cDNA library that contained the sequence of interest, and 2) for
use as probes to isolate a clone of the full-length coding sequence
for PRO302.
[1279] PCR primers (forward and reverse) were synthesized:
TABLE-US-00084 forward PCR primer 5'-GTCCGCAAGGATGCCTACATGTTC-3'
(SEQ ID NO:264) forward PCR primer 5'-GCAGAGGTGTCTAAGGTTG-3' (SEQ
ID NO:265) reverse PCR primer 5'-AGCTCTAGACCAATGCCAGCTTCC-3' (SEQ
ID NO:266)
[1280] Also, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA35953 sequence which had the
following nucleotide sequence. TABLE-US-00085 hybridization probe
5'-GCCACCAACTCCTGCAAGAACTTCTCAGAAC (SEQ ID NO:267)
TGCCCCTGGTCATG-3'
[1281] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pairs identified above. A
positive library was then used to isolate clones encoding the
PRO302 gene using the probe oligonucleotide and one of the PCR
primers.
[1282] RNA for construction of the cDNA libraries was isolated from
human fetal kidney tissue (LIB228).
[1283] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO302 [herein designated as
DNA40370-1217] (SEQ ID NO:254) and the derived protein sequence for
PRO302.
[1284] The entire nucleotide sequence of DNA40370-1217 is shown in
FIG. 89 (SEQ ID NO:254). Clone DNA40370-1217 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 34-36 and ending at the stop codon at
nucleotide positions 1390-1392 (FIG. 89). The predicted polypeptide
precursor is 452 amino acids long (FIG. 90). Various unique aspects
of the PRO302 protein are shown in FIG. 90. Clone DNA40370-1217 has
been deposited with the ATCC on Nov. 21, 1997 and is assigned ATCC
deposit no. ATCC 209485.
[1285] Based on the DNA35955 consensus sequence, oligonucleotides
were synthesized: 1) to identify by PCR a cDNA library that
contained the sequence of interest, and 2) for use as probes to
isolate a clone of the full-length coding sequence for PRO303.
[1286] A pair of PCR primers (forward and reverse) were
synthesized: TABLE-US-00086 forward PCR primer
5'-GGGGAATTCACCCTATGACATTGCC-3' (SEQ ID NO:268) reverse PCR primer
5'-GAATGCCCTGCAAGCATCAACTGG-3' (SEQ ID NO:269)
[1287] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA35955 sequence which
had the following nucleotide sequence: TABLE-US-00087 hybridization
probe 5'-GCACCTGTCACCTACACTAAACACATCCAGC (SEQ ID NO:270)
CCATCTGTCTCCAGGCCTC-3'
[1288] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification wit with the PCR primer pairs identified above. A
positive library was then used to. isolate clones encoding the
PRO303 gene using the probe oligonucleotide and one of the PCR
primers.
[1289] RNA for construction of the cDNA libraries was isolated from
human fetal lung tissue (LIB25).
[1290] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO303 [herein designated as
DNA42551-1217] (SEQ ID NO:256) and the derived protein sequence for
PRO303.
[1291] The entire nucleotide sequence of DNA42551-1217 is shown in
FIG. 91 (SEQ ID NO:256). Clone DNA42551-1217 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 20-22 and ending at the stop codon at
nucleotide positions 962-964 (FIG. 91). The predicted polypeptide
precursor is 314 amino acids long (FIG. 92). Various unique aspects
of the PRO303 protein are shown in FIG. 92. Clone DNA42551-1217 has
been deposited on Nov. 21, 1997 with the ATCC and is assigned ATCC
deposit no. ATCC 209483.
[1292] Based on the DNA35958 consensus sequence, oligonucleotides
were synthesized: 1) to identify by PCR a cDNA library that
contained the sequence of interest, and 2) for use as probes to
isolate a clone of the full-length coding sequence for PRO304.
[1293] Pairs of PCR primers (forward and reverse) were synthesized:
TABLE-US-00088 forward PCR primer 1
5'-GCGGAAGGGCAGAATGGGACTCCAAG-3' (SEQ ID NO:271) forward PCR primer
2 5'-CAGCCCTGCCACATGTGC-3' (SEQ ID NO:272) forward PCR primer 3
5'-TACTGGGTGGTCAGCAAC-3' (SEQ ID NO:273) reverse PCR primer
5'-GGCGAAGAGCAGGGTGAGACCCCG-3' (SEQ ID NO:274)
[1294] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA35958 sequence which
had the following nucleotide sequence TABLE-US-00089 hybridization
probe 5'-GCCCTCATCCTCTCTGGCAAATGCAGTTACA (SEQ ID NO:275)
GCCCGGAGCCCGAC-3'
[1295] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pairs identified above. A
positive library was then used to isolate clones encoding the
PRO304 gene using the probe oligonucleotide and one of the PCR
primers.
[1296] RNA for construction of the cDNA libraries was isolated from
22 week human fetal brain tissue (LIB 153).
[1297] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO304 [herein designated as
DNA39520-1217] (SEQ ID NO:258) and the derived protein sequence for
PRO304.
[1298] The entire nucleotide sequence of DNA39520-1217 is shown in
FIG. 93 (SEQ ID NO:258). Clone DNA39520-1217 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 34-36 and ending at the stop codon at
nucleotide positions 1702-1704 (FIG. 93). The predicted polypeptide
precursor is 556 amino acids long (FIG. 94). Various unique aspects
of the PRO304 protein are shown in FIG. 94. Clone DNA39520-1217 has
been deposited with ATCC on Nov. 21, 1997 and is assigned ATCC
deposit no. ATCC 209482.
[1299] Based on the DNA37160 consensus sequence, oligonucleotides
were synthesized. 1) to identify by PCR a cDNA library that
contained the sequence of interest, and 2) for use as probes to
isolate a clone of the full-length coding sequence for PRO307.
[1300] Pairs of PCR primers (forward and reverse) were synthesized:
TABLE-US-00090 forward PCR primer 1 5'-GGGCAGGGATTCCAGGGCTCC-3'
(SEQ ID NO:276) forward PCR primer 2 5'-GGCTATGACAGCAGGTTC-3' (SEQ
ID NO:277) forward PCR primer 3 5'-TGACAATGACCGACCAGG-3' (SEQ ID
NO:278) reverse PCR primer 5'-GCATCGCATTGCTGGTAGAGCAAG-3' (SEQ ID
NO:279)
[1301] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA37160 sequence which
had the following nucleotide sequence TABLE-US-00091 hybridization
probe 5'-TTACAGTGCCCCCTGGAAACCCACTTGGCCT (SEQ ID NO:280)
GCATACCGCCTCCC-3'
[1302] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pairs identified above. A
positive library was then used to isolate clones encoding the
PRO307 gene using the probe oligonucleotide and one of the PCR
primers.
[1303] RNA for construction of the cDNA libraries was isolated from
human fetal liver tissue (LIB229).
[1304] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO307 [herein designated as
DNA41225-1217] (SEQ ID NO:260) and the derived protein sequence for
PRO307.
[1305] The entire nucleotide sequence of DNA41225-1217 is shown in
FIG. 95 (SEQ ID NO:260). Clone DNA41225-1217 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 92-94 and ending at the stop codon at
nucleotide positions 1241 -1243 (FIG. 95). The predicted
polypeptide precursor is 383 amino acids long (FIG. 196). Various
unique aspects of the PRO307 protein are shown in FIG. 96. Clone
DNA41225-1217 has been deposited with ATCC on Nov. 21, 1997 and is
assigned ATCC deposit no. ATCC 209491.
[1306] Based on the DNA30895 consensus sequence, oligonucleotides
were synthesized: 1) to identify by PCR a cDNA library that
contained the sequence of interest, and 2) for use as probes to
isolate a clone of the full-length coding sequence for PRO343.
[1307] A pair of PCR primers (forward and reverse) were
synthesized: TABLE-US-00092 forward PCR primer
5'-CGTCTCGAGCGCTCCATACAGTTCCCTTGCC (SEQ ID NO:281) CCA-3'
[1308] TABLE-US-00093 reverse PCR primer
5'-TGGAGGGGGAGCGGGATGCTTGTCTGGGCGA (SEQ ID NO:282)
CTCCGGGGGCCCCCTCATGTGCCAGGTGGA-3'
[1309] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA30895 sequence which
had the following nucleotide sequence TABLE-US-00094 hybridization
probe 5'-CCCTCAGACCCTGCAGAAGCTGAAGGTTCCT (SEQ ID NO:283)
ATCATCGACTCGGAAGTCTGCAGCCATCTGTACT
GGCGGGGAGCAGGACAGGGACCCATCACTGAGGA CATGCTGTGTGCCGGCTACT-3'
[1310] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pairs identified above. A
positive library was then used to isolate clones encoding the
PRO343 gene using the probe oligonucleotide and one of the PCR
primers.
[1311] RNA for construction of the cDNA libraries was isolated from
human fetal lung tissue (LIB26).
[1312] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO343 [herein designated as
DNA43318-1217] (SEQ ID NO:262) and the derived protein sequence for
PRO343.
[1313] The entire nucleotide sequence of DNA43318-1217 is shown in
FIG. 97 (SEQ ID NO:262). Clone DNA43318-1217 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 53-55 and ending at the stop codon at
nucleotide positions 1004-1006 (FIG. 97). The predicted polypeptide
precursor is 317 amino acids long (FIG. 98). Various unique aspects
of the PRO343 protein are shown in FIG. 98. Clone DNA43318-1217 has
been deposited with ATCC on Nov. 21, 1997 and is assigned ATCC
deposit no. ATCC 209481.
Example 42
Isolation of cDNA Clones Encoding Human PRO328
[1314] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described in Example 1 above. This
consensus sequence is herein designated DNA35615. Based on the
DNA35615 consensus sequence, oligonucleotides were synthesized: 1)
to identify by PCR a cDNA library that contained the sequence of
interest, and 2) for use as probes to isolate a clone of the
full-length coding sequence for PRO328.
[1315] Forward and reverse PCR primers were synthesized:
TABLE-US-00095 forward PCR primer 5'-TCCTGCAGMCCTGATGC-3' (SEQ ID
NO:286) reverse PCR primer 5'-CTCATATTGCACACCAGTAATTCG-3' (SEQ ID
NO:287)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed from the consensus DNA35615 sequence which had the
following nucleotide sequence
[1316] hybridization probe TABLE-US-00096
5'-ATGAGGAGAAACGTTTGATGGTGGAGCTGCA (SEQ ID NO:288)
CAACCTCTACCGGG-3'
[1317] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones encoding the PRO328 gene
using the probe oligonucleotide and one of the PCR primers.
[1318] RNA for construction of the cDNA libraries was isolated from
human fetal kidney tissue.
[1319] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO328 [herein designated as
DNA40587-123 I] (SEQ ID NO:284) and the derived protein sequence
for PRO328.
[1320] The entire nucleotide sequence of DNA40587-1231 is shown in
FIG. 99 (SEQ ID NO:284). Clone DNA40587-1231 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 15-17 and ending at the stop codon at
nucleotide positions 1404-1406 (FIG. 99), The predicted polypeptide
precursor is 463 amino acids long (FIG. 100). Clone DNA40587-1231
has been deposited with ATCC and is assigned ATCC deposit no. ATCC
209438.
[1321] Analysis of the amino acid sequence of the full-length
PRO328 polypeptide suggests that portions of it possess significant
homology to the human glioblastoma protein and to the cysteine rich
secretory protein thereby indicating that PRO328 may be a novel
glioblastoma protein or cysteine rich secretory protein.
Example 43
Isolation of cDNA Clones Encoding Human PRO335, PRO331 or
PRO326
[1322] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described in Example 1 above. This
consensus sequence is herein designated DNA36685. Based on the
DNA36685 consensus sequence, and Incyte EST sequence no. 2228990,
oligonucleotides were synthesized: 1) to identify by PCR a cDNA
library that contained the sequence of interest, and 2) for use as
probes to isolate a clone of the full-length coding sequence for
PRO335, PRO331 or PRO326.
[1323] Forward and reverse PCR primers were synthesized for the
determination of PRO335: TABLE-US-00097 forward PCR primer
5'-GGAACCGAATCTCAGCTA-3' (SEQ ID NO:295) forward PCR primer
5'-CCTAAACTGAACTGGACCA-3' (SEQ ID NO:296) forward PCR primer
5'-GGCTGGAGACACTGAACCT-3' (SEQ ID NO:297) forward PCR primer
5'-ACAGCTGCACAGCTCAGAACAGTG-3' (SEQ ID NO:298) reverse PCR primer
5'-CATTCCCAGTATAAAAATTTTC-3' (SEQ ID NO:299) reverse PCR primer
5-GGGTCTTGGTGAATGAGG-3' (SEQ ID NO:300) reverse PCR primer
5'-GTGCCTCTCGGTTACCACCAATGG-3' (SEQ ID NO:301)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed for the determination of PRO335 which had the following
nucleotide sequence
[1324] hybridization probe TABLE-US-00098
5-GCGGCCACTGTTGGACCGAACTGTAACCAAGG (SEQ ID NO:302)
GAGAAACAGCCGTCCTAC-3'
[1325] Forward and reverse PCR primers were synthesized for the
determination of PRO331: TABLE-US-00099 forward PCR primer
5-GCCTTFGACAACCTTCAGTCACTAGTGG-3' (SEQ ID NO:303) reverse PCR
primer 5'-CCCCATGTGTCCATGACTGTTCCC-3' (SEQ ID NO:304)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed for the determination of PRO331 which had the following
nucleotide sequence
[1326] hybridization probe TABLE-US-00100
5'-TACTGCCTCATGACCTCT'TCACTCCCTTGC (SEQ ID NO:305)
ATCATCTTAGAGCGG-3'
[1327] Forward and reverse PCR primers were synthesized for the
determination of PRO326: TABLE-US-00101 forward PCR primer
5'-ACTCCAAGGAAATCGGATCCGTFC-3' (SEQ ID NO:306) reverse PCR primer
5'-TTAGCAGCTGAGGATGGGCACAAC-3' (SEQ ID NO:307)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed for the determination of PRO331 which had the following
nucleotide sequence
[1328] hybridization probe TABLE-US-00102
5'-GCCTTCACTGGTTTGGATGCATTGGAGCATC (SEQ ID NO:308)
TAGACCTGAGTGACAACGC-3'
[1329] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pairs identified above. A
positive library was then used to isolate clones encoding the
PRO335, PRO331 or PRO326 gene using the probe oligonucleotide and
one of the PCR primers.
[1330] RNA for construction of the cDNA libraries was isolated from
human fetal kidney tissue (PRO335 and PRO326) and human fetal brain
(PRO331).
[1331] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO335, PRO331 or PRO326
[herein designated as SEQ ID NOS:289, 291 and 293, respectively;
see FIGS. 101, 103 and 105, respectively], and the derived protein
sequence for PRO335, PRO331 or PRO326 (see FIGS. 102, 104 and 106,
respectively; SEQ ID NOS:290, 292 and 294, respectively).
[1332] The entire nucleotide sequences are shown in FIGS. 101, 103
and 105, deposited with the ATCC on Jun. 2, 1998, Nov. 7, 1997 and
Nov. 21, 1997, respectively.
[1333] Analysis of the amino acid sequence of the full-length
PRO335, PRO331 or PRO326 polypeptide suggests that portions of it
possess significant homology to the LIG-1 protein, thereby
indicating that PRO335, PRO331 and PRO326 may be a novel
LIG-1-related protein.
Example 44
Isolation of cDNA Clones Encoding Human PRO332
[1334] Based upon an ECD homology search performed as described in
Example 1 above, a consensus DNA sequence designated herein as
DNA36688 was assembled. Based on the DNA36688 consensus sequence,
oligonucleotides were synthesized to identify by PCR a cDNA library
that contained the sequence of interest and for use as probes to
isolate a clone of the full-length coding sequence for PRO332.
[1335] A pair of PCR primers (forward and reverse) were
synthesized: TABLE-US-00103 5'-GCATTGGCCGCGAGACTTTGCC-3' (SEQ ID
NO:311) 5'-GCGGCCACGGTCCTTGGAAATG-3' (SEQ ID NO:312)
[1336] A probe was also synthesized: TABLE-US-00104
5'-TGGAGGAGCTCAACCTCAGCTACAACCGCAT (SEQ ID NO:313)
CACCAGCCCACAGG-3'
[1337] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones encoding the PRO332 gene
using the probe oligonucleotide and one of the PCR primers.
[1338] RNA for construction of the cDNA libraries was isolated from
a human fetal liver library (LIB229).
[1339] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for DNA40982-1235 and the derived
protein sequence for PRO332.
[1340] The entire nucleotide sequence of DNA40982-1235 is shown in
FIG. 107 (SEQ ID NO:309). Clone DNA40982-1235 contains a single
open reading frame (with an apparent translational initiation site
at nucleotide positions 342-344, as indicated in FIG. 107). The
predicted polypeptide precursor is 642 amino acids long, and has a
calculated molecular weight of 72,067 (pI: 6.60). Clone
DNA40982-1235 has been deposited with ATCC and is assigned ATCC
deposit no. ATCC 209433.
[1341] Based on a BLAST and FastA sequence alignment analysis of
the full-length sequence, PRO332 shows about 30-40% amino acid
sequence identity with a series of known proteoglycan sequences,
including, for example, fibromodulin and fibromodulin precursor
sequences of various species (FMOD_BOVIN, FMOD_CHICK, FMOD RAT,
FMOD_MOUSE, FMOD_HUMAN, P_R36773), osteomodulin sequences
(AB000114.sub.--1, AB007848-1), decorin sequences
(CFU83141.sub.--1, OCU033941, P_R42266,
P-R42267,P-R42260,P-R89439), keratan sulfate proteoglycans
(BTU48360-1, AF0228901), corneal proteoglycan (AF0222561), and
bone/cartilage proteoglycans and proteoglycane precursors
(PGSI_BOVIN, PGS2_MOUSE, PGS2_HUMAN).
Example 45
Isolation of cDNA Clones Encoding Human PRO334
[1342] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described in Example 1 above. Based on the
consensus sequence, oligonucleotides were synthesized: 1) to
identify by PCR a cDNA library that contained the sequence of
interest and 2) for use as probes to isolate a clone of the
full-length coding sequence for PRO334.
[1343] Forward and reverse PCR primers were synthesized for the
determination of PRO334: TABLE-US-00105 forward PCR primer
5'-GATGGTTCCTGCTCAAGTGCCCTG-3' (SEQ ID NO:316) reverse PCR primer
5'-TTGCACTTGTAGGACCCACGTACG-3' (SEQ ID NO:317)
Additionally, a synthetic oligonucleotide hybridization probe was
constructed for the determination of PRO334 which had the following
nucleotide sequence
[1344] hybridization probe TABLE-US-00106
5'-CTGATGGGAGGACCTGTGTAGATGTTGATGA (SEQ ID NO:318)
ATGTGCTACAGGAAGAGCC-3'
[1345] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones encoding the PRO334 gene
using the probe oligonucleotide and one of the PCR primers.
[1346] Human fetal kidney cDNA libraries used to isolate the cDNA
clones were constructed by standard methods using commercially
available reagents such as those from Invitrogen, San Diego,
Calif.
[1347] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO334 [herein designated as
DNA41379-1236] (SEQ ID NO:314) and the derived protein sequence for
PRO334.
[1348] The entire nucleotide sequence of DNA41379-1236 (also
referred to as UNQ295) is shown in FIG. 109 (SEQ ID NO:314). Clone
DNA41379-1236 contains a single open reading frame with an apparent
translational initiation site at nucleotide positions 203-205 and
ending at the stop codon at nucleotide positions 1730-1732 (FIG.
109). The predicted polypeptide precursor is 509 amino acids long
(FIG. 110). Clone DNA413 79-1236 has been deposited with ATCC and
is assigned ATCC deposit no. ATCC 209488.
[1349] Analysis of the amino acid sequence of the full-length
PRO334 polypeptide suggests that portions of it possess significant
homology to the fibulin and fibrillin proteins, thereby indicating
that PRO334 may be a novel member of the EGF protein family.
Example 46
Isolation of cDNA Clones Encoding Human PRO346
[1350] A consensus DNA sequence was identified using phrap as
described in Example 1 above. Specifically, this consensus sequence
is herein designated DNA38240. Based on the DNA38240 consensus
sequence, oligonucleotides were synthesized: 1) to identify by PCR
a cDNA library that contained the sequence of interest, and 2) for
use as probes to isolate a clone of the full-length PRO346 coding
sequence.
[1351] RNA for construction of the cDNA libraries was isolated from
human fetal liver. The cDNA libraries used to isolated the cDNA
clones were constructed by standard methods using-commercially
available reagents (e.g., Invitrogen, San Diego, Calif.; Clontech,
etc.) The cDNA was primed with oligo dT containing a NotI site,
linked with blunt to Sall 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 Sfil site;
see, Holmes et al., Science, 253:1278-1280 (1991)) in the unique
XhoI and NotI sites.
[1352] A cDNA clone was sequenced in entirety. The entire
nucleotide sequence of DNA44167-1243 is shown in FIG. 111 (SEQ ID
NO:319). Clone DNA44167-1243 contains a single open reading frame
with an apparent translational initiation site at nucleotide
positions 64-66 (FIG. 111; SEQ ID NO:319). The predicted
polypeptide precursor is 450 amino acids long. Clone DNA44167-1243
has been deposited with ATCC and is assigned ATCC deposit no. ATCC
209434 (designation DNA44167-1243).
[1353] Based on a BLAST, BLAST-2 and FastA sequence alignment
analysis (using the ALIGN computer program) of the full-length
sequence, PRO346 shows amino acid sequence identity to
carcinoembryonic antigen (28%).
[1354] The oligonucleotide sequences used in the above procedure
were the following: TABLE-US-00107 OL12691 (38240.fl)
5'-GATCCTGTCACAAAGCCAGTGGTGC-3' (SEQ ID NO:321) OL12693 (38240.rl)
5'-CACTGACAGGGTTCCTCACCCAGG-3' (SEQ ID NO:322) OL12692 (38240.pl)
5'-CTCCCTCTGGGCTGTGGAGTATGTGGGGAAC (SEQ ID NO:323)
ATGACCCTGACATG-3'
Example 47
Isolation of cDNA Clones Encoding Human PRO268
[1355] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described in Example 1 above. This
consensus sequence is herein designated DNA35698. Based on the
DNA35698 consensus sequence, oligonucleotides were synthesized: 1)
to identify by PCR a cDNA library that contained the sequence of
interest, and 2) for use as probes to isolate a clone of the
full-length coding sequence for PRO268.
[1356] Forward and reverse PCR primers were synthesized:
TABLE-US-00108 forward PCR primer 1 5'-TGAGGTGGGCAAGCGGCGAAATG-3'
(SEQ ID NO:326) forward PCR primer 2 5'-TATGTGGATCAGGACGTGCC-3'
(SEQ ID NO:327) forward PCR primer 3 5'-TGCAGGGTTCAGTCTAGATTG-3'
(SEQ ID NO:328) reverse PCR primer 5-TTGAAGGACAAAGGCAATCTGCCAC-3'
(SEQ ID NO:329)
[1357] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus DNA35698 sequence which
had the following nucleotide sequence TABLE-US-00109 hybridization
probe 5'-GGAGTCTTGCAGTTCCCCTGGCAGTCCTGGT (SEQ ID NO:330)
GCTGTTGCTTTGGG-3'
[1358] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones encoding the PRO268 gene
using the probe oligonucleotide and one of the PCR primers.
[1359] RNA for construction of the cDNA libraries was isolated from
human fetal lung tissue.
[1360] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO268 [herein designated as
DNA39427-1179] (SEQ ID NO:324) and the derived protein sequence for
PRO268.
[1361] The entire nucleotide sequence of DNA39427-1179 is shown in
FIG. 113 (SEQ ID NO:324). Clone DNA39427-1179 contains a single
open reading frame with an apparent translational initiation site
at nucleotide positions 13-15 and ending at the stop codon at
nucleotide positions 853-855 (FIG. 113). The predicted polypeptide
precursor is 280 amino acids long (FIG. 114). Clone DNA39427-1179
has been deposited with ATCC and is assigned ATCC deposit no. ATCC
209395.
[1362] Analysis of the amino acid sequence of the full-length
PRO268 polypeptide suggests that it possess significant homology to
protein disulfide isomerase, thereby indicating that PRO268 may be
a novel protein disulfide isomerase.
Example 48
Isolation of cDNA Clones Encoding Human PRO330
[1363] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described in Example 1 above. This
consensus sequence is herein designated DNA35730. Based on the
DNA35730 consensus sequence, oligonucleotides were synthesized: 1)
to identify by PCR a cDNA library that contained the sequence of
interest, and 2) for use as probes to isolate a clone of the
full-length coding sequence for PRO330.
[1364] Forward and reverse PCR primers were synthesized:
TABLE-US-00110 forward PCR primer 1 5'-CCAGGCACAATTTCCAGA-3' (SEQ
ID NO:333) forward PCR primer 2 5'-GGACCCTTCTGTGTGCCAG-3' (SEQ ID
NO:334) reverse PCR primer 1 5'-GGTCTCAAGAACTCCTGTC-3' (SEQ ID
NO:335) reverse PCR primer 2 5'-ACACTCAGCATTGCCTGGTACTTG-3' (SEQ ID
NO:336)
[1365] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus sequence which had the
following nucleotide sequence TABLE-US-00111 hybridization probe
5'-GGGCACATGACTGACCTGATTTATGCAGA (SEQ ID NO:337)
GAAAGAGCTGGTGCAG-3'
[1366] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones encoding the PRO330 gene
using the probe oligonucleotide and one of the PCR primers.
[1367] RNA for construction of the cDNA libraries was isolated from
human fetal liver tissue.
[1368] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO330 [herein designated as
DNA40603-1232] (SEQ ID NO:331) and the derived protein sequence for
PRO330.
[1369] The entire nucleotide sequence of DNA40603-1232 is shown in
FIG. 115 (SEQ ID NO:331). Clone DNA40603-1232 contains a single
open reading frame with an apparent translational initiation site
at nucleotide positions 167-169 and ending at the stop codon at
nucleotide positions 1766-1768 (FIG. 115). The predicted
polypeptide precursor is 533 amino acids long (FIG. 116). Clone
DNA40603-1232 has been deposited with ATCC and is assigned ATCC
deposit no. ATCC 209486 on Nov. 21, 1997.
[1370] Analysis of the amino acid sequence of the full-length
PRO330 polypeptide suggests that portions of it possess significant
homology to the mouse prolyl 4-hydroxylase alpha subunit protein,
thereby indicating that PRO330 may be a novel prolyl 4-hydroxylase
alpha subunit polypeptide.
Example 49
Isolation of cDNA Clones Encoding Human PRO310
[1371] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described in Example 1 above. This
consensus sequence is herein designated DNA40553. Based on the
DNA40553 consensus sequence, oligonucleotides were synthesized: 1)
to identify by PCR a cDNA library that contained the sequence of
interest, and 2) for use as probes to isolate a clone of the
full-length coding sequence for PRO310.
[1372] Forward and reverse PCR primers were synthesized:
TABLE-US-00112 forward PCR primer 1 5'-TCCCCAAGCCGTTCTAGACGCGG-3'
(SEQ ID NO:342) forward PCR primer 2 5'-CTGGTTCTTCCTTGCACG-3' (SEQ
ID NO:343) reverse PCR primer 5'-GCCCAAATGCCCTAAGGCGGTATACCCC-3'
(SEQ ID NO:344)
[1373] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus sequence which had the
following nucleotide sequence TABLE-US-00113 hybridization probe
5'-GGGTGTGATGCTTGGAAGCATTTTCTGTGCT (SEQ ID NO:345)
TTGATCACTATGCTAGGAC-3'
[1374] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones encoding the PRO310 gene
using the probe oligonucleotide and one of the PCR primers.
[1375] RNA for construction of the cDNA libraries was isolated from
human fetal liver tissue.
[1376] DNA sequencing of the clones isolated as described above
gave the full-length DNA sequence for PRO310 [herein designated as
DNA43046-1225 (SEQ ID NO:340) and the derived protein sequence for
PRO310 (SEQ ID NO:341).
[1377] The entire nucleotide sequence of DNA43046-1225 is shown in
FIG. 119 (SEQ ID NO:340). Clone DNA43046-1225 contains a single
open reading frame with an apparent translational initiation site
at nucleotide positions 81-83 and ending at the stop codon at
nucleotide positions 1035-1037 (FIG. 119). The predicted
polypeptide precursor is 318 amino acids long (FIG. 120) and has a
calculated molecular weight of approximately 36,382 daltons. Clone
DNA43046-1225 has been deposited with ATCC and is assigned ATCC
deposit no. ATCC 209484.
[1378] Analysis of the amino acid sequence of the full-length
PRO310 polypeptide suggests that portions of it possess homology to
C. elegans proteins and to fringe, thereby indicating that PRO310
may be involved in development.
Example 50
Isolation of cDNA Clones Encoding Human PRO339
[1379] An expressed sequence tag (EST) DNA database (LIFESEQ.TM.,
Incyte Pharmaceuticals, Palo Alto, Calif.) was searched and ESTs
were identified. An assembly of Incyte clones and a consensus
sequence was formed using phrap as described in Example 1
above.
[1380] Forward and reverse PCR primers were synthesized based upon
the assembly-created consensus sequence: TABLE-US-00114 forward PCR
primer 1 5'-GGGATGCAGGTGGTGTCTCATGGGG-3' (SEQ ID NO:346) forward
PCR primer 2 5'-CCCTCATGTACCGGCTCC-3' (SEQ ID NO:347) forward PCR
primer 3 5'-GTGTGACACAGCGTGGGC-3' (SEQ ID NO:43) forward PCR primer
4 5'-GACCGGCAGGCTTCTGCG-3' (SEQ ID NO:44) reverse PCR primer 1
5'-CAGCAGCTTCAGCCACCAGGAGTGG-3' (SEQ ID NO:45) reverse PCR primer 2
5'-CTGAGCCGTGGGCTGCAGTCTCGC-3' (SEQ ID NO:46)
[1381] Additionally, a synthetic oligonucleotide hybridization
probe was constructed from the consensus sequence which had the
following nucleotide sequence TABLE-US-00115 hybridization probe
5'-CCGACTACGACTGGTTCTTCATCATGCAGGA (SEQ ID NO:47)
TGACACATATGTGC-3'
[1382] In order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pairs identified above. A
positive library was then used to isolate clones encoding the
PRO339 gene using the probe oligonucleotide and one of the PCR
primers.
[1383] RNA for construction of the cDNA libraries was isolated from
human fetal liver tissue.
[1384] A cDNA clone was sequenced in entirety. The entire
nucleotide sequence of DNA43466-1225 is shown in FIG. 117 (SEQ ID
NO:338). Clone DNA43466-1225 contains a single open reading frame
with an apparent translational initiation site at nucleotide
positions 333-335 and ending at the stop codon found at nucleotide
positions 2649-2651 (FIG. 117; SEQ ID NO:338). The predicted
polypeptide precursor is 772 amino acids long and has a calculated
molecular weight of approximately 86,226 daltons. Clone
DNA43466-1225 has been deposited with ATCC and is assigned ATCC
deposit no. ATCC 209490.
[1385] Based on a BLAST and FastA sequence alignment analysis
(using the ALIGN computer program) of the full-length sequence,
PRO339 has homology to C. elegans proteins and collagen-like
polymer sequences as well as to fringe, thereby indicating that
PRO339 may be involved in development or tissue growth.
Example 51
Isolation of cDNA Clones Encoding Human PRO244
[1386] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described in Example 1 above. Based on
this consensus sequence, oligonucleotides were synthesized to
identify by PCR a cDNA library that contained the sequence of
interest and for use as probes to isolate a clone of the
full-length coding sequence for PRO244.
[1387] A pair of PCR primers (forward and reverse) were
synthesized: TABLE-US-00116 (30923.f1) 5'-TTCAGCTTCTGGGATGTAGGG-3'
(SEQ ID NO:378) (30923.r1) 5'-TATTCCTACCATTTCACAAATCCG-3' (SEQ ID
NO:379)
[1388] A probe was also synthesized: TABLE-US-00117 (30923.p1)
5'-GGAGGACTGTGCCACCATGAGAGACTCTTCA (SEQ ID NO:380)
AACCCAAGGCAAAATTGG-3'
[1389] in order to screen several libraries for a source of a
full-length clone, DNA from the libraries was screened by PCR
amplification with the PCR primer pair identified above. A positive
library was then used to isolate clones encoding the PRO244 gene
using the probe oligonucleotide and one of the PCR primers.
[1390] RNA for construction of the cDNA libraries was isolated from
a human fetal kidney library. DNA sequencing of the clones isolated
as described above gave the full-length DNA sequence and the
derived protein sequence for PRO244.
[1391] The entire nucleotide sequence of PRO244 is shown in FIG.
121 (SEQ ID NO:376). Clone DNA35668-1171 contains a single open
reading frame with an apparent translational initiation site at
nucleotide positions 106108 (FIG. 121). The predicted polypeptide
precursor is 219 amino acids long. Clone DNA35668-1171 has been
deposited with ATCC (designated as DNA35663-1171) and is assigned
ATCC deposit no. ATCC209371. The protein has a cytoplasmic domain
(aa 1-20), a transmembrane domain (aa 21-46), and an extracellular
domain (aa 47-219), with a C-lectin domain at aa 55-206.
[1392] Based on a BLAST and FastA sequence alignment analysis of
the full-length sequence, PRO244 shows notable amino acid sequence
identity to hepatic lectin gallus gallus (43%), HIC hp120-binding
C-type lectin (42%), macrophage lectin 2 (HUMHML2-1, 41%), and
sequence PR32188 (44%).
Example 52
Use of PRO Polypeptide-Encoding Nucleic Acid as Hybridization
Probes
[1393] The following method describes use of a nucleotide sequence
encoding a PRO polypeptide as a hybridization probe.
[1394] DNA comprising the coding sequence of a PRO polypeptide of
interest as disclosed herein may be employed as a probe or used as
a basis from which to prepare probes to screen for homologous DNAs
(such as those encoding naturally-occurring variants of the PRO
polypeptide) in human tissue cDNA libraries or human tissue genomic
libraries.
[1395] Hybridization and washing of filters containing either
library DNAs is performed under the following high stringency
conditions. Hybridization of radiolabeled PRO polypeptide-encoding
nucleic; acid-derived probe to the filters is performed in a
solution of 50% formamide, 5.times.SSC, 0.1% SDS, 0.1% sodium
pyrophosphate, 50 mM sodium phosphate, pH 6.8, 2.times. Denhardt's
solution, and 10% dextran sulfate at 42.degree. C. for 20 hours.
Washing of the filters is performed in an aqueous solution of
0.1.times.SSC and 0.1% SDS at 42.degree. C.
[1396] DNAs having a desired sequence identity with the DNA
encoding full-length native sequence PRO polypeptide can then be
identified using standard techniques known in the art.
Example 53
Expression of PRO Polypeptides in E. coli
[1397] This example illustrates preparation of an unglycosylated
form of a desired PRO polypeptide by recombinant expression in E.
coli.
[1398] The DNA sequence encoding the desired PRO polypeptide is
initially amplified using selected PCR primers. The primers should
contain restriction enzyme sites which correspond to the
restriction enzyme sites on the selected expression vector. A
variety of expression vectors may be employed. An example of a
suitable vector is pBR322 (derived from E. coli; see Bolivar et
al., Gene, 2:95 (1977)) which contains genes for ampicillin and
tetracycline resistance. The vector is digested with restriction
enzyme and dephosphorylated. The PCR amplified sequences are then
ligated into the vector. The vector will preferably include
sequences which encode for an antibiotic resistance gene, a trp
promoter, a polyhis leader (including the first six STII codons,
polyhis sequence, and enterokinase cleavage site), the specific PRO
polypeptide coding region, lambda transcriptional terminator, and
an argU gene.
[1399] The ligation mixture is then used to transform a selected E.
coli strain using the methods described in Sambrook et al., supra.
Transformants are identified by their ability to grow on LB plates
and antibiotic resistant colonies are then selected. Plasmid DNA
can be isolated and confirmed by restriction analysis and DNA
sequencing.
[1400] 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.
[1401] After culturing the cells for several more hours, the cells
can be harvested by centrifugation. The cell pellet obtained by the
centrifugation can be solubilized using various agents known in the
art, and the solubilized PRO polypeptide can then be purified using
a metal chelating column under conditions that allow tight binding
of the protein.
[1402] PRO187, PRO317, PRO301, PRO224 and PRO238 were successfully
expressed in E. coli in a poly-His tagged form, using the following
procedure. The DNA encoding PRO187, PRO317, PRO301, PRO224 or
PRO238 was initially amplified using selected PCR primers. The
primers contained restriction enzyme sites which correspond to the
restriction enzyme sites on the selected expression vector, and
other useful sequences providing for efficient and reliable
translation initiation, rapid purification on a metal chelation
column, and proteolytic removal with enterokinase. The
PCR-amplified, poly-His tagged sequences were then ligated into an
expression vector, which was used to transform an E. coli host
based on strain 52 (W3110 fuhA(tonA) Ion galE rpoHts(htpRts)
clpP(lacily). 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 2H20, 1.07 g KCI, 5.36 g Difco yeast extract,
5.36 g Sheffield hycas 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.
[1403] 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 0.1 M 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 Ultracentrifuge for 30 min. The supernatant was diluted
with 3-5 volumes of metal chelate column buffer (6 M guanidine, 20
mM Tris, pH 7.4) and filtered through 0.22 micron filters to
clarify. Depending the clarified extract was loaded onto a 5 ml
Qiagen Ni--NTA metal chelate column equilibrated in the metal
chelate column buffer. The column was washed with additional buffer
containing 50 mM imidazole (Calbiochem, Utrol grade), pH 7.4. The
protein was eluted with buffer containing 250 mM imidazole.
Fractions containing the desired protein were pooled and stored at
4.degree. C. Protein concentration was estimated by its absorbance
at 280 nm using the calculated extinction coefficient based on its
amino acid sequence.
[1404] The proteins were refolded by diluting sample slowly into
freshly prepared refolding buffer consisting of: 20 mM Tris, pH
8.6, 0.3 M NaCl, 2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM
EDTA. Refolding volumes were chosen so that the final protein
concentration was between 50 to 100 micrograms/ml. The refolding
solution was stirred gently at 4.degree. C. for 12-36 hours. The
refolding reaction was quenched by the addition of TFA to a final
concentration of 0.4% (pH of approximately 3). Before further
purification of the protein, the solution was filtered through a
0.22 micron filter and acetonitrile was added to 2-10% final
concentration. The refolded protein was chromatographed on a Poros
R1/H reversed phase column using a mobile buffer of 0.1% TFA with
elution with a gradient of acetonitrile from 10 to 80%. Aliquots of
fractions with A280 absorbance were analyzed on SDS polyacrylamide
gets 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.
[1405] Fractions containing the desired folded PRO187, PRO317,
PRO301, PRO224 and PRO238 proteins, respectively, were pooled and
the acetonitrile removed using a gentle stream of nitrogen directed
at the solution. Proteins were formulated into 20 mM Hepes, pH 6.8
with 0.14 M sodium chloride and 4% mannitol by dialysis or by gel
filtration using G25 Superfine (Pharmacia) resins equilibrated in
the formulation buffer and sterile filtered.
Example 54
Expression of PRO Polypeptides in Mammalian Cells
[1406] This example illustrates preparation of a glycosylated form
of a desired PRO polypeptide by recombinant expression in mammalian
cells.
[1407] The vector, pRK5 (see EP 307,247, published Mar. 15, 1989),
is employed as the expression vector. Optionally, the PRO
polypeptide-encoding DNA is Heated into pRK5 with selected
restriction enzymes to allow insertion of the PRO polypeptide DNA
using ligation methods such as described in Sambrook et al., The
resulting vector is called pRK5-PRO polypeptide.
[1408] 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-HCI, 0.1 mM EDTA, 0.227 M
CaCl.sub.2. To this mixture is added, dropwise, 500 .mu.l of 50 mM
HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO.sub.4, and a precipitate
is allowed to form for 10 minutes at 25.degree. C. The precipitate
is suspended and added to the 293 cells and allowed to settle for
about four hours at 37.degree. C. The culture medium is aspirated
off and 2 ml of 20% glycerol in PBS is added for 30 seconds. The
293 cells are then washed with serum free medium, fresh medium is
added and the cells are incubated for about 5 days.
[1409] Approximately 24 hours after the transfections, the culture
medium is removed and replaced with culture medium (alone) or
culture medium containing 200 uCi/ml .sup.35S-cysteine and 200
uCi/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.
[1410] In an alternative technique, PRO polypeptide may be
introduced into 293 cells transiently using the dextran sulfate
method described by Somparyrac et al., Proc. Natl. Acad. Sci.
12:7575 (1981). 293 cells are grown to maximal density in a spinner
flask and -700 .mu.g pRK5-PRO polypeptide DNA is added. The cells
are first concentrated from the spinner flask by centrifugation and
washed with PBS. The DNA-dextran precipitate is incubated on the
cell pellet for four hours. The cells are treated with 20% glycerol
for 90 seconds, washed with tissue culture medium, and
re-introduced into the spinner flask containing tissue culture
medium, 5 .mu.g/ml bovine insulin and 0.1 .mu.g/ml bovine
transferrin. After about four days, the conditioned media is
centrifuged and filtered to remove cells and debris. The sample
containing expressed PRO polypeptide can then be concentrated and
purified by any selected method, such as dialysis and/or column
chromatography.
[1411] In another embodiment, PRO polypeptides; can be expressed in
CHO cells. The pRKS-PRO polypeptide can be transfected into CHO
cells using known reagents such as CaPO.sub.4 or DEAE-dextran. As
described above, the cell cultures can be incubated, and the medium
replaced with culture medium (alone) or medium containing a
radiolabel such as .sup.35S-methionine. After determining the
presence of PRO polypeptide, the culture medium may be replaced
with serum free medium. Preferably, the cultures are incubated for
about 6 days, and then the conditioned medium is harvested. The
medium containing the expressed PRO polypeptide can then be
concentrated and purified by any selected method.
[1412] Epitope-tagged PRO polypeptide may also be expressed in host
CHO cells. The PRO polypeptide may be subcloned out of the pRK5
vector. The subclone insert can undergo PCR to fuse in frame with a
selected epitope such as a poly-his tag into a Baculovirus
expression vector. The poly-his tagged PRO polypeptide insert can
then be subcloned into a SV40 driven vector containing a selection
marker such as DHFR for selection of stable clones. Finally, the
CHO cells can be transfected (as described above) with the SV40
driven vector. Labeling may be performed, as described above, to
verify expression. The culture medium containing the expressed
poly-His tagged PRO polypeptide can then be concentrated and
purified by any selected method, such as by Ni.sup.2+-chelate
affinity chromatography.
[1413] PRO211, PRO217, PRO230, PRO219, PRO245, PRO221, PRO258,
PRO301, PRO224, PRO222, PRO234, PRO229, PRO223, PRO328 and PRO332
were successfully expressed in CHO cells by both a transient and a
stable expression procedure. In addition, PRO232, PRO265, PRO246,
PRO228, PRO227, PRO220, PRO266, PRO269, PRO287, PRO214, PRO231,
PRO233, PRO238, PRO244, PRO235, PRO236, PRO262, PRO239, PRO257,
PRO260, PRO263, PRO270, PRO271, PRO272, PRO294, PRO295, PRO293,
PRO247, PRO303 and PRO268 were successfully transiently expressed
in CHO cells.
[1414] 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 IgG 1 constant region sequence containing
the hinge, CH2 and CH2 domains and/or is a poly-His tagged
form.
[1415] Following PCR amplification, the respective DNAs were
subcloned in a CHO expression vector using standard techniques as
described in Ausubel et al., Current Protocols of Molecular
Biology, Unit 3.16, John Wiley and Sons (1997). CHO expression
vectors are constructed to have compatible restriction sites 5' and
3' of the DNA of interest to allow the convenient shuttling of
cDNA's. The vector used expression in CHO cells is as described in
Lucas et al., Nucl. Acids Res. 24: 9 (1774-1779 (1996), and uses
the SV40 early promoter/enhancer to drive expression of the cDNA of
interest and dihydrofolate reductase (DHFR). DHFR expression
permits selection for stable maintenance of the plasmid following
transfection.
[1416] Twelve micrograms of the desired plasmid DNA were introduced
into approximately 10 million CHO cells using commercially
available transfection reagents Superfect.RTM. (Qiagen),
Dosper.RTM. or Fugene.RTM. (Boehringer Mannheim). The cells were
grown and described in Lucas et al., supra. Approximately
3.times.10-7 cells are frozen in an ampule for further growth and
production as described below.
[1417] The ampules containing the plasmid DNA were thawed by
placement into water bath and mixed by vortexing. The contents were
pipetted into a centrifuge tube containing 10 mLs of media and
centrifuged at 1000 rpm for 5 minutes. The supernatant was
aspirated and the cells were resuspended in 10 mL of selective
media (0.2 .mu.m filtered PS20 with 5% 0.2 .mu.m diafiltered fetal
bovine serum). The cells were then aliquoted into a 100 mL spinner
containing 90 mL of selective media. After 1-2 days, the cells were
transferred into a 250 mL spinner filled with 150 mL selective
growth medium and incubated at 37.degree. C. After another 2-3
days, a 250 mL, 500 mL and 2000 mL, spinners were seeded with
3.times.10.sup.5 cells/mL. The cell media was exchanged with fresh
media by centrifugation and resuspension in production medium.
Although any suitable CHO media may be employed, a production
medium described in U.S. Pat. No. 5,122,469, issued Jun. 16, 1992
was actually used. 3L production spinner is seeded at
1.2.times.10.sup.6 cells/mL. On day 0, the cell number pH were
determined. On day 1, the spinner was sampled and sparging with
filtered air was commenced. On day 2, the spinner was sampled, the
temperature shifted to 33.degree. C., and 30 mL of 500. g/L glucose
and 0.6 mL of 10% antifoam (e.g. 35% polydimethylsiloxane emulsion,
Dow Coming 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 centrifugation and filtering through a 0.22 .mu.m
filter. The filtrate was either stored at 4.degree. C. or
immediately loaded onto columns for purification.
[1418] For the poly-His tagged constructs, the proteins were
purified using a Ni--NTA column (Qiagen). Before purification,
imidazole was added to the conditioned media to a concentration of
5 mM. The conditioned media was pumped onto a 6 ml Ni--NTA column
equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl
and 5 mM imidazole at a flow rate of 4-5 ml/min. at 4.degree. C.
After loading, the column was washed with additional equilibration
buffer and the protein eluted with equilibration buffer containing
0.25 M imidazole. The highly purified protein was subsequently
desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl
and 4% mannitol, pH 6.8, with a 25 mL G25 Superfine (Pharmacia)
column and stored at -80.degree. C.
[1419] 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.
[1420] PRO211, PRO217, PRO230, PRO232, PRO187, PRO265, PRO219,
PRO246, PRO228, PRO533, PRO245, PRO221, PRO227, PRO220, PRO258,
PRO266, PRO269, PRO287, PRO214, PRO317, PRO301, PRO224, PRO222,
PRO234, PRO231, PRO229, PRO233, PRO238, PRO223, PRO235, PRO236,
PRO262, PRO239, PRO257, PRO260, PRO263, PRO270, PRO271, PRO272,
PRO294, PRO295, PRO293, PRO247, PRO304, PRO302, PRO307, PRO303,
PRO343, PRO328, PRO326, PRO331, PRO332, PRO334, PRO346, PRO268,
PRO330, PRO310 and PRO339 were also successfully transiently
expressed in COS cells.
Example 55
Expression of PRO Polypeptides in Yeast
[1421] The following method describes recombinant expression of a
desired PRO polypeptide in yeast.
[1422] First, yeast expression vectors are constructed for
intracellular production or secretion of PRO polypeptides from the
ADH2/GAPDH promoter. DNA encoding a desired PRO polypeptide, a
selected signal peptide and the promoter is inserted into suitable
restriction enzyme sites in the selected plasmid to direct
intracellular expression of the PRO polypeptide. For secretion, DNA
encoding the PRO polypeptide can be cloned into the selected
plasmid, together with DNA encoding the ADH2/GAPDH promoter, the
yeast alpha-factor secretory signal/leader sequence, and linker
sequences (if needed) for expression of the PRO polypeptide.
[1423] Yeast cells, such as yeast strain AB110, can then be
transformed with the expression plasmids described above and
cultured in selected fermentation media. The transformed yeast
supernatants can be analyzed by precipitation with 10%
trichloroacetic acid and separation by SDS-PAGE, followed by
staining of the gels with Coomassie Blue stain.
[1424] Recombinant PRO polypeptide can subsequently be isolated and
purified by removing the yeast cells from the fermentation medium
by centrifugation and then concentrating the medium using selected
cartridge filters. The concentrate containing the PRO polypeptide
may further be purified using selected column chromatography
resins.
Example 56
Expression of PRO Polypeptides in Baculovirus-Infected Insect
Cells
[1425] The following method describes recombinant expression of PRO
polypeptides in Baculovirus-infected insect cells.
[1426] 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 pVL 1393
(Novagen). Briefly, the PRO polypeptide or the desired portion of
the PRO polypeptide (such as the sequence encoding the
extracellular domain of a transmembrane protein) is amplified by
PCR with primers complementary to the 5' and 3' regions. The 5'
primer may incorporate flanking (selected) restriction enzyme
sites. The product is then digested with those selected restriction
enzymes and subcloned into the expression vector.
[1427] Recombinant baculovirus is generated by co-transfecting the
above plasmid and BaculoGold.TM. virus DNA (Pharmingen) into
Spodopterafrugiperda ("Sf9") cells (ATCC CRL 1711) using lipofectin
(commercially available from GIBCO-BRL). After 4-5 days of
incubation at 28.degree. C., the released viruses are harvested and
used for further amplifications. Viral infection and protein
expression is performed as described by O'Reilley et al.,
Baculovirus expression vectors: A laboratory Manual, Oxford: Oxford
University Press (1994).
[1428] 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% NP-40; 0.4 M KCl), and sonicated twice for 20 seconds on ice.
The sonicates, are cleared by centrifugation, and the supernatant
is diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl,
10% Glycerol, pH 7.8) and filtered through a 0.45 .mu.m filter. A
Ni.sup.2+-NTA agarose column (commercially available from Qiagen)
is prepared with a bed volume of 5 mL, washed with 25 mL of water
and equilibrated with 25 mL of loading buffer. The filtered cell
extract is loaded onto the column at 0.5 ML per minute. The column
is washed to baseline A.sub.280 with loading buffer, at which point
fraction collection is started. Next, the column is washed with a
secondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% Glycerol,
pH 6.0), which elutes nonspecifically bound protein. After reaching
A.sub.280 baseline again, the column is developed with a 0 to 500
mM Imidazole gradient in the secondary wash buffer. One mL
fractions are collected and analyzed by SDS-PAGE and silver
staining or western blot with Ni.sup.2+-NTA-conjugated to alkaline
phosphatase (Qiagen). Fractions containing the eluted
His.sub.10-tagged PRO polypeptide are pooled and dialyzed against
loading buffer.
[1429] 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.
[1430] PRO211, PRO217, PRO230, PRO187, PRO165, PRO246, PRO228,
PRO533, PRO245, PRO221, PRO220, PRO258, PRO266, PRO269, PRO287,
PRO214, PRO301, PRO224, PRO222, PRO234, PRO231, PRO229, PRO235,
PRO239, PRO257, PRO257, PRO294, PRO295, PRO328, PRO326, PRO331,
PRO334, PRO346 and PRO310 were successfully expressed in
baculovirus infected Sf9 or high5 insect cells. While the
expression was actually performed in a 0.5-2 L scale, it can be
readily scaled up for larger (e.g. 8 L) preparations. The proteins
were expressed as an IgG construct (immunoadhesin), in which the
protein extracellular region was fused to an IgG1 constant region
sequence containing the hinge, CH2 and CH3 domains and/or in
poly-His tagged forms.
[1431] Following PCR amplification, the respective coding sequences
were subcloned into a baculovirus expression vector (pb.PH.IgG for
IgG fusions and pb.PH.His.c for poly-His tagged proteins), and the
vector and Baculogold.RTM. baculovirus DNA (Pharmingen) were
co-transfected into 105 Spodoptera frugiperda ("Sf9") cells (ATCC
CRL 1711), using Lipofectin (Gibco BRL). pb.PH.IgG and pb.PH.His
are modifications of the commercially available baculovirus
expression vector pVL1393 (Pharmingen), with modified polylinker
regions to include the His or Fc tag sequences. The cells were
grown in Hink's TNM-FH medium supplemented with 10% FBS (Hyclone).
Cells were incubated for 5 days at 28.degree. C. The supernatant
was harvested and subsequently used for the first viral
amplification by infecting Sf9 cells in Hink's TNM-FH medium
supplemented with 10% FBS at an approximate multiplicity of
infection (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 CL-4B
beads (Pharmacia) for IgG tagged proteins followed by SDS-PAGE
analysis comparing to a known concentration of protein standard by
Coomassie blue staining.
[1432] 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.
[1433] The conditioned medium from the transfected cells (0.5 to 3
L) was harvested by centrifugation to remove the cells and filtered
through 0.22 micron filters. For the poly-His tagged constructs,
the protein construct were purified using a Ni--NTA column
(Qiagen). Before purification, imidazole was added to the
conditioned media to a concentration of 5 mM. The conditioned media
were pumped onto a 6 ml Ni--NTA column equilibrated in 20 mM Hepes,
pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow
rate of 4-5 ml/min. at 4.degree. C. After loading, the column was
washed with additional equilibration buffer and the protein eluted
with equilibration buffer containing 0.25 M imidazole. The highly
purified protein was subsequently desalted into a storage buffer
containing 10 mM Hepes, 0.14 M NaCt and 4% mannitol, pH 6.8, with a
25 ml G25 Superfine (Pharmacia) column and stored at -80.degree.
C.
[1434] Immunoadhesin (Fc containing) constructs of proteins were
purified from the conditioned media as follows. The conditioned
media were pumped onto a 5 ml Protein A column (Pharmacia) which
had been equilibrated in 20 mM Na phosphate buffer, pH 6.8. After
loading, the column was washed extensively with equilibration
buffer before elution with 100 mM citric acid, pH 3.5. The eluted
protein was immediately neutralized by collecting 1 ml fractions
into tubes containing 275 mL of 1 M Tris buffer, pH 9. The highly
purified protein was subsequently desalted into storage buffer as
described above for the poly-His tagged proteins. The homogeneity
of the proteins was verified by SDS polyacrylamide gel (PEG)
electrophoresis and N-terminal amino acid sequencing by Edman
degradation.
Example 57
Preparation of Antibodies that Bind to PRO Polypeptides
[1435] This example illustrates preparation of monoclonal
antibodies which can specifically bind to a PRO polypeptide.
[1436] Techniques for producing the monoclonal antibodies are known
in the art and are described, for instance, in Goding, supra.
Immunogens that may be employed include purified PRO polypeptide,
fusion proteins containing the PRO polypeptide, and cells
expressing recombinant PRO polypeptide on the cell surface.
Selection of the immunogen can be made by the skilled artisan
without undue experimentation.
[1437] Mice, such as Balb/c, are immunized with the PRO polypeptide
immunogen emulsified in complete Freund's adjuvant and injected
subcutaneously or intraperitoneally in an amount from 1-100
micrograms. Alternatively, the immunogen is emulsified in MPL-TDM
adjuvant (Ribi Immunochemical Research, Hamilton, Mont.) and
injected into the animal's hind foot pads. The immunized mice are
then boosted 10 to 12 days later with additional immunogen
emulsified in the selected adjuvant. Thereafter, for several weeks,
the mice may also be boosted with additional immunization
injections. Serum samples may be periodically obtained from the
mice by retro-orbital bleeding for testing in ELISA assays to
detect anti-PRO polypeptide antibodies.
[1438] 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 thymidine) medium to inhibit
proliferation of non-fused cells, myeloma hybrids, and spleen cell
hybrids.
[1439] 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.
[1440] The positive hybridoma cells can be injected
Intraperitoneally into syngeneic Balb/c mice to produce ascites
containing the anti-PRO polypeptide monoclonal antibodies.
Alternatively, the hybridoma cells can be grown in tissue culture
flasks or roller bottles. Purification of the monoclonal antibodies
produced in the ascites can be accomplished using ammonium sulfate
precipitation, followed by gel exclusion chromatography.
Alternatively, affinity chromatography based upon binding of
antibody to protein A or protein G can be employed.
Example 58
Chimeric PRO Polypeptides
[1441] PRO polypeptides may be expressed as chimeric proteins with
one or more additional polypeptide domains added to facilitate
protein purification. Such purification facilitating domains
include, but are not limited to, metal chelating peptides such as
histidine-tryptophan modules that allow purification on immobilized
metals, protein A domains that allow purification on immobilized
immunoglobulin, and the domain utilized in the FLAGS.TM.
extension/affinity purification system (Immunex. Corp., Seattle
Wash.). The inclusion of a cleavable linker sequence such as Factor
XA or enterokinase (Invitrogen, San Diego Calif.) between the
purification domain and the PRO polypeptide sequence may be useful
to facilitate expression of DNA encoding the PRO polypeptide.
Example 59
Purification of PRO Polypeptides Using Specific Antibodies
[1442] 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.
[1443] Polyclonal immunoglobulins are prepared from immune sera
either by precipitation with ammonium sulfate or by purification on
immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway,
N.J.). Likewise, monoclonal antibodies are prepared from mouse
ascites fluid by ammonium sulfate precipitation or chromatography
on immobilized Protein A. Partially purified immunoglobulin is
covalently attached to a chromatographic resin such as
CnBr-activated SEPHAROSE.TM. (Pharmacia LKB Biotechnology). The
antibody is coupled to the resin, the resin is blocked, and the
derivative resin is washed according to the manufacturer's
instructions.
[1444] 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.
[1445] A soluble PRO polypeptide-containing preparation is passed
over the immunoaffinity column, and the column is washed under
conditions that allow the preferential absorbance of PRO
polypeptide (e.g., high ionic strength buffers in the presence of
detergent). Then, the column is eluted under conditions that
disrupt antibody/PRO polypeptide binding (e.g., a low pH buffer
such as approximately pH 2-3, or a high concentration of a
chaotrope such as urea or thiocyanate ion), and PRO polypeptide is
collected.
Example 60
Drug Screening
[1446] This invention is particularly useful for screening
compounds by using PRO polypeptides or binding fragment thereof in
any of a variety of drug screening techniques. The PRO polypeptide
or fragment employed in such a test may either be free in solution,
affixed to a solid support, borne on a cell surface, or located
intracellularly. One method of drug screening utilizes eukaryotic
or prokaryotic host cells which are stably transformed with
recombinant nucleic acids expressing the PRO polypeptide or
fragment. Drugs are screened against such transformed cells in
competitive binding assays. Such cells, either in viable or fixed
form, can be used for standard binding assays. One may measure, for
example, the formation of complexes between PRO polypeptide or a
fragment and the agent being tested. Alternatively, one can examine
the diminution in complex formation between the PRO polypeptide and
its target cell or target receptors caused by the agent being
tested.
[1447] 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.
[1448] Another technique for drug screening provides high
throughput screening for compounds having suitable binding affinity
to a polypeptide and is described in detail in WO 84/03564,
published on Sep. 13, 1984. Briefly stated, large numbers of
different small peptide test compounds are synthesized on a solid
substrate, such as plastic pins or some other surface. As applied
to a PRO polypeptide, the peptide test compounds are reacted with
PRO polypeptide and washed. Bound PRO polypeptide is detected by
methods well known in the art. Purified PRO polypeptide can also be
coated directly onto plates for use in the aforementioned drug
screening techniques. In addition, non-neutralizing antibodies can
be used to capture the peptide and immobilize it on the solid
support.
[1449] This invention also contemplates the use of competitive drug
screening assays in which neutralizing antibodies capable of
binding PRO polypeptide specifically compete with a test compound
for binding to PRO polypeptide or fragments thereof. In this
manner, the antibodies can be used to detect the presence of any
peptide which shares one or more antigenic determinants with PRO
polypeptide.
Example 61
Rational Drug Design
[1450] 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 (c.f., Hodgson, Bio/Technology. 2:19-21
(1991)).
[1451] In one approach, the three-dimensional structure of the PRO
polypeptide, or of an PRO polypeptide-inhibitor complex, is
determined by x-ray crystallography, by computer modeling or, most
typically, by a combination of the two approaches. Both the shape
and charges of the PRO polypeptide must be ascertained to elucidate
the structure and to determine active site(s) of the molecule. Less
often, useful information regarding the structure of the PRO
polypeptide may be gained by modeling based on the structure of
homologous proteins. In both cases, relevant structural information
is used to design analogous PRO polypeptide-like molecules or to
identify efficient inhibitors. Useful examples of rational drug
design may include molecules which have improved activity or
stability as shown by Braxton and Wells, Biochemistry 31:7796-7801
(1992) or which act as inhibitors, agonists, or antagonists of
native peptides as shown by Athauda et al., J. Biochem.,
113:742-746 (1993).
[1452] 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.
[1453] By virtue of the present invention, sufficient amounts of
the PRO polypeptide may be made available to perform such
analytical studies as X-ray crystallography. In addition, knowledge
of the PRO polypeptide amino acid sequence provided herein will
provide guidance to those employing computer modeling techniques in
place of or in addition to x-ray crystallography.
Example 62
Diagnostic Test Using PRO317 Polypeptide-Specific Antibodies
[1454] Particular anti-PRO317 polypeptide antibodies are useful for
the diagnosis of prepathologic conditions, and chronic or acute
diseases such as gynecological diseases or ischemic diseases which
are characterized by differences in the amount or distribution of
PRO317, PRO317 has been found to be expressed in human kidney and
is thus likely to be associated with abnormalities or pathologies
which affect this organ. Further, since it, is so closely related
to EBAF-1, it is likely to affect the endometrium and other genital
tissues. Further, due to library sources of certain ESTs, it
appears that PRO317 may be involved as well in forming blood
vessels and hence to be a modulator of angiogenesis.
[1455] Diagnostic tests for PRO317 include methods utilizing the
antibody and a label to detect PRO317 in human body fluids,
tissues, or extracts of such tissues. The polypeptide and
antibodies of the present invention may be used with or without
modification. Frequently, the polypeptide and antibodies will be
labeled by joining them, either covalently or noncovalently, with a
substance which provides for a detectable signal. A wide variety of
labels and conjugation techniques are known and have been reported
extensively in both the scientific and patent literature. Suitable
labels include radionuclides, enzymes, substrates, cofactors,
inhibitors, fluorescent agents, chemiluminescent agents, magnetic
particles, and the like. Patents teaching the use of such labels
include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;
4,277,437; 4,275,149; and 4,366,241. Also, recombinant
immunoglobulins may be produced as shown in U.S. Pat.
No.4,816,567.
[1456] A variety of protocols for measuring soluble or
membrane-bound PRo317, using either polyclonal or monoclonal
antibodies specific for that PRO317, are known in the art. Examples
include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay
(RIA), radioreceptor assay (RRA), and fluorescent activated cell
sorting (FACS). A two-site monoclonal-based immunoassay utilizing
monoclonal antibodies reactive to two non-interfering epitopes on
PRO317 is preferred, but a competitive binding assay may be
employed. These assays are described, among other places, in Maddox
et al. J Exp. Med. 158:1211 (1983).
Example 63
Identification of PRO317 Receptors
[1457] Purified PRO317 is useful for characterization and
purification of specific cell surface receptors and other binding
molecules. Cells which respond to PRO317 by metabolic changes or
other specific responses are likely to express a receptor for
PRO317. Such receptors include, but are not limited to, receptors
associated with and activated by tyrosine and serine/threonine
kinases. See Kolodziejczyk and Hall, supra, for a review on known
receptors for the TGF- superfamily. Candidate receptors for this
superfamily fall into two primary groups, termed type I and type II
receptors. Both types are serine/threonine kinases. Upon activation
by the appropriate ligand, type I and type II receptors physically
interact to form hetero-oligomers and subsequently activate
intracellular signaling cascades, ultimately regulating gene
transcription and expression. In addition, TGF- binds to a third
receptor class, type 111, a membrane-anchored proteoglycan lacking
the kinase activity typical of signal transducing molecules.
[1458] PRO317 receptors or other PRO317-binding molecules may be
identified by interaction with radiolabeled PRO317. Radioactive
labels may be incorporated into PRO317 by various methods known in
the art. A preferred embodiment is the labeling of primary amino
groups in PRO317 With .sup.125I Bolton-Hunter reagent (Bolton and
Hunter, Biochem. J., 133:529 (1973)), which has been used to label
other polypeptides without concomitant loss of biological activity
(Hebert et al. J. Biol. Chem. 266:18989 (1991); McColl et al. J.
Immunol. 150:45504555 (1993)). Receptor-bearing cells are incubated
with labeled PRO317. The cells are then washed to removed unbound
PRO317, and receptor-bound PRO317 is quantified. The data obtained
using different concentrations of PRO317 are used to calculate
values for the number and affinity of receptors.
[1459] Labeled PRO317 is useful as a reagent for purification of
its specific receptor. In one embodiment of affinity purification,
PRO317 is covalently coupled to a chromatography column.
Receptor-bearing cells are extracted, and the extract is passed
over the column. The receptor binds to the column by virtue of its
biological affinity for PRO317. The receptor is recovered from the
column and subjected to N-terminal protein sequencing. This amino
acid sequence is then used to design degenerate oligonucleotide
probes for cloning the receptor gene.
[1460] In an alternative method, mRNA is obtained from
receptor-bearing cells and made into a cDNA library. The library is
transfected into a population of cells, and those cells expressing
the receptor are selected using fluorescently labeled PRO317. The
receptor is identified by recovering and sequencing recombinant DNA
from highly labeled cells.
[1461] In another alternative method, antibodies are raised against
the surface of receptor bearing cells, specifically monoclonal
antibodies. The monoclonal antibodies are screened to identify
those which inhibit the binding of labeled PRO317. These monoclonal
antibodies are then used in affinity purification or expression
cloning of the receptor.
[1462] Soluble receptors or other soluble binding molecules are
identified in a similar manner. Labeled PRO317 is incubated with
extracts or other appropriate materials derived from the uterus.
After incubation, PRO317 complexes larger than the size of purified
PRO317 are identified by a sizing technique such as size-exclusion
chromatography or density gradient centrifugation and are purified
by methods known in the art. The soluble receptors or binding
protein(s) are subjected to N-terminal sequencing to obtain
information sufficient for database identification, if the soluble
protein is known, or for cloning, if the soluble protein is
unknown.
Example 64
Determination of PRO317-Induced Cellular Response
[1463] The biological activity of PRO317 is measured, for example,
by binding of an PRO317 of the invention to an PRO317 receptor. A
test compound is screened as an antagonist for its ability to block
binding of PRO317 to the receptor. A test compound is screened as
an agonist of the PRO317 for its ability to bind an PRO317 receptor
and influence the same physiological events as PRO317 using, for
example, the KIRA-ELISA assay described by Sadick et al.,
Analytical Biochemistry, 235:207-214 (1996) in which activation of
a receptor tyrosine kinase is monitored by immuno-capture of the
activated receptor and quantitation of the level of ligand-induced
phosphorylation. The assay may be adapted to monitor PRO317-induced
receptor activation through the use of an PRO317 receptor-specific
antibody to capture the activated receptor. These techniques are
also applicable to other PRO polypeptides described herein.
Example 65
Use of PRO224 for Screening Compounds
[1464] PRO224 is expressed in a cell stripped of membrane proteins
and capable of expressing PRO224. Low density lipoproteins having a
detectable label are added to the cells and incubated for a
sufficient time for endocytosis. The cells are washed. The cells
are then analysed for label bound to the membrane and within the
cell after cell lysis. Detection of the low density lipoproteins
within the cell determines that PRO224 is within the family of low
density lipoprotein receptor proteins. Members found within this
family are then used for screening compounds which affect these
receptors, and particularly the uptake of cholesterol via these
receptors.
Example 66
Ability of PRO Polypeptides to Inhibit Vascular Endothelial Growth
Factor (VEGF) Stimulated Proliferation of Endothelial Cell Growth
(Assay 9)
[1465] 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.
[1466] Specifically, bovine adrenal cortical capillary endothelial
cells (ACE) (from primary culture, maximum of 12-14 passages) were
plated in 96-well plates at 500 cells/well per 100 microliter.
Assay media included low glucose DMEM, 10% calf serum, 2 mM
glutamine, and IX penicillin/streptomycin/fungizone. Control wells
included the following: (1) no ACE cells added; (2) ACE cells
alone; (3) ACE cells plus 5 ng/ml FGF; (4) ACE cells plus 3 ng/ml
VEGF; (5) ACE cells plus 3 ng/ml VEGF plus 1 ng/ml TGF-beta; and
(6) ACE cells plus 3 ng/ml VEGF plus 5 ng/ml LIF. The test samples,
poly-his tagged PRO polypeptides (in 100 microliter volumes), were
then added to the wells (at dilutions of 1%, 0.1% and 0.01%,
respectively). The cell cultures were incubated for 6-7 days at
37.degree. C./5% CO.sub.2. After the incubation, the media in the
wells was aspirated, and the cells were washed IX 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 IN NaOH. Optical
density (OD) was measured on a microplate reader at 405 nm.
[1467] 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 mn)
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).
[1468] The following polypeptides tested positive in this assay:
PRO211, PRO217, PRO187, PRO219, PRO246, PRO228, PRO245, PRO221,
PRO258, PRO301, PRO224, PRO272, PRO328, PRO331, PRO224, PRO328,
PRO272, PRO301, PRO331 and PRO214.
Example 67
Retinal Neuron Survival (Assay 52)
[1469] 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.
[1470] Sprague Dawley rat pups at postnatal day 7 (mixed
population: glia and retinal neuronal types) are killed by
decapitation following CO.sub.2 anesthesia and the eyes are removed
under sterile conditions. The neural retina is dissected away from
the pigment epithelium and other ocular tissue and then dissociated
into a single cell suspension using 0.25% trypsin in Ca.sup.2+,
Mg.sup.2+-free PBS. The retinas are incubated at 37.degree. C. for
7-10 minutes after which the trypsin is inactivated by adding 1 ml
soybean trypsin inhibitor. The cells are plated at 100,000 cells
per well in 96 well plates in DMEM/F12 supplemented with N2 and
with or without the specific test PRO polypeptide. Cells for all
experiments are grown at 37.degree. C. in a water saturated
atmosphere Of 5% CO.sub.2. After 2-3 days in culture, cells are
stained with calcein AM then fixed using 4% paraformaldehyde and
stained with DAPI for determination of total cell count. The total
cells (fluorescent) are quantified at 20.times. objective
magnification using CCD camera and NIH image software for
MacIntosh. Fields in the well are chosen at random.
[1471] 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.
[1472] The following PRO polypeptides tested positive in this assay
using polypeptide concentrations within the range of 0.01% to 1.0%
in the assay: PRO220 and PRO346.
Example 68
Rod Photoreceptor Cell Survival (Assay 56)
[1473] This assay shows that certain polypeptides of the invention
act to enhance the survival/proliferation of rod photoreceptor
cells and, therefore, are useful for the therapeutic treatment of
retinal disorders or injuries including, for example, treating
sight loss in mammals due to retinitis pigmentosum, AMD, etc.
Sprague Dawley rat pups at 7 day postnatal (mixed population: glia
and retinal neuronal cell types) are killed by decapitation
following CO.sub.2 anesthesis and the eyes are removed under
sterile conditions. The neural retina is dissected away form the
pigment epithelium and other ocular tissue and then dissociated
into a single cell suspension using 0.25% trypsin in Ca.sup.2+,
Mg.sup.2+-free PBS. The retinas are incubated at 37.degree. C. for
7-10 minutes after which the trypsin is inactivated by adding 1 ml
soybean trypsin inhibitor. The cells are plated at 100,000 cells
per well in 96 well plates in DMEM/F12 supplemented with N.sub.2.
Cells for all experiments are grown at 37.degree. C. in a water
saturated atmosphere of 5% CO.sub.2. After 2-3 days in culture,
cells are fixed using 4% paraformaldehyde, and then stained using
CellTracker Green CMFDA. Rho 4D2 (ascites or IgG 1:100), a
monoclonal antibody directed towards the visual pigment rhodopsin
is used to detect rod photoreceptor cells by indirect
immunofluorescence. The results are calculated as % survival: total
number of calcein-rhodopsin positive cells at 2-3 days in culture,
divided by the total number of rhodopsin positive cells at time 2-3
days in culture. The total cells (fluorescent) are quantified at
20.times. objective magnification using a CCD camera and NIH image
software for MacIntosh. Fields in the well are chosen at
random.
[1474] The following polypeptides tested positive in this assay:
PRO220 and PRO346.
Example 69
Induction of Endothelial Cell Apoptosis (Assay 73)
[1475] The ability of PRO polypeptides to induce apoptosis in
endothelial cells was tested in human venous umbilical vein
endothelial cells (HUVEC, Cell Systems). A positive test in the
assay is indicative of the usefulness of the polypeptide in
therapeutically treating tumors as well as vascular disorders where
inducing apoptosis of endothelial cells would be beneficial.
[1476] 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.104 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.
[1477] 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 pi of the diluted Annexin V-Biotin solution was
added to each well (except controls) to a final concentration of
1.0 .mu.g/ml. The samples were incubated for 10-15 minutes with
Annexin-Biotin prior to direct addition of .sup.35S-Streptavidin.
.sup.35S-Streptavidin was diluted in 2.times.Ca.sup.2+ Binding
buffer, 2.5% BSA and was added to all wells at a final
concentration of 3.times.104 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.
[1478] The following PRO polypeptides tested positive in this
assay: PRO211, PRO217 and PRO301.
Example 70
PDB12 Cell Inhibition (Assay 40)
[1479] This example demonstrates that various PRO polypeptides have
efficacy in inhibiting protein production by PDB12 pancreatic
ductal cells and are therefore, useful in the therapeutic treatment
of disorders which involve protein secretion by the pancreas,
including diabetes, and the like.
[1480] PDB 12 pancreatic ductal cells are plated on fibronectin
coated 96 well plates at 1.5.times.10.sup.3 cells per well in 100
WI 80 .mu.L of growth media. 100 .mu.L of growth media with the PRO
polypeptide test sample or negative control lacking the PRO
polypeptide is then added to well, for a final volume of 200 .mu.L.
Controls contain growth medium containing a protein shown to be
inactive in this assay. Cells are incubated for 4 days at
37.degree. C. 20 .mu.L of Alamar Blue Dye (AB) is then added to
each well and the flourescent reading is measured at 4 hours post
addition of AB, on a microtiter plate reader at 530 nm excitation
and 590 mn emission. The standard employed is cells without Bovine
Pituitary Extract (BPE) and with various concentrations of BPE.
Buffer or CM controls from unknowns are run 2 times on each 96 well
plate.
[1481] These assays allow one to calculate a percent decrease in
protein production by comparing the Alamar Blue Dye calculated
protein concentration produced by the PRO polypeptide-treated cells
with the Alamar Blue Dye calculated protein concentration produced
by the negative control cells. A percent decrease in protein
production of greater than or equal to 25% as compared to the
negative control cells is considered positive.
[1482] The following polypeptides tested positive in this assay:
PRO211, PRO287, PRO301 and PRO293.
Example 71
Stimulation of Adult Heart Hypertrophy (Assay 2)
[1483] This assay is designed to measure the ability of various PRO
polypeptides to stimulate hypertrophy of adult heart. PRO
polypeptides testing positive in this assay would be expected to be
useful for the therapeutic treatment of various cardiac
insufficiency disorders.
[1484] Ventricular myocytes freshly isolated from adult (250 g)
Sprague Dawley rats are plated at 2000 cell/well in 180 .mu.l
volume. Cells are isolated and plated on day 1, the PRO
polypeptide-containing test samples or growth medium only (negative
control) (20 .mu.l volume) is added on day 2 and the cells are then
fixed and stained on day 5. After staining, cell size is visualized
wherein cells showing no growth enhancement as compared to control
cells are given a value of 0.0, cells showing small to moderate
growth enhancement as compared to control cells are given a value
of 1.0 and cells showing large growth enhancement as compared to
control cells are given a value of 2.0. Any degree of growth
enhancement as compared to the negative control cells is considered
positive for the assay.
[1485] The following PRO polypeptides tested positive in this
assay: PRO287, PRO301, PRO293 and PRO303.
Example 72
PDB12 Cell Proliferation Assay 29)
[1486] This example demonstrates that various PRO polypeptides have
efficacy in inducing proliferation of PDB12 pancreatic ductal cells
and are, therefore, useful in the therapeutic treatment of
disorders which involve protein secretion by the pancreas,
including diabetes, and the like.
[1487] PDB12 pancreatic ductal cells are plated on fibronectin
coated 96 well plates at 1.5.times.10.sup.3 cells per well in 100
.mu.L/80 .mu.L of growth media. 100 .mu.L of growth media with the
PRO polypeptide test sample or negative control lacking the PRO
polypeptide is then added to well, for a final volume of 200 .mu.L.
Controls contain growth medium containing a protein shown to be
inactive in this assay. Cells are incubated for 4 days at
37.degree. C. 20 .mu.L of Alamar Blue Dye (AB) is then added to
each well and the flourescent reading is measured at 4 hours post
addition of AB, on a microtiter plate reader at 530 nm excitation
and 590 nm emission. The standard employed is cells without Bovine
Pituitary Extract (BPE) and with various concentrations of BPE.
Buffer or growth medium only controls from unknowns are run 2 times
on each 96 well plate.
[1488] Percent increase in protein production is calculated by
comparing the Alamar Blue Dye calculated protein concentration
produced by the PRO polypeptide-treated cells with the Alamar Blue
Dye calculated protein concentration produced by the negative
control cells. A percent increase in protein production of greater
than or equal to 25% as compared to the negative control cells is
considered positive.
[1489] The following PRO polypeptides tested positive in this
assay: PRO301 and PRO303.
Example 73
Enhancement of Heart Neonatal Hypertrophy (Assay 1)
[1490] 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.
[1491] Cardiac myocytes from 1-day old Harlan Sprague Dawley rats
were obtained. Cells (180 .mu.l at 7.5.times.10.sup.4/ml,
serum<0.1%, freshly isolated) are added on day 1 to 96-well
plates previously coated with DMEM/F12+4% FCS. Test samples
containing the test PRO polypeptide or growth medium only (negative
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.
[1492] The following polypeptides tested positive in this assay:
PRO224 and PRO231.
Example 74
Stimulatory Activity in Mixed Lymphocyte Reaction (MLR) Assay
(Assay 24)
[1493] 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.
[1494] 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.
[1495] More specifically, in one assay variant, peripheral blood
mononuclear cells (PBMC) are isolated from mammalian individuals,
for example a human volunteer, by leukopheresis (one donor will
supply stimulator PBMCs, the other donor will supply responder
PBMCs). If desired, the cells are frozen in fetal bovine serum and
DMSO after isolation. Frozen cells may be thawed overnight in assay
media (37.degree. C., 5% CO.sub.2 and then washed and resuspended
to 3.times.10.sup.6 cells/ml of assay media (RPMI; 10% fetal bovine
serum, 1% penicillin/streptomycin, 1% glutamine, 1% HEPES, 1%
non-essential amino acids, 1% pyruvate). The stimulator PBMCs are
prepared by irradiating the cells (about 3000 Rads).
[1496] The assay is prepared by plating in triplicate wells a
mixture of:
[1497] 100:1 of test sample diluted to 1% or to 0.1%,
[1498] 50:1 of irradiated stimulator cells, and
[1499] 50:1 of responder PBMC cells.
[1500] 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.
[1501] In another variant of this assay, PBMCs are isolated from
the spleens of Balb/c mice and C57B6 mice. The cells are teased
from freshly harvested spleens in assay media (RPMI; 10% fetal
bovine serum, 1% penicillin/streptomycin, 1% glutamine, 1% HEPES,
1% non-essential amino acids, 1% pyruvate) and the PBMCs are
isolated by overlaying these cells over Lympholyte M (Organon
Teknika), centrifuging at 2000 rpm for 20 minutes, collecting and
washing the mononuclear cell layer in assay media and resuspending
the cells to 1.times.10.sup.7 cells/ml of assay media. The assay is
then conducted as described above.
[1502] 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.
[1503] The following PRO polypeptides tested positive in this
assay: PRO245, PRO269, PRO217, PRO301; PRO266, PRO335, PRO331,
PRO533 and PRO326.
Example 75
Pericyte c-Fos Induction (Assay 93)
[1504] This assay shows that certain polypeptides of the invention
act to induce the expression of c-fos in pericyte cells and,
therefore, are useful not only as diagnostic markers for particular
types of pericyte-associated tumors but also for giving rise to
antagonists which would be expected to be useful for the
therapeutic treatment of pericyte-associated tumors. Specifically,
on day 1, pericytes are received from VEC Technologies and all but
5 ml of media is removed from flask. On day 2, the pericytes are
trypsinized, washed, spun and then plated onto 96 well plates. On
day 7, the media is removed and the pericytes are treated with 100
.mu.l of PRO polypeptide test samples and controls (positive
control=DME+5% serum.+-.PDGF at 500 ng/ml; negative control=protein
32). Replicates are averaged and SD/CV are determined. Fold
increase over Protein 32 (buffer control) value indicated by
chemiluminescence units (RLU) luminometer reading verses frequency
is plotted on a histogram. Two-fold above Protein 32 value is
considered positive for the assay. ASY Matrix: Growth media=low
glucose DMEM=20% FBS+1.times. pen strep+1.times. fungizone. Assay
Media=low glucose DMEM+5% FBS.
[1505] The following polypeptides tested positive in this assay:
PRO214, PRO219, PRO221 and PRO224.
Example 76
Ability of PRO Polypeptides to Stimulate the Release of
Proteoglycans: from Cartilage (Assay 97)
[1506] The ability of various PRO polypeptides to stimulate the
release of proteoglycans from cartilage tissue was tested as
follows.
[1507] 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/F121:1) 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
polypeptides were then added at 1% either alone or in combination
with 18 ng/ml interleukin-1.A-inverted., a known stimulator of
proteoglycan release from cartilage tissue. The supernatant was
then harvested and assayed for the amount of proteoglycans using
the 1,9-dimethyl-mediylene blue (DMB) colorimetric assay (Farndale
and Buttle, Biochem. Biophys. Acta 883:173-177 (1985)). A positive
result in this assay indicates that the test polypeptide will find
use, for example, in the treatment of sports-related joint
problems, articular cartilage defects, osteoarthritis or rheumatoid
arthritis.
[1508] When various PRO polypeptides were tested in the above
assay, the polypeptides demonstrated a marked ability to stimulate
release of proteoglycans from cartilage tissue both basally and
after stimulation with interleukin-a 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
sportsrelated joint problems, articular cartilage defects,
osteoarthritis or rheumatoid arthritis. The polypeptides testing
positive in this assay are : PRO211.
Example 77
Skin Vascular Permeability Assay (Assay 64)
[1509] 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 grains 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 firs
after injection. Each skin injection site is biopsied and fixed in
formalin. The skins are then prepared for histopathologic
evaluation. Each site is evaluated for inflammatory cell
infiltration into the skin. Sites with visible inflammatory cell
inflammation are scored as positive. Inflammatory cells may be
neutrophilic, eosinophilic, monocytic or lymphocytic. At least a
minimal perivascular infiltrate at the injection site is scored as
positive, no infiltrate at the site of injection is scored as
negative.
[1510] The following polypeptides tested positive in this assay:
PRO245, PRO217, PRO326, PRO266, PRO272, PRO301, PRO331 and
PRO335.
Example 78
Enhancement of Heart Neonatal Hypertrophy Induced by F2a (Assay
37)
[1511] 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.
[1512] Cardiac myocytes from 1-day old Harlan Sprague Dawley rats
were obtained. Cells (180 .mu.l at 7.5.times.10.sup.4/ml,
serum<0.1%, freshly isolated) are added on day 1 to 96-well
plates previously coated with DMEM/F12+4% FCS. Test samples
containing the test PRO polypeptide (20 .mu.l/well) are added
directly to the wells on day 1. PGF (20 .mu.l/well) is then added
on day 2 at a final concentration of 10.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.
[1513] 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/F 12 (with 2.44 gm
bicarbonate), 10 .mu.g/ml transferrin, 1 .mu.g/ml insulin, 1
.mu.g/ml aprotinin, 2 mmol/L glutamine, 100 U/ml penicillin G, 100
.mu.g/ml streptomycin. Protein buffer containing mannitol (4%) gave
a positive signal (score 3.5) at 1/10 (0.4%) and 1/100 (0.04%), but
not at 1/1000 (0.004%). Therefore the test sample buffer containing
mannitol is not run.
[1514] The following PRO polypeptides tested positive in this
assay: PRO224.
Example 79
Inhibitory Activity in Mixed Lymphocyte Reaction (MLR) Assay (Assay
67)
[1515] 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.
