U.S. patent application number 09/154750 was filed with the patent office on 2002-05-09 for p53-induced apoptosis.
Invention is credited to KINZLER, KENNETH W., POLYAK, KORNELIA, VOGELSTEIN, BERT.
Application Number | 20020055097 09/154750 |
Document ID | / |
Family ID | 26738417 |
Filed Date | 2002-05-09 |
United States Patent
Application |
20020055097 |
Kind Code |
A1 |
POLYAK, KORNELIA ; et
al. |
May 9, 2002 |
P53-INDUCED APOPTOSIS
Abstract
The most well-documented biochemical property of p53 is its
ability to transcriptionally activate genes. Many of the genes
which are activated by p53 expression prior to the onset of
apoptosis are predicted to encode proteins which could generate or
respond to oxidative stress, including one that is implicated in
apoptosis within plant meristems. p53 may result in apoptosis
through a three-step process: (I) the transcriptional induction of
specific redox-related genes; (ii) the formation of reactive oxygen
species (ROS); and (iii) the oxidative degradation of mitochondrial
components, rapidly leading to cell death. Transcription of other
genes is decreased by p53. Examination of the level of
transcription of p53-induced or, -repressed genes can be used to
determine p53 status, to diagnose cancer, and to evaluate
cytotoxicity or carcinogenicity of a test agent.
Inventors: |
POLYAK, KORNELIA;
(BROOKLINE, MA) ; VOGELSTEIN, BERT; (BALIMORE,
MD) ; KINZLER, KENNETH W.; (BELAIR, MD) |
Correspondence
Address: |
BANNER & WITCOFF
1001 G STREET NW
WASHINGTON
DC
200014597
|
Family ID: |
26738417 |
Appl. No.: |
09/154750 |
Filed: |
September 17, 1998 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60059153 |
Sep 17, 1997 |
|
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60079817 |
Mar 30, 1998 |
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Current U.S.
Class: |
435/6.13 ;
435/91.1; 435/91.2; 536/23.1 |
Current CPC
Class: |
C12Q 1/6886 20130101;
C12Q 1/6809 20130101; C12Q 1/6897 20130101; C12Q 2600/142 20130101;
C12Q 2600/136 20130101; C12Q 2539/103 20130101 |
Class at
Publication: |
435/6 ; 435/91.1;
435/91.2; 536/23.1 |
International
Class: |
C12Q 001/68; C07H
021/02; C07H 021/04; C12P 019/34 |
Goverment Interests
[0002] This invention was made using grant funds from the U.S.
National Institutes of Health (CA57345). Therefore the government
retains some rights in the present invention.
Claims
1. A method of diagnosing cancer or determining p53 status in a
sample suspected of being neoplastic, comprising the steps of:
comparing the level of transcription of an RNA transcript in a
first sample of a first tissue to the level of transcription of the
transcript in a second sample of a second tissue, wherein the first
tissue is suspected of being neoplastic and the second tissue is a
normal human tissue, wherein the first and second tissue are of the
same tissue type, and wherein the transcript is identified by a tag
selected from the group consisting of SEQ ID NOS:10, 15-22, 26, 27,
and 30; categorizing the first sample as neoplastic or as having a
mutant p53 when transcription is found to be the same or lower in
the first sample than in the second sample.
2. A method of diagnosing cancer or determining p53 status in a
sample suspected of being neoplastic, comprising the steps of:
comparing the level of transcription of an RNA transcript in a
first sample of a first tissue to the level of transcription of the
transcript in a second sample of a second tissue, wherein the first
tissue is suspected of being neoplastic and the second tissue is a
normal human tissue, wherein the first and second tissue are of the
same tissue type, and wherein the transcript is identified by a tag
selected from the group consisting SEQ ID NOS:37-67; categorizing
the first sample as neoplastic or as having a mutant p53 when
transcription is found to be the same or higher in the first sample
than in the second sample.
3. The method of claim 1 wherein a comparison of at least two of
the transcripts is performed.
4. The method of claim 2 wherein a comparison of at least two of
the transcripts is performed.
5. The method of claim 1 wherein a comparison of at least five of
the transcripts is performed.
6. The method of claim 2 wherein a comparison of at least five of
the transcripts is performed.
7. The method of claim 1 wherein a comparison of at least ten of
the transcripts is performed.
8. The method of claim 2 wherein a comparison of at least ten of
the transcripts is performed.
9. The method of claim 1 wherein at least one tag is selected from
the group consisting of SEQ ID NOS:15, 16, 17, 19, 21, 22, and
30.
10. An isolated and purified nucleic acid molecule which comprises
a SAGE tag selected from the group consisting of SEQ ID NOS:15, 16,
17, 19, 21, 22, and 30.
11. The nucleic acid molecule of claim 10 which is a cDNA
molecule.
12. The nucleic acid molecule of claim 10 wherein the SAGE tag is
located at the 3' end of the molecule.
13. An isolated nucleotide probe comprising at least 12 contiguous
nucleotides of a human nucleic acid molecule, wherein the human
nucleic acid molecule comprises a SAGE tag selected from the group
consisting of SEQ ID NOS:15, 16, 17, 19, 21, 22, and 30.
14. The probe of claim 13 which comprises the selected SAGE
tag.
15. A kit for evaluating toxicity or carcinogenicity of an agent,
comprising at least 2 probes according to claim 13.