[1516] 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
Institutes of Health, Published by John Wiley & Sons, Inc.
[1517] More specifically, in one assay variant, peripheral blood
mononuclear cells (PBMC are isolated from mammalian individuals,
for example a human volunteer, by leukopheresis (one donor will
supply stimulator PBMCs, the other donor will supply responder
PBMCs). If desired, the cells are frozen in fetal bovine serum and
DMSO after isolation. Frozen cells may be thawed overnight in assay
media (37.degree. C., 5% CO.sub.2) and then washed and resuspended
to 3.times.10.sup.6 cells/ml of assay media (RPMI; 10% fetal bovine
serum, 1% penicillin/streptomycin, 1% glutamine, 1% HEPES, 1%
non-essential amino acids, 1% pyruvate). The stimulator PBMCs are
prepared by irradiating the cells (about 3000 Rads).
[1518] The assay is prepared by plating in triplicate wells a
mixture of:
[1519] 100:1 oftest sample diluted to 1% or to 0.1%,
[1520] 50:1 of irradiated stimulator cells, and
[1521] 50:1 of responder PBMC cells.
[1522] 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.
[1523] In another variant of this assay, PBMCs are isolated from
the spleens of Balb/c mice and C57B6 mice. The cells are teased
from freshly harvested spleens in assay media (RPMI; 10% fetal
bovine serum, 1% penicillin/streptomycin, 1% glutamine, 1% HEPES,
1% non-essential amino acids, 1% pyruvate) and the PBMCs are
isolated by overlaying these cells over Lympholyte M (Organon
Teknika), centrifuging at 2000 rpm for 20 minutes, collecting and
washing the mononuclear cell layer in assay media and resuspending
the cells to 1.times.10.sup.7 cells/ml of assay media. The assay is
thlen conducted as described above.
[1524] 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.
[1525] The following polypeptide tested positive in this assay:
PRO235, PRO245 and PRO332.
Example 80
Induction of Endothelial Cell Apoptosis (ELISA) (Assay 109)
[1526] The ability of PRO polypeptides to induce apoptosis in
endothelial cells was tested in human venous umbilical vein
endothelial cells (HUVEC, Cell Systems) using a 96-well format, in
0% serum media supplemented with 100 ng/ml VEGF, 0.1% BSA, 1.times.
penn/strep. A positive result in this assay indicates the
usefulness of the polypeptide for therapeutically treating any of a
variety of conditions associated with undesired endothelial cell
growth including, for example, the inhibition of tumor growth. The
96-well plates used were manufactured by Falcon (No. 3072). Coating
of 96 well plates were prepared by allowing gelatinization to occur
for >30 minutes with 100 .mu.l of 0.2% gelatin in PBS solution.
The gelatin mix was aspirated thoroughly before plating HUVEC cells
at a final concentration of 2.times.10.sup.4 cells/ml in 10% serum
containing medium -100 .mu.l volume per well. The cells were grown
for 24 hours before adding test samples containing the PRO
polypeptide of interest.
[1527] To all wells, 100 .mu.l of 0% serum media (Cell Systems)
complemented with 100 ng/ml VEGF, 0.1% BSA, 1.times. perm/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.
[1528] ELISA was used to determine levels of apoptosis preparing
solutions according to the Boehringer Manual [Boehringer, Cell
Death Detection ELISA plus, Cat No. 1920 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. Cysts 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 lysate
in each well. The MTP was covered with adhesive foil and incubated
at room temperature 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.) into each well and incubated on an orbital
shaker at room temperature at 250 rpm until color development was
sufficient for a photometric analysis (approx. after 10-20
minutes). A 96 well reader was used to read the plates at 405 nm,
reference wavelength, 492 nm. The levels obtained for PIN 32
(control buffer) was set to 100%. Samples with levels>130% were
considered positive for induction of apoptosis.
[1529] The following PRO polypeptides tested positive in this
assay: PRO235.
Example 81
Human Venous Endothelial Cell Calcium Flux Assay (Assay 68)
[1530] 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.
[1531] Human venous umbilical vein endothelial cells (HUVEC, Cell
Systems) in growth media (50:50 without glycine, 1% glutamine, 110
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 3.times. with buffer
(described above) leaving 100 .mu.l/well. Test samples of the PRO
polypeptides were prepared on different 96-well plates at 5.times.
concentration in buffer. The positive control corresponded to 50
.mu.M ionomycin (5.times.); the negative control corresponded to
Protein 32. Cell plate and sample plates were run on a FLIPR
(Molecular Devices) machine. The FLIPR machine added 25 .mu.l of
test sample to the cells, and readings were taken every second for
one minute, then every 3 seconds for the next three minutes.
[1532] The fluorescence change from baseline to the maximum rise of
the curve (A change) was calculated, and replicates averaged. The
rate of fluorescence increase was monitored, and only those samples
which had a A change greater than 1000 and a rise within 60
seconds, were considered positive.
[1533] The following PRO polypeptides tested positive in the
present assay: PRO245.
Example 82
Fibroblast (BHK-21) Proliferation (Assay 98)
[1534] This assay shows that certain PRO polypeptides of the
invention act to induce proliferation of mammalian fibroblast cells
in culture and, therefore, function as useful growth factors in
mammalian systems. The assay is performed as follows. BHK-21
fibroblast cells plated in standard growth medium at 2500
cells/well in a total volume of 100 .mu.l. The PRO polypeptide,
.beta.-FGF (positive control) or nothing. (negative control) are
then added to the wells in the presence of 1 .mu.g/ml of heparin
for a total final volume of 200 .mu.l. The cells are then incubated
at 37.degree. C. for 6 to 7 days. After incubation, the media is
removed, the cells are washed with PBS and then an acid phosphatase
substrate reaction mixture (100 .mu.l/well) is added. The cells are
then incubated at 37.degree. C. for 2 hours. 10 RI per well of IN
NaOH is then added to stop the acid phosphatase reaction. The
plates are then read at OD 405 nm. A positive in the assay is acid
phosphatase activity which is at least 50% above the negative
control.
[1535] The following PRO polypeptide tested positive in this assay:
PRO258.
Example 83
Inhibition of Heart Adult Hypertrophy (Assn 42)
[1536] This assay is designed to measure the inhibition of heart
adult hypertrophy. PRO polypeptides testing positive in this assay
may find use in the therapeutic treatment of cardiac disorders
associated with cardiac hypertrophy.
[1537] Ventricular myocytes are freshly isolated from adult (250 g)
Harlan Sprague Dawley rats and the cells are plated at 2000/well in
180 pi volume. On day two, test samples (20 .mu.l) containing the
test PRO polypeptide are added. On day five, the cells are fixed
and then stained. An increase in ANP message can also be measured
by PCR from cells after a few hours. Results are based on a visual
score of cell size: 0=no inhibition, -1=small inhibition, -2=large
inhibition. A score of less than 0 is considered positive. Activity
reference corresponds to phenylephrin (PE) at 0.1 mM, as a positive
control. Assay media included: M199 (modified)-glutamine free,
NaHCO.sub.3, phenol red, supplemented with 100 nM insulin, 0.2%
BSA, 5 mM cretine, 2 mM L-carnitine, 5 mM taurine, 100 Wail
penicillin G, 100 .mu.g/ml streptomycin (CCT medium). Only inner 60
wells are used in 96 well plates. Of these, 6 wells are reserved
for negative and positive (PE) controls.
[1538] The following PRO polypeptides provided a score of less than
0 in the above assay: PRO269.
Example 84
Induction of c-fos in Endothelial Cells (Assay 34)
[1539] 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.
[1540] Human venous umbilical vein endothelial cells (HUVEC, Cell
Systems) in growth media (50% Ham's F12 w/o GHT: low glucose, and
50% DMEM without glycine: with NaHCO3, 1% glutamine, 10 mM HEPES,
10% FBS, 10 ng/ml bFGF) were plated on 96-well microtiter plates at
a cell density of 1.times.10.sup.4 cells/well. The day after
plating, the cells were starved by removing the growth media and
treating the cells with 100 .mu.l/well test samples and controls
(positive control=growth media; negative control=Protein 32
buffer=10 mM HEPES, 140 mM NaCl, 4% (w/v) mannitol, pH 6.8). The
cells were incubated for 30 minutes at 37.degree. C., in 5%
CO.sub.2. The samples were removed, and the first part of the bDNA
kit protocol (Chiron Diagnostics, cat. #6005-037) was followed,
where each capitalized reagent/buffer listed below was available
from the kit.
[1541] 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.
[1542] 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.
[1543] 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 twofold value over the negative buffer control.
Negative control=1.00 RLU at 1.00% dilution. Positive control=8.39
RLU at 1.00% dilution.
[1544] The following PRO polypeptides tested positive in this
assay: PRO287.
Example 85
Guinea Pig Vascular Leak (Assays 32 and 51)
[1545] 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.
[1546] Hairless guinea pigs weighing 350 grams or more were
anesthetized with Ketamine (75-80 mg/kg) and 5 mg/kg Xylazine
intramuscularly. Test samples containing the PRO polypeptide or a
physiological buffer without the test polypeptide are injected into
skin on the back of the test animals with 100 .mu.l per injection
site intradermally. There were approximately 16-24 injection sites
per animal. One in] 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 nun) of blue-colored
leaks from the site of injection at 1 and 6 hours post
administration of the test materials. The mm diameter of blueness
at the site of injection is observed and recorded as well as the
severity of the vascular leakage. Blemishes of at least 5 mm in
diameter are considered positive for the assay when testing
purified proteins, being indicative of the ability to induce
vascular leakage or permeability. A response greater than 7 mm
diameter is considered positive for conditioned media samples.
Human VEGF at 0.1 .mu.g/100 .mu.l is used as a positive control,
inducing a response of 15-23 nun diameter.
[1547] The following PRO polypeptides tested positive in this
assay: PRO302 and PRO533.
Example 86
Detection of Endothelial Cell Apoptosis (FACS) (Assay 96)
[1548] The ability of PRO polypeptides of the present invention to
induce apoptosis in endothelial cells was tested in human venous
umbilical vein endothelial cells (HUVEC, Cell Systems) in
gelatinized T175 flasks using HUVEC cells below passage 10. PRO
polypeptides testing positive in this assay are expected to be
useful for therapeutically treating conditions where apoptosis of
endothelial cells would be beneficial including, for example, the
therapeutic treatment of tumors.
[1549] On day one, the cells were split [420,000 cells per
gelatinized 6 cm dishes-(11.times.10.sup.3 cells/cm Falcon,
Primaria)] and grown in media containing serum (CS-C, Cell System)
overnight or for 16 hours to 24 hours.
[1550] On day 2, the cells were washed 1.times. with 5 ml PBS; 3 ml
of 0% serum medium was added with VEGF (100 ng/ml); and 30 .mu.l of
the PRO test compound (final dilution 1%) or 0% serum medium
(negative control) was added. The mixtures were incubated for 48
hours before harvesting.
[1551] The cells were then harvested for FACS analysis. The medium
was aspirated and the cells washed once with PBS. 5 ml of 1.times.
trypsin was added to the cells in a T-175 flask, and the cells were
allowed to stand until they were released from the plate (about
5-10 minutes). Trypsinization was stopped by adding 5 ml of growth
media. The cells were spun at 1000 rpm for 5 minutes at 4.degree.
C. The media was aspirated and the cells were resuspended in 10 ml
of 10% serum complemented medium (Cell Systems), 5 .mu.l of
Annexin-FITC (BioVison) added and chilled tubes were submitted for
FACS. A positive result was determined to be enhanced apoptosis in
the PRO polypeptide treated samples as compared to the negative
control.
[1552] The following PRO polypeptides tested positive in this
assay: PRO331.
Example 87
Induction of c-fos in Cortical Neurons (Assay 83)
[1553] 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.
[1554] Cortical neurons are dissociated and plated in growth medium
at 10,000 cells per well in 96 well plates. After approximately 2
cellular divisions, the cells are treated for 30 minutes with the
PRO polypeptide or nothing (negative control). The cells are then
fixed for 5 minutes with cold methanol and stained with an antibody
directed against phosphorylated CREB. mRNA levels are then
calculated using chemiluminescence. A positive in the assay is any
factor that results in at least a 2-fold increase in c-fos message
as compared to the negative controls.
[1555] The following PRO polypeptides tested positive in this
assay: PRO229 and PRO269.
Example 88
Stimulation of Endothelial Tube Formation (Assay 85)
[1556] This assay is designed to determine whether PRO polypeptides
show the ability to promote endothelial vacuole and lumen formation
in the absence of exogenous; growth factors. PRO polypeptides
testing positive in this assay would be expected to be useful for
the therapeutic treatment of disorders where endothelial vacuole
and/or lumen formation would be beneficial including, for example,
where the stimulation of pinocytosis, ion pumping, vascular
permeability and/or junctional formation would be beneficial.
[1557] HUVEC cells (passage<8 from primary) are mixed with type
I rat tail collagen (final concentration 2.6 mg/ml) at a density of
6.times.10-5 cells per ml and plated at 50 .mu.l per well of M199
culture media supplemented with 1% FBS and 1 .mu.M 6-FAM-FITC dye
to stain the vacuoles while they are forming and in the presence of
the PRO polypeptide. The cells are then incubated at 37.degree.
C./5% CO.sub.2 for 48 hours, fixed with 3.7% forrnalin at room
temperature for 10 minutes, washed 5 times with M199 medium and
then stained with Rh-Phalloidin at 4.degree. C. overnight followed
by nuclear staining with 4 .mu.M DAPI. A positive result in the
assay is when vacuoles are present in greater than 50% of the
cells.
[1558] The following PRO polypeptides tested positive in this
assay: PRO230.
Example 89
Detection of Polypeptides That Affect Glucose and/or FFA Uptake in
Skeletal Muscle (Assay 106)
[1559] 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.
[1560] In a 96 well format, PRO polypeptides to be assayed are
added to primary rat differentiated skeletal muscle, and allowed to
incubate overnight. Then fresh media with the PRO polypeptide and
insulin are added to the wells. The sample media is then monitored
to determine glucose and FFA uptake by the skeletal muscle cells.
The insulin will stimulate glucose and FFA uptake by the skeletal
muscle, and insulin in media without the PRO polypeptide is used as
a positive control, and a limit for scoring. As the PRO polypeptide
being tested may either stimulate or inhibit glucose and FFA
uptake, results are scored as positive in the assay if greater than
1.5 times or less than 0.5 times the insulin control.
[1561] The following PRO polypeptides tested positive as either
stimulators or inhibitors of glucose and/or FFA uptake in this
assay: PRO187, PRO211, PRO221, PRO222, PRO224, PRO230, PRO239,
PRO231, PRO245, PRO247, PRO258, PRO269, PRO328 and PRO533.
Example 90
Rod Photoreceptor Cell Survival Assay (Assay 46)
[1562] This assay shows that certain polypeptides of the invention
act to enhance the survival/proliferation of rod photoreceptor
cells and, therefore, are useful for the therapeutic treatment of
retinal disorders or injuries including, for example, treating
sight loss in mammals due to retinitis pigmentosum, AMD, etc.
[1563] Sprague Dawley rat pups (postnatal day 7, mixed population:
glia and netinal neural cell types) are killed by decapitation
following CO.sub.2 anesthesia and the eyes removed under sterile
conditions. The neural retina is dissected away from the pigment
epithelium and other ocular tissue and then dissociated into a
single cell suspension using 0.25% trypsin in Ca.sup.2+,
Mg.sup.2+-free PBS. The retinas are incubated at 37.degree. C. in
this solution for 7-10 minutes after which the trypsin is
inactivated by adding 1 ml soybean trypsin inhibitor. The cells are
plated at a density of approximately 10,000 cells/ml into 96 well
plates in DMEM/F12 supplemented with N.sub.2. Cells for all
experiments are grown at 37.degree. C. in a water saturated
atmosphere of 5% CO.sub.2. After 7-10 days in culture, the cells
are stained using calcein AM or CellTracker Green CMFDA and then
fixed using 4% paraformaldehyde. Rho 4D2 (ascities or IgG 1:100)
monoclonal antibody directed towards the visual pigment rhodopsin
is used to detect rod photoreceptor cells by indirect
immunofluorescence. The results are calculated as % survival: total
number of calcein-rhodopsin positive cells at 7-10 days in culture,
divided by the total number of rhodopsin positive cells at time
7-10 days in culture. The total cells (fluorescent) are quantified
at 20x objective magnification using a CCD camera and NIH image
software for Macintosh. Fields in the well are chosen at
random.
[1564] The following polypeptides tested positive in this assay:
PRO245.
Example 91
In Vitro Antitumor Assay (Assay 161)
[1565] The antiproliferative activity of various PRO polypeptides
was determined in the investigational, diseaseoriented 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]).
[1566] 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.
[1567] 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, an& 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 run was measured using a
computer-interfaced, 96-well microtiter plate reader.
[1568] 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: [1569] NSCL=non-small cell
lung carcinoma
[1570] CNS=central nervous system TABLE-US-00118 TABLE 7 Test
compound Tumor Cell Line Type Cell Line Designation PRO211 NSCL
HOP62 PRO211 Leukemia RPMI-8226 PRO211 Leukemia HL-60 (TB) PRO211
NSCL NCI-H522 PRO211 CNS SF-539 PRO211 Melanoma LOX IMVI PRO211
Breast MDA-MB-435 PRO211 Leukemia MOLT-4 PRO211 CNS U251 PRO211
Breast MCF7 PRO211 Leukemia HT-60 (TB) PRO211 Leukemia MOLT-4
PRO211 NSCL EKVX PRO211 NSCL NCI-H23 PRO211 NSCL NCI-H322M PRO211
NSCL NCI-H460 PRO211 Colon HCT-116 PRO211 Colon HT29 PRO211 CNS
SF-268 PRO211 CNS SF-295 PRO211 CNS SNB-19 PRO211 CNS U251 PRO211
Melanoma LOXIMVI PRO211 Melanoma SK-MEL-5 PRO211 Melanoma UACCr257
PRO211 Melanoma UACC-62 PRO211 Ovarian OVCAR-8 PRO211 Renal RXF 393
PRO211 Breast MCF7 PRO211 Breast NCI/ADR-REHS 578T PRO211 Breast
T-47D PRO211 Leukemia HL-60 (TB) PRO211 Leukemia SR PRO211 NSCL
NCI-1123 PRO211 Colon HCT-116 PRO211 Melanoma LOX-IMVI PRO211
Melanoma SK-MEL-5 PRO211 Breast T-47D PRO228 Leukemia MOLT-4 PRO228
NSCL EKVX PRO228 Colon KM12 PRO228 Melanoma UACC-62 PRO228 Ovarian
OVCAR-3 PRO228 Renal TKIO PRO228 Renal SN12C PRO228 Breast MCF7
PRO228 Leukemia CCRF-CEM PRO228 Leukemia HL-60 (TB) PRO228 Colon
COLO 205 PRO228 Colon HCT-15 PRO228 Colon KM12 PRO228 CNS SF-268
PRO228 CNS SNB-75 PRO228 Melanoma LOX-IMVI PRO228 Melanoma SK-MEL2
PRO228 Melanoma UACC-257 PRO228 Ovarian IGROVI PRO228 Ovarian
OVCAR-4 PRO228 Ovarian OVCAR-5 PRO228 Ovarian OVCAR-8 PRO228 Renal
786-0 PRO228 Renal CAKI-I PRO228 Renal RXF 393 PRO228 Renal TK-10
PRO228 Renal UO-31 PRO228 Prostate PC-3 PRO228 Prostate DU-145
PRO228 Breast MCF7 PRO228 Breast NCI/ADR-REHS 578T PRO228 Breast
MDA-MB435MDA-N PRO228 Breast T-47D PRO219 Leukemia SR PRO219 NSCL
NCI-H5222 PRO219 Breast MCF7 PRO219 Leukemia K-562; RPNH-8226
PRO219 NSCL HOP-62; NCI-H322M PRO219 NSCL NCI-H460 PRO219 Colon
HT29; KM12; HCT-116 PRO219 CNS SF-539; U251 PRO219 Prostate DU-145
PRO219 Breast MDA-N PRO219 Ovarian IGROV1 PRO219 NSCL NCI-H226
PRO219 Leukemia MOLT-4 PRO219 NSCL A549/ATCC; EKVX; NCI-H23 PRO219
Colon HCC-2998 PRO219 CNS SF-295; SNB-19 PRO219 Melanoma SK-MEL-2;
SK-MEL-5 PRO219 Melanoma UACC-257; UACC-62 PRO219 Ovarian OCAR-4;
SK-OV-3 PRO219 Renal 786-0; ACHN; CAKI-1; SN 12C PRO219 Renal
TK-10; UO-31 PRO219 Breast NCI/ADR-RES; BT-549; T-47D PRO219 Breast
MDA-MB-435 PRO221 Leukemia CCRF-CEM PRO221 Leukemia MOLT-4 PRO221
NSCL HOP-62 PRO221 Breast MDA-N PRO221 Leukemia RPMI-8226; SR
PRO221 NSCL NCI-H460 PRO221 Colon HCC-2998 PRO221 Ovarian IGROV1
PRO221 Renal TK-10 PRO221 Breast MCF7 PRO221 Leukemia K-562 PRO221
Breast MDA-MB-435 PRO224 Ovarian OVCAR-4 PRO224 Renal RXF 393
PRO224 Prostate DU-145 PRO224 NSCL HOP-62; NCI-H322M PRO224
Melanoma LOX IMVI PRO224 Ovarian OVCAR-8 PRO224 Leukemia SR PRO224
NSCL NCI-H460 PRO224 CNS SF-295 PRO224 Leukemia RPMI-8226 PRO224
Breast BT-549 PRO224 Leukemia CCRF-CEM; LH-60 (TB) PRO224 Colon
HCT-116 PRO224 Breast MDA-MB-435 PRO224 Leukemia HL-60 (TB) PRO224
Colon HCC-2998 PRO224 Prostate PC-3 PRO224 CNS U251 PRO224 Colon
HCT-15 PRO224 CNS SF-539 PRO224 Renal ACHN PRO328 Leukemia
RPMI-8226 PRO328 NSCL A549/ATCC; EKVX; HOP-62 PRO328 NSCL NCI-H23;
NCI-H322M PRO328 Colon HCT-15; KM12 PRO328 CNS SF-295; SF-539;
SNB-19; U251 PRO328 Melanoma M14; UACC-257; UCAA-62 PRO328 Renal
786-0; ACHN PRO328 Breast MCF7 PRO328 Leukemia SR PRO328 Colon
NCI-H23 PRO328 Melanoma SK-MEL-5 PRO328 Prostate DU-145 PRO328
Melanoma LOX IMVI PRO328 Breast MDA-MB-435 PRO328 Ovarian OVCAR-3
PRO328 Breast T-47D PRO301 NSCL NCI-H322M PRO301 Leukemia MOLT-4;
SR PRO301 NSCL A549/ATCC; EKVX; PRO301 NSCL NCI-H23; NCI-460;
NCI-H226 PRO301 Colon COLO 205; HCC-2998; PRO301 Colon HCT-15;
KM12; HT29; PRO301 Colon HCT-1 16 PRO301 CNS SF-268; SF-295; SNB-19
PRO301 Melanoma MALME-3M; SK-MEL-2; PRO301 Melanoma SK-MEL-5;
UACC-257 PRO301 Melanoma UACC-62 PRO301 Ovarian IGROV1; OVCAR-4
PRO301 Ovarian OVCAR-5 PRO301 Ovarian OVCAR-8; SK0OV-3 PRO301 Renal
ACHN; CAKI-1; TK-10; UO-31 PRO301 Prostate PC-3; DU-145 PRO301
Breast NCI/ADR-RES; HS 578T PRO301 Breast MDA-MB-435; MDA-N; T-47D
PRO301 Melanoma M14 PRO301 Leukemia CCRF-CEM; HL-60(TB); K-562
PRO301 Leukemia RPMI-8226 PRO301 Melanoma LOX IMVI PRO301 Renal
786-0; SN12C PRO301 Breast MCF7; MDA-MB-231/ATCC PRO301 Breast
BT-549 PRO301 NSCL HOP-62 PRO301 CNS SF-539 PRO301 Ovarian OVCAR-3
PRO326 NSCL NCI-H322M PRO326 CNS SF295 PRO326 CNS ST539 PRO326 CNS
U251
[1571] 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 92
Gene Amplification
[1572] 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.
[1573] The starting material for the screen was genomic DNA
isolated from a variety cancers. The DNA is quantitated precisely,
e.g., fluorometrically. As a negative control, DNA was isolated
from the cells of ten normal healthy individuals which was pooled
and used as assay controls for the gene copy in healthy individuals
(not shown). The 5' nuclease assay (for example, TaqMan.TM.) and
real time quantitative PCR (for example, ABI Prizm 7700 Sequence
Detection 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.
[1574] 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-encoding gene.
Regions of the PRO polypeptide encoding gene which are most likely
to contain unique nucleic acid sequences and which are least likely
to have spliced out introns are preferred for the primer and probe
derivation, e.g., 3'-untranslated regions. The sequences for the
primers and probes (forward, reverse and probe) used for the PRO
polypeptide gene amplification analysis were as follows:
TABLE-US-00119 PRO187 (DNA27864 1155) 27864.tm.p:
5'-GCAGATTTTGAGGACAGCCACCTCCA 3' (SEQ ID NO:381) 27864.tm.f.
5'-GGCCTTGCAGACAACCGT 3' (SEQ ID NO:382) 27864.tm.r: 5'
CAGACTGAGGGAGATCCGAGA 3' (SEQ ID NO:383) 27864.tm.p2:
5'-CAGCTGCCCTTCCCCAACCA 3' (SEQ ID NO:384) 27864.tm.f2:
5'-CATCAAGCGCCTCTACCA-3' (SEQ ID NO:385) 27864.tm.r2:
5'-CACAAACTCGAACT,GCTTCTG-3' (SEQ ID NO:386) PRO214 (DNA32286
1191): 32286.3utr-5: 5'-GGGCCATCACAGCTCCCT-3' (SEQ ID NO:387)
32286.3utr-3b: 5'-GGGATGTGGTGAACACAGAACA-3' (SEQ ID NO:388)
32286.3utr-probe: 5'-TGCCAGCTGCATGCTGCCAGTT-3' (SEQ ID NO:389)
PRO211 (DNA32292-1131): 32292.3utr-5: 5'-CAGAAGGATGTCCCGTGGAA-3'
(SEQ ID NO:390) 32292.3utr-3: 5'-GCCGCTGTCCACTGCAG-3' (SEQ ID
NO:391) 32292.3utr-probe.rc: 5'-GACGGCATCCTCAGGGCCACA-3' (SEQ ID
NO:392) PRO230 (DNA33223-1136): 33223.tm.p3:
5'-ATGTCCTCCATGCCCACGCG-3' (SEQ ID NO:393) 33223.tm.f3:
5'-GAGTGCGACATCGAGAGCTT-3' (SEQ ID NO:394) 33223.tm.r3:
5'-CCGCAGCCTCAGTGATGA-3' (SEQ ID NO:395) 33223.3utr-5:
5'-GAAGAGCACAGCTGCAGATCC-3' (SEQ ID NO:396) 33223.3utr-3:
5'-GAGGTGTCCTGGCTTTGGTAGT-3' (SEQ ID NO:397) 33223.3utr-probe:
5'-CCTCTGGCGCCCCCACTCAA-3' (SEQ ID NO:398) PRO317 (DNA33461-1199:
33461.tm.f: 5'-CCAGGAGAGCTGGCGATG-3' (SEQ ID NO:399) 33461.tm.r:
5'-GCAAATTCAGGGCTCACTAGAGA-3' (SEQ ID NO:400) 33461.tm.p:
5'-CACAGAGCATTMTCCATCAGCAGTTCAG-3' (SEQ ID NO:401) PRO246
(DNA35639-1172): 35639.3utr-5: 5'-GGCAGAGACTTCCAGTCACTGA-3' (SEQ ID
NO:402) 35639.3utr-3: 5'-GCCAAGGGTGGTGTTAGATAGG-3' (SEQ ID NO:403)
35639.3utr-probe: 5'-CAGGCCCCCTTGATCTGTACCCCA-3' (SEQ ID NO:404)
PRO533 (DNA49435-1219): 49435.tm.f: 5'-GGGACGTGCTTCTACAAGAACAG-3'
(SEQ ID NO:405) 49435.tm.r: 5'-CAGGCTTACAATGTTATGATCAGACA-3' (SEQ
ID NO:406) 49435.tm.p: 5'-TATTCAGAGTTTTCCATTGGCAGTGCCAGT (SEQ ID
NO:407) T-3' PRO343 (DNA43318-1217: 43318.tm.f1
5'-TCTACATCAGCCTCTCTGCGC-3' (SEQ ID NO:408) 43318.tm.p1
5'-CGATCTTCTCCACCCAGGAGCGG-3' (SEQ ID NO:409) 43318.tm.r1
5'-GGAGCTGCACCCCTTGC-3' (SEQ ID NO:237) PRO232 (DNA34435-1140):
34435.3utr-5: 5'-GCCAGGCCTCACATTCGT-3' (SEQ ID NO:410)
DNA34435.3utr-probe: 5'-CTCCCTGAATGGCAGCCTGAGCA-3' (SEQ ID NO:411)
DNA34435.3utr-3: 5'-AGGTGTTTATTAAGGGCCTACGCT-3' (SEQ ID NO:412)
PRO269 (DNA38260-1180): 38260.tm.f: 5'-CAGAGCAGAGGGTGCCTTG-3' (SEQ
ID NO:413 38260.tm.p: 5'-TGGCGGAGTCCCCTCTTGGCT-3' (SEQ ID NO:414)
38260.tm.r: 5'-CCCTGTTTCCCTATGCATCACT-3' (SEQ ID NO:415) PRO304
(DNA39520-1217): 39520.tm.f 5'-TCAACCCCTGACCCTTTCCTA-3' (SEQ ID
NO:416) 39520.tm.p: 5'-GGCAGGGGACAAGCCATCTCTCCT-3' (SEQ ID NO:417)
39520.tm.r: 5'-GGGACTGAACTGCCAGCTTC-3' (SEQ ID NO:418) PRO339
(DNA43466-1225): 43466.tm.f1: 5'-GGGCCCTAACCTCATTACCTTT-3' (SEQ ID
NO:419) 43466.tm.p1: 5'-TGTCTGCCTCAGCCCCAGGAAGG-3' (SEQ ID NO:420)
43466.tm.r1: 5'-TCTGTCCACCATCTTGCCTTG-3' (SEQ ID NO:421)
[1575] 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.
[1576] 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 f6r running the instrument and for
analyzing the data.
[1577] 5' Nuclease assay data are initially expressed as Ct, or the
threshold cycle. This is defined as the cycle at which the reporter
signal accumulates above the background level of fluorescence. The
.DELTA.Ct values are used as quantitative measurement of the
relative number of starting copies of a particular tar-get sequence
in a nucleic acid sample when comparing cancer DNA results to
normal human DNA results.
[1578] 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. TABLE-US-00120 TABLE 8 Primary Lung and
Colon Tumor Profiles Primary Tumor Stage Stage Other Stage Dukes
Stage T Stage N Stage Human lung tumor AdenoCa (SRCC724) [LTI] 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] IA T1 N0 Human colon
AdenoCa (SRCC742) [CT2] MI 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, RI B T3 N0 Human colon AdenoCa (SRCC747)
[CT14] pMO, RO B pT3 pN0 Human colon AdenoCa (SRCC748) [CT15] MI,
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, RI 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
[1579] 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 Qiagen, according to the
manufacturer's instructions and the description below.
[1580] Cell Culture Lysis:
[1581] Cells were washed and trypsinized at a concentration of
7.5.times.10.sup.8 per tip and pelleted by centrifuging at 1000 rpm
for 5 minutes at 4.degree. C., followed by washing again with 1/2
volume of PBS recentrifugation. The pellets were washed a third
time, the suspended cells collected and washed 2.times. with PBS.
The cells were then suspended into 10 ml PBS. Buffer C1 was
equilibrated at 4.degree. C. Qiagen protease #19155 was diluted
into 6.25 ml cold ddH.sub.2O to a final concentration of 20 mg/ml
and equilibrated at 4.degree. C. 10 ml of G2 Buffer was prepared by
diluting Qiagen RNAse A stock (100 mg/ml) to a final concentration
of 200 .mu.g/ml.
[1582] Buffer C1 (10 ml, 4.degree. C.) and ddH.sub.2O (40 ml,
4.degree. C.) were then added to the 10 ml of cell suspension,
mixed by inverting and incubated on ice for 10 minutes. The cell
nuclei were pelleted by centrifuging in a Beckman swinging bucket
rotor at 2500 rpm at 4.degree. C. for 15 minutes. The supernatant
was discarded and the nuclei were suspended with a vortex into 2 ml
Buffer C1 (at 4.degree. C.) and 6 ml ddH.sub.2O, followed by a
second 4.degree. C. centrifugation at 2500 rpm for 15 minutes. The
nuclei were then resuspended into the residual buffer using 200
.mu.l per tip. G2 buffer (10 ml) was added to the suspended nuclei
while gentle vortexing was applied. Upon completion of buffer
addition, vigorous vortexing was applied for 30 seconds. Qiagen
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.).
[1583] Solid Human Tumor Sample Preparation and Lysis:
[1584] 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 (I tip/preparation). The protease
-solution was freshly prepared by diluting into 6.25 ml cold
ddH.sub.2O to a final concentration of 20 mg/ml and stored at
4.degree. C. G2 buffer (20 ml) was prepared by diluting DNAse A to
a final concentration of 200 mg/ml (from 100 mg/ml stock). The
tumor tissue was homogenated in 19 ml G2 buffer for 60 seconds
using the large tip of the polytron in a laminar-flow TC hood in
order to avoid inhalation of aerosols, and held at room
temperature. Between samples, the polytron was cleaned by spinning
at 2.times.30 seconds each in 2L ddH.sub.2O, followed by G2 buffer
(50 ml). If tissue was still present on the generator tip, the
apparatus was disassembled and cleaned.
[1585] Qiagen protease (prepared as indicated above, 1.0 ml) was
added, followed by vortexing and incubation at 50.degree. C. for 3
hours. The incubation and centrifugation was repeated until the
lysates were clear (e.g., incubating additional 30-60 minutes,
pelleting at 3000.times.g for 10 min., 4.degree. C.).
[1586] Human Blood Preparation and Lysis:
[1587] Blood was drawn from healthy volunteers using standard
infectious agent protocols and citrated into 10 ml samples per tip.
Qiagen protease was freshly prepared by dilution into 6.25 ml cold
ddH.sub.2O to a final concentration of 20 mg/ml and stored at
4.degree. C. G2 buffer was prepared by diluting RNAse A to a final
concentration of 200 .mu.g/ml from 100 mg/ml stock. The blood (10
ml) was placed into a 50 ml conical tube and 10 ml C1 buffer and 30
ml ddH2O (both previously equilibrated to 4.degree. C.) were added,
and the components mixed by inverting and held on ice for 10
minutes. The nuclei were pelleted with a Beckman swinging bucket
rotor at 2500 rpm, 4.degree. C. for 15 minutes and the supernatant
discarded. With a vortex, the nuclei were suspended into 2 ml C1
buffer (4.degree. C.) and 6 ml ddH.sub.2O (4.degree. C.). Vortexing
was repeated until 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. Qiagen 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.).
[1588] Purification of Cleared Lysates:
[1589] (1) Isolation of Genomic DNA:
[1590] 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 silanized, autoclaved 30 ml Corex tubes with 15
ml QF buffer (50.degree. C.). Isopropanol (10.5 ml) was added to
each sample, the tubes covered with parafin and mixed by repeated
inversion until the DNA precipitated. Samples were pelleted by
centrifugation in the SS-34 rotor at 15,000 rpm for 10 minutes at
4.degree. C. The pellet location was marked, the supernatant
discarded, and 10 ml 70% ethanol (4.degree. C.) was added. Samples
were pelleted again by centrifugation on the SS-34 rotor at 10,000
rpm for 10 minutes at 4.degree. C. The pellet location was marked
and the supernatant discarded. The tubes were then placed on their
side in a drying rack and dried 10 minutes at 37.degree. C., taking
care not to overdry the samples.
[1591] 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.
[1592] (2) Quantitation of Genomic DNA and Preparation for Gene
Amplification Assay:
[1593] 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.
[1594] Fluorometric DNA quantitation was then performed on the
diluted material (20-600 ng/ml) using the manufacturer's guidelines
as modified below. This was accomplished by allowing a Hoeffer DyNA
Quant 200 fluorometer to warm-up for about 15 minutes. The Hoechst
dye working solution (#H33258, 10 .mu.l, prepared within 12 hours
of use) was diluted into 100 ml 1.times. TNE buffer. A 2 ml cuvette
was filled with the fluorometer solution, placed into the machine,
and the machine was zeroed. pGEM 3Zf(+) (2 .mu.l, lot #360851026)
was added to 2 ml of fluorometer solution and calibrated at 200
units. An additional 2 .mu.l of pGEM 3Zf(+) DNA was then tested and
the reading confirmed at 400.+-.10 units. Each sample was then read
at least in triplicate. When 3 samples were found to be within 10%
of each other, their average was taken and this value was used as
the quantification value.
[1595] The fluorometricly determined concentration was then used to
dilute each sample to 10 ng/.mu.l in ddH.sub.2O. 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.
[1596] Gene Amplification Assay:
[1597] 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.
TABLE-US-00121 TABLE 9 .DELTA.Ct values in lung and colon primary
tumors and cell line models Primary Tumors or Cell PRO lines PRO187
533 PRO214 PRO343 PRO211 PRO230 PRO246 PRO317 PRO232 PRO269 PRO304
PRO339 LT7 1.52 1.04 1.08 LT13 2.74 1.85 2.71 1.88 3.42 1.63 1.90
1.27 1.29 1.04 2.98 1.83 2.23 2.26 3.22 1.68 2.24 2.44 2.84 2.93
2.15 2.75 2.53 1.82 LT3 1.57 1.97 1.06 1.86 1.17 LT4 1.17 1.18 LT9
1.42 1.04 1.80 1.03 LT12 2.70 1.38 2.23 1.51 2.86 1.54 2.54 2.40
1.14 1.15 1.26 2.90 1.49 1.50 1.27 2.96 2.47 1.74 2.27 2.92 1.25
2.68 2.28 1.34 LT30 1.67 2.13 1.36 LT21 1.26 1.09 1.50 LT1-A 1.02
1.18 1.29 LT6 1.93 LT10 1.96 1.07 2.57 LT11 1.09 1.67 1.00 2.05
1.32 3.43 2.20 1.14 1.51 1.39 1.80 1.89 1.14 1.41 2.33 1.54 1.02
LT15 3.75 1.77 3.62 2.44 4.32 2.11 2.06 1.86 1.36 1.34 3.92 1.58
1.30 2.16 4.47 1.56 2.76 3.64 1.63 2.94 3.56 3.32 2.68 LT16 2.10
1.66 1.70 1.25 1.15 1.55 1.00 2.04 1.08 1.83 1.33 LT21 1.32 1.93
1.15 1.85 1.26 2.68 2.29 1.35 1.42 1.68 1.63 1.87 2.30 1.39 1.69
2.03 1.30 1.10 1.33 1.30 LT18 1.17 1.04 LT19 4.05 1.67 2.09 3.82
2.42 4.05 1.91 2.51 1.21 1.60 1.15 3.99 1.98 2.55 4.92 1.68 2.03
4.93 1.16 3.78 4.76 HF- 1.58 000840 Calu-1 1.08 SW900 1.86 CT2 3.56
2.49 1.95 1.42 2.75 3.49 2.36 CT3 2.06 1.15 1.34 CT8 1.01 1.48 1.29
1.58 CT10 1.81 1.84 1.88 1.00 1.88 1.49 1.55 C12 1.81 1.74 1.13
CT14 1.82 2.48 2.33 1.36 1.72 1.24 CT15 1.63 2.06 1.33 1.41 1.04
CT16 1.95 1.78 1.40 CT17 2.04 2.40 1.74 CT1 1.24 1.22 1.27 1.25
2.41 1.34 1.46 1.14 CT4 1.36 1.77 1.33 1.32 1.10 1.17 2.05 1.42
1.02 CT5 2.96 1.56 2.68 1.76 2.27 1.33 1.59 2.99 2.76 1.64 2.39 CT6
1.10 1.33 1.01 1.14 CT7 1.40 1.66 1.39 1.00 CT9 1.39 1.16 1.09 1.24
1.13 CT11 2.22 2.05 1.55 2.01 1.75 1.48 1.92 2.26 1.85 1.83 1.12
HF000539 1.57 SW620 1.14 HF000611 4.64 HF000733 1.93 2.33 HF000716
1.68 2.82 CT18 1.29
SUMMARY
[1598] Because amplification of the various DNA's as described
above occurs in various tumors, it is likely associated with tumor
formation and/or growth. As a result, antagonists (e.g.,
antibodies) directed against these polypeptides would be expected
to be useful in cancer therapy.
Example 94
Detection of PRO Polypeptides That Affect Glucose or FFA Uptake by
Prim=Rat Adipocytes (Assay 94)
[1599] 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.
[1600] In a 96 well format, PRO polypeptides to be assayed are
added to primary rat adipocytes, and allowed to incubate overnight.
Samples are taken at 4 and 16 hours and assayed for glycerol,
glucose and FFA uptake. After the 16 hour incubation, insulin is
added to the media and allowed to incubate for 4 hours. At this
time, a sample is taken and glycerol, glucose and FFA uptake is
measured. Media containing insulin without the PRO polypeptide is
used as a positive reference control. As the PRO polypeptide being
tested may either stimulate or inhibit glucose and FFA uptake,
results are scored as positive in the assay if greater than 1.5
times or less than 0.5 times the insulin control.
[1601] The following PRO polypeptides tested positive as
stimulators of glucose and/or FFA uptake in this assay: PRO221,
PRO235, PRO245, PRO295, PRO301 and PRO332.
[1602] The following PRO polypeptides tested positive as inhibitors
of glucose and/or FFA uptake in this assay: PRO214, PRO219, PRO228,
PRO222, PRO231 and PRO265.
Example 95
Chondrocyte Re-Differentiation Assay (Assay 110)
[1603] This assay shows that certain polypeptides of the invention
act to induce redifferentiation of chondrocytes, therefore, are
expected to be useful for the treatment of various bone and/or
cartilage disorders such as, for example, sports injuries and
arthritis. The assay is performed as follows. Porcine chondrocytes
are isolated by overnight collagenase digestion of articulary
cartilage of metacarpophalangeal joints of 4-6 month old female
pigs. The isolated cells are then seeded at 25,000 cells/cm.sup.2
in Ham F-12 containing 10% FBS and 4 .mu.g/ml gentamycin. lie
culture media is changed every third day and the cells are then
seeded in 96 well plates at 5,000 cells/well in 100 .mu.l of the
same media without serum and 100 .mu.l of the test PRO polypeptide,
5 nM staurosporin (positive control) or medium alone (negative
control) is added to give a final volume of 200 .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.
[1604] The following polypeptide tested positive in this assay:
PRO214, PRO219, PRO229, PRO222, PRO224, PRO230, PRO257, PRO272 and
PRO301.
Example 96
Fetal Hemoglobin Induction in an Ertyhroblastic Cell Line (Assay
107)
[1605] 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 uM hemin and as a
negative-control, the cells are untreated. After 5 days, cell
lysates are prepared and analyzed for the expression of gamma glob
in (a fetal marker). A positive in the assay is a gamma globin
level at least 2-fold above the negative control.
[1606] The following polypeptides tested positive in this assay:
PRO221 and PRO245.
Example 97
Mouse Kidney Mesangial Cell Proliferation Assay (Assay 92)
[1607] This assay shows that certain polypeptides of the invention
act to induce proliferation of mammalian kidney mesangial cells
and, therefore, are useful for treating kidney disorders associated
with decreased mesangial cell function such as Berger disease or
other nephropathies associated with Schonlein-Henoch purpura,
celiac disease, dermatitis herpetiformis or Crohn disease. The
assay is performed as follows. On day one, mouse kidney mesangial
cells are plated on a 96 well plate in growth media (3:1 mixture of
Dulbecco's modified Eagle's medium and Ham's F12 medium, 95% fetal
bovine serum, 5% supplemented with 14 mM HEPES) and grown
overnight. On day 2, PRO polypeptides are diluted at 2
concentrations (1% and 0.1%) in serum-free medium and added to the
cells. Control samples are serum-free medium alone. On day 4, 20 ul
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.
[1608] The following polypeptide tested positive in this assay:
PRO227.
Example 98
Proliferation of Rat Utricular Supporting Cells (Assay 54)
[1609] This assay shows that certain polypeptides of the invention
act as potent mitogens for inner ear supporting cells which are
auditory hair cell progenitors and, therefore, are useful for
inducing the regeneration of auditory hair cells and treating
hearing loss in mammals. The assay is performed as follows. Rat
UEC-4 utricular epithelial cells are aliquoted into 96 well plates
with a density of 3000 cells/well in 200 .mu.l of serum-containing
medium at 33.degree. C. The cells are cultured overnight and are
then switched to 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.
[1610] The following polypeptides tested positive in this assay:
PRO310 and PRO346.
Example 99
Chondrocyte Proliferation Assay (Assay 111)
[1611] 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.
[1612] Porcine chondrocytes are isolated by overnight collagenase
digestion of articular cartilage of the metacarpophalangeal joint
of 4-6 month old female pigs. The isolated cells are then seeded at
25,000 cells/cm.sup.2 in Ham F-12 containing 10% FBS and 4 .mu.g/ml
gentamycin. The culture media is changed every third day and the
cells are reseeded to 25,000 cells/cm.sup.2 every five days. On day
12, the cells are seeded in 96 well plates at 5,000 cellstwell in
100 ul of the same media without serum and 100 .mu.l of either
serum-free medium (negative control), staurosporin (final
concentration of 5 nM; positive control) or the test PRO
polypeptide are added to give a final volume of 200 .mu.l/well.
After 5 days at 37.degree. C., 20 .mu.l of Alamar blue is added to
each well and the plates are incubated for an additional 3 hours at
37.degree. C. The fluorescence is then measured in each well (Ex:
530 nm; Em: 590 nm). The fluorescence of a plate containing 200
.mu.l of the serum-free medium is measured to obtain the
background. A positive result in the assay is obtained when the
fluorescence of the PRO polypeptide treated sample is more like
that of the positive control than the negative control.
[1613] The following PRO polypeptides; tested positive in this
assay: PRO219, PRO222, PRO317, PRO257, PRO265, PRO287, PRO272 and
PRO533.
Example 100
Inhibition of Heart Neonatal Hypertrophy Induced by LIF+ET-1 (Assay
74)
[1614] This assay is designed to determine whether PRO polypeptides
of the present invention show the ability to inhibit neonatal heart
hypertrophy induced by LIF and endothelin-I (ET-1). A test compound
that provides a positive response in the present assay would be
useful for the therapeutic treatment of cardiac insufficiency
diseases or disorders characterized or associated with an undesired
hypertrophy of the cardiac muscle.
[1615] Cardiac myocytes from 1-day old Harlan Sprague Dawley rats
(180 .mu.l at 7.5.times.10.sup.4/ml, serum<0.1, freshly
isolated) are introduced on day 1 to 96-well plates previously
coated with DMEM/F1212+4% FCS. Test PRO polypeptide samples or
growth medium alone (negative control) are then added directly to
the wells on day 2 in 20 .mu.l volume. LIF+ET-1 are then added to
the wells on day 3. The cells are stained after an additional 2
days in culture and are then scored visually the next day. A
positive in the assay occurs when the PRO polypeptide treated
myocytes are visually smaller on the average or less numerous than
the untreated myocytes.
[1616] The following PRO polypeptides tested positive in this
assay: PRO238.
Example 101
Tissue Expression Distribution
[1617] Oligonucleotide probes were constructed from some of the PRO
polypeptide-encoding nucleotide sequences shown in the accompanying
figures for use in quantitative PCR amplification reactions. The
oligonucleotide probes were chosen so as to give an approximately
200-600 base pair amplified fragment from the 3' end of its
associated template in a standard PCR reaction. The oligonucleotide
probes were employed in standard quantitative PCR amplification
reactions with cDNA libraries isolated from different human adult
and/or fetal tissue sources and analyzed by agarose gel
electrophoresis so as to obtain a quantitative determination of the
level of expression of the PRO polypeptide-encoding nucleic acid in
the various tissues tested. Knowledge of the expression pattern or
the differential expression of the PRO polypeptide-encoding nucleic
acid in various different human tissue types provides a diagnostic
marker useful for tissue typing, with or without other
tissue-specific markers, for determining the primary tissue source
of a metastatic tumor, and the like. These assays provided the
following results. TABLE-US-00122 Tissues With Tissues Lacking DNA
Molecule Significant Expression Significant Expression
DNA34436-1238 lung, placenta, brain testis DNA35557-1137 lung,
kidney, brain placenta DNA35599-1168 kidney, brain liver, placenta
DNA35668-1171 liver, lung, kidney placenta, brain DNA36992-1168
liver, lung, kidney, brain placenta DNA39423-1182 kidney, brain
liver DNA40603-1232 liver brain, kidney, lung DNA40604-1187 liver
brain, kidney, lung DNA41379-1236 lung, brain liver DNA33206-1165
heart, spleen, substantia nigra, dendrocytes hippocampus,
cartilage, prostate, HUVEC DNA3.4431-1177 spleen, HUVEC, brain,
colon tumor, cartilage, heart, uterus prostate, THP-1 macrophages
DNA41225-1217 HUVEC, uterus, colon spleen, brain, heart, tumor,
cartilage, prostate IM-9 lymphoblasts
Example 102
In situ Hybridization
[1618] In situ hybridization is a powerful and versatile technique
for the detection and localization of nucleic acid sequences within
cell or tissue preparations. It may be useful, for example, to
identify sites of gene expression, analyze the tissue distribution
of transcription, identify and localize viral infection, follow
changes in specific mRNA synthesis and aid in chromosome
mapping.
[1619] 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 g/ml) for IS
minutes at 37.degree. C., and further processed for in situ
hybridization as described by Lu and Gillett, supra. A [.sup.33-P]
UTP-labeled antisense riboprobe was generated from a PCR product
and hybridized at 55.degree. C. overnight. The slides were dipped
in Kodak NTB2 nuclear track emulsion and exposed for 4 weeks.
.sup.33P-Riboyrobe Synthesis
[1620] 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:
[1621] 2.0 .mu.l 5.times. transcription buffer
[1622] 1.0 .mu.l DTT (100 mM)
[1623] 2.0 .mu.l NTP mix (2.5 mM: 10 .mu.; each of 10 mM GTP, CTP
& ATP+10 .mu.l H.sub.2O)
[1624] 1.0 .mu.l UTP (50 .mu.M)
[1625] 1.0 .mu.l Rnasin
[1626] 1.0 .mu.l DNA template (1 .mu.g)
[1627] 1.0 .mu.l H.sub.2O
[1628] 1.00 RNA polymerase (for PCR products T3=AS, T7=S,
usually)
[1629] The tubes were incubated at 37.degree. C. for one hour. 1.0
.mu.l RQI DNase were added, followed by incubation at 37.degree. C.
for 15 minutes. 90 .mu.l TE (10 mM Tris pH 7.6/1 mM EDTA pH 8.0)
were added, and the mixture was pipetted onto DE81 paper. The
remaining solution was loaded in a Microcon-50 ultrafiltration
unit, and spun using program 10 (6 minutes). The filtration unit
was inverted over a second tube and spun using program 2 (3
minutes). After the final recovery spin, 100 .mu.l TE were added. 1
.mu.l of the final product was pipetted on DE81 paper and counted
in 6 ml of Biofluor II.
[1630] The probe was run on a TBE/urea gel. 1-3 .mu.l of the probe
or 5 .mu.l of RNA Mrk III were added to 3 .mu.l of loading buffer.
After heating on a 95.degree. C. heat block for three minutes, the
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.
.sup.33P-Hybridization
[1631] A. Pretreatment of Frozen Sections
[1632] The slides were removed from the freezer, placed on
aluminium trays and thawed at room temperature for 5 minutes. The
trays were placed in 55.degree. C. incubator for five minutes to
reduce condensation. The slides were fixed for 10 minutes in 4%
paraformaldehyde on ice in the fume hood, and washed in
0.5.times.SSC for 5 minutes, at room temperature (25 ml
20.times.SSC+975 ml SQ H.sub.2O). After deproteination in 0.5
.mu.g/ml proteinase K for 10 minutes at 37.degree. C. (12.5 .mu.l
of 10 mg/ml stock in 250 ml prewarmed RNase-free RNAse buffer), the
sections were washed in 0.5.times.SSC for 10 minutes at room
temperature. The sections were dehydrated in 70%, 95%, 100%
ethanol, 2 minutes each.
[1633] B. Pretreatment of Paraffin-Embedded Sections
[1634] The slides were deparaffinized, placed in SQ H.sub.2O, and
rinsed twice in 2.times.SSC at room temperature, for 5 minutes each
time. The sections were deproteinated in 20 .mu.g/ml proteinase K
(500 .mu.l of 10 mg/ml in 250 ml RNase-free RNase buffer;
37.degree. C., 15 minutes)-human embryo, or 8.times. proteinase K
(100 .mu.l in 250 ml Rnase buffer, 37.degree. C., 30
minutes)-formalin tissues. Subsequent rinsing in 0.5.times.SSC and
dehydration were performed as described above.
[1635] C. Prehybridization
[1636] 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.
[1637] D. Hybridization
[1638] 1.0.times.10.sup.6 cpm probe and 1.0 .mu.l tRNA (50 mg/ml
stock) per slide were heated at 95.degree. C. for 3 minutes. The
slides were cooled on ice, and 48 .mu.l hybridization buffer were
added per slide. After vortexing, 50 .infin.l .sup.33P Mix Were
added to 50 .mu.l prehybridization on slide. The slides were
incubated overnight at 55.degree. C.
[1639] E. Washes
[1640] 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=4 L), followed by RNaseA treatment at 37.degree. C. for 30
minutes (500 .mu.l of 10 mg/ml in 250 ml Rnase buffer=20 .mu.g/ml),
The slides were washed 2.times.10 minutes with 2.times.SSC, EDTA at
room temperature. The stringency wash conditions were as follows: 2
hours at 55.degree. C., 0.1.times.SSC, EDTA (20 ml 20.times.SSC+16
ml EDTA, V.sub.f=4 L).
[1641] F. Olistonucleotides
[1642] In situ analysis was performed on a variety of DNA sequences
disclosed herein. The oligonucleotides employed for these analyses
are as follows. TABLE-US-00123 (1) DNA33094-1131 (PRO217) p1
5-GGATTCTAATACGACTCACTATAGGGCTCAGA (SEQ ID NO:348)
AAAGCGCAACAGAGAA-3' p2 5'-CTATGAAATTAACCCTCACTAAAGGGATGTC (SEQ ID
NO:349) TTCCATGCCAACCTTC-3' (2) DNA33223-1136 (PRO230) p1
5'-GGATTCTAATACGACTCACTATAGGGCGGCG (SEQ ID NO:350)
ATGTCCACTGGGGCTAC-3' p2 5'-CTATGAAATTAACCCTCACTAAAGGGACGAG (SEQ ID
NO:351) GAAGATGGGCGGATGGT-3' (3) DNA34435-1140 (PRO232) p1
5'-GGATTCTAATACGACTCACTATAGGGCACCC (SEQ ID NO:352)
ACGCGTCCGGCTGCTT-3' p2 5'-CTATGAAATTAACCCTCACTAAAGGGACGGG (SEQ ID
NO:353) GGACACCACGGACCAGA-3' (4) DNA35639-1172 (PRO246) p1
5'-GGATTCTAATACGACTCACTATAGGGCTTGC (SEQ ID NO:354)
TGCGGTTTTTGTTCCTG-3' p2 5'-CTATGAAATTAACCCTCACTAAAGGGAGCTG (SEQ ID
NO:355) CCGATCCCACTGGTATT-3' (5) DNA49435-1219 (PRO533) p1
5'-GGATTCTAATACGACTCACTATAGGGCGGAT (SEQ ID NO:356)
CCTGGCCGGCCTCTG-3' p2 5'-CTATGAAATTAACCCTCACTAAAGGGAGCCC (SEQ ID
NO:357) GGGCATGGTCTCAGTTA-3' (6) DNA35638-1141 (PRO245) p1
5'-GGATTCTAATACGACTCACTATAGGGCGGGA (SEQ ID NO:358)
AGATGGCGAGGAGGAG-3' p2 5'-CTATGAAATTAACCCTCACTAAAGGGACCAA (SEQ ID
NO:359) GGCCACAAACGGAAATC-3' (7) DNA33089-1132 (PRO221 p1
5'-GGATTCTAATACGACTCACTATAGGGCTGTG (SEQ ID NO:360)
CTTTCATTCTGCCAGTA-3' p2 5-ATGAAATTAACCCTCACTAAAGGGAGGGTACA (SEQ ID
NO:361) ATTAAGGGGTGGAT-3' (8) DNA35918-1174 (PRO258) p1
5'-GGATTCTAATACGACTCACTATAGGGCCCGC (SEQ ID NO:362)
CTCGCTCCTGCTCCTG-3' p2 5-ATGAAATTAACCCTCACTAAAGGGAGGATTGC (SEQ ID
NO:363) CGCGACCCTCACAG-3' (9) DNA32286-1191 (PRO214) p1
5'-GGATTCTAATACGACTCACTATAGGGCCCCT (SEQ ID NO:364)
CCTGCCTTCCCTGTCC-3' p2 5'-CTATGAAATTAACCCTCACTAAAGGGAGTGG (SEQ ID
NO:365) TGGCCGCGATTATCTGC-3' (10) DNA33221-1133 (PRO224) p1
5'-GGATTCTAATACGACTCACTATAGGGCGCAG (SEQ ID NO:366)
CGATGGCAGCGATGAGG-3' p2 5'-CTATGAAATTAACCCTCACTAAAGGGACAGA (SEQ ID
NO:367) CGGGGCAGAGGGAGTG-3' (11) DNA35557-1137 (PRO234) p1
5'-GGATTCTAATACGACTCACTATAGGGCCAGG (SEQ ID NO:368)
AGGCGTGAGGAGAAAC-3' p2 5'-CTATGAAATTAACCCTCACTAAAGGGAAAGA (SEQ ID
NO:369) CATGTCATCGGGAGTGG-3' (12) DNA33100-1159 (PRO229) p1
5'-GGATTCTAATACGACTCACTATAGGGCCGGG (SEQ ID NO:370)
TGGAGGTGGAACAGAAA-3' p2 5'-CTATGAAATTAACCCTCACTAAAGGGACACA (SEQ ID
NO:371) GACAGAGCCCCATACGC-3' (13) DNA34431-1177 (PRO263) p1
5'-GGATTCTAATACGACTCACTATAGGGCCAGG (SEQ ID NO:372)
GAAATCCGGATGTCTC-3' p2 5'-ATGAAATTAACCCTCACTAAAGGGAGTAAGG (SEQ ID
NO:373) GGATGCCACCGAGTA-3' (14) DNA38268-1188 (PRO295). p1
5'-GGATTCTAATACGACTCACTATAGGGCCAGC (SEQ ID NO:374)
TACCCGCAGGAGGAGG-3' p2 5'-CTATGAAATTAACCCTCACTAAAGGGATCCC (SEQ ID
NO:375) AGGTGATGAGGTCCAGA-3'
[1643] In situ analysis was performed on a variety of DNA sequences
disclosed herein. The results from these analyses are as
follows.
(1) DNA33094-1131 (PRO217)
[1644] Highly distinctive expression pattern, that does not
indicate an obvious biological function. In the human embryo it was
expressed in outer smooth muscle layer of the GI tract, respiratiry
cartilage, branching respiratory epithelium, osteoblasts, tendons,
gonad, in the optic nerve head and developing dermis. In the adult
expression was observed in the epidermal pegs of the chimp tongue,
the basal epithelial/myoepithelial cells of the prostate and
urinary bladder. Also expressed in the alveolar lining cells of the
adult lung, mesenchymal cells juxtaposed to erectile tissue in the
penis and the cerebral cortex (probably glial cells). In the
kidney, expression was only seen in disease, in cells surrounding
thyroidized renal tubules. [1645] 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. [1646] Adult human
tissues examined: Kidney (normal and end-stage), adrenal,
myocardium, aorta, spleen, lymph node, gall bladder, pancreas,
lung, skin, eye (inc. retina), prostate, bladder, liver (normal,
cirrhotic, acute failure). [1647] Non-human primate tissues
examined:
[1648] (a) Chimp Tissue: Salivary gland, stomach, thyroid,
parathyroid, skin, thymus, ovary, lymph node.
[1649] (b) Rhesus Monkey Tissues: Cerebral cortex, hippocampus,
cerebellum, penis.
(2) DNA33223-1136 (PRO230)
[1650] Sections show an intense signal associated with arterial and
venous vessels in the fetus. In arteries the signal appeared to be
confined to smooth-muscle/pericytic cells. The signal is also seen
in capillary vessels and in glomeruli. It is not clear whether or
not endothelial cells are expressing this mRNA. Expression is also
observed in epithelial cells in the fetal lens. Strong expression
was also seen in cells within placental trophoblastic villi, these
cells lie between the trophoblast and the fibroblast-like cells
that express HGF-uncertain histogenesis. In the adult, there was no
evidence of expression and the wall of the aorta and most vessels
appear to be negative. However, expression was seen over vascular
channels in the normal prostate and in the epithelium lining the
gallbladder. insurers expression was seen in the vessels of the
soft-tissue sarcoma and a renal cell carcinoma. In summary, this is
a molecule that shows relatively specific vascular expression in
the fetus as well as in some adult organs. Expression was also
observed in the fetal lens and the adult gallbladder.
[1651] In a secondary screen, vascular expression was observed,
similar to that observed above, seen in fetal blocks. Expression is
on vascular smooth muscle, rather than endothelium. Expression also
seen in smooth muscle of the developing oesophagus, so as reported
previously, this molecule is not vascular specific. Expression was
examined in 4 lung and 4 breast carcinomas. Substantial expression
was seen in vascular smooth muscle of at least 3/4 lung cancers and
2/4 breast cancers. In addition, in one breast carcinoma,
expression was observed in peritumoral stromal cells of uncertain
histogenesis (possibly myofibroblasts). No endothelial cell
expression was observed in this study.
(3) DNA34435-1140 (PRO232)
[1652] Strong expression in prostatic epithelium and bladder
epithelium, lower level of expression in bronchial epithelium. High
background/low level expression seen in a number of sites,
including among others, bone, blood, chondrosarcoma, adult heart
and fetal liver. It is felt that this level of signal represents
background, partly because signal at this level was seen over the
blood. All other tissues negative. [1653] Human fetal tissues
examined (E12-E16 weeks) include: Placenta, umbilical cord, liver,
kidney, adrenals, thyroid, lungs, heart, great vessels, oesophagus,
stomach, small intestine, spleen, thymus, pancreas, brain, eye,
spinal cord, body wall, pelvis, testis and lower limb. [1654] Adult
human tissues examined: Kidney (normal and end-stage), adrenal,
spleen, lymph node, pancreas, lung, eye (in c. retina), bladder,
liver (normal, cirrhotic, acute failure). [1655] Non-human primate
tissues examined:
[1656] Chimp Tissues: adrenal
[1657] Rhesus Monkey Tissues: Cerebral cortex, hippocampus
[1658] In a secondary screen, expression was observed in the
epithelium of the prostate, the superficial layers of the
urethelium of the urinary bladder, the urethelium lining the renal
pelvis and the urethelium of the ureter (1 out of 2 experiments).
The urethra of a rhesus monkey was negative; it is unclear whether
this represents a true lack of expression by the urethra, or if it
is the result of a failure of the probe to cross react with rhesus
tissue. The findings in the prostate and bladder are similar to
those previously described using an isotopic detection technique.
Expression of the mRNA for this antigen is NOT prostate epithelial
specific. The antigen may serve as a useful marker for urethelial
derived tissues. Expression in the superficial, post-mitotic cells,
of the urinary tract epithelium also suggest that it is unlikely to
represent a specific stem cell marker, as this would be expected to
be expressed specifically in basal epithelium. [1659] (4)
DNA35639-1172 (PR0246)
[1660] Strongly expressed in fetal vascular endothelium, including
tissues of the CNS. Lower level of expression in adult vasculature,
including the CNS. Not obviously expressed at higher levels in
tumor vascular endothelium. Signal also seen over bone matrix and
adult spleen, not obviously cell associated, probably related to
non-specific background at these sites. [1661] Human fetal tissues
examined (E12-E16 weeks) include: Placenta, umbilical cord, liver,
kidney, adrenals, thyroid, lungs, heart, great vessels, oesophagus,
stomach, small intestine, spleen, thymus, pancreas, brain, eye,
spinal cord, body wall, pelvis, testis and lower limb. [1662] Adult
human tissues examined: Kidney (normal and end-stage), adrenal,
spleen, lymph node, pancreas, lung, eye (inc. retina), bladder,
liver (normal, cirrhotic, acute failure). [1663] Non-human primate
tissues examined:
[1664] Chimp Tissues: adrenal
[1665] Rhesus Monkey Tissues: Cerebral cortex, hippocampus
(5) DNA49435-1219 (PR0533)
[1666] Moderate expression over cortical neurones in the fetal
brain. Expression over the inner aspect of the fetal retina,
possible expression in the developing lens. Expression over fetal
skin, cartilage, small intestine, placental villi and umbilical
cord. In adult tissues there is an extremely high level of
expression over the gallbladder epithelium. Moderate expression
over the adult kidney, gastric and colonic epithelia. Low-level
expression was observed over many cell types in many tissues, this
may be related to stickiness of the probe, these data should
therefore be interpreted with a degree of caution. [1667] Human
fetal tissues examined (E12-E16 weeks) include: Placenta, umbilical
cord, liver, kidney, adrenals, thyroid, lungs, heart, great
vessels, oesophagus, stomach, small intestine, spleen, thymus,
pancreas, brain, eye, spinal cord, body wall, pelvis, testis and
lower limb. [1668] Adult human tissues examined: Kidney (normal and
end-stage), adrenal, spleen, lymph node, pancreas, lung, eye (inc.
retina), bladder, liver (normal, cirrhotic, acute failure). [1669]
Non-human primate tissues examined:
[1670] Chimp Tissues: adrenal
[1671] Rhesus Monkey Tissues: Cerebral cortex, hippocampus,
cerebellum.
(6) DNA35638-1141 (PRO245)
[1672] Expression observed in the endothelium lining a subset of
fetal and placental vessels. Endothelial expression was confined to
these tissue blocks. Expression also observed over intermediate
trophoblast cells of placenta. Expression also observed tumor
vasculature but not in the vasculature of normal tissues of the
same type. All other tissues negative. [1673] 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. [1674] Adult tissues
examined: Liver, kidney, adrenal, myocardium, aorta, spleen, lymph
node, pancreas, lung, skin, cerebral cortex (rm), hippocampus(rm),
cerebellum(rm), penis, eye, bladder, stomach, gastric carcinoma,
colon, colonic carcinoma, thyroid (chimp), parathyroid (chimp)
ovary (chimp) and chondrosarcoma. Acetominophen induced liver
injury and hepatic cirrhosis (7) DNA33089-1132 (PRO221)
[1675] Specific expression over fetal cerebral white and grey
matter, as well as over neurones in the spinal cord. Probe appears
to cross react with mt. Low level of expression over cerebellar
neurones in adult rhesus brain. All other tissues negative. [1676]
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. [1677] Adult tissues examined: Liver, kidney, adrenal,
myocardium, aorta, spleen, lymph node, pancreas, lung, skin,
cerebral. cortex (rm), hippocampus(rm), cerebellum(rm), penis, eye,
bladder, stomach, gastric carcinoma, colon, colonic carcinoma and
chondrosarcoma. Acetominophen induced liver injury and hepatic
cirrhosis (8) DNA35918-1174 (PRO258)
[1678] Strong expression in the nervous system. In the rhesus
monkey brain expression is observed in cortical, hippocampal and
cerebellar neurones. Expression over spinal neurones in the fetal
spinal cord, the developing brain and the inner aspects of the
fetal retina. Expression over developing dorsal root and autonomic
ganglia as well as enteric nerves. Expression observed over
ganglion cells in the adult prostate. In the rat, there is strong
expression over the developing hind brain and spinal cord. Strong
expression over interstitial cells in the placental villi. All
other tissues were negative. [1679] 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. [1680] Adult tissues examined: Liver,
kidney, renal cell carcinoma, adrenal, aorta, spleen, lymph node,
pancreas, lung, myocardium, skin, cerebral cortex (rm),
hippocampus(rm), cerebellum(rm), bladder, prostate, stomach,
gastric carcinoma, colon, colonic carcinoma, thyroid (chimp),
pamthyroid (chimp) ovary (chimp) and chondrosarcoma. Acetominophen
induced liver injury and hepatic cirrhosis. (9) DNA32286-1191
(PR0214)
[1681] Fetal tissue: Low level throughout mesenchyme. Moderate
expression in placental stromal cells in membranous tissues and in
thyroid. Low level expression in cortical neurones. [1682] Adult
tissue: all negative. [1683] 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. [1684] Adult tissues examined include:
Liver, kidney, adrenal, myocardium, aorta, spleen, lymph node,
pancreas, lung and skin. (10) DNA33221-1133 (PR0224)
[1685] Expression limited to vascular endothelium in fetal spleen,
adult spleen, fetal liver, adult thyroid and adult lymph node
(chimp). Additional site of expression is the developing spinal
ganglia. All other tissues negative. [1686] Human fetal tissues
examined (E12-E16 weeks) include: Placenta, umbilical cord, liver,
kidney, adrenals, thyroid, lungs, heart, great vessels, oesophagus,
stomach, small all intestine, spleen, thymus, pancreas, brain, eye,
spinal cord, body wall, pelvis and lower limb. [1687] Adult human
tissues examined: Kidney (normal and end-stage), adrenal,
myocardium, aorta, spleen, lymph node, pancreas, lung, skin, eye
(inc. retina), bladder, liver (normal, cirrhotic, acute failure).
[1688] Non-human primate tissues examined:
[1689] Chimp Tissues: Salivary gland, stomach, thyroid,
parathyroid, skin, thymus, ovary, lymph node.
[1690] Rhesus Monkey Tissues: Cerebral cortex, hippocampus,
cerebellum, penis.
(11) DNA35557-1137 (PR0234)
[1691] Specific expression over developing motor neurones in
ventral aspect of the fetal spinal cord (will develop into ventral
horns of spinal cord). All other tissues negative. Possible role in
growth, differentiation and/or development of spinal motor neurons.
[1692] 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. [1693] Adult tissues examined: Liver, kidney, adrenal,
myocardium, aorta, spleen, lymph node, pancreas, lung, skin,
cerebral cortex (rm), hippocampus(rm), cerebellum(rm), penis, eye,
bladder, stomach, gastric carcinoma, colon, colonic carcinoma and
chondrosarcoma. Acetominophen induced liver injury and hepatic
cirrhosis (12) DNA33100-1159 (PRO229)
[1694] Striking expression in mononuclear phagocytes (macrophages)
of fetal and adult spleen, liver, lymph node and adult thymus (in
tingible body macrophages). The highest expression is in the
spleen. All other tissues negative. Localisation and homology are
entirely consistent with a role as a scavenger receptor for cells
of the reticuloendothelial system. Expression also observed in
placental mononuclear cells. [1695] 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. [1696] Adult human
tissues examined: Kidney (normal and end-stage), adrenal,
myocardium, aorta, spleen, lymph node, gall bladder, pancreas,
lung, skin, eye (inc. retina), prostate, bladder, liver (normal,
cirrhotic, acute failure). [1697] Non-human primate tissues
examined:
[1698] Chimp Tissues: Salivary gland, stomach, thyroid,
parathyroid, skin, thymus, ovary, lymph node.
[1699] Rhesus Monkey Tissues: Cerebral cortex, hippocampus,
cerebellum, penis.
(13) DNA34431-1177 (PRO263)
[1700] Widespread expression in human fetal tissues and placenta
over mononuclear cells, probably macrophages .+-. lymphocytes. The
cellular distribution follows a perivascular pattern in many
tissues. Strong expression also seen in epithelial cells of the
fetal adrenal cortex. All adult tissues were negative. [1701] 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. [1702]
Adult tissues examined: Liver, kidney, adrenal, spleen, lymph node,
pancreas, lung, skin, cerebral cortex (rm), hippocampus(rm),
cerebellum(rm), bladder, stomach, colon and colonic carcinoma.
Acetominophen induced liver injury and hepatic cirrhosis.
[1703] A secondary screen evidenced expression over stromal
mononuclear cells probably histiocytes.
(14) DNA38268-1188 (PRO295)
[1704] High expression over ganglion cells in human fetal spinal
ganglia and over large neurones in the anterior horns of the
developing spinal cord. In the adult there is expression in the
chimp adrenal medulla (neural), neurones of the rhesus monkey brain
(hippocampus [+++] and cerebral cortex) and neurones in ganglia in
the normal adult human prostate (the only section that contains
ganglion cells, ie expression in this cell type is presumed NOT to
be confined to the prostate). All other tissues negative. [1705]
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. [1706]
Adult human tissues examined: Kidney (normal and end-stage),
adrenal, spleen, lymph node, pancreas, lung, eye (inc. retina),
bladder, liver (normal, cirrhotic, acute failure). [1707] Non-human
primate tissues examined:
[1708] Chimp Tissues: adrenal
[1709] Rhesus Monkey Tissues: Cerebral cortex, hippocampus,
cerebellum.
Example 103
Isolation of cDNA clones Encoding Human PRO1868
[1710] A consensus DNA sequence was assembled relative to other EST
sequences using phrap as described in Example 1 above. This
consensus sequence is herein designated DNA49803. Based up an
observed homology between the DNA49803 consensus sequence and an
EST sequence contained within the Incyte EST clone no. 2994689,
Incyte EST clone no. 2994689 was purchased and its insert obtained
and sequenced. The sequence of that insert is shown in FIG. 123 and
is herein designated DNA77624-2515.
[1711] The entire nucleotide sequence of DNA77624-2515 is shown in
FIG. 123 (SEQ ID NO:422). Clone DNA77624-2515 contains a single
open reading frame with an apparent translational initiation site
at nucleotide positions 51-53 and ending at the stop codon at
nucleotide positions 981-983 (FIG. 123). The predicted polypeptide
precursor is 310 amino acids long (FIG. 124). The full-length PRO
1868 protein shown in FIG. 124 has an estimated molecular weight of
about 35,020 daltons and a pI of about 7.90. Analysis of the
full-length PRO 1868 sequence shown in FIG. 124 (SEQ ID NO:423)
evidences the presence of the following: a signal peptide from
about amino acid I to about amino acid 30, a transmembrane domain
from about amino acid 243 to about amino acid 263, potential
N-glycosylation sites from about amino acid 104 to about amino acid
107 and from about amino acid 192 to about amino acid 195, a cAMY-
and cGMP-dependent protein kinase phosphorylation site from about
amino acid 107 to about amino acid 110, casein kinase 11
phosphorylation sites from about amino acid 106 to about amino acid
109 and from about amino acid 296 to about amino acid 299, a
tyrosine kinase phosphorylation site from about amino acid 69 to
about amino acid 77 and potential N-myristolation sites from about
amino acid 26 to about amino acid 31, from about amino acid 215 to
about amino acid 220, from about amino acid 226 to about amino acid
231, from about amino acid 243 to about amino acid 248, from about
amino acid 244 to about amino acid 249 and from about amino acid
262 to about amino acid 267. Clone DNA77624-2515 has been deposited
with ATCC on Dec. 22, 1998 and is assigned ATCC deposit no.
203553.
[1712] An analysis of the Dayhoff database (version 35.45 SwissProt
35), using a WU-BLAST2 sequence alignment analysis of the
full-length sequence shown in FIG. 124 (SEQ ID NO:423), evidenced
significant homology between the PRO1868 amino acid sequence and
the following Dayhoff sequences: HGS_RC75, P_W61379, A33_HUMAN,
P_W14146, PW14158, AMAL_DROME, P_R77437, 138346, NCM2_HUMAN and
PTPD_HUMAN.
Example 104
Identification of Receptor/Ligand Interactions
[1713] 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
preparation 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.
[1714] The assay is performed as follows. A PRO polypeptide of the
present invention suspected of being a ligand for a receptor is
expressed as a fusion protein containing the Fc domain of human IgG
(an immunoadhesin). Receptor-ligand binding is detected by allowing
interaction of the immunoadhesin polypeptide with cells (e.g. Cos
cells) expressing candidate PRO polypeptide receptors and
visualization of bound immunoadhesin with fluorescent reagents
directed toward the Fc fusion domain and examination by microscope.
Cells expressing candidate receptors are produced by transient
transfection, in parallel, of defined subsets of a library of cDNA
expression vectors encoding PRO polypeptides that may function as
receptor molecules. Cells are then incubated for I hour in the
presence of the PRO polypeptide immunoadhesin being tested for
possible receptor binding. The cells are then washed and fixed with
paraformaldehyde. The cells are then incubated with fluorescent
conjugated antibody directed against the Fc portion of the PRO
polypeptide immunoadhesin (e.g. FITC conjugated goat anti-human-Fc
antibody). The cells are then washed again and examined by
microscope. A positive interaction is judged by the presence of
fluorescent labeling of cells transfected with cDNA encoding a
particular PRO polypeptide receptor or pool of receptors and an
absence of similar fluorescent labeling of similarly prepared cells
that have been transfected with other cDNA or pools of cDNA. If a
defined pool of cDNA expression vectors is judged to be positive
for interaction with a PRO polypeptide immunoadhesin, the
individual cDNA species that comprise the pool are tested
individually (the pool is "broken down") to determine the specific
cDNA that encodes a receptor able to interact with the PRO
polypeptide immunoadhesin.
[1715] 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.
[1716] Using these assays, the following receptor/ligand
interactions have been herein identified: PRO245 binds to
PRO1868.
Deposit of Material
[1717] The following materials have been deposited with the
American Type Culture Collection, 10801 University Boulevard,
Manassas, Va. USA (ATCC): TABLE-US-00124 Material ATCC Dep. No.
Deposit Date DNA32292-1131 ATCC 209258 Sep. 16, 1997 DNA33094-1131
ATCC 209256 Sep. 16, 1997 DNA33223-1136 ATCC 209264 Sep. 16, 1997
DNA34435-1140 ATCC 209250 Sep. 16, 1997 DNA27864-1155 ATCC 209375
Oct. 16, 1997 DNA36350-1158 ATCC 209378 Oct. 16, 1997 DNA32290-1164
ATCC 209384 Oct. 16, 1997 DNA35639-1172 ATCC 209396 Oct. 17, 1997
DNA33092-1202 ATCC 209420 Oct. 28, 1997 DNA49435-1219 ATCC 209480
Nov. 21, 1997 DNA35638-1141 ATCC 209265 Sep. 16, 1997 DNA32298-1132
ATCC 209257 Sep. 16, 1997 DNA33089-1132 ATCC 209262 Sep. 16, 1997
DNA33786-1132 ATCC 209253 Sep. 16, 1997 DNA35918-1174 ATCC 209402
Oct. 17, 1997 DNA37150-1178 ATCC 209401 Oct. 17, 1997 DNA38260-1180
ATCC 209397 Oct. 17, 1997 DNA39969-1185 ATCC 209400 Oct. 17, 1997
DNA32286-1191 ATCC 209385 Oct. 16, 1997 DNA33461-1199 ATCC 209367
Oct. 15, 1997 DNA40628-1216 ATCC 209432 Nov. 7, 1997 DNA33221-1133
ATCC 209263 Sep. 16, 1997 DNA33107-1135 ATCC 209251 Sep. 16, 1997
DNA35557-1137 ATCC 209255 Sep. 16, 1997 DNA34434-1139 ATCC 209252
Sep. 16, 1997 DNA33100-1159 ATCC 209373 Oct. 16, 1997 DNA35600-1162
ATCC 209370 Oct. 16, 1997 DNA34436-1238 ATCC 209523 Dec. 10, 1997
DNA33206-1165 ATCC 209372 Oct. 16, 1997 DNA35558-1167 ATCC 209374
Oct. 16, 1997 DNA35599-1168 ATCC 209373 Oct. 16, 1997 DNA36992-1168
ATCC 209382 Oct. 16, 1997 DNA34407-1169 ATCC 209383 Oct. 16, 1997
DNA35841-1173 ATCC 209403 Oct. 17, 1997 DNA33470-1175 ATCC 209398
Oct. 17, 1997 DNA34431-1177 ATCC 209399 Oct. 17, 1997 DNA39510-1181
ATCC 209392 Oct. 17, 1997 DNA39423-1182 ATCC 209387 Oct. 17, 1997
DNA40620-1183 ATCC 209388 Oct. 17, 1997 DNA40604-1187 ATCC 209394
Oct. 17, 1997 DNA38268-1188 ATCC 209421 Oct. 28, 1997 DNA37151-1193
ATCC 209393 Oct. 17, 1997 DNA35673-1201 ATCC 209418 Oct. 28, 1997
DNA40370-1217 ATCC 209485 Nov. 21, 1997 DNA42551-1217 ATCC 209483
Nov. 21, 1997 DNA39520-1217 ATCC 209482 Nov. 21, 1997 DNA41225-1217
ATCC 209491 Nov. 21, 1997 DNA43318-1217 ATCC 209481 Nov. 21, 1997
DNA40587-1231 ATCC 209438 Nov. 7, 1997 DNA41338-1234 ATCC 209927
Jun. 2, 1998 DNA40981-1234 ATCC 209439 Nov. 7, 1997 DNA37140-1234
ATCC 209489 Nov. 21, 1997 DNA40982-1235 ATCC 209433 Nov. 7, 1997
DNA41379-1236 ATCC 209488 Nov. 21, 1997 DNA44167-1243 ATCC 209434
Nov. 7, 1997 DNA39427-1179 ATCC 209395 Oct. 17, 1997 DNA40603-1232
ATCC 209486 Nov. 21, 1997 DNA43466-1225 ATCC 209490 Nov. 21, 1997
DNA43046-1225 ATCC 209484 Nov. 21, 1997 DNA35668-1171 ATCC 209371
Oct. 16, 1997 DNA77624-2515 ATCC 203553 Dec. 22, 1998
[1718] 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 there under (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 that all restrictions
imposed by the depositor on the availability to the public of the
deposited material will be irrevocably removed upon the granting of
the pertinent U.S. patent, 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 U.S.C. .sctn.122 and the
Commissioner's rules pursuant thereto (including 37 C.F.R.
.sctn.1.14 with particular reference to 886 OG 638).
[1719] 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.
[1720] The foregoing written specification is considered to be
sufficient to enable one skilled in the art to practice the
invention. The present invention is not to be limited in scope by
the construct deposited, since the deposited embodiment is intended
as a single illustration of certain aspects of the invention and
any constructs that are functionally equivalent are within the
scope of this invention. The deposit of material herein does not
constitute an admission that the written description herein
contained is inadequate to enable the practice of any aspect of the
invention, including the best mode thereof, nor is it to be
construed as limiting the scope of the claims to the specific
illustrations that it represents. Indeed, various modifications of
the invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description and fall within the scope of the appended claims.
Sequence CWU 1
1
423 1 1825 DNA Homo sapiens 1 actgcacctc ggttctatcg attgaattcc
ccggggatcc tctagagatc cctcgacctc 60 gacccacgcg tccgggccgg
agcagcacgg ccgcaggacc tggagctccg gctgcgtctt 120 cccgcagcgc
tacccgccat gcgcctgccg cgccgggccg cgctggggct cctgccgctt 180
ctgctgctgc tgccgcccgc gccggaggcc gccaagaagc cgacgccctg ccaccggtgc
240 cgggggctgg tggacaagtt taaccagggg atggtggaca ccgcaaagaa
gaactttggc 300 ggcgggaaca cggcttggga ggaaaagacg ctgtccaagt
acgagtccag cgagattcgc 360 ctgctggaga tcctggaggg gctgtgcgag
agcagcgact tcgaatgcaa tcagatgcta 420 gaggcgcagg aggagcacct
ggaggcctgg tggctgcagc tgaagagcga atatcctgac 480 ttattcgagt
ggttttgtgt gaagacactg aaagtgtgct gctctccagg aacctacggt 540
cccgactgtc tcgcatgcca gggcggatcc cagaggccct gcagcgggaa tggccactgc
600 agcggagatg ggagcagaca gggcgacggg tcctgccggt gccacatggg
gtaccagggc 660 ccgctgtgca ctgactgcat ggacggctac ttcagctcgc
tccggaacga gacccacagc 720 atctgcacag cctgtgacga gtcctgcaag
acgtgctcgg gcctgaccaa cagagactgc 780 ggcgagtgtg aagtgggctg
ggtgctggac gagggcgcct gtgtggatgt ggacgagtgt 840 gcggccgagc
cgcctccctg cagcgctgcg cagttctgta agaacgccaa cggctcctac 900
acgtgcgaag agtgtgactc cagctgtgtg ggctgcacag gggaaggccc aggaaactgt
960 aaagagtgta tctctggcta cgcgagggag cacggacagt gtgcagatgt
ggacgagtgc 1020 tcactagcag aaaaaacctg tgtgaggaaa aacgaaaact
gctacaatac tccagggagc 1080 tacgtctgtg tgtgtcctga cggcttcgaa
gaaacggaag atgcctgtgt gccgccggca 1140 gaggctgaag ccacagaagg
agaaagcccg acacagctgc cctcccgcga agacctgtaa 1200 tgtgccggac
ttacccttta aattattcag aaggatgtcc cgtggaaaat gtggccctga 1260
ggatgccgtc tcctgcagtg gacagcggcg gggagaggct gcctgctctc taacggttga
1320 ttctcatttg tcccttaaac agctgcattt cttggttgtt cttaaacaga
cttgtatatt 1380 ttgatacagt tctttgtaat aaaattgacc attgtaggta
atcaggagga aaaaaaaaaa 1440 aaaaaaaaaa aaagggcggc cgcgactcta
gagtcgacct gcagaagctt ggccgccatg 1500 gcccaacttg tttattgcag
cttataatgg ttacaaataa agcaatagca tcacaaattt 1560 cacaaataaa
gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt 1620
atcttatcat gtctggatcg ggaattaatt cggcgcagca ccatggcctg aaataacctc
1680 tgaaagagga acttggttag gtaccttctg aggcggaaag aaccagctgt
ggaatgtgtg 1740 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
agaagtatgc aagcatgcat 1800 ctcaattagt cagcaaccca gtttt 1825 2 353
PRT Homo sapiens 2 Met Arg Leu Pro Arg Arg Ala Ala Leu Gly Leu Leu
Pro Leu Leu Leu 1 5 10 15 Leu Leu Pro Pro Ala Pro Glu Ala Ala Lys
Lys Pro Thr Pro Cys His 20 25 30 Arg Cys Arg Gly Leu Val Asp Lys
Phe Asn Gln Gly Met Val Asp Thr 35 40 45 Ala Lys Lys Asn Phe Gly
Gly Gly Asn Thr Ala Trp Glu Glu Lys Thr 50 55 60 Leu Ser Lys Tyr
Glu Ser Ser Glu Ile Arg Leu Leu Glu Ile Leu Glu 65 70 75 80 Gly Leu
Cys Glu Ser Ser Asp Phe Glu Cys Asn Gln Met Leu Glu Ala 85 90 95
Gln Glu Glu His Leu Glu Ala Trp Trp Leu Gln Leu Lys Ser Glu Tyr 100
105 110 Pro Asp Leu Phe Glu Trp Phe Cys Val Lys Thr Leu Lys Val Cys
Cys 115 120 125 Ser Pro Gly Thr Tyr Gly Pro Asp Cys Leu Ala Cys Gln
Gly Gly Ser 130 135 140 Gln Arg Pro Cys Ser Gly Asn Gly His Cys Ser
Gly Asp Gly Ser Arg 145 150 155 160 Gln Gly Asp Gly Ser Cys Arg Cys
His Met Gly Tyr Gln Gly Pro Leu 165 170 175 Cys Thr Asp Cys Met Asp
Gly Tyr Phe Ser Ser Leu Arg Asn Glu Thr 180 185 190 His Ser Ile Cys
Thr Ala Cys Asp Glu Ser Cys Lys Thr Cys Ser Gly 195 200 205 Leu Thr
Asn Arg Asp Cys Gly Glu Cys Glu Val Gly Trp Val Leu Asp 210 215 220
Glu Gly Ala Cys Val Asp Val Asp Glu Cys Ala Ala Glu Pro Pro Pro 225
230 235 240 Cys Ser Ala Ala Gln Phe Cys Lys Asn Ala Asn Gly Ser Tyr
Thr Cys 245 250 255 Glu Glu Cys Asp Ser Ser Cys Val Gly Cys Thr Gly
Glu Gly Pro Gly 260 265 270 Asn Cys Lys Glu Cys Ile Ser Gly Tyr Ala
Arg Glu His Gly Gln Cys 275 280 285 Ala Asp Val Asp Glu Cys Ser Leu
Ala Glu Lys Thr Cys Val Arg Lys 290 295 300 Asn Glu Asn Cys Tyr Asn
Thr Pro Gly Ser Tyr Val Cys Val Cys Pro 305 310 315 320 Asp Gly Phe
Glu Glu Thr Glu Asp Ala Cys Val Pro Pro Ala Glu Ala 325 330 335 Glu
Ala Thr Glu Gly Glu Ser Pro Thr Gln Leu Pro Ser Arg Glu Asp 340 345
350 Leu 3 2206 DNA Homo sapiens 3 caggtccaac tgcacctcgg ttctatcgat
tgaattcccc ggggatcctc tagagatccc 60 tcgacctcga cccacgcgtc
cgccaggccg ggaggcgacg cgcccagccg tctaaacggg 120 aacagccctg
gctgagggag ctgcagcgca gcagagtatc tgacggcgcc aggttgcgta 180
ggtgcggcac gaggagtttt cccggcagcg aggaggtcct gagcagcatg gcccggagga
240 gcgccttccc tgccgccgcg ctctggctct ggagcatcct cctgtgcctg
ctggcactgc 300 gggcggaggc cgggccgccg caggaggaga gcctgtacct
atggatcgat gctcaccagg 360 caagagtact cataggattt gaagaagata
tcctgattgt ttcagagggg aaaatggcac 420 cttttacaca tgatttcaga
aaagcgcaac agagaatgcc agctattcct gtcaatatcc 480 attccatgaa
ttttacctgg caagctgcag ggcaggcaga atacttctat gaattcctgt 540
ccttgcgctc cctggataaa ggcatcatgg cagatccaac cgtcaatgtc cctctgctgg
600 gaacagtgcc tcacaaggca tcagttgttc aagttggttt cccatgtctt
ggaaaacagg 660 atggggtggc agcatttgaa gtggatgtga ttgttatgaa
ttctgaaggc aacaccattc 720 tccaaacacc tcaaaatgct atcttcttta
aaacatgtca acaagctgag tgcccaggcg 780 ggtgccgaaa tggaggcttt
tgtaatgaaa gacgcatctg cgagtgtcct gatgggttcc 840 acggacctca
ctgtgagaaa gccctttgta ccccacgatg tatgaatggt ggactttgtg 900
tgactcctgg tttctgcatc tgcccacctg gattctatgg agtgaactgt gacaaagcaa
960 actgctcaac cacctgcttt aatggaggga cctgtttcta ccctggaaaa
tgtatttgcc 1020 ctccaggact agagggagag cagtgtgaaa tcagcaaatg
cccacaaccc tgtcgaaatg 1080 gaggtaaatg cattggtaaa agcaaatgta
agtgttccaa aggttaccag ggagacctct 1140 gttcaaagcc tgtctgcgag
cctggctgtg gtgcacatgg aacctgccat gaacccaaca 1200 aatgccaatg
tcaagaaggt tggcatggaa gacactgcaa taaaaggtac gaagccagcc 1260
tcatacatgc cctgaggcca gcaggcgccc agctcaggca gcacacgcct tcacttaaaa
1320 aggccgagga gcggcgggat ccacctgaat ccaattacat ctggtgaact
ccgacatctg 1380 aaacgtttta agttacacca agttcatagc ctttgttaac
ctttcatgtg ttgaatgttc 1440 aaataatgtt cattacactt aagaatactg
gcctgaattt tattagcttc attataaatc 1500 actgagctga tatttactct
tccttttaag ttttctaagt acgtctgtag catgatggta 1560 tagattttct
tgtttcagtg ctttgggaca gattttatat tatgtcaatt gatcaggtta 1620
aaattttcag tgtgtagttg gcagatattt tcaaaattac aatgcattta tggtgtctgg
1680 gggcagggga acatcagaaa ggttaaattg ggcaaaaatg cgtaagtcac
aagaatttgg 1740 atggtgcagt taatgttgaa gttacagcat ttcagatttt
attgtcagat atttagatgt 1800 ttgttacatt tttaaaaatt gctcttaatt
tttaaactct caatacaata tattttgacc 1860 ttaccattat tccagagatt
cagtattaaa aaaaaaaaaa ttacactgtg gtagtggcat 1920 ttaaacaata
taatatattc taaacacaat gaaataggga atataatgta tgaacttttt 1980
gcattggctt gaagcaatat aatatattgt aaacaaaaca cagctcttac ctaataaaca
2040 ttttatactg tttgtatgta taaaataaag gtgctgcttt agttttttgg
aaaaaaaaaa 2100 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa gggcggccgc
gactctagag tcgacctgca 2160 gaagcttggc cgccatggcc caacttgttt
attgcagctt ataatg 2206 4 379 PRT Homo sapiens 4 Met Ala Arg Arg Ser
Ala Phe Pro Ala Ala Ala Leu Trp Leu Trp Ser 1 5 10 15 Ile Leu Leu
Cys Leu Leu Ala Leu Arg Ala Glu Ala Gly Pro Pro Gln 20 25 30 Glu
Glu Ser Leu Tyr Leu Trp Ile Asp Ala His Gln Ala Arg Val Leu 35 40
45 Ile Gly Phe Glu Glu Asp Ile Leu Ile Val Ser Glu Gly Lys Met Ala
50 55 60 Pro Phe Thr His Asp Phe Arg Lys Ala Gln Gln Arg Met Pro
Ala Ile 65 70 75 80 Pro Val Asn Ile His Ser Met Asn Phe Thr Trp Gln
Ala Ala Gly Gln 85 90 95 Ala Glu Tyr Phe Tyr Glu Phe Leu Ser Leu
Arg Ser Leu Asp Lys Gly 100 105 110 Ile Met Ala Asp Pro Thr Val Asn
Val Pro Leu Leu Gly Thr Val Pro 115 120 125 His Lys Ala Ser Val Val
Gln Val Gly Phe Pro Cys Leu Gly Lys Gln 130 135 140 Asp Gly Val Ala
Ala Phe Glu Val Asp Val Ile Val Met Asn Ser Glu 145 150 155 160 Gly
Asn Thr Ile Leu Gln Thr Pro Gln Asn Ala Ile Phe Phe Lys Thr 165 170
175 Cys Gln Gln Ala Glu Cys Pro Gly Gly Cys Arg Asn Gly Gly Phe Cys
180 185 190 Asn Glu Arg Arg Ile Cys Glu Cys Pro Asp Gly Phe His Gly
Pro His 195 200 205 Cys Glu Lys Ala Leu Cys Thr Pro Arg Cys Met Asn
Gly Gly Leu Cys 210 215 220 Val Thr Pro Gly Phe Cys Ile Cys Pro Pro
Gly Phe Tyr Gly Val Asn 225 230 235 240 Cys Asp Lys Ala Asn Cys Ser
Thr Thr Cys Phe Asn Gly Gly Thr Cys 245 250 255 Phe Tyr Pro Gly Lys
Cys Ile Cys Pro Pro Gly Leu Glu Gly Glu Gln 260 265 270 Cys Glu Ile
Ser Lys Cys Pro Gln Pro Cys Arg Asn Gly Gly Lys Cys 275 280 285 Ile
Gly Lys Ser Lys Cys Lys Cys Ser Lys Gly Tyr Gln Gly Asp Leu 290 295
300 Cys Ser Lys Pro Val Cys Glu Pro Gly Cys Gly Ala His Gly Thr Cys
305 310 315 320 His Glu Pro Asn Lys Cys Gln Cys Gln Glu Gly Trp His
Gly Arg His 325 330 335 Cys Asn Lys Arg Tyr Glu Ala Ser Leu Ile His
Ala Leu Arg Pro Ala 340 345 350 Gly Ala Gln Leu Arg Gln His Thr Pro
Ser Leu Lys Lys Ala Glu Glu 355 360 365 Arg Arg Asp Pro Pro Glu Ser
Asn Tyr Ile Trp 370 375 5 45 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide probe 5 agggagcacg
gacagtgtgc agatgtggac gagtgctcac tagca 45 6 21 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 6 agagtgtatc tctggctacg c 21 7 22 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 7 taagtccggc acattacagg tc 22 8 49 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 8 cccacgatgt atgaatggtg gactttgtgt gactcctggt
ttctgcatc 49 9 22 DNA Artificial Sequence Description of Artificial
Sequence Synthetic oligonucleotide probe 9 aaagacgcat ctgcgagtgt cc
22 10 23 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide probe 10 tgctgatttc acactgctct ccc 23 11
2197 DNA Homo sapiens 11 cggacgcgtg ggcgtccggc ggtcgcagag
ccaggaggcg gaggcgcgcg ggccagcctg 60 ggccccagcc cacaccttca
ccagggccca ggagccacca tgtggcgatg tccactgggg 120 ctactgctgt
tgctgccgct ggctggccac ttggctctgg gtgcccagca gggtcgtggg 180
cgccgggagc tagcaccggg tctgcacctg cggggcatcc gggacgcggg aggccggtac
240 tgccaggagc aggacctgtg ctgccgcggc cgtgccgacg actgtgccct
gccctacctg 300 ggcgccatct gttactgtga cctcttctgc aaccgcacgg
tctccgactg ctgccctgac 360 ttctgggact tctgcctcgg cgtgccaccc
ccttttcccc cgatccaagg atgtatgcat 420 ggaggtcgta tctatccagt
cttgggaacg tactgggaca actgtaaccg ttgcacctgc 480 caggagaaca
ggcagtggca tggtggatcc agacatgatc aaagccatca accagggcaa 540
ctatggctgg caggctggga accacagcgc cttctggggc atgaccctgg atgagggcat
600 tcgctaccgc ctgggcacca tccgcccatc ttcctcggtc atgaacatgc
atgaaattta 660 tacagtgctg aacccagggg aggtgcttcc cacagccttc
gaggcctctg agaagtggcc 720 caacctgatt catgagcctc ttgaccaagg
caactgtgca ggctcctggg ccttctccac 780 agcagctgtg gcatccgatc
gtgtctcaat ccattctctg ggacacatga cgcctgtcct 840 gtcgccccag
aacctgctgt cttgtgacac ccaccagcag cagggctgcc gcggtgggcg 900
tctcgatggt gcctggtggt tcctgcgtcg ccgaggggtg gtgtctgacc actgctaccc
960 cttctcgggc cgtgaacgag acgaggctgg ccctgcgccc ccctgtatga
tgcacagccg 1020 agccatgggt cggggcaagc gccaggccac tgcccactgc
cccaacagct atgttaataa 1080 caatgacatc taccaggtca ctcctgtcta
ccgcctcggc tccaacgaca aggagatcat 1140 gaaggagctg atggagaatg
gccctgtcca agccctcatg gaggtgcatg aggacttctt 1200 cctatacaag
ggaggcatct acagccacac gccagtgagc cttgggaggc cagagagata 1260
ccgccggcat gggacccact cagtcaagat cacaggatgg ggagaggaga cgctgccaga
1320 tggaaggacg ctcaaatact ggactgcggc caactcctgg ggcccagcct
ggggcgagag 1380 gggccacttc cgcatcgtgc gcggcgtcaa tgagtgcgac
atcgagagct tcgtgctggg 1440 cgtctggggc cgcgtgggca tggaggacat
gggtcatcac tgaggctgcg ggcaccacgc 1500 ggggtccggc ctgggatcca
ggctaagggc cggcggaaga ggccccaatg gggcggtgac 1560 cccagcctcg
cccgacagag cccggggcgc aggcgggcgc cagggcgcta atcccggcgc 1620
gggttccgct gacgcagcgc cccgcctggg agccgcgggc aggcgagact ggcggagccc
1680 ccagacctcc cagtggggac ggggcagggc ctggcctggg aagagcacag
ctgcagatcc 1740 caggcctctg gcgcccccac tcaagactac caaagccagg
acacctcaag tctccagccc 1800 caatacccca ccccaatccc gtattctttt
tttttttttt ttagacaggg tcttgctccg 1860 ttgcccaggt tggagtgcag
tggcccatca gggctcactg taacctccga ctcctgggtt 1920 caagtgaccc
tcccacctca gcctctcaag tagctgggac tacaggtgca ccaccacacc 1980
tggctaattt ttgtattttt tgtaaagagg ggggtctcac tgtgttgccc aggctggttt
2040 cgaactcctg ggctcaagcg gtccacctgc ctccgcctcc caaagtgctg
ggattgcagg 2100 catgagccac tgcacccagc cctgtattct tattcttcag
atatttattt ttcttttcac 2160 tgttttaaaa taaaaccaaa gtattgataa aaaaaaa
2197 12 164 PRT Homo sapiens 12 Met Trp Arg Cys Pro Leu Gly Leu Leu
Leu Leu Leu Pro Leu Ala Gly 1 5 10 15 His Leu Ala Leu Gly Ala Gln
Gln Gly Arg Gly Arg Arg Glu Leu Ala 20 25 30 Pro Gly Leu His Leu
Arg Gly Ile Arg Asp Ala Gly Gly Arg Tyr Cys 35 40 45 Gln Glu Gln
Asp Leu Cys Cys Arg Gly Arg Ala Asp Asp Cys Ala Leu 50 55 60 Pro
Tyr Leu Gly Ala Ile Cys Tyr Cys Asp Leu Phe Cys Asn Arg Thr 65 70
75 80 Val Ser Asp Cys Cys Pro Asp Phe Trp Asp Phe Cys Leu Gly Val
Pro 85 90 95 Pro Pro Phe Pro Pro Ile Gln Gly Cys Met His Gly Gly
Arg Ile Tyr 100 105 110 Pro Val Leu Gly Thr Tyr Trp Asp Asn Cys Asn
Arg Cys Thr Cys Gln 115 120 125 Glu Asn Arg Gln Trp His Gly Gly Ser
Arg His Asp Gln Ser His Gln 130 135 140 Pro Gly Gln Leu Trp Leu Ala
Gly Trp Glu Pro Gln Arg Leu Leu Gly 145 150 155 160 His Asp Pro Gly
13 533 DNA Homo sapiens modified_base (33)..(33) a, t, c or g
modified_base (37)..(37) a, t, c or g modified_base (80)..(80) a,
t, c or g modified_base (94)..(94) a, t, c or g modified_base
(144)..(144) a, t, c or g modified_base (188)..(188) a, t, c or g
13 aggctccttg gccctttttc cacagcaagc ttntgcnatc ccgattcgtt
gtctcaaatc 60 caattctctt gggacacatn acgcctgtcc tttngcccca
gaacctgctg tcttgtacac 120 ccaccagcag cagggctgcc gcgntgggcg
tctcgatggt gcctggtggt tcctgcgtcg 180 ccgagggntg gtgtctgacc
actgctaccc cttctcgggc cgtgaacgag acgaggctgg 240 ccctgcgccc
ccctgtatga tgcacagccg agccatgggt cggggcaagc gccaggccac 300
tgcccactgc cccaacagct atgttaataa caatgacatc taccaggtca ctcctgtcta
360 ccgcctcggc tccaacgaca aggagatcat gaaggagctg atggagaatg
gccctgtcca 420 agccctcatg gaggtgcatg aggacttctt cctatacaag
ggaggcatct acagccacac 480 gccagtgagc cttgggaggc cagagagata
ccgccggcat gggacccact cag 533 14 24 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide probe
14 ttcgaggcct ctgagaagtg gccc 24 15 22 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide probe
15 ggcggtatct ctctggcctc cc 22 16 50 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide probe
16 ttctccacag cagctgtggc atccgatcgt gtctcaatcc attctctggg 50 17 960
DNA Homo sapiens 17 gctgcttgcc ctgttgatgg caggcttggc cctgcagcca
ggcactgccc tgctgtgcta 60 ctcctgcaaa gcccaggtga gcaacgagga
ctgcctgcag gtggagaact gcacccagct 120 gggggagcag tgctggaccg
cgcgcatccg cgcagttggc ctcctgaccg tcatcagcaa 180 aggctgcagc
ttgaactgcg tggatgactc acaggactac tacgtgggca agaagaacat 240
cacgtgctgt gacaccgact tgtgcaacgc cagcggggcc catgccctgc agccggctgc
300 cgccatcctt gcgctgctcc ctgcactcgg cctgctgctc tggggacccg
gccagctata 360 ggctctgggg ggccccgctg cagcccacac tgggtgtggt
gccccaggcc tctgtgccac 420 tcctcacaga cctggcccag tgggagcctg
tcctggttcc tgaggcacat cctaacgcaa 480 gtctgaccat gtatgtctgc
acccctgtcc cccaccctga ccctcccatg gccctctcca 540 ggactcccac
ccggcagatc agctctagtg acacagatcc gcctgcagat ggcccctcca 600
accctctctg ctgctgtttc catggcccag cattctccac ccttaaccct gtgctcaggc
660 acctcttccc ccaggaagcc ttccctgccc accccatcta tgacttgagc
caggtctggt 720 ccgtggtgtc ccccgcaccc agcaggggac aggcactcag
gagggcccag taaaggctga 780 gatgaagtgg actgagtaga actggaggac
aagagtcgac gtgagttcct gggagtctcc 840 agagatgggg cctggaggcc
tggaggaagg ggccaggcct cacattcgtg gggctccctg 900 aatggcagcc
tgagcacagc gtaggccctt aataaacacc tgttggataa gccaaaaaaa 960 18 189
PRT
Homo sapiens 18 Met Thr His Arg Thr Thr Thr Trp Ala Arg Arg Thr Ser
Arg Ala Val 1 5 10 15 Thr Pro Thr Cys Ala Thr Pro Ala Gly Pro Met
Pro Cys Ser Arg Leu 20 25 30 Pro Pro Ser Leu Arg Cys Ser Leu His
Ser Ala Cys Cys Ser Gly Asp 35 40 45 Pro Ala Ser Tyr Arg Leu Trp
Gly Ala Pro Leu Gln Pro Thr Leu Gly 50 55 60 Val Val Pro Gln Ala
Ser Val Pro Leu Leu Thr Asp Leu Ala Gln Trp 65 70 75 80 Glu Pro Val
Leu Val Pro Glu Ala His Pro Asn Ala Ser Leu Thr Met 85 90 95 Tyr
Val Cys Thr Pro Val Pro His Pro Asp Pro Pro Met Ala Leu Ser 100 105
110 Arg Thr Pro Thr Arg Gln Ile Ser Ser Ser Asp Thr Asp Pro Pro Ala
115 120 125 Asp Gly Pro Ser Asn Pro Leu Cys Cys Cys Phe His Gly Pro
Ala Phe 130 135 140 Ser Thr Leu Asn Pro Val Leu Arg His Leu Phe Pro
Gln Glu Ala Phe 145 150 155 160 Pro Ala His Pro Ile Tyr Asp Leu Ser
Gln Val Trp Ser Val Val Ser 165 170 175 Pro Ala Pro Ser Arg Gly Gln
Ala Leu Arg Arg Ala Gln 180 185 19 24 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide probe
19 tgctgtgcta ctcctgcaaa gccc 24 20 24 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide probe
20 tgcacaagtc ggtgtcacag cacg 24 21 44 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide probe
21 agcaacgagg actgcctgca ggtggagaac tgcacccagc tggg 44 22 1200 DNA
Homo sapiens 22 cccacgcgtc cgaacctctc cagcgatggg agccgcccgc
ctgctgccca acctcactct 60 gtgcttacag ctgctgattc tctgctgtca
aactcagtac gtgagggacc agggcgccat 120 gaccgaccag ctgagcaggc
ggcagatccg cgagtaccaa ctctacagca ggaccagtgg 180 caagcacgtg
caggtcaccg ggcgtcgcat ctccgccacc gccgaggacg gcaacaagtt 240
tgccaagctc atagtggaga cggacacgtt tggcagccgg gttcgcatca aaggggctga
300 gagtgagaag tacatctgta tgaacaagag gggcaagctc atcgggaagc
ccagcgggaa 360 gagcaaagac tgcgtgttca cggagatcgt gctggagaac
aactatacgg ccttccagaa 420 cgcccggcac gagggctggt tcatggcctt
cacgcggcag gggcggcccc gccaggcttc 480 ccgcagccgc cagaaccagc
gcgaggccca cttcatcaag cgcctctacc aaggccagct 540 gcccttcccc
aaccacgccg agaagcagaa gcagttcgag tttgtgggct ccgcccccac 600
ccgccggacc aagcgcacac ggcggcccca gcccctcacg tagtctggga ggcagggggc
660 agcagcccct gggccgcctc cccacccctt tcccttctta atccaaggac
tgggctgggg 720 tggcgggagg ggagccagat ccccgaggga ggaccctgag
ggccgcgaag catccgagcc 780 cccagctggg aaggggcagg ccggtgcccc
aggggcggct ggcacagtgc ccccttcccg 840 gacgggtggc aggccctgga
gaggaactga gtgtcaccct gatctcaggc caccagcctc 900 tgccggcctc
ccagccgggc tcctgaagcc cgctgaaagg tcagcgactg aaggccttgc 960
agacaaccgt ctggaggtgg ctgtcctcaa aatctgcttc tcggatctcc ctcagtctgc
1020 ccccagcccc caaactcctc ctggctagac tgtaggaagg gacttttgtt
tgtttgtttg 1080 tttcaggaaa aaagaaaggg agagagagga aaatagaggg
ttgtccactc ctcacattcc 1140 acgacccagg cctgcacccc acccccaact
cccagccccg gaataaaacc attttcctgc 1200 23 205 PRT Homo sapiens 23
Met Gly Ala Ala Arg Leu Leu Pro Asn Leu Thr Leu Cys Leu Gln Leu 1 5
10 15 Leu Ile Leu Cys Cys Gln Thr Gln Tyr Val Arg Asp Gln Gly Ala
Met 20 25 30 Thr Asp Gln Leu Ser Arg Arg Gln Ile Arg Glu Tyr Gln
Leu Tyr Ser 35 40 45 Arg Thr Ser Gly Lys His Val Gln Val Thr Gly
Arg Arg Ile Ser Ala 50 55 60 Thr Ala Glu Asp Gly Asn Lys Phe Ala
Lys Leu Ile Val Glu Thr Asp 65 70 75 80 Thr Phe Gly Ser Arg Val Arg
Ile Lys Gly Ala Glu Ser Glu Lys Tyr 85 90 95 Ile Cys Met Asn Lys
Arg Gly Lys Leu Ile Gly Lys Pro Ser Gly Lys 100 105 110 Ser Lys Asp
Cys Val Phe Thr Glu Ile Val Leu Glu Asn Asn Tyr Thr 115 120 125 Ala
Phe Gln Asn Ala Arg His Glu Gly Trp Phe Met Ala Phe Thr Arg 130 135
140 Gln Gly Arg Pro Arg Gln Ala Ser Arg Ser Arg Gln Asn Gln Arg Glu
145 150 155 160 Ala His Phe Ile Lys Arg Leu Tyr Gln Gly Gln Leu Pro
Phe Pro Asn 165 170 175 His Ala Glu Lys Gln Lys Gln Phe Glu Phe Val
Gly Ser Ala Pro Thr 180 185 190 Arg Arg Thr Lys Arg Thr Arg Arg Pro
Gln Pro Leu Thr 195 200 205 24 28 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide probe
24 cagtacgtga gggaccaggg cgccatga 28 25 24 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide probe
25 ccggtgacct gcacgtgctt gcca 24 26 41 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide probe
modified_base (21)..(21) a, t, c or g 26 gcggatctgc cgcctgctca
nctggtcggt catggcgccc t 41 27 2479 DNA Homo sapiens 27 acttgccatc
acctgttgcc agtgtggaaa aattctccct gttgaatttt ttgcacatgg 60
aggacagcag caaagagggc aacacaggct gataagacca gagacagcag ggagattatt
120 ttaccatacg ccctcaggac gttccctcta gctggagttc tggacttcaa
cagaacccca 180 tccagtcatt ttgattttgc tgtttatttt ttttttcttt
ttctttttcc caccacattg 240 tattttattt ccgtacttca gaaatgggcc
tacagaccac aaagtggccc agccatgggg 300 cttttttcct gaagtcttgg
cttatcattt ccctggggct ctactcacag gtgtccaaac 360 tcctggcctg
ccctagtgtg tgccgctgcg acaggaactt tgtctactgt aatgagcgaa 420
gcttgacctc agtgcctctt gggatcccgg agggcgtaac cgtactctac ctccacaaca
480 accaaattaa taatgctgga tttcctgcag aactgcacaa tgtacagtcg
gtgcacacgg 540 tctacctgta tggcaaccaa ctggacgaat tccccatgaa
ccttcccaag aatgtcagag 600 ttctccattt gcaggaaaac aatattcaga
ccatttcacg ggctgctctt gcccagctct 660 tgaagcttga agagctgcac
ctggatgaca actccatatc cacagtgggg gtggaagacg 720 gggccttccg
ggaggctatt agcctcaaat tgttgttttt gtctaagaat cacctgagca 780
gtgtgcctgt tgggcttcct gtggacttgc aagagctgag agtggatgaa aatcgaattg
840 ctgtcatatc cgacatggcc ttccagaatc tcacgagctt ggagcgtctt
attgtggacg 900 ggaacctcct gaccaacaag ggtatcgccg agggcacctt
cagccatctc accaagctca 960 aggaattttc aattgtacgt aattcgctgt
cccaccctcc tcccgatctc ccaggtacgc 1020 atctgatcag gctctatttg
caggacaacc agataaacca cattcctttg acagccttct 1080 caaatctgcg
taagctggaa cggctggata tatccaacaa ccaactgcgg atgctgactc 1140
aaggggtttt tgataatctc tccaacctga agcagctcac tgctcggaat aacccttggt
1200 tttgtgactg cagtattaaa tgggtcacag aatggctcaa atatatccct
tcatctctca 1260 acgtgcgggg tttcatgtgc caaggtcctg aacaagtccg
ggggatggcc gtcagggaat 1320 taaatatgaa tcttttgtcc tgtcccacca
cgacccccgg cctgcctctc ttcaccccag 1380 ccccaagtac agcttctccg
accactcagc ctcccaccct ctctattcca aaccctagca 1440 gaagctacac
gcctccaact cctaccacat cgaaacttcc cacgattcct gactgggatg 1500
gcagagaaag agtgacccca cctatttctg aacggatcca gctctctatc cattttgtga
1560 atgatacttc cattcaagtc agctggctct ctctcttcac cgtgatggca
tacaaactca 1620 catgggtgaa aatgggccac agtttagtag ggggcatcgt
tcaggagcgc atagtcagcg 1680 gtgagaagca acacctgagc ctggttaact
tagagccccg atccacctat cggatttgtt 1740 tagtgccact ggatgctttt
aactaccgcg cggtagaaga caccatttgt tcagaggcca 1800 ccacccatgc
ctcctatctg aacaacggca gcaacacagc gtccagccat gagcagacga 1860
cgtcccacag catgggctcc ccctttctgc tggcgggctt gatcgggggc gcggtgatat
1920 ttgtgctggt ggtcttgctc agcgtctttt gctggcatat gcacaaaaag
gggcgctaca 1980 cctcccagaa gtggaaatac aaccggggcc ggcggaaaga
tgattattgc gaggcaggca 2040 ccaagaagga caactccatc ctggagatga
cagaaaccag ttttcagatc gtctccttaa 2100 ataacgatca actccttaaa
ggagatttca gactgcagcc catttacacc ccaaatgggg 2160 gcattaatta
cacagactgc catatcccca acaacatgcg atactgcaac agcagcgtgc 2220
cagacctgga gcactgccat acgtgacagc cagaggccca gcgttatcaa ggcggacaat
2280 tagactcttg agaacacact cgtgtgtgca cataaagaca cgcagattac
atttgataaa 2340 tgttacacag atgcatttgt gcatttgaat actctgtaat
ttatacggtg tactatataa 2400 tgggatttaa aaaaagtgct atcttttcta
tttcaagtta attacaaaca gttttgtaac 2460 tctttgcttt ttaaatctt 2479 28
660 PRT Homo sapiens 28 Met Gly Leu Gln Thr Thr Lys Trp Pro Ser His
Gly Ala Phe Phe Leu 1 5 10 15 Lys Ser Trp Leu Ile Ile Ser Leu Gly
Leu Tyr Ser Gln Val Ser Lys 20 25 30 Leu Leu Ala Cys Pro Ser Val
Cys Arg Cys Asp Arg Asn Phe Val Tyr 35 40 45 Cys Asn Glu Arg Ser
Leu Thr Ser Val Pro Leu Gly Ile Pro Glu Gly 50 55 60 Val Thr Val
Leu Tyr Leu His Asn Asn Gln Ile Asn Asn Ala Gly Phe 65 70 75 80 Pro
Ala Glu Leu His Asn Val Gln Ser Val His Thr Val Tyr Leu Tyr 85 90
95 Gly Asn Gln Leu Asp Glu Phe Pro Met Asn Leu Pro Lys Asn Val Arg
100 105 110 Val Leu His Leu Gln Glu Asn Asn Ile Gln Thr Ile Ser Arg
Ala Ala 115 120 125 Leu Ala Gln Leu Leu Lys Leu Glu Glu Leu His Leu
Asp Asp Asn Ser 130 135 140 Ile Ser Thr Val Gly Val Glu Asp Gly Ala
Phe Arg Glu Ala Ile Ser 145 150 155 160 Leu Lys Leu Leu Phe Leu Ser
Lys Asn His Leu Ser Ser Val Pro Val 165 170 175 Gly Leu Pro Val Asp
Leu Gln Glu Leu Arg Val Asp Glu Asn Arg Ile 180 185 190 Ala Val Ile
Ser Asp Met Ala Phe Gln Asn Leu Thr Ser Leu Glu Arg 195 200 205 Leu
Ile Val Asp Gly Asn Leu Leu Thr Asn Lys Gly Ile Ala Glu Gly 210 215
220 Thr Phe Ser His Leu Thr Lys Leu Lys Glu Phe Ser Ile Val Arg Asn
225 230 235 240 Ser Leu Ser His Pro Pro Pro Asp Leu Pro Gly Thr His
Leu Ile Arg 245 250 255 Leu Tyr Leu Gln Asp Asn Gln Ile Asn His Ile
Pro Leu Thr Ala Phe 260 265 270 Ser Asn Leu Arg Lys Leu Glu Arg Leu
Asp Ile Ser Asn Asn Gln Leu 275 280 285 Arg Met Leu Thr Gln Gly Val
Phe Asp Asn Leu Ser Asn Leu Lys Gln 290 295 300 Leu Thr Ala Arg Asn
Asn Pro Trp Phe Cys Asp Cys Ser Ile Lys Trp 305 310 315 320 Val Thr
Glu Trp Leu Lys Tyr Ile Pro Ser Ser Leu Asn Val Arg Gly 325 330 335
Phe Met Cys Gln Gly Pro Glu Gln Val Arg Gly Met Ala Val Arg Glu 340
345 350 Leu Asn Met Asn Leu Leu Ser Cys Pro Thr Thr Thr Pro Gly Leu
Pro 355 360 365 Leu Phe Thr Pro Ala Pro Ser Thr Ala Ser Pro Thr Thr
Gln Pro Pro 370 375 380 Thr Leu Ser Ile Pro Asn Pro Ser Arg Ser Tyr
Thr Pro Pro Thr Pro 385 390 395 400 Thr Thr Ser Lys Leu Pro Thr Ile
Pro Asp Trp Asp Gly Arg Glu Arg 405 410 415 Val Thr Pro Pro Ile Ser
Glu Arg Ile Gln Leu Ser Ile His Phe Val 420 425 430 Asn Asp Thr Ser
Ile Gln Val Ser Trp Leu Ser Leu Phe Thr Val Met 435 440 445 Ala Tyr
Lys Leu Thr Trp Val Lys Met Gly His Ser Leu Val Gly Gly 450 455 460
Ile Val Gln Glu Arg Ile Val Ser Gly Glu Lys Gln His Leu Ser Leu 465
470 475 480 Val Asn Leu Glu Pro Arg Ser Thr Tyr Arg Ile Cys Leu Val
Pro Leu 485 490 495 Asp Ala Phe Asn Tyr Arg Ala Val Glu Asp Thr Ile
Cys Ser Glu Ala 500 505 510 Thr Thr His Ala Ser Tyr Leu Asn Asn Gly
Ser Asn Thr Ala Ser Ser 515 520 525 His Glu Gln Thr Thr Ser His Ser
Met Gly Ser Pro Phe Leu Leu Ala 530 535 540 Gly Leu Ile Gly Gly Ala
Val Ile Phe Val Leu Val Val Leu Leu Ser 545 550 555 560 Val Phe Cys
Trp His Met His Lys Lys Gly Arg Tyr Thr Ser Gln Lys 565 570 575 Trp
Lys Tyr Asn Arg Gly Arg Arg Lys Asp Asp Tyr Cys Glu Ala Gly 580 585
590 Thr Lys Lys Asp Asn Ser Ile Leu Glu Met Thr Glu Thr Ser Phe Gln
595 600 605 Ile Val Ser Leu Asn Asn Asp Gln Leu Leu Lys Gly Asp Phe
Arg Leu 610 615 620 Gln Pro Ile Tyr Thr Pro Asn Gly Gly Ile Asn Tyr
Thr Asp Cys His 625 630 635 640 Ile Pro Asn Asn Met Arg Tyr Cys Asn
Ser Ser Val Pro Asp Leu Glu 645 650 655 His Cys His Thr 660 29 21
DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide probe 29 cggtctacct gtatggcaac c 21 30 22
DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide probe 30 gcaggacaac cagataaacc ac 22 31
22 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide probe 31 acgcagattt gagaaggctg tc 22 32
46 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide probe 32 ttcacgggct gctcttgccc agctcttgaa
gcttgaagag ctgcac 46 33 3449 DNA Homo sapiens 33 acttggagca
agcggcggcg gcggagacag aggcagaggc agaagctggg gctccgtcct 60
cgcctcccac gagcgatccc cgaggagagc cgcggccctc ggcgaggcga agaggccgac
120 gaggaagacc cgggtggctg cgcccctgcc tcgcttccca ggcgccggcg
gctgcagcct 180 tgcccctctt gctcgccttg aaaatggaaa agatgctcgc
aggctgcttt ctgctgatcc 240 tcggacagat cgtcctcctc cctgccgagg
ccagggagcg gtcacgtggg aggtccatct 300 ctaggggcag acacgctcgg
acccacccgc agacggccct tctggagagt tcctgtgaga 360 acaagcgggc
agacctggtt ttcatcattg acagctctcg cagtgtcaac acccatgact 420
atgcaaaggt caaggagttc atcgtggaca tcttgcaatt cttggacatt ggtcctgatg
480 tcacccgagt gggcctgctc caatatggca gcactgtcaa gaatgagttc
tccctcaaga 540 ccttcaagag gaagtccgag gtggagcgtg ctgtcaagag
gatgcggcat ctgtccacgg 600 gcaccatgac tgggctggcc atccagtatg
ccctgaacat cgcattctca gaagcagagg 660 gggcccggcc cctgagggag
aatgtgccac gggtcataat gatcgtgaca gatgggagac 720 ctcaggactc
cgtggccgag gtggctgcta aggcacggga cacgggcatc ctaatctttg 780
ccattggtgt gggccaggta gacttcaaca ccttgaagtc cattgggagt gagccccatg
840 aggaccatgt cttccttgtg gccaatttca gccagattga gacgctgacc
tccgtgttcc 900 agaagaagtt gtgcacggcc cacatgtgca gcaccctgga
gcataactgt gcccacttct 960 gcatcaacat ccctggctca tacgtctgca
ggtgcaaaca aggctacatt ctcaactcgg 1020 atcagacgac ttgcagaatc
caggatctgt gtgccatgga ggaccacaac tgtgagcagc 1080 tctgtgtgaa
tgtgccgggc tccttcgtct gccagtgcta cagtggctac gccctggctg 1140
aggatgggaa gaggtgtgtg gctgtggact actgtgcctc agaaaaccac ggatgtgaac
1200 atgagtgtgt aaatgctgat ggctcctacc tttgccagtg ccatgaagga
tttgctctta 1260 acccagatga aaaaacgtgc acaaggatca actactgtgc
actgaacaaa ccgggctgtg 1320 agcatgagtg cgtcaacatg gaggagagct
actactgccg ctgccaccgt ggctacactc 1380 tggaccccaa tggcaaaacc
tgcagccgag tggaccactg tgcacagcag gaccatggct 1440 gtgagcagct
gtgtctgaac acggaggatt ccttcgtctg ccagtgctca gaaggcttcc 1500
tcatcaacga ggacctcaag acctgctccc gggtggatta ctgcctgctg agtgaccatg
1560 gttgtgaata ctcctgtgtc aacatggaca gatcctttgc ctgtcagtgt
cctgagggac 1620 acgtgctccg cagcgatggg aagacgtgtg caaaattgga
ctcttgtgct ctgggggacc 1680 acggttgtga acattcgtgt gtaagcagtg
aagattcgtt tgtgtgccag tgctttgaag 1740 gttatatact ccgtgaagat
ggaaaaacct gcagaaggaa agatgtctgc caagctatag 1800 accatggctg
tgaacacatt tgtgtgaaca gtgacgactc atacacgtgc gagtgcttgg 1860
agggattccg gctcgctgag gatgggaaac gctgccgaag gaaggatgtc tgcaaatcaa
1920 cccaccatgg ctgcgaacac atttgtgtta ataatgggaa ttcctacatc
tgcaaatgct 1980 cagagggatt tgttctagct gaggacggaa gacggtgcaa
gaaatgcact gaaggcccaa 2040 ttgacctggt ctttgtgatc gatggatcca
agagtcttgg agaagagaat tttgaggtcg 2100 tgaagcagtt tgtcactgga
attatagatt ccttgacaat ttcccccaaa gccgctcgag 2160 tggggctgct
ccagtattcc acacaggtcc acacagagtt cactctgaga aacttcaact 2220
cagccaaaga catgaaaaaa gccgtggccc acatgaaata catgggaaag ggctctatga
2280 ctgggctggc cctgaaacac atgtttgaga gaagttttac ccaaggagaa
ggggccaggc 2340 ccctttccac aagggtgccc agagcagcca ttgtgttcac
cgacggacgg gctcaggatg 2400 acgtctccga gtgggccagt aaagccaagg
ccaatggtat cactatgtat gctgttgggg 2460 taggaaaagc cattgaggag
gaactacaag agattgcctc tgagcccaca aacaagcatc 2520 tcttctatgc
cgaagacttc agcacaatgg atgagataag tgaaaaactc aagaaaggca 2580
tctgtgaagc tctagaagac tccgatggaa gacaggactc tccagcaggg gaactgccaa
2640 aaacggtcca acagccaaca gaatctgagc cagtcaccat aaatatccaa
gacctacttt 2700 cctgttctaa ttttgcagtg caacacagat atctgtttga
agaagacaat cttttacggt 2760 ctacacaaaa gctttcccat tcaacaaaac
cttcaggaag ccctttggaa gaaaaacacg 2820 atcaatgcaa atgtgaaaac
cttataatgt tccagaacct tgcaaacgaa gaagtaagaa 2880 aattaacaca
gcgcttagaa gaaatgacac agagaatgga agccctggaa aatcgcctga 2940
gatacagatg aagattagaa atcgcgacac atttgtagtc attgtatcac ggattacaat
3000 gaacgcagtg cagagcccca aagctcaggc tattgttaaa tcaataatgt
tgtgaagtaa 3060 aacaatcagt actgagaaac ctggtttgcc acagaacaaa
gacaagaagt atacactaac 3120 ttgtataaat ttatctagga aaaaaatcct
tcagaattct aagatgaatt
taccaggtga 3180 gaatgaataa gctatgcaag gtattttgta atatactgtg
gacacaactt gcttctgcct 3240 catcctgcct tagtgtgcaa tctcatttga
ctatacgata aagtttgcac agtcttactt 3300 ctgtagaaca ctggccatag
gaaatgctgt ttttttgtac tggactttac cttgatatat 3360 gtatatggat
gtatgcataa aatcatagga catatgtact tgtggaacaa gttggatttt 3420
ttatacaata ttaaaattca ccacttcag 3449 34 915 PRT Homo sapiens 34 Met
Glu Lys Met Leu Ala Gly Cys Phe Leu Leu Ile Leu Gly Gln Ile 1 5 10
15 Val Leu Leu Pro Ala Glu Ala Arg Glu Arg Ser Arg Gly Arg Ser Ile
20 25 30 Ser Arg Gly Arg His Ala Arg Thr His Pro Gln Thr Ala Leu
Leu Glu 35 40 45 Ser Ser Cys Glu Asn Lys Arg Ala Asp Leu Val Phe
Ile Ile Asp Ser 50 55 60 Ser Arg Ser Val Asn Thr His Asp Tyr Ala
Lys Val Lys Glu Phe Ile 65 70 75 80 Val Asp Ile Leu Gln Phe Leu Asp
Ile Gly Pro Asp Val Thr Arg Val 85 90 95 Gly Leu Leu Gln Tyr Gly
Ser Thr Val Lys Asn Glu Phe Ser Leu Lys 100 105 110 Thr Phe Lys Arg
Lys Ser Glu Val Glu Arg Ala Val Lys Arg Met Arg 115 120 125 His Leu
Ser Thr Gly Thr Met Thr Gly Leu Ala Ile Gln Tyr Ala Leu 130 135 140
Asn Ile Ala Phe Ser Glu Ala Glu Gly Ala Arg Pro Leu Arg Glu Asn 145
150 155 160 Val Pro Arg Val Ile Met Ile Val Thr Asp Gly Arg Pro Gln
Asp Ser 165 170 175 Val Ala Glu Val Ala Ala Lys Ala Arg Asp Thr Gly
Ile Leu Ile Phe 180 185 190 Ala Ile Gly Val Gly Gln Val Asp Phe Asn
Thr Leu Lys Ser Ile Gly 195 200 205 Ser Glu Pro His Glu Asp His Val
Phe Leu Val Ala Asn Phe Ser Gln 210 215 220 Ile Glu Thr Leu Thr Ser
Val Phe Gln Lys Lys Leu Cys Thr Ala His 225 230 235 240 Met Cys Ser
Thr Leu Glu His Asn Cys Ala His Phe Cys Ile Asn Ile 245 250 255 Pro
Gly Ser Tyr Val Cys Arg Cys Lys Gln Gly Tyr Ile Leu Asn Ser 260 265
270 Asp Gln Thr Thr Cys Arg Ile Gln Asp Leu Cys Ala Met Glu Asp His
275 280 285 Asn Cys Glu Gln Leu Cys Val Asn Val Pro Gly Ser Phe Val
Cys Gln 290 295 300 Cys Tyr Ser Gly Tyr Ala Leu Ala Glu Asp Gly Lys
Arg Cys Val Ala 305 310 315 320 Val Asp Tyr Cys Ala Ser Glu Asn His
Gly Cys Glu His Glu Cys Val 325 330 335 Asn Ala Asp Gly Ser Tyr Leu
Cys Gln Cys His Glu Gly Phe Ala Leu 340 345 350 Asn Pro Asp Glu Lys
Thr Cys Thr Arg Ile Asn Tyr Cys Ala Leu Asn 355 360 365 Lys Pro Gly
Cys Glu His Glu Cys Val Asn Met Glu Glu Ser Tyr Tyr 370 375 380 Cys
Arg Cys His Arg Gly Tyr Thr Leu Asp Pro Asn Gly Lys Thr Cys 385 390
395 400 Ser Arg Val Asp His Cys Ala Gln Gln Asp His Gly Cys Glu Gln
Leu 405 410 415 Cys Leu Asn Thr Glu Asp Ser Phe Val Cys Gln Cys Ser
Glu Gly Phe 420 425 430 Leu Ile Asn Glu Asp Leu Lys Thr Cys Ser Arg
Val Asp Tyr Cys Leu 435 440 445 Leu Ser Asp His Gly Cys Glu Tyr Ser
Cys Val Asn Met Asp Arg Ser 450 455 460 Phe Ala Cys Gln Cys Pro Glu
Gly His Val Leu Arg Ser Asp Gly Lys 465 470 475 480 Thr Cys Ala Lys
Leu Asp Ser Cys Ala Leu Gly Asp His Gly Cys Glu 485 490 495 His Ser
Cys Val Ser Ser Glu Asp Ser Phe Val Cys Gln Cys Phe Glu 500 505 510
Gly Tyr Ile Leu Arg Glu Asp Gly Lys Thr Cys Arg Arg Lys Asp Val 515
520 525 Cys Gln Ala Ile Asp His Gly Cys Glu His Ile Cys Val Asn Ser
Asp 530 535 540 Asp Ser Tyr Thr Cys Glu Cys Leu Glu Gly Phe Arg Leu
Ala Glu Asp 545 550 555 560 Gly Lys Arg Cys Arg Arg Lys Asp Val Cys
Lys Ser Thr His His Gly 565 570 575 Cys Glu His Ile Cys Val Asn Asn
Gly Asn Ser Tyr Ile Cys Lys Cys 580 585 590 Ser Glu Gly Phe Val Leu
Ala Glu Asp Gly Arg Arg Cys Lys Lys Cys 595 600 605 Thr Glu Gly Pro
Ile Asp Leu Val Phe Val Ile Asp Gly Ser Lys Ser 610 615 620 Leu Gly
Glu Glu Asn Phe Glu Val Val Lys Gln Phe Val Thr Gly Ile 625 630 635
640 Ile Asp Ser Leu Thr Ile Ser Pro Lys Ala Ala Arg Val Gly Leu Leu
645 650 655 Gln Tyr Ser Thr Gln Val His Thr Glu Phe Thr Leu Arg Asn
Phe Asn 660 665 670 Ser Ala Lys Asp Met Lys Lys Ala Val Ala His Met
Lys Tyr Met Gly 675 680 685 Lys Gly Ser Met Thr Gly Leu Ala Leu Lys
His Met Phe Glu Arg Ser 690 695 700 Phe Thr Gln Gly Glu Gly Ala Arg
Pro Leu Ser Thr Arg Val Pro Arg 705 710 715 720 Ala Ala Ile Val Phe
Thr Asp Gly Arg Ala Gln Asp Asp Val Ser Glu 725 730 735 Trp Ala Ser
Lys Ala Lys Ala Asn Gly Ile Thr Met Tyr Ala Val Gly 740 745 750 Val
Gly Lys Ala Ile Glu Glu Glu Leu Gln Glu Ile Ala Ser Glu Pro 755 760
765 Thr Asn Lys His Leu Phe Tyr Ala Glu Asp Phe Ser Thr Met Asp Glu
770 775 780 Ile Ser Glu Lys Leu Lys Lys Gly Ile Cys Glu Ala Leu Glu
Asp Ser 785 790 795 800 Asp Gly Arg Gln Asp Ser Pro Ala Gly Glu Leu
Pro Lys Thr Val Gln 805 810 815 Gln Pro Thr Glu Ser Glu Pro Val Thr
Ile Asn Ile Gln Asp Leu Leu 820 825 830 Ser Cys Ser Asn Phe Ala Val
Gln His Arg Tyr Leu Phe Glu Glu Asp 835 840 845 Asn Leu Leu Arg Ser
Thr Gln Lys Leu Ser His Ser Thr Lys Pro Ser 850 855 860 Gly Ser Pro
Leu Glu Glu Lys His Asp Gln Cys Lys Cys Glu Asn Leu 865 870 875 880
Ile Met Phe Gln Asn Leu Ala Asn Glu Glu Val Arg Lys Leu Thr Gln 885
890 895 Arg Leu Glu Glu Met Thr Gln Arg Met Glu Ala Leu Glu Asn Arg
Leu 900 905 910 Arg Tyr Arg 915 35 23 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide probe
35 gtgaccctgg ttgtgaatac tcc 23 36 22 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide probe
36 acagccatgg tctatagctt gg 22 37 45 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide probe
37 gcctgtcagt gtcctgaggg acacgtgctc cgcagcgatg ggaag 45 38 1813 DNA
Homo sapiens 38 ggagccgccc tgggtgtcag cggctcggct cccgcgcacg
ctccggccgt cgcgcagcct 60 cggcacctgc aggtccgtgc gtcccgcggc
tggcgcccct gactccgtcc cggccaggga 120 gggccatgat ttccctcccg
gggcccctgg tgaccaactt gctgcggttt ttgttcctgg 180 ggctgagtgc
cctcgcgccc ccctcgcggg cccagctgca actgcacttg cccgccaacc 240
ggttgcaggc ggtggaggga ggggaagtgg tgcttccagc gtggtacacc ttgcacgggg
300 aggtgtcttc atcccagcca tgggaggtgc cctttgtgat gtggttcttc
aaacagaaag 360 aaaaggagga tcaggtgttg tcctacatca atggggtcac
aacaagcaaa cctggagtat 420 ccttggtcta ctccatgccc tcccggaacc
tgtccctgcg gctggagggt ctccaggaga 480 aagactctgg cccctacagc
tgctccgtga atgtgcaaga caaacaaggc aaatctaggg 540 gccacagcat
caaaacctta gaactcaatg tactggttcc tccagctcct ccatcctgcc 600
gtctccaggg tgtgccccat gtgggggcaa acgtgaccct gagctgccag tctccaagga
660 gtaagcccgc tgtccaatac cagtgggatc ggcagcttcc atccttccag
actttctttg 720 caccagcatt agatgtcatc cgtgggtctt taagcctcac
caacctttcg tcttccatgg 780 ctggagtcta tgtctgcaag gcccacaatg
aggtgggcac tgcccaatgt aatgtgacgc 840 tggaagtgag cacagggcct
ggagctgcag tggttgctgg agctgttgtg ggtaccctgg 900 ttggactggg
gttgctggct gggctggtcc tcttgtacca ccgccggggc aaggccctgg 960
aggagccagc caatgatatc aaggaggatg ccattgctcc ccggaccctg ccctggccca
1020 agagctcaga cacaatctcc aagaatggga ccctttcctc tgtcacctcc
gcacgagccc 1080 tccggccacc ccatggccct cccaggcctg gtgcattgac
ccccacgccc agtctctcca 1140 gccaggccct gccctcacca agactgccca
cgacagatgg ggcccaccct caaccaatat 1200 cccccatccc tggtggggtt
tcttcctctg gcttgagccg catgggtgct gtgcctgtga 1260 tggtgcctgc
ccagagtcaa gctggctctc tggtatgatg accccaccac tcattggcta 1320
aaggatttgg ggtctctcct tcctataagg gtcacctcta gcacagaggc ctgagtcatg
1380 ggaaagagtc acactcctga cccttagtac tctgccccca cctctcttta
ctgtgggaaa 1440 accatctcag taagacctaa gtgtccagga gacagaagga
gaagaggaag tggatctgga 1500 attgggagga gcctccaccc acccctgact
cctccttatg aagccagctg ctgaaattag 1560 ctactcacca agagtgaggg
gcagagactt ccagtcactg agtctcccag gcccccttga 1620 tctgtacccc
acccctatct aacaccaccc ttggctccca ctccagctcc ctgtattgat 1680
ataacctgtc aggctggctt ggttaggttt tactggggca gaggataggg aatctcttat
1740 taaaactaac atgaaatatg tgttgttttc atttgcaaat ttaaataaag
atacataatg 1800 tttgtatgaa aaa 1813 39 390 PRT Homo sapiens 39 Met
Ile Ser Leu Pro Gly Pro Leu Val Thr Asn Leu Leu Arg Phe Leu 1 5 10
15 Phe Leu Gly Leu Ser Ala Leu Ala Pro Pro Ser Arg Ala Gln Leu Gln
20 25 30 Leu His Leu Pro Ala Asn Arg Leu Gln Ala Val Glu Gly Gly
Glu Val 35 40 45 Val Leu Pro Ala Trp Tyr Thr Leu His Gly Glu Val
Ser Ser Ser Gln 50 55 60 Pro Trp Glu Val Pro Phe Val Met Trp Phe
Phe Lys Gln Lys Glu Lys 65 70 75 80 Glu Asp Gln Val Leu Ser Tyr Ile
Asn Gly Val Thr Thr Ser Lys Pro 85 90 95 Gly Val Ser Leu Val Tyr
Ser Met Pro Ser Arg Asn Leu Ser Leu Arg 100 105 110 Leu Glu Gly Leu
Gln Glu Lys Asp Ser Gly Pro Tyr Ser Cys Ser Val 115 120 125 Asn Val
Gln Asp Lys Gln Gly Lys Ser Arg Gly His Ser Ile Lys Thr 130 135 140
Leu Glu Leu Asn Val Leu Val Pro Pro Ala Pro Pro Ser Cys Arg Leu 145
150 155 160 Gln Gly Val Pro His Val Gly Ala Asn Val Thr Leu Ser Cys
Gln Ser 165 170 175 Pro Arg Ser Lys Pro Ala Val Gln Tyr Gln Trp Asp
Arg Gln Leu Pro 180 185 190 Ser Phe Gln Thr Phe Phe Ala Pro Ala Leu
Asp Val Ile Arg Gly Ser 195 200 205 Leu Ser Leu Thr Asn Leu Ser Ser
Ser Met Ala Gly Val Tyr Val Cys 210 215 220 Lys Ala His Asn Glu Val
Gly Thr Ala Gln Cys Asn Val Thr Leu Glu 225 230 235 240 Val Ser Thr
Gly Pro Gly Ala Ala Val Val Ala Gly Ala Val Val Gly 245 250 255 Thr
Leu Val Gly Leu Gly Leu Leu Ala Gly Leu Val Leu Leu Tyr His 260 265
270 Arg Arg Gly Lys Ala Leu Glu Glu Pro Ala Asn Asp Ile Lys Glu Asp
275 280 285 Ala Ile Ala Pro Arg Thr Leu Pro Trp Pro Lys Ser Ser Asp
Thr Ile 290 295 300 Ser Lys Asn Gly Thr Leu Ser Ser Val Thr Ser Ala
Arg Ala Leu Arg 305 310 315 320 Pro Pro His Gly Pro Pro Arg Pro Gly
Ala Leu Thr Pro Thr Pro Ser 325 330 335 Leu Ser Ser Gln Ala Leu Pro
Ser Pro Arg Leu Pro Thr Thr Asp Gly 340 345 350 Ala His Pro Gln Pro
Ile Ser Pro Ile Pro Gly Gly Val Ser Ser Ser 355 360 365 Gly Leu Ser
Arg Met Gly Ala Val Pro Val Met Val Pro Ala Gln Ser 370 375 380 Gln
Ala Gly Ser Leu Val 385 390 40 22 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide probe
40 agggtctcca ggagaaagac tc 22 41 24 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide probe
41 attgtgggcc ttgcagacat agac 24 42 50 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide probe
42 ggccacagca tcaaaacctt agaactcaat gtactggttc ctccagctcc 50 43 18
DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide probe 43 gtgtgacaca gcgtgggc 18 44 18 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 44 gaccggcagg cttctgcg 18 45 25 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 45 cagcagcttc agccaccagg agtgg 25 46 24 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 46 ctgagccgtg ggctgcagtc tcgc 24 47 45 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 47 ccgactacga ctggttcttc atcatgcagg
atgacacata tgtgc 45 48 2822 DNA Homo sapiens 48 cgccaccact
gcggccaccg ccaatgaaac gcctcccgct cctagtggtt ttttccactt 60
tgttgaattg ttcctatact caaaattgca ccaagacacc ttgtctccca aatgcaaaat
120 gtgaaatacg caatggaatt gaagcctgct attgcaacat gggattttca
ggaaatggtg 180 tcacaatttg tgaagatgat aatgaatgtg gaaatttaac
tcagtcctgt ggcgaaaatg 240 ctaattgcac taacacagaa ggaagttatt
attgtatgtg tgtacctggc ttcagatcca 300 gcagtaacca agacaggttt
atcactaatg atggaaccgt ctgtatagaa aatgtgaatg 360 caaactgcca
tttagataat gtctgtatag ctgcaaatat taataaaact ttaacaaaaa 420
tcagatccat aaaagaacct gtggctttgc tacaagaagt ctatagaaat tctgtgacag
480 atctttcacc aacagatata attacatata tagaaatatt agctgaatca
tcttcattac 540 taggttacaa gaacaacact atctcagcca aggacaccct
ttctaactca actcttactg 600 aatttgtaaa aaccgtgaat aattttgttc
aaagggatac atttgtagtt tgggacaagt 660 tatctgtgaa tcataggaga
acacatctta caaaactcat gcacactgtt gaacaagcta 720 ctttaaggat
atcccagagc ttccaaaaga ccacagagtt tgatacaaat tcaacggata 780
tagctctcaa agttttcttt tttgattcat ataacatgaa acatattcat cctcatatga
840 atatggatgg agactacata aatatatttc caaagagaaa agctgcatat
gattcaaatg 900 gcaatgttgc agttgcattt ttatattata agagtattgg
tcctttgctt tcatcatctg 960 acaacttctt attgaaacct caaaattatg
ataattctga agaggaggaa agagtcatat 1020 cttcagtaat ttcagtctca
atgagctcaa acccacccac attatatgaa cttgaaaaaa 1080 taacatttac
attaagtcat cgaaaggtca cagataggta taggagtcta tgtgcatttt 1140
ggaattactc acctgatacc atgaatggca gctggtcttc agagggctgt gagctgacat
1200 actcaaatga gacccacacc tcatgccgct gtaatcacct gacacatttt
gcaattttga 1260 tgtcctctgg tccttccatt ggtattaaag attataatat
tcttacaagg atcactcaac 1320 taggaataat tatttcactg atttgtcttg
ccatatgcat ttttaccttc tggttcttca 1380 gtgaaattca aagcaccagg
acaacaattc acaaaaatct ttgctgtagc ctatttcttg 1440 ctgaacttgt
ttttcttgtt gggatcaata caaatactaa taagctcttc tgttcaatca 1500
ttgccggact gctacactac ttctttttag ctgcttttgc atggatgtgc attgaaggca
1560 tacatctcta tctcattgtt gtgggtgtca tctacaacaa gggatttttg
cacaagaatt 1620 tttatatctt tggctatcta agcccagccg tggtagttgg
attttcggca gcactaggat 1680 acagatatta tggcacaacc aaagtatgtt
ggcttagcac cgaaaacaac tttatttgga 1740 gttttatagg accagcatgc
ctaatcattc ttgttaatct cttggctttt ggagtcatca 1800 tatacaaagt
ttttcgtcac actgcagggt tgaaaccaga agttagttgc tttgagaaca 1860
taaggtcttg tgcaagagga gccctcgctc ttctgttcct tctcggcacc acctggatct
1920 ttggggttct ccatgttgtg cacgcatcag tggttacagc ttacctcttc
acagtcagca 1980 atgctttcca ggggatgttc atttttttat tcctgtgtgt
tttatctaga aagattcaag 2040 aagaatatta cagattgttc aaaaatgtcc
cctgttgttt tggatgttta aggtaaacat 2100 agagaatggt ggataattac
aactgcacaa aaataaaaat tccaagctgt ggatgaccaa 2160 tgtataaaaa
tgactcatca aattatccaa ttattaacta ctagacaaaa agtattttaa 2220
atcagttttt ctgtttatgc tataggaact gtagataata aggtaaaatt atgtatcata
2280 tagatatact atgtttttct atgtgaaata gttctgtcaa aaatagtatt
gcagatattt 2340 ggaaagtaat tggtttctca ggagtgatat cactgcaccc
aaggaaagat tttctttcta 2400 acacgagaag tatatgaatg tcctgaagga
aaccactggc ttgatatttc tgtgactcgt 2460 gttgcctttg aaactagtcc
cctaccacct cggtaatgag ctccattaca gaaagtggaa 2520 cataagagaa
tgaaggggca gaatatcaaa cagtgaaaag ggaatgataa gatgtatttt 2580
gaatgaactg ttttttctgt agactagctg agaaattgtt gacataaaat aaagaattga
2640 agaaacacat tttaccattt tgtgaattgt tctgaactta aatgtccact
aaaacaactt 2700 agacttctgt ttgctaaatc tgtttctttt tctaatattc
taaaaaaaaa aaaaaggttt 2760 acctccacaa attgaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2820 aa 2822 49 690 PRT Homo
sapiens 49 Met Lys Arg Leu Pro Leu Leu Val Val Phe Ser Thr Leu Leu
Asn Cys 1 5 10 15 Ser Tyr Thr Gln Asn Cys Thr Lys Thr Pro Cys Leu
Pro Asn Ala Lys 20 25 30
Cys Glu Ile Arg Asn Gly Ile Glu Ala Cys Tyr Cys Asn Met Gly Phe 35
40 45 Ser Gly Asn Gly Val Thr Ile Cys Glu Asp Asp Asn Glu Cys Gly
Asn 50 55 60 Leu Thr Gln Ser Cys Gly Glu Asn Ala Asn Cys Thr Asn
Thr Glu Gly 65 70 75 80 Ser Tyr Tyr Cys Met Cys Val Pro Gly Phe Arg
Ser Ser Ser Asn Gln 85 90 95 Asp Arg Phe Ile Thr Asn Asp Gly Thr
Val Cys Ile Glu Asn Val Asn 100 105 110 Ala Asn Cys His Leu Asp Asn
Val Cys Ile Ala Ala Asn Ile Asn Lys 115 120 125 Thr Leu Thr Lys Ile
Arg Ser Ile Lys Glu Pro Val Ala Leu Leu Gln 130 135 140 Glu Val Tyr
Arg Asn Ser Val Thr Asp Leu Ser Pro Thr Asp Ile Ile 145 150 155 160
Thr Tyr Ile Glu Ile Leu Ala Glu Ser Ser Ser Leu Leu Gly Tyr Lys 165
170 175 Asn Asn Thr Ile Ser Ala Lys Asp Thr Leu Ser Asn Ser Thr Leu
Thr 180 185 190 Glu Phe Val Lys Thr Val Asn Asn Phe Val Gln Arg Asp
Thr Phe Val 195 200 205 Val Trp Asp Lys Leu Ser Val Asn His Arg Arg
Thr His Leu Thr Lys 210 215 220 Leu Met His Thr Val Glu Gln Ala Thr
Leu Arg Ile Ser Gln Ser Phe 225 230 235 240 Gln Lys Thr Thr Glu Phe
Asp Thr Asn Ser Thr Asp Ile Ala Leu Lys 245 250 255 Val Phe Phe Phe
Asp Ser Tyr Asn Met Lys His Ile His Pro His Met 260 265 270 Asn Met
Asp Gly Asp Tyr Ile Asn Ile Phe Pro Lys Arg Lys Ala Ala 275 280 285
Tyr Asp Ser Asn Gly Asn Val Ala Val Ala Phe Leu Tyr Tyr Lys Ser 290
295 300 Ile Gly Pro Leu Leu Ser Ser Ser Asp Asn Phe Leu Leu Lys Pro
Gln 305 310 315 320 Asn Tyr Asp Asn Ser Glu Glu Glu Glu Arg Val Ile
Ser Ser Val Ile 325 330 335 Ser Val Ser Met Ser Ser Asn Pro Pro Thr
Leu Tyr Glu Leu Glu Lys 340 345 350 Ile Thr Phe Thr Leu Ser His Arg
Lys Val Thr Asp Arg Tyr Arg Ser 355 360 365 Leu Cys Ala Phe Trp Asn
Tyr Ser Pro Asp Thr Met Asn Gly Ser Trp 370 375 380 Ser Ser Glu Gly
Cys Glu Leu Thr Tyr Ser Asn Glu Thr His Thr Ser 385 390 395 400 Cys
Arg Cys Asn His Leu Thr His Phe Ala Ile Leu Met Ser Ser Gly 405 410
415 Pro Ser Ile Gly Ile Lys Asp Tyr Asn Ile Leu Thr Arg Ile Thr Gln
420 425 430 Leu Gly Ile Ile Ile Ser Leu Ile Cys Leu Ala Ile Cys Ile
Phe Thr 435 440 445 Phe Trp Phe Phe Ser Glu Ile Gln Ser Thr Arg Thr
Thr Ile His Lys 450 455 460 Asn Leu Cys Cys Ser Leu Phe Leu Ala Glu
Leu Val Phe Leu Val Gly 465 470 475 480 Ile Asn Thr Asn Thr Asn Lys
Leu Phe Cys Ser Ile Ile Ala Gly Leu 485 490 495 Leu His Tyr Phe Phe
Leu Ala Ala Phe Ala Trp Met Cys Ile Glu Gly 500 505 510 Ile His Leu
Tyr Leu Ile Val Val Gly Val Ile Tyr Asn Lys Gly Phe 515 520 525 Leu
His Lys Asn Phe Tyr Ile Phe Gly Tyr Leu Ser Pro Ala Val Val 530 535
540 Val Gly Phe Ser Ala Ala Leu Gly Tyr Arg Tyr Tyr Gly Thr Thr Lys
545 550 555 560 Val Cys Trp Leu Ser Thr Glu Asn Asn Phe Ile Trp Ser
Phe Ile Gly 565 570 575 Pro Ala Cys Leu Ile Ile Leu Val Asn Leu Leu
Ala Phe Gly Val Ile 580 585 590 Ile Tyr Lys Val Phe Arg His Thr Ala
Gly Leu Lys Pro Glu Val Ser 595 600 605 Cys Phe Glu Asn Ile Arg Ser
Cys Ala Arg Gly Ala Leu Ala Leu Leu 610 615 620 Phe Leu Leu Gly Thr
Thr Trp Ile Phe Gly Val Leu His Val Val His 625 630 635 640 Ala Ser
Val Val Thr Ala Tyr Leu Phe Thr Val Ser Asn Ala Phe Gln 645 650 655
Gly Met Phe Ile Phe Leu Phe Leu Cys Val Leu Ser Arg Lys Ile Gln 660
665 670 Glu Glu Tyr Tyr Arg Leu Phe Lys Asn Val Pro Cys Cys Phe Gly
Cys 675 680 685 Leu Arg 690 50 589 DNA Homo sapiens modified_base
(61)..(61) a, t, c or g 50 tggaaacata tcctccctca tatgaatatg
gatggagact acataaatat atttccaaag 60 ngaaaagccg gcatatggat
tcaaatggca atgttgcagt tgcattttta tattataaga 120 gtattggtcc
ctttgctttc atcatctgac aacttcttat tgaaacctca aaattatgat 180
aattctgaag aggaggaaag agtcatatct tcagtaattt cagtctcaat gagctcaaac
240 ccacccacat tatatgaact tgaaaaaata acatttacat taagtcatcg
aaaggtcaca 300 gataggtata ggagtctatg tggcattttg gaatactcac
ctgataccat gaatggcagc 360 tggtcttcag agggctgtga gctgacatac
tcaaatgaga cccacacctc atgccgctgt 420 aatcacctga cacattttgc
aattttgatg tcctctggtc cttccattgg tattaaagat 480 tataatattc
ttacaaggat cactcaacta ggaataatta tttcactgat ttgtcttgcc 540
atatgcattt ttaccttctg gttcttcagt gaaattcaaa gcaccagga 589 51 20 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 51 ggtaatgagc tccattacag 20 52 18 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 52 ggagtagaaa gcgcatgg 18 53 22 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 53 cacctgatac catgaatggc ag 22 54 18 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 54 cgagctcgaa ttaattcg 18 55 18 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 55 ggatctcctg agctcagg 18 56 23 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 56 cctagttgag tgatccttgt aag 23 57 50 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 57 atgagaccca cacctcatgc cgctgtaatc
acctgacaca ttttgcaatt 50 58 2137 DNA Homo sapiens 58 gctcccagcc
aagaacctcg gggccgctgc gcggtgggga ggagttcccc gaaacccggc 60
cgctaagcga ggcctcctcc tcccgcagat ccgaacggcc tgggcggggt caccccggct
120 gggacaagaa gccgccgcct gcctgcccgg gcccggggag ggggctgggg
ctggggccgg 180 aggcggggtg tgagtgggtg tgtgcggggg gcggaggctt
gatgcaatcc cgataagaaa 240 tgctcgggtg tcttgggcac ctacccgtgg
ggcccgtaag gcgctactat ataaggctgc 300 cggcccggag ccgccgcgcc
gtcagagcag gagcgctgcg tccaggatct agggccacga 360 ccatcccaac
ccggcactca cagccccgca gcgcatcccg gtcgccgccc agcctcccgc 420
acccccatcg ccggagctgc gccgagagcc ccagggaggt gccatgcgga gcgggtgtgt
480 ggtggtccac gtatggatcc tggccggcct ctggctggcc gtggccgggc
gccccctcgc 540 cttctcggac gcggggcccc acgtgcacta cggctggggc
gaccccatcc gcctgcggca 600 cctgtacacc tccggccccc acgggctctc
cagctgcttc ctgcgcatcc gtgccgacgg 660 cgtcgtggac tgcgcgcggg
gccagagcgc gcacagtttg ctggagatca aggcagtcgc 720 tctgcggacc
gtggccatca agggcgtgca cagcgtgcgg tacctctgca tgggcgccga 780
cggcaagatg caggggctgc ttcagtactc ggaggaagac tgtgctttcg aggaggagat
840 ccgcccagat ggctacaatg tgtaccgatc cgagaagcac cgcctcccgg
tctccctgag 900 cagtgccaaa cagcggcagc tgtacaagaa cagaggcttt
cttccactct ctcatttcct 960 gcccatgctg cccatggtcc cagaggagcc
tgaggacctc aggggccact tggaatctga 1020 catgttctct tcgcccctgg
agaccgacag catggaccca tttgggcttg tcaccggact 1080 ggaggccgtg
aggagtccca gctttgagaa gtaactgaga ccatgcccgg gcctcttcac 1140
tgctgccagg ggctgtggta cctgcagcgt gggggacgtg cttctacaag aacagtcctg
1200 agtccacgtt ctgtttagct ttaggaagaa acatctagaa gttgtacata
ttcagagttt 1260 tccattggca gtgccagttt ctagccaata gacttgtctg
atcataacat tgtaagcctg 1320 tagcttgccc agctgctgcc tgggccccca
ttctgctccc tcgaggttgc tggacaagct 1380 gctgcactgt ctcagttctg
cttgaatacc tccatcgatg gggaactcac ttcctttgga 1440 aaaattctta
tgtcaagctg aaattctcta attttttctc atcacttccc caggagcagc 1500
cagaagacag gcagtagttt taatttcagg aacaggtgat ccactctgta aaacagcagg
1560 taaatttcac tcaaccccat gtgggaattg atctatatct ctacttccag
ggaccatttg 1620 cccttcccaa atccctccag gccagaactg actggagcag
gcatggccca ccaggcttca 1680 ggagtagggg aagcctggag ccccactcca
gccctgggac aacttgagaa ttccccctga 1740 ggccagttct gtcatggatg
ctgtcctgag aataacttgc tgtcccggtg tcacctgctt 1800 ccatctccca
gcccaccagc cctctgccca cctcacatgc ctccccatgg attggggcct 1860
cccaggcccc ccaccttatg tcaacctgca cttcttgttc aaaaatcagg aaaagaaaag
1920 atttgaagac cccaagtctt gtcaataact tgctgtgtgg aagcagcggg
ggaagaccta 1980 gaaccctttc cccagcactt ggttttccaa catgatattt
atgagtaatt tattttgata 2040 tgtacatctc ttattttctt acattattta
tgcccccaaa ttatatttat gtatgtaagt 2100 gaggtttgtt ttgtatatta
aaatggagtt tgtttgt 2137 59 216 PRT Homo sapiens 59 Met Arg Ser Gly
Cys Val Val Val His Val Trp Ile Leu Ala Gly Leu 1 5 10 15 Trp Leu
Ala Val Ala Gly Arg Pro Leu Ala Phe Ser Asp Ala Gly Pro 20 25 30
His Val His Tyr Gly Trp Gly Asp Pro Ile Arg Leu Arg His Leu Tyr 35
40 45 Thr Ser Gly Pro His Gly Leu Ser Ser Cys Phe Leu Arg Ile Arg
Ala 50 55 60 Asp Gly Val Val Asp Cys Ala Arg Gly Gln Ser Ala His
Ser Leu Leu 65 70 75 80 Glu Ile Lys Ala Val Ala Leu Arg Thr Val Ala
Ile Lys Gly Val His 85 90 95 Ser Val Arg Tyr Leu Cys Met Gly Ala
Asp Gly Lys Met Gln Gly Leu 100 105 110 Leu Gln Tyr Ser Glu Glu Asp
Cys Ala Phe Glu Glu Glu Ile Arg Pro 115 120 125 Asp Gly Tyr Asn Val
Tyr Arg Ser Glu Lys His Arg Leu Pro Val Ser 130 135 140 Leu Ser Ser
Ala Lys Gln Arg Gln Leu Tyr Lys Asn Arg Gly Phe Leu 145 150 155 160
Pro Leu Ser His Phe Leu Pro Met Leu Pro Met Val Pro Glu Glu Pro 165
170 175 Glu Asp Leu Arg Gly His Leu Glu Ser Asp Met Phe Ser Ser Pro
Leu 180 185 190 Glu Thr Asp Ser Met Asp Pro Phe Gly Leu Val Thr Gly
Leu Glu Ala 195 200 205 Val Arg Ser Pro Ser Phe Glu Lys 210 215 60
26 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide probe 60 atccgcccag atggctacaa tgtgta 26
61 42 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide probe 61 gcctcccggt ctccctgagc agtgccaaac
agcggcagtg ta 42 62 22 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide probe 62 ccagtccggt
gacaagccca aa 22 63 1295 DNA Homo sapiens 63 cccagaagtt caagggcccc
cggcctcctg cgctcctgcc gccgggaccc tcgacctcct 60 cagagcagcc
ggctgccgcc ccgggaagat ggcgaggagg agccgccacc gcctcctcct 120
gctgctgctg cgctacctgg tggtcgccct gggctatcat aaggcctatg ggttttctgc
180 cccaaaagac caacaagtag tcacagcagt agagtaccaa gaggctattt
tagcctgcaa 240 aaccccaaag aagactgttt cctccagatt agagtggaag
aaactgggtc ggagtgtctc 300 ctttgtctac tatcaacaga ctcttcaagg
tgattttaaa aatcgagctg agatgataga 360 tttcaatatc cggatcaaaa
atgtgacaag aagtgatgcg gggaaatatc gttgtgaagt 420 tagtgcccca
tctgagcaag gccaaaacct ggaagaggat acagtcactc tggaagtatt 480
agtggctcca gcagttccat catgtgaagt accctcttct gctctgagtg gaactgtggt
540 agagctacga tgtcaagaca aagaagggaa tccagctcct gaatacacat
ggtttaagga 600 tggcatccgt ttgctagaaa atcccagact tggctcccaa
agcaccaaca gctcatacac 660 aatgaataca aaaactggaa ctctgcaatt
taatactgtt tccaaactgg acactggaga 720 atattcctgt gaagcccgca
attctgttgg atatcgcagg tgtcctggga aacgaatgca 780 agtagatgat
ctcaacataa gtggcatcat agcagccgta gtagttgtgg ccttagtgat 840
ttccgtttgt ggccttggtg tatgctatgc tcagaggaaa ggctactttt caaaagaaac
900 ctccttccag aagagtaatt cttcatctaa agccacgaca atgagtgaaa
atgtgcagtg 960 gctcacgcct gtaatcccag cactttggaa ggccgcggcg
ggcggatcac gaggtcagga 1020 gttctagacc agtctggcca atatggtgaa
accccatctc tactaaaata caaaaattag 1080 ctgggcatgg tggcatgtgc
ctgcagttcc agctgcttgg gagacaggag aatcacttga 1140 acccgggagg
cggaggttgc agtgagctga gatcacgcca ctgcagtcca gcctgggtaa 1200
cagagcaaga ttccatctca aaaaataaaa taaataaata aataaatact ggtttttacc
1260 tgtagaattc ttacaataaa tatagcttga tattc 1295 64 312 PRT Homo
sapiens 64 Met Ala Arg Arg Ser Arg His Arg Leu Leu Leu Leu Leu Leu
Arg Tyr 1 5 10 15 Leu Val Val Ala Leu Gly Tyr His Lys Ala Tyr Gly
Phe Ser Ala Pro 20 25 30 Lys Asp Gln Gln Val Val Thr Ala Val Glu
Tyr Gln Glu Ala Ile Leu 35 40 45 Ala Cys Lys Thr Pro Lys Lys Thr
Val Ser Ser Arg Leu Glu Trp Lys 50 55 60 Lys Leu Gly Arg Ser Val
Ser Phe Val Tyr Tyr Gln Gln Thr Leu Gln 65 70 75 80 Gly Asp Phe Lys
Asn Arg Ala Glu Met Ile Asp Phe Asn Ile Arg Ile 85 90 95 Lys Asn
Val Thr Arg Ser Asp Ala Gly Lys Tyr Arg Cys Glu Val Ser 100 105 110
Ala Pro Ser Glu Gln Gly Gln Asn Leu Glu Glu Asp Thr Val Thr Leu 115
120 125 Glu Val Leu Val Ala Pro Ala Val Pro Ser Cys Glu Val Pro Ser
Ser 130 135 140 Ala Leu Ser Gly Thr Val Val Glu Leu Arg Cys Gln Asp
Lys Glu Gly 145 150 155 160 Asn Pro Ala Pro Glu Tyr Thr Trp Phe Lys
Asp Gly Ile Arg Leu Leu 165 170 175 Glu Asn Pro Arg Leu Gly Ser Gln
Ser Thr Asn Ser Ser Tyr Thr Met 180 185 190 Asn Thr Lys Thr Gly Thr
Leu Gln Phe Asn Thr Val Ser Lys Leu Asp 195 200 205 Thr Gly Glu Tyr
Ser Cys Glu Ala Arg Asn Ser Val Gly Tyr Arg Arg 210 215 220 Cys Pro
Gly Lys Arg Met Gln Val Asp Asp Leu Asn Ile Ser Gly Ile 225 230 235
240 Ile Ala Ala Val Val Val Val Ala Leu Val Ile Ser Val Cys Gly Leu
245 250 255 Gly Val Cys Tyr Ala Gln Arg Lys Gly Tyr Phe Ser Lys Glu
Thr Ser 260 265 270 Phe Gln Lys Ser Asn Ser Ser Ser Lys Ala Thr Thr
Met Ser Glu Asn 275 280 285 Val Gln Trp Leu Thr Pro Val Ile Pro Ala
Leu Trp Lys Ala Ala Ala 290 295 300 Gly Gly Ser Arg Gly Gln Glu Phe
305 310 65 22 DNA Artificial Sequence Description of Artificial
Sequence Synthetic oligonucleotide probe 65 atcgttgtga agttagtgcc
cc 22 66 23 DNA Artificial Sequence Description of Artificial
Sequence Synthetic oligonucleotide probe 66 acctgcgata tccaacagaa
ttg 23 67 48 DNA Artificial Sequence Description of Artificial
Sequence Synthetic oligonucleotide probe 67 ggaagaggat acagtcactc
tggaagtatt agtggctcca gcagttcc 48 68 2639 DNA Homo sapiens 68