16. The kit of claim 15 which comprises at least 5 of said
probes.
17. The kit of claim 15 which comprises at least 10 of said
probes.
18. The kit of claim 15 which comprises at least 20 of said
probes.
19. The kit of claim 15 which comprises at least 30 of said
probes.
20. A kit for evaluating cytotoxicity or carcinogenicity,
comprising at least 2 probes according to claim 14.
21. A method for evaluating cytotoxicity or carcinogenicity of an
agent, comprising the steps of: contacting a test agent with a
human cell; determining the level of transcription of a transcript
in the human cell after contacting with the agent; wherein an agent
which increases the level of a transcript identified by a SAGE tag
selected from the group consisting of SEQ ID NOS:10, 15-22, 26, 27,
and 30, or an agent which decreases the level of a transcript
identified by a SAGE tag selected from the group consisting of SEQ
ID NOS:37-67 is a potential cytotoxin or carcinogen.
22. A method to determine the neoplastic status or p53 status of a
cell comprising: comparing ROS levels in a first sample of a first
tissue to the level in a second sample of a second tissue, wherein
the first tissue is or is suspected of being neoplastic and the
second tissue is a normal human tissue; wherein elevated levels of
ROS in the first sample indicate expression of p53 and low levels
of ROS indicate lack of expression of p53, wherein lack of
expression of p53 is an indicator of neoplasia.
23. A DNA construct for screening drugs as anti-neoplastic agents
comprising: a reporter gene under the control of a PIG-3 promoter,
wherein the reporter gene is 3' and covalently linked to the PIG-3
promoter, wherein the PIG-3 promoter comprises the sequence
CAGCTTGCCCACCCATGCTC (SEQ ID NO:1).
24. A method of diagnosing cancer or determining p53 status in a
sample suspected of being neoplastic, comprising the steps of;
treating cells of a test sample with a DNA-damaging agent;
comparing the level of transcription of an RNA transcript in cells
of the sample to the level of transcription of the transcript in
cells of the sample which are not subject to said treating, wherein
the transcript is identified by a tag selected from the group
consisting of SEQ ID NOS:10, 15-22, 26, 27, and 30; categorizing
the sample as neoplastic or as having a mutant p53 when
transcription is found to be the same or lower in the treated cells
than in the untreated cells.
25. A method of diagnosing cancer or determining p53 status in a
sample suspected of being neoplastic, comprising the steps of:
treating cells of a test sample with a DNA-damaging agent;
comparing the level of transcription of an RNA transcript in the
cells to the level of transcription of the transcript in cells of
the sample which are not subject to said treating, wherein the
transcript is identified by a tag selected from the group
consisting of SEQ ID NOS:37-67; categorizing the sample as
neoplastic or as having a mutant p53 when transcription is found to
be the same or higher in the treated cells than in the untreated
cells.
26. The method of claim 24 wherein a comparison of at least two of
the transcripts is performed.
27. The method of claim 25 wherein a comparison of at least two of
the transcripts is performed.
28. The method of claim 24 wherein a comparison of at least five of
the transcripts is performed.
29. The method of claim 25 wherein a comparison of at least five of
the transcripts is performed.
30. The method of claim 24 wherein a comparison of at least ten of
the transcripts is performed.
31. The method of claim 25 wherein a comparison of at least ten of
the transcripts is performed.
32. The method of claim 24 wherein at least one tag is selected
from the group consisting of SEQ ID NOS:15-17, 19, 21, 22, and
30.
33. The method of claim 1 wherein the first and second samples are
treated with a DNA-damaging agent prior to said step of
comparing.
34. The method of claim 2 wherein the first and second samples are
treated with a DNA-damaging agent prior to said step of
comparing.
35. A preparation of antibodies which specifically bind to a PIG
protein having an amino acid sequence selected from the group
consisting of SEQ ID NOS:81, 83, 84, 86, 87, and 88.
36. The preparation of antibodies of claim 35 wherein the
antibodies are monoclonal.
37. The preparation of antibodies of claim 35 wherein the
antibodies are polyclonal.
38. The preparation of antibodies of claim 35 wherein the
antibodies are affinity purified.
39. A method of diagnosing cancer or determining p53 status in a
sample suspected of being neoplastic, comprising the steps of:
comparing the level of a PIG protein having an amino acid sequence
selected from the group consisting of SEQ ID NOS:79-88 and the
amino acid sequence encoded by SEQ ID NO:72 in a first sample of a
first tissue to the level of the PIG protein in a second sample of
a second tissue, wherein the first tissue is suspected of being
neoplastic and the second tissue is a normal human tissue, wherein
the first and second tissue are of the same tissue type; and
categorizing the first sample as neoplastic or as having a mutant
p53 when the level of the PIG protein is found to be the same or
lower in the first sample than in the second sample.
40. A method of diagnosing cancer or determining p53 status in a
sample suspected of being neoplastic, comprising the steps of:
comparing the level of a protein of Table 2 in a first sample of a
first tissue to the level of the protein of Table 2 in a second
sample of a second tissue, wherein the first tissue is suspected of
being neoplastic and the second tissue is a normal human tissue,
wherein the first and second tissue are of the same tissue type;
and categorizing the first sample as neoplastic or as having a
mutant p53 when the level of the protein of Table 2 is found to be
the same or higher in the first sample than in the second
sample.