gacatcggag gtgggctagc actgaaactg cttttcaaga cgaggaagag gaggagaaag
60 agaaagaaga ggaagatgtt gggcaacatt tatttaacat gctccacagc
ccggaccctg 120 gcatcatgct gctattcctg caaatactga agaagcatgg
gatttaaata ttttacttct 180 aaataaatga attactcaat ctcctatgac
catctataca tactccacct tcaaaaagta 240 catcaatatt atatcattaa
ggaaatagta accttctctt ctccaatatg catgacattt 300 ttggacaatg
caattgtggc actggcactt atttcagtga agaaaaactt tgtggttcta 360
tggcattcat catttgacaa atgcaagcat cttccttatc aatcagctcc tattgaactt
420 actagcactg actgtggaat ccttaagggc ccattacatt tctgaagaag
aaagctaaga 480 tgaaggacat gccactccga attcatgtgc tacttggcct
agctatcact acactagtac 540 aagctgtaga taaaaaagtg gattgtccac
ggttatgtac gtgtgaaatc aggccttggt 600 ttacacccag atccatttat
atggaagcat ctacagtgga ttgtaatgat ttaggtcttt 660 taactttccc
agccagattg ccagctaaca cacagattct tctcctacag actaacaata 720
ttgcaaaaat tgaatactcc acagactttc cagtaaacct tactggcctg gatttatctc
780 aaaacaattt atcttcagtc accaatatta atgtaaaaaa gatgcctcag
ctcctttctg 840 tgtacctaga ggaaaacaaa cttactgaac tgcctgaaaa
atgtctgtcc gaactgagca 900 acttacaaga actctatatt aatcacaact
tgctttctac aatttcacct ggagccttta 960 ttggcctaca taatcttctt
cgacttcatc tcaattcaaa tagattgcag atgatcaaca 1020 gtaagtggtt
tgatgctctt ccaaatctag agattctgat gattggggaa aatccaatta 1080
tcagaatcaa agacatgaac tttaagcctc ttatcaatct tcgcagcctg gttatagctg
1140 gtataaacct cacagaaata ccagataacg ccttggttgg actggaaaac
ttagaaagca 1200 tctcttttta cgataacagg cttattaaag taccccatgt
tgctcttcaa aaagttgtaa 1260 atctcaaatt tttggatcta aataaaaatc
ctattaatag aatacgaagg ggtgatttta 1320 gcaatatgct acacttaaaa
gagttgggga taaataatat gcctgagctg atttccatcg 1380 atagtcttgc
tgtggataac ctgccagatt taagaaaaat agaagctact aacaacccta 1440
gattgtctta cattcacccc aatgcatttt tcagactccc caagctggaa tcactcatgc
1500 tgaacagcaa tgctctcagt gccctgtacc atggtaccat tgagtctctg
ccaaacctca 1560 aggaaatcag catacacagt aaccccatca ggtgtgactg
tgtcatccgt tggatgaaca 1620 tgaacaaaac caacattcga ttcatggagc
cagattcact gttttgcgtg gacccacctg 1680 aattccaagg tcagaatgtt
cggcaagtgc atttcaggga catgatggaa atttgtctcc 1740 ctcttatagc
tcctgagagc tttccttcta atctaaatgt agaagctggg agctatgttt 1800
cctttcactg tagagctact gcagaaccac agcctgaaat ctactggata acaccttctg
1860 gtcaaaaact cttgcctaat accctgacag acaagttcta tgtccattct
gagggaacac 1920 tagatataaa tggcgtaact cccaaagaag ggggtttata
tacttgtata gcaactaacc 1980 tagttggcgc tgacttgaag tctgttatga
tcaaagtgga tggatctttt ccacaagata 2040 acaatggctc tttgaatatt
aaaataagag atattcaggc caattcagtt ttggtgtcct 2100 ggaaagcaag
ttctaaaatt ctcaaatcta gtgttaaatg gacagccttt gtcaagactg 2160
aaaattctca tgctgcgcaa agtgctcgaa taccatctga tgtcaaggta tataatctta
2220 ctcatctgaa tccatcaact gagtataaaa tttgtattga tattcccacc
atctatcaga 2280 aaaacagaaa aaaatgtgta aatgtcacca ccaaaggttt
gcaccctgat caaaaagagt 2340 atgaaaagaa taataccaca acacttatgg
cctgtcttgg aggccttctg gggattattg 2400 gtgtgatatg tcttatcagc
tgcctctctc cagaaatgaa ctgtgatggt ggacacagct 2460 atgtgaggaa
ttacttacag aaaccaacct ttgcattagg tgagctttat cctcctctga 2520
taaatctctg ggaagcagga aaagaaaaaa gtacatcact gaaagtaaaa gcaactgtta
2580 taggtttacc aacaaatatg tcctaaaaac caccaaggaa acctactcca
aaaatgaac 2639 69 708 PRT Homo sapiens 69 Met Lys Asp Met Pro Leu
Arg Ile His Val Leu Leu Gly Leu Ala Ile 1 5 10 15 Thr Thr Leu Val
Gln Ala Val Asp Lys Lys Val Asp Cys Pro Arg Leu 20 25 30 Cys Thr
Cys Glu Ile Arg Pro Trp Phe Thr Pro Arg Ser Ile Tyr Met 35 40 45
Glu Ala Ser Thr Val Asp Cys Asn Asp Leu Gly Leu Leu Thr Phe Pro 50
55 60 Ala Arg Leu Pro Ala Asn Thr Gln Ile Leu Leu Leu Gln Thr Asn
Asn 65 70 75 80 Ile Ala Lys Ile Glu Tyr Ser Thr Asp Phe Pro Val Asn
Leu Thr Gly 85 90 95 Leu Asp Leu Ser Gln Asn Asn Leu Ser Ser Val
Thr Asn Ile Asn Val 100 105 110 Lys Lys Met Pro Gln Leu Leu Ser Val
Tyr Leu Glu Glu Asn Lys Leu 115 120 125 Thr Glu Leu Pro Glu Lys Cys
Leu Ser Glu Leu Ser Asn Leu Gln Glu 130 135 140 Leu Tyr Ile Asn His
Asn Leu Leu Ser Thr Ile Ser Pro Gly Ala Phe 145 150 155 160 Ile Gly
Leu His Asn Leu Leu Arg Leu His Leu Asn Ser Asn Arg Leu 165 170 175
Gln Met Ile Asn Ser Lys Trp Phe Asp Ala Leu Pro Asn Leu Glu Ile 180
185 190 Leu Met Ile Gly Glu Asn Pro Ile Ile Arg Ile Lys Asp Met Asn
Phe 195 200 205 Lys Pro Leu Ile Asn Leu Arg Ser Leu Val Ile Ala Gly
Ile Asn Leu 210 215 220 Thr Glu Ile Pro Asp Asn Ala Leu Val Gly Leu
Glu Asn Leu Glu Ser 225 230 235 240 Ile Ser Phe Tyr Asp Asn Arg Leu
Ile Lys Val Pro His Val Ala Leu 245 250 255 Gln Lys Val Val Asn Leu
Lys Phe Leu Asp Leu Asn Lys Asn Pro Ile 260 265 270 Asn Arg Ile Arg
Arg Gly Asp Phe Ser Asn Met Leu His Leu Lys Glu 275 280 285 Leu Gly
Ile Asn Asn Met Pro Glu Leu Ile Ser Ile Asp Ser Leu Ala 290 295 300
Val Asp Asn Leu Pro Asp Leu Arg Lys Ile Glu Ala Thr Asn Asn Pro 305
310 315 320 Arg Leu Ser Tyr Ile His Pro Asn Ala Phe Phe Arg Leu Pro
Lys Leu 325 330 335 Glu Ser Leu Met Leu Asn Ser Asn Ala Leu Ser Ala
Leu Tyr His Gly 340 345 350 Thr Ile Glu Ser Leu Pro Asn Leu Lys Glu
Ile Ser Ile His Ser Asn 355 360 365 Pro Ile Arg Cys Asp Cys Val Ile
Arg Trp Met Asn Met Asn Lys Thr 370 375 380 Asn Ile Arg Phe Met Glu
Pro Asp Ser Leu Phe Cys Val Asp Pro Pro 385 390 395 400 Glu Phe Gln
Gly Gln Asn Val Arg Gln Val His Phe Arg Asp Met Met 405 410 415 Glu
Ile Cys Leu Pro Leu Ile Ala Pro Glu Ser Phe Pro Ser Asn Leu 420 425
430 Asn Val Glu Ala Gly Ser Tyr Val Ser Phe His Cys Arg Ala Thr Ala
435 440 445 Glu Pro Gln Pro Glu Ile Tyr Trp Ile Thr Pro Ser Gly Gln
Lys Leu 450 455 460 Leu Pro Asn Thr Leu Thr Asp Lys Phe Tyr Val His
Ser Glu Gly Thr 465 470 475 480 Leu Asp Ile Asn Gly Val Thr Pro Lys
Glu Gly Gly Leu Tyr Thr Cys 485 490 495 Ile Ala Thr Asn Leu Val Gly
Ala Asp Leu Lys Ser Val Met Ile Lys 500 505 510 Val Asp Gly Ser Phe
Pro Gln Asp Asn Asn Gly Ser Leu Asn Ile Lys 515 520 525 Ile Arg Asp
Ile Gln Ala Asn Ser Val Leu Val Ser Trp Lys Ala Ser 530 535 540 Ser
Lys Ile Leu Lys Ser Ser Val Lys Trp Thr Ala Phe Val Lys Thr 545 550
555 560 Glu Asn Ser His Ala Ala Gln Ser Ala Arg Ile Pro Ser Asp Val
Lys 565 570 575 Val Tyr Asn Leu Thr His Leu Asn Pro Ser Thr Glu Tyr
Lys Ile Cys 580 585 590 Ile Asp Ile Pro Thr Ile Tyr Gln Lys Asn Arg
Lys Lys Cys Val Asn 595 600 605 Val Thr Thr Lys Gly Leu His Pro Asp
Gln Lys Glu Tyr Glu Lys Asn 610 615 620 Asn Thr Thr Thr Leu Met Ala
Cys Leu Gly Gly Leu Leu Gly Ile Ile 625 630 635 640 Gly Val Ile Cys
Leu Ile Ser Cys Leu Ser Pro Glu Met Asn Cys Asp 645 650 655 Gly Gly
His Ser Tyr Val Arg Asn Tyr Leu Gln Lys Pro Thr Phe Ala 660 665 670
Leu Gly Glu Leu Tyr Pro Pro Leu Ile Asn Leu Trp Glu Ala Gly Lys 675
680 685 Glu Lys Ser Thr Ser Leu Lys Val Lys Ala Thr Val Ile Gly Leu
Pro 690 695 700 Thr Asn Met Ser 705 70 1305 DNA Homo sapiens 70
gcccgggact ggcgcaaggt gcccaagcaa ggaaagaaat aatgaagaga cacatgtgtt
60 agctgcagcc ttttgaaaca cgcaagaagg aaatcaatag tgtggacagg
gctggaacct 120 ttaccacgct tgttggagta gatgaggaat gggctcgtga
ttatgctgac attccagcat 180 gaatctggta gacctgtggt taacccgttc
cctctccatg tgtctcctcc tacaaagttt 240 tgttcttatg atactgtgct
ttcattctgc cagtatgtgt cccaagggct gtctttgttc 300 ttcctctggg
ggtttaaatg tcacctgtag caatgcaaat ctcaaggaaa tacctagaga 360
tcttcctcct gaaacagtct tactgtatct ggactccaat cagatcacat ctattcccaa
420 tgaaattttt aaggacctcc atcaactgag agttctcaac ctgtccaaaa
atggcattga 480 gtttatcgat gagcatgcct tcaaaggagt agctgaaacc
ttgcagactc tggacttgtc 540 cgacaatcgg attcaaagtg tgcacaaaaa
tgccttcaat aacctgaagg ccagggccag 600 aattgccaac aacccctggc
actgcgactg tactctacag caagttctga ggagcatggc 660 gtccaatcat
gagacagccc acaacgtgat ctgtaaaacg tccgtgttgg atgaacatgc 720
tggcagacca ttcctcaatg ctgccaacga cgctgacctt tgtaacctcc ctaaaaaaac
780 taccgattat gccatgctgg tcaccatgtt tggctggttc actatggtga
tctcatatgt 840 ggtatattat gtgaggcaaa atcaggagga tgcccggaga
cacctcgaat acttgaaatc 900 cctgccaagc aggcagaaga aagcagatga
acctgatgat attagcactg tggtatagtg 960 tccaaactga ctgtcattga
gaaagaaaga aagtagtttg cgattgcagt agaaataagt 1020 ggtttacttc
tcccatccat tgtaaacatt tgaaactttg tatttcagtt ttttttgaat 1080
tatgccactg ctgaactttt aacaaacact acaacataaa taatttgagt ttaggtgatc
1140 caccccttaa ttgtaccccc gatggtatat ttctgagtaa gctactatct
gaacattagt 1200 tagatccatc tcactattta ataatgaaat ttattttttt
aatttaaaag caaataaaag 1260 cttaactttg aaccatggga aaaaaaaaaa
aaaaaaaaaa aaaca 1305 71 259 PRT Homo sapiens 71 Met Asn Leu Val
Asp Leu Trp Leu Thr Arg Ser Leu Ser Met Cys Leu 1 5 10 15 Leu Leu
Gln Ser Phe Val Leu Met Ile Leu Cys Phe His Ser Ala Ser 20 25 30
Met Cys Pro Lys Gly Cys Leu Cys Ser Ser Ser Gly Gly Leu Asn Val 35
40 45 Thr Cys Ser Asn Ala Asn Leu Lys Glu Ile Pro Arg Asp Leu Pro
Pro 50 55 60 Glu Thr Val Leu Leu Tyr Leu Asp Ser Asn Gln Ile Thr
Ser Ile Pro 65 70 75 80 Asn Glu Ile Phe Lys Asp Leu His Gln Leu Arg
Val Leu Asn Leu Ser 85 90 95 Lys Asn Gly Ile Glu Phe Ile Asp Glu
His Ala Phe Lys Gly Val Ala 100 105 110 Glu Thr Leu Gln Thr Leu Asp
Leu Ser Asp Asn Arg Ile Gln Ser Val 115 120 125 His Lys Asn Ala Phe
Asn Asn Leu Lys Ala Arg Ala Arg Ile Ala Asn 130 135 140 Asn Pro Trp
His Cys Asp Cys Thr Leu Gln Gln Val Leu Arg Ser Met 145 150 155 160
Ala Ser Asn His Glu Thr Ala His Asn Val Ile Cys Lys Thr Ser Val 165
170 175 Leu Asp Glu His Ala Gly Arg Pro Phe Leu Asn Ala Ala Asn Asp
Ala 180 185 190 Asp Leu Cys Asn Leu Pro Lys Lys Thr Thr Asp Tyr Ala
Met Leu Val 195 200 205 Thr Met Phe Gly Trp Phe Thr Met Val Ile Ser
Tyr Val Val Tyr Tyr 210 215 220 Val Arg Gln Asn Gln Glu Asp Ala Arg
Arg His Leu Glu Tyr Leu Lys 225 230 235 240 Ser Leu Pro Ser Arg Gln
Lys Lys Ala Asp Glu Pro Asp Asp Ile Ser 245 250 255 Thr Val Val 72
2290 DNA Homo sapiens 72 accgagccga gcggaccgaa ggcgcgcccg
agatgcaggt gagcaagagg atgctggcgg 60 ggggcgtgag gagcatgccc
agccccctcc tggcctgctg gcagcccatc ctcctgctgg 120 tgctgggctc
agtgctgtca ggctcggcca cgggctgccc gccccgctgc gagtgctccg 180
cccaggaccg cgctgtgctg tgccaccgca agtgctttgt ggcagtcccc gagggcatcc
240 ccaccgagac gcgcctgctg gacctaggca agaaccgcat caaaacgctc
aaccaggacg 300 agttcgccag cttcccgcac ctggaggagc tggagctcaa
cgagaacatc gtgagcgccg 360 tggagcccgg cgccttcaac aacctcttca
acctccggac gctgggtctc cgcagcaacc 420 gcctgaagct catcccgcta
ggcgtcttca ctggcctcag caacctgacc aagcaggaca 480 tcagcgagaa
caagatcgtt atcctactgg actacatgtt tcaggacctg tacaacctca 540
agtcactgga ggttggcgac aatgacctcg tctacatctc tcaccgcgcc ttcagcggcc
600 tcaacagcct ggagcagctg acgctggaga aatgcaacct gacctccatc
cccaccgagg 660 cgctgtccca cctgcacggc ctcatcgtcc tgaggctccg
gcacctcaac atcaatgcca 720 tccgggacta ctccttcaag aggctgtacc
gactcaaggt cttggagatc tcccactggc 780 cctacttgga caccatgaca
cccaactgcc tctacggcct caacctgacg tccctgtcca 840 tcacacactg
caatctgacc gctgtgccct acctggccgt ccgccaccta gtctatctcc 900
gcttcctcaa cctctcctac aaccccatca gcaccattga gggctccatg ttgcatgagc
960 tgctccggct gcaggagatc cagctggtgg gcgggcagct ggccgtggtg
gagccctatg 1020 ccttccgcgg cctcaactac ctgcgcgtgc tcaatgtctc
tggcaaccag ctgaccacac 1080 tggaggaatc agtcttccac tcggtgggca
acctggagac actcatcctg gactccaacc 1140 cgctggcctg cgactgtcgg
ctcctgtggg tgttccggcg ccgctggcgg ctcaacttca 1200 accggcagca
gcccacgtgc gccacgcccg agtttgtcca gggcaaggag ttcaaggact 1260
tccctgatgt gctactgccc aactacttca cctgccgccg cgcccgcatc cgggaccgca
1320 aggcccagca ggtgtttgtg gacgagggcc acacggtgca gtttgtgtgc
cgggccgatg 1380 gcgacccgcc gcccgccatc ctctggctct caccccgaaa
gcacctggtc tcagccaaga 1440 gcaatgggcg gctcacagtc ttccctgatg
gcacgctgga ggtgcgctac gcccaggtac 1500 aggacaacgg cacgtacctg
tgcatcgcgg ccaacgcggg cggcaacgac tccatgcccg 1560 cccacctgca
tgtgcgcagc tactcgcccg actggcccca tcagcccaac aagaccttcg 1620
ctttcatctc caaccagccg ggcgagggag aggccaacag cacccgcgcc actgtgcctt
1680 tccccttcga catcaagacc ctcatcatcg ccaccaccat gggcttcatc
tctttcctgg 1740 gcgtcgtcct cttctgcctg gtgctgctgt ttctctggag
ccggggcaag ggcaacacaa 1800 agcacaacat cgagatcgag tatgtgcccc
gaaagtcgga cgcaggcatc agctccgccg 1860 acgcgccccg caagttcaac
atgaagatga tatgaggccg gggcgggggg cagggacccc 1920 cgggcggccg
ggcaggggaa ggggcctggt cgccacctgc tcactctcca gtccttccca 1980
cctcctccct acccttctac acacgttctc tttctccctc ccgcctccgt cccctgctgc
2040 cccccgccag ccctcaccac ctgccctcct tctaccagga cctcagaagc
ccagacctgg 2100 ggaccccacc tacacagggg cattgacaga ctggagttga
aagccgacga accgacacgc 2160 ggcagagtca ataattcaat aaaaaagtta
cgaactttct ctgtaacttg ggtttcaata 2220 attatggatt tttatgaaaa
cttgaaataa taaaaagaga aaaaaactaa aaaaaaaaaa 2280 aaaaaaaaaa 2290 73
620 PRT Homo sapiens 73 Met Gln Val Ser Lys Arg Met Leu Ala Gly Gly
Val Arg Ser Met Pro 1 5 10 15 Ser Pro Leu Leu Ala Cys Trp Gln Pro
Ile Leu Leu Leu Val Leu Gly 20 25 30 Ser Val Leu Ser Gly Ser Ala
Thr Gly Cys Pro Pro Arg Cys Glu Cys 35 40 45 Ser Ala Gln Asp Arg
Ala Val Leu Cys His Arg Lys Cys Phe Val Ala 50 55 60 Val Pro Glu
Gly Ile Pro Thr Glu Thr Arg Leu Leu Asp Leu Gly Lys 65 70 75 80 Asn
Arg Ile Lys Thr Leu Asn Gln Asp Glu Phe Ala Ser Phe Pro His 85 90
95 Leu Glu Glu Leu Glu Leu Asn Glu Asn Ile Val Ser Ala Val Glu Pro
100 105 110 Gly Ala Phe Asn Asn Leu Phe Asn Leu Arg Thr Leu Gly Leu
Arg Ser 115 120 125 Asn Arg Leu Lys Leu Ile Pro Leu Gly Val Phe Thr
Gly Leu Ser Asn 130 135 140 Leu Thr Lys Gln Asp Ile Ser Glu Asn Lys
Ile Val Ile Leu Leu Asp 145 150 155 160 Tyr Met Phe Gln Asp Leu Tyr
Asn Leu Lys Ser Leu Glu Val Gly Asp 165 170 175 Asn Asp Leu Val Tyr
Ile Ser His Arg Ala Phe Ser Gly Leu Asn Ser 180 185 190 Leu Glu Gln
Leu Thr Leu Glu Lys Cys Asn Leu Thr Ser Ile Pro Thr 195 200 205 Glu
Ala Leu Ser His Leu His Gly Leu Ile Val Leu Arg Leu Arg His 210 215
220 Leu Asn Ile Asn Ala Ile Arg Asp Tyr Ser Phe Lys Arg Leu Tyr Arg
225 230 235 240 Leu Lys Val Leu Glu Ile Ser His Trp Pro Tyr Leu Asp
Thr Met Thr 245 250 255 Pro Asn Cys Leu Tyr Gly Leu Asn Leu Thr Ser
Leu Ser Ile Thr His 260 265 270 Cys Asn Leu Thr Ala Val Pro Tyr Leu
Ala Val Arg His Leu Val Tyr 275 280 285 Leu Arg Phe Leu Asn Leu Ser
Tyr Asn Pro Ile Ser Thr Ile Glu Gly 290 295 300 Ser Met Leu His Glu
Leu Leu Arg Leu Gln Glu Ile Gln Leu Val Gly 305 310 315 320 Gly Gln
Leu Ala Val Val Glu Pro Tyr Ala Phe Arg Gly Leu Asn Tyr 325 330 335
Leu Arg Val Leu Asn Val Ser Gly Asn Gln Leu Thr Thr Leu Glu Glu 340
345 350 Ser Val Phe His Ser Val Gly Asn Leu Glu Thr Leu Ile Leu Asp
Ser 355 360 365 Asn Pro Leu Ala Cys Asp Cys Arg Leu Leu Trp Val Phe
Arg Arg Arg 370 375 380 Trp Arg Leu Asn Phe Asn Arg Gln Gln Pro Thr
Cys Ala Thr Pro Glu 385 390 395 400 Phe Val Gln Gly Lys Glu Phe Lys
Asp Phe Pro Asp Val Leu Leu Pro 405 410 415 Asn Tyr Phe Thr Cys Arg
Arg Ala Arg Ile Arg Asp Arg Lys Ala Gln 420 425 430 Gln Val Phe Val
Asp Glu Gly His Thr Val Gln Phe Val Cys Arg Ala 435 440 445 Asp Gly
Asp Pro Pro Pro Ala Ile Leu Trp Leu Ser Pro Arg Lys His 450 455 460
Leu Val Ser Ala Lys Ser Asn Gly Arg Leu Thr Val Phe Pro Asp Gly 465
470 475 480 Thr Leu Glu Val Arg Tyr Ala Gln Val Gln Asp Asn Gly Thr
Tyr Leu 485 490 495 Cys Ile Ala Ala Asn Ala Gly Gly Asn Asp Ser Met
Pro Ala His Leu 500 505 510 His Val Arg Ser Tyr Ser Pro Asp Trp Pro
His Gln Pro Asn Lys Thr 515 520 525 Phe Ala Phe Ile Ser Asn Gln Pro
Gly Glu Gly Glu Ala Asn Ser Thr 530 535 540 Arg Ala Thr Val Pro Phe
Pro Phe Asp Ile Lys Thr Leu Ile Ile Ala 545 550 555 560 Thr Thr Met
Gly Phe Ile Ser Phe Leu Gly Val Val Leu Phe Cys Leu 565 570 575 Val
Leu Leu Phe Leu Trp Ser Arg Gly Lys Gly Asn Thr Lys His Asn 580 585
590 Ile Glu Ile Glu Tyr Val Pro Arg Lys Ser Asp Ala Gly Ile Ser Ser
595 600 605 Ala Asp Ala Pro Arg Lys Phe Asn Met Lys Met Ile 610 615
620 74 22 DNA Artificial Sequence Description of Artificial
Sequence Synthetic oligonucleotide probe 74 tcacctggag cctttattgg
cc 22 75 23 DNA Artificial Sequence Description of Artificial
Sequence Synthetic oligonucleotide probe 75 ataccagcta taaccaggct
gcg 23 76 52 DNA Artificial Sequence Description of Artificial
Sequence Synthetic oligonucleotide probe 76 caacagtaag tggtttgatg
ctcttccaaa tctagagatt ctgatgattg
50 gg 52 77 22 DNA Artificial Sequence Description of Artificial
Sequence Synthetic oligonucleotide probe 77 ccatgtgtct cctcctacaa
ag 22 78 23 DNA Artificial Sequence Description of Artificial
Sequence Synthetic oligonucleotide probe 78 gggaatagat gtgatctgat
tgg 23 79 50 DNA Artificial Sequence Description of Artificial
Sequence Synthetic oligonucleotide probe 79 cacctgtagc aatgcaaatc
tcaaggaaat acctagagat cttcctcctg 50 80 22 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide probe
80 agcaaccgcc tgaagctcat cc 22 81 24 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide probe
81 aaggcgcggt gaaagatgta gacg 24 82 50 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide probe
82 gactacatgt ttcaggacct gtacaacctc aagtcactgg aggttggcga 50 83
1685 DNA Homo sapiens 83 cccacgcgtc cgcacctcgg ccccgggctc
cgaagcggct cgggggcgcc ctttcggtca 60 acatcgtagt ccaccccctc
cccatcccca gcccccgggg attcaggctc gccagcgccc 120 agccagggag
ccggccggga agcgcgatgg gggccccagc cgcctcgctc ctgctcctgc 180
tcctgctgtt cgcctgctgc tgggcgcccg gcggggccaa cctctcccag gacgacagcc
240 agccctggac atctgatgaa acagtggtgg ctggtggcac cgtggtgctc
aagtgccaag 300 tgaaagatca cgaggactca tccctgcaat ggtctaaccc
tgctcagcag actctctact 360 ttggggagaa gagagccctt cgagataatc
gaattcagct ggttacctct acgccccacg 420 agctcagcat cagcatcagc
aatgtggccc tggcagacga gggcgagtac acctgctcaa 480 tcttcactat
gcctgtgcga actgccaagt ccctcgtcac tgtgctagga attccacaga 540
agcccatcat cactggttat aaatcttcat tacgggaaaa agacacagcc accctaaact
600 gtcagtcttc tgggagcaag cctgcagccc ggctcacctg gagaaagggt
gaccaagaac 660 tccacggaga accaacccgc atacaggaag atcccaatgg
taaaaccttc actgtcagca 720 gctcggtgac attccaggtt acccgggagg
atgatggggc gagcatcgtg tgctctgtga 780 accatgaatc tctaaaggga
gctgacagat ccacctctca acgcattgaa gttttataca 840 caccaactgc
gatgattagg ccagaccctc cccatcctcg tgagggccag aagctgttgc 900
tacactgtga gggtcgcggc aatccagtcc cccagcagta cctatgggag aaggagggca
960 gtgtgccacc cctgaagatg acccaggaga gtgccctgat cttccctttc
ctcaacaaga 1020 gtgacagtgg cacctacggc tgcacagcca ccagcaacat
gggcagctac aaggcctact 1080 acaccctcaa tgttaatgac cccagtccgg
tgccctcctc ctccagcacc taccacgcca 1140 tcatcggtgg gatcgtggct
ttcattgtct tcctgctgct catcatgctc atcttccttg 1200 gccactactt
gatccggcac aaaggaacct acctgacaca tgaggcaaaa ggctccgacg 1260
atgctccaga cgcggacacg gccatcatca atgcagaagg cgggcagtca ggaggggacg
1320 acaagaagga atatttcatc tagaggcgcc tgcccacttc ctgcgccccc
caggggccct 1380 gtggggactg ctggggccgt caccaacccg gacttgtaca
gagcaaccgc agggccgccc 1440 ctcccgcttg ctccccagcc cacccacccc
cctgtacaga atgtctgctt tgggtgcggt 1500 tttgtactcg gtttggaatg
gggagggagg agggcggggg gaggggaggg ttgccctcag 1560 ccctttccgt
ggcttctctg catttgggtt attattattt ttgtaacaat cccaaatcaa 1620
atctgtctcc aggctggaga ggcaggagcc ctggggtgag aaaagcaaaa aacaaacaaa
1680 aaaca 1685 84 398 PRT Homo sapiens 84 Met Gly Ala Pro Ala Ala
Ser Leu Leu Leu Leu Leu Leu Leu Phe Ala 1 5 10 15 Cys Cys Trp Ala
Pro Gly Gly Ala Asn Leu Ser Gln Asp Asp Ser Gln 20 25 30 Pro Trp
Thr Ser Asp Glu Thr Val Val Ala Gly Gly Thr Val Val Leu 35 40 45
Lys Cys Gln Val Lys Asp His Glu Asp Ser Ser Leu Gln Trp Ser Asn 50
55 60 Pro Ala Gln Gln Thr Leu Tyr Phe Gly Glu Lys Arg Ala Leu Arg
Asp 65 70 75 80 Asn Arg Ile Gln Leu Val Thr Ser Thr Pro His Glu Leu
Ser Ile Ser 85 90 95 Ile Ser Asn Val Ala Leu Ala Asp Glu Gly Glu
Tyr Thr Cys Ser Ile 100 105 110 Phe Thr Met Pro Val Arg Thr Ala Lys
Ser Leu Val Thr Val Leu Gly 115 120 125 Ile Pro Gln Lys Pro Ile Ile
Thr Gly Tyr Lys Ser Ser Leu Arg Glu 130 135 140 Lys Asp Thr Ala Thr
Leu Asn Cys Gln Ser Ser Gly Ser Lys Pro Ala 145 150 155 160 Ala Arg
Leu Thr Trp Arg Lys Gly Asp Gln Glu Leu His Gly Glu Pro 165 170 175
Thr Arg Ile Gln Glu Asp Pro Asn Gly Lys Thr Phe Thr Val Ser Ser 180
185 190 Ser Val Thr Phe Gln Val Thr Arg Glu Asp Asp Gly Ala Ser Ile
Val 195 200 205 Cys Ser Val Asn His Glu Ser Leu Lys Gly Ala Asp Arg
Ser Thr Ser 210 215 220 Gln Arg Ile Glu Val Leu Tyr Thr Pro Thr Ala
Met Ile Arg Pro Asp 225 230 235 240 Pro Pro His Pro Arg Glu Gly Gln
Lys Leu Leu Leu His Cys Glu Gly 245 250 255 Arg Gly Asn Pro Val Pro
Gln Gln Tyr Leu Trp Glu Lys Glu Gly Ser 260 265 270 Val Pro Pro Leu
Lys Met Thr Gln Glu Ser Ala Leu Ile Phe Pro Phe 275 280 285 Leu Asn
Lys Ser Asp Ser Gly Thr Tyr Gly Cys Thr Ala Thr Ser Asn 290 295 300
Met Gly Ser Tyr Lys Ala Tyr Tyr Thr Leu Asn Val Asn Asp Pro Ser 305
310 315 320 Pro Val Pro Ser Ser Ser Ser Thr Tyr His Ala Ile Ile Gly
Gly Ile 325 330 335 Val Ala Phe Ile Val Phe Leu Leu Leu Ile Met Leu
Ile Phe Leu Gly 340 345 350 His Tyr Leu Ile Arg His Lys Gly Thr Tyr
Leu Thr His Glu Ala Lys 355 360 365 Gly Ser Asp Asp Ala Pro Asp Ala
Asp Thr Ala Ile Ile Asn Ala Glu 370 375 380 Gly Gly Gln Ser Gly Gly
Asp Asp Lys Lys Glu Tyr Phe Ile 385 390 395 85 22 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 85 gctaggaatt ccacagaagc cc 22 86 22 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 86 aacctggaat gtcaccgagc tg 22 87 26 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 87 cctagcacag tgacgaggga cttggc 26 88 50 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 88 aagacacagc caccctaaac tgtcagtctt
ctgggagcaa gcctgcagcc 50 89 50 DNA Artificial Sequence Description
of Artificial Sequence Synthetic oligonucleotide probe 89
gccctggcag acgagggcga gtacacctgc tcaatcttca ctatgcctgt 50 90 2755
DNA Homo sapiens 90 gggggttagg gaggaaggaa tccaccccca cccccccaaa
cccttttctt ctcctttcct 60 ggcttcggac attggagcac taaatgaact
tgaattgtgt ctgtggcgag caggatggtc 120 gctgttactt tgtgatgaga
tcggggatga attgctcgct ttaaaaatgc tgctttggat 180 tctgttgctg
gagacgtctc tttgttttgc cgctggaaac gttacagggg acgtttgcaa 240
agagaagatc tgttcctgca atgagataga aggggaccta cacgtagact gtgaaaaaaa
300 gggcttcaca agtctgcagc gtttcactgc cccgacttcc cagttttacc
atttatttct 360 gcatggcaat tccctcactc gacttttccc taatgagttc
gctaactttt ataatgcggt 420 tagtttgcac atggaaaaca atggcttgca
tgaaatcgtt ccgggggctt ttctggggct 480 gcagctggtg aaaaggctgc
acatcaacaa caacaagatc aagtcttttc gaaagcagac 540 ttttctgggg
ctggacgatc tggaatatct ccaggctgat tttaatttat tacgagatat 600
agacccgggg gccttccagg acttgaacaa gctggaggtg ctcattttaa atgacaatct
660 catcagcacc ctacctgcca acgtgttcca gtatgtgccc atcacccacc
tcgacctccg 720 gggtaacagg ctgaaaacgc tgccctatga ggaggtcttg
gagcaaatcc ctggtattgc 780 ggagatcctg ctagaggata acccttggga
ctgcacctgt gatctgctct ccctgaaaga 840 atggctggaa aacattccca
agaatgccct gatcggccga gtggtctgcg aagcccccac 900 cagactgcag
ggtaaagacc tcaatgaaac caccgaacag gacttgtgtc ctttgaaaaa 960
ccgagtggat tctagtctcc cggcgccccc tgcccaagaa gagacctttg ctcctggacc
1020 cctgccaact cctttcaaga caaatgggca agaggatcat gccacaccag
ggtctgctcc 1080 aaacggaggt acaaagatcc caggcaactg gcagatcaaa
atcagaccca cagcagcgat 1140 agcgacgggt agctccagga acaaaccctt
agctaacagt ttaccctgcc ctgggggctg 1200 cagctgcgac cacatcccag
ggtcgggttt aaagatgaac tgcaacaaca ggaacgtgag 1260 cagcttggct
gatttgaagc ccaagctctc taacgtgcag gagcttttcc tacgagataa 1320
caagatccac agcatccgaa aatcgcactt tgtggattac aagaacctca ttctgttgga
1380 tctgggcaac aataacatcg ctactgtaga gaacaacact ttcaagaacc
ttttggacct 1440 caggtggcta tacatggata gcaattacct ggacacgctg
tcccgggaga aattcgcggg 1500 gctgcaaaac ctagagtacc tgaacgtgga
gtacaacgct atccagctca tcctcccggg 1560 cactttcaat gccatgccca
aactgaggat cctcattctc aacaacaacc tgctgaggtc 1620 cctgcctgtg
gacgtgttcg ctggggtctc gctctctaaa ctcagcctgc acaacaatta 1680
cttcatgtac ctcccggtgg caggggtgct ggaccagtta acctccatca tccagataga
1740 cctccacgga aacccctggg agtgctcctg cacaattgtg cctttcaagc
agtgggcaga 1800 acgcttgggt tccgaagtgc tgatgagcga cctcaagtgt
gagacgccgg tgaacttctt 1860 tagaaaggat ttcatgctcc tctccaatga
cgagatctgc cctcagctgt acgctaggat 1920 ctcgcccacg ttaacttcgc
acagtaaaaa cagcactggg ttggcggaga ccgggacgca 1980 ctccaactcc
tacctagaca ccagcagggt gtccatctcg gtgttggtcc cgggactgct 2040
gctggtgttt gtcacctccg ccttcaccgt ggtgggcatg ctcgtgttta tcctgaggaa
2100 ccgaaagcgg tccaagagac gagatgccaa ctcctccgcg tccgagatta
attccctaca 2160 gacagtctgt gactcttcct actggcacaa tgggccttac
aacgcagatg gggcccacag 2220 agtgtatgac tgtggctctc actcgctctc
agactaagac cccaacccca ataggggagg 2280 gcagagggaa ggcgatacat
ccttccccac cgcaggcacc ccgggggctg gaggggcgtg 2340 tacccaaatc
cccgcgccat cagcctggat gggcataagt agataaataa ctgtgagctc 2400
gcacaaccga aagggcctga ccccttactt agctccctcc ttgaaacaaa gagcagactg
2460 tggagagctg ggagagcgca gccagctcgc tctttgctga gagccccttt
tgacagaaag 2520 cccagcacga ccctgctgga agaactgaca gtgccctcgc
cctcggcccc ggggcctgtg 2580 gggttggatg ccgcggttct atacatatat
acatatatcc acatctatat agagagatag 2640 atatctattt ttcccctgtg
gattagcccc gtgatggctc cctgttggct acgcagggat 2700 gggcagttgc
acgaaggcat gaatgtattg taaataagta actttgactt ctgac 2755 91 696 PRT
Homo sapiens 91 Met Leu Leu Trp Ile Leu Leu Leu Glu Thr Ser Leu Cys
Phe Ala Ala 1 5 10 15 Gly Asn Val Thr Gly Asp Val Cys Lys Glu Lys
Ile Cys Ser Cys Asn 20 25 30 Glu Ile Glu Gly Asp Leu His Val Asp
Cys Glu Lys Lys Gly Phe Thr 35 40 45 Ser Leu Gln Arg Phe Thr Ala
Pro Thr Ser Gln Phe Tyr His Leu Phe 50 55 60 Leu His Gly Asn Ser
Leu Thr Arg Leu Phe Pro Asn Glu Phe Ala Asn 65 70 75 80 Phe Tyr Asn
Ala Val Ser Leu His Met Glu Asn Asn Gly Leu His Glu 85 90 95 Ile
Val Pro Gly Ala Phe Leu Gly Leu Gln Leu Val Lys Arg Leu His 100 105
110 Ile Asn Asn Asn Lys Ile Lys Ser Phe Arg Lys Gln Thr Phe Leu Gly
115 120 125 Leu Asp Asp Leu Glu Tyr Leu Gln Ala Asp Phe Asn Leu Leu
Arg Asp 130 135 140 Ile Asp Pro Gly Ala Phe Gln Asp Leu Asn Lys Leu
Glu Val Leu Ile 145 150 155 160 Leu Asn Asp Asn Leu Ile Ser Thr Leu
Pro Ala Asn Val Phe Gln Tyr 165 170 175 Val Pro Ile Thr His Leu Asp
Leu Arg Gly Asn Arg Leu Lys Thr Leu 180 185 190 Pro Tyr Glu Glu Val
Leu Glu Gln Ile Pro Gly Ile Ala Glu Ile Leu 195 200 205 Leu Glu Asp
Asn Pro Trp Asp Cys Thr Cys Asp Leu Leu Ser Leu Lys 210 215 220 Glu
Trp Leu Glu Asn Ile Pro Lys Asn Ala Leu Ile Gly Arg Val Val 225 230
235 240 Cys Glu Ala Pro Thr Arg Leu Gln Gly Lys Asp Leu Asn Glu Thr
Thr 245 250 255 Glu Gln Asp Leu Cys Pro Leu Lys Asn Arg Val Asp Ser
Ser Leu Pro 260 265 270 Ala Pro Pro Ala Gln Glu Glu Thr Phe Ala Pro
Gly Pro Leu Pro Thr 275 280 285 Pro Phe Lys Thr Asn Gly Gln Glu Asp
His Ala Thr Pro Gly Ser Ala 290 295 300 Pro Asn Gly Gly Thr Lys Ile
Pro Gly Asn Trp Gln Ile Lys Ile Arg 305 310 315 320 Pro Thr Ala Ala
Ile Ala Thr Gly Ser Ser Arg Asn Lys Pro Leu Ala 325 330 335 Asn Ser
Leu Pro Cys Pro Gly Gly Cys Ser Cys Asp His Ile Pro Gly 340 345 350
Ser Gly Leu Lys Met Asn Cys Asn Asn Arg Asn Val Ser Ser Leu Ala 355
360 365 Asp Leu Lys Pro Lys Leu Ser Asn Val Gln Glu Leu Phe Leu Arg
Asp 370 375 380 Asn Lys Ile His Ser Ile Arg Lys Ser His Phe Val Asp
Tyr Lys Asn 385 390 395 400 Leu Ile Leu Leu Asp Leu Gly Asn Asn Asn
Ile Ala Thr Val Glu Asn 405 410 415 Asn Thr Phe Lys Asn Leu Leu Asp
Leu Arg Trp Leu Tyr Met Asp Ser 420 425 430 Asn Tyr Leu Asp Thr Leu
Ser Arg Glu Lys Phe Ala Gly Leu Gln Asn 435 440 445 Leu Glu Tyr Leu
Asn Val Glu Tyr Asn Ala Ile Gln Leu Ile Leu Pro 450 455 460 Gly Thr
Phe Asn Ala Met Pro Lys Leu Arg Ile Leu Ile Leu Asn Asn 465 470 475
480 Asn Leu Leu Arg Ser Leu Pro Val Asp Val Phe Ala Gly Val Ser Leu
485 490 495 Ser Lys Leu Ser Leu His Asn Asn Tyr Phe Met Tyr Leu Pro
Val Ala 500 505 510 Gly Val Leu Asp Gln Leu Thr Ser Ile Ile Gln Ile
Asp Leu His Gly 515 520 525 Asn Pro Trp Glu Cys Ser Cys Thr Ile Val
Pro Phe Lys Gln Trp Ala 530 535 540 Glu Arg Leu Gly Ser Glu Val Leu
Met Ser Asp Leu Lys Cys Glu Thr 545 550 555 560 Pro Val Asn Phe Phe
Arg Lys Asp Phe Met Leu Leu Ser Asn Asp Glu 565 570 575 Ile Cys Pro
Gln Leu Tyr Ala Arg Ile Ser Pro Thr Leu Thr Ser His 580 585 590 Ser
Lys Asn Ser Thr Gly Leu Ala Glu Thr Gly Thr His Ser Asn Ser 595 600
605 Tyr Leu Asp Thr Ser Arg Val Ser Ile Ser Val Leu Val Pro Gly Leu
610 615 620 Leu Leu Val Phe Val Thr Ser Ala Phe Thr Val Val Gly Met
Leu Val 625 630 635 640 Phe Ile Leu Arg Asn Arg Lys Arg Ser Lys Arg
Arg Asp Ala Asn Ser 645 650 655 Ser Ala Ser Glu Ile Asn Ser Leu Gln
Thr Val Cys Asp Ser Ser Tyr 660 665 670 Trp His Asn Gly Pro Tyr Asn
Ala Asp Gly Ala His Arg Val Tyr Asp 675 680 685 Cys Gly Ser His Ser
Leu Ser Asp 690 695 92 22 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide probe 92 gttggatctg
ggcaacaata ac 22 93 24 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide probe 93 attgttgtgc
aggctgagtt taag 24 94 45 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide probe 94 ggtggctata
catggatagc aattacctgg acacgctgtc ccggg 45 95 2226 DNA Homo sapiens
95 agtcgactgc gtcccctgta cccggcgcca gctgtgttcc tgaccccaga
ataactcagg 60 gctgcaccgg gcctggcagc gctccgcaca catttcctgt
cgcggcctaa gggaaactgt 120 tggccgctgg gcccgcgggg ggattcttgg
cagttggggg gtccgtcggg agcgagggcg 180 gaggggaagg gagggggaac
cgggttgggg aagccagctg tagagggcgg tgaccgcgct 240 ccagacacag
ctctgcgtcc tcgagcggga cagatccaag ttgggagcag ctctgcgtgc 300
ggggcctcag agaatgaggc cggcgttcgc cctgtgcctc ctctggcagg cgctctggcc
360 cgggccgggc ggcggcgaac accccactgc cgaccgtgct ggctgctcgg
cctcgggggc 420 ctgctacagc ctgcaccacg ctaccatgaa gcggcaggcg
gccgaggagg cctgcatcct 480 gcgaggtggg gcgctcagca ccgtgcgtgc
gggcgccgag ctgcgcgctg tgctcgcgct 540 cctgcgggca ggcccagggc
ccggaggggg ctccaaagac ctgctgttct gggtcgcact 600 ggagcgcagg
cgttcccact gcaccctgga gaacgagcct ttgcggggtt tctcctggct 660
gtcctccgac cccggcggtc tcgaaagcga cacgctgcag tgggtggagg agccccaacg
720 ctcctgcacc gcgcggagat gcgcggtact ccaggccacc ggtggggtcg
agcccgcagg 780 ctggaaggag atgcgatgcc acctgcgcgc caacggctac
ctgtgcaagt accagtttga 840 ggtcttgtgt cctgcgccgc gccccggggc
cgcctctaac ttgagctatc gcgcgccctt 900 ccagctgcac agcgccgctc
tggacttcag tccacctggg accgaggtga gtgcgctctg 960 ccggggacag
ctcccgatct cagttacttg catcgcggac gaaatcggcg ctcgctggga 1020
caaactctcg ggcgatgtgt tgtgtccctg ccccgggagg tacctccgtg ctggcaaatg
1080 cgcagagctc cctaactgcc tagacgactt gggaggcttt gcctgcgaat
gtgctacggg 1140 cttcgagctg gggaaggacg gccgctcttg tgtgaccagt
ggggaaggac agccgaccct 1200 tggggggacc ggggtgccca ccaggcgccc
gccggccact gcaaccagcc ccgtgccgca 1260 gagaacatgg ccaatcaggg
tcgacgagaa gctgggagag acaccacttg tccctgaaca 1320 agacaattca
gtaacatcta ttcctgagat tcctcgatgg ggatcacaga gcacgatgtc 1380
tacccttcaa atgtcccttc aagccgagtc aaaggccact atcaccccat cagggagcgt
1440 gatttccaag tttaattcta cgacttcctc tgccactcct caggctttcg
actcctcctc 1500 tgccgtggtc
ttcatatttg tgagcacagc agtagtagtg ttggtgatct tgaccatgac 1560
agtactgggg cttgtcaagc tctgctttca cgaaagcccc tcttcccagc caaggaagga
1620 gtctatgggc ccgccgggcc tggagagtga tcctgagccc gctgctttgg
gctccagttc 1680 tgcacattgc acaaacaatg gggtgaaagt cggggactgt
gatctgcggg acagagcaga 1740 gggtgccttg ctggcggagt cccctcttgg
ctctagtgat gcatagggaa acaggggaca 1800 tgggcactcc tgtgaacagt
ttttcacttt tgatgaaacg gggaaccaag aggaacttac 1860 ttgtgtaact
gacaatttct gcagaaatcc cccttcctct aaattccctt tactccactg 1920
aggagctaaa tcagaactgc acactccttc cctgatgata gaggaagtgg aagtgccttt
1980 aggatggtga tactggggga ccgggtagtg ctggggagag atattttctt
atgtttattc 2040 ggagaatttg gagaagtgat tgaacttttc aagacattgg
aaacaaatag aacacaatat 2100 aatttacatt aaaaaataat ttctaccaaa
atggaaagga aatgttctat gttgttcagg 2160 ctaggagtat attggttcga
aatcccaggg aaaaaaataa aaataaaaaa ttaaaggatt 2220 gttgat 2226 96 490
PRT Homo sapiens 96 Met Arg Pro Ala Phe Ala Leu Cys Leu Leu Trp Gln
Ala Leu Trp Pro 1 5 10 15 Gly Pro Gly Gly Gly Glu His Pro Thr Ala
Asp Arg Ala Gly Cys Ser 20 25 30 Ala Ser Gly Ala Cys Tyr Ser Leu
His His Ala Thr Met Lys Arg Gln 35 40 45 Ala Ala Glu Glu Ala Cys
Ile Leu Arg Gly Gly Ala Leu Ser Thr Val 50 55 60 Arg Ala Gly Ala
Glu Leu Arg Ala Val Leu Ala Leu Leu Arg Ala Gly 65 70 75 80 Pro Gly
Pro Gly Gly Gly Ser Lys Asp Leu Leu Phe Trp Val Ala Leu 85 90 95
Glu Arg Arg Arg Ser His Cys Thr Leu Glu Asn Glu Pro Leu Arg Gly 100
105 110 Phe Ser Trp Leu Ser Ser Asp Pro Gly Gly Leu Glu Ser Asp Thr
Leu 115 120 125 Gln Trp Val Glu Glu Pro Gln Arg Ser Cys Thr Ala Arg
Arg Cys Ala 130 135 140 Val Leu Gln Ala Thr Gly Gly Val Glu Pro Ala
Gly Trp Lys Glu Met 145 150 155 160 Arg Cys His Leu Arg Ala Asn Gly
Tyr Leu Cys Lys Tyr Gln Phe Glu 165 170 175 Val Leu Cys Pro Ala Pro
Arg Pro Gly Ala Ala Ser Asn Leu Ser Tyr 180 185 190 Arg Ala Pro Phe
Gln Leu His Ser Ala Ala Leu Asp Phe Ser Pro Pro 195 200 205 Gly Thr
Glu Val Ser Ala Leu Cys Arg Gly Gln Leu Pro Ile Ser Val 210 215 220
Thr Cys Ile Ala Asp Glu Ile Gly Ala Arg Trp Asp Lys Leu Ser Gly 225
230 235 240 Asp Val Leu Cys Pro Cys Pro Gly Arg Tyr Leu Arg Ala Gly
Lys Cys 245 250 255 Ala Glu Leu Pro Asn Cys Leu Asp Asp Leu Gly Gly
Phe Ala Cys Glu 260 265 270 Cys Ala Thr Gly Phe Glu Leu Gly Lys Asp
Gly Arg Ser Cys Val Thr 275 280 285 Ser Gly Glu Gly Gln Pro Thr Leu
Gly Gly Thr Gly Val Pro Thr Arg 290 295 300 Arg Pro Pro Ala Thr Ala
Thr Ser Pro Val Pro Gln Arg Thr Trp Pro 305 310 315 320 Ile Arg Val
Asp Glu Lys Leu Gly Glu Thr Pro Leu Val Pro Glu Gln 325 330 335 Asp
Asn Ser Val Thr Ser Ile Pro Glu Ile Pro Arg Trp Gly Ser Gln 340 345
350 Ser Thr Met Ser Thr Leu Gln Met Ser Leu Gln Ala Glu Ser Lys Ala
355 360 365 Thr Ile Thr Pro Ser Gly Ser Val Ile Ser Lys Phe Asn Ser
Thr Thr 370 375 380 Ser Ser Ala Thr Pro Gln Ala Phe Asp Ser Ser Ser
Ala Val Val Phe 385 390 395 400 Ile Phe Val Ser Thr Ala Val Val Val
Leu Val Ile Leu Thr Met Thr 405 410 415 Val Leu Gly Leu Val Lys Leu
Cys Phe His Glu Ser Pro Ser Ser Gln 420 425 430 Pro Arg Lys Glu Ser
Met Gly Pro Pro Gly Leu Glu Ser Asp Pro Glu 435 440 445 Pro Ala Ala
Leu Gly Ser Ser Ser Ala His Cys Thr Asn Asn Gly Val 450 455 460 Lys
Val Gly Asp Cys Asp Leu Arg Asp Arg Ala Glu Gly Ala Leu Leu 465 470
475 480 Ala Glu Ser Pro Leu Gly Ser Ser Asp Ala 485 490 97 24 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 97 tggaaggaga tgcgatgcca cctg 24 98 20 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 98 tgaccagtgg ggaaggacag 20 99 20 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 99 acagagcaga gggtgccttg 20 100 24 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 100 tcagggacaa gtggtgtctc tccc 24 101 24 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 101 tcagggaagg agtgtgcagt tctg 24 102 50 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 102 acagctcccg atctcagtta cttgcatcgc
ggacgaaatc ggcgctcgct 50 103 2026 DNA Homo sapiens 103 cggacgcgtg
ggattcagca gtggcctgtg gctgccagag cagctcctca ggggaaacta 60
agcgtcgagt cagacggcac cataatcgcc tttaaaagtg cctccgccct gccggccgcg
120 tatcccccgg ctacctgggc cgccccgcgg cggtgcgcgc gtgagaggga
gcgcgcgggc 180 agccgagcgc cggtgtgagc cagcgctgct gccagtgtga
gcggcggtgt gagcgcggtg 240 ggtgcggagg ggcgtgtgtg ccggcgcgcg
cgccgtgggg tgcaaacccc gagcgtctac 300 gctgccatga ggggcgcgaa
cgcctgggcg ccactctgcc tgctgctggc tgccgccacc 360 cagctctcgc
ggcagcagtc cccagagaga cctgttttca catgtggtgg cattcttact 420
ggagagtctg gatttattgg cagtgaaggt tttcctggag tgtaccctcc aaatagcaaa
480 tgtacttgga aaatcacagt tcccgaagga aaagtagtcg ttctcaattt
ccgattcata 540 gacctcgaga gtgacaacct gtgccgctat gactttgtgg
atgtgtacaa tggccatgcc 600 aatggccagc gcattggccg cttctgtggc
actttccggc ctggagccct tgtgtccagt 660 ggcaacaaga tgatggtgca
gatgatttct gatgccaaca cagctggcaa tggcttcatg 720 gccatgttct
ccgctgctga accaaacgaa agaggggatc agtattgtgg aggactcctt 780
gacagacctt ccggctcttt taaaaccccc aactggccag accgggatta ccctgcagga
840 gtcacttgtg tgtggcacat tgtagcccca aagaatcagc ttatagaatt
aaagtttgag 900 aagtttgatg tggagcgaga taactactgc cgatatgatt
atgtggctgt gtttaatggc 960 ggggaagtca acgatgctag aagaattgga
aagtattgtg gtgatagtcc acctgcgcca 1020 attgtgtctg agagaaatga
acttcttatt cagtttttat cagacttaag tttaactgca 1080 gatgggttta
ttggtcacta catattcagg ccaaaaaaac tgcctacaac tacagaacag 1140
cctgtcacca ccacattccc tgtaaccacg ggtttaaaac ccaccgtggc cttgtgtcaa
1200 caaaagtgta gacggacggg gactctggag ggcaattatt gttcaagtga
ctttgtatta 1260 gccggcactg ttatcacaac catcactcgc gatgggagtt
tgcacgccac agtctcgatc 1320 atcaacatct acaaagaggg aaatttggcg
attcagcagg cgggcaagaa catgagtgcc 1380 aggctgactg tcgtctgcaa
gcagtgccct ctcctcagaa gaggtctaaa ttacattatt 1440 atgggccaag
taggtgaaga tgggcgaggc aaaatcatgc caaacagctt tatcatgatg 1500
ttcaagacca agaatcagaa gctcctggat gccttaaaaa ataagcaatg ttaacagtga
1560 actgtgtcca tttaagctgt attctgccat tgcctttgaa agatctatgt
tctctcagta 1620 gaaaaaaaaa tacttataaa attacatatt ctgaaagagg
attccgaaag atgggactgg 1680 ttgactcttc acatgatgga ggtatgaggc
ctccgagata gctgagggaa gttctttgcc 1740 tgctgtcaga ggagcagcta
tctgattgga aacctgccga cttagtgcgg tgataggaag 1800 ctaaaagtgt
caagcgttga cagcttggaa gcgtttattt atacatctct gtaaaaggat 1860
attttagaat tgagttgtgt gaagatgtca aaaaaagatt ttagaagtgc aatatttata
1920 gtgttatttg tttcaccttc aagcctttgc cctgaggtgt tacaatcttg
tcttgcgttt 1980 tctaaatcaa tgcttaataa aatattttta aaggaaaaaa aaaaaa
2026 104 415 PRT Homo sapiens 104 Met Arg Gly Ala Asn Ala Trp Ala
Pro Leu Cys Leu Leu Leu Ala Ala 1 5 10 15 Ala Thr Gln Leu Ser Arg
Gln Gln Ser Pro Glu Arg Pro Val Phe Thr 20 25 30 Cys Gly Gly Ile
Leu Thr Gly Glu Ser Gly Phe Ile Gly Ser Glu Gly 35 40 45 Phe Pro
Gly Val Tyr Pro Pro Asn Ser Lys Cys Thr Trp Lys Ile Thr 50 55 60
Val Pro Glu Gly Lys Val Val Val Leu Asn Phe Arg Phe Ile Asp Leu 65
70 75 80 Glu Ser Asp Asn Leu Cys Arg Tyr Asp Phe Val Asp Val Tyr
Asn Gly 85 90 95 His Ala Asn Gly Gln Arg Ile Gly Arg Phe Cys Gly
Thr Phe Arg Pro 100 105 110 Gly Ala Leu Val Ser Ser Gly Asn Lys Met
Met Val Gln Met Ile Ser 115 120 125 Asp Ala Asn Thr Ala Gly Asn Gly
Phe Met Ala Met Phe Ser Ala Ala 130 135 140 Glu Pro Asn Glu Arg Gly
Asp Gln Tyr Cys Gly Gly Leu Leu Asp Arg 145 150 155 160 Pro Ser Gly
Ser Phe Lys Thr Pro Asn Trp Pro Asp Arg Asp Tyr Pro 165 170 175 Ala
Gly Val Thr Cys Val Trp His Ile Val Ala Pro Lys Asn Gln Leu 180 185
190 Ile Glu Leu Lys Phe Glu Lys Phe Asp Val Glu Arg Asp Asn Tyr Cys
195 200 205 Arg Tyr Asp Tyr Val Ala Val Phe Asn Gly Gly Glu Val Asn
Asp Ala 210 215 220 Arg Arg Ile Gly Lys Tyr Cys Gly Asp Ser Pro Pro
Ala Pro Ile Val 225 230 235 240 Ser Glu Arg Asn Glu Leu Leu Ile Gln
Phe Leu Ser Asp Leu Ser Leu 245 250 255 Thr Ala Asp Gly Phe Ile Gly
His Tyr Ile Phe Arg Pro Lys Lys Leu 260 265 270 Pro Thr Thr Thr Glu
Gln Pro Val Thr Thr Thr Phe Pro Val Thr Thr 275 280 285 Gly Leu Lys
Pro Thr Val Ala Leu Cys Gln Gln Lys Cys Arg Arg Thr 290 295 300 Gly
Thr Leu Glu Gly Asn Tyr Cys Ser Ser Asp Phe Val Leu Ala Gly 305 310
315 320 Thr Val Ile Thr Thr Ile Thr Arg Asp Gly Ser Leu His Ala Thr
Val 325 330 335 Ser Ile Ile Asn Ile Tyr Lys Glu Gly Asn Leu Ala Ile
Gln Gln Ala 340 345 350 Gly Lys Asn Met Ser Ala Arg Leu Thr Val Val
Cys Lys Gln Cys Pro 355 360 365 Leu Leu Arg Arg Gly Leu Asn Tyr Ile
Ile Met Gly Gln Val Gly Glu 370 375 380 Asp Gly Arg Gly Lys Ile Met
Pro Asn Ser Phe Ile Met Met Phe Lys 385 390 395 400 Thr Lys Asn Gln
Lys Leu Leu Asp Ala Leu Lys Asn Lys Gln Cys 405 410 415 105 22 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 105 ccgattcata gacctcgaga gt 22 106 22 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 106 gtcaaggagt cctccacaat ac 22 107 45 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 107 gtgtacaatg gccatgccaa tggccagcgc
attggccgct tctgt 45 108 1838 DNA Homo sapiens 108 cggacgcgtg
ggcggacgcg tgggcggccc acggcgcccg cgggctgggg cggtcgcttc 60
ttccttctcc gtggcctacg agggtcccca gcctgggtaa agatggcccc atggcccccg
120 aagggcctag tcccagctgt gctctggggc ctcagcctct tcctcaacct
cccaggacct 180 atctggctcc agccctctcc acctccccag tcttctcccc
cgcctcagcc ccatccgtgt 240 catacctgcc ggggactggt tgacagcttt
aacaagggcc tggagagaac catccgggac 300 aactttggag gtggaaacac
tgcctgggag gaagagaatt tgtccaaata caaagacagt 360 gagacccgcc
tggtagaggt gctggagggt gtgtgcagca agtcagactt cgagtgccac 420
cgcctgctgg agctgagtga ggagctggtg gagagctggt ggtttcacaa gcagcaggag
480 gccccggacc tcttccagtg gctgtgctca gattccctga agctctgctg
ccccgcaggc 540 accttcgggc cctcctgcct tccctgtcct gggggaacag
agaggccctg cggtggctac 600 gggcagtgtg aaggagaagg gacacgaggg
ggcagcgggc actgtgactg ccaagccggc 660 tacgggggtg aggcctgtgg
ccagtgtggc cttggctact ttgaggcaga acgcaacgcc 720 agccatctgg
tatgttcggc ttgttttggc ccctgtgccc gatgctcagg acctgaggaa 780
tcaaactgtt tgcaatgcaa gaagggctgg gccctgcatc acctcaagtg tgtagacatt
840 gatgagtgtg gcacagaggg agccaactgt ggagctgacc aattctgcgt
gaacactgag 900 ggctcctatg agtgccgaga ctgtgccaag gcctgcctag
gctgcatggg ggcagggcca 960 ggtcgctgta agaagtgtag ccctggctat
cagcaggtgg gctccaagtg tctcgatgtg 1020 gatgagtgtg agacagaggt
gtgtccggga gagaacaagc agtgtgaaaa caccgagggc 1080 ggttatcgct
gcatctgtgc cgagggctac aagcagatgg aaggcatctg tgtgaaggag 1140
cagatcccag agtcagcagg cttcttctca gagatgacag aagacgagtt ggtggtgctg
1200 cagcagatgt tctttggcat catcatctgt gcactggcca cgctggctgc
taagggcgac 1260 ttggtgttca ccgccatctt cattggggct gtggcggcca
tgactggcta ctggttgtca 1320 gagcgcagtg accgtgtgct ggagggcttc
atcaagggca gataatcgcg gccaccacct 1380 gtaggacctc ctcccaccca
cgctgccccc agagcttggg ctgccctcct gctggacact 1440 caggacagct
tggtttattt ttgagagtgg ggtaagcacc cctacctgcc ttacagagca 1500
gcccaggtac ccaggcccgg gcagacaagg cccctggggt aaaaagtagc cctgaaggtg
1560 gataccatga gctcttcacc tggcggggac tggcaggctt cacaatgtgt
gaatttcaaa 1620 agtttttcct taatggtggc tgctagagct ttggcccctg
cttaggatta ggtggtcctc 1680 acaggggtgg ggccatcaca gctccctcct
gccagctgca tgctgccagt tcctgttctg 1740 tgttcaccac atccccacac
cccattgcca cttatttatt catctcagga aataaagaaa 1800 ggtcttggaa
agttaaaaaa aaaaaaaaaa aaaaaaaa 1838 109 420 PRT Homo sapiens 109
Met Ala Pro Trp Pro Pro Lys Gly Leu Val Pro Ala Val Leu Trp Gly 1 5
10 15 Leu Ser Leu Phe Leu Asn Leu Pro Gly Pro Ile Trp Leu Gln Pro
Ser 20 25 30 Pro Pro Pro Gln Ser Ser Pro Pro Pro Gln Pro His Pro
Cys His Thr 35 40 45 Cys Arg Gly Leu Val Asp Ser Phe Asn Lys Gly
Leu Glu Arg Thr Ile 50 55 60 Arg Asp Asn Phe Gly Gly Gly Asn Thr
Ala Trp Glu Glu Glu Asn Leu 65 70 75 80 Ser Lys Tyr Lys Asp Ser Glu
Thr Arg Leu Val Glu Val Leu Glu Gly 85 90 95 Val Cys Ser Lys Ser
Asp Phe Glu Cys His Arg Leu Leu Glu Leu Ser 100 105 110 Glu Glu Leu
Val Glu Ser Trp Trp Phe His Lys Gln Gln Glu Ala Pro 115 120 125 Asp
Leu Phe Gln Trp Leu Cys Ser Asp Ser Leu Lys Leu Cys Cys Pro 130 135
140 Ala Gly Thr Phe Gly Pro Ser Cys Leu Pro Cys Pro Gly Gly Thr Glu
145 150 155 160 Arg Pro Cys Gly Gly Tyr Gly Gln Cys Glu Gly Glu Gly
Thr Arg Gly 165 170 175 Gly Ser Gly His Cys Asp Cys Gln Ala Gly Tyr
Gly Gly Glu Ala Cys 180 185 190 Gly Gln Cys Gly Leu Gly Tyr Phe Glu
Ala Glu Arg Asn Ala Ser His 195 200 205 Leu Val Cys Ser Ala Cys Phe
Gly Pro Cys Ala Arg Cys Ser Gly Pro 210 215 220 Glu Glu Ser Asn Cys
Leu Gln Cys Lys Lys Gly Trp Ala Leu His His 225 230 235 240 Leu Lys
Cys Val Asp Ile Asp Glu Cys Gly Thr Glu Gly Ala Asn Cys 245 250 255
Gly Ala Asp Gln Phe Cys Val Asn Thr Glu Gly Ser Tyr Glu Cys Arg 260
265 270 Asp Cys Ala Lys Ala Cys Leu Gly Cys Met Gly Ala Gly Pro Gly
Arg 275 280 285 Cys Lys Lys Cys Ser Pro Gly Tyr Gln Gln Val Gly Ser
Lys Cys Leu 290 295 300 Asp Val Asp Glu Cys Glu Thr Glu Val Cys Pro
Gly Glu Asn Lys Gln 305 310 315 320 Cys Glu Asn Thr Glu Gly Gly Tyr
Arg Cys Ile Cys Ala Glu Gly Tyr 325 330 335 Lys Gln Met Glu Gly Ile
Cys Val Lys Glu Gln Ile Pro Glu Ser Ala 340 345 350 Gly Phe Phe Ser
Glu Met Thr Glu Asp Glu Leu Val Val Leu Gln Gln 355 360 365 Met Phe
Phe Gly Ile Ile Ile Cys Ala Leu Ala Thr Leu Ala Ala Lys 370 375 380
Gly Asp Leu Val Phe Thr Ala Ile Phe Ile Gly Ala Val Ala Ala Met 385
390 395 400 Thr Gly Tyr Trp Leu Ser Glu Arg Ser Asp Arg Val Leu Glu
Gly Phe 405 410 415 Ile Lys Gly Arg 420 110 50 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 110 cctggctatc agcaggtggg ctccaagtgt
ctcgatgtgg atgagtgtga 50 111 22 DNA Artificial Sequence Description
of Artificial Sequence Synthetic oligonucleotide probe 111
attctgcgtg aacactgagg gc 22 112 22 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide probe
112 atctgcttgt agccctcggc ac 22 113 1616 DNA Homo sapiens
modified_base (1461)..(1461) a, t, c or g 113 tgagaccctc ctgcagcctt
ctcaagggac agccccactc tgcctcttgc tcctccaggg 60 cagcaccatg
cagcccctgt ggctctgctg ggcactctgg gtgttgcccc tggccagccc 120
cggggccgcc ctgaccgggg agcagctcct gggcagcctg ctgcggcagc tgcagctcaa
180 agaggtgccc accctggaca
gggccgacat ggaggagctg gtcatcccca cccacgtgag 240 ggcccagtac
gtggccctgc tgcagcgcag ccacggggac cgctcccgcg gaaagaggtt 300
cagccagagc ttccgagagg tggccggcag gttcctggcg ttggaggcca gcacacacct
360 gctggtgttc ggcatggagc agcggctgcc gcccaacagc gagctggtgc
aggccgtgct 420 gcggctcttc caggagccgg tccccaaggc cgcgctgcac
aggcacgggc ggctgtcccc 480 gcgcagcgcc cgggcccggg tgaccgtcga
gtggctgcgc gtccgcgacg acggctccaa 540 ccgcacctcc ctcatcgact
ccaggctggt gtccgtccac gagagcggct ggaaggcctt 600 cgacgtgacc
gaggccgtga acttctggca gcagctgagc cggccccggc agccgctgct 660
gctacaggtg tcggtgcaga gggagcatct gggcccgctg gcgtccggcg cccacaagct
720 ggtccgcttt gcctcgcagg gggcgccagc cgggcttggg gagccccagc
tggagctgca 780 caccctggac cttggggact atggagctca gggcgactgt
gaccctgaag caccaatgac 840 cgagggcacc cgctgctgcc gccaggagat
gtacattgac ctgcagggga tgaagtgggc 900 cgagaactgg gtgctggagc
ccccgggctt cctggcttat gagtgtgtgg gcacctgccg 960 gcagcccccg
gaggccctgg ccttcaagtg gccgtttctg gggcctcgac agtgcatcgc 1020
ctcggagact gactcgctgc ccatgatcgt cagcatcaag gagggaggca ggaccaggcc
1080 ccaggtggtc agcctgccca acatgagggt gcagaagtgc agctgtgcct
cggatggtgc 1140 gctcgtgcca aggaggctcc agccataggc gcctagtgta
gccatcgagg gacttgactt 1200 gtgtgtgttt ctgaagtgtt cgagggtacc
aggagagctg gcgatgactg aactgctgat 1260 ggacaaatgc tctgtgctct
ctagtgagcc ctgaatttgc ttcctctgac aagttacctc 1320 acctaatttt
tgcttctcag gaatgagaat ctttggccac tggagagccc ttgctcagtt 1380
ttctctattc ttattattca ctgcactata ttctaagcac ttacatgtgg agatactgta
1440 acctgagggc agaaagccca ntgtgtcatt gtttacttgt cctgtcactg
gatctgggct 1500 aaagtcctcc accaccactc tggacctaag acctggggtt
aagtgtgggt tgtgcatccc 1560 caatccagat aataaagact ttgtaaaaca
tgaataaaac acattttatt ctaaaa 1616 114 366 PRT Homo sapiens 114 Met
Gln Pro Leu Trp Leu Cys Trp Ala Leu Trp Val Leu Pro Leu Ala 1 5 10
15 Ser Pro Gly Ala Ala Leu Thr Gly Glu Gln Leu Leu Gly Ser Leu Leu
20 25 30 Arg Gln Leu Gln Leu Lys Glu Val Pro Thr Leu Asp Arg Ala
Asp Met 35 40 45 Glu Glu Leu Val Ile Pro Thr His Val Arg Ala Gln
Tyr Val Ala Leu 50 55 60 Leu Gln Arg Ser His Gly Asp Arg Ser Arg
Gly Lys Arg Phe Ser Gln 65 70 75 80 Ser Phe Arg Glu Val Ala Gly Arg
Phe Leu Ala Leu Glu Ala Ser Thr 85 90 95 His Leu Leu Val Phe Gly
Met Glu Gln Arg Leu Pro Pro Asn Ser Glu 100 105 110 Leu Val Gln Ala
Val Leu Arg Leu Phe Gln Glu Pro Val Pro Lys Ala 115 120 125 Ala Leu
His Arg His Gly Arg Leu Ser Pro Arg Ser Ala Arg Ala Arg 130 135 140
Val Thr Val Glu Trp Leu Arg Val Arg Asp Asp Gly Ser Asn Arg Thr 145
150 155 160 Ser Leu Ile Asp Ser Arg Leu Val Ser Val His Glu Ser Gly
Trp Lys 165 170 175 Ala Phe Asp Val Thr Glu Ala Val Asn Phe Trp Gln
Gln Leu Ser Arg 180 185 190 Pro Arg Gln Pro Leu Leu Leu Gln Val Ser
Val Gln Arg Glu His Leu 195 200 205 Gly Pro Leu Ala Ser Gly Ala His
Lys Leu Val Arg Phe Ala Ser Gln 210 215 220 Gly Ala Pro Ala Gly Leu
Gly Glu Pro Gln Leu Glu Leu His Thr Leu 225 230 235 240 Asp Leu Gly
Asp Tyr Gly Ala Gln Gly Asp Cys Asp Pro Glu Ala Pro 245 250 255 Met
Thr Glu Gly Thr Arg Cys Cys Arg Gln Glu Met Tyr Ile Asp Leu 260 265
270 Gln Gly Met Lys Trp Ala Glu Asn Trp Val Leu Glu Pro Pro Gly Phe
275 280 285 Leu Ala Tyr Glu Cys Val Gly Thr Cys Arg Gln Pro Pro Glu
Ala Leu 290 295 300 Ala Phe Lys Trp Pro Phe Leu Gly Pro Arg Gln Cys
Ile Ala Ser Glu 305 310 315 320 Thr Asp Ser Leu Pro Met Ile Val Ser
Ile Lys Glu Gly Gly Arg Thr 325 330 335 Arg Pro Gln Val Val Ser Leu
Pro Asn Met Arg Val Gln Lys Cys Ser 340 345 350 Cys Ala Ser Asp Gly
Ala Leu Val Pro Arg Arg Leu Gln Pro 355 360 365 115 21 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 115 aggactgcca taacttgcct g 21 116 22 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 116 ataggagttg aagcagcgct gc 22 117 45 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 117 tgtgtggaca tagacgagtg ccgctaccgc
tactgccagc accgc 45 118 1857 DNA Homo sapiens 118 gtctgttccc
aggagtcctt cggcggctgt tgtgtcagtg gcctgatcgc gatggggaca 60
aaggcgcaag tcgagaggaa actgttgtgc ctcttcatat tggcgatcct gttgtgctcc
120 ctggcattgg gcagtgttac agtgcactct tctgaacctg aagtcagaat
tcctgagaat 180 aatcctgtga agttgtcctg tgcctactcg ggcttttctt
ctccccgtgt ggagtggaag 240 tttgaccaag gagacaccac cagactcgtt
tgctataata acaagatcac agcttcctat 300 gaggaccggg tgaccttctt
gccaactggt atcaccttca agtccgtgac acgggaagac 360 actgggacat
acacttgtat ggtctctgag gaaggcggca acagctatgg ggaggtcaag 420
gtcaagctca tcgtgcttgt gcctccatcc aagcctacag ttaacatccc ctcctctgcc
480 accattggga accgggcagt gctgacatgc tcagaacaag atggttcccc
accttctgaa 540 tacacctggt tcaaagatgg gatagtgatg cctacgaatc
ccaaaagcac ccgtgccttc 600 agcaactctt cctatgtcct gaatcccaca
acaggagagc tggtctttga tcccctgtca 660 gcctctgata ctggagaata
cagctgtgag gcacggaatg ggtatgggac acccatgact 720 tcaaatgctg
tgcgcatgga agctgtggag cggaatgtgg gggtcatcgt ggcagccgtc 780
cttgtaaccc tgattctcct gggaatcttg gtttttggca tctggtttgc ctatagccga
840 ggccactttg acagaacaaa gaaagggact tcgagtaaga aggtgattta
cagccagcct 900 agtgcccgaa gtgaaggaga attcaaacag acctcgtcat
tcctggtgtg agcctggtcg 960 gctcaccgcc tatcatctgc atttgcctta
ctcaggtgct accggactct ggcccctgat 1020 gtctgtagtt tcacaggatg
ccttatttgt cttctacacc ccacagggcc ccctacttct 1080 tcggatgtgt
ttttaataat gtcagctatg tgccccatcc tccttcatgc cctccctccc 1140
tttcctacca ctgctgagtg gcctggaact tgtttaaagt gtttattccc catttctttg
1200 agggatcagg aaggaatcct gggtatgcca ttgacttccc ttctaagtag
acagcaaaaa 1260 tggcgggggt cgcaggaatc tgcactcaac tgcccacctg
gctggcaggg atctttgaat 1320 aggtatcttg agcttggttc tgggctcttt
ccttgtgtac tgacgaccag ggccagctgt 1380 tctagagcgg gaattagagg
ctagagcggc tgaaatggtt gtttggtgat gacactgggg 1440 tccttccatc
tctggggccc actctcttct gtcttcccat gggaagtgcc actgggatcc 1500
ctctgccctg tcctcctgaa tacaagctga ctgacattga ctgtgtctgt ggaaaatggg
1560 agctcttgtt gtggagagca tagtaaattt tcagagaact tgaagccaaa
aggatttaaa 1620 accgctgctc taaagaaaag aaaactggag gctgggcgca
gtggctcacg cctgtaatcc 1680 cagaggctga ggcaggcgga tcacctgagg
tcgggagttc gggatcagcc tgaccaacat 1740 ggagaaaccc tactggaaat
acaaagttag ccaggcatgg tggtgcatgc ctgtagtccc 1800 agctgctcag
gagcctggca acaagagcaa aactccagct caaaaaaaaa aaaaaaa 1857 119 299
PRT Homo sapiens 119 Met Gly Thr Lys Ala Gln Val Glu Arg Lys Leu
Leu Cys Leu Phe Ile 1 5 10 15 Leu Ala Ile Leu Leu Cys Ser Leu Ala
Leu Gly Ser Val Thr Val His 20 25 30 Ser Ser Glu Pro Glu Val Arg
Ile Pro Glu Asn Asn Pro Val Lys Leu 35 40 45 Ser Cys Ala Tyr Ser
Gly Phe Ser Ser Pro Arg Val Glu Trp Lys Phe 50 55 60 Asp Gln Gly
Asp Thr Thr Arg Leu Val Cys Tyr Asn Asn Lys Ile Thr 65 70 75 80 Ala
Ser Tyr Glu Asp Arg Val Thr Phe Leu Pro Thr Gly Ile Thr Phe 85 90
95 Lys Ser Val Thr Arg Glu Asp Thr Gly Thr Tyr Thr Cys Met Val Ser
100 105 110 Glu Glu Gly Gly Asn Ser Tyr Gly Glu Val Lys Val Lys Leu
Ile Val 115 120 125 Leu Val Pro Pro Ser Lys Pro Thr Val Asn Ile Pro
Ser Ser Ala Thr 130 135 140 Ile Gly Asn Arg Ala Val Leu Thr Cys Ser
Glu Gln Asp Gly Ser Pro 145 150 155 160 Pro Ser Glu Tyr Thr Trp Phe
Lys Asp Gly Ile Val Met Pro Thr Asn 165 170 175 Pro Lys Ser Thr Arg
Ala Phe Ser Asn Ser Ser Tyr Val Leu Asn Pro 180 185 190 Thr Thr Gly
Glu Leu Val Phe Asp Pro Leu Ser Ala Ser Asp Thr Gly 195 200 205 Glu
Tyr Ser Cys Glu Ala Arg Asn Gly Tyr Gly Thr Pro Met Thr Ser 210 215
220 Asn Ala Val Arg Met Glu Ala Val Glu Arg Asn Val Gly Val Ile Val
225 230 235 240 Ala Ala Val Leu Val Thr Leu Ile Leu Leu Gly Ile Leu
Val Phe Gly 245 250 255 Ile Trp Phe Ala Tyr Ser Arg Gly His Phe Asp
Arg Thr Lys Lys Gly 260 265 270 Thr Ser Ser Lys Lys Val Ile Tyr Ser
Gln Pro Ser Ala Arg Ser Glu 275 280 285 Gly Glu Phe Lys Gln Thr Ser
Ser Phe Leu Val 290 295 120 24 DNA Artificial Sequence Description
of Artificial Sequence Synthetic oligonucleotide probe 120
tcgcggagct gtgttctgtt tccc 24 121 50 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide probe
121 tgatcgcgat ggggacaaag gcgcaagctc gagaggaaac tgttgtgcct 50 122
20 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide probe 122 acacctggtt caaagatggg 20 123 24
DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide probe 123 taggaagagt tgctgaaggc acgg 24
124 20 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide probe 124 ttgccttact caggtgctac 20 125 20
DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide probe 125 actcagcagt ggtaggaaag 20 126
1210 DNA Homo sapiens 126 cagcgcgtgg ccggcgccgc tgtggggaca
gcatgagcgg cggttggatg gcgcaggttg 60 gagcgtggcg aacaggggct
ctgggcctgg cgctgctgct gctgctcggc ctcggactag 120 gcctggaggc
cgccgcgagc ccgctttcca ccccgacctc tgcccaggcc gcaggcccca 180
gctcaggctc gtgcccaccc accaagttcc agtgccgcac cagtggctta tgcgtgcccc
240 tcacctggcg ctgcgacagg gacttggact gcagcgatgg cagcgatgag
gaggagtgca 300 ggattgagcc atgtacccag aaagggcaat gcccaccgcc
ccctggcctc ccctgcccct 360 gcaccggcgt cagtgactgc tctgggggaa
ctgacaagaa actgcgcaac tgcagccgcc 420 tggcctgcct agcaggcgag
ctccgttgca cgctgagcga tgactgcatt ccactcacgt 480 ggcgctgcga
cggccaccca gactgtcccg actccagcga cgagctcggc tgtggaacca 540
atgagatcct cccggaaggg gatgccacaa ccatggggcc ccctgtgacc ctggagagtg
600 tcacctctct caggaatgcc acaaccatgg ggccccctgt gaccctggag
agtgtcccct 660 ctgtcgggaa tgccacatcc tcctctgccg gagaccagtc
tggaagccca actgcctatg 720 gggttattgc agctgctgcg gtgctcagtg
caagcctggt caccgccacc ctcctccttt 780 tgtcctggct ccgagcccag
gagcgcctcc gcccactggg gttactggtg gccatgaagg 840 agtccctgct
gctgtcagaa cagaagacct cgctgccctg aggacaagca cttgccacca 900
ccgtcactca gccctgggcg tagccggaca ggaggagagc agtgatgcgg atgggtaccc
960 gggcacacca gccctcagag acctgagttc ttctggccac gtggaacctc
gaacccgagc 1020 tcctgcagaa gtggccctgg agattgaggg tccctggaca
ctccctatgg agatccgggg 1080 agctaggatg gggaacctgc cacagccaga
actgaggggc tggccccagg cagctcccag 1140 ggggtagaac ggccctgtgc
ttaagacact ccctgctgcc ccgtctgagg gtggcgatta 1200 aagttgcttc 1210
127 282 PRT Homo sapiens 127 Met Ser Gly Gly Trp Met Ala Gln Val
Gly Ala Trp Arg Thr Gly Ala 1 5 10 15 Leu Gly Leu Ala Leu Leu Leu
Leu Leu Gly Leu Gly Leu Gly Leu Glu 20 25 30 Ala Ala Ala Ser Pro
Leu Ser Thr Pro Thr Ser Ala Gln Ala Ala Gly 35 40 45 Pro Ser Ser
Gly Ser Cys Pro Pro Thr Lys Phe Gln Cys Arg Thr Ser 50 55 60 Gly
Leu Cys Val Pro Leu Thr Trp Arg Cys Asp Arg Asp Leu Asp Cys 65 70
75 80 Ser Asp Gly Ser Asp Glu Glu Glu Cys Arg Ile Glu Pro Cys Thr
Gln 85 90 95 Lys Gly Gln Cys Pro Pro Pro Pro Gly Leu Pro Cys Pro
Cys Thr Gly 100 105 110 Val Ser Asp Cys Ser Gly Gly Thr Asp Lys Lys
Leu Arg Asn Cys Ser 115 120 125 Arg Leu Ala Cys Leu Ala Gly Glu Leu
Arg Cys Thr Leu Ser Asp Asp 130 135 140 Cys Ile Pro Leu Thr Trp Arg
Cys Asp Gly His Pro Asp Cys Pro Asp 145 150 155 160 Ser Ser Asp Glu
Leu Gly Cys Gly Thr Asn Glu Ile Leu Pro Glu Gly 165 170 175 Asp Ala
Thr Thr Met Gly Pro Pro Val Thr Leu Glu Ser Val Thr Ser 180 185 190
Leu Arg Asn Ala Thr Thr Met Gly Pro Pro Val Thr Leu Glu Ser Val 195
200 205 Pro Ser Val Gly Asn Ala Thr Ser Ser Ser Ala Gly Asp Gln Ser
Gly 210 215 220 Ser Pro Thr Ala Tyr Gly Val Ile Ala Ala Ala Ala Val
Leu Ser Ala 225 230 235 240 Ser Leu Val Thr Ala Thr Leu Leu Leu Leu
Ser Trp Leu Arg Ala Gln 245 250 255 Glu Arg Leu Arg Pro Leu Gly Leu
Leu Val Ala Met Lys Glu Ser Leu 260 265 270 Leu Leu Ser Glu Gln Lys
Thr Ser Leu Pro 275 280 128 24 DNA Artificial Sequence Description
of Artificial Sequence Synthetic oligonucleotide probe 128
aagttccagt gccgcaccag tggc 24 129 24 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide probe
129 ttggttccac agccgagctc gtcg 24 130 50 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide probe
130 gaggaggagt gcaggattga gccatgtacc cagaaagggc aatgcccacc 50 131
1843 DNA Homo sapiens modified_base (1837)..(1837) a, t, c or g 131
cccacgcgtc cggtctcgct cgctcgcgca gcggcggcag cagaggtcgc gcacagatgc
60 gggttagact ggcgggggga ggaggcggag gagggaagga agctgcatgc
atgagaccca 120 cagactcttg caagctggat gccctctgtg gatgaaagat
gtatcatgga atgaacccga 180 gcaatggaga tggatttcta gagcagcagc
agcagcagca gcaacctcag tccccccaga 240 gactcttggc cgtgatcctg
tggtttcagc tggcgctgtg cttcggccct gcacagctca 300 cgggcgggtt
cgatgacctt caagtgtgtg ctgaccccgg cattcccgag aatggcttca 360
ggacccccag cggaggggtt ttctttgaag gctctgtagc ccgatttcac tgccaagacg
420 gattcaagct gaagggcgct acaaagagac tgtgtttgaa gcattttaat
ggaaccctag 480 gctggatccc aagtgataat tccatctgtg tgcaagaaga
ttgccgtatc cctcaaatcg 540 aagatgctga gattcataac aagacatata
gacatggaga gaagctaatc atcacttgtc 600 atgaaggatt caagatccgg
taccccgacc tacacaatat ggtttcatta tgtcgcgatg 660 atggaacgtg
gaataatctg cccatctgtc aaggctgcct gagacctcta gcctcttcta 720
atggctatgt aaacatctct gagctccaga cctccttccc ggtggggact gtgatctcct
780 atcgctgctt tcccggattt aaacttgatg ggtctgcgta tcttgagtgc
ttacaaaacc 840 ttatctggtc gtccagccca ccccggtgcc ttgctctgga
agcccaagtc tgtccactac 900 ctccaatggt gagtcacgga gatttcgtct
gccacccgcg gccttgtgag cgctacaacc 960 acggaactgt ggtggagttt
tactgcgatc ctggctacag cctcaccagc gactacaagt 1020 acatcacctg
ccagtatgga gagtggtttc cttcttatca agtctactgc atcaaatcag 1080
agcaaacgtg gcccagcacc catgagaccc tcctgaccac gtggaagatt gtggcgttca
1140 cggcaaccag tgtgctgctg gtgctgctgc tcgtcatcct ggccaggatg
ttccagacca 1200 agttcaaggc ccactttccc cccagggggc ctccccggag
ttccagcagt gaccctgact 1260 ttgtggtggt agacggcgtg cccgtcatgc
tcccgtccta tgacgaagct gtgagtggcg 1320 gcttgagtgc cttaggcccc
gggtacatgg cctctgtggg ccagggctgc cccttacccg 1380 tggacgacca
gagcccccca gcataccccg gctcagggga cacggacaca ggcccagggg 1440
agtcagaaac ctgtgacagc gtctcaggct cttctgagct gctccaaagt ctgtattcac
1500 ctcccaggtg ccaagagagc acccaccctg cttcggacaa ccctgacata
attgccagca 1560 cggcagagga ggtggcatcc accagcccag gcatccatca
tgcccactgg gtgttgttcc 1620 taagaaactg attgattaaa aaatttccca
aagtgtcctg aagtgtctct tcaaatacat 1680 gttgatctgt ggagttgatt
cctttccttc tcttggtttt agacaaatgt aaacaaagct 1740 ctgatcctta
aaattgctat gctgatagag tggtgagggc tggaagcttg atcaagtcct 1800
gtttcttctt gacacagact gattaaaaat taaaagnaaa aaa 1843 132 490 PRT
Homo sapiens 132 Met Tyr His Gly Met Asn Pro Ser Asn Gly Asp Gly
Phe Leu Glu Gln 1 5 10 15 Gln Gln Gln Gln Gln Gln Pro Gln Ser Pro
Gln Arg Leu Leu Ala Val 20 25 30 Ile Leu Trp Phe Gln Leu Ala Leu
Cys Phe Gly Pro Ala Gln Leu Thr 35 40 45 Gly Gly Phe Asp Asp Leu
Gln Val Cys Ala Asp Pro Gly Ile Pro Glu 50 55 60 Asn Gly Phe Arg
Thr Pro Ser Gly Gly Val Phe Phe Glu Gly Ser Val 65 70 75 80 Ala Arg
Phe His Cys Gln Asp Gly Phe Lys Leu Lys Gly Ala Thr Lys 85 90 95
Arg Leu Cys Leu Lys His Phe Asn Gly Thr Leu Gly Trp Ile Pro Ser 100
105 110 Asp Asn Ser Ile Cys Val Gln Glu Asp Cys Arg Ile Pro Gln Ile
Glu 115 120 125
Asp Ala Glu Ile His Asn Lys Thr Tyr Arg His Gly Glu Lys Leu Ile 130
135 140 Ile Thr Cys His Glu Gly Phe Lys Ile Arg Tyr Pro Asp Leu His
Asn 145 150 155 160 Met Val Ser Leu Cys Arg Asp Asp Gly Thr Trp Asn
Asn Leu Pro Ile 165 170 175 Cys Gln Gly Cys Leu Arg Pro Leu Ala Ser
Ser Asn Gly Tyr Val Asn 180 185 190 Ile Ser Glu Leu Gln Thr Ser Phe
Pro Val Gly Thr Val Ile Ser Tyr 195 200 205 Arg Cys Phe Pro Gly Phe
Lys Leu Asp Gly Ser Ala Tyr Leu Glu Cys 210 215 220 Leu Gln Asn Leu
Ile Trp Ser Ser Ser Pro Pro Arg Cys Leu Ala Leu 225 230 235 240 Glu
Ala Gln Val Cys Pro Leu Pro Pro Met Val Ser His Gly Asp Phe 245 250
255 Val Cys His Pro Arg Pro Cys Glu Arg Tyr Asn His Gly Thr Val Val
260 265 270 Glu Phe Tyr Cys Asp Pro Gly Tyr Ser Leu Thr Ser Asp Tyr
Lys Tyr 275 280 285 Ile Thr Cys Gln Tyr Gly Glu Trp Phe Pro Ser Tyr
Gln Val Tyr Cys 290 295 300 Ile Lys Ser Glu Gln Thr Trp Pro Ser Thr
His Glu Thr Leu Leu Thr 305 310 315 320 Thr Trp Lys Ile Val Ala Phe
Thr Ala Thr Ser Val Leu Leu Val Leu 325 330 335 Leu Leu Val Ile Leu
Ala Arg Met Phe Gln Thr Lys Phe Lys Ala His 340 345 350 Phe Pro Pro
Arg Gly Pro Pro Arg Ser Ser Ser Ser Asp Pro Asp Phe 355 360 365 Val
Val Val Asp Gly Val Pro Val Met Leu Pro Ser Tyr Asp Glu Ala 370 375
380 Val Ser Gly Gly Leu Ser Ala Leu Gly Pro Gly Tyr Met Ala Ser Val
385 390 395 400 Gly Gln Gly Cys Pro Leu Pro Val Asp Asp Gln Ser Pro
Pro Ala Tyr 405 410 415 Pro Gly Ser Gly Asp Thr Asp Thr Gly Pro Gly
Glu Ser Glu Thr Cys 420 425 430 Asp Ser Val Ser Gly Ser Ser Glu Leu
Leu Gln Ser Leu Tyr Ser Pro 435 440 445 Pro Arg Cys Gln Glu Ser Thr
His Pro Ala Ser Asp Asn Pro Asp Ile 450 455 460 Ile Ala Ser Thr Ala
Glu Glu Val Ala Ser Thr Ser Pro Gly Ile His 465 470 475 480 His Ala
His Trp Val Leu Phe Leu Arg Asn 485 490 133 23 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 133 atctcctatc gctgctttcc cgg 23 134 23 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 134 agccaggatc gcagtaaaac tcc 23 135 50 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 135 atttaaactt gatgggtctg cgtatcttga
gtgcttacaa aaccttatct 50 136 1815 DNA Homo sapiens 136 cccacgcgtc
cgctccgcgc cctccccccc gcctcccgtg cggtccgtcg gtggcctaga 60
gatgctgctg ccgcggttgc agttgtcgcg cacgcctctg cccgccagcc cgctccaccg
120 ccgtagcgcc cgagtgtcgg ggggcgcacc cgagtcgggc catgaggccg
ggaaccgcgc 180 tacaggccgt gctgctggcc gtgctgctgg tggggctgcg
ggccgcgacg ggtcgcctgc 240 tgagtgcctc ggatttggac ctcagaggag
ggcagccagt ctgccgggga gggacacaga 300 ggccttgtta taaagtcatt
tacttccatg atacttctcg aagactgaac tttgaggaag 360 ccaaagaagc
ctgcaggagg gatggaggcc agctagtcag catcgagtct gaagatgaac 420
agaaactgat agaaaagttc attgaaaacc tcttgccatc tgatggtgac ttctggattg
480 ggctcaggag gcgtgaggag aaacaaagca atagcacagc ctgccaggac
ctttatgctt 540 ggactgatgg cagcatatca caatttagga actggtatgt
ggatgagccg tcctgcggca 600 gcgaggtctg cgtggtcatg taccatcagc
catcggcacc cgctggcatc ggaggcccct 660 acatgttcca gtggaatgat
gaccggtgca acatgaagaa caatttcatt tgcaaatatt 720 ctgatgagaa
accagcagtt ccttctagag aagctgaagg tgaggaaaca gagctgacaa 780
cacctgtact tccagaagaa acacaggaag aagatgccaa aaaaacattt aaagaaagta
840 gagaagctgc cttgaatctg gcctacatcc taatccccag cattcccctt
ctcctcctcc 900 ttgtggtcac cacagttgta tgttgggttt ggatctgtag
aaaaagaaaa cgggagcagc 960 cagaccctag cacaaagaag caacacacca
tctggccctc tcctcaccag ggaaacagcc 1020 cggacctaga ggtctacaat
gtcataagaa aacaaagcga agctgactta gctgagaccc 1080 ggccagacct
gaagaatatt tcattccgag tgtgttcggg agaagccact cccgatgaca 1140
tgtcttgtga ctatgacaac atggctgtga acccatcaga aagtgggttt gtgactctgg
1200 tgagcgtgga gagtggattt gtgaccaatg acatttatga gttctcccca
gaccaaatgg 1260 ggaggagtaa ggagtctgga tgggtggaaa atgaaatata
tggttattag gacatataaa 1320 aaactgaaac tgacaacaat ggaaaagaaa
tgataagcaa aatcctctta ttttctataa 1380 ggaaaataca cagaaggtct
atgaacaagc ttagatcagg tcctgtggat gagcatgtgg 1440 tccccacgac
ctcctgttgg acccccacgt tttggctgta tcctttatcc cagccagtca 1500
tccagctcga ccttatgaga aggtaccttg cccaggtctg gcacatagta gagtctcaat
1560 aaatgtcact tggttggttg tatctaactt ttaagggaca gagctttacc
tggcagtgat 1620 aaagatgggc tgtggagctt ggaaaaccac ctctgttttc
cttgctctat acagcagcac 1680 atattatcat acagacagaa aatccagaat
cttttcaaag cccacatatg gtagcacagg 1740 ttggcctgtg catcggcaat
tctcatatct gtttttttca aagaataaaa tcaaataaag 1800 agcaggaaaa aaaaa
1815 137 382 PRT Homo sapiens 137 Met Arg Pro Gly Thr Ala Leu Gln
Ala Val Leu Leu Ala Val Leu Leu 1 5 10 15 Val Gly Leu Arg Ala Ala
Thr Gly Arg Leu Leu Ser Ala Ser Asp Leu 20 25 30 Asp Leu Arg Gly
Gly Gln Pro Val Cys Arg Gly Gly Thr Gln Arg Pro 35 40 45 Cys Tyr
Lys Val Ile Tyr Phe His Asp Thr Ser Arg Arg Leu Asn Phe 50 55 60
Glu Glu Ala Lys Glu Ala Cys Arg Arg Asp Gly Gly Gln Leu Val Ser 65
70 75 80 Ile Glu Ser Glu Asp Glu Gln Lys Leu Ile Glu Lys Phe Ile
Glu Asn 85 90 95 Leu Leu Pro Ser Asp Gly Asp Phe Trp Ile Gly Leu
Arg Arg Arg Glu 100 105 110 Glu Lys Gln Ser Asn Ser Thr Ala Cys Gln
Asp Leu Tyr Ala Trp Thr 115 120 125 Asp Gly Ser Ile Ser Gln Phe Arg
Asn Trp Tyr Val Asp Glu Pro Ser 130 135 140 Cys Gly Ser Glu Val Cys
Val Val Met Tyr His Gln Pro Ser Ala Pro 145 150 155 160 Ala Gly Ile
Gly Gly Pro Tyr Met Phe Gln Trp Asn Asp Asp Arg Cys 165 170 175 Asn
Met Lys Asn Asn Phe Ile Cys Lys Tyr Ser Asp Glu Lys Pro Ala 180 185
190 Val Pro Ser Arg Glu Ala Glu Gly Glu Glu Thr Glu Leu Thr Thr Pro
195 200 205 Val Leu Pro Glu Glu Thr Gln Glu Glu Asp Ala Lys Lys Thr
Phe Lys 210 215 220 Glu Ser Arg Glu Ala Ala Leu Asn Leu Ala Tyr Ile
Leu Ile Pro Ser 225 230 235 240 Ile Pro Leu Leu Leu Leu Leu Val Val
Thr Thr Val Val Cys Trp Val 245 250 255 Trp Ile Cys Arg Lys Arg Lys
Arg Glu Gln Pro Asp Pro Ser Thr Lys 260 265 270 Lys Gln His Thr Ile
Trp Pro Ser Pro His Gln Gly Asn Ser Pro Asp 275 280 285 Leu Glu Val
Tyr Asn Val Ile Arg Lys Gln Ser Glu Ala Asp Leu Ala 290 295 300 Glu
Thr Arg Pro Asp Leu Lys Asn Ile Ser Phe Arg Val Cys Ser Gly 305 310
315 320 Glu Ala Thr Pro Asp Asp Met Ser Cys Asp Tyr Asp Asn Met Ala
Val 325 330 335 Asn Pro Ser Glu Ser Gly Phe Val Thr Leu Val Ser Val
Glu Ser Gly 340 345 350 Phe Val Thr Asn Asp Ile Tyr Glu Phe Ser Pro
Asp Gln Met Gly Arg 355 360 365 Ser Lys Glu Ser Gly Trp Val Glu Asn
Glu Ile Tyr Gly Tyr 370 375 380 138 50 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide probe
138 gttcattgaa aacctcttgc catctgatgg tgacttctgg attgggctca 50 139
24 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide probe 139 aagccaaaga agcctgcagg aggg 24
140 24 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide probe 140 cagtccaagc ataaaggtcc tggc 24
141 1514 DNA Homo sapiens 141 ggggtctccc tcagggccgg gaggcacagc
ggtccctgct tgctgaaggg ctggatgtac 60 gcatccgcag gttcccgcgg
acttgggggc gcccgctgag ccccggcgcc cgcagaagac 120 ttgtgtttgc
ctcctgcagc ctcaacccgg agggcagcga gggcctacca ccatgatcac 180
tggtgtgttc agcatgcgct tgtggacccc agtgggcgtc ctgacctcgc tggcgtactg
240 cctgcaccag cggcgggtgg ccctggccga gctgcaggag gccgatggcc
agtgtccggt 300 cgaccgcagc ctgctgaagt tgaaaatggt gcaggtcgtg
tttcgacacg gggctcggag 360 tcctctcaag ccgctcccgc tggaggagca
ggtagagtgg aacccccagc tattagaggt 420 cccaccccaa actcagtttg
attacacagt caccaatcta gctggtggtc cgaaaccata 480 ttctccttac
gactctcaat accatgagac caccctgaag gggggcatgt ttgctgggca 540
gctgaccaag gtgggcatgc agcaaatgtt tgccttggga gagagactga ggaagaacta
600 tgtggaagac attccctttc tttcaccaac cttcaaccca caggaggtct
ttattcgttc 660 cactaacatt tttcggaatc tggagtccac ccgttgtttg
ctggctgggc ttttccagtg 720 tcagaaagaa ggacccatca tcatccacac
tgatgaagca gattcagaag tcttgtatcc 780 caactaccaa agctgctgga
gcctgaggca gagaaccaga ggccggaggc agactgcctc 840 tttacagcca
ggaatctcag aggatttgaa aaaggtgaag gacaggatgg gcattgacag 900
tagtgataaa gtggacttct tcatcctcct ggacaacgtg gctgccgagc aggcacacaa
960 cctcccaagc tgccccatgc tgaagagatt tgcacggatg atcgaacaga
gagctgtgga 1020 cacatccttg tacatactgc ccaaggaaga cagggaaagt
cttcagatgg cagtaggccc 1080 attcctccac atcctagaga gcaacctgct
gaaagccatg gactctgcca ctgcccccga 1140 caagatcaga aagctgtatc
tctatgcggc tcatgatgtg accttcatac cgctcttaat 1200 gaccctgggg
atttttgacc acaaatggcc accgtttgct gttgacctga ccatggaact 1260
ttaccagcac ctggaatcta aggagtggtt tgtgcagctc tattaccacg ggaaggagca
1320 ggtgccgaga ggttgccctg atgggctctg cccgctggac atgttcttga
atgccatgtc 1380 agtttatacc ttaagcccag aaaaatacca tgcactctgc
tctcaaactc aggtgatgga 1440 agttggaaat gaagagtaac tgatttataa
aagcaggatg tgttgatttt aaaataaagt 1500 gcctttatac aatg 1514 142 428
PRT Homo sapiens 142 Met Ile Thr Gly Val Phe Ser Met Arg Leu Trp
Thr Pro Val Gly Val 1 5 10 15 Leu Thr Ser Leu Ala Tyr Cys Leu His
Gln Arg Arg Val Ala Leu Ala 20 25 30 Glu Leu Gln Glu Ala Asp Gly
Gln Cys Pro Val Asp Arg Ser Leu Leu 35 40 45 Lys Leu Lys Met Val
Gln Val Val Phe Arg His Gly Ala Arg Ser Pro 50 55 60 Leu Lys Pro
Leu Pro Leu Glu Glu Gln Val Glu Trp Asn Pro Gln Leu 65 70 75 80 Leu
Glu Val Pro Pro Gln Thr Gln Phe Asp Tyr Thr Val Thr Asn Leu 85 90
95 Ala Gly Gly Pro Lys Pro Tyr Ser Pro Tyr Asp Ser Gln Tyr His Glu
100 105 110 Thr Thr Leu Lys Gly Gly Met Phe Ala Gly Gln Leu Thr Lys
Val Gly 115 120 125 Met Gln Gln Met Phe Ala Leu Gly Glu Arg Leu Arg
Lys Asn Tyr Val 130 135 140 Glu Asp Ile Pro Phe Leu Ser Pro Thr Phe
Asn Pro Gln Glu Val Phe 145 150 155 160 Ile Arg Ser Thr Asn Ile Phe
Arg Asn Leu Glu Ser Thr Arg Cys Leu 165 170 175 Leu Ala Gly Leu Phe
Gln Cys Gln Lys Glu Gly Pro Ile Ile Ile His 180 185 190 Thr Asp Glu
Ala Asp Ser Glu Val Leu Tyr Pro Asn Tyr Gln Ser Cys 195 200 205 Trp
Ser Leu Arg Gln Arg Thr Arg Gly Arg Arg Gln Thr Ala Ser Leu 210 215
220 Gln Pro Gly Ile Ser Glu Asp Leu Lys Lys Val Lys Asp Arg Met Gly
225 230 235 240 Ile Asp Ser Ser Asp Lys Val Asp Phe Phe Ile Leu Leu
Asp Asn Val 245 250 255 Ala Ala Glu Gln Ala His Asn Leu Pro Ser Cys
Pro Met Leu Lys Arg 260 265 270 Phe Ala Arg Met Ile Glu Gln Arg Ala
Val Asp Thr Ser Leu Tyr Ile 275 280 285 Leu Pro Lys Glu Asp Arg Glu
Ser Leu Gln Met Ala Val Gly Pro Phe 290 295 300 Leu His Ile Leu Glu
Ser Asn Leu Leu Lys Ala Met Asp Ser Ala Thr 305 310 315 320 Ala Pro
Asp Lys Ile Arg Lys Leu Tyr Leu Tyr Ala Ala His Asp Val 325 330 335
Thr Phe Ile Pro Leu Leu Met Thr Leu Gly Ile Phe Asp His Lys Trp 340
345 350 Pro Pro Phe Ala Val Asp Leu Thr Met Glu Leu Tyr Gln His Leu
Glu 355 360 365 Ser Lys Glu Trp Phe Val Gln Leu Tyr Tyr His Gly Lys
Glu Gln Val 370 375 380 Pro Arg Gly Cys Pro Asp Gly Leu Cys Pro Leu
Asp Met Phe Leu Asn 385 390 395 400 Ala Met Ser Val Tyr Thr Leu Ser
Pro Glu Lys Tyr His Ala Leu Cys 405 410 415 Ser Gln Thr Gln Val Met
Glu Val Gly Asn Glu Glu 420 425 143 24 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide probe
143 ccaactacca aagctgctgg agcc 24 144 24 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide probe
144 gcagctctat taccacggga agga 24 145 24 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide probe
145 tccttcccgt ggtaatagag ctgc 24 146 45 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide probe
146 ggcagagaac cagaggccgg aggagactgc ctctttacag ccagg 45 147 1686
DNA Homo sapiens 147 ctcctcttaa catacttgca gctaaaacta aatattgctg
cttggggacc tccttctagc 60 cttaaatttc agctcatcac cttcacctgc
cttggtcatg gctctgctat tctccttgat 120 ccttgccatt tgcaccagac
ctggattcct agcgtctcca tctggagtgc ggctggtggg 180 gggcctccac
cgctgtgaag ggcgggtgga ggtggaacag aaaggccagt ggggcaccgt 240
gtgtgatgac ggctgggaca ttaaggacgt ggctgtgttg tgccgggagc tgggctgtgg
300 agctgccagc ggaaccccta gtggtatttt gtatgagcca ccagcagaaa
aagagcaaaa 360 ggtcctcatc caatcagtca gttgcacagg aacagaagat
acattggctc agtgtgagca 420 agaagaagtt tatgattgtt cacatgatga
agatgctggg gcatcgtgtg agaacccaga 480 gagctctttc tccccagtcc
cagagggtgt caggctggct gacggccctg ggcattgcaa 540 gggacgcgtg
gaagtgaagc accagaacca gtggtatacc gtgtgccaga caggctggag 600
cctccgggcc gcaaaggtgg tgtgccggca gctgggatgt gggagggctg tactgactca
660 aaaacgctgc aacaagcatg cctatggccg aaaacccatc tggctgagcc
agatgtcatg 720 ctcaggacga gaagcaaccc ttcaggattg cccttctggg
ccttggggga agaacacctg 780 caaccatgat gaagacacgt gggtcgaatg
tgaagatccc tttgacttga gactagtagg 840 aggagacaac ctctgctctg
ggcgactgga ggtgctgcac aagggcgtat ggggctctgt 900 ctgtgatgac
aactggggag aaaaggagga ccaggtggta tgcaagcaac tgggctgtgg 960
gaagtccctc tctccctcct tcagagaccg gaaatgctat ggccctgggg ttggccgcat
1020 ctggctggat aatgttcgtt gctcagggga ggagcagtcc ctggagcagt
gccagcacag 1080 attttggggg tttcacgact gcacccacca ggaagatgtg
gctgtcatct gctcagtgta 1140 ggtgggcatc atctaatctg ttgagtgcct
gaatagaaga aaaacacaga agaagggagc 1200 atttactgtc tacatgactg
catgggatga acactgatct tcttctgccc ttggactggg 1260 acttatactt
ggtgcccctg attctcaggc cttcagagtt ggatcagaac ttacaacatc 1320
aggtctagtt ctcaggccat cagacatagt ttggaactac atcaccacct ttcctatgtc
1380 tccacattgc acacagcaga ttcccagcct ccataattgt gtgtatcaac
tacttaaata 1440 cattctcaca cacacacaca cacacacaca cacacacaca
cacacataca ccatttgtcc 1500 tgtttctctg aagaactctg acaaaataca
gattttggta ctgaaagaga ttctagagga 1560 acggaatttt aaggataaat
tttctgaatt ggttatgggg tttctgaaat tggctctata 1620 atctaattag
atataaaatt ctggtaactt tatttacaat aataaagata gcactatgtg 1680 ttcaaa
1686 148 347 PRT Homo sapiens 148 Met Ala Leu Leu Phe Ser Leu Ile
Leu Ala Ile Cys Thr Arg Pro Gly 1 5 10 15 Phe Leu Ala Ser Pro Ser
Gly Val Arg Leu Val Gly Gly Leu His Arg 20 25 30 Cys Glu Gly Arg
Val Glu Val Glu Gln Lys Gly Gln Trp Gly Thr Val 35 40 45 Cys Asp
Asp Gly Trp Asp Ile Lys Asp Val Ala Val Leu Cys Arg Glu 50 55 60
Leu Gly Cys Gly Ala Ala Ser Gly Thr Pro Ser Gly Ile Leu Tyr Glu 65
70 75 80 Pro Pro Ala Glu Lys Glu Gln Lys Val Leu Ile Gln Ser Val
Ser Cys 85 90 95 Thr Gly Thr Glu Asp Thr Leu Ala Gln Cys Glu Gln
Glu Glu Val Tyr 100 105 110 Asp Cys Ser His Asp Glu Asp Ala Gly Ala
Ser Cys Glu Asn Pro Glu 115 120 125 Ser Ser Phe Ser Pro Val Pro Glu
Gly Val Arg Leu Ala Asp Gly Pro 130 135 140 Gly His Cys Lys Gly Arg
Val Glu Val Lys His Gln Asn Gln Trp Tyr 145 150 155 160 Thr Val Cys
Gln Thr Gly Trp
Ser Leu Arg Ala Ala Lys Val Val Cys 165 170 175 Arg Gln Leu Gly Cys
Gly Arg Ala Val Leu Thr Gln Lys Arg Cys Asn 180 185 190 Lys His Ala
Tyr Gly Arg Lys Pro Ile Trp Leu Ser Gln Met Ser Cys 195 200 205 Ser
Gly Arg Glu Ala Thr Leu Gln Asp Cys Pro Ser Gly Pro Trp Gly 210 215
220 Lys Asn Thr Cys Asn His Asp Glu Asp Thr Trp Val Glu Cys Glu Asp
225 230 235 240 Pro Phe Asp Leu Arg Leu Val Gly Gly Asp Asn Leu Cys
Ser Gly Arg 245 250 255 Leu Glu Val Leu His Lys Gly Val Trp Gly Ser
Val Cys Asp Asp Asn 260 265 270 Trp Gly Glu Lys Glu Asp Gln Val Val
Cys Lys Gln Leu Gly Cys Gly 275 280 285 Lys Ser Leu Ser Pro Ser Phe
Arg Asp Arg Lys Cys Tyr Gly Pro Gly 290 295 300 Val Gly Arg Ile Trp
Leu Asp Asn Val Arg Cys Ser Gly Glu Glu Gln 305 310 315 320 Ser Leu
Glu Gln Cys Gln His Arg Phe Trp Gly Phe His Asp Cys Thr 325 330 335
His Gln Glu Asp Val Ala Val Ile Cys Ser Val 340 345 149 24 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 149 ttcagctcat caccttcacc tgcc 24 150 24 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 150 ggctcataca aaataccact aggg 24 151 50 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 151 gggcctccac cgctgtgaag ggcgggtgga
ggtggaacag aaaggccagt 50 152 1427 DNA Homo sapiens 152 actgcactcg
gttctatcga ttgaattccc cggggatcct ctagagatcc ctcgacctcg 60
acccacgcgt ccgcggacgc gtgggcggac gcgtgggccg gctaccagga agagtctgcc
120 gaaggtgaag gccatggact tcatcacctc cacagccatc ctgcccctgc
tgttcggctg 180 cctgggcgtc ttcggcctct tccggctgct gcagtgggtg
cgcgggaagg cctacctgcg 240 gaatgctgtg gtggtgatca caggcgccac
ctcagggctg ggcaaagaat gtgcaaaagt 300 cttctatgct gcgggtgcta
aactggtgct ctgtggccgg aatggtgggg ccctagaaga 360 gctcatcaga
gaacttaccg cttctcatgc caccaaggtg cagacacaca agccttactt 420
ggtgaccttc gacctcacag actctggggc catagttgca gcagcagctg agatcctgca
480 gtgctttggc tatgtcgaca tacttgtcaa caatgctggg atcagctacc
gtggtaccat 540 catggacacc acagtggatg tggacaagag ggtcatggag
acaaactact ttggcccagt 600 tgctctaacg aaagcactcc tgccctccat
gatcaagagg aggcaaggcc acattgtcgc 660 catcagcagc atccagggca
agatgagcat tccttttcga tcagcatatg cagcctccaa 720 gcacgcaacc
caggctttct ttgactgtct gcgtgccgag atggaacagt atgaaattga 780
ggtgaccgtc atcagccccg gctacatcca caccaacctc tctgtaaatg ccatcaccgc
840 ggatggatct aggtatggag ttatggacac caccacagcc cagggccgaa
gccctgtgga 900 ggtggcccag gatgttcttg ctgctgtggg gaagaagaag
aaagatgtga tcctggctga 960 cttactgcct tccttggctg tttatcttcg
aactctggct cctgggctct tcttcagcct 1020 catggcctcc agggccagaa
aagagcggaa atccaagaac tcctagtact ctgaccagcc 1080 agggccaggg
cagagaagca gcactcttag gcttgcttac tctacaaggg acagttgcat 1140
ttgttgagac tttaatggag atttgtctca caagtgggaa agactgaaga aacacatctc
1200 gtgcagatct gctggcagag gacaatcaaa aacgacaaca agcttcttcc
cagggtgagg 1260 ggaaacactt aaggaataaa tatggagctg gggtttaaca
ctaaaaacta gaaataaaca 1320 tctcaaacag taaaaaaaaa aaaaaagggc
ggccgcgact ctagagtcga cctgcagaag 1380 cttggccgcc atggcccaac
ttgtttattg cagcttataa tggttac 1427 153 310 PRT Homo sapiens 153 Met
Asp Phe Ile Thr Ser Thr Ala Ile Leu Pro Leu Leu Phe Gly Cys 1 5 10
15 Leu Gly Val Phe Gly Leu Phe Arg Leu Leu Gln Trp Val Arg Gly Lys
20 25 30 Ala Tyr Leu Arg Asn Ala Val Val Val Ile Thr Gly Ala Thr
Ser Gly 35 40 45 Leu Gly Lys Glu Cys Ala Lys Val Phe Tyr Ala Ala
Gly Ala Lys Leu 50 55 60 Val Leu Cys Gly Arg Asn Gly Gly Ala Leu
Glu Glu Leu Ile Arg Glu 65 70 75 80 Leu Thr Ala Ser His Ala Thr Lys
Val Gln Thr His Lys Pro Tyr Leu 85 90 95 Val Thr Phe Asp Leu Thr
Asp Ser Gly Ala Ile Val Ala Ala Ala Ala 100 105 110 Glu Ile Leu Gln
Cys Phe Gly Tyr Val Asp Ile Leu Val Asn Asn Ala 115 120 125 Gly Ile
Ser Tyr Arg Gly Thr Ile Met Asp Thr Thr Val Asp Val Asp 130 135 140
Lys Arg Val Met Glu Thr Asn Tyr Phe Gly Pro Val Ala Leu Thr Lys 145
150 155 160 Ala Leu Leu Pro Ser Met Ile Lys Arg Arg Gln Gly His Ile
Val Ala 165 170 175 Ile Ser Ser Ile Gln Gly Lys Met Ser Ile Pro Phe
Arg Ser Ala Tyr 180 185 190 Ala Ala Ser Lys His Ala Thr Gln Ala Phe
Phe Asp Cys Leu Arg Ala 195 200 205 Glu Met Glu Gln Tyr Glu Ile Glu
Val Thr Val Ile Ser Pro Gly Tyr 210 215 220 Ile His Thr Asn Leu Ser
Val Asn Ala Ile Thr Ala Asp Gly Ser Arg 225 230 235 240 Tyr Gly Val
Met Asp Thr Thr Thr Ala Gln Gly Arg Ser Pro Val Glu 245 250 255 Val
Ala Gln Asp Val Leu Ala Ala Val Gly Lys Lys Lys Lys Asp Val 260 265
270 Ile Leu Ala Asp Leu Leu Pro Ser Leu Ala Val Tyr Leu Arg Thr Leu
275 280 285 Ala Pro Gly Leu Phe Phe Ser Leu Met Ala Ser Arg Ala Arg
Lys Glu 290 295 300 Arg Lys Ser Lys Asn Ser 305 310 154 24 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 154 ggtgctaaac tggtgctctg tggc 24 155 20 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 155 cagggcaaga tgagcattcc 20 156 24 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 156 tcatactgtt ccatctcggc acgc 24 157 50 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 157 aatggtgggg ccctagaaga gctcatcaga
gaactcaccg cttctcatgc 50 158 1771 DNA Homo sapiens 158 cccacgcgtc
cgctggtgtt agatcgagca accctctaaa agcagtttag agtggtaaaa 60
aaaaaaaaaa acacaccaaa cgctcgcagc cacaaaaggg atgaaatttc ttctggacat
120 cctcctgctt ctcccgttac tgatcgtctg ctccctagag tccttcgtga
agctttttat 180 tcctaagagg agaaaatcag tcaccggcga aatcgtgctg
attacaggag ctgggcatgg 240 aattgggaga ctgactgcct atgaatttgc
taaacttaaa agcaagctgg ttctctggga 300 tataaataag catggactgg
aggaaacagc tgccaaatgc aagggactgg gtgccaaggt 360 tcataccttt
gtggtagact gcagcaaccg agaagatatt tacagctctg caaagaaggt 420
gaaggcagaa attggagatg ttagtatttt agtaaataat gctggtgtag tctatacatc
480 agatttgttt gctacacaag atcctcagat tgaaaagact tttgaagtta
atgtacttgc 540 acatttctgg actacaaagg catttcttcc tgcaatgacg
aagaataacc atggccatat 600 tgtcactgtg gcttcggcag ctggacatgt
ctcggtcccc ttcttactgg cttactgttc 660 aagcaagttt gctgctgttg
gatttcataa aactttgaca gatgaactgg ctgccttaca 720 aataactgga
gtcaaaacaa catgtctgtg tcctaatttc gtaaacactg gcttcatcaa 780
aaatccaagt acaagtttgg gacccactct ggaacctgag gaagtggtaa acaggctgat
840 gcatgggatt ctgactgagc agaagatgat ttttattcca tcttctatag
cttttttaac 900 aacattggaa aggatccttc ctgagcgttt cctggcagtt
ttaaaacgaa aaatcagtgt 960 taagtttgat gcagttattg gatataaaat
gaaagcgcaa taagcaccta gttttctgaa 1020 aactgattta ccaggtttag
gttgatgtca tctaatagtg ccagaatttt aatgtttgaa 1080 cttctgtttt
ttctaattat ccccatttct tcaatatcat ttttgaggct ttggcagtct 1140
tcatttacta ccacttgttc tttagccaaa agctgattac atatgatata aacagagaaa
1200 tacctttaga ggtgacttta aggaaaatga agaaaaagaa ccaaaatgac
tttattaaaa 1260 taatttccaa gattatttgt ggctcacctg aaggctttgc
aaaatttgta ccataaccgt 1320 ttatttaaca tatattttta tttttgattg
cacttaaatt ttgtataatt tgtgtttctt 1380 tttctgttct acataaaatc
agaaacttca agctctctaa ataaaatgaa ggactatatc 1440 tagtggtatt
tcacaatgaa tatcatgaac tctcaatggg taggtttcat cctacccatt 1500
gccactctgt ttcctgagag atacctcaca ttccaatgcc aaacatttct gcacagggaa
1560 gctagaggtg gatacacgtg ttgcaagtat aaaagcatca ctgggattta
aggagaattg 1620 agagaatgta cccacaaatg gcagcaataa taaatggatc
acacttaaaa aaaaaaaaaa 1680 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1740 aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa a 1771 159 300 PRT Homo sapiens 159 Met Lys Phe Leu Leu
Asp Ile Leu Leu Leu Leu Pro Leu Leu Ile Val 1 5 10 15 Cys Ser Leu
Glu Ser Phe Val Lys Leu Phe Ile Pro Lys Arg Arg Lys 20 25 30 Ser
Val Thr Gly Glu Ile Val Leu Ile Thr Gly Ala Gly His Gly Ile 35 40
45 Gly Arg Leu Thr Ala Tyr Glu Phe Ala Lys Leu Lys Ser Lys Leu Val
50 55 60 Leu Trp Asp Ile Asn Lys His Gly Leu Glu Glu Thr Ala Ala
Lys Cys 65 70 75 80 Lys Gly Leu Gly Ala Lys Val His Thr Phe Val Val
Asp Cys Ser Asn 85 90 95 Arg Glu Asp Ile Tyr Ser Ser Ala Lys Lys
Val Lys Ala Glu Ile Gly 100 105 110 Asp Val Ser Ile Leu Val Asn Asn
Ala Gly Val Val Tyr Thr Ser Asp 115 120 125 Leu Phe Ala Thr Gln Asp
Pro Gln Ile Glu Lys Thr Phe Glu Val Asn 130 135 140 Val Leu Ala His
Phe Trp Thr Thr Lys Ala Phe Leu Pro Ala Met Thr 145 150 155 160 Lys
Asn Asn His Gly His Ile Val Thr Val Ala Ser Ala Ala Gly His 165 170
175 Val Ser Val Pro Phe Leu Leu Ala Tyr Cys Ser Ser Lys Phe Ala Ala
180 185 190 Val Gly Phe His Lys Thr Leu Thr Asp Glu Leu Ala Ala Leu
Gln Ile 195 200 205 Thr Gly Val Lys Thr Thr Cys Leu Cys Pro Asn Phe
Val Asn Thr Gly 210 215 220 Phe Ile Lys Asn Pro Ser Thr Ser Leu Gly
Pro Thr Leu Glu Pro Glu 225 230 235 240 Glu Val Val Asn Arg Leu Met
His Gly Ile Leu Thr Glu Gln Lys Met 245 250 255 Ile Phe Ile Pro Ser
Ser Ile Ala Phe Leu Thr Thr Leu Glu Arg Ile 260 265 270 Leu Pro Glu
Arg Phe Leu Ala Val Leu Lys Arg Lys Ile Ser Val Lys 275 280 285 Phe
Asp Ala Val Ile Gly Tyr Lys Met Lys Ala Gln 290 295 300 160 23 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 160 ggtgaaggca gaaattggag atg 23 161 24 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 161 atcccatgca tcagcctgtt tacc 24 162 48 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 162 gctggtgtag tctatacatc agatttgttt
gctacacaag atcctcag 48 163 2076 DNA Homo sapiens 163 cccacgcgtc
cgcggacgcg tgggtcgact agttctagat cgcgagcggc cgcccgcggc 60
tcagggagga gcaccgactg cgccgcaccc tgagagatgg ttggtgccat gtggaaggtg
120 attgtttcgc tggtcctgtt gatgcctggc ccctgtgatg ggctgtttcg
ctccctatac 180 agaagtgttt ccatgccacc taagggagac tcaggacagc
cattatttct caccccttac 240 attgaagctg ggaagatcca aaaaggaaga
gaattgagtt tggtcggccc tttcccagga 300 ctgaacatga agagttatgc
cggcttcctc accgtgaata agacttacaa cagcaacctc 360 ttcttctggt
tcttcccagc tcagatacag ccagaagatg ccccagtagt tctctggcta 420
cagggtgggc cgggaggttc atccatgttt ggactctttg tggaacatgg gccttatgtt
480 gtcacaagta acatgacctt gcgtgacaga gacttcccct ggaccacaac
gctctccatg 540 ctttacattg acaatccagt gggcacaggc ttcagtttta
ctgatgatac ccacggatat 600 gcagtcaatg aggacgatgt agcacgggat
ttatacagtg cactaattca gtttttccag 660 atatttcctg aatataaaaa
taatgacttt tatgtcactg gggagtctta tgcagggaaa 720 tatgtgccag
ccattgcaca cctcatccat tccctcaacc ctgtgagaga ggtgaagatc 780
aacctgaacg gaattgctat tggagatgga tattctgatc ccgaatcaat tatagggggc
840 tatgcagaat tcctgtacca aattggcttg ttggatgaga agcaaaaaaa
gtacttccag 900 aagcagtgcc atgaatgcat agaacacatc aggaagcaga
actggtttga ggcctttgaa 960 atactggata aactactaga tggcgactta
acaagtgatc cttcttactt ccagaatgtt 1020 acaggatgta gtaattacta
taactttttg cggtgcacgg aacctgagga tcagctttac 1080 tatgtgaaat
ttttgtcact cccagaggtg agacaagcca tccacgtggg gaatcagact 1140
tttaatgatg gaactatagt tgaaaagtac ttgcgagaag atacagtaca gtcagttaag
1200 ccatggttaa ctgaaatcat gaataattat aaggttctga tctacaatgg
ccaactggac 1260 atcatcgtgg cagctgccct gacagagcgc tccttgatgg
gcatggactg gaaaggatcc 1320 caggaataca agaaggcaga aaaaaaagtt
tggaagatct ttaaatctga cagtgaagtg 1380 gctggttaca tccggcaagc
gggtgacttc catcaggtaa ttattcgagg tggaggacat 1440 attttaccct
atgaccagcc tctgagagct tttgacatga ttaatcgatt catttatgga 1500
aaaggatggg atccttatgt tggataaact accttcccaa aagagaacat cagaggtttt
1560 cattgctgaa aagaaaatcg taaaaacaga aaatgtcata ggaataaaaa
aattatcttt 1620 tcatatctgc aagatttttt tcatcaataa aaattatcct
tgaaacaagt gagcttttgt 1680 ttttgggggg agatgtttac tacaaaatta
acatgagtac atgagtaaga attacattat 1740 ttaacttaaa ggatgaaagg
tatggatgat gtgacactga gacaagatgt ataaatgaaa 1800 ttttagggtc
ttgaatagga agttttaatt tcttctaaga gtaagtgaaa agtgcagttg 1860
taacaaacaa agctgtaaca tctttttctg ccaataacag aagtttggca tgccgtgaag
1920 gtgtttggaa atattattgg ataagaatag ctcaattatc ccaaataaat
ggatgaagct 1980 ataatagttt tggggaaaag attctcaaat gtataaagtc
ttagaacaaa agaattcttt 2040 gaaataaaaa tattatatat aaaagtaaaa aaaaaa
2076 164 476 PRT Homo sapiens 164 Met Val Gly Ala Met Trp Lys Val
Ile Val Ser Leu Val Leu Leu Met 1 5 10 15 Pro Gly Pro Cys Asp Gly
Leu Phe Arg Ser Leu Tyr Arg Ser Val Ser 20 25 30 Met Pro Pro Lys
Gly Asp Ser Gly Gln Pro Leu Phe Leu Thr Pro Tyr 35 40 45 Ile Glu
Ala Gly Lys Ile Gln Lys Gly Arg Glu Leu Ser Leu Val Gly 50 55 60
Pro Phe Pro Gly Leu Asn Met Lys Ser Tyr Ala Gly Phe Leu Thr Val 65
70 75 80 Asn Lys Thr Tyr Asn Ser Asn Leu Phe Phe Trp Phe Phe Pro
Ala Gln 85 90 95 Ile Gln Pro Glu Asp Ala Pro Val Val Leu Trp Leu
Gln Gly Gly Pro 100 105 110 Gly Gly Ser Ser Met Phe Gly Leu Phe Val
Glu His Gly Pro Tyr Val 115 120 125 Val Thr Ser Asn Met Thr Leu Arg
Asp Arg Asp Phe Pro Trp Thr Thr 130 135 140 Thr Leu Ser Met Leu Tyr
Ile Asp Asn Pro Val Gly Thr Gly Phe Ser 145 150 155 160 Phe Thr Asp
Asp Thr His Gly Tyr Ala Val Asn Glu Asp Asp Val Ala 165 170 175 Arg
Asp Leu Tyr Ser Ala Leu Ile Gln Phe Phe Gln Ile Phe Pro Glu 180 185
190 Tyr Lys Asn Asn Asp Phe Tyr Val Thr Gly Glu Ser Tyr Ala Gly Lys
195 200 205 Tyr Val Pro Ala Ile Ala His Leu Ile His Ser Leu Asn Pro
Val Arg 210 215 220 Glu Val Lys Ile Asn Leu Asn Gly Ile Ala Ile Gly
Asp Gly Tyr Ser 225 230 235 240 Asp Pro Glu Ser Ile Ile Gly Gly Tyr
Ala Glu Phe Leu Tyr Gln Ile 245 250 255 Gly Leu Leu Asp Glu Lys Gln
Lys Lys Tyr Phe Gln Lys Gln Cys His 260 265 270 Glu Cys Ile Glu His
Ile Arg Lys Gln Asn Trp Phe Glu Ala Phe Glu 275 280 285 Ile Leu Asp
Lys Leu Leu Asp Gly Asp Leu Thr Ser Asp Pro Ser Tyr 290 295 300 Phe
Gln Asn Val Thr Gly Cys Ser Asn Tyr Tyr Asn Phe Leu Arg Cys 305 310
315 320 Thr Glu Pro Glu Asp Gln Leu Tyr Tyr Val Lys Phe Leu Ser Leu
Pro 325 330 335 Glu Val Arg Gln Ala Ile His Val Gly Asn Gln Thr Phe
Asn Asp Gly 340 345 350 Thr Ile Val Glu Lys Tyr Leu Arg Glu Asp Thr
Val Gln Ser Val Lys 355 360 365 Pro Trp Leu Thr Glu Ile Met Asn Asn
Tyr Lys Val Leu Ile Tyr Asn 370 375 380 Gly Gln Leu Asp Ile Ile Val
Ala Ala Ala Leu Thr Glu Arg Ser Leu 385 390 395 400 Met Gly Met Asp
Trp Lys Gly Ser Gln Glu Tyr Lys Lys Ala Glu Lys 405 410 415 Lys Val
Trp Lys Ile Phe Lys Ser Asp Ser Glu Val Ala Gly Tyr Ile 420 425 430
Arg Gln Ala Gly Asp Phe His Gln Val Ile Ile Arg Gly Gly Gly His 435
440 445 Ile Leu Pro Tyr Asp Gln Pro Leu Arg Ala Phe Asp Met Ile Asn
Arg 450 455 460 Phe Ile Tyr Gly Lys Gly Trp Asp Pro Tyr Val Gly 465
470 475 165 24 DNA Artificial Sequence Description of Artificial
Sequence Synthetic oligonucleotide probe 165 ttccatgcca cctaagggag
actc 24 166 24 DNA Artificial Sequence Description of Artificial
Sequence Synthetic oligonucleotide probe
166 tggatgaggt gtgcaatggc tggc 24 167 24 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide probe
167 agctctcaga ggctggtcat aggg 24 168 50 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide probe
168 gtcggccctt tcccaggact gaacatgaag agttatgccg gcttcctcac 50 169
2477 DNA Homo sapiens 169 cgagggcttt tccggctccg gaatggcaca
tgtgggaatc ccagtcttgt tggctacaac 60 atttttccct ttcctaacaa
gttctaacag ctgttctaac agctagtgat caggggttct 120 tcttgctgga
gaagaaaggg ctgagggcag agcagggcac tctcactcag ggtgaccagc 180
tccttgcctc tctgtggata acagagcatg agaaagtgaa gagatgcagc ggagtgaggt
240 gatggaagtc taaaatagga aggaattttg tgtgcaatat cagactctgg
gagcagttga 300 cctggagagc ctgggggagg gcctgcctaa caagctttca
aaaaacagga gcgacttcca 360 ctgggctggg ataagacgtg ccggtaggat
agggaagact gggtttagtc ctaatatcaa 420 attgactggc tgggtgaact
tcaacagcct tttaacctct ctgggagatg aaaacgatgg 480 cttaaggggc
cagaaataga gatgctttgt aaaataaaat tttaaaaaaa gcaagtattt 540
tatagcataa aggctagaga ccaaaataga taacaggatt ccctgaacat tcctaagagg
600 gagaaagtat gttaaaaata gaaaaaccaa aatgcagaag gaggagactc
acagagctaa 660 accaggatgg ggaccctggg tcaggccagc ctctttgctc
ctcccggaaa ttatttttgg 720 tctgaccact ctgccttgtg ttttgcagaa
tcatgtgagg gccaaccggg gaaggtggag 780 cagatgagca cacacaggag
ccgtctcctc accgccgccc ctctcagcat ggaacagagg 840 cagccctggc
cccgggccct ggaggtggac agccgctctg tggtcctgct ctcagtggtc 900
tgggtgctgc tggccccccc agcagccggc atgcctcagt tcagcacctt ccactctgag
960 aatcgtgact ggaccttcaa ccacttgacc gtccaccaag ggacgggggc
cgtctatgtg 1020 ggggccatca accgggtcta taagctgaca ggcaacctga
ccatccaggt ggctcataag 1080 acagggccag aagaggacaa caagtctcgt
tacccgcccc tcatcgtgca gccctgcagc 1140 gaagtgctca ccctcaccaa
caatgtcaac aagctgctca tcattgacta ctctgagaac 1200 cgcctgctgg
cctgtgggag cctctaccag ggggtctgca agctgctgcg gctggatgac 1260
ctcttcatcc tggtggagcc atcccacaag aaggagcact acctgtccag tgtcaacaag
1320 acgggcacca tgtacggggt gattgtgcgc tctgagggtg aggatggcaa
gctcttcatc 1380 ggcacggctg tggatgggaa gcaggattac ttcccgaccc
tgtccagccg gaagctgccc 1440 cgagaccctg agtcctcagc catgctcgac
tatgagctac acagcgattt tgtctcctct 1500 ctcatcaaga tcccttcaga
caccctggcc ctggtctccc actttgacat cttctacatc 1560 tacggctttg
ctagtggggg ctttgtctac tttctcactg tccagcccga gacccctgag 1620
ggtgtggcca tcaactccgc tggagacctc ttctacacct cacgcatcgt gcggctctgc
1680 aaggatgacc ccaagttcca ctcatacgtg tccctgccct tcggctgcac
ccgggccggg 1740 gtggaatacc gcctcctgca ggctgcttac ctggccaagc
ctggggactc actggcccag 1800 gccttcaata tcaccagcca ggacgatgta
ctctttgcca tcttctccaa agggcagaag 1860 cagtatcacc acccgcccga
tgactctgcc ctgtgtgcct tccctatccg ggccatcaac 1920 ttgcagatca
aggagcgcct gcagtcctgc taccagggcg agggcaacct ggagctcaac 1980
tggctgctgg ggaaggacgt ccagtgcacg aaggcgcctg tccccatcga tgataacttc
2040 tgtggactgg acatcaacca gcccctggga ggctcaactc cagtggaggg
cctgaccctg 2100 tacaccacca gcagggaccg catgacctct gtggcctcct
acgtttacaa cggctacagc 2160 gtggtttttg tggggactaa gagtggcaag
ctgaaaaagg taagagtcta tgagttcaga 2220 tgctccaatg ccattcacct
cctcagcaaa gagtccctct tggaaggtag ctattggtgg 2280 agatttaact
ataggcaact ttattttctt ggggaacaaa ggtgaaatgg ggaggtaaga 2340
aggggttaat tttgtgactt agcttctagc tacttcctcc agccatcagt cattgggtat
2400 gtaaggaatg caagcgtatt tcaatatttc ccaaacttta agaaaaaact
ttaagaaggt 2460 acatctgcaa aagcaaa 2477 170 552 PRT Homo sapiens
170 Met Gly Thr Leu Gly Gln Ala Ser Leu Phe Ala Pro Pro Gly Asn Tyr
1 5 10 15 Phe Trp Ser Asp His Ser Ala Leu Cys Phe Ala Glu Ser Cys
Glu Gly 20 25 30 Gln Pro Gly Lys Val Glu Gln Met Ser Thr His Arg
Ser Arg Leu Leu 35 40 45 Thr Ala Ala Pro Leu Ser Met Glu Gln Arg
Gln Pro Trp Pro Arg Ala 50 55 60 Leu Glu Val Asp Ser Arg Ser Val
Val Leu Leu Ser Val Val Trp Val 65 70 75 80 Leu Leu Ala Pro Pro Ala
Ala Gly Met Pro Gln Phe Ser Thr Phe His 85 90 95 Ser Glu Asn Arg
Asp Trp Thr Phe Asn His Leu Thr Val His Gln Gly 100 105 110 Thr Gly
Ala Val Tyr Val Gly Ala Ile Asn Arg Val Tyr Lys Leu Thr 115 120 125
Gly Asn Leu Thr Ile Gln Val Ala His Lys Thr Gly Pro Glu Glu Asp 130
135 140 Asn Lys Ser Arg Tyr Pro Pro Leu Ile Val Gln Pro Cys Ser Glu
Val 145 150 155 160 Leu Thr Leu Thr Asn Asn Val Asn Lys Leu Leu Ile
Ile Asp Tyr Ser 165 170 175 Glu Asn Arg Leu Leu Ala Cys Gly Ser Leu
Tyr Gln Gly Val Cys Lys 180 185 190 Leu Leu Arg Leu Asp Asp Leu Phe
Ile Leu Val Glu Pro Ser His Lys 195 200 205 Lys Glu His Tyr Leu Ser
Ser Val Asn Lys Thr Gly Thr Met Tyr Gly 210 215 220 Val Ile Val Arg
Ser Glu Gly Glu Asp Gly Lys Leu Phe Ile Gly Thr 225 230 235 240 Ala
Val Asp Gly Lys Gln Asp Tyr Phe Pro Thr Leu Ser Ser Arg Lys 245 250
255 Leu Pro Arg Asp Pro Glu Ser Ser Ala Met Leu Asp Tyr Glu Leu His
260 265 270 Ser Asp Phe Val Ser Ser Leu Ile Lys Ile Pro Ser Asp Thr
Leu Ala 275 280 285 Leu Val Ser His Phe Asp Ile Phe Tyr Ile Tyr Gly
Phe Ala Ser Gly 290 295 300 Gly Phe Val Tyr Phe Leu Thr Val Gln Pro
Glu Thr Pro Glu Gly Val 305 310 315 320 Ala Ile Asn Ser Ala Gly Asp
Leu Phe Tyr Thr Ser Arg Ile Val Arg 325 330 335 Leu Cys Lys Asp Asp
Pro Lys Phe His Ser Tyr Val Ser Leu Pro Phe 340 345 350 Gly Cys Thr
Arg Ala Gly Val Glu Tyr Arg Leu Leu Gln Ala Ala Tyr 355 360 365 Leu
Ala Lys Pro Gly Asp Ser Leu Ala Gln Ala Phe Asn Ile Thr Ser 370 375
380 Gln Asp Asp Val Leu Phe Ala Ile Phe Ser Lys Gly Gln Lys Gln Tyr
385 390 395 400 His His Pro Pro Asp Asp Ser Ala Leu Cys Ala Phe Pro
Ile Arg Ala 405 410 415 Ile Asn Leu Gln Ile Lys Glu Arg Leu Gln Ser
Cys Tyr Gln Gly Glu 420 425 430 Gly Asn Leu Glu Leu Asn Trp Leu Leu
Gly Lys Asp Val Gln Cys Thr 435 440 445 Lys Ala Pro Val Pro Ile Asp
Asp Asn Phe Cys Gly Leu Asp Ile Asn 450 455 460 Gln Pro Leu Gly Gly
Ser Thr Pro Val Glu Gly Leu Thr Leu Tyr Thr 465 470 475 480 Thr Ser
Arg Asp Arg Met Thr Ser Val Ala Ser Tyr Val Tyr Asn Gly 485 490 495
Tyr Ser Val Val Phe Val Gly Thr Lys Ser Gly Lys Leu Lys Lys Val 500
505 510 Arg Val Tyr Glu Phe Arg Cys Ser Asn Ala Ile His Leu Leu Ser
Lys 515 520 525 Glu Ser Leu Leu Glu Gly Ser Tyr Trp Trp Arg Phe Asn
Tyr Arg Gln 530 535 540 Leu Tyr Phe Leu Gly Glu Gln Arg 545 550 171
20 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide probe 171 tggaataccg cctcctgcag 20 172 24
DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide probe 172 cttctgccct ttggagaaga tggc 24
173 43 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide probe 173 ggactcactg gcccaggcct
tcaatatcac cagccaggac gat 43 174 3106 DNA Homo sapiens
modified_base (1683)..(1683) a, t, c or g 174 aggctcccgc gcgcggctga
gtgcggactg gagtgggaac ccgggtcccc gcgcttagag 60 aacacgcgat
gaccacgtgg agcctccggc ggaggccggc ccgcacgctg ggactcctgc 120
tgctggtcgt cttgggcttc ctggtgctcc gcaggctgga ctggagcacc ctggtccctc
180 tgcggctccg ccatcgacag ctggggctgc aggccaaggg ctggaacttc
atgctggagg 240 attccacctt ctggatcttc gggggctcca tccactattt
ccgtgtgccc agggagtact 300 ggagggaccg cctgctgaag atgaaggcct
gtggcttgaa caccctcacc acctatgttc 360 cgtggaacct gcatgagcca
gaaagaggca aatttgactt ctctgggaac ctggacctgg 420 aggccttcgt
cctgatggcc gcagagatcg ggctgtgggt gattctgcgt ccaggcccct 480
acatctgcag tgagatggac ctcgggggct tgcccagctg gctactccaa gaccctggca
540 tgaggctgag gacaacttac aagggcttca ccgaagcagt ggacctttat
tttgaccacc 600 tgatgtccag ggtggtgcca ctccagtaca agcgtggggg
acctatcatt gccgtgcagg 660 tggagaatga atatggttcc tataataaag
accccgcata catgccctac gtcaagaagg 720 cactggagga ccgtggcatt
gtggaactgc tcctgacttc agacaacaag gatgggctga 780 gcaaggggat
tgtccaggga gtcttggcca ccatcaactt gcagtcaaca cacgagctgc 840
agctactgac cacctttctc ttcaacgtcc aggggactca gcccaagatg gtgatggagt
900 actggacggg gtggtttgac tcgtggggag gccctcacaa tatcttggat
tcttctgagg 960 ttttgaaaac cgtgtctgcc attgtggacg ccggctcctc
catcaacctc tacatgttcc 1020 acggaggcac caactttggc ttcatgaatg
gagccatgca cttccatgac tacaagtcag 1080 atgtcaccag ctatgactat
gatgctgtgc tgacagaagc cggcgattac acggccaagt 1140 acatgaagct
tcgagacttc ttcggctcca tctcaggcat ccctctccct cccccacctg 1200
accttcttcc caagatgccg tatgagccct taacgccagt cttgtacctg tctctgtggg
1260 acgccctcaa gtacctgggg gagccaatca agtctgaaaa gcccatcaac
atggagaacc 1320 tgccagtcaa tgggggaaat ggacagtcct tcgggtacat
tctctatgag accagcatca 1380 cctcgtctgg catcctcagt ggccacgtgc
atgatcgggg gcaggtgttt gtgaacacag 1440 tatccatagg attcttggac
tacaagacaa cgaagattgc tgtccccctg atccagggtt 1500 acaccgtgct
gaggatcttg gtggagaatc gtgggcgagt caactatggg gagaatattg 1560
atgaccagcg caaaggctta attggaaatc tctatctgaa tgattcaccc ctgaaaaact
1620 tcagaatcta tagcctggat atgaagaaga gcttctttca gaggttcggc
ctggacaaat 1680 ggngttccct cccagaaaca cccacattac ctgctttctt
cttgggtagc ttgtccatca 1740 gctccacgcc ttgtgacacc tttctgaagc
tggagggctg ggagaagggg gttgtattca 1800 tcaatggcca gaaccttgga
cgttactgga acattggacc ccagaagacg ctttacctcc 1860 caggtccctg
gttgagcagc ggaatcaacc aggtcatcgt ttttgaggag acgatggcgg 1920
gccctgcatt acagttcacg gaaacccccc acctgggcag gaaccagtac attaagtgag
1980 cggtggcacc ccctcctgct ggtgccagtg ggagactgcc gcctcctctt
gacctgaagc 2040 ctggtggctg ctgccccacc cctcactgca aaagcatctc
cttaagtagc aacctcaggg 2100 actgggggct acagtctgcc cctgtctcag
ctcaaaaccc taagcctgca gggaaaggtg 2160 ggatggctct gggcctggct
ttgttgatga tggctttcct acagccctgc tcttgtgccg 2220 aggctgtcgg
gctgtctcta gggtgggagc agctaatcag atcgcccagc ctttggccct 2280
cagaaaaagt gctgaaacgt gcccttgcac cggacgtcac agccctgcga gcatctgctg
2340 gactcaggcg tgctctttgc tggttcctgg gaggcttggc cacatccctc
atggccccat 2400 tttatccccg aaatcctggg tgtgtcacca gtgtagaggg
tggggaaggg gtgtctcacc 2460 tgagctgact ttgttcttcc ttcacaacct
tctgagcctt ctttgggatt ctggaaggaa 2520 ctcggcgtga gaaacatgtg
acttcccctt tcccttccca ctcgctgctt cccacagggt 2580 gacaggctgg
gctggagaaa cagaaatcct caccctgcgt cttcccaagt tagcaggtgt 2640
ctctggtgtt cagtgaggag gacatgtgag tcctggcaga agccatggcc catgtctgca
2700 catccaggga ggaggacaga aggcccagct cacatgtgag tcctggcaga
agccatggcc 2760 catgtctgca catccaggga ggaggacaga aggcccagct
cacatgtgag tcctggcaga 2820 agccatggcc catgtctgca catccaggga
ggaggacaga aggcccagct cacatgtgag 2880 tcctggcaga agccatggcc
catgtctgca catccaggga ggaggacaga aggcccagct 2940 cagtggcccc
cgctccccac cccccacgcc cgaacagcag gggcagagca gccctccttc 3000
gaagtgtgtc caagtccgca tttgagcctt gttctggggc ccagcccaac acctggcttg
3060 ggctcactgt cctgagttgc agtaaagcta taaccttgaa tcacaa 3106 175
636 PRT Homo sapiens MOD_RES (539) Any amino acid 175 Met Thr Thr
Trp Ser Leu Arg Arg Arg Pro Ala Arg Thr Leu Gly Leu 1 5 10 15 Leu
Leu Leu Val Val Leu Gly Phe Leu Val Leu Arg Arg Leu Asp Trp 20 25
30 Ser Thr Leu Val Pro Leu Arg Leu Arg His Arg Gln Leu Gly Leu Gln
35 40 45 Ala Lys Gly Trp Asn Phe Met Leu Glu Asp Ser Thr Phe Trp
Ile Phe 50 55 60 Gly Gly Ser Ile His Tyr Phe Arg Val Pro Arg Glu
Tyr Trp Arg Asp 65 70 75 80 Arg Leu Leu Lys Met Lys Ala Cys Gly Leu
Asn Thr Leu Thr Thr Tyr 85 90 95 Val Pro Trp Asn Leu His Glu Pro
Glu Arg Gly Lys Phe Asp Phe Ser 100 105 110 Gly Asn Leu Asp Leu Glu
Ala Phe Val Leu Met Ala Ala Glu Ile Gly 115 120 125 Leu Trp Val Ile
Leu Arg Pro Gly Pro Tyr Ile Cys Ser Glu Met Asp 130 135 140 Leu Gly
Gly Leu Pro Ser Trp Leu Leu Gln Asp Pro Gly Met Arg Leu 145 150 155
160 Arg Thr Thr Tyr Lys Gly Phe Thr Glu Ala Val Asp Leu Tyr Phe Asp
165 170 175 His Leu Met Ser Arg Val Val Pro Leu Gln Tyr Lys Arg Gly
Gly Pro 180 185 190 Ile Ile Ala Val Gln Val Glu Asn Glu Tyr Gly Ser
Tyr Asn Lys Asp 195 200 205 Pro Ala Tyr Met Pro Tyr Val Lys Lys Ala
Leu Glu Asp Arg Gly Ile 210 215 220 Val Glu Leu Leu Leu Thr Ser Asp
Asn Lys Asp Gly Leu Ser Lys Gly 225 230 235 240 Ile Val Gln Gly Val
Leu Ala Thr Ile Asn Leu Gln Ser Thr His Glu 245 250 255 Leu Gln Leu
Leu Thr Thr Phe Leu Phe Asn Val Gln Gly Thr Gln Pro 260 265 270 Lys
Met Val Met Glu Tyr Trp Thr Gly Trp Phe Asp Ser Trp Gly Gly 275 280
285 Pro His Asn Ile Leu Asp Ser Ser Glu Val Leu Lys Thr Val Ser Ala
290 295 300 Ile Val Asp Ala Gly Ser Ser Ile Asn Leu Tyr Met Phe His
Gly Gly 305 310 315 320 Thr Asn Phe Gly Phe Met Asn Gly Ala Met His
Phe His Asp Tyr Lys 325 330 335 Ser Asp Val Thr Ser Tyr Asp Tyr Asp
Ala Val Leu Thr Glu Ala Gly 340 345 350 Asp Tyr Thr Ala Lys Tyr Met
Lys Leu Arg Asp Phe Phe Gly Ser Ile 355 360 365 Ser Gly Ile Pro Leu
Pro Pro Pro Pro Asp Leu Leu Pro Lys Met Pro 370 375 380 Tyr Glu Pro
Leu Thr Pro Val Leu Tyr Leu Ser Leu Trp Asp Ala Leu 385 390 395 400
Lys Tyr Leu Gly Glu Pro Ile Lys Ser Glu Lys Pro Ile Asn Met Glu 405
410 415 Asn Leu Pro Val Asn Gly Gly Asn Gly Gln Ser Phe Gly Tyr Ile
Leu 420 425 430 Tyr Glu Thr Ser Ile Thr Ser Ser Gly Ile Leu Ser Gly
His Val His 435 440 445 Asp Arg Gly Gln Val Phe Val Asn Thr Val Ser
Ile Gly Phe Leu Asp 450 455 460 Tyr Lys Thr Thr Lys Ile Ala Val Pro
Leu Ile Gln Gly Tyr Thr Val 465 470 475 480 Leu Arg Ile Leu Val Glu
Asn Arg Gly Arg Val Asn Tyr Gly Glu Asn 485 490 495 Ile Asp Asp Gln
Arg Lys Gly Leu Ile Gly Asn Leu Tyr Leu Asn Asp 500 505 510 Ser Pro
Leu Lys Asn Phe Arg Ile Tyr Ser Leu Asp Met Lys Lys Ser 515 520 525
Phe Phe Gln Arg Phe Gly Leu Asp Lys Trp Xaa Ser Leu Pro Glu Thr 530
535 540 Pro Thr Leu Pro Ala Phe Phe Leu Gly Ser Leu Ser Ile Ser Ser
Thr 545 550 555 560 Pro Cys Asp Thr Phe Leu Lys Leu Glu Gly Trp Glu
Lys Gly Val Val 565 570 575 Phe Ile Asn Gly Gln Asn Leu Gly Arg Tyr
Trp Asn Ile Gly Pro Gln 580 585 590 Lys Thr Leu Tyr Leu Pro Gly Pro
Trp Leu Ser Ser Gly Ile Asn Gln 595 600 605 Val Ile Val Phe Glu Glu
Thr Met Ala Gly Pro Ala Leu Gln Phe Thr 610 615 620 Glu Thr Pro His
Leu Gly Arg Asn Gln Tyr Ile Lys 625 630 635 176 2505 DNA Homo
sapiens 176 ggggacgcgg agctgagagg ctccgggcta gctaggtgta ggggtggacg
ggtcccagga 60 ccctggtgag ggttctctac ttggccttcg gtgggggtca
agacgcaggc acctacgcca 120 aaggggagca aagccgggct cggcccgagg
cccccaggac ctccatctcc caatgttgga 180 ggaatccgac acgtgacggt
ctgtccgccg tctcagacta gaggagcgct gtaaacgcca 240 tggctcccaa
gaagctgtcc tgccttcgtt ccctgctgct gccgctcagc ctgacgctac 300
tgctgcccca ggcagacact cggtcgttcg tagtggatag gggtcatgac cggtttctcc
360 tagacggggc cccgttccgc tatgtgtctg gcagcctgca ctactttcgg
gtaccgcggg 420 tgctttgggc cgaccggctt ttgaagatgc gatggagcgg
cctcaacgcc atacagtttt 480 atgtgccctg gaactaccac gagccacagc
ctggggtcta taactttaat ggcagccggg 540 acctcattgc ctttctgaat
gaggcagctc tagcgaacct gttggtcata ctgagaccag 600 gaccttacat
ctgtgcagag tgggagatgg ggggtctccc atcctggttg cttcgaaaac 660
ctgaaattca tctaagaacc tcagatccag acttccttgc cgcagtggac tcctggttca
720 aggtcttgct gcccaagata tatccatggc tttatcacaa tgggggcaac
atcattagca 780 ttcaggtgga gaatgaatat ggtagctaca gagcctgtga
cttcagctac atgaggcact 840 tggctgggct cttccgtgca ctgctaggag
aaaagatctt gctcttcacc acagatgggc 900 ctgaaggact caagtgtggc
tccctccggg gactctatac cactgtagat tttggcccag 960 ctgacaacat
gaccaaaatc tttaccctgc ttcggaagta tgaaccccat gggccattgg 1020
taaactctga gtactacaca ggctggctgg attactgggg ccagaatcac tccacacggt
1080 ctgtgtcagc tgtaaccaaa ggactagaga acatgctcaa gttgggagcc
agtgtgaaca 1140 tgtacatgtt ccatggaggt accaactttg gatattggaa
tggtgccgat aagaagggac 1200 gcttccttcc gattactacc agctatgact
atgatgcacc tatatctgaa gcaggggacc 1260 ccacacctaa gctttttgct
cttcgagatg tcatcagcaa gttccaggaa gttcctttgg 1320 gacctttacc
tcccccgagc cccaagatga tgcttggacc tgtgactctg cacctggttg 1380
ggcatttact ggctttccta gacttgcttt gcccccgtgg gcccattcat tcaatcttgc
1440 caatgacctt tgaggctgtc aagcaggacc atggcttcat gttgtaccga
acctatatga 1500 cccataccat ttttgagcca acaccattct gggtgccaaa
taatggagtc catgaccgtg 1560 cctatgtgat ggtggatggg gtgttccagg
gtgttgtgga gcgaaatatg agagacaaac 1620 tatttttgac ggggaaactg
gggtccaaac tggatatctt ggtggagaac atggggaggc 1680 tcagctttgg
gtctaacagc agtgacttca agggcctgtt gaagccacca attctggggc 1740
aaacaatcct tacccagtgg atgatgttcc ctctgaaaat tgataacctt gtgaagtggt
1800 ggtttcccct ccagttgcca aaatggccat atcctcaagc tccttctggc
cccacattct 1860 actccaaaac atttccaatt ttaggctcag ttggggacac
atttctatat ctacctggat 1920 ggaccaaggg ccaagtctgg atcaatgggt
ttaacttggg ccggtactgg acaaagcagg 1980 ggccacaaca gaccctctac
gtgccaagat tcctgctgtt tcctagggga gccctcaaca 2040 aaattacatt
gctggaacta gaagatgtac ctctccagcc ccaagtccaa tttttggata 2100
agcctatcct caatagcact agtactttgc acaggacaca tatcaattcc ctttcagctg
2160 atacactgag tgcctctgaa ccaatggagt taagtgggca ctgaaaggta
ggccgggcat 2220 ggtggctcat gcctgtaatc ccagcacttt gggaggctga
gacgggtgga ttacctgagg 2280 tcaggacttc aagaccagcc tggccaacat
ggtgaaaccc cgtctccact aaaaatacaa 2340 aaattagccg ggcgtgatgg
tgggcacctc taatcccagc tacttgggag gctgagggca 2400 ggagaattgc
ttgaatccag gaggcagagg ttgcagtgag tggaggttgt accactgcac 2460
tccagcctgg ctgacagtga gacactccat ctcaaaaaaa aaaaa 2505 177 654 PRT
Homo sapiens 177 Met Ala Pro Lys Lys Leu Ser Cys Leu Arg Ser Leu
Leu Leu Pro Leu 1 5 10 15 Ser Leu Thr Leu Leu Leu Pro Gln Ala Asp
Thr Arg Ser Phe Val Val 20 25 30 Asp Arg Gly His Asp Arg Phe Leu
Leu Asp Gly Ala Pro Phe Arg Tyr 35 40 45 Val Ser Gly Ser Leu His
Tyr Phe Arg Val Pro Arg Val Leu Trp Ala 50 55 60 Asp Arg Leu Leu
Lys Met Arg Trp Ser Gly Leu Asn Ala Ile Gln Phe 65 70 75 80 Tyr Val
Pro Trp Asn Tyr His Glu Pro Gln Pro Gly Val Tyr Asn Phe 85 90 95
Asn Gly Ser Arg Asp Leu Ile Ala Phe Leu Asn Glu Ala Ala Leu Ala 100
105 110 Asn Leu Leu Val Ile Leu Arg Pro Gly Pro Tyr Ile Cys Ala Glu
Trp 115 120 125 Glu Met Gly Gly Leu Pro Ser Trp Leu Leu Arg Lys Pro
Glu Ile His 130 135 140 Leu Arg Thr Ser Asp Pro Asp Phe Leu Ala Ala
Val Asp Ser Trp Phe 145 150 155 160 Lys Val Leu Leu Pro Lys Ile Tyr
Pro Trp Leu Tyr His Asn Gly Gly 165 170 175 Asn Ile Ile Ser Ile Gln
Val Glu Asn Glu Tyr Gly Ser Tyr Arg Ala 180 185 190 Cys Asp Phe Ser
Tyr Met Arg His Leu Ala Gly Leu Phe Arg Ala Leu 195 200 205 Leu Gly
Glu Lys Ile Leu Leu Phe Thr Thr Asp Gly Pro Glu Gly Leu 210 215 220
Lys Cys Gly Ser Leu Arg Gly Leu Tyr Thr Thr Val Asp Phe Gly Pro 225
230 235 240 Ala Asp Asn Met Thr Lys Ile Phe Thr Leu Leu Arg Lys Tyr
Glu Pro 245 250 255 His Gly Pro Leu Val Asn Ser Glu Tyr Tyr Thr Gly
Trp Leu Asp Tyr 260 265 270 Trp Gly Gln Asn His Ser Thr Arg Ser Val
Ser Ala Val Thr Lys Gly 275 280 285 Leu Glu Asn Met Leu Lys Leu Gly
Ala Ser Val Asn Met Tyr Met Phe 290 295 300 His Gly Gly Thr Asn Phe
Gly Tyr Trp Asn Gly Ala Asp Lys Lys Gly 305 310 315 320 Arg Phe Leu
Pro Ile Thr Thr Ser Tyr Asp Tyr Asp Ala Pro Ile Ser 325 330 335 Glu
Ala Gly Asp Pro Thr Pro Lys Leu Phe Ala Leu Arg Asp Val Ile 340 345
350 Ser Lys Phe Gln Glu Val Pro Leu Gly Pro Leu Pro Pro Pro Ser Pro
355 360 365 Lys Met Met Leu Gly Pro Val Thr Leu His Leu Val Gly His
Leu Leu 370 375 380 Ala Phe Leu Asp Leu Leu Cys Pro Arg Gly Pro Ile
His Ser Ile Leu 385 390 395 400 Pro Met Thr Phe Glu Ala Val Lys Gln
Asp His Gly Phe Met Leu Tyr 405 410 415 Arg Thr Tyr Met Thr His Thr
Ile Phe Glu Pro Thr Pro Phe Trp Val 420 425 430 Pro Asn Asn Gly Val
His Asp Arg Ala Tyr Val Met Val Asp Gly Val 435 440 445 Phe Gln Gly
Val Val Glu Arg Asn Met Arg Asp Lys Leu Phe Leu Thr 450 455 460 Gly
Lys Leu Gly Ser Lys Leu Asp Ile Leu Val Glu Asn Met Gly Arg 465 470
475 480 Leu Ser Phe Gly Ser Asn Ser Ser Asp Phe Lys Gly Leu Leu Lys
Pro 485 490 495 Pro Ile Leu Gly Gln Thr Ile Leu Thr Gln Trp Met Met
Phe Pro Leu 500 505 510 Lys Ile Asp Asn Leu Val Lys Trp Trp Phe Pro
Leu Gln Leu Pro Lys 515 520 525 Trp Pro Tyr Pro Gln Ala Pro Ser Gly
Pro Thr Phe Tyr Ser Lys Thr 530 535 540 Phe Pro Ile Leu Gly Ser Val
Gly Asp Thr Phe Leu Tyr Leu Pro Gly 545 550 555 560 Trp Thr Lys Gly
Gln Val Trp Ile Asn Gly Phe Asn Leu Gly Arg Tyr 565 570 575 Trp Thr
Lys Gln Gly Pro Gln Gln Thr Leu Tyr Val Pro Arg Phe Leu 580 585 590
Leu Phe Pro Arg Gly Ala Leu Asn Lys Ile Thr Leu Leu Glu Leu Glu 595
600 605 Asp Val Pro Leu Gln Pro Gln Val Gln Phe Leu Asp Lys Pro Ile
Leu 610 615 620 Asn Ser Thr Ser Thr Leu His Arg Thr His Ile Asn Ser
Leu Ser Ala 625 630 635 640 Asp Thr Leu Ser Ala Ser Glu Pro Met Glu
Leu Ser Gly His 645 650 178 24 DNA Artificial Sequence Description
of Artificial Sequence Synthetic oligonucleotide probe 178
tggctactcc aagaccctgg catg 24 179 24 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide probe
179 tggacaaatc cccttgctca gccc 24 180 50 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide probe
180 gggcttcacc gaagcagtgg acctttattt tgaccacctg atgtccaggg 50 181
22 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide probe 181 ccagctatga ctatgatgca cc 22 182
24 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide probe 182 tggcacccag aatggtgttg gctc 24
183 50 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide probe 183 cgagatgtca tcagcaagtt
ccaggaagtt cctttgggac ctttacctcc 50 184 1947 DNA Homo sapiens 184
gctttgaaca cgtctgcaag cccaaagttg agcatctgat tggttatgag gtatttgagt
60 gcacccacaa tatggcttac atgttgaaaa agcttctcat cagttacata
tccattattt 120 gtgtttatgg ctttatctgc ctctacactc tcttctggtt
attcaggata cctttgaagg 180 aatattcttt cgaaaaagtc agagaagaga
gcagttttag tgacattcca gatgtcaaaa 240 acgattttgc gttccttctt
cacatggtag accagtatga ccagctatat tccaagcgtt 300 ttggtgtgtt
cttgtcagaa gttagtgaaa ataaacttag ggaaattagt ttgaaccatg 360
agtggacatt tgaaaaactc aggcagcaca tttcacgcaa cgcccaggac aagcaggagt
420 tgcatctgtt catgctgtcg ggggtgcccg atgctgtctt tgacctcaca
gacctggatg 480 tgctaaagct tgaactaatt ccagaagcta aaattcctgc
taagatttct caaatgacta 540 acctccaaga gctccacctc tgccactgcc
ctgcaaaagt tgaacagact gcttttagct 600 ttcttcgcga tcacttgaga
tgccttcacg tgaagttcac tgatgtggct gaaattcctg 660 cctgggtgta
tttgctcaaa aaccttcgag agttgtactt aataggcaat ttgaactctg 720
aaaacaataa gatgatagga cttgaatctc tccgagagtt gcggcacctt aagattctcc
780 acgtgaagag caatttgacc aaagttccct ccaacattac agatgtggct
ccacatctta 840 caaagttagt cattcataat gacggcacta aactcttggt
actgaacagc cttaagaaaa 900 tgatgaatgt cgctgagctg gaactccaga
actgtgagct agagagaatc ccacatgcta 960 ttttcagcct ctctaattta
caggaactgg atttaaagtc caataacatt cgcacaattg 1020 aggaaatcat
cagtttccag catttaaaac gactgacttg tttaaaatta tggcataaca 1080
aaattgttac tattcctccc tctattaccc atgtcaaaaa cttggagtca ctttatttct
1140 ctaacaacaa gctcgaatcc ttaccagtgg cagtatttag tttacagaaa
ctcagatgct 1200 tagatgtgag ctacaacaac atttcaatga ttccaataga
aataggattg cttcagaacc 1260 tgcagcattt gcatatcact gggaacaaag
tggacattct gccaaaacaa ttgtttaaat 1320 gcataaagtt gaggactttg
aatctgggac agaactgcat cacctcactc ccagagaaag 1380 ttggtcagct
ctcccagctc actcagctgg agctgaaggg gaactgcttg gaccgcctgc 1440
cagcccagct gggccagtgt cggatgctca agaaaagcgg gcttgttgtg gaagatcacc
1500 tttttgatac cctgccactc gaagtcaaag aggcattgaa tcaagacata
aatattccct 1560 ttgcaaatgg gatttaaact aagataatat atgcacagtg
atgtgcagga acaacttcct 1620 agattgcaag tgctcacgta caagttatta
caagataatg cattttagga gtagatacat 1680 cttttaaaat aaaacagaga
ggatgcatag aaggctgata gaagacataa ctgaatgttc 1740 aatgtttgta
gggttttaag tcattcattt ccaaatcatt tttttttttc ttttggggaa 1800
agggaaggaa aaattataat cactaatctt ggttcttttt aaattgtttg taacttggat
1860 gctgccgcta ctgaatgttt acaaattgct tgcctgctaa agtaaatgat
taaattgaca 1920 ttttcttact aaaaaaaaaa aaaaaaa 1947 185 501 PRT Homo
sapiens 185 Met Ala Tyr Met Leu Lys Lys Leu Leu Ile Ser Tyr Ile Ser
Ile Ile 1 5 10 15 Cys Val Tyr Gly Phe Ile Cys Leu Tyr Thr Leu Phe
Trp Leu Phe Arg 20 25 30 Ile Pro Leu Lys Glu Tyr Ser Phe Glu Lys
Val Arg Glu Glu Ser Ser 35 40 45 Phe Ser Asp Ile Pro Asp Val Lys
Asn Asp Phe Ala Phe Leu Leu His 50 55 60 Met Val Asp Gln Tyr Asp
Gln Leu Tyr Ser Lys Arg Phe Gly Val Phe 65 70 75 80 Leu Ser Glu Val
Ser Glu Asn Lys Leu Arg Glu Ile Ser Leu Asn His 85 90 95 Glu Trp
Thr Phe Glu Lys Leu Arg Gln His Ile Ser Arg Asn Ala Gln 100 105 110
Asp Lys Gln Glu Leu His Leu Phe Met Leu Ser Gly Val Pro Asp Ala 115
120 125 Val Phe Asp Leu Thr Asp Leu Asp Val Leu Lys Leu Glu Leu Ile
Pro 130 135 140 Glu Ala Lys Ile Pro Ala Lys Ile Ser Gln Met Thr Asn
Leu Gln Glu 145 150 155 160 Leu His Leu Cys His Cys Pro Ala Lys Val
Glu Gln Thr Ala Phe Ser 165 170 175 Phe Leu Arg Asp His Leu Arg Cys
Leu His Val Lys Phe Thr Asp Val 180 185 190 Ala Glu Ile Pro Ala Trp
Val Tyr Leu Leu Lys Asn Leu Arg Glu Leu 195 200 205 Tyr Leu Ile Gly
Asn Leu Asn Ser Glu Asn Asn Lys Met Ile Gly Leu 210 215 220 Glu Ser
Leu Arg Glu Leu Arg His Leu Lys Ile Leu His Val Lys Ser 225 230 235
240 Asn Leu Thr Lys Val Pro Ser Asn Ile Thr Asp Val Ala Pro His Leu
245 250 255 Thr Lys Leu Val Ile His Asn Asp Gly Thr Lys Leu Leu Val
Leu Asn 260 265 270 Ser Leu Lys Lys Met Met Asn Val Ala Glu Leu Glu
Leu Gln Asn Cys 275 280 285 Glu Leu Glu Arg Ile Pro His Ala Ile Phe
Ser Leu Ser Asn Leu Gln 290 295 300 Glu Leu Asp Leu Lys Ser Asn Asn
Ile Arg Thr Ile Glu Glu Ile Ile 305 310 315 320 Ser Phe Gln His Leu
Lys Arg Leu Thr Cys Leu Lys Leu Trp His Asn 325 330 335 Lys Ile Val
Thr Ile Pro Pro Ser Ile Thr His Val Lys Asn Leu Glu 340 345 350 Ser
Leu Tyr Phe Ser Asn Asn Lys Leu Glu Ser Leu Pro Val Ala Val 355 360
365 Phe Ser Leu Gln Lys Leu Arg Cys Leu Asp Val Ser Tyr Asn Asn Ile
370 375 380 Ser Met Ile Pro Ile Glu Ile Gly Leu Leu Gln Asn Leu Gln
His Leu 385 390 395 400 His Ile Thr Gly Asn Lys Val Asp Ile Leu Pro
Lys Gln Leu Phe Lys 405 410 415 Cys Ile Lys Leu Arg Thr Leu Asn Leu
Gly Gln Asn Cys Ile Thr Ser 420 425 430 Leu Pro Glu Lys Val Gly Gln
Leu Ser Gln Leu Thr Gln Leu Glu Leu 435 440 445 Lys Gly Asn Cys Leu
Asp Arg Leu Pro Ala Gln Leu Gly Gln Cys Arg 450 455 460 Met Leu Lys
Lys Ser Gly Leu Val Val Glu Asp His Leu Phe Asp Thr 465 470 475 480
Leu Pro Leu Glu Val Lys Glu Ala Leu Asn Gln Asp Ile Asn Ile Pro 485
490 495 Phe Ala Asn Gly Ile 500 186 21 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide probe
186 cctccctcta ttacccatgt c 21 187 24 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide probe
187 gaccaacttt ctctgggagt gagg 24 188 47 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide probe
188 gtcactttat ttctctaaca acaagctcga atccttacca gtggcag 47 189 2917
DNA Homo sapiens 189 cccacgcgtc cggccttctc tctggacttt gcatttccat