41. The method of claim 39 wherein the level of the PIG protein is
measured using an antibody which specifically binds to a PIG
protein having an amino acid sequence selected from the group
consisting of SEQ ID NOS:79-88 and the amino acid sequence encoded
by SEQ ID NO:72.
42. The method of claim 40 wherein the level of the protein is
measuring using an antibody which specifically binds to a protein
selected from the group of proteins shown in Table 2.
43. The method of claim 39 wherein a comparison of the levels of at
least two PIG proteins is performed.
44. The method of claim 40 wherein a comparison of the levels of at
least two proteins of Table 2 is performed.
45. The method of claim 39 wherein a comparison of the levels of at
least five PIG proteins is performed.
46. The method of claim 40 wherein a comparison of the levels of at
least five proteins of Table 2 is performed.
47. The method of claim 39 wherein a comparison of the levels of at
least at least ten PIG proteins is performed.
48. The method of claim 40 wherein a comparison of the levels of at
least ten proteins of Table 2 is performed.
49. The method of claim 39 wherein the first and second samples are
treated with a DNA-damaging agent prior to said step of
comparing.
50. The method of claim 40 wherein the first and second samples are
treated with a DNA-damaging agent prior to said step of
comparing.
51. A kit for evaluating toxicity or carcinogenicity of an agent,
comprising at least 2 antibodies according to claim 35.
52. A method for evaluating cytotoxicity or carcinogenicity of an
agent, comprising the steps of: contacting a test agent with a
human cell; determining the level of a PIG protein having an amino
acid sequence selected from the group consisting of SEQ ID
NOS:79-88 and the amino acid sequence encoded by SEQ ID NO:72 or of
a protein of Table 2 in the human cell after contacting with the
agent; wherein an agent which increases the level of the PIG
protein, or an agent which decreases the level of the protein of
Table 2 is identified as a potential cytotoxin or carcinogen.
53. The method of claim 52 wherein the level of the PIG protein is
measuring using an antibody which specifically binds to a protein
having an amino acid sequence selected from the group consisting of
SEQ ID NOS:79-88 and the amino acid sequence encoded by SEQ ID
NO:72.
54. A method of diagnosing cancer or determining p53 status in a
sample suspected of being neoplastic, comprising the steps of:
treating cells of a test sample with a DNA-damaging agent;
comparing the level of a PIG protein having an amino acid sequence
selected from the group consisting of SEQ ID NOS:79-88 and the
amino acid sequence encoded by SEQ ID NO:72 in cells of the sample
to the level of the PIG protein in cells of the sample which are
not subject to said treating; and categorizing the sample as
neoplastic or as having a mutant p53 when the level of the PIG
protein is found to be the same or lower in the treated cells than
in the untreated cells.
55. A method of diagnosing cancer or determining p53 status in a
sample suspected of being neoplastic, comprising the steps of:
treating cells of a test sample with a DNA-damaging agent;
comparing the level of a protein of Table 2 in cells of the sample
to the level of the protein of Table 2 in cells of the sample which
are not subject to said treating; and categorizing the sample as
neoplastic or as having a mutant p53 when the level of the protein
of Table 2 is found to be the same or higher in the treated cells
than in the untreated cells.
56. The method of claim 54 wherein the level of the PIG protein is
measured using an antibody which specifically binds to a protein
having an amino acid sequence selected from the group consisting of
SEQ ID NOS:81, 83, 84, 86, 87, and 88.
57. The method of claim 55 wherein the level of the protein of
Table 2 is measured using an antibody which specifically binds to a
protein selected from the group consisting of the proteins shown in
Table 2.
58. The method of claim 54 wherein a comparison of the levels of at
least two PIG proteins is performed.
59. The method of claim 55 wherein a comparison of the levels of at
least two proteins of Table 2 is performed.
60. The method of claim 54 wherein a comparison of the levels of at
least five PIG proteins is performed.
61. The method of claim 55 wherein a comparison of the levels of at
least five proteins of Table 2 is performed.
62. The method of claim 54 wherein a comparison of the levels of at
least ten PIG proteins is performed.
63. The method of claim 55 wherein a comparison of the levels of at
least ten proteins of Table 2 is performed.
64. The method of claim 54 wherein at least one antibody is an
antibody which specifically binds to a protein having an amino acid
sequence selected from the group consisting of SEQ ID NOS:81, 83,
84, 86, 87, and 88.
Description
[0001] This application claims the benefit of co-pending
provisional applications Serial No. 60/059,153 filed Sep. 17, 1997
and Serial No. 60/079,817 filed Mar. 27 1998. These two
applications are incorporated by reference herein.
TECHNICAL FIELD OF THE INVENTION
[0003] This invention is related to genes and proteins involved in
cell cycle control and tumorigenesis. These genes can be used
diagnostically and therapeutically because of their role in
cancers.
BACKGROUND OF THE INVENTION
[0004] The inactivation of the p53 gene in a large fraction of
human cancers has inspired an intense search for the encoded
protein's physiologic and biologic properties. Expression of p53
induces either a stable growth arrest or programmed cell death
(apoptosis). In human colorectal cancers (CRC), the growth arrest
is dependent on the transcriptional induction of p21WAF1/CIP1(1),
but the biochemical mechanisms underlying the development of
p53-dependent apoptosis are largely unknown (2). Thus, there is a
continuing need in the art for discovering new genes which are
regulated by p53 and genes which are related to cell cycle control
and tumorigenesis.
SUMMARY OF THE INVENTION
[0005] It is an object of the present invention to provide methods
of diagnosing cancer or determining p53 status in a sample
suspected of being neoplastic.
[0006] It is another object of the present invention to provide an
isolated and purified nucleic acid molecule which is identified by
a SAGE tag.
[0007] It is an object of the present invention to provide an
isolated nucleotide probe comprising at least 12 nucleotides of a
rat nucleic acid molecule identified by a SAGE tag.
[0008] Another object of the invention is to provide methods and
kits for evaluating cytotoxicity or carcinogenicity of an
agent.
[0009] It is still another object of the invention to provide a DNA
construct useful for screening drugs as anti-neoplastic agents.
[0010] It is even another object of the invention to provide a
preparation of antibodies.
[0011] These and other objects of the invention are provided by one
or more of the embodiments described below. One embodiment of the
invention provides a method of diagnosing cancer or determining p53
status in a sample suspected of being neoplastic. The level of
transcription of an RNA transcript in a first sample of a first
tissue is compared to the level of transcription of the transcript
in a second sample of a second tissue. The first tissue is
suspected of being neoplastic and the second tissue is a normal
human tissue. The first and second tissue are of the same tissue
type. The transcript is identified by a tag selected from the group
consisting of SEQ ID NOS:10, 15-22, 26, 27, and 30. The first
sample is characterized as neoplastic or as having a mutant p53
when transcription is found to be the same or lower in the first
sample than in the second sample.
[0012] Another embodiment of the invention provides a method of
diagnosing cancer or determining p53 status in a sample suspected
of being neoplastic. The level of transcription of an RNA
transcript in a first sample of a first tissue is compared to the
level of transcription of the transcript in a second sample of a
second tissue. The first tissue is suspected of being neoplastic,
and the second tissue is a normal human tissue. The first and
second tissue are of the same tissue type. The transcript is
identified by a tag selected from the group consisting of SEQ ID
NOS:37-67. The first sample is categorized as neoplastic or as
having a mutant p53 when transcription is found to be the same or
higher in the first sample than in the second sample.
[0013] Yet another embodiment of the invention provides an isolated
and purified nucleic acid molecule which comprises a SAGE tag
selected from the group consisting of SEQ ID NOS:15, 16, 17, 19,
21, 22, and 30.
[0014] Even another embodiment of the invention provides an
isolated nucleotide probe comprising at least 12 contiguous
nucleotides of a human nucleic acid molecule. The human nucleic
acid molecule comprises a SAGE tag selected from the group
consisting of SEQ ID NOS: 15, 16, 17, 19, 21, 22, and 30.
[0015] A further embodiment of the invention provides a kit for
evaluating toxicity or carcinogenicity of an agent. The kit
comprises at least 2 probes. The probes comprise at least 12
contiguous nucleotides of a human nucleic acid molecule. The human
nucleic acid molecule comprises a SAGE tag selected from the group
consisting of SEQ ID NOS:15, 16, 17, 19, 21, 22, and 30.
[0016] Another embodiment of the invention provides a kit for
evaluating cytotoxicity or carcinogenicity. The kit comprises at
least 2 probes. The probes comprise a SAGE tag selected from the
group consisting of SEQ ID NOS:15, 16, 17, 19, 21, 22, and 30.
[0017] Even another embodiment of the invention provides a method
for evaluating cytotoxicity or carcinogenicity of an agent. A test
agent is contacted with a human cell. The level of transcription of
a transcript in the human cell after contacting with the agent is
determined. An agent which increases the level of a transcript
identified by a SAGE tag selected from the group consisting of SEQ
ID NOS:10, 15-22, 26, 27, and 30, or an agent which decreases the
level of a transcript identified by a SAGE tag selected from the
group consisting of SEQ ID NOS:37-67 is a potential cytotoxin or
carcinogen.
[0018] Another embodiment of the invention provides a method to
determine the neoplastic status or p53 status of a cell. ROS levels
in a first sample of a first tissue are compared to ROS levels in a
second sample of a second tissue. The first tissue is or is
suspected of being neoplastic, and the second tissue is a normal
human tissue. Elevated levels of ROS in the first sample indicate
expression of p53, and low levels of ROS in the first sample
indicate lack of expression of p53. Lack of expression of p53 is an
indicator of neoplasia.
[0019] Still another embodiment of the invention provides a DNA
construct for screening drugs as anti-neoplastic agents. The DNA
construct comprises a reporter gene under the control of a PIG-3
promoter. The reporter gene is 3' and covalently linked to the
PIG-3 promoter. The PIG-3 promoter comprises the sequence
CAGCTTGCCCACCCATGCTC (SEQ ID NO: 1).
[0020] A further embodiment of the invention provides a method of
diagnosing cancer or determining p53 status in a sample suspected
of being neoplastic. Cells of a test sample are treated with a
DNA-damaging agent. The level of transcription of an RNA transcript
in cells of the sample is compared to the level of transcription of
the transcript in cells of the sample which are not subject to said
treating. The transcript is identified by a tag selected from the
group consisting of SEQ ID NOS: 10, 15-22, 26, 27, and 30. The
sample is characterized as neoplastic or as having a mutant p53
when transcription is found to be the same or lower in the treated
cells than in the untreated cells.