tccttttcat tgacaaactg 60 acttttttta tttctttttt tccatctctg
ggccagcttg ggatcctagg ccgccctggg 120 aagacatttg tgttttacac
acataaggat ctgtgtttgg ggtttcttct tcctcccctg 180 acattggcat
tgcttagtgg ttgtgtgggg agggagacca cgtgggctca gtgcttgctt 240
gcacttatct gcctaggtac atcgaagtct tttgacctcc atacagtgat tatgcctgtc
300 atcgctggtg gtatcctggc ggccttgctc ctgctgatag ttgtcgtgct
ctgtctttac 360 ttcaaaatac acaacgcgct aaaagctgca aaggaacctg
aagctgtggc tgtaaaaaat 420 cacaacccag acaaggtgtg gtgggccaag
aacagccagg ccaaaaccat tgccacggag 480 tcttgtcctg ccctgcagtg
ctgtgaagga tatagaatgt gtgccagttt tgattccctg 540 ccaccttgct
gttgcgacat aaatgagggc ctctgagtta ggaaaggctc ccttctcaaa 600
gcagagccct gaagacttca atgatgtcaa tgaggccacc tgtttgtgat gtgcaggcac
660 agaagaaagg cacagctccc catcagtttc atggaaaata actcagtgcc
tgctgggaac 720 cagctgctgg agatccctac agagagcttc cactgggggc
aacccttcca ggaaggagtt 780 ggggagagag aaccctcact gtggggaatg
ctgataaacc agtcacacag ctgctctatt 840 ctcacacaaa tctacccctt
gcgtggctgg aactgacgtt tccctggagg tgtccagaaa 900 gctgatgtaa
cacagagcct ataaaagctg tcggtcctta aggctgccca gcgccttgcc 960
aaaatggagc ttgtaagaag gctcatgcca ttgaccctct taattctctc ctgtttggcg
1020 gagctgacaa tggcggaggc tgaaggcaat gcaagctgca cagtcagtct
agggggtgcc 1080 aatatggcag agacccacaa agccatgatc ctgcaactca
atcccagtga gaactgcacc 1140 tggacaatag aaagaccaga aaacaaaagc
atcagaatta tcttttccta tgtccagctt 1200 gatccagatg gaagctgtga
aagtgaaaac attaaagtct ttgacggaac ctccagcaat 1260 gggcctctgc
tagggcaagt ctgcagtaaa aacgactatg ttcctgtatt tgaatcatca 1320
tccagtacat tgacgtttca aatagttact gactcagcaa gaattcaaag aactgtcttt
1380 gtcttctact acttcttctc tcctaacatc tctattccaa actgtggcgg
ttacctggat 1440 accttggaag gatccttcac cagccccaat tacccaaagc
cgcatcctga gctggcttat 1500 tgtgtgtggc acatacaagt ggagaaagat
tacaagataa aactaaactt caaagagatt 1560 ttcctagaaa tagacaaaca
gtgcaaattt gattttcttg ccatctatga tggcccctcc 1620 accaactctg
gcctgattgg acaagtctgt ggccgtgtga ctcccacctt cgaatcgtca 1680
tcaaactctc tgactgtcgt gttgtctaca gattatgcca attcttaccg gggattttct
1740 gcttcctaca cctcaattta tgcagaaaac atcaacacta catctttaac
ttgctcttct 1800 gacaggatga gagttattat aagcaaatcc tacctagagg
cttttaactc taatgggaat 1860 aacttgcaac taaaagaccc aacttgcaga
ccaaaattat caaatgttgt ggaattttct 1920 gtccctctta atggatgtgg
tacaatcaga aaggtagaag atcagtcaat tacttacacc 1980 aatataatca
ccttttctgc atcctcaact tctgaagtga tcacccgtca gaaacaactc 2040
cagattattg tgaagtgtga aatgggacat aattctacag tggagataat atacataaca
2100 gaagatgatg taatacaaag tcaaaatgca ctgggcaaat ataacaccag
catggctctt 2160 tttgaatcca attcatttga aaagactata cttgaatcac
catattatgt ggatttgaac 2220 caaactcttt ttgttcaagt tagtctgcac
acctcagatc caaatttggt ggtgtttctt 2280 gatacctgta gagcctctcc
cacctctgac tttgcatctc caacctacga cctaatcaag 2340 agtggatgta
gtcgagatga aacttgtaag gtgtatccct tatttggaca ctatgggaga 2400
ttccagttta atgcctttaa attcttgaga agtatgagct ctgtgtatct gcagtgtaaa
2460 gttttgatat gtgatagcag tgaccaccag tctcgctgca atcaaggttg
tgtctccaga 2520 agcaaacgag acatttcttc atataaatgg aaaacagatt
ccatcatagg acccattcgt 2580 ctgaaaaggg atcgaagtgc aagtggcaat
tcaggatttc agcatgaaac acatgcggaa 2640 gaaactccaa accagccttt
caacagtgtg catctgtttt ccttcatggt tctagctctg 2700 aatgtggtga
ctgtagcgac aatcacagtg aggcattttg taaatcaacg ggcagactac 2760
aaataccaga agctgcagaa ctattaacta acaggtccaa ccctaagtga gacatgtttc
2820 tccaggatgc caaaggaaat gctacctcgt ggctacacat attatgaata
aatgaggaag 2880 ggcctgaaag tgacacacag gcctgcatgt aaaaaaa 2917 190
607 PRT Homo sapiens 190 Met Glu Leu Val Arg Arg Leu Met Pro Leu
Thr Leu Leu Ile Leu Ser 1 5 10 15 Cys Leu Ala Glu Leu Thr Met Ala
Glu Ala Glu Gly
Asn Ala Ser Cys 20 25 30 Thr Val Ser Leu Gly Gly Ala Asn Met Ala
Glu Thr His Lys Ala Met 35 40 45 Ile Leu Gln Leu Asn Pro Ser Glu
Asn Cys Thr Trp Thr Ile Glu Arg 50 55 60 Pro Glu Asn Lys Ser Ile
Arg Ile Ile Phe Ser Tyr Val Gln Leu Asp 65 70 75 80 Pro Asp Gly Ser
Cys Glu Ser Glu Asn Ile Lys Val Phe Asp Gly Thr 85 90 95 Ser Ser
Asn Gly Pro Leu Leu Gly Gln Val Cys Ser Lys Asn Asp Tyr 100 105 110
Val Pro Val Phe Glu Ser Ser Ser Ser Thr Leu Thr Phe Gln Ile Val 115
120 125 Thr Asp Ser Ala Arg Ile Gln Arg Thr Val Phe Val Phe Tyr Tyr
Phe 130 135 140 Phe Ser Pro Asn Ile Ser Ile Pro Asn Cys Gly Gly Tyr
Leu Asp Thr 145 150 155 160 Leu Glu Gly Ser Phe Thr Ser Pro Asn Tyr
Pro Lys Pro His Pro Glu 165 170 175 Leu Ala Tyr Cys Val Trp His Ile
Gln Val Glu Lys Asp Tyr Lys Ile 180 185 190 Lys Leu Asn Phe Lys Glu
Ile Phe Leu Glu Ile Asp Lys Gln Cys Lys 195 200 205 Phe Asp Phe Leu
Ala Ile Tyr Asp Gly Pro Ser Thr Asn Ser Gly Leu 210 215 220 Ile Gly
Gln Val Cys Gly Arg Val Thr Pro Thr Phe Glu Ser Ser Ser 225 230 235
240 Asn Ser Leu Thr Val Val Leu Ser Thr Asp Tyr Ala Asn Ser Tyr Arg
245 250 255 Gly Phe Ser Ala Ser Tyr Thr Ser Ile Tyr Ala Glu Asn Ile
Asn Thr 260 265 270 Thr Ser Leu Thr Cys Ser Ser Asp Arg Met Arg Val
Ile Ile Ser Lys 275 280 285 Ser Tyr Leu Glu Ala Phe Asn Ser Asn Gly
Asn Asn Leu Gln Leu Lys 290 295 300 Asp Pro Thr Cys Arg Pro Lys Leu
Ser Asn Val Val Glu Phe Ser Val 305 310 315 320 Pro Leu Asn Gly Cys
Gly Thr Ile Arg Lys Val Glu Asp Gln Ser Ile 325 330 335 Thr Tyr Thr
Asn Ile Ile Thr Phe Ser Ala Ser Ser Thr Ser Glu Val 340 345 350 Ile
Thr Arg Gln Lys Gln Leu Gln Ile Ile Val Lys Cys Glu Met Gly 355 360
365 His Asn Ser Thr Val Glu Ile Ile Tyr Ile Thr Glu Asp Asp Val Ile
370 375 380 Gln Ser Gln Asn Ala Leu Gly Lys Tyr Asn Thr Ser Met Ala
Leu Phe 385 390 395 400 Glu Ser Asn Ser Phe Glu Lys Thr Ile Leu Glu
Ser Pro Tyr Tyr Val 405 410 415 Asp Leu Asn Gln Thr Leu Phe Val Gln
Val Ser Leu His Thr Ser Asp 420 425 430 Pro Asn Leu Val Val Phe Leu
Asp Thr Cys Arg Ala Ser Pro Thr Ser 435 440 445 Asp Phe Ala Ser Pro
Thr Tyr Asp Leu Ile Lys Ser Gly Cys Ser Arg 450 455 460 Asp Glu Thr
Cys Lys Val Tyr Pro Leu Phe Gly His Tyr Gly Arg Phe 465 470 475 480
Gln Phe Asn Ala Phe Lys Phe Leu Arg Ser Met Ser Ser Val Tyr Leu 485
490 495 Gln Cys Lys Val Leu Ile Cys Asp Ser Ser Asp His Gln Ser Arg
Cys 500 505 510 Asn Gln Gly Cys Val Ser Arg Ser Lys Arg Asp Ile Ser
Ser Tyr Lys 515 520 525 Trp Lys Thr Asp Ser Ile Ile Gly Pro Ile Arg
Leu Lys Arg Asp Arg 530 535 540 Ser Ala Ser Gly Asn Ser Gly Phe Gln
His Glu Thr His Ala Glu Glu 545 550 555 560 Thr Pro Asn Gln Pro Phe
Asn Ser Val His Leu Phe Ser Phe Met Val 565 570 575 Leu Ala Leu Asn
Val Val Thr Val Ala Thr Ile Thr Val Arg His Phe 580 585 590 Val Asn
Gln Arg Ala Asp Tyr Lys Tyr Gln Lys Leu Gln Asn Tyr 595 600 605 191
21 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide probe 191 tctctattcc aaactgtggc g 21 192
22 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide probe 192 tttgatgacg attcgaaggt gg 22 193
47 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide probe 193 ggaaggatcc ttcaccagcc
ccaattaccc aaagccgcat cctgagc 47 194 2362 DNA Homo sapiens 194
gacggaagaa cagcgctccc gaggccgcgg gagcctgcag agaggacagc cggcctgcgc
60 cgggacatgc ggccccagga gctccccagg ctcgcgttcc cgttgctgct
gttgctgttg 120 ctgctgctgc cgccgccgcc gtgccctgcc cacagcgcca
cgcgcttcga ccccacctgg 180 gagtccctgg acgcccgcca gctgcccgcg
tggtttgacc aggccaagtt cggcatcttc 240 atccactggg gagtgttttc
cgtgcccagc ttcggtagcg agtggttctg gtggtattgg 300 caaaaggaaa
agataccgaa gtatgtggaa tttatgaaag ataattaccc tcctagtttc 360
aaatatgaag attttggacc actatttaca gcaaaatttt ttaatgccaa ccagtgggca
420 gatatttttc aggcctctgg tgccaaatac attgtcttaa cttccaaaca
tcatgaaggc 480 tttaccttgt gggggtcaga atattcgtgg aactggaatg
ccatagatga ggggcccaag 540 agggacattg tcaaggaact tgaggtagcc
attaggaaca gaactgacct gcgttttgga 600 ctgtactatt ccctttttga
atggtttcat ccgctcttcc ttgaggatga atccagttca 660 ttccataagc
ggcaatttcc agtttctaag acattgccag agctctatga gttagtgaac 720
aactatcagc ctgaggttct gtggtcggat ggtgacggag gagcaccgga tcaatactgg
780 aacagcacag gcttcttggc ctggttatat aatgaaagcc cagttcgggg
cacagtagtc 840 accaatgatc gttggggagc tggtagcatc tgtaagcatg
gtggcttcta tacctgcagt 900 gatcgttata acccaggaca tcttttgcca
cataaatggg aaaactgcat gacaatagac 960 aaactgtcct ggggctatag
gagggaagct ggaatctctg actatcttac aattgaagaa 1020 ttggtgaagc
aacttgtaga gacagtttca tgtggaggaa atcttttgat gaatattggg 1080
cccacactag atggcaccat ttctgtagtt tttgaggagc gactgaggca agtggggtcc
1140 tggctaaaag tcaatggaga agctatttat gaaacctata cctggcgatc
ccagaatgac 1200 actgtcaccc cagatgtgtg gtacacatcc aagcctaaag
aaaaattagt ctatgccatt 1260 tttcttaaat ggcccacatc aggacagctg
ttccttggcc atcccaaagc tattctgggg 1320 gcaacagagg tgaaactact
gggccatgga cagccactta actggatttc tttggagcaa 1380 aatggcatta
tggtagaact gccacagcta accattcatc agatgccgtg taaatggggc 1440
tgggctctag ccctaactaa tgtgatctaa agtgcagcag agtggctgat gctgcaagtt
1500 atgtctaagg ctaggaacta tcaggtgtct ataattgtag cacatggaga
aagcaatgta 1560 aactggataa gaaaattatt tggcagttca gccctttccc
tttttcccac taaatttttc 1620 ttaaattacc catgtaacca ttttaactct
ccagtgcact ttgccattaa agtctcttca 1680 cattgatttg tttccatgtg
tgactcagag gtgagaattt tttcacatta tagtagcaag 1740 gaattggtgg
tattatggac cgaactgaaa attttatgtt gaagccatat cccccatgat 1800
tatatagtta tgcatcactt aatatgggga tattttctgg gaaatgcatt gctagtcaat
1860 ttttttttgt gccaacatca tagagtgtat ttacaaaatc ctagatggca
tagcctacta 1920 cacacctaat gtgtatggta tagactgttg ctcctaggct
acagacatat acagcatgtt 1980 actgaatact gtaggcaata gtaacagtgg
tatttgtata tcgaaacata tggaaacata 2040 gagaaggtac agtaaaaata
ctgtaaaata aatggtgcac ctgtataggg cacttaccac 2100 gaatggagct
tacaggactg gaagttgctc tgggtgagtc agtgagtgaa tgtgaaggcc 2160
taggacatta ttgaacactg ccagacgtta taaatactgt atgcttaggc tacactacat
2220 ttataaaaaa aagtttttct ttcttcaatt ataaattaac ataagtgtac
tgtaacttta 2280 caaacgtttt aatttttaaa acctttttgg ctcttttgta
ataacactta gcttaaaaca 2340 taaactcatt gtgcaaatgt aa 2362 195 467
PRT Homo sapiens 195 Met Arg Pro Gln Glu Leu Pro Arg Leu Ala Phe
Pro Leu Leu Leu Leu 1 5 10 15 Leu Leu Leu Leu Leu Pro Pro Pro Pro
Cys Pro Ala His Ser Ala Thr 20 25 30 Arg Phe Asp Pro Thr Trp Glu
Ser Leu Asp Ala Arg Gln Leu Pro Ala 35 40 45 Trp Phe Asp Gln Ala
Lys Phe Gly Ile Phe Ile His Trp Gly Val Phe 50 55 60 Ser Val Pro
Ser Phe Gly Ser Glu Trp Phe Trp Trp Tyr Trp Gln Lys 65 70 75 80 Glu
Lys Ile Pro Lys Tyr Val Glu Phe Met Lys Asp Asn Tyr Pro Pro 85 90
95 Ser Phe Lys Tyr Glu Asp Phe Gly Pro Leu Phe Thr Ala Lys Phe Phe
100 105 110 Asn Ala Asn Gln Trp Ala Asp Ile Phe Gln Ala Ser Gly Ala
Lys Tyr 115 120 125 Ile Val Leu Thr Ser Lys His His Glu Gly Phe Thr
Leu Trp Gly Ser 130 135 140 Glu Tyr Ser Trp Asn Trp Asn Ala Ile Asp
Glu Gly Pro Lys Arg Asp 145 150 155 160 Ile Val Lys Glu Leu Glu Val
Ala Ile Arg Asn Arg Thr Asp Leu Arg 165 170 175 Phe Gly Leu Tyr Tyr
Ser Leu Phe Glu Trp Phe His Pro Leu Phe Leu 180 185 190 Glu Asp Glu
Ser Ser Ser Phe His Lys Arg Gln Phe Pro Val Ser Lys 195 200 205 Thr
Leu Pro Glu Leu Tyr Glu Leu Val Asn Asn Tyr Gln Pro Glu Val 210 215
220 Leu Trp Ser Asp Gly Asp Gly Gly Ala Pro Asp Gln Tyr Trp Asn Ser
225 230 235 240 Thr Gly Phe Leu Ala Trp Leu Tyr Asn Glu Ser Pro Val
Arg Gly Thr 245 250 255 Val Val Thr Asn Asp Arg Trp Gly Ala Gly Ser
Ile Cys Lys His Gly 260 265 270 Gly Phe Tyr Thr Cys Ser Asp Arg Tyr
Asn Pro Gly His Leu Leu Pro 275 280 285 His Lys Trp Glu Asn Cys Met
Thr Ile Asp Lys Leu Ser Trp Gly Tyr 290 295 300 Arg Arg Glu Ala Gly
Ile Ser Asp Tyr Leu Thr Ile Glu Glu Leu Val 305 310 315 320 Lys Gln
Leu Val Glu Thr Val Ser Cys Gly Gly Asn Leu Leu Met Asn 325 330 335
Ile Gly Pro Thr Leu Asp Gly Thr Ile Ser Val Val Phe Glu Glu Arg 340
345 350 Leu Arg Gln Val Gly Ser Trp Leu Lys Val Asn Gly Glu Ala Ile
Tyr 355 360 365 Glu Thr Tyr Thr Trp Arg Ser Gln Asn Asp Thr Val Thr
Pro Asp Val 370 375 380 Trp Tyr Thr Ser Lys Pro Lys Glu Lys Leu Val
Tyr Ala Ile Phe Leu 385 390 395 400 Lys Trp Pro Thr Ser Gly Gln Leu
Phe Leu Gly His Pro Lys Ala Ile 405 410 415 Leu Gly Ala Thr Glu Val
Lys Leu Leu Gly His Gly Gln Pro Leu Asn 420 425 430 Trp Ile Ser Leu
Glu Gln Asn Gly Ile Met Val Glu Leu Pro Gln Leu 435 440 445 Thr Ile
His Gln Met Pro Cys Lys Trp Gly Trp Ala Leu Ala Leu Thr 450 455 460
Asn Val Ile 465 196 23 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide probe 196 tggtttgacc
aggccaagtt cgg 23 197 24 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide probe 197 ggattcatcc
tcaaggaaga gcgg 24 198 24 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide probe 198 aacttgcagc
atcagccact ctgc 24 199 45 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide probe 199 ttccgtgccc
agcttcggta gcgagtggtt ctggtggtat tggca 45 200 2372 DNA Homo sapiens
200 agcagggaaa tccggatgtc tcggttatga agtggagcag tgagtgtgag
cctcaacata 60 gttccagaac tctccatccg gactagttat tgagcatctg
cctctcatat caccagtggc 120 catctgaggt gtttccctgg ctctgaaggg
gtaggcacga tggccaggtg cttcagcctg 180 gtgttgcttc tcacttccat
ctggaccacg aggctcctgg tccaaggctc tttgcgtgca 240 gaagagcttt
ccatccaggt gtcatgcaga attatgggga tcacccttgt gagcaaaaag 300
gcgaaccagc agctgaattt cacagaagct aaggaggcct gtaggctgct gggactaagt
360 ttggccggca aggaccaagt tgaaacagcc ttgaaagcta gctttgaaac
ttgcagctat 420 ggctgggttg gagatggatt cgtggtcatc tctaggatta
gcccaaaccc caagtgtggg 480 aaaaatgggg tgggtgtcct gatttggaag
gttccagtga gccgacagtt tgcagcctat 540 tgttacaact catctgatac
ttggactaac tcgtgcattc cagaaattat caccaccaaa 600 gatcccatat
tcaacactca aactgcaaca caaacaacag aatttattgt cagtgacagt 660
acctactcgg tggcatcccc ttactctaca atacctgccc ctactactac tcctcctgct
720 ccagcttcca cttctattcc acggagaaaa aaattgattt gtgtcacaga
agtttttatg 780 gaaactagca ccatgtctac agaaactgaa ccatttgttg
aaaataaagc agcattcaag 840 aatgaagctg ctgggtttgg aggtgtcccc
acggctctgc tagtgcttgc tctcctcttc 900 tttggtgctg cagctggtct
tggattttgc tatgtcaaaa ggtatgtgaa ggccttccct 960 tttacaaaca
agaatcagca gaaggaaatg atcgaaacca aagtagtaaa ggaggagaag 1020
gccaatgata gcaaccctaa tgaggaatca aagaaaactg ataaaaaccc agaagagtcc
1080 aagagtccaa gcaaaactac cgtgcgatgc ctggaagctg aagtttagat
gagacagaaa 1140 tgaggagaca cacctgaggc tggtttcttt catgctcctt
accctgcccc agctggggaa 1200 atcaaaaggg ccaaagaacc aaagaagaaa
gtccaccctt ggttcctaac tggaatcagc 1260 tcaggactgc cattggacta
tggagtgcac caaagagaat gcccttctcc ttattgtaac 1320 cctgtctgga
tcctatcctc ctacctccaa agcttcccac ggcctttcta gcctggctat 1380
gtcctaataa tatcccactg ggagaaagga gttttgcaaa gtgcaaggac ctaaaacatc
1440 tcatcagtat ccagtggtaa aaaggcctcc tggctgtctg aggctaggtg
ggttgaaagc 1500 caaggagtca ctgagaccaa ggctttctct actgattccg
cagctcagac cctttcttca 1560 gctctgaaag agaaacacgt atcccacctg
acatgtcctt ctgagcccgg taagagcaaa 1620 agaatggcag aaaagtttag
cccctgaaag ccatggagat tctcataact tgagacctaa 1680 tctctgtaaa
gctaaaataa agaaatagaa caaggctgag gatacgacag tacactgtca 1740
gcagggactg taaacacaga cagggtcaaa gtgttttctc tgaacacatt gagttggaat
1800 cactgtttag aacacacaca cttacttttt ctggtctcta ccactgctga
tattttctct 1860 aggaaatata cttttacaag taacaaaaat aaaaactctt
ataaatttct atttttatct 1920 gagttacaga aatgattact aaggaagatt
actcagtaat ttgtttaaaa agtaataaaa 1980 ttcaacaaac atttgctgaa
tagctactat atgtcaagtg ctgtgcaagg tattacactc 2040 tgtaattgaa
tattattcct caaaaaattg cacatagtag aacgctatct gggaagctat 2100
ttttttcagt tttgatattt ctagcttatc tacttccaaa ctaattttta tttttgctga
2160 gactaatctt attcattttc tctaatatgg caaccattat aaccttaatt
tattattaac 2220 atacctaaga agtacattgt tacctctata taccaaagca
cattttaaaa gtgccattaa 2280 caaatgtatc actagccctc ctttttccaa
caagaaggga ctgagagatg cagaaatatt 2340 tgtgacaaaa aattaaagca
tttagaaaac tt 2372 201 322 PRT Artificial sequence Synthetic
protein 201 Met Ala Arg Cys Phe Ser Leu Val Leu Leu Leu Thr Ser Ile
Trp Thr 1 5 10 15 Thr Arg Leu Leu Val Gln Gly Ser Leu Arg Ala Glu
Glu Leu Ser Ile 20 25 30 Gln Val Ser Cys Arg Ile Met Gly Ile Thr
Leu Val Ser Lys Lys Ala 35 40 45 Asn Gln Gln Leu Asn Phe Thr Glu
Ala Lys Glu Ala Cys Arg Leu Leu 50 55 60 Gly Leu Ser Leu Ala Gly
Lys Asp Gln Val Glu Thr Ala Leu Lys Ala 65 70 75 80 Ser Phe Glu Thr
Cys Ser Tyr Gly Trp Val Gly Asp Gly Phe Val Val 85 90 95 Ile Ser
Arg Ile Ser Pro Asn Pro Lys Cys Gly Lys Asn Gly Val Gly 100 105 110
Val Leu Ile Trp Lys Val Pro Val Ser Arg Gln Phe Ala Ala Tyr Cys 115
120 125 Tyr Asn Ser Ser Asp Thr Trp Thr Asn Ser Cys Ile Pro Glu Ile
Ile 130 135 140 Thr Thr Lys Asp Pro Ile Phe Asn Thr Gln Thr Ala Thr
Gln Thr Thr 145 150 155 160 Glu Phe Ile Val Ser Asp Ser Thr Tyr Ser
Val Ala Ser Pro Tyr Ser 165 170 175 Thr Ile Pro Ala Pro Thr Thr Thr
Pro Pro Ala Pro Ala Ser Thr Ser 180 185 190 Ile Pro Arg Arg Lys Lys
Leu Ile Cys Val Thr Glu Val Phe Met Glu 195 200 205 Thr Ser Thr Met
Ser Thr Glu Thr Glu Pro Phe Val Glu Asn Lys Ala 210 215 220 Ala Phe
Lys Asn Glu Ala Ala Gly Phe Gly Gly Val Pro Thr Ala Leu 225 230 235
240 Leu Val Leu Ala Leu Leu Phe Phe Gly Ala Ala Ala Gly Leu Gly Phe
245 250 255 Cys Tyr Val Lys Arg Tyr Val Lys Ala Phe Pro Phe Thr Asn
Lys Asn 260 265 270 Gln Gln Lys Glu Met Ile Glu Thr Lys Val Val Lys
Glu Glu Lys Ala 275 280 285 Asn Asp Ser Asn Pro Asn Glu Glu Ser Lys
Lys Thr Asp Lys Asn Pro 290 295 300 Glu Glu Ser Lys Ser Pro Ser Lys
Thr Thr Val Arg Cys Leu Glu Ala 305 310 315 320 Glu Val 202 24 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 202 gagctttcca tccaggtgtc atgc 24 203 22 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 203 gtcagtgaca gtacctactc gg 22 204 24 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 204 tggagcagga ggagtagtag tagg 24 205 50 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 205 aggaggcctg taggctgctg ggactaagtt
tggccggcaa ggaccaagtt 50 206 1620 DNA Homo
sapiens modified_base (973)..(973) a, t, c or g modified_base
(977)..(977) a, t, c or g modified_base (996)..(996) a, t, c or g
modified_base (1003)..(1003) a, t, c or g 206 agatggcggt cttggcacct
ctaattgctc tcgtgtattc ggtgccgcga ctttcacgat 60 ggctcgccca
accttactac cttctgtcgg ccctgctctc tgctgccttc ctactcgtga 120
ggaaactgcc gccgctctgc cacggtctgc ccacccaacg cgaagacggt aacccgtgtg
180 actttgactg gagagaagtg gagatcctga tgtttctcag tgccattgtg
atgatgaaga 240 accgcagatc catcactgtg gagcaacata taggcaacat
tttcatgttt agtaaagtgg 300 ccaacacaat tcttttcttc cgcttggata
ttcgcatggg cctactttac atcacactct 360 gcatagtgtt cctgatgacg
tgcaaacccc ccctatatat gggccctgag tatatcaagt 420 acttcaatga
taaaaccatt gatgaggaac tagaacggga caagagggtc acttggattg 480
tggagttctt tgccaattgg tctaatgact gccaatcatt tgcccctatc tatgctgacc
540 tctcccttaa atacaactgt acagggctaa attttgggaa ggtggatgtt
ggacgctata 600 ctgatgttag tacgcggtac aaagtgagca catcacccct
caccaagcaa ctccctaccc 660 tgatcctgtt ccaaggtggc aaggaggcaa
tgcggcggcc acagattgac aagaaaggac 720 gggctgtctc atggaccttc
tctgaggaga atgtgatccg agaatttaac ttaaatgagc 780 tataccagcg
ggccaagaaa ctatcaaagg ctggagacaa tatccctgag gagcagcctg 840
tggcttcaac ccccaccaca gtgtcagatg gggaaaacaa gaaggataaa taagatcctc
900 actttggcag tgcttcctct cctgtcaatt ccaggctctt tccataacca
caagcctgag 960 gctgcagcct ttnattnatg ttttcccttt ggctgngact
ggntggggca gcatgcagct 1020 tctgatttta aagaggcatc tagggaattg
tcaggcaccc tacaggaagg cctgccatgc 1080 tgtggccaac tgtttcactg
gagcaagaaa gagatctcat aggacggagg gggaaatggt 1140 ttccctccaa
gcttgggtca gtgtgttaac tgcttatcag ctattcagac atctccatgg 1200
tttctccatg aaactctgtg gtttcatcat tccttcttag ttgacctgca cagcttggtt
1260 agacctagat ttaaccctaa ggtaagatgc tggggtatag aacgctaaga
attttccccc 1320 aaggactctt gcttccttaa gcccttctgg cttcgtttat
ggtcttcatt aaaagtataa 1380 gcctaacttt gtcgctagtc ctaaggagaa
acctttaacc acaaagtttt tatcattgaa 1440 gacaatattg aacaaccccc
tattttgtgg ggattgagaa ggggtgaata gaggcttgag 1500 actttccttt
gtgtggtagg acttggagga gaaatcccct ggactttcac taaccctctg 1560
acatactccc cacacccagt tgatggcttt ccgtaataaa aagattggga tttccttttg
1620 207 296 PRT Homo sapiens 207 Met Ala Val Leu Ala Pro Leu Ile
Ala Leu Val Tyr Ser Val Pro Arg 1 5 10 15 Leu Ser Arg Trp Leu Ala
Gln Pro Tyr Tyr Leu Leu Ser Ala Leu Leu 20 25 30 Ser Ala Ala Phe
Leu Leu Val Arg Lys Leu Pro Pro Leu Cys His Gly 35 40 45 Leu Pro
Thr Gln Arg Glu Asp Gly Asn Pro Cys Asp Phe Asp Trp Arg 50 55 60
Glu Val Glu Ile Leu Met Phe Leu Ser Ala Ile Val Met Met Lys Asn 65
70 75 80 Arg Arg Ser Ile Thr Val Glu Gln His Ile Gly Asn Ile Phe
Met Phe 85 90 95 Ser Lys Val Ala Asn Thr Ile Leu Phe Phe Arg Leu
Asp Ile Arg Met 100 105 110 Gly Leu Leu Tyr Ile Thr Leu Cys Ile Val
Phe Leu Met Thr Cys Lys 115 120 125 Pro Pro Leu Tyr Met Gly Pro Glu
Tyr Ile Lys Tyr Phe Asn Asp Lys 130 135 140 Thr Ile Asp Glu Glu Leu
Glu Arg Asp Lys Arg Val Thr Trp Ile Val 145 150 155 160 Glu Phe Phe
Ala Asn Trp Ser Asn Asp Cys Gln Ser Phe Ala Pro Ile 165 170 175 Tyr
Ala Asp Leu Ser Leu Lys Tyr Asn Cys Thr Gly Leu Asn Phe Gly 180 185
190 Lys Val Asp Val Gly Arg Tyr Thr Asp Val Ser Thr Arg Tyr Lys Val
195 200 205 Ser Thr Ser Pro Leu Thr Lys Gln Leu Pro Thr Leu Ile Leu
Phe Gln 210 215 220 Gly Gly Lys Glu Ala Met Arg Arg Pro Gln Ile Asp
Lys Lys Gly Arg 225 230 235 240 Ala Val Ser Trp Thr Phe Ser Glu Glu
Asn Val Ile Arg Glu Phe Asn 245 250 255 Leu Asn Glu Leu Tyr Gln Arg
Ala Lys Lys Leu Ser Lys Ala Gly Asp 260 265 270 Asn Ile Pro Glu Glu
Gln Pro Val Ala Ser Thr Pro Thr Thr Val Ser 275 280 285 Asp Gly Glu
Asn Lys Lys Asp Lys 290 295 208 24 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide probe
208 gcttggatat tcgcatgggc ctac 24 209 20 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide probe
209 tggagacaat atccctgagg 20 210 24 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide probe
210 aacagttggc cacagcatgg cagg 24 211 50 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide probe
211 ccattgatga ggaactagaa cgggacaaga gggtcacttg gattgtggag 50 212
1985 DNA Homo sapiens 212 ggacagctcg cggcccccga gagctctagc
cgtcgaggag ctgcctgggg acgtttgccc 60 tggggcccca gcctggcccg
ggtcaccctg gcatgaggag atgggcctgt tgctcctggt 120 cccattgctc
ctgctgcccg gctcctacgg actgcccttc tacaacggct tctactactc 180
caacagcgcc aacgaccaga acctaggcaa cggtcatggc aaagacctcc ttaatggagt
240 gaagctggtg gtggagacac ccgaggagac cctgttcacc taccaagggg
ccagtgtgat 300 cctgccctgc cgctaccgct acgagccggc cctggtctcc
ccgcggcgtg tgcgtgtcaa 360 atggtggaag ctgtcggaga acggggcccc
agagaaggac gtgctggtgg ccatcgggct 420 gaggcaccgc tcctttgggg
actaccaagg ccgcgtgcac ctgcggcagg acaaagagca 480 tgacgtctcg
ctggagatcc aggatctgcg gctggaggac tatgggcgtt accgctgtga 540
ggtcattgac gggctggagg atgaaagcgg tctggtggag ctggagctgc ggggtgtggt
600 ctttccttac cagtccccca acgggcgcta ccagttcaac ttccacgagg
gccagcaggt 660 ctgtgcagag caggctgcgg tggtggcctc ctttgagcag
ctcttccggg cctgggagga 720 gggcctggac tggtgcaacg cgggctggct
gcaggatgct acggtgcagt accccatcat 780 gttgccccgg cagccctgcg
gtggcccagg cctggcacct ggcgtgcgaa gctacggccc 840 ccgccaccgc
cgcctgcacc gctatgatgt attctgcttc gctactgccc tcaaggggcg 900
ggtgtactac ctggagcacc ctgagaagct gacgctgaca gaggcaaggg aggcctgcca
960 ggaagatgat gccacgatcg ccaaggtggg acagctcttt gccgcctgga
agttccatgg 1020 cctggaccgc tgcgacgctg gctggctggc agatggcagc
gtccgctacc ctgtggttca 1080 cccgcatcct aactgtgggc ccccagagcc
tggggtccga agctttggct tccccgaccc 1140 gcagagccgc ttgtacggtg
tttactgcta ccgccagcac taggacctgg ggccctcccc 1200 tgccgcattc
cctcactggc tgtgtattta ttgagtggtt cgttttccct tgtgggttgg 1260
agccatttta actgttttta tacttctcaa tttaaatttt ctttaaacat ttttttacta
1320 ttttttgtaa agcaaacaga acccaatgcc tccctttgct cctggatgcc
ccactccagg 1380 aatcatgctt gctcccctgg gccatttgcg gttttgtggg
cttctggagg gttccccgcc 1440 atccaggctg gtctccctcc cttaaggagg
ttggtgccca gagtgggcgg tggcctgtct 1500 agaatgccgc cgggagtccg
ggcatggtgg gcacagttct ccctgcccct cagcctgggg 1560 gaagaagagg
gcctcggggg cctccggagc tgggctttgg gcctctcctg cccacctcta 1620
cttctctgtg aagccgctga ccccagtctg cccactgagg ggctagggct ggaagccagt
1680 tctaggcttc caggcgaaat ctgagggaag gaagaaactc ccctccccgt
tccccttccc 1740 ctctcggttc caaagaatct gttttgttgt catttgtttc
tcctgtttcc ctgtgtgggg 1800 aggggccctc aggtgtgtgt actttggaca
ataaatggtg ctatgactgc cttccgccaa 1860 aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1920 aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1980 aaaaa
1985 213 360 PRT Homo sapiens 213 Met Gly Leu Leu Leu Leu Val Pro
Leu Leu Leu Leu Pro Gly Ser Tyr 1 5 10 15 Gly Leu Pro Phe Tyr Asn
Gly Phe Tyr Tyr Ser Asn Ser Ala Asn Asp 20 25 30 Gln Asn Leu Gly
Asn Gly His Gly Lys Asp Leu Leu Asn Gly Val Lys 35 40 45 Leu Val
Val Glu Thr Pro Glu Glu Thr Leu Phe Thr Tyr Gln Gly Ala 50 55 60
Ser Val Ile Leu Pro Cys Arg Tyr Arg Tyr Glu Pro Ala Leu Val Ser 65
70 75 80 Pro Arg Arg Val Arg Val Lys Trp Trp Lys Leu Ser Glu Asn
Gly Ala 85 90 95 Pro Glu Lys Asp Val Leu Val Ala Ile Gly Leu Arg
His Arg Ser Phe 100 105 110 Gly Asp Tyr Gln Gly Arg Val His Leu Arg
Gln Asp Lys Glu His Asp 115 120 125 Val Ser Leu Glu Ile Gln Asp Leu
Arg Leu Glu Asp Tyr Gly Arg Tyr 130 135 140 Arg Cys Glu Val Ile Asp
Gly Leu Glu Asp Glu Ser Gly Leu Val Glu 145 150 155 160 Leu Glu Leu
Arg Gly Val Val Phe Pro Tyr Gln Ser Pro Asn Gly Arg 165 170 175 Tyr
Gln Phe Asn Phe His Glu Gly Gln Gln Val Cys Ala Glu Gln Ala 180 185
190 Ala Val Val Ala Ser Phe Glu Gln Leu Phe Arg Ala Trp Glu Glu Gly
195 200 205 Leu Asp Trp Cys Asn Ala Gly Trp Leu Gln Asp Ala Thr Val
Gln Tyr 210 215 220 Pro Ile Met Leu Pro Arg Gln Pro Cys Gly Gly Pro
Gly Leu Ala Pro 225 230 235 240 Gly Val Arg Ser Tyr Gly Pro Arg His
Arg Arg Leu His Arg Tyr Asp 245 250 255 Val Phe Cys Phe Ala Thr Ala
Leu Lys Gly Arg Val Tyr Tyr Leu Glu 260 265 270 His Pro Glu Lys Leu
Thr Leu Thr Glu Ala Arg Glu Ala Cys Gln Glu 275 280 285 Asp Asp Ala
Thr Ile Ala Lys Val Gly Gln Leu Phe Ala Ala Trp Lys 290 295 300 Phe
His Gly Leu Asp Arg Cys Asp Ala Gly Trp Leu Ala Asp Gly Ser 305 310
315 320 Val Arg Tyr Pro Val Val His Pro His Pro Asn Cys Gly Pro Pro
Glu 325 330 335 Pro Gly Val Arg Ser Phe Gly Phe Pro Asp Pro Gln Ser
Arg Leu Tyr 340 345 350 Gly Val Tyr Cys Tyr Arg Gln His 355 360 214
18 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide probe 214 tgcttcgcta ctgccctc 18 215 18
DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide probe 215 ttcccttgtg ggttggag 18 216 18
DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide probe 216 agggctggaa gccagttc 18 217 18
DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide probe 217 agccagtgag gaaatgcg 18 218 24
DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide probe 218 tgtccaaagt acacacacct gagg 24
219 45 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide probe 219 gatgccacga tcgccaaggt
gggacagctc tttgccgcct ggaag 45 220 1503 DNA Homo sapiens 220
ggagagcgga gcgaagctgg ataacagggg accgatgatg tggcgaccat cagttctgct
60 gcttctgttg ctactgaggc acggggccca ggggaagcca tccccagacg
caggccctca 120 tggccagggg agggtgcacc aggcggcccc cctgagcgac
gctccccatg atgacgccca 180 cgggaacttc cagtacgacc atgaggcttt
cctgggacgg gaagtggcca aggaattcga 240 ccaactcacc ccagaggaaa
gccaggcccg tctggggcgg atcgtggacc gcatggaccg 300 cgcgggggac
ggcgacggct gggtgtcgct ggccgagctt cgcgcgtgga tcgcgcacac 360
gcagcagcgg cacatacggg actcggtgag cgcggcctgg gacacgtacg acacggaccg
420 cgacgggcgt gtgggttggg aggagctgcg caacgccacc tatggccact
acgcgcccgg 480 tgaagaattt catgacgtgg aggatgcaga gacctacaaa
aagatgctgg ctcgggacga 540 gcggcgtttc cgggtggccg accaggatgg
ggactcgatg gccactcgag aggagctgac 600 agccttcctg caccccgagg
agttccctca catgcgggac atcgtgattg ctgaaaccct 660 ggaggacctg
gacagaaaca aagatggcta tgtccaggtg gaggagtaca tcgcggatct 720
gtactcagcc gagcctgggg aggaggagcc ggcgtgggtg cagacggaga ggcagcagtt
780 ccgggacttc cgggatctga acaaggatgg gcacctggat gggagtgagg
tgggccactg 840 ggtgctgccc cctgcccagg accagcccct ggtggaagcc
aaccacctgc tgcacgagag 900 cgacacggac aaggatgggc ggctgagcaa
agcggaaatc ctgggtaatt ggaacatgtt 960 tgtgggcagt caggccacca
actatggcga ggacctgacc cggcaccacg atgagctgtg 1020 agcaccgcgc
acctgccaca gcctcagagg cccgcacaat gaccggagga ggggccgctg 1080
tggtctggcc ccctccctgt ccaggccccg caggaggcag atgcagtccc aggcatcctc
1140 ctgcccctgg gctctcaggg accccctggg tcggcttctg tccctgtcac
acccccaacc 1200 ccagggaggg gctgtcatag tcccagagga taagcaatac
ctatttctga ctgagtctcc 1260 cagcccagac ccagggaccc ttggccccaa
gctcagctct aagaaccgcc ccaacccctc 1320 cagctccaaa tctgagcctc
caccacatag actgaaactc ccctggcccc agccctctcc 1380 tgcctggcct
ggcctgggac acctcctctc tgccaggagg caataaaagc cagcgccggg 1440
accttgaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
1500 aaa 1503 221 328 PRT Homo sapiens 221 Met Met Trp Arg Pro Ser
Val Leu Leu Leu Leu Leu Leu Leu Arg His 1 5 10 15 Gly Ala Gln Gly
Lys Pro Ser Pro Asp Ala Gly Pro His Gly Gln Gly 20 25 30 Arg Val
His Gln Ala Ala Pro Leu Ser Asp Ala Pro His Asp Asp Ala 35 40 45
His Gly Asn Phe Gln Tyr Asp His Glu Ala Phe Leu Gly Arg Glu Val 50
55 60 Ala Lys Glu Phe Asp Gln Leu Thr Pro Glu Glu Ser Gln Ala Arg
Leu 65 70 75 80 Gly Arg Ile Val Asp Arg Met Asp Arg Ala Gly Asp Gly
Asp Gly Trp 85 90 95 Val Ser Leu Ala Glu Leu Arg Ala Trp Ile Ala
His Thr Gln Gln Arg 100 105 110 His Ile Arg Asp Ser Val Ser Ala Ala
Trp Asp Thr Tyr Asp Thr Asp 115 120 125 Arg Asp Gly Arg Val Gly Trp
Glu Glu Leu Arg Asn Ala Thr Tyr Gly 130 135 140 His Tyr Ala Pro Gly
Glu Glu Phe His Asp Val Glu Asp Ala Glu Thr 145 150 155 160 Tyr Lys
Lys Met Leu Ala Arg Asp Glu Arg Arg Phe Arg Val Ala Asp 165 170 175
Gln Asp Gly Asp Ser Met Ala Thr Arg Glu Glu Leu Thr Ala Phe Leu 180
185 190 His Pro Glu Glu Phe Pro His Met Arg Asp Ile Val Ile Ala Glu
Thr 195 200 205 Leu Glu Asp Leu Asp Arg Asn Lys Asp Gly Tyr Val Gln
Val Glu Glu 210 215 220 Tyr Ile Ala Asp Leu Tyr Ser Ala Glu Pro Gly
Glu Glu Glu Pro Ala 225 230 235 240 Trp Val Gln Thr Glu Arg Gln Gln
Phe Arg Asp Phe Arg Asp Leu Asn 245 250 255 Lys Asp Gly His Leu Asp
Gly Ser Glu Val Gly His Trp Val Leu Pro 260 265 270 Pro Ala Gln Asp
Gln Pro Leu Val Glu Ala Asn His Leu Leu His Glu 275 280 285 Ser Asp
Thr Asp Lys Asp Gly Arg Leu Ser Lys Ala Glu Ile Leu Gly 290 295 300
Asn Trp Asn Met Phe Val Gly Ser Gln Ala Thr Asn Tyr Gly Glu Asp 305
310 315 320 Leu Thr Arg His His Asp Glu Leu 325 222 20 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 222 cgcaggccct catggccagg 20 223 18 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 223 gaaatcctgg gtaattgg 18 224 23 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 224 gtgcgcggtg ctcacagctc atc 23 225 44 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide probe 225 cccccctgag cgacgctccc ccatgatgac
gcccacggga actt 44 226 2403 DNA Homo sapiens 226 ggggccttgc
cttccgcact cgggcgcagc cgggtggatc tcgagcaggt gcggagcccc 60
gggcggcggg cgcgggtgcg agggatccct gacgcctctg tccctgtttc tttgtcgctc
120 ccagcctgtc tgtcgtcgtt ttggcgcccc cgcctccccg cggtgcgggg
ttgcacaccg 180 atcctgggct tcgctcgatt tgccgccgag gcgcctccca
gacctagagg ggcgctggcc 240 tggagcagcg ggtcgtctgt gtcctctctc
ctctgcgccg cgcccgggga tccgaagggt 300 gcggggctct gaggaggtga
cgcgcggggc ctcccgcacc ctggccttgc ccgcattctc 360 cctctctccc
aggtgtgagc agcctatcag tcaccatgtc cgcagcctgg atcccggctc 420
tcggcctcgg tgtgtgtctg ctgctgctgc cggggcccgc gggcagcgag ggagccgctc
480 ccattgctat cacatgtttt accagaggct tggacatcag gaaagagaaa
gcagatgtcc 540 tctgcccagg gggctgccct cttgaggaat tctctgtgta
tgggaacata gtatatgctt 600 ctgtatcgag catatgtggg gctgctgtcc
acaggggagt aatcagcaac tcagggggac 660 ctgtacgagt ctatagccta
cctggtcgag aaaactattc ctcagtagat gccaatggca 720 tccagtctca
aatgctttct agatggtctg cttctttcac agtaactaaa ggcaaaagta 780
gtacacagga ggccacagga caagcagtgt ccacagcaca tccaccaaca ggtaaacgac
840 taaagaaaac acccgagaag aaaactggca ataaagattg taaagcagac
attgcatttc 900 tgattgatgg aagctttaat attgggcagc gccgatttaa
tttacagaag aattttgttg 960 gaaaagtggc tctaatgttg ggaattggaa
cagaaggacc acatgtgggc cttgttcaag 1020 ccagtgaaca tcccaaaata
gaattttact tgaaaaactt tacatcagcc aaagatgttt 1080 tgtttgccat
aaaggaagta ggtttcagag ggggtaattc caatacagga aaagccttga 1140
agcatactgc tcagaaattc ttcacggtag atgctggagt aagaaaaggg atccccaaag
1200 tggtggtggt atttattgat ggttggcctt ctgatgacat cgaggaagca
ggcattgtgg 1260 ccagagagtt tggtgtcaat gtatttatag tttctgtggc
caagcctatc cctgaagaac 1320 tggggatggt
tcaggatgtc acatttgttg acaaggctgt ctgtcggaat aatggcttct 1380
tctcttacca catgcccaac tggtttggca ccacaaaata cgtaaagcct ctggtacaga
1440 agctgtgcac tcatgaacaa atgatgtgca gcaagacctg ttataactca
gtgaacattg 1500 cctttctaat tgatggctcc agcagtgttg gagatagcaa
tttccgcctc atgcttgaat 1560 ttgtttccaa catagccaag acttttgaaa
tctcggacat tggtgccaag atagctgctg 1620 tacagtttac ttatgatcag
cgcacggagt tcagtttcac tgactatagc accaaagaga 1680 atgtcctagc
tgtcatcaga aacatccgct atatgagtgg tggaacagct actggtgatg 1740
ccatttcctt cactgttaga aatgtgtttg gccctataag ggagagcccc aacaagaact
1800 tcctagtaat tgtcacagat gggcagtcct atgatgatgt ccaaggccct
gcagctgctg 1860 cacatgatgc aggaatcact atcttctctg ttggtgtggc
ttgggcacct ctggatgacc 1920 tgaaagatat ggcttctaaa ccgaaggagt
ctcacgcttt cttcacaaga gagttcacag 1980 gattagaacc aattgtttct
gatgtcatca gaggcatttg tagagatttc ttagaatccc 2040 agcaataatg
gtaacatttt gacaactgaa agaaaaagta caaggggatc cagtgtgtaa 2100
attgtattct cataatactg aaatgcttta gcatactaga atcagataca aaactattaa
2160 gtatgtcaac agccatttag gcaaataagc actcctttaa agccgctgcc
ttctggttac 2220 aatttacagt gtactttgtt aaaaacactg ctgaggcttc
ataatcatgg ctcttagaaa 2280 ctcaggaaag aggagataat gtggattaaa
accttaagag ttctaaccat gcctactaaa 2340 tgtacagata tgcaaattcc
atagctcaat aaaagaatct gatacttaga ccaaaaaaaa 2400 aaa 2403 227 550
PRT Homo sapiens 227 Met Ser Ala Ala Trp Ile Pro Ala Leu Gly Leu
Gly Val Cys Leu Leu 1 5 10 15 Leu Leu Pro Gly Pro Ala Gly Ser Glu
Gly Ala Ala Pro Ile Ala Ile 20 25 30 Thr Cys Phe Thr Arg Gly Leu
Asp Ile Arg Lys Glu Lys Ala Asp Val 35 40 45 Leu Cys Pro Gly Gly
Cys Pro Leu Glu Glu Phe Ser Val Tyr Gly Asn 50 55 60 Ile Val Tyr
Ala Ser Val Ser Ser Ile Cys Gly Ala Ala Val His Arg 65 70 75 80 Gly
Val Ile Ser Asn Ser Gly Gly Pro Val Arg Val Tyr Ser Leu Pro 85 90
95 Gly Arg Glu Asn Tyr Ser Ser Val Asp Ala Asn Gly Ile Gln Ser Gln
100 105 110 Met Leu Ser Arg Trp Ser Ala Ser Phe Thr Val Thr Lys Gly
Lys Ser 115 120 125 Ser Thr Gln Glu Ala Thr Gly Gln Ala Val Ser Thr
Ala His Pro Pro 130 135 140 Thr Gly Lys Arg Leu Lys Lys Thr Pro Glu
Lys Lys Thr Gly Asn Lys 145 150 155 160 Asp Cys Lys Ala Asp Ile Ala
Phe Leu Ile Asp Gly Ser Phe Asn Ile 165 170 175 Gly Gln Arg Arg Phe
Asn Leu Gln Lys Asn Phe Val Gly Lys Val Ala 180 185 190 Leu Met Leu
Gly Ile Gly Thr Glu Gly Pro His Val Gly Leu Val Gln 195 200 205 Ala
Ser Glu His Pro Lys Ile Glu Phe Tyr Leu Lys Asn Phe Thr Ser 210 215
220 Ala Lys Asp Val Leu Phe Ala Ile Lys Glu Val Gly Phe Arg Gly Gly
225 230 235 240 Asn Ser Asn Thr Gly Lys Ala Leu Lys His Thr Ala Gln
Lys Phe Phe 245 250 255 Thr Val Asp Ala Gly Val Arg Lys Gly Ile Pro
Lys Val Val Val Val 260 265 270 Phe Ile Asp Gly Trp Pro Ser Asp Asp
Ile Glu Glu Ala Gly Ile Val 275 280 285 Ala Arg Glu Phe Gly Val Asn
Val Phe Ile Val Ser Val Ala Lys Pro 290 295 300 Ile Pro Glu Glu Leu
Gly Met Val Gln Asp Val Thr Phe Val Asp Lys 305 310 315 320 Ala Val
Cys Arg Asn Asn Gly Phe Phe Ser Tyr His Met Pro Asn Trp 325 330 335
Phe Gly Thr Thr Lys Tyr Val Lys Pro Leu Val Gln Lys Leu Cys Thr 340
345 350 His Glu Gln Met Met Cys Ser Lys Thr Cys Tyr Asn Ser Val Asn
Ile 355 360 365 Ala Phe Leu Ile Asp Gly Ser Ser Ser Val Gly Asp Ser
Asn Phe Arg 370 375 380 Leu Met Leu Glu Phe Val Ser Asn Ile Ala Lys
Thr Phe Glu Ile Ser 385 390 395 400 Asp Ile Gly Ala Lys Ile Ala Ala
Val Gln Phe Thr Tyr Asp Gln Arg 405 410 415 Thr Glu Phe Ser Phe Thr
Asp Tyr Ser Thr Lys Glu Asn Val Leu Ala 420 425 430 Val Ile Arg Asn
Ile Arg Tyr Met Ser Gly Gly Thr Ala Thr Gly Asp 435 440 445 Ala Ile
Ser Phe Thr Val Arg Asn Val Phe Gly Pro Ile Arg Glu Ser 450 455 460
Pro Asn Lys Asn Phe Leu Val Ile Val Thr Asp Gly Gln Ser Tyr Asp 465
470 475 480 Asp Val Gln Gly Pro Ala Ala Ala Ala His Asp Ala Gly Ile
Thr Ile 485 490 495 Phe Ser Val Gly Val Ala Trp Ala Pro Leu Asp Asp
Leu Lys Asp Met 500 505 510 Ala Ser Lys Pro Lys Glu Ser His Ala Phe
Phe Thr Arg Glu Phe Thr 515 520 525 Gly Leu Glu Pro Ile Val Ser Asp
Val Ile Arg Gly Ile Cys Arg Asp 530 535 540 Phe Leu Glu Ser Gln Gln
545 550 228 18 DNA Artificial Sequence Description of Artificial
Sequence Synthetic oligonucleotide probe 228 tggtctcgca caccgatc 18
229 18 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide probe 229 ctgctgtcca caggggag 18 230 18
DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide probe 230 ccttgaagca tactgctc 18 231 18
DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide probe 231 gagatagcaa tttccgcc 18 232 18
DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide probe 232 ttcctcaaga gggcagcc 18 233 24
DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide probe 233 cttggcacca atgtccgaga tttc 24
234 45 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide probe 234 gctctgagga aggtgacgcg
cggggcctcc gaacccttgg ccttg 45 235 2586 DNA Homo sapiens 235
cgccgcgctc ccgcacccgc ggcccgccca ccgcgccgct cccgcatctg cacccgcagc
60 ccggcggcct cccggcggga gcgagcagat ccagtccggc ccgcagcgca
actcggtcca 120 gtcggggcgg cggctgcggg cgcagagcgg agatgcagcg
gcttggggcc accctgctgt 180 gcctgctgct ggcggcggcg gtccccacgg
cccccgcgcc cgctccgacg gcgacctcgg 240 ctccagtcaa gcccggcccg
gctctcagct acccgcagga ggaggccacc ctcaatgaga 300 tgttccgcga
ggttgaggaa ctgatggagg acacgcagca caaattgcgc agcgcggtgg 360
aagagatgga ggcagaagaa gctgctgcta aagcatcatc agaagtgaac ctggcaaact
420 tacctcccag ctatcacaat gagaccaaca cagacacgaa ggttggaaat
aataccatcc 480 atgtgcaccg agaaattcac aagataacca acaaccagac
tggacaaatg gtcttttcag 540 agacagttat cacatctgtg ggagacgaag
aaggcagaag gagccacgag tgcatcatcg 600 acgaggactg tgggcccagc
atgtactgcc agtttgccag cttccagtac acctgccagc 660 catgccgggg
ccagaggatg ctctgcaccc gggacagtga gtgctgtgga gaccagctgt 720
gtgtctgggg tcactgcacc aaaatggcca ccaggggcag caatgggacc atctgtgaca
780 accagaggga ctgccagccg gggctgtgct gtgccttcca gagaggcctg
ctgttccctg 840 tgtgcacacc cctgcccgtg gagggcgagc tttgccatga
ccccgccagc cggcttctgg 900 acctcatcac ctgggagcta gagcctgatg
gagccttgga ccgatgccct tgtgccagtg 960 gcctcctctg ccagccccac
agccacagcc tggtgtatgt gtgcaagccg accttcgtgg 1020 ggagccgtga
ccaagatggg gagatcctgc tgcccagaga ggtccccgat gagtatgaag 1080
ttggcagctt catggaggag gtgcgccagg agctggagga cctggagagg agcctgactg
1140 aagagatggc gctgggggag cctgcggctg ccgccgctgc actgctggga
ggggaagaga 1200 tttagatctg gaccaggctg tgggtagatg tgcaatagaa
atagctaatt tatttcccca 1260 ggtgtgtgct ttaggcgtgg gctgaccagg
cttcttccta catcttcttc ccagtaagtt 1320 tcccctctgg cttgacagca
tgaggtgttg tgcatttgtt cagctccccc aggctgttct 1380 ccaggcttca
cagtctggtg cttgggagag tcaggcaggg ttaaactgca ggagcagttt 1440
gccacccctg tccagattat tggctgcttt gcctctacca gttggcagac agccgtttgt
1500 tctacatggc tttgataatt gtttgagggg aggagatgga aacaatgtgg
agtctccctc 1560 tgattggttt tggggaaatg tggagaagag tgccctgctt
tgcaaacatc aacctggcaa 1620 aaatgcaaca aatgaatttt ccacgcagtt
ctttccatgg gcataggtaa gctgtgcctt 1680 cagctgttgc agatgaaatg
ttctgttcac cctgcattac atgtgtttat tcatccagca 1740 gtgttgctca
gctcctacct ctgtgccagg gcagcatttt catatccaag atcaattccc 1800
tctctcagca cagcctgggg agggggtcat tgttctcctc gtccatcagg gatctcagag
1860 gctcagagac tgcaagctgc ttgcccaagt cacacagcta gtgaagacca
gagcagtttc 1920 atctggttgt gactctaagc tcagtgctct ctccactacc
ccacaccagc cttggtgcca 1980 ccaaaagtgc tccccaaaag gaaggagaat
gggatttttc ttgaggcatg cacatctgga 2040 attaaggtca aactaattct
cacatccctc taaaagtaaa ctactgttag gaacagcagt 2100 gttctcacag
tgtggggcag ccgtccttct aatgaagaca atgatattga cactgtccct 2160
ctttggcagt tgcattagta actttgaaag gtatatgact gagcgtagca tacaggttaa
2220 cctgcagaaa cagtacttag gtaattgtag ggcgaggatt ataaatgaaa
tttgcaaaat 2280 cacttagcag caactgaaga caattatcaa ccacgtggag
aaaatcaaac cgagcagggc 2340 tgtgtgaaac atggttgtaa tatgcgactg
cgaacactga actctacgcc actccacaaa 2400 tgatgttttc aggtgtcatg
gactgttgcc accatgtatt catccagagt tcttaaagtt 2460 taaagttgca
catgattgta taagcatgct ttctttgagt tttaaattat gtataaacat 2520
aagttgcatt tagaaatcaa gcataaatca cttcaactgc aaaaaaaaaa aaaaaaaaaa
2580 aaaaaa 2586 236 350 PRT Homo sapiens 236 Met Gln Arg Leu Gly
Ala Thr Leu Leu Cys Leu Leu Leu Ala Ala Ala 1 5 10 15 Val Pro Thr
Ala Pro Ala Pro Ala Pro Thr Ala Thr Ser Ala Pro Val 20 25 30 Lys
Pro Gly Pro Ala Leu Ser Tyr Pro Gln Glu Glu Ala Thr Leu Asn 35 40
45 Glu Met Phe Arg Glu Val Glu Glu Leu Met Glu Asp Thr Gln His Lys
50 55 60 Leu Arg Ser Ala Val Glu Glu Met Glu Ala Glu Glu Ala Ala
Ala Lys 65 70 75 80 Ala Ser Ser Glu Val Asn Leu Ala Asn Leu Pro Pro
Ser Tyr His Asn 85 90 95 Glu Thr Asn Thr Asp Thr Lys Val Gly Asn
Asn Thr Ile His Val His 100 105 110 Arg Glu Ile His Lys Ile Thr Asn
Asn Gln Thr Gly Gln Met Val Phe 115 120 125 Ser Glu Thr Val Ile Thr
Ser Val Gly Asp Glu Glu Gly Arg Arg Ser 130 135 140 His Glu Cys Ile
Ile Asp Glu Asp Cys Gly Pro Ser Met Tyr Cys Gln 145 150 155 160 Phe
Ala Ser Phe Gln Tyr Thr Cys Gln Pro Cys Arg Gly Gln Arg Met 165 170
175 Leu Cys Thr Arg Asp Ser Glu Cys Cys Gly Asp Gln Leu Cys Val Trp
180 185 190 Gly His Cys Thr Lys Met Ala Thr Arg Gly Ser Asn Gly Thr
Ile Cys 195 200 205 Asp Asn Gln Arg Asp Cys Gln Pro Gly Leu Cys Cys
Ala Phe Gln Arg 210 215 220 Gly Leu Leu Phe Pro Val Cys Thr Pro Leu
Pro Val Glu Gly Glu Leu 225 230 235 240 Cys His Asp Pro Ala Ser Arg
Leu Leu Asp Leu Ile Thr Trp Glu Leu 245 250 255 Glu Pro Asp Gly Ala
Leu Asp Arg Cys Pro Cys Ala Ser Gly Leu Leu 260 265 270 Cys Gln Pro
His Ser His Ser Leu Val Tyr Val Cys Lys Pro Thr Phe 275 280 285 Val
Gly Ser Arg Asp Gln Asp Gly Glu Ile Leu Leu Pro Arg Glu Val 290 295
300 Pro Asp Glu Tyr Glu Val Gly Ser Phe Met Glu Glu Val Arg Gln Glu
305 310 315 320 Leu Glu Asp Leu Glu Arg Ser Leu Thr Glu Glu Met Ala
Leu Gly Glu 325 330 335 Pro Ala Ala Ala Ala Ala Ala Leu Leu Gly Gly
Glu Glu Ile 340 345 350 237 17 DNA Artificial Sequence Synthetic
oligonucleotide probe 237 ggagctgcac cccttgc 17 238 49 DNA
Artificial Sequence Synthetic Oligonucleotide Probe 238 ggaggactgt
gccaccatga gagactcttc aaacccaagg caaaattgg 49 239 24 DNA Artificial
Sequence Synthetic Oligonucleotide Probe 239 gcagagcgga gatgcagcgg
cttg 24 240 18 DNA Artificial Sequence Synthetic Oligonucleotide
Probe 240 ttggcagctt catggagg 18 241 18 DNA Artificial Sequence
Synthetic Oligonucleotide Probe 241 cctgggcaaa aatgcaac 18 242 24
DNA Artificial Sequence Synthetic Oligonucleotide Probe 242
ctccagctcc tggcgcacct cctc 24 243 45 DNA Artificial Sequence
Synthetic Oligonucleotide Probe 243 ggctctcagc taccgcgcag
gagcgaggcc accctcaatg agatg 45 244 3679 DNA Homo Sapien 244
aaggaggctg ggaggaaaga ggtaagaaag gttagagaac ctacctcaca 50
tctctctggg ctcagaagga ctctgaagat aacaataatt tcagcccatc 100
cactctcctt ccctcccaaa cacacatgtg catgtacaca cacacataca 150
cacacataca ccttcctctc cttcactgaa gactcacagt cactcactct 200
gtgagcaggt catagaaaag gacactaaag ccttaaggac aggcctggcc 250
attacctctg cagctccttt ggcttgttga gtcaaaaaac atgggagggg 300
ccaggcacgg tgactcacac ctgtaatccc agcattttgg gagaccgagg 350
tgagcagatc acttgaggtc aggagttcga gaccagcctg gccaacatgg 400
agaaaccccc atctctacta aaaatacaaa aattagccag gagtggtggc 450
aggtgcctgt aatcccagct actcaggtgg ctgagccagg agaatcgctt 500
gaatccagga ggcggaggat gcagtcagct gagtgcaccg ctgcactcca 550
gcctgggtga cagaatgaga ctctgtctca aacaaacaaa cacgggagga 600
ggggtagata ctgcttctct gcaacctcct taactctgca tcctcttctt 650
ccagggctgc ccctgatggg gcctggcaat gactgagcag gcccagcccc 700
agaggacaag gaagagaagg catattgagg agggcaagaa gtgacgcccg 750
gtgtagaatg actgccctgg gagggtggtt ccttgggccc tggcagggtt 800
gctgaccctt accctgcaaa acacaaagag caggactcca gactctcctt 850
gtgaatggtc ccctgccctg cagctccacc atgaggcttc tcgtggcccc 900
actcttgcta gcttgggtgg ctggtgccac tgccactgtg cccgtggtac 950
cctggcatgt tccctgcccc cctcagtgtg cctgccagat ccggccctgg 1000
tatacgcccc gctcgtccta ccgcgaggct accactgtgg actgcaatga 1050
cctattcctg acggcagtcc ccccggcact ccccgcaggc acacagaccc 1100
tgctcctgca gagcaacagc attgtccgtg tggaccagag tgagctgggc 1150
tacctggcca atctcacaga gctggacctg tcccagaaca gcttttcgga 1200
tgcccgagac tgtgatttcc atgccctgcc ccagctgctg agcctgcacc 1250
tagaggagaa ccagctgacc cggctggagg accacagctt tgcagggctg 1300
gccagcctac aggaactcta tctcaaccac aaccagctct accgcatcgc 1350
ccccagggcc ttttctggcc tcagcaactt gctgcggctg cacctcaact 1400
ccaacctcct gagggccatt gacagccgct ggtttgaaat gctgcccaac 1450
ttggagatac tcatgattgg cggcaacaag gtagatgcca tcctggacat 1500
gaacttccgg cccctggcca acctgcgtag cctggtgcta gcaggcatga 1550
acctgcggga gatctccgac tatgccctgg aggggctgca aagcctggag 1600
agcctctcct tctatgacaa ccagctggcc cgggtgccca ggcgggcact 1650
ggaacaggtg cccgggctca agttcctaga cctcaacaag aacccgctcc 1700
agcgggtagg gccgggggac tttgccaaca tgctgcacct taaggagctg 1750
ggactgaaca acatggagga gctggtctcc atcgacaagt ttgccctggt 1800
gaacctcccc gagctgacca agctggacat caccaataac ccacggctgt 1850
ccttcatcca cccccgcgcc ttccaccacc tgccccagat ggagaccctc 1900
atgctcaaca acaacgctct cagtgccttg caccagcaga cggtggagtc 1950
cctgcccaac ctgcaggagg taggtctcca cggcaacccc atccgctgtg 2000
actgtgtcat ccgctgggcc aatgccacgg gcacccgtgt ccgcttcatc 2050
gagccgcaat ccaccctgtg tgcggagcct ccggacctcc agcgcctccc 2100
ggtccgtgag gtgcccttcc gggagatgac ggaccactgt ttgcccctca 2150
tctccccacg aagcttcccc ccaagcctcc aggtagccag tggagagagc 2200
atggtgctgc attgccgggc actggccgaa cccgaacccg agatctactg 2250
ggtcactcca gctgggcttc gactgacacc tgcccatgca ggcaggaggt 2300
accgggtgta ccccgagggg accctggagc tgcggagggt gacagcagaa 2350
gaggcagggc tatacacctg tgtggcccag aacctggtgg gggctgacac 2400
taagacggtt agtgtggttg tgggccgtgc tctcctccag ccaggcaggg 2450
acgaaggaca ggggctggag ctccgggtgc aggagaccca cccctatcac 2500
atcctgctat cttgggtcac cccacccaac acagtgtcca ccaacctcac 2550
ctggtccagt gcctcctccc tccggggcca gggggccaca gctctggccc 2600
gcctgcctcg gggaacccac agctacaaca ttacccgcct ccttcaggcc 2650
acggagtact gggcctgcct gcaagtggcc tttgctgatg cccacaccca 2700
gttggcttgt gtatgggcca ggaccaaaga ggccacttct tgccacagag 2750
ccttagggga tcgtcctggg ctcattgcca tcctggctct cgctgtcctt 2800
ctcctggcag ctgggctagc ggcccacctt ggcacaggcc aacccaggaa 2850
gggtgtgggt gggaggcggc ctctccctcc agcctgggct ttctggggct 2900
ggagtgcccc ttctgtccgg gttgtgtctg ctcccctcgt cctgccctgg 2950
aatccaggga ggaagctgcc cagatcctca gaaggggaga cactgttgcc 3000
accattgtct caaaattctt gaagctcagc ctgttctcag cagtagagaa 3050
atcactagga ctacttttta ccaaaagaga agcagtctgg gccagatgcc 3100
ctgccaggaa agggacatgg acccacgtgc ttgaggcctg
gcagctgggc 3150 caagacagat ggggctttgt ggccctgggg gtgcttctgc
agccttgaaa 3200 aagttgccct tacctcctag ggtcacctct gctgccattc
tgaggaacat 3250 ctccaaggaa caggagggac tttggctaga gcctcctgcc
tccccatctt 3300 ctctctgccc agaggctcct gggcctggct tggctgtccc
ctacctgtgt 3350 ccccgggctg caccccttcc tcttctcttt ctctgtacag
tctcagttgc 3400 ttgctcttgt gcctcctggg caagggctga aggaggccac
tccatctcac 3450 ctcggggggc tgccctcaat gtgggagtga ccccagccag
atctgaagga 3500 catttgggag agggatgccc aggaacgcct catctcagca
gcctgggctc 3550 ggcattccga agctgacttt ctataggcaa ttttgtacct
ttgtggagaa 3600 atgtgtcacc tcccccaacc cgattcactc ttttctcctg
ttttgtaaaa 3650 aataaaaata aataataaca ataaaaaaa 3679 245 713 PRT
Homo Sapien 245 Met Arg Leu Leu Val Ala Pro Leu Leu Leu Ala Trp Val
Ala Gly 1 5 10 15 Ala Thr Ala Thr Val Pro Val Val Pro Trp His Val
Pro Cys Pro 20 25 30 Pro Gln Cys Ala Cys Gln Ile Arg Pro Trp Tyr
Thr Pro Arg Ser 35 40 45 Ser Tyr Arg Glu Ala Thr Thr Val Asp Cys
Asn Asp Leu Phe Leu 50 55 60 Thr Ala Val Pro Pro Ala Leu Pro Ala
Gly Thr Gln Thr Leu Leu 65 70 75 Leu Gln Ser Asn Ser Ile Val Arg
Val Asp Gln Ser Glu Leu Gly 80 85 90 Tyr Leu Ala Asn Leu Thr Glu
Leu Asp Leu Ser Gln Asn Ser Phe 95 100 105 Ser Asp Ala Arg Asp Cys
Asp Phe His Ala Leu Pro Gln Leu Leu 110 115 120 Ser Leu His Leu Glu
Glu Asn Gln Leu Thr Arg Leu Glu Asp His 125 130 135 Ser Phe Ala Gly
Leu Ala Ser Leu Gln Glu Leu Tyr Leu Asn His 140 145 150 Asn Gln Leu
Tyr Arg Ile Ala Pro Arg Ala Phe Ser Gly Leu Ser 155 160 165 Asn Leu
Leu Arg Leu His Leu Asn Ser Asn Leu Leu Arg Ala Ile 170 175 180 Asp
Ser Arg Trp Phe Glu Met Leu Pro Asn Leu Glu Ile Leu Met 185 190 195
Ile Gly Gly Asn Lys Val Asp Ala Ile Leu Asp Met Asn Phe Arg 200 205
210 Pro Leu Ala Asn Leu Arg Ser Leu Val Leu Ala Gly Met Asn Leu 215
220 225 Arg Glu Ile Ser Asp Tyr Ala Leu Glu Gly Leu Gln Ser Leu Glu
230 235 240 Ser Leu Ser Phe Tyr Asp Asn Gln Leu Ala Arg Val Pro Arg
Arg 245 250 255 Ala Leu Glu Gln Val Pro Gly Leu Lys Phe Leu Asp Leu
Asn Lys 260 265 270 Asn Pro Leu Gln Arg Val Gly Pro Gly Asp Phe Ala
Asn Met Leu 275 280 285 His Leu Lys Glu Leu Gly Leu Asn Asn Met Glu
Glu Leu Val Ser 290 295 300 Ile Asp Lys Phe Ala Leu Val Asn Leu Pro
Glu Leu Thr Lys Leu 305 310 315 Asp Ile Thr Asn Asn Pro Arg Leu Ser
Phe Ile His Pro Arg Ala 320 325 330 Phe His His Leu Pro Gln Met Glu
Thr Leu Met Leu Asn Asn Asn 335 340 345 Ala Leu Ser Ala Leu His Gln
Gln Thr Val Glu Ser Leu Pro Asn 350 355 360 Leu Gln Glu Val Gly Leu
His Gly Asn Pro Ile Arg Cys Asp Cys 365 370 375 Val Ile Arg Trp Ala
Asn Ala Thr Gly Thr Arg Val Arg Phe Ile 380 385 390 Glu Pro Gln Ser
Thr Leu Cys Ala Glu Pro Pro Asp Leu Gln Arg 395 400 405 Leu Pro Val
Arg Glu Val Pro Phe Arg Glu Met Thr Asp His Cys 410 415 420 Leu Pro
Leu Ile Ser Pro Arg Ser Phe Pro Pro Ser Leu Gln Val 425 430 435 Ala
Ser Gly Glu Ser Met Val Leu His Cys Arg Ala Leu Ala Glu 440 445 450
Pro Glu Pro Glu Ile Tyr Trp Val Thr Pro Ala Gly Leu Arg Leu 455 460
465 Thr Pro Ala His Ala Gly Arg Arg Tyr Arg Val Tyr Pro Glu Gly 470
475 480 Thr Leu Glu Leu Arg Arg Val Thr Ala Glu Glu Ala Gly Leu Tyr
485 490 495 Thr Cys Val Ala Gln Asn Leu Val Gly Ala Asp Thr Lys Thr
Val 500 505 510 Ser Val Val Val Gly Arg Ala Leu Leu Gln Pro Gly Arg
Asp Glu 515 520 525 Gly Gln Gly Leu Glu Leu Arg Val Gln Glu Thr His
Pro Tyr His 530 535 540 Ile Leu Leu Ser Trp Val Thr Pro Pro Asn Thr
Val Ser Thr Asn 545 550 555 Leu Thr Trp Ser Ser Ala Ser Ser Leu Arg
Gly Gln Gly Ala Thr 560 565 570 Ala Leu Ala Arg Leu Pro Arg Gly Thr
His Ser Tyr Asn Ile Thr 575 580 585 Arg Leu Leu Gln Ala Thr Glu Tyr
Trp Ala Cys Leu Gln Val Ala 590 595 600 Phe Ala Asp Ala His Thr Gln
Leu Ala Cys Val Trp Ala Arg Thr 605 610 615 Lys Glu Ala Thr Ser Cys
His Arg Ala Leu Gly Asp Arg Pro Gly 620 625 630 Leu Ile Ala Ile Leu
Ala Leu Ala Val Leu Leu Leu Ala Ala Gly 635 640 645 Leu Ala Ala His
Leu Gly Thr Gly Gln Pro Arg Lys Gly Val Gly 650 655 660 Gly Arg Arg
Pro Leu Pro Pro Ala Trp Ala Phe Trp Gly Trp Ser 665 670 675 Ala Pro
Ser Val Arg Val Val Ser Ala Pro Leu Val Leu Pro Trp 680 685 690 Asn
Pro Gly Arg Lys Leu Pro Arg Ser Ser Glu Gly Glu Thr Leu 695 700 705
Leu Pro Pro Leu Ser Gln Asn Ser 710 246 22 DNA Artificial Sequence
Synthetic Oligonucleotide Probe 246 aacaaggtaa gatgccatcc tg 22 247
24 DNA Artificial Sequence Synthetic Oligonucleotide Probe 247
aaacttgtcg atggagacca gctc 24 248 45 DNA Artificial Sequence
Synthetic Oligonucleotide Probe 248 aggggctgca aagcctggag
agcctctcct tctatgacaa ccagc 45 249 3401 DNA Homo Sapien 249
gcaagccaag gcgctgtttg agaaggtgaa gaagttccgg acccatgtgg 50
aggaggggga cattgtgtac cgcctctaca tgcggcagac catcatcaag 100
gtgatcaagt tcatcctcat catctgctac accgtctact acgtgcacaa 150
catcaagttc gacgtggact gcaccgtgga cattgagagc ctgacgggct 200
accgcaccta ccgctgtgcc caccccctgg ccacactctt caagatcctg 250
gcgtccttct acatcagcct agtcatcttc tacggcctca tctgcatgta 300
cacactgtgg tggatgctac ggcgctccct caagaagtac tcgtttgagt 350
cgatccgtga ggagagcagc tacagcgaca tccccgacgt caagaacgac 400
ttcgccttca tgctgcacct cattgaccaa tacgacccgc tctactccaa 450
gcgcttcgcc gtcttcctgt cggaggtgag tgagaacaag ctgcggcagc 500
tgaacctcaa caacgagtgg acgctggaca agctccggca gcggctcacc 550
aagaacgcgc aggacaagct ggagctgcac ctgttcatgc tcagtggcat 600
ccctgacact gtgtttgacc tggtggagct ggaggtcctc aagctggagc 650
tgatccccga cgtgaccatc ccgcccagca ttgcccagct cacgggcctc 700
aaggagctgt ggctctacca cacagcggcc aagattgaag cgcctgcgct 750
ggccttcctg cgcgagaacc tgcgggcgct gcacatcaag ttcaccgaca 800
tcaaggagat cccgctgtgg atctatagcc tgaagacact ggaggagctg 850
cacctgacgg gcaacctgag cgcggagaac aaccgctaca tcgtcatcga 900
cgggctgcgg gagctcaaac gcctcaaggt gctgcggctc aagagcaacc 950
taagcaagct gccacaggtg gtcacagatg tgggcgtgca cctgcagaag 1000
ctgtccatca acaatgaggg caccaagctc atcgtcctca acagcctcaa 1050
gaagatggcg aacctgactg agctggagct gatccgctgc gacctggagc 1100
gcatccccca ctccatcttc agcctccaca acctgcagga gattgacctc 1150
aaggacaaca acctcaagac catcgaggag atcatcagct tccagcacct 1200
gcaccgcctc acctgcctta agctgtggta caaccacatc gcctacatcc 1250
ccatccagat cggcaacctc accaacctgg agcgcctcta cctgaaccgc 1300
aacaagatcg agaagatccc cacccagctc ttctactgcc gcaagctgcg 1350
ctacctggac ctcagccaca acaacctgac cttcctccct gccgacatcg 1400
gcctcctgca gaacctccag aacctagcca tcacggccaa ccggatcgag 1450
acgctccctc cggagctctt ccagtgccgg aagctgcggg ccctgcacct 1500
gggcaacaac gtgctgcagt cactgccctc cagggtgggc gagctgacca 1550
acctgacgca gatcgagctg cggggcaacc ggctggagtg cctgcctgtg 1600
gagctgggcg agtgcccact gctcaagcgc agcggcttgg tggtggagga 1650
ggacctgttc aacacactgc cacccgaggt gaaggagcgg ctgtggaggg 1700
ctgacaagga gcaggcctga gcgaggccgg cccagcacag caagcagcag 1750
gaccgctgcc cagtcctcag gcccggaggg gcaggcctag cttctcccag 1800
aactcccgga cagccaggac agcctcgcgg ctgggcagga gcctggggcc 1850
gcttgtgagt caggccagag cgagaggaca gtatctgtgg ggctggcccc 1900
ttttctccct ctgagactca cgtcccccag ggcaagtgct tgtggaggag 1950
agcaagtctc aagagcgcag tatttggata atcagggtct cctccctgga 2000
ggccagctct gccccagggg ctgagctgcc accagaggtc ctgggaccct 2050
cactttagtt cttggtattt atttttctcc atctcccacc tccttcatcc 2100
agataactta tacattccca agaaagttca gcccagatgg aaggtgttca 2150
gggaaaggtg ggctgccttt tccccttgtc cttatttagc gatgccgccg 2200
ggcatttaac acccacctgg acttcagcag agtggtccgg ggcgaaccag 2250
ccatgggacg gtcacccagc agtgccgggc tgggctctgc ggtgcggtcc 2300
acgggagagc aggcctccag ctggaaaggc caggcctgga gcttgcctct 2350
tcagtttttg tggcagtttt agttttttgt tttttttttt tttaatcaaa 2400
aaacaatttt ttttaaaaaa aagctttgaa aatggatggt ttgggtatta 2450
aaaagaaaaa aaaaacttaa aaaaaaaaag acactaacgg ccagtgagtt 2500
ggagtctcag ggcagggtgg cagtttccct tgagcaaagc agccagacgt 2550
tgaactgtgt ttcctttccc tgggcgcagg gtgcagggtg tcttccggat 2600
ctggtgtgac cttggtccag gagttctatt tgttcctggg gagggaggtt 2650
tttttgtttg ttttttgggt ttttttggtg tcttgttttc tttctcctcc 2700
atgtgtcttg gcaggcactc atttctgtgg ctgtcggcca gagggaatgt 2750
tctggagctg ccaaggaggg aggagactcg ggttggctaa tccccggatg 2800
aacggtgctc cattcgcacc tcccctcctc gtgcctgccc tgcctctcca 2850
cgcacagtgt taaggagcca agaggagcca cttcgcccag actttgtttc 2900
cccacctcct gcggcatggg tgtgtccagt gccaccgctg gcctccgctg 2950
cttccatcag ccctgtcgcc acctggtcct tcatgaagag cagacactta 3000
gaggctggtc gggaatgggg aggtcgcccc tgggagggca ggcgttggtt 3050
ccaagccggt tcccgtccct ggcgcctgga gtgcacacag cccagtcggc 3100
acctggtggc tggaagccaa cctgctttag atcactcggg tccccacctt 3150
agaagggtcc ccgccttaga tcaatcacgt ggacactaag gcacgtttta 3200
gagtctcttg tcttaatgat tatgtccatc cgtctgtccg tccatttgtg 3250
ttttctgcgt cgtgtcattg gatataatcc tcagaaataa tgcacactag 3300
cctctgacaa ccatgaagca aaaatccgtt acatgtgggt ctgaacttgt 3350
agactcggtc acagtatcaa ataaaatcta taacagaaaa aaaaaaaaaa 3400 a 3401
250 546 PRT Homo Sapien 250 Met Arg Gln Thr Ile Ile Lys Val Ile Lys
Phe Ile Leu Ile Ile 1 5 10 15 Cys Tyr Thr Val Tyr Tyr Val His Asn
Ile Lys Phe Asp Val Asp 20 25 30 Cys Thr Val Asp Ile Glu Ser Leu
Thr Gly Tyr Arg Thr Tyr Arg 35 40 45 Cys Ala His Pro Leu Ala Thr
Leu Phe Lys Ile Leu Ala Ser Phe 50 55 60 Tyr Ile Ser Leu Val Ile
Phe Tyr Gly Leu Ile Cys Met Tyr Thr 65 70 75 Leu Trp Trp Met Leu
Arg Arg Ser Leu Lys Lys Tyr Ser Phe Glu 80 85 90 Ser Ile Arg Glu
Glu Ser Ser Tyr Ser Asp Ile Pro Asp Val Lys 95 100 105 Asn Asp Phe
Ala Phe Met Leu His Leu Ile Asp Gln Tyr Asp Pro 110 115 120 Leu Tyr
Ser Lys Arg Phe Ala Val Phe Leu Ser Glu Val Ser Glu 125 130 135 Asn
Lys Leu Arg Gln Leu Asn Leu Asn Asn Glu Trp Thr Leu Asp 140 145 150
Lys Leu Arg Gln Arg Leu Thr Lys Asn Ala Gln Asp Lys Leu Glu 155 160
165 Leu His Leu Phe Met Leu Ser Gly Ile Pro Asp Thr Val Phe Asp 170
175 180 Leu Val Glu Leu Glu Val Leu Lys Leu Glu Leu Ile Pro Asp Val
185 190 195 Thr Ile Pro Pro Ser Ile Ala Gln Leu Thr Gly Leu Lys Glu
Leu 200 205 210 Trp Leu Tyr His Thr Ala Ala Lys Ile Glu Ala Pro Ala
Leu Ala 215 220 225 Phe Leu Arg Glu Asn Leu Arg Ala Leu His Ile Lys