[0021] Another embodiment of the invention provides a method of
diagnosing cancer or determining p53 status in a sample suspected
of being neoplastic. Cells of a test sample are treated with a
DNA-damaging agent. The level of transcription of an RNA transcript
in the cells is compared to the level of transcription of the
transcript in cells of the sample which are not subject to said
treating. The transcript is identified by a tag selected from the
group consisting SEQ ID NOS:37-67. The sample is categorized as
neoplastic or as having a mutant p53 when transcription is found to
be the same or higher in the treated cells than in the untreated
cells.
[0022] Even another embodiment of the invention provides a
preparation of antibodies which specifically bind to a PIG protein
having an amino acid sequence selected from the group consisting of
SEQ ID NOS:81, 83, 84, 86, 87, and 88.
[0023] Still another embodiment of the invention provides a method
of diagnosing cancer or determining p53 status in a sample
suspected of being neoplastic. The level of a PIG protein having an
amino acid sequence selected from the group consisting of SEQ ID
NOS:79-88 and the amino acid sequence encoded by SEQ ID NO:72 in a
first sample of a first tissue is compared to the level of the PIG
protein in a second sample of a second tissue. The first tissue is
suspected of being neoplastic, and the second tissue is a normal
human tissue. The first and second tissue are of the same tissue
type. The first sample is categorized as neoplastic or as having a
mutant p53 when the level of the PIG protein is found to be the
same or lower in the first sample than in the second sample.
[0024] Yet another embodiment of the invention provides a method of
diagnosing cancer or determining p53 status in a sample suspected
of being neoplastic. The level of a protein of Table 2 in a first
sample of a first tissue is compared to the level of the protein of
Table 2 in a second sample of a second tissue. The first tissue is
suspected of being neoplastic, and the second tissue is a normal
human tissue. The first and second tissue are of the same tissue
type. The first sample is categorized as neoplastic or as having a
mutant p53 when the level of the protein of Table 2 is found to be
the same or higher in the first sample than in the second
sample.
[0025] Even another embodiment of the invention provides a kit for
evaluating toxicity or carcinogenicity of an agent. The kit
comprises at least 2 antibodies which specifically bind to a PIG
protein having an amino acid sequence selected from the group
consisting of SEQ ID NOS:81, 83, 84, 86, 87, and 88.
[0026] Still another embodiment of the invention provides a method
for evaluating cytotoxicity or carcinogenicity of an agent. A test
agent is contacted with a human cell. The level of a PIG protein
having an amino acid sequence selected from the group consisting of
SEQ ID NOS:79-88 and the amino acid sequence encoded by SEQ ID
NO:72 or of a protein of Table 2 in the human cell is determined
after contacting with the agent. An agent which increases the level
of the PIG protein or an agent which decreases the level of the
protein of Table 2 is identified as a potential cytotoxin or
carcinogen.
[0027] A further embodiment of the invention provides a method of
diagnosing cancer or determining p53 status in a sample suspected
of being neoplastic. Cells of a test sample are treated with a
DNA-damaging agent. The level of a PIG protein having an amino acid
sequence selected from the group consisting of SEQ ID NOS:79-88 and
the amino acid sequence encoded by SEQ ID NO:72 in cells of the
sample is compared to the level of the PIG protein in cells of the
sample which are not subject to said treating. The sample is
categorized as neoplastic or as having a mutant p53 when the level
of the PIG protein is found to be the same or lower in the treated
cells than in the untreated cells.
[0028] Even another embodiment of the invention provides a method
of diagnosing cancer or determining p53 status in a sample
suspected of being neoplastic. Cells of a test sample are treated
with a DNA-damaging agent. The level of a protein of Table 2 in
cells of the sample is compared to the level of the protein of
Table 2 in cells of the sample which are not subject to said
treating. The sample is categorized as neoplastic or as having a
mutant p53 when the level of the protein of Table 2 is found to be
the same or higher in the treated cells than in the untreated
cells.
[0029] These and other embodiments of the invention provide the art
with tools for assessing p53 status in cells, which can provide
diagnostic and prognostic information useful in the evaluation of
patients and the management of cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] FIG. 1A. Summary of SAGE data. For each of 7,202 different
transcripts identified, the ratio of their abundances in two
libraries is plotted. The y-axis indicates the number of tags
expressed at the ratio indicated on the x-axis. Bars representing
tags exhibiting less than 5-fold differences in expression are
shown in green, and those induced or repressed more than 8-fold are
shown in blue and red, respectively. FIG. 1B. Northern blot
analysis after Ad-p53 infection. Representative Northern blots are
shown for several transcripts identified by SAGE to be expressed at
higher levels in p53-expressing cells at the indicated times post
infection. Uninfected cells (column marked "0") and cells infected
with Ad-lacZ for 48 hrs (column marked "B") were included for
comparison. EF1 is a control transcript expressed at relatively
equal levels in cells 16 hours after infection with Ad-p53 and
Ad-lacZ. The SAGE tag abundances (16 hours after infection) are
included at the right.
[0031] FIG. 2A. Schematic of PIG3 gene, illustrating intron-exon
structure and promoter region. Numbers refer to nucleotides
relative to the 5' end of the cDNA. The fragments used for the
luciferase constructs had their distal ends at the Eag I site
within exon 1 and their 5' ends at either the Apa LI or Nsi I sites
(FULL and DEL, respectively). The 53-binding site located at
nucleotides 328-308 is indicated, with the upper case letters
corresponding to the highly conserved residues that were altered in
one of the oligonucleotides used for immunoprecipitation. FIG. 2B.
p53-induction of the PIG3 promoter. Fragments encompassing 5.6 or
0.7 kb (FULL and DEL, respectively) of the PIG3 gene promoter were
cloned upstream of a luciferase reporter and transfected into the
indicated cell types in the presence of wt and mutant p53
expression vectors. The levels of luciferase activity were
determined in cell lysates 24 hours after transfection. FIG. 2C. In
vitro binding assay with end-labeled fragments containing wild type
(WT) and mutant (M) p53 binding sites. A fragment containing
thirteen copies of a p53 binding site from the WAF1 promoter region
3026 was used as a control (C). The "input" lanes contained 0.5% of
the amount of fragment used in the binding assays.
[0032] FIG. 3. Sequences of selected genes identified through SAGE.
In each case, the indicated gene is compared to the homologue from
the non-human species that revealed a clue to its possible
function. The amino acid sequences were aligned using Macaw Version
2.0.3, and the most significant similarities are indicated by
shading. With the exception of PIG6, the cloned human sequences
appeared to be full length with respect to the coding region.
Accession numbers are provided in Table 1.
[0033] FIG. 4. Oxidative stress and mitochondrial damage in
p53-mediated apoptosis. FIG. 4A. DLD-1 cells were infected with
Ad-p53 or control (Ad-lacZ) viruses and harvested after 27, 35, or
42 hours. Cells were incubated with CM-DCF-DA, a probe of ROS, or
NAO, a probe of the mitochondrial membrane cardiolipin, and
analyzed by flow cytometry. The mean fluorescence of the control
cells is indicated by vertical lines in each box. The pro-oxidant
drug menadione was used as a positive control to induce oxidative
stress. An increase in ROS and a decrease in cardiolipin
concentration could be clearly observed by cytometry at 27 hours
and increased as the p53-expressing cells entered apoptosis. FIG.
4B. Time course of apoptosis-related events following p53
expression. Cells were infected with Ad-p53 at 0 hours and PIG3
expression (.circle-solid.) was quantitated by densitometry of
Northern blots. ROS production (.largecircle.) was assessed with
lucigenin; glutathione depletion exhibited a similar time course
(not shown). Cardiolipin concentration (.DELTA.) was assessed with
nonyl-acridine orange staining. Caspase activation
(.tangle-solidup.) was assessed by cleavage of PARP, and chromatin
condensation/fragmentation (.diamond.) was assessed by staining
with DAPI.
DETAILED DESCRIPTION
[0034] The most well-documented biochemical property of p53 is its
ability to transcriptionally activate genes. Of 7,202 transcripts
induced by p53 expression prior to the onset of apoptosis, only 14
(0.19%) are found at markedly higher levels in p53-expressing cells
than in control cells. The genes encoding these transcripts are
termed PIGS (p53-induced genes). Many of these genes are predicted
to encode proteins which could generate or respond to oxidative
stress, including one that is implicated in apoptosis within plant
meristems. Thus, p53 may result in apoptosis through a three-step
process: (I) the transcriptional induction of specific
redox-related genes; (ii) the formation of reactive oxygen species
(ROS); and (iii) the oxidative degradation of mitochondrial
components, rapidly leading to in cell death.
[0035] Using the SAGE tags disclosed in Tables 1 and 2, transcripts
can be evaluated for enhanced or reduced expression, respectively.
A SAGE tag is a short sequence tag, preferably 10 or 11 base pairs,
which is generated from defined positions within each mRNA
molecule. Expression patterns are deduced from the abundance of
individual tags. The altered expression can provide an indication
of the status of the p53 genes in the cells, which themselves
reflect the neoplastic status of cells. While the presence of
wild-type p53 is not determinative of normalcy, the presence of
mutant p53 is an indication of neoplasia.
[0036] The tags which are shown in Table 1 identify transcripts
which are enhanced by p53; the tags of Table 2 identify transcripts
which are decreased by p53. Wild-type p53 is required for these
modulations. Thus failure to so-modulate is an indication of mutant
p53 in the cell. Similarly, DNA-damaging agents which cause
apoptosis do so via wild-type p53. In the absence of wild-type p53
these agents cannot induce transcription of the Table 1
tag-identified transcripts nor can they decrease transcription of
the Table 2 tag-identified transcripts. Thus, analysis of the
status of these transcripts can provide an indication of the
presence or absence of wild-type p53.
[0037] Cells can be compared from suspect tissues to normal
tissues. Similarly, a suspect or test tissue sample can be treated
with a DNA-damaging agent and the response of the cells in the
tissue assessed. The response assessed is the induction or
reduction in the transcripts identified by the tags. Tags
"identify" transcripts by hybridization to them. This hybridization
can be determined using any method of measuring transcription,
including but not limited to Northern blots, quantitative RT PCR,
etc. Conditions for optimizing hybridization signals and minimizing
background are known in the art and can be selected by the skilled
artisan. Preferably an assay is done with at least two, five, or
ten of the transcripts which are known to be modulated by p53. More
preferably one or more of the tags used is selected from SEQ ID
NOS: 15-17, 19, 21, 22, or 30. Suitable DNA-damaging agents include
adriamycin, mitomycin, alkylating agents, and .gamma.- and
UV-radiation.