Phe Thr Asp 230 235 240 Ile Lys Glu Ile Pro Leu Trp Ile Tyr Ser Leu
Lys Thr Leu Glu 245 250 255 Glu Leu His Leu Thr Gly Asn Leu Ser Ala
Glu Asn Asn Arg Tyr 260 265 270 Ile Val Ile Asp Gly Leu Arg Glu Leu
Lys Arg Leu Lys Val Leu 275 280 285 Arg Leu Lys Ser Asn Leu Ser Lys
Leu Pro Gln Val Val Thr Asp 290 295 300 Val Gly Val His Leu Gln Lys
Leu Ser Ile Asn Asn Glu Gly Thr 305 310 315 Lys Leu Ile Val Leu Asn
Ser Leu Lys Lys Met Ala Asn Leu Thr 320 325 330 Glu Leu Glu Leu Ile
Arg Cys Asp Leu Glu Arg Ile Pro His Ser 335 340 345 Ile Phe Ser Leu
His Asn Leu Gln Glu Ile Asp Leu Lys Asp Asn 350 355 360 Asn Leu Lys
Thr Ile Glu Glu Ile Ile Ser Phe Gln His Leu His 365 370 375 Arg Leu
Thr Cys Leu Lys Leu Trp Tyr Asn His Ile Ala Tyr Ile 380 385 390 Pro
Ile Gln Ile Gly Asn Leu Thr Asn Leu Glu Arg Leu Tyr Leu 395 400 405
Asn Arg Asn Lys Ile Glu Lys Ile Pro Thr Gln Leu Phe Tyr Cys 410 415
420 Arg Lys Leu Arg Tyr Leu Asp Leu Ser His Asn Asn Leu Thr Phe 425
430 435 Leu Pro Ala Asp Ile Gly Leu Leu Gln Asn Leu Gln Asn Leu Ala
440 445 450 Ile Thr Ala Asn Arg Ile Glu Thr Leu Pro Pro Glu Leu Phe
Gln 455 460 465 Cys Arg Lys Leu Arg Ala Leu His Leu Gly Asn Asn Val
Leu Gln 470 475 480 Ser Leu Pro Ser Arg Val Gly Glu Leu Thr Asn Leu
Thr Gln Ile 485 490 495 Glu Leu Arg Gly Asn Arg Leu Glu Cys Leu Pro
Val Glu Leu Gly 500 505 510 Glu Cys Pro Leu Leu Lys Arg Ser Gly Leu
Val Val Glu Glu Asp 515 520 525 Leu Phe Asn Thr Leu Pro Pro Glu Val
Lys Glu Arg Leu Trp Arg 530 535 540 Ala Asp Lys Glu Gln Ala 545 251
20 DNA Artificial Sequence Synthetic Oligonucleotide Probe 251
caacaatgag ggcaccaagc 20 252 24 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 252 gatggctagg ttctggaggt tctg 24 253 47 DNA
Artificial Sequence Synthetic Oligonucleotide Probe 253 caacctgcag
gagattgacc tcaaggacaa caacctcaag accatcg 47 254 1650 DNA Homo
Sapien 254 gcctgttgct gatgctgccg tgcggtactt gtcatggagc tggcactgcg
50 gcgctctccc gtcccgcggt ggttgctgct gctgccgctg ctgctgggcc 100
tgaacgcagg agctgtcatt gactggccca cagaggaggg caaggaagta 150
tgggattatg tgacggtccg caaggatgcc tacatgttct ggtggctcta 200
ttatgccacc aactcctgca agaacttctc agaactgccc ctggtcatgt 250
ggcttcaggg cggtccaggc ggttctagca ctggatttgg aaactttgag 300
gaaattgggc cccttgacag tgatctcaaa ccacggaaaa ccacctggct 350
ccaggctgcc agtctcctat ttgtggataa tcccgtgggc actgggttca 400
gttatgtgaa tggtagtggt gcctatgcca aggacctggc tatggtggct 450
tcagacatga tggttctcct gaagaccttc ttcagttgcc acaaagaatt 500
ccagacagtt ccattctaca ttttctcaga gtcctatgga ggaaaaatgg 550
cagctggcat tggtctagag ctttataagg ccattcagcg agggaccatc 600
aagtgcaact ttgcgggggt tgccttgggt gattcctgga tctcccctgt 650
tgattcggtg ctctcctggg gaccttacct gtacagcatg tctcttctcg 700
aagacaaagg tctggcagag gtgtctaagg ttgcagagca agtactgaat 750
gccgtaaata aggggctcta cagagaggcc acagagctgt gggggaaagc 800
agaaatgatc attgaacaga acacagatgg ggtgaacttc tataacatct 850
taactaaaag cactcccacg tctacaatgg agtcgagtct agaattcaca 900
cagagccacc tagtttgtct ttgtcagcgc cacgtgagac acctacaacg 950
agatgcctta agccagctca tgaatggccc catcagaaag
aagctcaaaa 1000 ttattcctga ggatcaatcc tggggaggcc aggctaccaa
cgtctttgtg 1050 aacatggagg aggacttcat gaagccagtc attagcattg
tggacgagtt 1100 gctggaggca gggatcaacg tgacggtgta taatggacag
ctggatctca 1150 tcgtagatac catgggtcag gaggcctggg tgcggaaact
gaagtggcca 1200 gaactgccta aattcagtca gctgaagtgg aaggccctgt
acagtgaccc 1250 taaatctttg gaaacatctg cttttgtcaa gtcctacaag
aaccttgctt 1300 tctactggat tctgaaagct ggtcatatgg ttccttctga
ccaaggggac 1350 atggctctga agatgatgag actggtgact cagcaagaat
aggatggatg 1400 gggctggaga tgagctggtt tggccttggg gcacagagct
gagctgaggc 1450 cgctgaagct gtaggaagcg ccattcttcc ctgtatctaa
ctggggctgt 1500 gatcaagaag gttctgacca gcttctgcag aggataaaat
cattgtctct 1550 ggaggcaatt tggaaattat ttctgcttct taaaaaaacc
taagattttt 1600 taaaaaattg atttgttttg atcaaaataa aggatgataa
tagatattaa 1650 255 452 PRT Homo Sapien 255 Met Glu Leu Ala Leu Arg
Arg Ser Pro Val Pro Arg Trp Leu Leu 1 5 10 15 Leu Leu Pro Leu Leu
Leu Gly Leu Asn Ala Gly Ala Val Ile Asp 20 25 30 Trp Pro Thr Glu
Glu Gly Lys Glu Val Trp Asp Tyr Val Thr Val 35 40 45 Arg Lys Asp
Ala Tyr Met Phe Trp Trp Leu Tyr Tyr Ala Thr Asn 50 55 60 Ser Cys
Lys Asn Phe Ser Glu Leu Pro Leu Val Met Trp Leu Gln 65 70 75 Gly
Gly Pro Gly Gly Ser Ser Thr Gly Phe Gly Asn Phe Glu Glu 80 85 90
Ile Gly Pro Leu Asp Ser Asp Leu Lys Pro Arg Lys Thr Thr Trp 95 100
105 Leu Gln Ala Ala Ser Leu Leu Phe Val Asp Asn Pro Val Gly Thr 110
115 120 Gly Phe Ser Tyr Val Asn Gly Ser Gly Ala Tyr Ala Lys Asp Leu
125 130 135 Ala Met Val Ala Ser Asp Met Met Val Leu Leu Lys Thr Phe
Phe 140 145 150 Ser Cys His Lys Glu Phe Gln Thr Val Pro Phe Tyr Ile
Phe Ser 155 160 165 Glu Ser Tyr Gly Gly Lys Met Ala Ala Gly Ile Gly
Leu Glu Leu 170 175 180 Tyr Lys Ala Ile Gln Arg Gly Thr Ile Lys Cys
Asn Phe Ala Gly 185 190 195 Val Ala Leu Gly Asp Ser Trp Ile Ser Pro
Val Asp Ser Val Leu 200 205 210 Ser Trp Gly Pro Tyr Leu Tyr Ser Met
Ser Leu Leu Glu Asp Lys 215 220 225 Gly Leu Ala Glu Val Ser Lys Val
Ala Glu Gln Val Leu Asn Ala 230 235 240 Val Asn Lys Gly Leu Tyr Arg
Glu Ala Thr Glu Leu Trp Gly Lys 245 250 255 Ala Glu Met Ile Ile Glu
Gln Asn Thr Asp Gly Val Asn Phe Tyr 260 265 270 Asn Ile Leu Thr Lys
Ser Thr Pro Thr Ser Thr Met Glu Ser Ser 275 280 285 Leu Glu Phe Thr
Gln Ser His Leu Val Cys Leu Cys Gln Arg His 290 295 300 Val Arg His
Leu Gln Arg Asp Ala Leu Ser Gln Leu Met Asn Gly 305 310 315 Pro Ile
Arg Lys Lys Leu Lys Ile Ile Pro Glu Asp Gln Ser Trp 320 325 330 Gly
Gly Gln Ala Thr Asn Val Phe Val Asn Met Glu Glu Asp Phe 335 340 345
Met Lys Pro Val Ile Ser Ile Val Asp Glu Leu Leu Glu Ala Gly 350 355
360 Ile Asn Val Thr Val Tyr Asn Gly Gln Leu Asp Leu Ile Val Asp 365
370 375 Thr Met Gly Gln Glu Ala Trp Val Arg Lys Leu Lys Trp Pro Glu
380 385 390 Leu Pro Lys Phe Ser Gln Leu Lys Trp Lys Ala Leu Tyr Ser
Asp 395 400 405 Pro Lys Ser Leu Glu Thr Ser Ala Phe Val Lys Ser Tyr
Lys Asn 410 415 420 Leu Ala Phe Tyr Trp Ile Leu Lys Ala Gly His Met
Val Pro Ser 425 430 435 Asp Gln Gly Asp Met Ala Leu Lys Met Met Arg
Leu Val Thr Gln 440 445 450 Gln Glu 256 1100 DNA Homo Sapien 256
ggccgcggga gaggaggcca tgggcgcgcg cggggcgctg ctgctggcgc 50
tgctgctggc tcgggctgga ctcaggaagc cggagtcgca ggaggcggcg 100
ccgttatcag gaccatgcgg ccgacgggtc atcacgtcgc gcatcgtggg 150
tggagaggac gccgaactcg ggcgttggcc gtggcagggg agcctgcgcc 200
tgtgggattc ccacgtatgc ggagtgagcc tgctcagcca ccgctgggca 250
ctcacggcgg cgcactgctt tgaaacctat agtgacctta gtgatccctc 300
cgggtggatg gtccagtttg gccagctgac ttccatgcca tccttctgga 350
gcctgcaggc ctactacacc cgttacttcg tatcgaatat ctatctgagc 400
cctcgctacc tggggaattc accctatgac attgccttgg tgaagctgtc 450
tgcacctgtc acctacacta aacacatcca gcccatctgt ctccaggcct 500
ccacatttga gtttgagaac cggacagact gctgggtgac tggctggggg 550
tacatcaaag aggatgaggc actgccatct ccccacaccc tccaggaagt 600
tcaggtcgcc atcataaaca actctatgtg caaccacctc ttcctcaagt 650
acagtttccg caaggacatc tttggagaca tggtttgtgc tggcaacgcc 700
caaggcggga aggatgcctg cttcggtgac tcaggtggac ccttggcctg 750
taacaagaat ggactgtggt atcagattgg agtcgtgagc tggggagtgg 800
gctgtggtcg gcccaatcgg cccggtgtct acaccaatat cagccaccac 850
tttgagtgga tccagaagct gatggcccag agtggcatgt cccagccaga 900
cccctcctgg ccactactct ttttccctct tctctgggct ctcccactcc 950
tggggccggt ctgagcctac ctgagcccat gcagcctggg gccactgcca 1000
agtcaggccc tggttctctt ctgtcttgtt tggtaataaa cacattccag 1050
ttgatgcctt gcagggcatt cttcaaaaaa aaaaaaaaaa aaaaaaaaaa 1100 257 314
PRT Homo Sapien 257 Met Gly Ala Arg Gly Ala Leu Leu Leu Ala Leu Leu
Leu Ala Arg 1 5 10 15 Ala Gly Leu Arg Lys Pro Glu Ser Gln Glu Ala
Ala Pro Leu Ser 20 25 30 Gly Pro Cys Gly Arg Arg Val Ile Thr Ser
Arg Ile Val Gly Gly 35 40 45 Glu Asp Ala Glu Leu Gly Arg Trp Pro
Trp Gln Gly Ser Leu Arg 50 55 60 Leu Trp Asp Ser His Val Cys Gly
Val Ser Leu Leu Ser His Arg 65 70 75 Trp Ala Leu Thr Ala Ala His
Cys Phe Glu Thr Tyr Ser Asp Leu 80 85 90 Ser Asp Pro Ser Gly Trp
Met Val Gln Phe Gly Gln Leu Thr Ser 95 100 105 Met Pro Ser Phe Trp
Ser Leu Gln Ala Tyr Tyr Thr Arg Tyr Phe 110 115 120 Val Ser Asn Ile
Tyr Leu Ser Pro Arg Tyr Leu Gly Asn Ser Pro 125 130 135 Tyr Asp Ile
Ala Leu Val Lys Leu Ser Ala Pro Val Thr Tyr Thr 140 145 150 Lys His
Ile Gln Pro Ile Cys Leu Gln Ala Ser Thr Phe Glu Phe 155 160 165 Glu
Asn Arg Thr Asp Cys Trp Val Thr Gly Trp Gly Tyr Ile Lys 170 175 180
Glu Asp Glu Ala Leu Pro Ser Pro His Thr Leu Gln Glu Val Gln 185 190
195 Val Ala Ile Ile Asn Asn Ser Met Cys Asn His Leu Phe Leu Lys 200
205 210 Tyr Ser Phe Arg Lys Asp Ile Phe Gly Asp Met Val Cys Ala Gly
215 220 225 Asn Ala Gln Gly Gly Lys Asp Ala Cys Phe Gly Asp Ser Gly
Gly 230 235 240 Pro Leu Ala Cys Asn Lys Asn Gly Leu Trp Tyr Gln Ile
Gly Val 245 250 255 Val Ser Trp Gly Val Gly Cys Gly Arg Pro Asn Arg
Pro Gly Val 260 265 270 Tyr Thr Asn Ile Ser His His Phe Glu Trp Ile
Gln Lys Leu Met 275 280 285 Ala Gln Ser Gly Met Ser Gln Pro Asp Pro
Ser Trp Pro Leu Leu 290 295 300 Phe Phe Pro Leu Leu Trp Ala Leu Pro
Leu Leu Gly Pro Val 305 310 258 2427 DNA Homo Sapien 258 cccacgcgtc
cgcggacgcg tgggaagggc agaatgggac tccaagcctg 50 cctcctaggg
ctctttgccc tcatcctctc tggcaaatgc agttacagcc 100 cggagcccga
ccagcggagg acgctgcccc caggctgggt gtccctgggc 150 cgtgcggacc
ctgaggaaga gctgagtctc acctttgccc tgagacagca 200 gaatgtggaa
agactctcgg agctggtgca ggctgtgtcg gatcccagct 250 ctcctcaata
cggaaaatac ctgaccctag agaatgtggc tgatctggtg 300 aggccatccc
cactgaccct ccacacggtg caaaaatggc tcttggcagc 350 cggagcccag
aagtgccatt ctgtgatcac acaggacttt ctgacttgct 400 ggctgagcat
ccgacaagca gagctgctgc tccctggggc tgagtttcat 450 cactatgtgg
gaggacctac ggaaacccat gttgtaaggt ccccacatcc 500 ctaccagctt
ccacaggcct tggcccccca tgtggacttt gtggggggac 550 tgcaccgttt
tcccccaaca tcatccctga ggcaacgtcc tgagccgcag 600 gtgacaggga
ctgtaggcct gcatctgggg gtaaccccct ctgtgatccg 650 taagcgatac
aacttgacct cacaagacgt gggctctggc accagcaata 700 acagccaagc
ctgtgcccag ttcctggagc agtatttcca tgactcagac 750 ctggctcagt
tcatgcgcct cttcggtggc aactttgcac atcaggcatc 800 agtagcccgt
gtggttggac aacagggccg gggccgggcc gggattgagg 850 ccagtctaga
tgtgcagtac ctgatgagtg ctggtgccaa catctccacc 900 tgggtctaca
gtagccctgg ccggcatgag ggacaggagc ccttcctgca 950 gtggctcatg
ctgctcagta atgagtcagc cctgccacat gtgcatactg 1000 tgagctatgg
agatgatgag gactccctca gcagcgccta catccagcgg 1050 gtcaacactg
agctcatgaa ggctgccgct cggggtctca ccctgctctt 1100 cgcctcaggt
gacagtgggg ccgggtgttg gtctgtctct ggaagacacc 1150 agttccgccc
taccttccct gcctccagcc cctatgtcac cacagtggga 1200 ggcacatcct
tccaggaacc tttcctcatc acaaatgaaa ttgttgacta 1250 tatcagtggt
ggtggcttca gcaatgtgtt cccacggcct tcataccagg 1300 aggaagctgt
aacgaagttc ctgagctcta gcccccacct gccaccatcc 1350 agttacttca
atgccagtgg ccgtgcctac ccagatgtgg ctgcactttc 1400 tgatggctac
tgggtggtca gcaacagagt gcccattcca tgggtgtccg 1450 gaacctcggc
ctctactcca gtgtttgggg ggatcctatc cttgatcaat 1500 gagcacagga
tccttagtgg ccgcccccct cttggctttc tcaacccaag 1550 gctctaccag
cagcatgggg caggtctctt tgatgtaacc cgtggctgcc 1600 atgagtcctg
tctggatgaa gaggtagagg gccagggttt ctgctctggt 1650 cctggctggg
atcctgtaac aggctgggga acaccaactt cccagctttg 1700 ctgaagactc
tactcaaccc ctgacccttt cctatcagga gagatggctt 1750 gtcccctgcc
ctgaagctgg cagttcagtc ccttattctg ccctgttgga 1800 agccctgctg
aaccctcaac tattgactgc tgcagacagc ttatctccct 1850 aaccctgaaa
tgctgtgagc ttgacttgac tcccaaccct accatgctcc 1900 atcatactca
ggtctcccta ctcctgcctt agattcctca ataagatgct 1950 gtaactagca
ttttttgaat gcctctccct ccgcatctca tctttctctt 2000 ttcaatcagg
cttttccaaa gggttgtata cagactctgt gcactatttc 2050 acttgatatt
cattccccaa ttcactgcaa ggagacctct actgtcaccg 2100 tttactcttt
cctaccctga catccagaaa caatggcctc cagtgcatac 2150 ttctcaatct
ttgctttatg gcctttccat catagttgcc cactccctct 2200 ccttacttag
cttccaggtc ttaacttctc tgactactct tgtcttcctc 2250 tctcatcaat
ttctgcttct tcatggaatg ctgaccttca ttgctccatt 2300 tgtagatttt
tgctcttctc agtttactca ttgtcccctg gaacaaatca 2350 ctgacatcta
caaccattac catctcacta aataagactt tctatccaat 2400 aatgattgat
acctcaaatg taaaaaa 2427 259 556 PRT Homo Sapien 259 Met Gly Leu Gln
Ala Cys Leu Leu Gly Leu Phe Ala Leu Ile Leu 1 5 10 15 Ser Gly Lys
Cys Ser Tyr Ser Pro Glu Pro Asp Gln Arg Arg Thr 20 25 30 Leu Pro
Pro Gly Trp Val Ser Leu Gly Arg Ala Asp Pro Glu Glu 35 40 45 Glu
Leu Ser Leu Thr Phe Ala Leu Arg Gln Gln Asn Val Glu Arg 50 55 60
Leu Ser Glu Leu Val Gln Ala Val Ser Asp Pro Ser Ser Pro Gln 65 70
75 Tyr Gly Lys Tyr Leu Thr Leu Glu Asn Val Ala Asp Leu Val Arg 80
85 90 Pro Ser Pro Leu Thr Leu His Thr Val Gln Lys Trp Leu Leu Ala
95 100 105 Ala Gly Ala Gln Lys Cys His Ser Val Ile Thr Gln Asp Phe
Leu 110 115 120 Thr Cys Trp Leu Ser Ile Arg Gln Ala Glu Leu Leu Leu
Pro Gly 125 130 135 Ala Glu Phe His His Tyr Val Gly Gly Pro Thr Glu
Thr His Val 140 145 150 Val Arg Ser Pro His Pro Tyr Gln Leu Pro Gln
Ala Leu Ala Pro 155 160 165 His Val Asp Phe Val Gly Gly Leu His Arg
Phe Pro Pro Thr Ser 170 175 180 Ser Leu Arg Gln Arg Pro Glu Pro Gln
Val Thr Gly Thr Val Gly 185 190 195 Leu His Leu Gly Val Thr Pro Ser
Val Ile Arg Lys Arg Tyr Asn 200 205 210 Leu Thr Ser Gln Asp Val Gly
Ser Gly Thr Ser Asn Asn Ser Gln 215 220 225 Ala Cys Ala Gln Phe Leu
Glu Gln Tyr Phe His Asp Ser Asp Leu 230 235 240 Ala Gln Phe Met Arg
Leu Phe Gly Gly Asn Phe Ala His Gln Ala 245 250 255 Ser Val Ala Arg
Val Val Gly Gln Gln Gly Arg Gly Arg Ala Gly 260 265 270 Ile Glu Ala
Ser Leu Asp Val Gln Tyr Leu Met Ser Ala Gly Ala 275 280 285 Asn Ile
Ser Thr Trp Val Tyr Ser Ser Pro Gly Arg His Glu Gly 290 295 300 Gln
Glu Pro Phe Leu Gln Trp Leu Met Leu Leu Ser Asn Glu Ser 305 310 315
Ala Leu Pro His Val His Thr Val Ser Tyr Gly Asp Asp Glu Asp 320 325
330 Ser Leu Ser Ser Ala Tyr Ile Gln Arg Val Asn Thr Glu Leu Met 335
340 345 Lys Ala Ala Ala Arg Gly Leu Thr Leu Leu Phe Ala Ser Gly Asp
350 355 360 Ser Gly Ala Gly Cys Trp Ser Val Ser Gly Arg His Gln Phe
Arg 365 370 375 Pro Thr Phe Pro Ala Ser Ser Pro Tyr Val Thr Thr Val
Gly Gly 380 385 390 Thr Ser Phe Gln Glu Pro Phe Leu Ile Thr Asn Glu
Ile Val Asp 395 400 405 Tyr Ile Ser Gly Gly Gly Phe Ser Asn Val Phe
Pro Arg Pro Ser 410 415 420 Tyr Gln Glu Glu Ala Val Thr Lys Phe Leu
Ser Ser Ser Pro His 425 430 435 Leu Pro Pro Ser Ser Tyr Phe Asn Ala
Ser Gly Arg Ala Tyr Pro 440 445 450 Asp Val Ala Ala Leu Ser Asp Gly
Tyr Trp Val Val Ser Asn Arg 455 460 465 Val Pro Ile Pro Trp Val Ser
Gly Thr Ser Ala Ser Thr Pro Val 470 475 480 Phe Gly Gly Ile Leu Ser
Leu Ile Asn Glu His Arg Ile Leu Ser 485 490 495 Gly Arg Pro Pro Leu
Gly Phe Leu Asn Pro Arg Leu Tyr Gln Gln 500 505 510 His Gly Ala Gly
Leu Phe Asp Val Thr Arg Gly Cys His Glu Ser 515 520 525 Cys Leu Asp
Glu Glu Val Glu Gly Gln Gly Phe Cys Ser Gly Pro 530 535 540 Gly Trp
Asp Pro Val Thr Gly Trp Gly Thr Pro Thr Ser Gln Leu 545 550 555 Cys
260 1638 DNA Homo Sapien 260 gccgcgcgct ctctcccggc gcccacacct
gtctgagcgg cgcagcgagc 50 cgcggcccgg gcgggctgct cggcgcggaa
cagtgctcgg catggcaggg 100 attccagggc tcctcttcct tctcttcttt
ctgctctgtg ctgttgggca 150 agtgagccct tacagtgccc cctggaaacc
cacttggcct gcataccgcc 200 tccctgtcgt cttgccccag tctaccctca
atttagccaa gccagacttt 250 ggagccgaag ccaaattaga agtatcttct
tcatgtggac cccagtgtca 300 taagggaact ccactgccca cttacgaaga
ggccaagcaa tatctgtctt 350 atgaaacgct ctatgccaat ggcagccgca
cagagacgca ggtgggcatc 400 tacatcctca gcagtagtgg agatggggcc
caacaccgag actcagggtc 450 ttcaggaaag tctcgaagga agcggcagat
ttatggctat gacagcaggt 500 tcagcatttt tgggaaggac ttcctgctca
actacccttt ctcaacatca 550 gtgaagttat ccacgggctg caccggcacc
ctggtggcag agaagcatgt 600 cctcacagct gcccactgca tacacgatgg
aaaaacctat gtgaaaggaa 650 cccagaagct tcgagtgggc ttcctaaagc
ccaagtttaa agatggtggt 700 cgaggggcca acgactccac ttcagccatg
cccgagcaga tgaaatttca 750 gtggatccgg gtgaaacgca cccatgtgcc
caagggttgg atcaagggca 800 atgccaatga catcggcatg gattatgatt
atgccctcct ggaactcaaa 850 aagccccaca agagaaaatt tatgaagatt
ggggtgagcc ctcctgctaa 900 gcagctgcca gggggcagaa ttcacttctc
tggttatgac aatgaccgac 950 caggcaattt ggtgtatcgc ttctgtgacg
tcaaagacga gacctatgac 1000 ttgctctacc agcaatgcga tgcccagcca
ggggccagcg ggtctggggt 1050 ctatgtgagg atgtggaaga gacagcagca
gaagtgggag cgaaaaatta 1100 ttggcatttt ttcagggcac cagtgggtgg
acatgaatgg ttccccacag 1150 gatttcaacg tggctgtcag aatcactcct
ctcaaatatg cccagatttg 1200 ctattggatt aaaggaaact acctggattg
tagggagggg tgacacagtg 1250 ttccctcctg gcagcaatta agggtcttca
tgttcttatt ttaggagagg 1300 ccaaattgtt ttttgtcatt ggcgtgcaca
cgtgtgtgtg tgtgtgtgtg 1350 tgtgtgtaag gtgtcttata atcttttacc
tatttcttac aattgcaaga 1400 tgactggctt tactatttga aaactggttt
gtgtatcata tcatatatca 1450 tttaagcagt ttgaaggcat acttttgcat
agaaataaaa aaaatactga 1500 tttggggcaa tgaggaatat ttgacaatta
agttaatctt cacgtttttg 1550 caaactttga tttttatttc atctgaactt
gtttcaaaga tttatattaa 1600 atatttggca tacaagagat atgaaaaaaa
aaaaaaaa 1638 261 383 PRT Homo Sapien 261 Met Ala Gly Ile Pro Gly
Leu Leu Phe Leu Leu Phe Phe Leu Leu 1 5 10 15 Cys Ala Val Gly Gln
Val Ser Pro Tyr Ser Ala Pro Trp Lys Pro 20 25 30 Thr Trp Pro Ala
Tyr Arg Leu Pro Val Val Leu Pro Gln Ser Thr 35 40 45 Leu Asn Leu
Ala Lys Pro Asp Phe Gly Ala Glu Ala Lys Leu Glu 50 55 60 Val Ser
Ser Ser Cys Gly Pro Gln Cys His Lys Gly Thr Pro Leu 65 70 75 Pro
Thr Tyr Glu Glu Ala Lys Gln Tyr Leu Ser Tyr Glu Thr Leu 80 85 90
Tyr Ala Asn Gly Ser Arg Thr Glu Thr Gln Val Gly Ile Tyr Ile 95 100
105 Leu Ser Ser Ser Gly Asp Gly Ala Gln His Arg Asp Ser Gly Ser 110
115 120 Ser Gly Lys Ser Arg Arg Lys Arg Gln Ile Tyr Gly Tyr Asp Ser
125 130 135 Arg Phe Ser Ile Phe Gly Lys Asp Phe Leu Leu Asn Tyr Pro
Phe 140 145 150 Ser Thr Ser Val Lys Leu Ser Thr Gly Cys Thr Gly Thr
Leu Val 155 160 165 Ala Glu Lys His Val Leu Thr Ala Ala His Cys Ile
His Asp Gly 170 175 180 Lys Thr Tyr Val Lys Gly Thr Gln Lys Leu Arg
Val Gly Phe Leu 185 190 195 Lys Pro Lys Phe Lys Asp Gly Gly Arg Gly
Ala Asn Asp Ser Thr 200 205 210 Ser Ala Met Pro Glu Gln Met Lys Phe
Gln Trp Ile Arg Val Lys 215 220 225 Arg Thr His Val Pro Lys Gly Trp
Ile Lys Gly Asn Ala Asn Asp 230 235 240 Ile Gly Met Asp Tyr Asp Tyr
Ala Leu Leu Glu Leu Lys Lys Pro 245 250 255 His Lys Arg Lys Phe Met
Lys Ile Gly Val Ser Pro Pro Ala Lys 260 265 270 Gln Leu Pro Gly Gly
Arg Ile His Phe Ser Gly Tyr Asp Asn Asp 275 280 285 Arg Pro Gly Asn
Leu Val Tyr Arg Phe Cys Asp Val Lys Asp Glu 290 295 300 Thr Tyr Asp
Leu Leu Tyr Gln Gln Cys Asp Ala Gln Pro Gly Ala 305 310 315 Ser Gly
Ser Gly Val Tyr Val Arg Met Trp Lys Arg Gln Gln Gln 320 325 330 Lys
Trp Glu Arg Lys Ile Ile Gly Ile Phe Ser Gly His Gln Trp 335 340 345
Val Asp Met Asn Gly Ser Pro Gln Asp Phe Asn Val Ala Val Arg 350 355
360 Ile Thr Pro Leu Lys Tyr Ala Gln Ile Cys Tyr Trp Ile Lys Gly 365
370 375 Asn Tyr Leu Asp Cys Arg Glu Gly 380 262 1378 DNA Homo
Sapien 262 gcatcgccct gggtctctcg agcctgctgc ctgctccccc gccccaccag
50 ccatggtggt ttctggagcg cccccagccc tgggtggggg ctgtctcggc 100
accttcacct ccctgctgct gctggcgtcg acagccatcc tcaatgcggc 150
caggatacct gttcccccag cctgtgggaa gccccagcag ctgaaccggg 200
ttgtgggcgg cgaggacagc actgacagcg agtggccctg gatcgtgagc 250
atccagaaga atgggaccca ccactgcgca ggttctctgc tcaccagccg 300
ctgggtgatc actgctgccc actgtttcaa ggacaacctg aacaaaccat 350
acctgttctc tgtgctgctg ggggcctggc agctggggaa ccctggctct 400
cggtcccaga aggtgggtgt tgcctgggtg gagccccacc ctgtgtattc 450
ctggaaggaa ggtgcctgtg cagacattgc cctggtgcgt ctcgagcgct 500
ccatacagtt ctcagagcgg gtcctgccca tctgcctacc tgatgcctct 550
atccacctcc ctccaaacac ccactgctgg atctcaggct gggggagcat 600
ccaagatgga gttcccttgc cccaccctca gaccctgcag aagctgaagg 650
ttcctatcat cgactcggaa gtctgcagcc atctgtactg gcggggagca 700
ggacagggac ccatcactga ggacatgctg tgtgccggct acttggaggg 750
ggagcgggat gcttgtctgg gcgactccgg gggccccctc atgtgccagg 800
tggacggcgc ctggctgctg gccggcatca tcagctgggg cgagggctgt 850
gccgagcgca acaggcccgg ggtctacatc agcctctctg cgcaccgctc 900
ctgggtggag aagatcgtgc aaggggtgca gctccgcggg cgcgctcagg 950
ggggtggggc cctcagggca ccgagccagg gctctggggc cgccgcgcgc 1000
tcctagggcg cagcgggacg cggggctcgg atctgaaagg cggccagatc 1050
cacatctgga tctggatctg cggcggcctc gggcggtttc ccccgccgta 1100
aataggctca tctacctcta cctctggggg cccggacggc tgctgcggaa 1150
aggaaacccc ctccccgacc cgcccgacgg cctcaggccc ccctccaagg 1200
catcaggccc cgcccaacgg cctcatgtcc ccgcccccac gacttccggc 1250
cccgcccccg ggccccagcg cttttgtgta tataaatgtt aatgattttt 1300
ataggtattt gtaaccctgc ccacatatct tatttattcc tccaatttca 1350
ataaattatt tattctccaa aaaaaaaa 1378 263 317 PRT Homo Sapien 263 Met
Val Val Ser Gly Ala Pro Pro Ala Leu Gly Gly Gly Cys Leu 1 5 10 15
Gly Thr Phe Thr Ser Leu Leu Leu Leu Ala Ser Thr Ala Ile Leu 20 25
30 Asn Ala Ala Arg Ile Pro Val Pro Pro Ala Cys Gly Lys Pro Gln 35
40 45 Gln Leu Asn Arg Val Val Gly Gly Glu Asp Ser Thr Asp Ser Glu
50 55 60 Trp Pro Trp Ile Val Ser Ile Gln Lys Asn Gly Thr His His
Cys 65 70 75 Ala Gly Ser Leu Leu Thr Ser Arg Trp Val Ile Thr Ala
Ala His 80 85 90 Cys Phe Lys Asp Asn Leu Asn Lys Pro Tyr Leu Phe
Ser Val Leu 95 100 105 Leu Gly Ala Trp Gln Leu Gly Asn Pro Gly Ser
Arg Ser Gln Lys 110 115 120 Val Gly Val Ala Trp Val Glu Pro His Pro
Val Tyr Ser Trp Lys 125 130 135 Glu Gly Ala Cys Ala Asp Ile Ala Leu
Val Arg Leu Glu Arg Ser 140 145 150 Ile Gln Phe Ser Glu Arg Val Leu
Pro Ile Cys Leu Pro Asp Ala 155 160 165 Ser Ile His Leu Pro Pro Asn
Thr His Cys Trp Ile Ser Gly Trp 170 175 180 Gly Ser Ile Gln Asp Gly
Val Pro Leu Pro His Pro Gln Thr Leu 185 190 195 Gln Lys Leu Lys Val
Pro Ile Ile Asp Ser Glu Val Cys Ser His 200 205 210 Leu Tyr Trp Arg
Gly Ala Gly Gln Gly Pro Ile Thr Glu Asp Met 215 220 225 Leu Cys Ala
Gly Tyr Leu Glu Gly Glu Arg Asp Ala Cys Leu Gly 230 235 240 Asp Ser
Gly Gly Pro Leu Met Cys Gln Val Asp Gly Ala Trp Leu 245 250 255 Leu
Ala Gly Ile Ile Ser Trp Gly Glu Gly Cys Ala Glu Arg Asn 260 265 270
Arg Pro Gly Val Tyr Ile Ser Leu Ser Ala His Arg Ser Trp Val 275 280
285 Glu Lys Ile Val Gln Gly Val Gln Leu Arg Gly Arg Ala Gln Gly 290
295 300 Gly Gly Ala Leu Arg Ala Pro Ser Gln Gly Ser Gly Ala Ala Ala
305 310 315 Arg Ser 264 24 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 264 gtccgcaagg atgcctacat gttc 24 265 19 DNA
Artificial Sequence Synthetic Oligonucleotide Probe 265 gcagaggtgt
ctaaggttg 19 266 24 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 266 agctctagac caatgccagc ttcc 24 267 45 DNA
Artificial Sequence Synthetic Oligonucleotide Probe 267 gccaccaact
cctgcaagaa cttctcagaa ctgcccctgg tcatg 45 268 25 DNA Artificial
Sequence Synthetic Oligonucleotide Probe 268 ggggaattca ccctatgaca
ttgcc 25 269 24 DNA Artificial Sequence Synthetic Oligonucleotide
Probe 269 gaatgccctg caagcatcaa ctgg 24 270 50 DNA Artificial
Sequence Synthetic Oligonucleotide Probe 270 gcacctgtca cctacactaa
acacatccag cccatctgtc tccaggcctc 50 271 26 DNA Artificial Sequence
Synthetic Oligonucleotide Probe 271 gcggaagggc agaatgggac tccaag 26
272 18 DNA Artificial Sequence Synthetic Oligonucleotide Probe 272
cagccctgcc acatgtgc 18 273 18 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 273 tactgggtgg tcagcaac 18 274 24 DNA
Artificial Sequence Synthetic Oligonucleotide Probe 274 ggcgaagagc
agggtgagac cccg 24 275 45 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 275 gccctcatcc tctctggcaa atgcagttac
agcccggagc ccgac 45 276 21 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 276 gggcagggat tccagggctc c 21 277 18 DNA
Artificial Sequence Synthetic Oligonucleotide Probe 277 ggctatgaca
gcaggttc 18 278 18 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 278 tgacaatgac cgaccagg 18 279 24 DNA
Artificial Sequence Synthetic Oligonucleotide Probe 279 gcatcgcatt
gctggtagag caag 24 280 45 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 280 ttacagtgcc ccctggaaac ccacttggcc
tgcataccgc ctccc 45 281 34 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 281 cgtctcgagc gctccataca gttcccttgc ccca 34
282 61 DNA Artificial Sequence Synthetic Oligonucleotide Probe 282
tggaggggga gcgggatgct tgtctgggcg actccggggg ccccctcatg 50
tgccaggtgg a 61 283 119 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 283 ccctcagacc ctgcagaagc tgaaggttcc
tatcatcgac tcggaagtct 50 gcagccatct gtactggcgg ggagcaggac
agggacccat cactgaggac 100 atgctgtgtg ccggctact 119 284 1875 DNA
Homo Sapien 284 gacggctggc caccatgcac ggctcctgca gtttcctgat
gcttctgctg 50 ccgctactgc tactgctggt ggccaccaca ggccccgttg
gagccctcac 100 agatgaggag aaacgtttga tggtggagct gcacaacctc
taccgggccc 150 aggtatcccc gacggcctca gacatgctgc acatgagatg
ggacgaggag 200 ctggccgcct tcgccaaggc ctacgcacgg cagtgcgtgt
ggggccacaa 250 caaggagcgc gggcgccgcg gcgagaatct gttcgccatc
acagacgagg 300 gcatggacgt gccgctggcc atggaggagt ggcaccacga
gcgtgagcac 350 tacaacctca gcgccgccac ctgcagccca ggccagatgt
gcggccacta 400 cacgcaggtg gtatgggcca agacagagag gatcggctgt
ggttcccact 450 tctgtgagaa gctccagggt gttgaggaga ccaacatcga
attactggtg 500 tgcaactatg agcctccggg gaacgtgaag gggaaacggc
cctaccagga 550 ggggactccg tgctcccaat gtccctctgg ctaccactgc
aagaactccc 600 tctgtgaacc catcggaagc ccggaagatg ctcaggattt
gccttacctg 650 gtaactgagg ccccatcctt ccgggcgact gaagcatcag
actctaggaa 700 aatgggtact ccttcttccc tagcaacggg gattccggct
ttcttggtaa 750 cagaggtctc aggctccctg gcaaccaagg ctctgcctgc
tgtggaaacc 800 caggccccaa cttccttagc aacgaaagac ccgccctcca
tggcaacaga 850 ggctccacct tgcgtaacaa ctgaggtccc ttccattttg
gcagctcaca 900 gcctgccctc cttggatgag gagccagtta ccttccccaa
atcgacccat 950 gttcctatcc caaaatcagc agacaaagtg acagacaaaa
caaaagtgcc 1000 ctctaggagc ccagagaact ctctggaccc caagatgtcc
ctgacagggg 1050 caagggaact cctaccccat gcccaggagg aggctgaggc
tgaggctgag 1100 ttgcctcctt ccagtgaggt cttggcctca gtttttccag
cccaggacaa 1150 gccaggtgag ctgcaggcca cactggacca cacggggcac
acctcctcca 1200 agtccctgcc caatttcccc aatacctctg ccaccgctaa
tgccacgggt 1250 gggcgtgccc tggctctgca gtcgtccttg ccaggtgcag
agggccctga 1300 caagcctagc gttgtgtcag ggctgaactc gggccctggt
catgtgtggg 1350 gccctctcct gggactactg ctcctgcctc ctctggtgtt
ggctggaatc 1400 ttctgaatgg gataccactc aaagggtgaa gaggtcagct
gtcctcctgt 1450 catcttcccc accctgtccc cagcccctaa acaagatact
tcttggttaa 1500 ggccctccgg aagggaaagg ctacggggca tgtgcctcat
cacaccatcc 1550 atcctggagg cacaaggcct ggctggctgc gagctcagga
ggccgcctga 1600 ggactgcaca ccgggcccac acctctcctg cccctccctc
ctgagtcctg 1650 ggggtgggag gatttgaggg agctcactgc ctacctggcc
tggggctgtc 1700 tgcccacaca gcatgtgcgc tctccctgag tgcctgtgta
gctggggatg 1750 gggattccta ggggcagatg aaggacaagc cccactggag
tggggttctt 1800 tgagtggggg aggcagggac gagggaagga aagtaactcc
tgactctcca 1850 ataaaaacct gtccaacctg tgaaa 1875 285 463 PRT Homo
Sapien 285 Met His Gly Ser Cys Ser Phe Leu Met Leu Leu Leu Pro Leu
Leu 1 5 10 15 Leu Leu Leu Val Ala Thr Thr Gly Pro Val Gly Ala Leu
Thr Asp 20 25 30 Glu Glu Lys Arg Leu Met Val Glu Leu His Asn Leu
Tyr Arg Ala 35 40 45 Gln Val Ser Pro Thr Ala Ser Asp Met Leu His
Met Arg Trp Asp 50 55 60 Glu Glu Leu Ala Ala Phe Ala Lys Ala Tyr
Ala Arg Gln Cys Val 65 70 75 Trp Gly His Asn Lys Glu Arg Gly Arg
Arg Gly Glu Asn Leu Phe 80 85 90 Ala Ile Thr Asp Glu Gly Met Asp
Val Pro Leu Ala Met Glu Glu 95 100 105 Trp His His Glu Arg Glu His
Tyr Asn Leu Ser Ala Ala Thr Cys 110 115 120 Ser Pro Gly Gln Met Cys
Gly His Tyr Thr Gln Val Val Trp Ala 125 130 135 Lys Thr Glu Arg Ile
Gly Cys Gly Ser His Phe Cys Glu Lys Leu 140 145 150 Gln Gly Val Glu
Glu Thr Asn Ile Glu Leu Leu Val Cys Asn Tyr 155 160 165 Glu Pro Pro
Gly Asn Val Lys Gly Lys Arg Pro Tyr Gln Glu Gly 170 175 180 Thr Pro
Cys Ser Gln Cys Pro Ser Gly Tyr His Cys Lys Asn Ser 185 190 195 Leu
Cys Glu Pro Ile Gly Ser Pro Glu Asp Ala Gln Asp Leu Pro 200 205 210
Tyr Leu Val Thr Glu Ala Pro Ser Phe Arg Ala Thr Glu Ala Ser 215 220
225 Asp Ser Arg Lys Met Gly Thr Pro Ser Ser Leu Ala Thr Gly Ile 230
235 240 Pro Ala Phe Leu Val Thr Glu Val Ser Gly Ser Leu Ala Thr Lys
245 250 255 Ala Leu Pro Ala Val Glu Thr Gln Ala Pro Thr Ser Leu Ala
Thr 260 265 270 Lys Asp Pro Pro Ser Met Ala Thr Glu Ala Pro Pro Cys
Val Thr 275 280 285 Thr Glu Val Pro Ser Ile Leu Ala Ala His Ser Leu
Pro Ser Leu 290 295 300 Asp Glu Glu Pro Val Thr Phe Pro Lys Ser Thr
His Val Pro Ile 305 310 315 Pro Lys Ser Ala Asp Lys Val Thr Asp Lys
Thr Lys Val Pro Ser 320 325 330 Arg Ser Pro Glu Asn Ser Leu Asp Pro
Lys Met Ser Leu Thr Gly 335 340 345 Ala Arg Glu Leu Leu Pro His Ala
Gln Glu Glu Ala Glu Ala Glu 350 355 360 Ala Glu Leu Pro Pro Ser Ser
Glu Val Leu Ala Ser Val Phe Pro 365 370 375 Ala Gln Asp Lys Pro Gly
Glu Leu Gln Ala Thr Leu Asp His Thr 380 385 390 Gly His Thr Ser Ser
Lys Ser Leu Pro Asn Phe Pro Asn Thr Ser 395 400 405 Ala Thr Ala Asn
Ala Thr Gly Gly Arg Ala Leu Ala Leu Gln Ser 410 415 420 Ser Leu Pro
Gly Ala Glu Gly Pro Asp Lys Pro Ser Val Val Ser 425 430 435 Gly Leu
Asn Ser Gly Pro Gly His Val Trp Gly Pro Leu Leu Gly 440 445 450 Leu
Leu Leu Leu Pro Pro Leu Val Leu Ala Gly Ile Phe 455 460 286 19 DNA
Artificial Sequence Synthetic Oligonucleotide Probe 286 tcctgcagtt
tcctgatgc
19 287 24 DNA Artificial Sequence Synthetic Oligonucleotide Probe
287 ctcatattgc acaccagtaa ttcg 24 288 45 DNA Artificial Sequence
Synthetic Oligonucleotide Probe 288 atgaggagaa acgtttgatg
gtggagctgc acaacctcta ccggg 45 289 3662 DNA Homo Sapien 289
gtaactgaag tcaggctttt catttgggaa gccccctcaa cagaattcgg 50
tcattctcca agttatggtg gacgtacttc tgttgttctc cctctgcttg 100
ctttttcaca ttagcagacc ggacttaagt cacaacagat tatctttcat 150
caaggcaagt tccatgagcc accttcaaag ccttcgagaa gtgaaactga 200
acaacaatga attggagacc attccaaatc tgggaccagt ctcggcaaat 250
attacacttc tctccttggc tggaaacagg attgttgaaa tactccctga 300
acatctgaaa gagtttcagt cccttgaaac tttggacctt agcagcaaca 350
atatttcaga gctccaaact gcatttccag ccctacagct caaatatctg 400
tatctcaaca gcaaccgagt cacatcaatg gaacctgggt attttgacaa 450
tttggccaac acactccttg tgttaaagct gaacaggaac cgaatctcag 500
ctatcccacc caagatgttt aaactgcccc aactgcaaca tctcgaattg 550
aaccgaaaca agattaaaaa tgtagatgga ctgacattcc aaggccttgg 600
tgctctgaag tctctgaaaa tgcaaagaaa tggagtaacg aaacttatgg 650
atggagcttt ttgggggctg agcaacatgg aaattttgca gctggaccat 700
aacaacctaa cagagattac caaaggctgg ctttacggct tgctgatgct 750
gcaggaactt catctcagcc aaaatgccat caacaggatc agccctgatg 800
cctgggagtt ctgccagaag ctcagtgagc tggacctaac tttcaatcac 850
ttatcaaggt tagatgattc aagcttcctt ggcctaagct tactaaatac 900
actgcacatt gggaacaaca gagtcagcta cattgctgat tgtgccttcc 950
gggggctttc cagtttaaag actttggatc tgaagaacaa tgaaatttcc 1000
tggactattg aagacatgaa tggtgctttc tctgggcttg acaaactgag 1050
gcgactgata ctccaaggaa atcggatccg ttctattact aaaaaagcct 1100
tcactggttt ggatgcattg gagcatctag acctgagtga caacgcaatc 1150
atgtctttac aaggcaatgc attttcacaa atgaagaaac tgcaacaatt 1200
gcatttaaat acatcaagcc ttttgtgcga ttgccagcta aaatggctcc 1250
cacagtgggt ggcggaaaac aactttcaga gctttgtaaa tgccagttgt 1300
gcccatcctc agctgctaaa aggaagaagc atttttgctg ttagcccaga 1350
tggctttgtg tgtgatgatt ttcccaaacc ccagatcacg gttcagccag 1400
aaacacagtc ggcaataaaa ggttccaatt tgagtttcat ctgctcagct 1450
gccagcagca gtgattcccc aatgactttt gcttggaaaa aagacaatga 1500
actactgcat gatgctgaaa tggaaaatta tgcacacctc cgggcccaag 1550
gtggcgaggt gatggagtat accaccatcc ttcggctgcg cgaggtggaa 1600
tttgccagtg aggggaaata tcagtgtgtc atctccaatc actttggttc 1650
atcctactct gtcaaagcca agcttacagt aaatatgctt ccctcattca 1700
ccaagacccc catggatctc accatccgag ctggggccat ggcacgcttg 1750
gagtgtgctg ctgtggggca cccagccccc cagatagcct ggcagaagga 1800
tgggggcaca gacttcccag ctgcacggga gagacgcatg catgtgatgc 1850
ccgaggatga cgtgttcttt atcgtggatg tgaagataga ggacattggg 1900
gtatacagct gcacagctca gaacagtgca ggaagtattt cagcaaatgc 1950
aactctgact gtcctagaaa caccatcatt tttgcggcca ctgttggacc 2000
gaactgtaac caagggagaa acagccgtcc tacagtgcat tgctggagga 2050
agccctcccc ctaaactgaa ctggaccaaa gatgatagcc cattggtggt 2100
aaccgagagg cacttttttg cagcaggcaa tcagcttctg attattgtgg 2150
actcagatgt cagtgatgct gggaaataca catgtgagat gtctaacacc 2200
cttggcactg agagaggaaa cgtgcgcctc agtgtgatcc ccactccaac 2250
ctgcgactcc cctcagatga cagccccatc gttagacgat gacggatggg 2300
ccactgtggg tgtcgtgatc atagccgtgg tttgctgtgt ggtgggcacg 2350
tcactcgtgt gggtggtcat catataccac acaaggcgga ggaatgaaga 2400
ttgcagcatt accaacacag atgagaccaa cttgccagca gatattccta 2450
gttatttgtc atctcaggga acgttagctg acaggcagga tgggtacgtg 2500
tcttcagaaa gtggaagcca ccaccagttt gtcacatctt caggtgctgg 2550
atttttctta ccacaacatg acagtagtgg gacctgccat attgacaata 2600
gcagtgaagc tgatgtggaa gctgccacag atctgttcct ttgtccgttt 2650
ttgggatcca caggccctat gtatttgaag ggaaatgtgt atggctcaga 2700
tccttttgaa acatatcata caggttgcag tcctgaccca agaacagttt 2750
taatggacca ctatgagccc agttacataa agaaaaagga gtgctaccca 2800
tgttctcatc cttcagaaga atcctgcgaa cggagcttca gtaatatatc 2850
gtggccttca catgtgagga agctacttaa cactagttac tctcacaatg 2900
aaggacctgg aatgaaaaat ctgtgtctaa acaagtcctc tttagatttt 2950
agtgcaaatc cagagccagc gtcggttgcc tcgagtaatt ctttcatggg 3000
tacctttgga aaagctctca ggagacctca cctagatgcc tattcaagct 3050
ttggacagcc atcagattgt cagccaagag ccttttattt gaaagctcat 3100
tcttccccag acttggactc tgggtcagag gaagatggga aagaaaggac 3150
agattttcag gaagaaaatc acatttgtac ctttaaacag actttagaaa 3200
actacaggac tccaaatttt cagtcttatg acttggacac atagactgaa 3250
tgagaccaaa ggaaaagctt aacatactac ctcaagtgaa cttttattta 3300
aaagagagag aatcttatgt tttttaaatg gagttatgaa ttttaaaagg 3350
ataaaaatgc tttatttata cagatgaacc aaaattacaa aaagttatga 3400
aaatttttat actgggaatg atgctcatat aagaatacct ttttaaacta 3450
ttttttaact ttgttttatg caaaaaagta tcttacgtaa attaatgata 3500
taaatcatga ttattttatg tatttttata atgccagatt tctttttatg 3550
gaaaatgagt tactaaagca ttttaaataa tacctgcctt gtaccatttt 3600
ttaaatagaa gttacttcat tatattttgc acattatatt taataaaatg 3650
tgtcaatttg aa 3662 290 1059 PRT Homo Sapien 290 Met Val Asp Val Leu
Leu Leu Phe Ser Leu Cys Leu Leu Phe His 1 5 10 15 Ile Ser Arg Pro
Asp Leu Ser His Asn Arg Leu Ser Phe Ile Lys 20 25 30 Ala Ser Ser
Met Ser His Leu Gln Ser Leu Arg Glu Val Lys Leu 35 40 45 Asn Asn
Asn Glu Leu Glu Thr Ile Pro Asn Leu Gly Pro Val Ser 50 55 60 Ala
Asn Ile Thr Leu Leu Ser Leu Ala Gly Asn Arg Ile Val Glu 65 70 75
Ile Leu Pro Glu His Leu Lys Glu Phe Gln Ser Leu Glu Thr Leu 80 85
90 Asp Leu Ser Ser Asn Asn Ile Ser Glu Leu Gln Thr Ala Phe Pro 95
100 105 Ala Leu Gln Leu Lys Tyr Leu Tyr Leu Asn Ser Asn Arg Val Thr
110 115 120 Ser Met Glu Pro Gly Tyr Phe Asp Asn Leu Ala Asn Thr Leu
Leu 125 130 135 Val Leu Lys Leu Asn Arg Asn Arg Ile Ser Ala Ile Pro
Pro Lys 140 145 150 Met Phe Lys Leu Pro Gln Leu Gln His Leu Glu Leu
Asn Arg Asn 155 160 165 Lys Ile Lys Asn Val Asp Gly Leu Thr Phe Gln
Gly Leu Gly Ala 170 175 180 Leu Lys Ser Leu Lys Met Gln Arg Asn Gly
Val Thr Lys Leu Met 185 190 195 Asp Gly Ala Phe Trp Gly Leu Ser Asn
Met Glu Ile Leu Gln Leu 200 205 210 Asp His Asn Asn Leu Thr Glu Ile
Thr Lys Gly Trp Leu Tyr Gly 215 220 225 Leu Leu Met Leu Gln Glu Leu
His Leu Ser Gln Asn Ala Ile Asn 230 235 240 Arg Ile Ser Pro Asp Ala
Trp Glu Phe Cys Gln Lys Leu Ser Glu 245 250 255 Leu Asp Leu Thr Phe
Asn His Leu Ser Arg Leu Asp Asp Ser Ser 260 265 270 Phe Leu Gly Leu
Ser Leu Leu Asn Thr Leu His Ile Gly Asn Asn 275 280 285 Arg Val Ser
Tyr Ile Ala Asp Cys Ala Phe Arg Gly Leu Ser Ser 290 295 300 Leu Lys
Thr Leu Asp Leu Lys Asn Asn Glu Ile Ser Trp Thr Ile 305 310 315 Glu
Asp Met Asn Gly Ala Phe Ser Gly Leu Asp Lys Leu Arg Arg 320 325 330
Leu Ile Leu Gln Gly Asn Arg Ile Arg Ser Ile Thr Lys Lys Ala 335 340
345 Phe Thr Gly Leu Asp Ala Leu Glu His Leu Asp Leu Ser Asp Asn 350
355 360 Ala Ile Met Ser Leu Gln Gly Asn Ala Phe Ser Gln Met Lys Lys
365 370 375 Leu Gln Gln Leu His Leu Asn Thr Ser Ser Leu Leu Cys Asp
Cys 380 385 390 Gln Leu Lys Trp Leu Pro Gln Trp Val Ala Glu Asn Asn
Phe Gln 395 400 405 Ser Phe Val Asn Ala Ser Cys Ala His Pro Gln Leu
Leu Lys Gly 410 415 420 Arg Ser Ile Phe Ala Val Ser Pro Asp Gly Phe
Val Cys Asp Asp 425 430 435 Phe Pro Lys Pro Gln Ile Thr Val Gln Pro
Glu Thr Gln Ser Ala 440 445 450 Ile Lys Gly Ser Asn Leu Ser Phe Ile
Cys Ser Ala Ala Ser Ser 455 460 465 Ser Asp Ser Pro Met Thr Phe Ala
Trp Lys Lys Asp Asn Glu Leu 470 475 480 Leu His Asp Ala Glu Met Glu
Asn Tyr Ala His Leu Arg Ala Gln 485 490 495 Gly Gly Glu Val Met Glu
Tyr Thr Thr Ile Leu Arg Leu Arg Glu 500 505 510 Val Glu Phe Ala Ser
Glu Gly Lys Tyr Gln Cys Val Ile Ser Asn 515 520 525 His Phe Gly Ser
Ser Tyr Ser Val Lys Ala Lys Leu Thr Val Asn 530 535 540 Met Leu Pro
Ser Phe Thr Lys Thr Pro Met Asp Leu Thr Ile Arg 545 550 555 Ala Gly
Ala Met Ala Arg Leu Glu Cys Ala Ala Val Gly His Pro 560 565 570 Ala
Pro Gln Ile Ala Trp Gln Lys Asp Gly Gly Thr Asp Phe Pro 575 580 585
Ala Ala Arg Glu Arg Arg Met His Val Met Pro Glu Asp Asp Val 590 595
600 Phe Phe Ile Val Asp Val Lys Ile Glu Asp Ile Gly Val Tyr Ser 605
610 615 Cys Thr Ala Gln Asn Ser Ala Gly Ser Ile Ser Ala Asn Ala Thr
620 625 630 Leu Thr Val Leu Glu Thr Pro Ser Phe Leu Arg Pro Leu Leu
Asp 635 640 645 Arg Thr Val Thr Lys Gly Glu Thr Ala Val Leu Gln Cys
Ile Ala 650 655 660 Gly Gly Ser Pro Pro Pro Lys Leu Asn Trp Thr Lys
Asp Asp Ser 665 670 675 Pro Leu Val Val Thr Glu Arg His Phe Phe Ala
Ala Gly Asn Gln 680 685 690 Leu Leu Ile Ile Val Asp Ser Asp Val Ser
Asp Ala Gly Lys Tyr 695 700 705 Thr Cys Glu Met Ser Asn Thr Leu Gly
Thr Glu Arg Gly Asn Val 710 715 720 Arg Leu Ser Val Ile Pro Thr Pro
Thr Cys Asp Ser Pro Gln Met 725 730 735 Thr Ala Pro Ser Leu Asp Asp
Asp Gly Trp Ala Thr Val Gly Val 740 745 750 Val Ile Ile Ala Val Val
Cys Cys Val Val Gly Thr Ser Leu Val 755 760 765 Trp Val Val Ile Ile
Tyr His Thr Arg Arg Arg Asn Glu Asp Cys 770 775 780 Ser Ile Thr Asn
Thr Asp Glu Thr Asn Leu Pro Ala Asp Ile Pro 785 790 795 Ser Tyr Leu
Ser Ser Gln Gly Thr Leu Ala Asp Arg Gln Asp Gly 800 805 810 Tyr Val
Ser Ser Glu Ser Gly Ser His His Gln Phe Val Thr Ser 815 820 825 Ser
Gly Ala Gly Phe Phe Leu Pro Gln His Asp Ser Ser Gly Thr 830 835 840
Cys His Ile Asp Asn Ser Ser Glu Ala Asp Val Glu Ala Ala Thr 845 850
855 Asp Leu Phe Leu Cys Pro Phe Leu Gly Ser Thr Gly Pro Met Tyr 860
865 870 Leu Lys Gly Asn Val Tyr Gly Ser Asp Pro Phe Glu Thr Tyr His
875 880 885 Thr Gly Cys Ser Pro Asp Pro Arg Thr Val Leu Met Asp His
Tyr 890 895 900 Glu Pro Ser Tyr Ile Lys Lys Lys Glu Cys Tyr Pro Cys
Ser His 905 910 915 Pro Ser Glu Glu Ser Cys Glu Arg Ser Phe Ser Asn
Ile Ser Trp 920 925 930 Pro Ser His Val Arg Lys Leu Leu Asn Thr Ser
Tyr Ser His Asn 935 940 945 Glu Gly Pro Gly Met Lys Asn Leu Cys Leu
Asn Lys Ser Ser Leu 950 955 960 Asp Phe Ser Ala Asn Pro Glu Pro Ala
Ser Val Ala Ser Ser Asn 965 970 975 Ser Phe Met Gly Thr Phe Gly Lys
Ala Leu Arg Arg Pro His Leu 980 985 990 Asp Ala Tyr Ser Ser Phe Gly
Gln Pro Ser Asp Cys Gln Pro Arg 995 1000 1005 Ala Phe Tyr Leu Lys
Ala His Ser Ser Pro Asp Leu Asp Ser Gly 1010 1015 1020 Ser Glu Glu
Asp Gly Lys Glu Arg Thr Asp Phe Gln Glu Glu Asn 1025 1030 1035 His
Ile Cys Thr Phe Lys Gln Thr Leu Glu Asn Tyr Arg Thr Pro 1040 1045
1050 Asn Phe Gln Ser Tyr Asp Leu Asp Thr 1055 291 2906 DNA Homo
Sapien 291 ggggagagga attgaccatg taaaaggaga cttttttttt tggtggtggt
50 ggctgttggg tgccttgcaa aaatgaagga tgcaggacgc agctttctcc 100
tggaaccgaa cgcaatggat aaactgattg tgcaagagag aaggaagaac 150
gaagcttttt cttgtgagcc ctggatctta acacaaatgt gtatatgtgc 200
acacagggag cattcaagaa tgaaataaac cagagttaga cccgcggggg 250
ttggtgtgtt ctgacataaa taaataatct taaagcagct gttcccctcc 300
ccacccccaa aaaaaaggat gattggaaat gaagaaccga ggattcacaa 350
agaaaaaagt atgttcattt ttctctataa aggagaaagt gagccaagga 400
gatatttttg gaatgaaaag tttggggctt ttttagtaaa gtaaagaact 450
ggtgtggtgg tgttttcctt tctttttgaa tttcccacaa gaggagagga 500
aattaataat acatctgcaa agaaatttca gagaagaaaa gttgaccgcg 550
gcagattgag gcattgattg ggggagagaa accagcagag cacagttgga 600
tttgtgccta tgttgactaa aattgacgga taattgcagt tggatttttc 650
ttcatcaacc tccttttttt taaattttta ttccttttgg tatcaagatc 700
atgcgttttc tcttgttctt aaccacctgg atttccatct ggatgttgct 750
gtgatcagtc tgaaatacaa ctgtttgaat tccagaagga ccaacaccag 800
ataaattatg aatgttgaac aagatgacct tacatccaca gcagataatg 850
ataggtccta ggtttaacag ggccctattt gaccccctgc ttgtggtgct 900
gctggctctt caacttcttg tggtggctgg tctggtgcgg gctcagacct 950
gcccttctgt gtgctcctgc agcaaccagt tcagcaaggt gatttgtgtt 1000
cggaaaaacc tgcgtgaggt tccggatggc atctccacca acacacggct 1050
gctgaacctc catgagaacc aaatccagat catcaaagtg aacagcttca 1100
agcacttgag gcacttggaa atcctacagt tgagtaggaa ccatatcaga 1150
accattgaaa ttggggcttt caatggtctg gcgaacctca acactctgga 1200
actctttgac aatcgtctta ctaccatccc gaatggagct tttgtatact 1250
tgtctaaact gaaggagctc tggttgcgaa acaaccccat tgaaagcatc 1300
ccttcttatg cttttaacag aattccttct ttgcgccgac tagacttagg 1350
ggaattgaaa agactttcat acatctcaga aggtgccttt gaaggtctgt 1400
ccaacttgag gtatttgaac cttgccatgt gcaaccttcg ggaaatccct 1450
aacctcacac cgctcataaa actagatgag ctggatcttt ctgggaatca 1500
tttatctgcc atcaggcctg gctctttcca gggtttgatg caccttcaaa 1550
aactgtggat gatacagtcc cagattcaag tgattgaacg gaatgccttt 1600
gacaaccttc agtcactagt ggagatcaac ctggcacaca ataatctaac 1650
attactgcct catgacctct tcactccctt gcatcatcta gagcggatac 1700
atttacatca caacccttgg aactgtaact gtgacatact gtggctcagc 1750
tggtggataa aagacatggc cccctcgaac acagcttgtt gtgcccggtg 1800
taacactcct cccaatctaa aggggaggta cattggagag ctcgaccaga 1850
attacttcac atgctatgct ccggtgattg tggagccccc tgcagacctc 1900
aatgtcactg aaggcatggc agctgagctg aaatgtcggg cctccacatc 1950
cctgacatct gtatcttgga ttactccaaa tggaacagtc atgacacatg 2000
gggcgtacaa agtgcggata gctgtgctca gtgatggtac gttaaatttc 2050
acaaatgtaa ctgtgcaaga tacaggcatg tacacatgta tggtgagtaa 2100
ttccgttggg aatactactg cttcagccac cctgaatgtt actgcagcaa 2150
ccactactcc tttctcttac ttttcaaccg tcacagtaga gactatggaa 2200
ccgtctcagg atgaggcacg gaccacagat aacaatgtgg gtcccactcc 2250
agtggtcgac tgggagacca ccaatgtgac cacctctctc acaccacaga 2300
gcacaaggtc gacagagaaa accttcacca tcccagtgac tgatataaac 2350
agtgggatcc caggaattga tgaggtcatg aagactacca aaatcatcat 2400
tgggtgtttt gtggccatca cactcatggc tgcagtgatg ctggtcattt 2450
tctacaagat gaggaagcag caccatcggc aaaaccatca cgccccaaca 2500
aggactgttg aaattattaa tgtggatgat gagattacgg gagacacacc 2550
catggaaagc cacctgccca tgcctgctat cgagcatgag cacctaaatc 2600
actataactc atacaaatct cccttcaacc acacaacaac agttaacaca 2650
ataaattcaa tacacagttc agtgcatgaa ccgttattga tccgaatgaa 2700
ctctaaagac aatgtacaag agactcaaat ctaaaacatt tacagagtta 2750
caaaaaacaa acaatcaaaa aaaaagacag tttattaaaa atgacacaaa 2800
tgactgggct aaatctactg tttcaaaaaa
gtgtctttac aaaaaaacaa 2850 aaaagaaaag aaatttattt attaaaaatt
ctattgtgat ctaaagcaga 2900 caaaaa 2906 292 640 PRT Homo Sapien 292
Met Leu Asn Lys Met Thr Leu His Pro Gln Gln Ile Met Ile Gly 1 5 10
15 Pro Arg Phe Asn Arg Ala Leu Phe Asp Pro Leu Leu Val Val Leu 20
25 30 Leu Ala Leu Gln Leu Leu Val Val Ala Gly Leu Val Arg Ala Gln
35 40 45 Thr Cys Pro Ser Val Cys Ser Cys Ser Asn Gln Phe Ser Lys
Val 50 55 60 Ile Cys Val Arg Lys Asn Leu Arg Glu Val Pro Asp Gly
Ile Ser 65 70 75 Thr Asn Thr Arg Leu Leu Asn Leu His Glu Asn Gln
Ile Gln Ile 80 85 90 Ile Lys Val Asn Ser Phe Lys His Leu Arg His
Leu Glu Ile Leu 95 100 105 Gln Leu Ser Arg Asn His Ile Arg Thr Ile
Glu Ile Gly Ala Phe 110 115 120 Asn Gly Leu Ala Asn Leu Asn Thr Leu
Glu Leu Phe Asp Asn Arg 125 130 135 Leu Thr Thr Ile Pro Asn Gly Ala
Phe Val Tyr Leu Ser Lys Leu 140 145 150 Lys Glu Leu Trp Leu Arg Asn
Asn Pro Ile Glu Ser Ile Pro Ser 155 160 165 Tyr Ala Phe Asn Arg Ile
Pro Ser Leu Arg Arg Leu Asp Leu Gly 170 175 180 Glu Leu Lys Arg Leu
Ser Tyr Ile Ser Glu Gly Ala Phe Glu Gly 185 190 195 Leu Ser Asn Leu
Arg Tyr Leu Asn Leu Ala Met Cys Asn Leu Arg 200 205 210 Glu Ile Pro
Asn Leu Thr Pro Leu Ile Lys Leu Asp Glu Leu Asp 215 220 225 Leu Ser
Gly Asn His Leu Ser Ala Ile Arg Pro Gly Ser Phe Gln 230 235 240 Gly
Leu Met His Leu Gln Lys Leu Trp Met Ile Gln Ser Gln Ile 245 250 255
Gln Val Ile Glu Arg Asn Ala Phe Asp Asn Leu Gln Ser Leu Val 260 265
270 Glu Ile Asn Leu Ala His Asn Asn Leu Thr Leu Leu Pro His Asp 275
280 285 Leu Phe Thr Pro Leu His His Leu Glu Arg Ile His Leu His His
290 295 300 Asn Pro Trp Asn Cys Asn Cys Asp Ile Leu Trp Leu Ser Trp
Trp 305 310 315 Ile Lys Asp Met Ala Pro Ser Asn Thr Ala Cys Cys Ala
Arg Cys 320 325 330 Asn Thr Pro Pro Asn Leu Lys Gly Arg Tyr Ile Gly
Glu Leu Asp 335 340 345 Gln Asn Tyr Phe Thr Cys Tyr Ala Pro Val Ile
Val Glu Pro Pro 350 355 360 Ala Asp Leu Asn Val Thr Glu Gly Met Ala
Ala Glu Leu Lys Cys 365 370 375 Arg Ala Ser Thr Ser Leu Thr Ser Val
Ser Trp Ile Thr Pro Asn 380 385 390 Gly Thr Val Met Thr His Gly Ala
Tyr Lys Val Arg Ile Ala Val 395 400 405 Leu Ser Asp Gly Thr Leu Asn
Phe Thr Asn Val Thr Val Gln Asp 410 415 420 Thr Gly Met Tyr Thr Cys
Met Val Ser Asn Ser Val Gly Asn Thr 425 430 435 Thr Ala Ser Ala Thr
Leu Asn Val Thr Ala Ala Thr Thr Thr Pro 440 445 450 Phe Ser Tyr Phe
Ser Thr Val Thr Val Glu Thr Met Glu Pro Ser 455 460 465 Gln Asp Glu
Ala Arg Thr Thr Asp Asn Asn Val Gly Pro Thr Pro 470 475 480 Val Val
Asp Trp Glu Thr Thr Asn Val Thr Thr Ser Leu Thr Pro 485 490 495 Gln
Ser Thr Arg Ser Thr Glu Lys Thr Phe Thr Ile Pro Val Thr 500 505 510
Asp Ile Asn Ser Gly Ile Pro Gly Ile Asp Glu Val Met Lys Thr 515 520
525 Thr Lys Ile Ile Ile Gly Cys Phe Val Ala Ile Thr Leu Met Ala 530
535 540 Ala Val Met Leu Val Ile Phe Tyr Lys Met Arg Lys Gln His His
545 550 555 Arg Gln Asn His His Ala Pro Thr Arg Thr Val Glu Ile Ile
Asn 560 565 570 Val Asp Asp Glu Ile Thr Gly Asp Thr Pro Met Glu Ser
His Leu 575 580 585 Pro Met Pro Ala Ile Glu His Glu His Leu Asn His
Tyr Asn Ser 590 595 600 Tyr Lys Ser Pro Phe Asn His Thr Thr Thr Val
Asn Thr Ile Asn 605 610 615 Ser Ile His Ser Ser Val His Glu Pro Leu
Leu Ile Arg Met Asn 620 625 630 Ser Lys Asp Asn Val Gln Glu Thr Gln
Ile 635 640 293 4053 DNA Homo Sapien 293 agccgacgct gctcaagctg
caactctgtt gcagttggca gttcttttcg 50 gtttccctcc tgctgtttgg
gggcatgaaa gggcttcgcc gccgggagta 100 aaagaaggaa ttgaccgggc
agcgcgaggg aggagcgcgc acgcgaccgc 150 gagggcgggc gtgcaccctc
ggctggaagt ttgtgccggg ccccgagcgc 200 gcgccggctg ggagcttcgg
gtagagacct aggccgctgg accgcgatga 250 gcgcgccgag cctccgtgcg
cgcgccgcgg ggttggggct gctgctgtgc 300 gcggtgctgg ggcgcgctgg
ccggtccgac agcggcggtc gcggggaact 350 cgggcagccc tctggggtag
ccgccgagcg cccatgcccc actacctgcc 400 gctgcctcgg ggacctgctg
gactgcagtc gtaagcggct agcgcgtctt 450 cccgagccac tcccgtcctg
ggtcgctcgg ctggacttaa gtcacaacag 500 attatctttc atcaaggcaa
gttccatgag ccaccttcaa agccttcgag 550 aagtgaaact gaacaacaat
gaattggaga ccattccaaa tctgggacca 600 gtctcggcaa atattacact
tctctccttg gctggaaaca ggattgttga 650 aatactccct gaacatctga
aagagtttca gtcccttgaa actttggacc 700 ttagcagcaa caatatttca
gagctccaaa ctgcatttcc agccctacag 750 ctcaaatatc tgtatctcaa
cagcaaccga gtcacatcaa tggaacctgg 800 gtattttgac aatttggcca
acacactcct tgtgttaaag ctgaacagga 850 accgaatctc agctatccca
cccaagatgt ttaaactgcc ccaactgcaa 900 catctcgaat tgaaccgaaa
caagattaaa aatgtagatg gactgacatt 950 ccaaggcctt ggtgctctga
agtctctgaa aatgcaaaga aatggagtaa 1000 cgaaacttat ggatggagct
ttttgggggc tgagcaacat ggaaattttg 1050 cagctggacc ataacaacct
aacagagatt accaaaggct ggctttacgg 1100 cttgctgatg ctgcaggaac
ttcatctcag ccaaaatgcc atcaacagga 1150 tcagccctga tgcctgggag
ttctgccaga agctcagtga gctggaccta 1200 actttcaatc acttatcaag
gttagatgat tcaagcttcc ttggcctaag 1250 cttactaaat acactgcaca
ttgggaacaa cagagtcagc tacattgctg 1300 attgtgcctt ccgggggctt
tccagtttaa agactttgga tctgaagaac 1350 aatgaaattt cctggactat
tgaagacatg aatggtgctt tctctgggct 1400 tgacaaactg aggcgactga
tactccaagg aaatcggatc cgttctatta 1450 ctaaaaaagc cttcactggt
ttggatgcat tggagcatct agacctgagt 1500 gacaacgcaa tcatgtcttt
acaaggcaat gcattttcac aaatgaagaa 1550 actgcaacaa ttgcatttaa
atacatcaag ccttttgtgc gattgccagc 1600 taaaatggct cccacagtgg
gtggcggaaa acaactttca gagctttgta 1650 aatgccagtt gtgcccatcc
tcagctgcta aaaggaagaa gcatttttgc 1700 tgttagccca gatggctttg
tgtgtgatga ttttcccaaa ccccagatca 1750 cggttcagcc agaaacacag
tcggcaataa aaggttccaa tttgagtttc 1800 atctgctcag ctgccagcag
cagtgattcc ccaatgactt ttgcttggaa 1850 aaaagacaat gaactactgc
atgatgctga aatggaaaat tatgcacacc 1900 tccgggccca aggtggcgag
gtgatggagt ataccaccat ccttcggctg 1950 cgcgaggtgg aatttgccag
tgaggggaaa tatcagtgtg tcatctccaa 2000 tcactttggt tcatcctact
ctgtcaaagc caagcttaca gtaaatatgc 2050 ttccctcatt caccaagacc
cccatggatc tcaccatccg agctggggcc 2100 atggcacgct tggagtgtgc
tgctgtgggg cacccagccc cccagatagc 2150 ctggcagaag gatgggggca
cagacttccc agctgcacgg gagagacgca 2200 tgcatgtgat gcccgaggat
gacgtgttct ttatcgtgga tgtgaagata 2250 gaggacattg gggtatacag
ctgcacagct cagaacagtg caggaagtat 2300 ttcagcaaat gcaactctga
ctgtcctaga aacaccatca tttttgcggc 2350 cactgttgga ccgaactgta
accaagggag aaacagccgt cctacagtgc 2400 attgctggag gaagccctcc
ccctaaactg aactggacca aagatgatag 2450 cccattggtg gtaaccgaga
ggcacttttt tgcagcaggc aatcagcttc 2500 tgattattgt ggactcagat
gtcagtgatg ctgggaaata cacatgtgag 2550 atgtctaaca cccttggcac
tgagagagga aacgtgcgcc tcagtgtgat 2600 ccccactcca acctgcgact
cccctcagat gacagcccca tcgttagacg 2650 atgacggatg ggccactgtg
ggtgtcgtga tcatagccgt ggtttgctgt 2700 gtggtgggca cgtcactcgt
gtgggtggtc atcatatacc acacaaggcg 2750 gaggaatgaa gattgcagca
ttaccaacac agatgagacc aacttgccag 2800 cagatattcc tagttatttg
tcatctcagg gaacgttagc tgacaggcag 2850 gatgggtacg tgtcttcaga
aagtggaagc caccaccagt ttgtcacatc 2900 ttcaggtgct ggatttttct
taccacaaca tgacagtagt gggacctgcc 2950 atattgacaa tagcagtgaa
gctgatgtgg aagctgccac agatctgttc 3000 ctttgtccgt ttttgggatc
cacaggccct atgtatttga agggaaatgt 3050 gtatggctca gatccttttg
aaacatatca tacaggttgc agtcctgacc 3100 caagaacagt tttaatggac
cactatgagc ccagttacat aaagaaaaag 3150 gagtgctacc catgttctca
tccttcagaa gaatcctgcg aacggagctt 3200 cagtaatata tcgtggcctt
cacatgtgag gaagctactt aacactagtt 3250 actctcacaa tgaaggacct
ggaatgaaaa atctgtgtct aaacaagtcc 3300 tctttagatt ttagtgcaaa
tccagagcca gcgtcggttg cctcgagtaa 3350 ttctttcatg ggtacctttg
gaaaagctct caggagacct cacctagatg 3400 cctattcaag ctttggacag
ccatcagatt gtcagccaag agccttttat 3450 ttgaaagctc attcttcccc
agacttggac tctgggtcag aggaagatgg 3500 gaaagaaagg acagattttc
aggaagaaaa tcacatttgt acctttaaac 3550 agactttaga aaactacagg
actccaaatt ttcagtctta tgacttggac 3600 acatagactg aatgagacca
aaggaaaagc ttaacatact acctcaagtg 3650 aacttttatt taaaagagag
agaatcttat gttttttaaa tggagttatg 3700 aattttaaaa ggataaaaat
gctttattta tacagatgaa ccaaaattac 3750 aaaaagttat gaaaattttt
atactgggaa tgatgctcat ataagaatac 3800 ctttttaaac tattttttaa
ctttgtttta tgcaaaaaag tatcttacgt 3850 aaattaatga tataaatcat
gattatttta tgtattttta taatgccaga 3900 tttcttttta tggaaaatga
gttactaaag cattttaaat aatacctgcc 3950 ttgtaccatt ttttaaatag
aagttacttc attatatttt gcacattata 4000 tttaataaaa tgtgtcaatt
tgaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4050 aaa 4053 294 1119 PRT Homo
Sapien 294 Met Ser Ala Pro Ser Leu Arg Ala Arg Ala Ala Gly Leu Gly
Leu 1 5 10 15 Leu Leu Cys Ala Val Leu Gly Arg Ala Gly Arg Ser Asp
Ser Gly 20 25 30 Gly Arg Gly Glu Leu Gly Gln Pro Ser Gly Val Ala
Ala Glu Arg 35 40 45 Pro Cys Pro Thr Thr Cys Arg Cys Leu Gly Asp
Leu Leu Asp Cys 50 55 60 Ser Arg Lys Arg Leu Ala Arg Leu Pro Glu
Pro Leu Pro Ser Trp 65 70 75 Val Ala Arg Leu Asp Leu Ser His Asn
Arg Leu Ser Phe Ile Lys 80 85 90 Ala Ser Ser Met Ser His Leu Gln
Ser Leu Arg Glu Val Lys Leu 95 100 105 Asn Asn Asn Glu Leu Glu Thr
Ile Pro Asn Leu Gly Pro Val Ser 110 115 120 Ala Asn Ile Thr Leu Leu
Ser Leu Ala Gly Asn Arg Ile Val Glu 125 130 135 Ile Leu Pro Glu His
Leu Lys Glu Phe Gln Ser Leu Glu Thr Leu 140 145 150 Asp Leu Ser Ser
Asn Asn Ile Ser Glu Leu Gln Thr Ala Phe Pro 155 160 165 Ala Leu Gln
Leu Lys Tyr Leu Tyr Leu Asn Ser Asn Arg Val Thr 170 175 180 Ser Met
Glu Pro Gly Tyr Phe Asp Asn Leu Ala Asn Thr Leu Leu 185 190 195 Val
Leu Lys Leu Asn Arg Asn Arg Ile Ser Ala Ile Pro Pro Lys 200 205 210
Met Phe Lys Leu Pro Gln Leu Gln His Leu Glu Leu Asn Arg Asn 215 220
225 Lys Ile Lys Asn Val Asp Gly Leu Thr Phe Gln Gly Leu Gly Ala 230
235 240 Leu Lys Ser Leu Lys Met Gln Arg Asn Gly Val Thr Lys Leu Met
245 250 255 Asp Gly Ala Phe Trp Gly Leu Ser Asn Met Glu Ile Leu Gln
Leu 260 265 270 Asp His Asn Asn Leu Thr Glu Ile Thr Lys Gly Trp Leu
Tyr Gly 275 280 285 Leu Leu Met Leu Gln Glu Leu His Leu Ser Gln Asn
Ala Ile Asn 290 295 300 Arg Ile Ser Pro Asp Ala Trp Glu Phe Cys Gln
Lys Leu Ser Glu 305 310 315 Leu Asp Leu Thr Phe Asn His Leu Ser Arg
Leu Asp Asp Ser Ser 320 325 330 Phe Leu Gly Leu Ser Leu Leu Asn Thr
Leu His Ile Gly Asn Asn 335 340 345 Arg Val Ser Tyr Ile Ala Asp Cys
Ala Phe Arg Gly Leu Ser Ser 350 355 360 Leu Lys Thr Leu Asp Leu Lys
Asn Asn Glu Ile Ser Trp Thr Ile 365 370 375 Glu Asp Met Asn Gly Ala
Phe Ser Gly Leu Asp Lys Leu Arg Arg 380 385 390 Leu Ile Leu Gln Gly
Asn Arg Ile Arg Ser Ile Thr Lys Lys Ala 395 400 405 Phe Thr Gly Leu
Asp Ala Leu Glu His Leu Asp Leu Ser Asp Asn 410 415 420 Ala Ile Met
Ser Leu Gln Gly Asn Ala Phe Ser Gln Met Lys Lys 425 430 435 Leu Gln
Gln Leu His Leu Asn Thr Ser Ser Leu Leu Cys Asp Cys 440 445 450 Gln
Leu Lys Trp Leu Pro Gln Trp Val Ala Glu Asn Asn Phe Gln 455 460 465
Ser Phe Val Asn Ala Ser Cys Ala His Pro Gln Leu Leu Lys Gly 470 475
480 Arg Ser Ile Phe Ala Val Ser Pro Asp Gly Phe Val Cys Asp Asp 485
490 495 Phe Pro Lys Pro Gln Ile Thr Val Gln Pro Glu Thr Gln Ser Ala
500 505 510 Ile Lys Gly Ser Asn Leu Ser Phe Ile Cys Ser Ala Ala Ser
Ser 515 520 525 Ser Asp Ser Pro Met Thr Phe Ala Trp Lys Lys Asp Asn
Glu Leu 530 535 540 Leu His Asp Ala Glu Met Glu Asn Tyr Ala His Leu
Arg Ala Gln 545 550 555 Gly Gly Glu Val Met Glu Tyr Thr Thr Ile Leu
Arg Leu Arg Glu 560 565 570 Val Glu Phe Ala Ser Glu Gly Lys Tyr Gln
Cys Val Ile Ser Asn 575 580 585 His Phe Gly Ser Ser Tyr Ser Val Lys
Ala Lys Leu Thr Val Asn 590 595 600 Met Leu Pro Ser Phe Thr Lys Thr
Pro Met Asp Leu Thr Ile Arg 605 610 615 Ala Gly Ala Met Ala Arg Leu
Glu Cys Ala Ala Val Gly His Pro 620 625 630 Ala Pro Gln Ile Ala Trp
Gln Lys Asp Gly Gly Thr Asp Phe Pro 635 640 645 Ala Ala Arg Glu Arg
Arg Met His Val Met Pro Glu Asp Asp Val 650 655 660 Phe Phe Ile Val
Asp Val Lys Ile Glu Asp Ile Gly Val Tyr Ser 665 670 675 Cys Thr Ala
Gln Asn Ser Ala Gly Ser Ile Ser Ala Asn Ala Thr 680 685 690 Leu Thr
Val Leu Glu Thr Pro Ser Phe Leu Arg Pro Leu Leu Asp 695 700 705 Arg
Thr Val Thr Lys Gly Glu Thr Ala Val Leu Gln Cys Ile Ala 710 715 720
Gly Gly Ser Pro Pro Pro Lys Leu Asn Trp Thr Lys Asp Asp Ser 725 730
735 Pro Leu Val Val Thr Glu Arg His Phe Phe Ala Ala Gly Asn Gln 740
745 750 Leu Leu Ile Ile Val Asp Ser Asp Val Ser Asp Ala Gly Lys Tyr
755 760 765 Thr Cys Glu Met Ser Asn Thr Leu Gly Thr Glu Arg Gly Asn
Val 770 775 780 Arg Leu Ser Val Ile Pro Thr Pro Thr Cys Asp Ser Pro
Gln Met 785 790 795 Thr Ala Pro Ser Leu Asp Asp Asp Gly Trp Ala Thr
Val Gly Val 800 805 810 Val Ile Ile Ala Val Val Cys Cys Val Val Gly
Thr Ser Leu Val 815 820 825 Trp Val Val Ile Ile Tyr His Thr Arg Arg
Arg Asn Glu Asp Cys 830 835 840 Ser Ile Thr Asn Thr Asp Glu Thr Asn
Leu Pro Ala Asp Ile Pro 845 850 855 Ser Tyr Leu Ser Ser Gln Gly Thr
Leu Ala Asp Arg Gln Asp Gly 860 865 870 Tyr Val Ser Ser Glu Ser Gly
Ser His His Gln
Phe Val Thr Ser 875 880 885 Ser Gly Ala Gly Phe Phe Leu Pro Gln His
Asp Ser Ser Gly Thr 890 895 900 Cys His Ile Asp Asn Ser Ser Glu Ala
Asp Val Glu Ala Ala Thr 905 910 915 Asp Leu Phe Leu Cys Pro Phe Leu
Gly Ser Thr Gly Pro Met Tyr 920 925 930 Leu Lys Gly Asn Val Tyr Gly
Ser Asp Pro Phe Glu Thr Tyr His 935 940 945 Thr Gly Cys Ser Pro Asp
Pro Arg Thr Val Leu Met Asp His Tyr 950 955 960 Glu Pro Ser Tyr Ile
Lys Lys Lys Glu Cys Tyr Pro Cys Ser His 965 970 975 Pro Ser Glu Glu
Ser Cys Glu Arg Ser Phe Ser Asn Ile Ser Trp 980 985 990 Pro Ser His
Val Arg Lys Leu Leu Asn Thr Ser Tyr Ser His Asn 995 1000 1005 Glu
Gly Pro Gly Met Lys Asn Leu Cys Leu Asn Lys Ser Ser Leu 1010 1015
1020 Asp Phe Ser Ala Asn Pro Glu Pro Ala Ser Val Ala Ser Ser Asn
1025 1030 1035 Ser Phe Met Gly Thr Phe Gly Lys Ala Leu Arg Arg Pro
His Leu 1040 1045 1050 Asp Ala Tyr Ser Ser Phe Gly Gln Pro Ser Asp
Cys Gln Pro Arg 1055 1060 1065 Ala Phe Tyr Leu Lys Ala His Ser Ser