[0038] Isolated and purified nucleic acid molecules which include a
SAGE tag particularly SEQ ID NOS:15-17, 19, 21, 22, or 30, are also
provided. These can be made using the SAGE tags to isolate a full
length RNA, which is then reverse transcribed using reverse
transcriptase to form cDNA. Alternatively the SAGE tags can be used
to identify clones from cDNA libraries using hybridization. The
SAGE tags can also be used as primers to generate PCR products
which contain the SAGE tags. Any such method known in the art can
be used. Isolated and purified nucleic acid molecules are free of
other nucleic acid molecules with which they are found in cells.
Preferably they are also free of the genes to which they are
adjacent in the chromosome.
[0039] Nucleotide probes are typically less than full length genes
and can be labeled so that they can be used in hybridization
experiments. Such probes are typically at least 12 contiguous
nucleotides in length. Probes of the invention can comprise a SAGE
tag of Tables 1 and 2, particularly SEQ ID NOS: 15-17, 19, 21, 22,
or 30, or can comprise a different portion of a transcript or cDNA
molecule identified by such SAGE tags.
[0040] Kits can be formulated for evaluating toxicity or
carcinogenicity of test agents. The kits comprise at least 2, 3, 4,
5, 6, 7, 8, 9, or 10 probes which are complementary to the
transcripts identified by the SAGE tags of Table 1 and 2. Just as
DNA-damaging agents induce apoptosis via p53, which can be measured
by measuring the induction or repression of expression of specific
transcripts, so can other as yet unknown agents. Such agents which
cause DNA damage are likely to be toxic or carcinogenic. Thus,
human cells can be contacted with a test agent, and the levels of
one or more transcripts identified by a SAGE tag in Table 1 or 2
can be measured. If the agent causes the modulation which is caused
by the introduction of wild-type p53 or the modulation which is
caused by DNA-damaging agents in wild-type p53-containing cells,
then the agent is a suspected carcinogen or toxic agent.
[0041] Reactive Oxygen Species (ROS) production can also be used as
an indicator of p53 status and hence neoplasia. Levels of ROS can
be determined and compared between cells of a tissue which is
suspected of being neoplastic and normal cells. Elevated levels of
ROS indicate expression of p53, and low levels indicate lack of p53
expression. These levels can be measured after contacting the cells
with an agent which induces DNA damage. Alternatively a test sample
can be tested before and after treatment with DNA damaging agents.
The ability to induce ROS indicates a wild-type p53. Any method for
measuring ROS can be used, including but not limited to
carboxymethyl dichlorofluorescein diacetate and flow cytometry,
nonylacridine orange as a probe for cardiolipin, lucigenin
chemiluminescence, and intracellular glutathione.
[0042] DNA constructs which contain a reporter gene under the
transcriptional control of a PIG promoter can be used to test
agents for the ability to induce apoptosis. Such agents have
potential use as anti-neoplastic agents. One such construct
contains the PIG-3 promoter which contains the p53-binding site
CAGCTTGCCCACCCATGCTC (SEQ ID NO:1). Other PIG promoters can be used
similarly.
[0043] PIG-specific antibodies can be used in assays to determine
the status of the p53 gene in cells similar to those described
above employing SAGE tags. Proteins or polypeptides encoded by PIGs
1-7 and 9-12 (PIG proteins) can be purified by any method known in
the art or produced by recombinant DNA methods or by synthetic
chemical methods and used as immunogens, to obtain a preparation of
antibodies which specifically bind to a PIG protein, preferably to
PIG 3, 6, 7, 10, 11, or 12. The antibodies can be used to detect
wild-type PIG proteins in human tissue and fractions thereof
[0044] Preparations of polyclonal or monoclonal PIG antibodies can
be made using standard methods known in the art. The antibodies
specifically bind to epitopes present in PIG proteins. Preferably,
the PIG epitopes are not present in other human proteins.
Typically, at least 6, 8, 10, or 12 contiguous amino acids are
required to form an epitope. However, epitopes which involve
non-contiguous amino acids may require more, e.g., at least 15, 25,
or 50 amino acids. Antibodies which specifically bind to PIG
proteins provide a detection signal at least 5-, 10-, or 20-fold
higher than a detection signal provided with other proteins when
used in Western blots or other immunochemical assays. Preferably,
antibodies which specifically bind PIG proteins do not detect other
proteins in immunochemical assays and can immunoprecipitate PIG
proteins from solution.
[0045] Antibodies which specifically bind to PIG proteins,
particularly to PIG 3, 6, 7, 10, 11, or 12, can be purified by
methods well known in the art. Preferably, the antibodies are
affinity purified, by passing antiserum over a column to which a
PIG protein or polypeptide is bound. The bound antibodies can then
be eluted from the column, for example, using a buffer with a high
salt concentration.
[0046] As disclosed above, wild-type p53 is required to modulate
the level of transcripts identified in Tables 1 and 2, and the
presence of mutant p53 is an indication of neoplasia. For example,
wild-type p53 increases transcription of genes shown in Table 1 and
decreases transcription of genes shown in Table 2. The status of
the p53 gene in a tissue suspected of being neoplastic can be
determined by comparing the levels of one or more of the products
of genes whose transcription is modulated by wild-type p53 in the
suspect tissue with the level of a PIG protein in a tissue which is
normal.