Pro Asp Leu Asp Ser Gly 1070 1075 1080 Ser Glu Glu Asp Gly Lys Glu
Arg Thr Asp Phe Gln Glu Glu Asn 1085 1090 1095 His Ile Cys Thr Phe
Lys Gln Thr Leu Glu Asn Tyr Arg Thr Pro 1100 1105 1110 Asn Phe Gln
Ser Tyr Asp Leu Asp Thr 1115 295 18 DNA Artificial Sequence
Synthetic Oligonucleotide Probe 295 ggaaccgaat ctcagcta 18 296 19
DNA Artificial Sequence Synthetic Oligonucleotide Probe 296
cctaaactga actggacca 19 297 19 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 297 ggctggagac actgaacct 19 298 24 DNA
Artificial Sequence Synthetic Oligonucleotide Probe 298 acagctgcac
agctcagaac agtg 24 299 22 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 299 cattcccagt ataaaaattt tc 22 300 18 DNA
Artificial Sequence Synthetic Oligonucleotide Probe 300 gggtcttggt
gaatgagg 18 301 24 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 301 gtgcctctcg gttaccacca atgg 24 302 50 DNA
Artificial Sequence Synthetic Oligonucleotide Probe 302 gcggccactg
ttggaccgaa ctgtaaccaa gggagaaaca gccgtcctac 50 303 28 DNA
Artificial Sequence Synthetic Oligonucleotide Probe 303 gcctttgaca
accttcagtc actagtgg 28 304 24 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 304 ccccatgtgt ccatgactgt tccc 24 305 45 DNA
Artificial Sequence Synthetic Oligonucleotide Probe 305 tactgcctca
tgacctcttc actcccttgc atcatcttag agcgg 45 306 24 DNA Artificial
Sequence Synthetic Oligonucleotide Probe 306 actccaagga aatcggatcc
gttc 24 307 24 DNA Artificial Sequence Synthetic oligonucleotide
probe 307 ttagcagctg aggatgggca caac 24 308 24 DNA Artificial
Sequence Synthetic Oligonucleotide Probe 308 actccaagga aatcggatcc
gttc 24 309 50 DNA Artificial Sequence Synthetic Oligonucleotide
Probe 309 gccttcactg gtttggatgc attggagcat ctagacctga gtgacaacgc 50
310 3296 DNA Homo Sapien 310 caaaacttgc gtcgcggaga gcgcccagct
tgacttgaat ggaaggagcc 50 cgagcccgcg gagcgcagct gagactgggg
gagcgcgttc ggcctgtggg 100 gcgccgctcg gcgccggggc gcagcaggga
aggggaagct gtggtctgcc 150 ctgctccacg aggcgccact ggtgtgaacc
gggagagccc ctgggtggtc 200 ccgtccccta tccctccttt atatagaaac
cttccacact gggaaggcag 250 cggcgaggca ggagggctca tggtgagcaa
ggaggccggc tgatctgcag 300 gcgcacagca ttccgagttt acagattttt
acagatacca aatggaaggc 350 gaggaggcag aacagcctgc ctggttccat
cagccctggc gcccaggcgc 400 atctgactcg gcaccccctg caggcaccat
ggcccagagc cgggtgctgc 450 tgctcctgct gctgctgccg ccacagctgc
acctgggacc tgtgcttgcc 500 gtgagggccc caggatttgg ccgaagtggc
ggccacagcc tgagccccga 550 agagaacgaa tttgcggagg aggagccggt
gctggtactg agccctgagg 600 agcccgggcc tggcccagcc gcggtcagct
gcccccgaga ctgtgcctgt 650 tcccaggagg gcgtcgtgga ctgtggcggt
attgacctgc gtgagttccc 700 gggggacctg cctgagcaca ccaaccacct
atctctgcag aacaaccagc 750 tggaaaagat ctaccctgag gagctctccc
ggctgcaccg gctggagaca 800 ctgaacctgc aaaacaaccg cctgacttcc
cgagggctcc cagagaaggc 850 gtttgagcat ctgaccaacc tcaattacct
gtacttggcc aataacaagc 900 tgaccttggc accccgcttc ctgccaaacg
ccctgatcag tgtggacttt 950 gctgccaact atctcaccaa gatctatggg
ctcacctttg gccagaagcc 1000 aaacttgagg tctgtgtacc tgcacaacaa
caagctggca gacgccgggc 1050 tgccggacaa catgttcaac ggctccagca
acgtcgaggt cctcatcctg 1100 tccagcaact tcctgcgcca cgtgcccaag
cacctgccgc ctgccctgta 1150 caagctgcac ctcaagaaca acaagctgga
gaagatcccc ccgggggcct 1200 tcagcgagct gagcagcctg cgcgagctat
acctgcagaa caactacctg 1250 actgacgagg gcctggacaa cgagaccttc
tggaagctct ccagcctgga 1300 gtacctggat ctgtccagca acaacctgtc
tcgggtccca gctgggctgc 1350 cgcgcagcct ggtgctgctg cacttggaga
agaacgccat ccggagcgtg 1400 gacgcgaatg tgctgacccc catccgcagc
ctggagtacc tgctgctgca 1450 cagcaaccag ctgcgggagc agggcatcca
cccactggcc ttccagggcc 1500 tcaagcggtt gcacacggtg cacctgtaca
acaacgcgct ggagcgcgtg 1550 cccagtggcc tgcctcgccg cgtgcgcacc
ctcatgatcc tgcacaacca 1600 gatcacaggc attggccgcg aagactttgc
caccacctac ttcctggagg 1650 agctcaacct cagctacaac cgcatcacca
gcccacaggt gcaccgcgac 1700 gccttccgca agctgcgcct gctgcgctcg
ctggacctgt cgggcaaccg 1750 gctgcacacg ctgccacctg ggctgcctcg
aaatgtccat gtgctgaagg 1800 tcaagcgcaa tgagctggct gccttggcac
gaggggcgct ggcgggcatg 1850 gctcagctgc gtgagctgta cctcaccagc
aaccgactgc gcagccgagc 1900 cctgggcccc cgtgcctggg tggacctcgc
ccatctgcag ctgctggaca 1950 tcgccgggaa tcagctcaca gagatccccg
aggggctccc cgagtcactt 2000 gagtacctgt acctgcagaa caacaagatt
agtgcggtgc ccgccaatgc 2050 cttcgactcc acgcccaacc tcaaggggat
ctttctcagg tttaacaagc 2100 tggctgtggg ctccgtggtg gacagtgcct
tccggaggct gaagcacctg 2150 caggtcttgg acattgaagg caacttagag
tttggtgaca tttccaagga 2200 ccgtggccgc ttggggaagg aaaaggagga
ggaggaagag gaggaggagg 2250 aggaagagga aacaagatag tgacaaggtg
atgcagatgt gacctaggat 2300 gatggaccgc cggactcttt tctgcagcac
acgcctgtgt gctgtgagcc 2350 ccccactctg ccgtgctcac acagacacac
ccagctgcac acatgaggca 2400 tcccacatga cacgggctga cacagtctca
tatccccacc ccttcccacg 2450 gcgtgtccca cggccagaca catgcacaca
catcacaccc tcaaacaccc 2500 agctcagcca cacacaacta ccctccaaac
caccacagtc tctgtcacac 2550 ccccactacc gctgccacgc cctctgaatc
atgcagggaa gggtctgccc 2600 ctgccctggc acacacaggc acccattccc
tccccctgct gacatgtgta 2650 tgcgtatgca tacacaccac acacacacac
atgcacaagt catgtgcgaa 2700 cagccctcca aagcctatgc cacagacagc
tcttgcccca gccagaatca 2750 gccatagcag ctcgccgtct gccctgtcca
tctgtccgtc cgttccctgg 2800 agaagacaca agggtatcca tgctctgtgg
ccaggtgcct gccaccctct 2850 ggaactcaca aaagctggct tttattcctt
tcccatccta tggggacagg 2900 agccttcagg actgctggcc tggcctggcc
caccctgctc ctccaggtgc 2950 tgggcagtca ctctgctaag agtccctccc
tgccacgccc tggcaggaca 3000 caggcacttt tccaatgggc aagcccagtg
gaggcaggat gggagagccc 3050 cctgggtgct gctggggcct tggggcagga
gtgaagcaga ggtgatgggg 3100 ctgggctgag ccagggagga aggacccagc
tgcacctagg agacaccttt 3150 gttcttcagg cctgtggggg aagttccggg
tgcctttatt ttttattctt 3200 ttctaaggaa aaaaatgata aaaatctcaa
agctgatttt tcttgttata 3250 gaaaaactaa tataaaagca ttatccctat
ccctgcaaaa aaaaaa 3296 311 22 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 311 gcattggccg cgagactttg cc 22 312 22 DNA
Artificial Sequence Synthetic Oligonucleotide Probe 312 gcggccacgg
tccttggaaa tg 22 313 45 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 313 tggaggagct caacctcagc tacaaccgca
tcaccagccc acagg 45 314 3003 DNA Homo Sapien 314 gggagggggc
tccgggcgcc gcgcagcaga cctgctccgg ccgcgcgcct 50 cgccgctgtc
ctccgggagc ggcagcagta gcccgggcgg cgagggctgg 100 gggttcctcg
agactctcag aggggcgcct cccatcggcg cccaccaccc 150 caacctgttc
ctcgcgcgcc actgcgctgc gccccaggac ccgctgccca 200 acatggattt
tctcctggcg ctggtgctgg tatcctcgct ctacctgcag 250 gcggccgccg
agttcgacgg gaggtggccc aggcaaatag tgtcatcgat 300 tggcctatgt
cgttatggtg ggaggattga ctgctgctgg ggctgggctc 350 gccagtcttg
gggacagtgt cagcctgtgt gccaaccacg atgcaaacat 400 ggtgaatgta
tcgggccaaa caagtgcaag tgtcatcctg gttatgctgg 450 aaaaacctgt
aatcaagatc taaatgagtg tggcctgaag ccccggccct 500 gtaagcacag
gtgcatgaac acttacggca gctacaagtg ctactgtctc 550 aacggatata
tgctcatgcc ggatggttcc tgctcaagtg ccctgacctg 600 ctccatggca
aactgtcagt atggctgtga tgttgttaaa ggacaaatac 650 ggtgccagtg
cccatcccct ggcctgcacc tggctcctga tgggaggacc 700 tgtgtagatg
ttgatgaatg tgctacagga agagcctcct gccctagatt 750 taggcaatgt
gtcaacactt ttgggagcta catctgcaag tgtcataaag 800 gcttcgatct
catgtatatt ggaggcaaat atcaatgtca tgacatagac 850 gaatgctcac
ttggtcagta tcagtgcagc agctttgctc gatgttataa 900 cgtacgtggg
tcctacaagt gcaaatgtaa agaaggatac cagggtgatg 950 gactgacttg
tgtgtatatc ccaaaagtta tgattgaacc ttcaggtcca 1000 attcatgtac
caaagggaaa tggtaccatt ttaaagggtg acacaggaaa 1050 taataattgg
attcctgatg ttggaagtac ttggtggcct ccgaagacac 1100 catatattcc
tcctatcatt accaacaggc ctacttctaa gccaacaaca 1150 agacctacac
caaagccaac accaattcct actccaccac caccaccacc 1200 cctgccaaca
gagctcagaa cacctctacc acctacaacc ccagaaaggc 1250 caaccaccgg
actgacaact atagcaccag ctgccagtac acctccagga 1300 gggattacag
ttgacaacag ggtacagaca gaccctcaga aacccagagg 1350 agatgtgttc
agtgttctgg tacacagttg taattttgac catggacttt 1400 gtggatggat
cagggagaaa gacaatgact tgcactggga accaatcagg 1450 gacccagcag
gtggacaata tctgacagtg tcggcagcca aagccccagg 1500 gggaaaagct
gcacgcttgg tgctacctct cggccgcctc atgcattcag 1550 gggacctgtg
cctgtcattc aggcacaagg tgacggggct gcactctggc 1600 acactccagg
tgtttgtgag aaaacacggt gcccacggag cagccctgtg 1650 gggaagaaat
ggtggccatg gctggaggca aacacagatc accttgcgag 1700 gggctgacat
caagagcgaa tcacaaagat gattaaaggg ttggaaaaaa 1750 agatctatga
tggaaaatta aaggaactgg gattattgag cctggagaag 1800 agaagactga
ggggcaaacc attgatggtt ttcaagtata tgaagggttg 1850 gcacagagag
ggtggcgacc agctgttctc catatgcact aagaatagaa 1900 caagaggaaa
ctggcttaga ctagagtata agggagcatt tcttggcagg 1950 ggccattgtt
agaatacttc ataaaaaaag aagtgtgaaa atctcagtat 2000 ctctctctct
ttctaaaaaa ttagataaaa atttgtctat ttaagatggt 2050 taaagatgtt
cttacccaag gaaaagtaac aaattataga atttcccaaa 2100 agatgttttg
atcctactag tagtatgcag tgaaaatctt tagaactaaa 2150 taatttggac
aaggcttaat ttaggcattt ccctcttgac ctcctaatgg 2200 agagggattg
aaaggggaag agcccaccaa atgctgagct cactgaaata 2250 tctctccctt
atggcaatcc tagcagtatt aaagaaaaaa ggaaactatt 2300 tattccaaat
gagagtatga tggacagata ttttagtatc tcagtaatgt 2350 cctagtgtgg
cggtggtttt caatgtttct tcatggtaaa ggtataagcc 2400 tttcatttgt
tcaatggatg atgtttcaga tttttttttt tttaagagat 2450 ccttcaagga
acacagttca gagagatttt catcgggtgc attctctctg 2500 cttcgtgtgt
gacaagttat cttggctgct gagaaagagt gccctgcccc 2550 acaccggcag
acctttcctt cacctcatca gtatgattca gtttctctta 2600 tcaattggac
tctcccaggt tccacagaac agtaatattt tttgaacaat 2650 aggtacaata
gaaggtcttc tgtcatttaa cctggtaaag gcagggctgg 2700 agggggaaaa
taaatcatta agcctttgag taacggcaga atatatggct 2750 gtagatccat
ttttaatggt tcatttcctt tatggtcata taactgcaca 2800 gctgaagatg
aaaggggaaa ataaatgaaa attttacttt tcgatgccaa 2850 tgatacattg
cactaaactg atggaagaag ttatccaaag tactgtataa 2900 catcttgttt
attatttaat gttttctaaa ataaaaaatg ttagtggttt 2950 tccaaatggc
ctaataaaaa caattatttg taaataaaaa cactgttagt 3000 aat 3003 315 509
PRT Homo Sapien 315 Met Asp Phe Leu Leu Ala Leu Val Leu Val Ser Ser
Leu Tyr Leu 1 5 10 15 Gln Ala Ala Ala Glu Phe Asp Gly Arg Trp Pro
Arg Gln Ile Val 20 25 30 Ser Ser Ile Gly Leu Cys Arg Tyr Gly Gly
Arg Ile Asp Cys Cys 35 40 45 Trp Gly Trp Ala Arg Gln Ser Trp Gly
Gln Cys Gln Pro Val Cys 50 55 60 Gln Pro Arg Cys Lys His Gly Glu
Cys Ile Gly Pro Asn Lys Cys 65 70 75 Lys Cys His Pro Gly Tyr Ala
Gly Lys Thr Cys Asn Gln Asp Leu 80 85 90 Asn Glu Cys Gly Leu Lys
Pro Arg Pro Cys Lys His Arg Cys Met 95 100 105 Asn Thr Tyr Gly Ser
Tyr Lys Cys Tyr Cys Leu Asn Gly Tyr Met 110 115 120 Leu Met Pro Asp
Gly Ser Cys Ser Ser Ala Leu Thr Cys Ser Met 125 130 135 Ala Asn Cys
Gln Tyr Gly Cys Asp Val Val Lys Gly Gln Ile Arg 140 145 150 Cys Gln
Cys Pro Ser Pro Gly Leu His Leu Ala Pro Asp Gly Arg 155 160 165 Thr
Cys Val Asp Val Asp Glu Cys Ala Thr Gly Arg Ala Ser Cys 170 175 180
Pro Arg Phe Arg Gln Cys Val Asn Thr Phe Gly Ser Tyr Ile Cys 185 190
195 Lys Cys His Lys Gly Phe Asp Leu Met Tyr Ile Gly Gly Lys Tyr 200
205 210 Gln Cys His Asp Ile Asp Glu Cys Ser Leu Gly Gln Tyr Gln Cys
215 220 225 Ser Ser Phe Ala Arg Cys Tyr Asn Val Arg Gly Ser Tyr Lys
Cys 230 235 240 Lys Cys Lys Glu Gly Tyr Gln Gly Asp Gly Leu Thr Cys
Val Tyr 245 250 255 Ile Pro Lys Val Met Ile Glu Pro Ser Gly Pro Ile
His Val Pro 260 265 270 Lys Gly Asn Gly Thr Ile Leu Lys Gly Asp Thr
Gly Asn Asn Asn 275 280 285 Trp Ile Pro Asp Val Gly Ser Thr Trp Trp
Pro Pro Lys Thr Pro 290 295 300 Tyr Ile Pro Pro Ile Ile Thr Asn Arg
Pro Thr Ser Lys Pro Thr 305 310 315 Thr Arg Pro Thr Pro Lys Pro Thr
Pro Ile Pro Thr Pro Pro Pro 320 325 330 Pro Pro Pro Leu Pro Thr Glu
Leu Arg Thr Pro Leu Pro Pro Thr 335 340 345 Thr Pro Glu Arg Pro Thr
Thr Gly Leu Thr Thr Ile Ala Pro Ala 350 355 360 Ala Ser Thr Pro Pro
Gly Gly Ile Thr Val Asp Asn Arg Val Gln 365 370 375 Thr Asp Pro Gln
Lys Pro Arg Gly Asp Val Phe Ser Val Leu Val 380 385 390 His Ser Cys
Asn Phe Asp His Gly Leu Cys Gly Trp Ile Arg Glu 395 400 405 Lys Asp
Asn Asp Leu His Trp Glu Pro Ile Arg Asp Pro Ala Gly 410 415 420 Gly
Gln Tyr Leu Thr Val Ser Ala Ala Lys Ala Pro Gly Gly Lys 425 430 435
Ala Ala Arg Leu Val Leu Pro Leu Gly Arg Leu Met His Ser Gly 440 445
450 Asp Leu Cys Leu Ser Phe Arg His Lys Val Thr Gly Leu His Ser 455
460 465 Gly Thr Leu Gln Val Phe Val Arg Lys His Gly Ala His Gly Ala
470 475 480 Ala Leu Trp Gly Arg Asn Gly Gly His Gly Trp Arg Gln Thr
Gln 485 490 495 Ile Thr Leu Arg Gly Ala Asp Ile Lys Ser Glu Ser Gln
Arg 500 505 316 24 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 316 gatggttcct gctcaagtgc cctg 24 317 24 DNA
Artificial Sequence Synthetic Oligonucleotide Probe 317 ttgcacttgt
aggacccacg tacg 24 318 50 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 318
ctgatgggag gacctgtgta gatgttgatg aatgtgctac aggaagagcc 50 319 2110
DNA Homo Sapien 319 cttctttgaa aaggattatc acctgatcag gttctctctg
catttgcccc 50 tttagattgt gaaatgtggc tcaaggtctt cacaactttc
ctttcctttg 100 caacaggtgc ttgctcgggg ctgaaggtga cagtgccatc
acacactgtc 150 catggcgtca gaggtcaggc cctctaccta cccgtccact
atggcttcca 200 cactccagca tcagacatcc agatcatatg gctatttgag
agaccccaca 250 caatgcccaa atacttactg ggctctgtga ataagtctgt
ggttcctgac 300 ttggaatacc aacacaagtt caccatgatg ccacccaatg
catctctgct 350 tatcaaccca ctgcagttcc ctgatgaagg caattacatc
gtgaaggtca 400 acattcaggg aaatggaact ctatctgcca gtcagaagat
acaagtcacg 450 gttgatgatc ctgtcacaaa gccagtggtg cagattcatc
ctccctctgg 500 ggctgtggag tatgtgggga acatgaccct gacatgccat
gtggaagggg 550 gcactcggct agcttaccaa tggctaaaaa atgggagacc
tgtccacacc 600 agctccacct actccttttc tccccaaaac aatacccttc
atattgctcc 650 agtaaccaag gaagacattg ggaattacag ctgcctggtg
aggaaccctg 700 tcagtgaaat ggaaagtgat atcattatgc ccatcatata
ttatggacct 750 tatggacttc aagtgaattc tgataaaggg ctaaaagtag
gggaagtgtt 800 tactgttgac cttggagagg ccatcctatt tgattgttct
gctgattctc 850 atccccccaa cacctactcc tggattagga ggactgacaa
tactacatat 900 atcattaagc atgggcctcg cttagaagtt gcatctgaga
aagtagccca 950 gaagacaatg gactatgtgt gctgtgctta caacaacata
accggcaggc 1000 aagatgaaac tcatttcaca gttatcatca cttccgtagg
actggagaag 1050 cttgcacaga aaggaaaatc attgtcacct ttagcaagta
taactggaat 1100 atcactattt ttgattatat ccatgtgtct tctcttccta
tggaaaaaat 1150 atcaacccta caaagttata aaacagaaac tagaaggcag
gccagaaaca 1200 gaatacagga aagctcaaac attttcaggc catgaagatg
ctctggatga 1250 cttcggaata tatgaatttg ttgcttttcc agatgtttct
ggtgtttcca 1300 ggattccaag caggtctgtt ccagcctctg attgtgtatc
ggggcaagat 1350 ttgcacagta cagtgtatga agttattcag cacatccctg
cccagcagca 1400 agaccatcca gagtgaactt tcatgggcta aacagtacat
tcgagtgaaa 1450 ttctgaagaa acattttaag gaaaaacagt ggaaaagtat
attaatctgg 1500 aatcagtgaa gaaaccagga ccaacacctc ttactcatta
ttcctttaca 1550 tgcagaatag aggcatttat gcaaattgaa ctgcaggttt
ttcagcatat 1600 acacaatgtc ttgtgcaaca gaaaaacatg ttggggaaat
attcctcagt 1650 ggagagtcgt tctcatgctg acggggagaa cgaaagtgac
aggggtttcc 1700 tcataagttt tgtatgaaat atctctacaa acctcaatta
gttctactct 1750 acactttcac tatcatcaac actgagacta tcctgtctca
cctacaaatg 1800 tggaaacttt acattgttcg atttttcagc agactttgtt
ttattaaatt 1850 tttattagtg ttaagaatgc taaatttatg tttcaatttt
atttccaaat 1900 ttctatcttg ttatttgtac aacaaagtaa taaggatggt
tgtcacaaaa 1950 acaaaactat gccttctctt ttttttcaat caccagtagt
atttttgaga 2000 agacttgtga acacttaagg aaatgactat taaagtctta
tttttatttt 2050 tttcaaggaa agatggattc aaataaatta ttctgttttt
gcttttaaaa 2100 aaaaaaaaaa 2110 320 450 PRT Homo Sapien 320 Met Trp
Leu Lys Val Phe Thr Thr Phe Leu Ser Phe Ala Thr Gly 1 5 10 15 Ala
Cys Ser Gly Leu Lys Val Thr Val Pro Ser His Thr Val His 20 25 30
Gly Val Arg Gly Gln Ala Leu Tyr Leu Pro Val His Tyr Gly Phe 35 40
45 His Thr Pro Ala Ser Asp Ile Gln Ile Ile Trp Leu Phe Glu Arg 50
55 60 Pro His Thr Met Pro Lys Tyr Leu Leu Gly Ser Val Asn Lys Ser
65 70 75 Val Val Pro Asp Leu Glu Tyr Gln His Lys Phe Thr Met Met
Pro 80 85 90 Pro Asn Ala Ser Leu Leu Ile Asn Pro Leu Gln Phe Pro
Asp Glu 95 100 105 Gly Asn Tyr Ile Val Lys Val Asn Ile Gln Gly Asn
Gly Thr Leu 110 115 120 Ser Ala Ser Gln Lys Ile Gln Val Thr Val Asp
Asp Pro Val Thr 125 130 135 Lys Pro Val Val Gln Ile His Pro Pro Ser
Gly Ala Val Glu Tyr 140 145 150 Val Gly Asn Met Thr Leu Thr Cys His
Val Glu Gly Gly Thr Arg 155 160 165 Leu Ala Tyr Gln Trp Leu Lys Asn
Gly Arg Pro Val His Thr Ser 170 175 180 Ser Thr Tyr Ser Phe Ser Pro
Gln Asn Asn Thr Leu His Ile Ala 185 190 195 Pro Val Thr Lys Glu Asp
Ile Gly Asn Tyr Ser Cys Leu Val Arg 200 205 210 Asn Pro Val Ser Glu
Met Glu Ser Asp Ile Ile Met Pro Ile Ile 215 220 225 Tyr Tyr Gly Pro
Tyr Gly Leu Gln Val Asn Ser Asp Lys Gly Leu 230 235 240 Lys Val Gly
Glu Val Phe Thr Val Asp Leu Gly Glu Ala Ile Leu 245 250 255 Phe Asp
Cys Ser Ala Asp Ser His Pro Pro Asn Thr Tyr Ser Trp 260 265 270 Ile
Arg Arg Thr Asp Asn Thr Thr Tyr Ile Ile Lys His Gly Pro 275 280 285
Arg Leu Glu Val Ala Ser Glu Lys Val Ala Gln Lys Thr Met Asp 290 295
300 Tyr Val Cys Cys Ala Tyr Asn Asn Ile Thr Gly Arg Gln Asp Glu 305
310 315 Thr His Phe Thr Val Ile Ile Thr Ser Val Gly Leu Glu Lys Leu
320 325 330 Ala Gln Lys Gly Lys Ser Leu Ser Pro Leu Ala Ser Ile Thr
Gly 335 340 345 Ile Ser Leu Phe Leu Ile Ile Ser Met Cys Leu Leu Phe
Leu Trp 350 355 360 Lys Lys Tyr Gln Pro Tyr Lys Val Ile Lys Gln Lys
Leu Glu Gly 365 370 375 Arg Pro Glu Thr Glu Tyr Arg Lys Ala Gln Thr
Phe Ser Gly His 380 385 390 Glu Asp Ala Leu Asp Asp Phe Gly Ile Tyr
Glu Phe Val Ala Phe 395 400 405 Pro Asp Val Ser Gly Val Ser Arg Ile
Pro Ser Arg Ser Val Pro 410 415 420 Ala Ser Asp Cys Val Ser Gly Gln
Asp Leu His Ser Thr Val Tyr 425 430 435 Glu Val Ile Gln His Ile Pro
Ala Gln Gln Gln Asp His Pro Glu 440 445 450 321 25 DNA Artificial
Sequence Synthetic Oligonucleotide Probe 321 gatcctgtca caaagccagt
ggtgc 25 322 24 DNA Artificial Sequence Synthetic Oligonucleotide
Probe 322 cactgacagg gttcctcacc cagg 24 323 45 DNA Artificial
Sequence Synthetic Oligonucleotide Probe 323 ctccctctgg gctgtggagt
atgtggggaa catgaccctg acatg 45 324 2397 DNA Homo Sapien 324
gcaagcggcg aaatggcgcc ctccgggagt cttgcagttc ccctggcagt 50
cctggtgctg ttgctttggg gtgctccctg gacgcacggg cggcggagca 100
acgttcgcgt catcacggac gagaactgga gagaactgct ggaaggagac 150
tggatgatag aattttatgc cccgtggtgc cctgcttgtc aaaatcttca 200
accggaatgg gaaagttttg ctgaatgggg agaagatctt gaggttaata 250
ttgcgaaagt agatgtcaca gagcagccag gactgagtgg acggtttatc 300
ataactgctc ttcctactat ttatcattgt aaagatggtg aatttaggcg 350
ctatcagggt ccaaggacta agaaggactt cataaacttt ataagtgata 400
aagagtggaa gagtattgag cccgtttcat catggtttgg tccaggttct 450
gttctgatga gtagtatgtc agcactcttt cagctatcta tgtggatcag 500
gacgtgccat aactacttta ttgaagacct tggattgcca gtgtggggat 550
catatactgt ttttgcttta gcaactctgt tttccggact gttattagga 600
ctctgtatga tatttgtggc agattgcctt tgtccttcaa aaaggcgcag 650
accacagcca tacccatacc cttcaaaaaa attattatca gaatctgcac 700
aacctttgaa aaaagtggag gaggaacaag aggcggatga agaagatgtt 750
tcagaagaag aagctgaaag taaagaagga acaaacaaag actttccaca 800
gaatgccata agacaacgct ctctgggtcc atcattggcc acagataaat 850
cctagttaaa ttttatagtt atcttaatat tatgattttg ataaaaacag 900
aagattgatc attttgtttg gtttgaagtg aactgtgact tttttgaata 950
ttgcagggtt cagtctagat tgtcattaaa ttgaagagtc tacattcaga 1000
acataaaagc actaggtata caagtttgaa atatgattta agcacagtat 1050
gatggtttaa atagttctct aatttttgaa aaatcgtgcc aagcaataag 1100
atttatgtat atttgtttaa taataaccta tttcaagtct gagttttgaa 1150
aatttacatt tcccaagtat tgcattattg aggtatttaa gaagattatt 1200
ttagagaaaa atatttctca tttgatataa tttttctctg tttcactgtg 1250
tgaaaaaaag aagatatttc ccataaatgg gaagtttgcc cattgtctca 1300
agaaatgtgt atttcagtga caatttcgtg gtctttttag aggtatattc 1350
caaaatttcc ttgtattttt aggttatgca actaataaaa actaccttac 1400
attaattaat tacagttttc tacacatggt aatacaggat atgctactga 1450
tttaggaagt ttttaagttc atggtattct cttgattcca acaaagtttg 1500
attttctctt gtatttttct tacttactat gggttacatt ttttattttt 1550
caaattggat gataatttct tggaaacatt ttttatgttt tagtaaacag 1600
tatttttttg ttgtttcaaa ctgaagttta ctgagagatc catcaaattg 1650
aacaatctgt tgtaatttaa aattttggcc acttttttca gattttacat 1700
cattcttgct gaacttcaac ttgaaattgt tttttttttc tttttggatg 1750
tgaaggtgaa cattcctgat ttttgtctga tgtgaaaaag ccttggtatt 1800
ttacattttg aaaattcaaa gaagcttaat ataaaagttt gcattctact 1850
caggaaaaag catcttcttg tatatgtctt aaatgtattt ttgtcctcat 1900
atacagaaag ttcttaattg attttacagt ctgtaatgct tgatgtttta 1950
aaataataac atttttatat tttttaaaag acaaacttca tattatcctg 2000
tgttctttcc tgactggtaa tattgtgtgg gatttcacag gtaaaagtca 2050
gtaggatgga acattttagt gtatttttac tccttaaaga gctagaatac 2100
atagttttca ccttaaaaga agggggaaaa tcataaatac aatgaatcaa 2150
ctgaccatta cgtagtagac aatttctgta atgtcccctt ctttctaggc 2200
tctgttgctg tgtgaatcca ttagatttac agtatcgtaa tatacaagtt 2250
ttctttaaag ccctctcctt tagaatttaa aatattgtac cattaaagag 2300
tttggatgtg taacttgtga tgccttagaa aaatatccta agcacaaaat 2350
aaacctttct aaccacttca ttaaagctga aaaaaaaaaa aaaaaaa 2397 325 280
PRT Homo Sapien 325 Met Ala Pro Ser Gly Ser Leu Ala Val Pro Leu Ala
Val Leu Val 1 5 10 15 Leu Leu Leu Trp Gly Ala Pro Trp Thr His Gly
Arg Arg Ser Asn 20 25 30 Val Arg Val Ile Thr Asp Glu Asn Trp Arg
Glu Leu Leu Glu Gly 35 40 45 Asp Trp Met Ile Glu Phe Tyr Ala Pro
Trp Cys Pro Ala Cys Gln 50 55 60 Asn Leu Gln Pro Glu Trp Glu Ser
Phe Ala Glu Trp Gly Glu Asp 65 70 75 Leu Glu Val Asn Ile Ala Lys
Val Asp Val Thr Glu Gln Pro Gly 80 85 90 Leu Ser Gly Arg Phe Ile
Ile Thr Ala Leu Pro Thr Ile Tyr His 95 100 105 Cys Lys Asp Gly Glu
Phe Arg Arg Tyr Gln Gly Pro Arg Thr Lys 110 115 120 Lys Asp Phe Ile
Asn Phe Ile Ser Asp Lys Glu Trp Lys Ser Ile 125 130 135 Glu Pro Val
Ser Ser Trp Phe Gly Pro Gly Ser Val Leu Met Ser 140 145 150 Ser Met
Ser Ala Leu Phe Gln Leu Ser Met Trp Ile Arg Thr Cys 155 160 165 His
Asn Tyr Phe Ile Glu Asp Leu Gly Leu Pro Val Trp Gly Ser 170 175 180
Tyr Thr Val Phe Ala Leu Ala Thr Leu Phe Ser Gly Leu Leu Leu 185 190
195 Gly Leu Cys Met Ile Phe Val Ala Asp Cys Leu Cys Pro Ser Lys 200
205 210 Arg Arg Arg Pro Gln Pro Tyr Pro Tyr Pro Ser Lys Lys Leu Leu
215 220 225 Ser Glu Ser Ala Gln Pro Leu Lys Lys Val Glu Glu Glu Gln
Glu 230 235 240 Ala Asp Glu Glu Asp Val Ser Glu Glu Glu Ala Glu Ser
Lys Glu 245 250 255 Gly Thr Asn Lys Asp Phe Pro Gln Asn Ala Ile Arg
Gln Arg Ser 260 265 270 Leu Gly Pro Ser Leu Ala Thr Asp Lys Ser 275
280 326 23 DNA Artificial Sequence Synthetic Oligonucleotide Probe
326 tgaggtgggc aagcggcgaa atg 23 327 20 DNA Artificial Sequence
Synthetic Oligonucleotide Probe 327 tatgtggatc aggacgtgcc 20 328 21
DNA Artificial Sequence Synthetic Oligonucleotide Probe 328
tgcagggttc agtctagatt g 21 329 25 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 329 ttgaaggaca aaggcaatct gccac 25 330 45 DNA
Artificial Sequence Synthetic Oligonucleotide Probe 330 ggagtcttgc
agttcccctg gcagtcctgg tgctgttgct ttggg 45 331 2168 DNA Homo Sapien
331 gcgagtgtcc agctgcggag acccgtgata attcgttaac taattcaaca 50
aacgggaccc ttctgtgtgc cagaaaccgc aagcagttgc taacccagtg 100
ggacaggcgg attggaagag cgggaaggtc ctggcccaga gcagtgtgac 150
acttccctct gtgaccatga aactctgggt gtctgcattg ctgatggcct 200
ggtttggtgt cctgagctgt gtgcaggccg aattcttcac ctctattggg 250
cacatgactg acctgattta tgcagagaaa gagctggtgc agtctctgaa 300
agagtacatc cttgtggagg aagccaagct ttccaagatt aagagctggg 350
ccaacaaaat ggaagccttg actagcaagt cagctgctga tgctgagggc 400
tacctggctc accctgtgaa tgcctacaaa ctggtgaagc ggctaaacac 450
agactggcct gcgctggagg accttgtcct gcaggactca gctgcaggtt 500
ttatcgccaa cctctctgtg cagcggcagt tcttccccac tgatgaggac 550
gagataggag ctgccaaagc cctgatgaga cttcaggaca catacaggct 600
ggacccaggc acaatttcca gaggggaact tccaggaacc aagtaccagg 650
caatgctgag tgtggatgac tgctttggga tgggccgctc ggcctacaat 700
gaaggggact attatcatac ggtgttgtgg atggagcagg tgctaaagca 750
gcttgatgcc ggggaggagg ccaccacaac caagtcacag gtgctggact 800
acctcagcta tgctgtcttc cagttgggtg atctgcaccg tgccctggag 850
ctcacccgcc gcctgctctc ccttgaccca agccacgaac gagctggagg 900
gaatctgcgg tactttgagc agttattgga ggaagagaga gaaaaaacgt 950
taacaaatca gacagaagct gagctagcaa ccccagaagg catctatgag 1000
aggcctgtgg actacctgcc tgagagggat gtttacgaga gcctctgtcg 1050
tggggagggt gtcaaactga caccccgtag acagaagagg cttttctgta 1100
ggtaccacca tggcaacagg gccccacagc tgctcattgc ccccttcaaa 1150
gaggaggacg agtgggacag cccgcacatc gtcaggtact acgatgtcat 1200
gtctgatgag gaaatcgaga ggatcaagga gatcgcaaaa cctaaacttg 1250
cacgagccac cgttcgtgat cccaagacag gagtcctcac tgtcgccagc 1300
taccgggttt ccaaaagctc ctggctagag gaagatgatg accctgttgt 1350
ggcccgagta aatcgtcgga tgcagcatat cacagggtta acagtaaaga 1400
ctgcagaatt gttacaggtt gcaaattatg gagtgggagg acagtatgaa 1450
ccgcacttcg acttctctag gcgacctttt gacagcggcc tcaaaacaga 1500
ggggaatagg ttagcgacgt ttcttaacta catgagtgat gtagaagctg 1550
gtggtgccac cgtcttccct gatctggggg ctgcaatttg gcctaagaag 1600
ggtacagctg tgttctggta caacctcttg cggagcgggg aaggtgacta 1650
ccgaacaaga catgctgcct gccctgtgct tgtgggctgc aagtgggtct 1700
ccaataagtg gttccatgaa cgaggacagg agttcttgag accttgtgga 1750
tcaacagaag ttgactgaca tccttttctg tccttcccct tcctggtcct 1800
tcagcccatg tcaacgtgac agacaccttt gtatgttcct ttgtatgttc 1850
ctatcaggct gatttttgga gaaatgaatg tttgtctgga gcagagggag 1900
accatactag ggcgactcct gtgtgactga agtcccagcc cttccattca 1950
gcctgtgcca tccctggccc caaggctagg atcaaagtgg ctgcagcaga 2000
gttagctgtc tagcgcctag caaggtgcct ttgtacctca ggtgttttag 2050
gtgtgagatg tttcagtgaa ccaaagttct gataccttgt ttacatgttt 2100
gtttttatgg catttctatc tattgtggct ttaccaaaaa ataaaatgtc 2150
cctaccagaa aaaaaaaa 2168 332 533 PRT Homo Sapien 332 Met Lys Leu
Trp Val Ser Ala Leu Leu Met Ala Trp Phe Gly Val 1 5 10 15 Leu Ser
Cys Val Gln Ala Glu Phe Phe Thr Ser Ile Gly His Met 20 25 30 Thr
Asp Leu Ile Tyr Ala Glu Lys Glu Leu Val Gln Ser Leu Lys 35 40 45
Glu Tyr Ile Leu Val Glu Glu Ala Lys Leu Ser Lys Ile Lys Ser 50 55
60 Trp Ala Asn Lys Met Glu Ala Leu Thr Ser Lys Ser Ala Ala Asp 65
70 75 Ala Glu Gly Tyr Leu Ala His Pro Val Asn Ala Tyr Lys Leu Val
80 85 90 Lys Arg Leu Asn Thr Asp Trp Pro Ala Leu Glu Asp Leu Val
Leu 95 100 105 Gln Asp Ser Ala Ala Gly Phe Ile Ala Asn Leu Ser Val
Gln Arg 110 115 120 Gln Phe Phe Pro Thr Asp Glu Asp Glu Ile Gly Ala
Ala Lys Ala 125 130 135 Leu Met Arg Leu Gln Asp Thr Tyr Arg Leu Asp
Pro Gly Thr Ile 140 145 150 Ser Arg Gly Glu Leu Pro Gly Thr Lys Tyr
Gln Ala Met Leu Ser
155 160 165 Val Asp Asp Cys Phe Gly Met Gly Arg Ser Ala Tyr Asn Glu
Gly 170 175 180 Asp Tyr Tyr His Thr Val Leu Trp Met Glu Gln Val Leu
Lys Gln 185 190 195 Leu Asp Ala Gly Glu Glu Ala Thr Thr Thr Lys Ser
Gln Val Leu 200 205 210 Asp Tyr Leu Ser Tyr Ala Val Phe Gln Leu Gly
Asp Leu His Arg 215 220 225 Ala Leu Glu Leu Thr Arg Arg Leu Leu Ser
Leu Asp Pro Ser His 230 235 240 Glu Arg Ala Gly Gly Asn Leu Arg Tyr
Phe Glu Gln Leu Leu Glu 245 250 255 Glu Glu Arg Glu Lys Thr Leu Thr
Asn Gln Thr Glu Ala Glu Leu 260 265 270 Ala Thr Pro Glu Gly Ile Tyr
Glu Arg Pro Val Asp Tyr Leu Pro 275 280 285 Glu Arg Asp Val Tyr Glu
Ser Leu Cys Arg Gly Glu Gly Val Lys 290 295 300 Leu Thr Pro Arg Arg
Gln Lys Arg Leu Phe Cys Arg Tyr His His 305 310 315 Gly Asn Arg Ala
Pro Gln Leu Leu Ile Ala Pro Phe Lys Glu Glu 320 325 330 Asp Glu Trp
Asp Ser Pro His Ile Val Arg Tyr Tyr Asp Val Met 335 340 345 Ser Asp
Glu Glu Ile Glu Arg Ile Lys Glu Ile Ala Lys Pro Lys 350 355 360 Leu
Ala Arg Ala Thr Val Arg Asp Pro Lys Thr Gly Val Leu Thr 365 370 375
Val Ala Ser Tyr Arg Val Ser Lys Ser Ser Trp Leu Glu Glu Asp 380 385
390 Asp Asp Pro Val Val Ala Arg Val Asn Arg Arg Met Gln His Ile 395
400 405 Thr Gly Leu Thr Val Lys Thr Ala Glu Leu Leu Gln Val Ala Asn
410 415 420 Tyr Gly Val Gly Gly Gln Tyr Glu Pro His Phe Asp Phe Ser
Arg 425 430 435 Arg Pro Phe Asp Ser Gly Leu Lys Thr Glu Gly Asn Arg
Leu Ala 440 445 450 Thr Phe Leu Asn Tyr Met Ser Asp Val Glu Ala Gly
Gly Ala Thr 455 460 465 Val Phe Pro Asp Leu Gly Ala Ala Ile Trp Pro
Lys Lys Gly Thr 470 475 480 Ala Val Phe Trp Tyr Asn Leu Leu Arg Ser
Gly Glu Gly Asp Tyr 485 490 495 Arg Thr Arg His Ala Ala Cys Pro Val
Leu Val Gly Cys Lys Trp 500 505 510 Val Ser Asn Lys Trp Phe His Glu
Arg Gly Gln Glu Phe Leu Arg 515 520 525 Pro Cys Gly Ser Thr Glu Val
Asp 530 333 18 DNA Artificial Sequence Synthetic Oligonucleotide
Probe 333 ccaggcacaa tttccaga 18 334 19 DNA Artificial Sequence
Synthetic Oligonucleotide Probe 334 ggacccttct gtgtgccag 19 335 19
DNA Artificial Sequence Synthetic Oligonucleotide Probe 335
ggtctcaaga actcctgtc 19 336 24 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 336 acactcagca ttgcctggta cttg 24 337 45 DNA
Artificial Sequence Synthetic Oligonucleotide Probe 337 gggcacatga
ctgacctgat ttatgcagag aaagagctgg tgcag 45 338 2789 DNA Homo Sapien
338 gcagtattga gttttacttc ctcctctttt tagtggaaga cagaccataa 50
tcccagtgtg agtgaaattg attgtttcat ttattaccgt tttggctggg 100
ggttagttcc gacaccttca cagttgaaga gcaggcagaa ggagttgtga 150
agacaggaca atcttcttgg ggatgctggt cctggaagcc agcgggcctt 200
gctctgtctt tggcctcatt gaccccaggt tctctggtta aaactgaaag 250
cctactactg gcctggtgcc catcaatcca ttgatccttg aggctgtgcc 300
cctggggcac ccacctggca gggcctacca ccatgcgact gagctccctg 350
ttggctctgc tgcggccagc gcttcccctc atcttagggc tgtctctggg 400
gtgcagcctg agcctcctgc gggtttcctg gatccagggg gagggagaag 450
atccctgtgt cgaggctgta ggggagcgag gagggccaca gaatccagat 500
tcgagagctc ggctagacca aagtgatgaa gacttcaaac cccggattgt 550
cccctactac agggacccca acaagcccta caagaaggtg ctcaggactc 600
ggtacatcca gacagagctg ggctcccgtg agcggttgct ggtggctgtc 650
ctgacctccc gagctacact gtccactttg gccgtggctg tgaaccgtac 700
ggtggcccat cacttccctc ggttactcta cttcactggg cagcgggggg 750
cccgggctcc agcagggatg caggtggtgt ctcatgggga tgagcggccc 800
gcctggctca tgtcagagac cctgcgccac cttcacacac actttggggc 850
cgactacgac tggttcttca tcatgcagga tgacacatat gtgcaggccc 900
cccgcctggc agcccttgct ggccacctca gcatcaacca agacctgtac 950
ttaggccggg cagaggagtt cattggcgca ggcgagcagg cccggtactg 1000
tcatgggggc tttggctacc tgttgtcacg gagtctcctg cttcgtctgc 1050
ggccacatct ggatggctgc cgaggagaca ttctcagtgc ccgtcctgac 1100
gagtggcttg gacgctgcct cattgactct ctgggcgtcg gctgtgtctc 1150
acagcaccag gggcagcagt atcgctcatt tgaactggcc aaaaataggg 1200
accctgagaa ggaagggagc tcggctttcc tgagtgcctt cgccgtgcac 1250
cctgtctccg aaggtaccct catgtaccgg ctccacaaac gcttcagcgc 1300
tctggagttg gagcgggctt acagtgaaat agaacaactg caggctcaga 1350
tccggaacct gaccgtgctg acccccgaag gggaggcagg gctgagctgg 1400
cccgttgggc tccctgctcc tttcacacca cactctcgct ttgaggtgct 1450
gggctgggac tacttcacag agcagcacac cttctcctgt gcagatgggg 1500
ctcccaagtg cccactacag ggggctagca gggcggacgt gggtgatgcg 1550
ttggagactg ccctggagca gctcaatcgg cgctatcagc cccgcctgcg 1600
cttccagaag cagcgactgc tcaacggcta tcggcgcttc gacccagcac 1650
ggggcatgga gtacaccctg gacctgctgt tggaatgtgt gacacagcgt 1700
gggcaccggc gggccctggc tcgcagggtc agcctgctgc ggccactgag 1750
ccgggtggaa atcctaccta tgccctatgt cactgaggcc acccgagtgc 1800
agctggtgct gccactcctg gtggctgaag ctgctgcagc cccggctttc 1850
ctcgaggcgt ttgcagccaa tgtcctggag ccacgagaac atgcattgct 1900
caccctgttg ctggtctacg ggccacgaga aggtggccgt ggagctccag 1950
acccatttct tggggtgaag gctgcagcag cggagttaga gcgacggtac 2000
cctgggacga ggctggcctg gctcgctgtg cgagcagagg ccccttccca 2050
ggtgcgactc atggacgtgg tctcgaagaa gcaccctgtg gacactctct 2100
tcttccttac caccgtgtgg acaaggcctg ggcccgaagt cctcaaccgc 2150
tgtcgcatga atgccatctc tggctggcag gccttctttc cagtccattt 2200
ccaggagttc aatcctgccc tgtcaccaca gagatcaccc ccagggcccc 2250
cgggggctgg ccctgacccc ccctcccctc ctggtgctga cccctcccgg 2300
ggggctccta taggggggag atttgaccgg caggcttctg cggagggctg 2350
cttctacaac gctgactacc tggcggcccg agcccggctg gcaggtgaac 2400
tggcaggcca ggaagaggag gaagccctgg aggggctgga ggtgatggat 2450
gttttcctcc ggttctcagg gctccacctc tttcgggccg tagagccagg 2500
gctggtgcag aagttctccc tgcgagactg cagcccacgg ctcagtgaag 2550
aactctacca ccgctgccgc ctcagcaacc tggaggggct agggggccgt 2600
gcccagctgg ctatggctct ctttgagcag gagcaggcca atagcactta 2650
gcccgcctgg gggccctaac ctcattacct ttcctttgtc tgcctcagcc 2700
ccaggaaggg caaggcaaga tggtggacag atagagaatt gttgctgtat 2750
tttttaaata tgaaaatgtt attaaacatg tcttctgcc 2789 339 772 PRT Homo
Sapien 339 Met Arg Leu Ser Ser Leu Leu Ala Leu Leu Arg Pro Ala Leu
Pro 1 5 10 15 Leu Ile Leu Gly Leu Ser Leu Gly Cys Ser Leu Ser Leu
Leu Arg 20 25 30 Val Ser Trp Ile Gln Gly Glu Gly Glu Asp Pro Cys
Val Glu Ala 35 40 45 Val Gly Glu Arg Gly Gly Pro Gln Asn Pro Asp
Ser Arg Ala Arg 50 55 60 Leu Asp Gln Ser Asp Glu Asp Phe Lys Pro
Arg Ile Val Pro Tyr 65 70 75 Tyr Arg Asp Pro Asn Lys Pro Tyr Lys
Lys Val Leu Arg Thr Arg 80 85 90 Tyr Ile Gln Thr Glu Leu Gly Ser
Arg Glu Arg Leu Leu Val Ala 95 100 105 Val Leu Thr Ser Arg Ala Thr
Leu Ser Thr Leu Ala Val Ala Val 110 115 120 Asn Arg Thr Val Ala His
His Phe Pro Arg Leu Leu Tyr Phe Thr 125 130 135 Gly Gln Arg Gly Ala
Arg Ala Pro Ala Gly Met Gln Val Val Ser 140 145 150 His Gly Asp Glu
Arg Pro Ala Trp Leu Met Ser Glu Thr Leu Arg 155 160 165 His Leu His
Thr His Phe Gly Ala Asp Tyr Asp Trp Phe Phe Ile 170 175 180 Met Gln
Asp Asp Thr Tyr Val Gln Ala Pro Arg Leu Ala Ala Leu 185 190 195 Ala
Gly His Leu Ser Ile Asn Gln Asp Leu Tyr Leu Gly Arg Ala 200 205 210
Glu Glu Phe Ile Gly Ala Gly Glu Gln Ala Arg Tyr Cys His Gly 215 220
225 Gly Phe Gly Tyr Leu Leu Ser Arg Ser Leu Leu Leu Arg Leu Arg 230
235 240 Pro His Leu Asp Gly Cys Arg Gly Asp Ile Leu Ser Ala Arg Pro
245 250 255 Asp Glu Trp Leu Gly Arg Cys Leu Ile Asp Ser Leu Gly Val
Gly 260 265 270 Cys Val Ser Gln His Gln Gly Gln Gln Tyr Arg Ser Phe
Glu Leu 275 280 285 Ala Lys Asn Arg Asp Pro Glu Lys Glu Gly Ser Ser
Ala Phe Leu 290 295 300 Ser Ala Phe Ala Val His Pro Val Ser Glu Gly
Thr Leu Met Tyr 305 310 315 Arg Leu His Lys Arg Phe Ser Ala Leu Glu
Leu Glu Arg Ala Tyr 320 325 330 Ser Glu Ile Glu Gln Leu Gln Ala Gln
Ile Arg Asn Leu Thr Val 335 340 345 Leu Thr Pro Glu Gly Glu Ala Gly
Leu Ser Trp Pro Val Gly Leu 350 355 360 Pro Ala Pro Phe Thr Pro His
Ser Arg Phe Glu Val Leu Gly Trp 365 370 375 Asp Tyr Phe Thr Glu Gln
His Thr Phe Ser Cys Ala Asp Gly Ala 380 385 390 Pro Lys Cys Pro Leu
Gln Gly Ala Ser Arg Ala Asp Val Gly Asp 395 400 405 Ala Leu Glu Thr
Ala Leu Glu Gln Leu Asn Arg Arg Tyr Gln Pro 410 415 420 Arg Leu Arg
Phe Gln Lys Gln Arg Leu Leu Asn Gly Tyr Arg Arg 425 430 435 Phe Asp
Pro Ala Arg Gly Met Glu Tyr Thr Leu Asp Leu Leu Leu 440 445 450 Glu
Cys Val Thr Gln Arg Gly His Arg Arg Ala Leu Ala Arg Arg 455 460 465
Val Ser Leu Leu Arg Pro Leu Ser Arg Val Glu Ile Leu Pro Met 470 475
480 Pro Tyr Val Thr Glu Ala Thr Arg Val Gln Leu Val Leu Pro Leu 485
490 495 Leu Val Ala Glu Ala Ala Ala Ala Pro Ala Phe Leu Glu Ala Phe
500 505 510 Ala Ala Asn Val Leu Glu Pro Arg Glu His Ala Leu Leu Thr
Leu 515 520 525 Leu Leu Val Tyr Gly Pro Arg Glu Gly Gly Arg Gly Ala
Pro Asp 530 535 540 Pro Phe Leu Gly Val Lys Ala Ala Ala Ala Glu Leu
Glu Arg Arg 545 550 555 Tyr Pro Gly Thr Arg Leu Ala Trp Leu Ala Val
Arg Ala Glu Ala 560 565 570 Pro Ser Gln Val Arg Leu Met Asp Val Val
Ser Lys Lys His Pro 575 580 585 Val Asp Thr Leu Phe Phe Leu Thr Thr
Val Trp Thr Arg Pro Gly 590 595 600 Pro Glu Val Leu Asn Arg Cys Arg
Met Asn Ala Ile Ser Gly Trp 605 610 615 Gln Ala Phe Phe Pro Val His
Phe Gln Glu Phe Asn Pro Ala Leu 620 625 630 Ser Pro Gln Arg Ser Pro
Pro Gly Pro Pro Gly Ala Gly Pro Asp 635 640 645 Pro Pro Ser Pro Pro
Gly Ala Asp Pro Ser Arg Gly Ala Pro Ile 650 655 660 Gly Gly Arg Phe
Asp Arg Gln Ala Ser Ala Glu Gly Cys Phe Tyr 665 670 675 Asn Ala Asp
Tyr Leu Ala Ala Arg Ala Arg Leu Ala Gly Glu Leu 680 685 690 Ala Gly
Gln Glu Glu Glu Glu Ala Leu Glu Gly Leu Glu Val Met 695 700 705 Asp
Val Phe Leu Arg Phe Ser Gly Leu His Leu Phe Arg Ala Val 710 715 720
Glu Pro Gly Leu Val Gln Lys Phe Ser Leu Arg Asp Cys Ser Pro 725 730
735 Arg Leu Ser Glu Glu Leu Tyr His Arg Cys Arg Leu Ser Asn Leu 740
745 750 Glu Gly Leu Gly Gly Arg Ala Gln Leu Ala Met Ala Leu Phe Glu
755 760 765 Gln Glu Gln Ala Asn Ser Thr 770 340 1572 DNA Homo
Sapien 340 cggagtggtg cgccaacgtg agaggaaacc cgtgcgcggc tgcgctttcc
50 tgtccccaag ccgttctaga cgcgggaaaa atgctttctg aaagcagctc 100
ctttttgaag ggtgtgatgc ttggaagcat tttctgtgct ttgatcacta 150
tgctaggaca cattaggatt ggtcatggaa atagaatgca ccaccatgag 200
catcatcacc tacaagctcc taacaaagaa gatatcttga aaatttcaga 250
ggatgagcgc atggagctca gtaagagctt tcgagtatac tgtattatcc 300
ttgtaaaacc caaagatgtg agtctttggg ctgcagtaaa ggagacttgg 350
accaaacact gtgacaaagc agagttcttc agttctgaaa atgttaaagt 400
gtttgagtca attaatatgg acacaaatga catgtggtta atgatgagaa 450
aagcttacaa atacgccttt gataagtata gagaccaata caactggttc 500
ttccttgcac gccccactac gtttgctatc attgaaaacc taaagtattt 550
tttgttaaaa aaggatccat cacagccttt ctatctaggc cacactataa 600
aatctggaga ccttgaatat gtgggtatgg aaggaggaat tgtcttaagt 650
gtagaatcaa tgaaaagact taacagcctt ctcaatatcc cagaaaagtg 700
tcctgaacag ggagggatga tttggaagat atctgaagat aaacagctag 750
cagtttgcct gaaatatgct ggagtatttg cagaaaatgc agaagatgct 800
gatggaaaag atgtatttaa taccaaatct gttgggcttt ctattaaaga 850
ggcaatgact tatcacccca accaggtagt agaaggctgt tgttcagata 900
tggctgttac ttttaatgga ctgactccaa atcagatgca tgtgatgatg 950
tatggggtat accgccttag ggcatttggg catattttca atgatgcatt 1000
ggttttctta cctccaaatg gttctgacaa tgactgagaa gtggtagaaa 1050
agcgtgaata tgatctttgt ataggacgtg tgttgtcatt atttgtagta 1100
gtaactacat atccaataca gctgtatgtt tctttttctt ttctaatttg 1150
gtggcactgg tataaccaca cattaaagtc agtagtacat ttttaaatga 1200
gggtggtttt tttctttaaa acacatgaac attgtaaatg tgttggaaag 1250
aagtgtttta agaataataa ttttgcaaat aaactattaa taaatattat 1300
atgtgataaa ttctaaatta tgaacattag aaatctgtgg ggcacatatt 1350
tttgctgatt ggttaaaaaa ttttaacagg tctttagcgt tctaagatat 1400
gcaaatgata tctctagttg tgaatttgtg attaaagtaa aacttttagc 1450
tgtgtgttcc ctttacttct aatactgatt tatgttctaa gcctccccaa 1500
gttccaatgg atttgccttc tcaaaatgta caactaagca actaaagaaa 1550
attaaagtga aagttgaaaa at 1572 341 318 PRT Homo Sapien 341 Met Leu
Ser Glu Ser Ser Ser Phe Leu Lys Gly Val Met Leu Gly 1 5 10 15 Ser
Ile Phe Cys Ala Leu Ile Thr Met Leu Gly His Ile Arg Ile 20 25 30
Gly His Gly Asn Arg Met His His His Glu His His His Leu Gln 35 40
45 Ala Pro Asn Lys Glu Asp Ile Leu Lys Ile Ser Glu Asp Glu Arg 50
55 60 Met Glu Leu Ser Lys Ser Phe Arg Val Tyr Cys Ile Ile Leu Val
65 70 75 Lys Pro Lys Asp Val Ser Leu Trp Ala Ala Val Lys Glu Thr
Trp 80 85 90 Thr Lys His Cys Asp Lys Ala Glu Phe Phe Ser Ser Glu
Asn Val 95 100 105 Lys Val Phe Glu Ser Ile Asn Met Asp Thr Asn Asp
Met Trp Leu 110 115 120 Met Met Arg Lys Ala Tyr Lys Tyr Ala Phe Asp
Lys Tyr Arg Asp 125 130 135 Gln Tyr Asn Trp Phe Phe Leu Ala Arg Pro
Thr Thr Phe Ala Ile 140 145 150 Ile Glu Asn Leu Lys Tyr Phe Leu Leu
Lys Lys Asp Pro Ser Gln 155 160 165 Pro Phe Tyr Leu Gly His Thr Ile
Lys Ser Gly Asp Leu Glu Tyr 170 175 180 Val Gly Met Glu Gly Gly Ile
Val Leu Ser Val Glu Ser Met Lys 185 190 195 Arg Leu Asn Ser Leu Leu
Asn Ile Pro Glu Lys Cys Pro Glu Gln 200 205 210 Gly Gly Met Ile Trp
Lys Ile Ser Glu Asp Lys Gln Leu Ala Val 215 220 225 Cys Leu Lys Tyr
Ala Gly Val Phe Ala Glu Asn Ala Glu Asp Ala 230 235 240 Asp Gly Lys
Asp Val Phe Asn
Thr Lys Ser Val Gly Leu Ser Ile 245 250 255 Lys Glu Ala Met Thr Tyr
His Pro Asn Gln Val Val Glu Gly Cys 260 265 270 Cys Ser Asp Met Ala
Val Thr Phe Asn Gly Leu Thr Pro Asn Gln 275 280 285 Met His Val Met
Met Tyr Gly Val Tyr Arg Leu Arg Ala Phe Gly 290 295 300 His Ile Phe
Asn Asp Ala Leu Val Phe Leu Pro Pro Asn Gly Ser 305 310 315 Asp Asn
Asp 342 23 DNA Artificial Sequence Synthetic Oligonucleotide Probe
342 tccccaagcc gttctagacg cgg 23 343 18 DNA Artificial Sequence
Synthetic Oligonucleotide Probe 343 ctggttcttc cttgcacg 18 344 28
DNA Artificial Sequence Synthetic Oligonucleotide Probe 344
gcccaaatgc cctaaggcgg tatacccc 28 345 50 DNA Artificial Sequence
Synthetic Oligonucleotide Probe 345 gggtgtgatg cttggaagca
ttttctgtgc tttgatcact atgctaggac 50 346 25 DNA Artificial Sequence
Synthetic Oligonucleotide Probe 346 gggatgcagg tggtgtctca tgggg 25
347 18 DNA Artificial Sequence Synthetic Oligonucleotide Probe 347
ccctcatgta ccggctcc 18 348 48 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 348 ggattctaat acgactcact atagggctca
gaaaagcgca acagagaa 48 349 47 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 349 ctatgaaatt aaccctcact aaagggatgt
cttccatgcc aaccttc 47 350 48 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 350 ggattctaat acgactcact atagggcggc
gatgtccact ggggctac 48 351 48 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 351 ctatgaaatt aaccctcact aaagggacga
ggaagatggg cggatggt 48 352 47 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 352 ggattctaat acgactcact atagggcacc
cacgcgtccg gctgctt 47 353 48 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 353 ctatgaaatt aaccctcact aaagggacgg
gggacaccac ggaccaga 48 354 48 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 354 ggattctaat acgactcact atagggcttg
ctgcggtttt tgttcctg 48 355 48 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 355 ctatgaaatt aaccctcact aaagggagct
gccgatccca ctggtatt 48 356 46 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 356 ggattctaat acgactcact atagggcgga
tcctggccgg cctctg 46 357 48 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 357 ctatgaaatt aaccctcact aaagggagcc
cgggcatggt ctcagtta 48 358 47 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 358 ggattctaat acgactcact atagggcggg
aagatggcga ggaggag 47 359 48 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 359 ctatgaaatt aaccctcact aaagggacca
aggccacaaa cggaaatc 48 360 48 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 360 ggattctaat acgactcact atagggctgt
gctttcattc tgccagta 48 361 48 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 361 ctatgaaatt aaccctcact aaagggaggg
tacaattaag gggtggat 48 362 47 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 362 ggattctaat acgactcact atagggcccg
cctcgctcct gctcctg 47 363 48 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 363 ctatgaaatt aaccctcact aaagggagga
ttgccgcgac cctcacag 48 364 47 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 364 ggattctaat acgactcact atagggcccc
tcctgccttc cctgtcc 47 365 48 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 365 ctatgaaatt aaccctcact aaagggagtg
gtggccgcga ttatctgc 48 366 48 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 366 ggattctaat acgactcact atagggcgca
gcgatggcag cgatgagg 48 367 47 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 367 ctatgaaatt aaccctcact aaagggacag
acggggcaga gggagtg 47 368 47 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 368 ggattctaat acgactcact atagggccag
gaggcgtgag gagaaac 47 369 48 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 369 ctatgaaatt aaccctcact aaagggaaag
acatgtcatc gggagtgg 48 370 48 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 370 ggattctaat acgactcact atagggccgg
gtggaggtgg aacagaaa 48 371 48 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 371 ctatgaaatt aaccctcact aaagggacac
agacagagcc ccatacgc 48 372 47 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 372 ggattctaat acgactcact atagggccag
ggaaatccgg atgtctc 47 373 48 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 373 ctatgaaatt aaccctcact aaagggagta
aggggatgcc accgagta 48 374 47 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 374 ggattctaat acgactcact atagggccag
ctacccgcag gaggagg 47 375 48 DNA Artificial Sequence Synthetic
Oligonucleotide Probe 375 ctatgaaatt aaccctcact aaagggatcc
caggtgatga ggtccaga 48 376 997 DNA Homo Sapien 376 cccacgcgtc
cgatcttacc aacaaaacac tcctgaggag aaagaaagag 50 agggagggag
agaaaaagag agagagagaa acaaaaaacc aaagagagag 100 aaaaaatgaa
ttcatctaaa tcatctgaaa cacaatgcac agagagagga 150 tgcttctctt
cccaaatgtt cttatggact gttgctggga tccccatcct 200 atttctcagt
gcctgtttca tcaccagatg tgttgtgaca tttcgcatct 250 ttcaaacctg
tgatgagaaa aagtttcagc tacctgagaa tttcacagag 300 ctctcctgct
acaattatgg atcaggttca gtcaagaatt gttgtccatt 350 gaactgggaa
tattttcaat ccagctgcta cttcttttct actgacacca 400 tttcctgggc
gttaagttta aagaactgct cagccatggg ggctcacctg 450 gtggttatca
actcacagga ggagcaggaa ttcctttcct acaagaaacc 500 taaaatgaga
gagtttttta ttggactgtc agaccaggtt gtcgagggtc 550 agtggcaatg
ggtggacggc acacctttga caaagtctct gagcttctgg 600 gatgtagggg
agcccaacaa catagctacc ctggaggact gtgccaccat 650 gagagactct
tcaaacccaa ggcaaaattg gaatgatgta acctgtttcc 700 tcaattattt
tcggatttgt gaaatggtag gaataaatcc tttgaacaaa 750 ggaaaatctc
tttaagaaca gaaggcacaa ctcaaatgtg taaagaagga 800 agagcaagaa
catggccaca cccaccgccc cacacgagaa atttgtgcgc 850 tgaacttcaa
aggacttcat aagtatttgt tactctgata caaataaaaa 900 taagtagttt
taaatgttaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 950 aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa 997 377 219 PRT Homo
Sapien 377 Met Asn Ser Ser Lys Ser Ser Glu Thr Gln Cys Thr Glu Arg
Gly 1 5 10 15 Cys Phe Ser Ser Gln Met Phe Leu Trp Thr Val Ala Gly
Ile Pro 20 25 30 Ile Leu Phe Leu Ser Ala Cys Phe Ile Thr Arg Cys
Val Val Thr 35 40 45 Phe Arg Ile Phe Gln Thr Cys Asp Glu Lys Lys
Phe Gln Leu Pro 50 55 60 Glu Asn Phe Thr Glu Leu Ser Cys Tyr Asn
Tyr Gly Ser Gly Ser 65 70 75 Val Lys Asn Cys Cys Pro Leu Asn Trp
Glu Tyr Phe Gln Ser Ser 80 85 90 Cys Tyr Phe Phe Ser Thr Asp Thr
Ile Ser Trp Ala Leu Ser Leu 95 100 105 Lys Asn Cys Ser Ala Met Gly
Ala His Leu Val Val Ile Asn Ser 110 115 120 Gln Glu Glu Gln Glu Phe
Leu Ser Tyr Lys Lys Pro Lys Met Arg 125 130 135 Glu Phe Phe Ile Gly
Leu Ser Asp Gln Val Val Glu Gly Gln Trp 140 145 150 Gln Trp Val Asp
Gly Thr Pro Leu Thr Lys Ser Leu Ser Phe Trp 155 160 165 Asp Val Gly
Glu Pro Asn Asn Ile Ala Thr Leu Glu Asp Cys Ala 170 175 180 Thr Met
Arg Asp Ser Ser Asn Pro Arg Gln Asn Trp Asn Asp Val 185 190 195 Thr
Cys Phe Leu Asn Tyr Phe Arg Ile Cys Glu Met Val Gly Ile 200 205 210
Asn Pro Leu Asn Lys Gly Lys Ser Leu 215 378 21 DNA Artificial
Sequence Synthetic Oligonucleotide Probe 378 ttcagcttct gggatgtagg
g 21 379 24 DNA Artificial Sequence Synthetic Oligonucleotide Probe
379 tattcctacc atttcacaaa tccg 24 380 49 DNA Artificial Sequence
Synthetic oligonucleotide probe 380 ggaggactgt gccaccatga
gagactcttc aaacccaagg caaaattgg 49 381 26 DNA Artificial Sequence
Synthetic oligonucleotide probe 381 gcagattttg aggacagcca cctcca 26
382 18 DNA Artificial Sequence Synthetic oligonucleotide probe 382
ggccttgcag acaaccgt 18 383 21 DNA Artificial Sequence Synthetic
oligonucleotide probe 383 cagactgagg gagatccgag a 21 384 20 DNA
Artificial Sequence Synthetic oligonucleotide probe 384 cagctgccct
tccccaacca 20 385 18 DNA Artificial Sequence Synthetic
oligonucleotide probe 385 catcaagcgc ctctacca 18 386 21 DNA
Artificial Sequence Synthetic oligonucleotide probe 386 cacaaactcg
aactgcttct g 21 387 18 DNA Artificial Sequence Synthetic
oligonucleotide probe 387 gggccatcac agctccct 18 388 22 DNA
Artificial Sequence Synthetic oligonucleotide probe 388 gggatgtggt
gaacacagaa ca 22 389 22 DNA Artificial Sequence Synthetic
oligonucleotide probe 389 tgccagctgc atgctgccag tt 22 390 20 DNA
Artificial Sequence Synthetic oligonucleotide probe 390 cagaaggatg
tcccgtggaa 20 391 17 DNA Artificial Sequence Synthetic
oligonucleotide probe 391 gccgctgtcc actgcag 17 392 21 DNA
Artificial Sequence Synthetic oligonucleotide probe 392 gacggcatcc
tcagggccac a 21 393 20 DNA Artificial Sequence Synthetic
oligonucleotide probe 393 atgtcctcca tgcccacgcg 20 394 20 DNA
Artificial Sequence Synthetic oligonucleotide probe 394 gagtgcgaca
tcgagagctt 20 395 18 DNA Artificial Sequence Synthetic
oligonucleotide probe 395 ccgcagcctc agtgatga 18 396 21 DNA
Artificial Sequence Synthetic oligonucleotide probe 396 gaagagcaca
gctgcagatc c 21 397 22 DNA Artificial Sequence Synthetic
oligonucleotide probe 397 gaggtgtcct ggctttggta gt 22 398 20 DNA
Artificial Sequence Synthetic oligonucleotide probe 398 cctctggcgc
ccccactcaa 20 399 18 DNA Artificial Sequence Synthetic
oligonucleotide probe 399 ccaggagagc tggcgatg 18 400 23 DNA
Artificial Sequence Synthetic oligonucleotide probe 400 gcaaattcag
ggctcactag aga 23 401 29 DNA Artificial Sequence Synthetic
oligonucleotide probe 401 cacagagcat ttgtccatca gcagttcag 29 402 22
DNA Artificial Sequence Synthetic oligonucleotide probe 402
ggcagagact tccagtcact ga 22 403 22 DNA Artificial Sequence
Synthetic oligonucleotide probe 403 gccaagggtg gtgttagata gg 22 404
24 DNA Artificial Sequence Synthetic oligonucleotide probe 404
caggccccct tgatctgtac ccca 24 405 23 DNA Artificial Sequence
Synthetic oligonucleotide probe 405 gggacgtgct tctacaagaa cag 23
406 26 DNA Artificial Sequence Synthetic oligonucleotide probe 406
caggcttaca atgttatgat cagaca 26 407 31 DNA Artificial Sequence
Synthetic oligonucleotide probe 407 tattcagagt tttccattgg
cagtgccagt t 31 408 21 DNA Artificial Sequence Synthetic
oligonucleotide probe 408 tctacatcag cctctctgcg c 21 409 23 DNA
Artificial Sequence Synthetic oligonucleotide probe 409 cgatcttctc
cacccaggag cgg 23 410 18 DNA Artificial Sequence Synthetic
oligonucleotide probe 410 gccaggcctc acattcgt 18 411 23 DNA
Artificial Sequence Synthetic oligonucleotide probe 411 ctccctgaat
ggcagcctga gca 23 412 24 DNA Artificial Sequence Synthetic
oligonucleotide probe 412 aggtgtttat taagggccta cgct 24 413 19 DNA
Artificial Sequence Synthetic oligonucleotide probe 413 cagagcagag
ggtgccttg 19 414 21 DNA Artificial Sequence Synthetic
oligonucleotide probe 414 tggcggagtc ccctcttggc t 21 415 22 DNA
Artificial Sequence Synthetic oligonucleotide probe 415 ccctgtttcc
ctatgcatca ct 22 416 21 DNA Artificial Sequence Synthetic
oligonucleotide probe 416 tcaacccctg accctttcct a 21 417 24 DNA
Artificial Sequence Synthetic oligonucleotide probe 417 ggcaggggac
aagccatctc tcct 24 418 20 DNA Artificial Sequence Synthetic
oligonucleotide probe 418 gggactgaac tgccagcttc 20 419 22 DNA
Artificial Sequence Synthetic oligonucleotide probe 419 gggccctaac
ctcattacct tt 22 420 23 DNA Artificial Sequence Synthetic
oligonucleotide probe 420 tgtctgcctc agccccagga agg 23 421 21 DNA
Artificial Sequence Synthetic oligonucleotide probe 421 tctgtccacc
atcttgcctt g 21 422 3554 DNA Homo Sapien 422 gggactacaa gccgcgccgc
gctgccgctg gcccctcagc aaccctcgac 50 atggcgctga ggcggccacc
gcgactccgg ctctgcgctc ggctgcctga 100 cttcttcctg ctgctgcttt
tcaggggctg cctgataggg gctgtaaatc 150 tcaaatccag caatcgaacc
ccagtggtac aggaatttga aagtgtggaa 200 ctgtcttgca tcattacgga
ttcgcagaca agtgacccca ggatcgagtg 250 gaagaaaatt caagatgaac
aaaccacata tgtgtttttt gacaacaaaa 300 ttcagggaga cttggcgggt
cgtgcagaaa tactggggaa gacatccctg 350 aagatctgga atgtgacacg
gagagactca gccctttatc gctgtgaggt 400 cgttgctcga aatgaccgca
aggaaattga tgagattgtg atcgagttaa 450 ctgtgcaagt gaagccagtg
acccctgtct gtagagtgcc gaaggctgta 500 ccagtaggca agatggcaac
actgcactgc caggagagtg agggccaccc 550 ccggcctcac tacagctggt
atcgcaatga tgtaccactg cccacggatt 600 ccagagccaa tcccagattt
cgcaattctt ctttccactt aaactctgaa 650 acaggcactt tggtgttcac
tgctgttcac aaggacgact ctgggcagta 700 ctactgcatt gcttccaatg
acgcaggctc agccaggtgt gaggagcagg 750 agatggaagt ctatgacctg
aacattggcg gaattattgg gggggttctg 800 gttgtccttg ctgtactggc
cctgatcacg ttgggcatct gctgtgcata 850 cagacgtggc tacttcatca
acaataaaca ggatggagaa agttacaaga 900 acccagggaa accagatgga
gttaactaca tccgcactga cgaggagggc 950 gacttcagac acaagtcatc
gtttgtgatc tgagacccgc ggtgtggctg 1000 agagcgcaca gagcgcacgt
gcacatacct ctgctagaaa ctcctgtcaa 1050 ggcagcgaga gctgatgcac
tcggacagag ctagacactc attcagaagc 1100 ttttcgtttt ggccaaagtt
gaccactact cttcttactc taacaagcca 1150 catgaataga agaattttcc
tcaagatgga cccggtaaat ataaccacaa 1200 ggaagcgaaa ctgggtgcgt
tcactgagtt gggttcctaa tctgtttctg 1250 gcctgattcc cgcatgagta
ttagggtgat cttaaagagt ttgctcacgt 1300 aaacgcccgt gctgggccct
gtgaagccag catgttcacc actggtcgtt 1350 cagcagccac gacagcacca
tgtgagatgg cgaggtggct ggacagcacc 1400 agcagcgcat cccggcggga
acccagaaaa ggcttcttac acagcagcct 1450 tacttcatcg gcccacagac
accaccgcag tttcttctta aaggctctgc 1500 tgatcggtgt tgcagtgtcc
attgtggaga agctttttgg atcagcattt 1550 tgtaaaaaca accaaaatca
ggaaggtaaa ttggttgctg gaagagggat 1600 cttgcctgag gaaccctgct
tgtccaacag ggtgtcagga tttaaggaaa 1650 accttcgtct taggctaagt
ctgaaatggt actgaaatat gcttttctat 1700 gggtcttgtt tattttataa
aattttacat ctaaattttt gctaaggatg 1750 tattttgatt attgaaaaga
aaatttctat ttaaactgta aatatattgt 1800 catacaatgt taaataacct
atttttttaa aaaagttcaa cttaaggtag 1850 aagttccaag ctactagtgt
taaattggaa aatatcaata attaagagta 1900 ttttacccaa ggaatcctct
catggaagtt tactgtgatg ttccttttct 1950 cacacaagtt ttagcctttt
tcacaaggga actcatactg tctacacatc 2000 agaccatagt tgcttaggaa
acctttaaaa attccagtta agcaatgttg 2050 aaatcagttt gcatctcttc
aaaagaaacc tctcaggtta gctttgaact 2100 gcctcttcct
gagatgacta ggacagtctg tacccagagg ccacccagaa 2150 gccctcagat
gtacatacac agatgccagt cagctcctgg ggttgcgcca 2200 ggcgcccccg
ctctagctca ctgttgcctc gctgtctgcc aggaggccct 2250 gccatccttg
ggccctggca gtggctgtgt cccagtgagc tttactcacg 2300 tggcccttgc
ttcatccagc acagctctca ggtgggcact gcagggacac 2350 tggtgtcttc
catgtagcgt cccagctttg ggctcctgta acagacctct 2400 ttttggttat
ggatggctca caaaataggg cccccaatgc tatttttttt 2450 ttttaagttt
gtttaattat ttgttaagat tgtctaaggc caaaggcaat 2500 tgcgaaatca
agtctgtcaa gtacaataac atttttaaaa gaaaatggat 2550 cccactgttc
ctctttgcca cagagaaagc acccagacgc cacaggctct 2600 gtcgcatttc
aaaacaaacc atgatggagt ggcggccagt ccagcctttt 2650 aaagaacgtc
aggtggagca gccaggtgaa aggcctggcg gggaggaaag 2700 tgaaacgcct
gaatcaaaag cagttttcta attttgactt taaatttttc 2750 atccgccgga
gacactgctc ccatttgtgg ggggacatta gcaacatcac 2800 tcagaagcct
gtgttcttca agagcaggtg ttctcagcct cacatgccct 2850 gccgtgctgg
actcaggact gaagtgctgt aaagcaagga gctgctgaga 2900 aggagcactc
cactgtgtgc ctggagaatg gctctcacta ctcaccttgt 2950 ctttcagctt
ccagtgtctt gggtttttta tactttgaca gctttttttt 3000 aattgcatac
atgagactgt gttgactttt tttagttatg tgaaacactt 3050 tgccgcaggc
cgcctggcag aggcaggaaa tgctccagca gtggctcagt 3100 gctccctggt
gtctgctgca tggcatcctg gatgcttagc atgcaagttc 3150 cctccatcat
tgccaccttg gtagagaggg atggctcccc accctcagcg 3200 ttggggattc
acgctccagc ctccttcttg gttgtcatag tgatagggta 3250 gccttattgc
cccctcttct tataccctaa aaccttctac actagtgcca 3300 tgggaaccag
gtctgaaaaa gtagagagaa gtgaaagtag agtctgggaa 3350 gtagctgcct
ataactgaga ctagacggaa aaggaatact cgtgtatttt 3400 aagatatgaa
tgtgactcaa gactcgaggc cgatacgagg ctgtgattct 3450 gcctttggat
ggatgttgct gtacacagat gctacagact tgtactaaca 3500 caccgtaatt
tggcatttgt ttaacctcat ttataaaagc ttcaaaaaaa 3550 ccca 3554 423 310
PRT Homo Sapien 423 Met Ala Leu Arg Arg Pro Pro Arg Leu Arg Leu Cys
Ala Arg Leu 1 5 10 15 Pro Asp Phe Phe Leu Leu Leu Leu Phe Arg Gly
Cys Leu Ile Gly 20 25 30 Ala Val Asn Leu Lys Ser Ser Asn Arg Thr
Pro Val Val Gln Glu 35 40 45 Phe Glu Ser Val Glu Leu Ser Cys Ile
Ile Thr Asp Ser Gln Thr 50 55 60 Ser Asp Pro Arg Ile Glu Trp Lys
Lys Ile Gln Asp Glu Gln Thr 65 70 75 Thr Tyr Val Phe Phe Asp Asn
Lys Ile Gln Gly Asp Leu Ala Gly 80 85 90 Arg Ala Glu Ile Leu Gly
Lys Thr Ser Leu Lys Ile Trp Asn Val 95 100 105 Thr Arg Arg Asp Ser
Ala Leu Tyr Arg Cys Glu Val Val Ala Arg 110 115 120 Asn Asp Arg Lys
Glu Ile Asp Glu Ile Val Ile Glu Leu Thr Val 125 130 135 Gln Val Lys
Pro Val Thr Pro Val Cys Arg Val Pro Lys Ala Val 140 145 150 Pro Val
Gly Lys Met Ala Thr Leu His Cys Gln Glu Ser Glu Gly 155 160 165 His
Pro Arg Pro His Tyr Ser Trp Tyr Arg Asn Asp Val Pro Leu 170 175 180
Pro Thr Asp Ser Arg Ala Asn Pro Arg Phe Arg Asn Ser Ser Phe 185 190
195 His Leu Asn Ser Glu Thr Gly Thr Leu Val Phe Thr Ala Val His 200
205 210 Lys Asp Asp Ser Gly Gln Tyr Tyr Cys Ile Ala Ser Asn Asp Ala
215 220 225 Gly Ser Ala Arg Cys Glu Glu Gln Glu Met Glu Val Tyr Asp
Leu 230 235 240 Asn Ile Gly Gly Ile Ile Gly Gly Val Leu Val Val Leu
Ala Val 245 250 255 Leu Ala Leu Ile Thr Leu Gly Ile Cys Cys Ala Tyr
Arg Arg Gly 260 265 270 Tyr Phe Ile Asn Asn Lys Gln Asp Gly Glu Ser
Tyr Lys Asn Pro 275 280 285 Gly Lys Pro Asp Gly Val Asn Tyr Ile Arg
Thr Asp Glu Glu Gly 290 295 300 Asp Phe Arg His Lys Ser Ser Phe Val
Ile 305 310
* * * * *