[0047] Such comparisons can be made by any methods known in the
art. Preferably, antibodies which specifically bind to the protein
products of the modulated genes are used, for example in
radioimmunoassays or immunocytochemical methods, as is known in the
art. Antibodies which specifically bind to the proteins of Table 2
can be used to measure the levels of the proteins of Table 2.
Antibodies which specifically bind to PIGs 1-7 and 9-12,
particularly those which specifically bind to PIG 3, 6, 7, 10, 11,
and 12, can be used to measure the levels of PIG proteins.
[0048] The same or a lower level of a PIG protein in the suspect
tissue indicates the presence of mutant p53. Similarly, the same or
a higher level of a protein of Table 2 in the suspect tissue
indicates the presence of mutant p53. The levels of two, 3, 4, 5,
6, 7, 8, 9, or 10 or more proteins can be compared. Detection of
binding of PIG-specific antibodies to PIG proteins, or of
antibodies which specifically bind to the proteins of Table 2, can
also be used to determine if a suspect tissue contains a wild-type
or mutant p53 gene after treatment with DNA-damaging agents.
[0049] Antibodies of the invention which specifically bind to PIG
3, 6, 7, 10, 11, or 12 can be provided in kits, for evaluating
cytotoxicity or carcinogenicity of test agents, as described above.
A kit can contain one, 2, 3, 4, 5, or 6 of the antibodies of the
invention.
[0050] The above disclosure generally describes the present
invention. A more complete understanding can be obtained by
reference to the following specific examples which are provided
herein for purposes of illustration only, and are not intended to
limit the scope of the invention. The following methods were used
in the examples reported below.
Methods
[0051] Cells and RNA. All cell lines used in this study were
obtained from the American Type Culture Collection and were
cultured in McCoy's medium supplemented with 10% fetal bovine serum
(FBS). Cells were infected with recombinant adenoviruses containing
either the p53 gene or the .beta.-galactosidase gene (26) at a
multiplicity of infection of 10-100. RNA was purified from cells at
various times after infection using the MessageMaker Kit
(Gibco/BRL). Northern blot analysis was performed as described
(26).
[0052] SAGE. SAGE was performed as previously described (3, 27).
Briefly, polyadenylated RNA was converted to double-stranded cDNA
with a BRL synthesis kit using the manufacturer's protocol with the
inclusion of primer biotin-5'-T18-3'. The cDNA was cleaved with
NlaIII, and the 3'-terminal cDNA fragments were bound to
streptavidin-coated magnetic beads (Dynal). After ligation of
oligonucleotides containing recognition sites for BsmFI, the
linkered-cDNA was released from the beads by digestion with BsmFI.
The released tags were ligated to one another, concatemerized, and
cloned into the Sph I site of pZero 21.0 (Invitrogen). Colonies
were screened with PCR using M13 forward and M13 reverse primers.
PCR products containing inserts of greater than 300 bp (>20
tags) were sequenced with the TaqFS DyePrimer kit and analyzed
using a 377 ABI automated sequencer (Perkin Elmer).
[0053] Statistical analysis. 53,022 and 51,853 tags were identified
from DLD-1 cells infected with Ad-p53 and Ad-lacZ, respectively.
The two libraries were compared using the SAGE program group (3).
Corrections for tags containing linker sequences and other
potential artifacts were made as described previously (27). Of
104,875 total tags identified, 3,181 were excluded from analysis on
this basis. Monte Carlo simulations revealed that the computational
analyses had a >99% probability of detecting a transcript
expressed at an abundance of 0.00005 in either RNA sample.
[0054] cDNA clones. Cellular mRNA from Ad-p53-infected cells was
used to prepare cDNA as described for the SAGE libraries, except
that the 3' primer contained an additional M13 forward sequence
between the olio-DT tract and the biotinylated 5' residue. To
determine the sequence of the transcript from which an individual
tag was derived, this cDNA was used as a template for PCR,
employing an M13 forward primer and a primer containing the tag
sequence. In other cases, mRNA from Ad-p53 -infected cells was used
to construct a cDNA library in the ZAP Express vector (Stratagene)
and the library was screened by hybridization with oligonucleotides
corresponding to tags, as described (3). Of 14 tags identified by
SAGE as differentially expressed in p53-expressing cells, 8
corresponding genes could be identified simply by searching public
databases, particularly those including expressed sequence tags. In
5 cases, one of the two strategies described above was used to
obtain the corresponding PIG. In one of the 14 cases (PIG13), no
cDNA clone could be recovered corresponding to the tag
sequence.
[0055] Analysis of PIG3 genomic structure. An arrayed BAC library
(Research Genetics) was screened by PCR using the following primers
derived from the 5' end of the PIG3 gene:
5'-GGC-CAG-GAG-TAA-GTA-ACT-3' (SEQ ID NO:2) and
5'-GCC-CTG-GTC-TGC-CGC-GGA-3' (SEQ ID NO:3). Eco RI fragments
encompassing the PIG3 coding sequences were subcloned into pBR322
and partially sequenced to determine the intron-exon borders. A 6.1
kb Apa LI fragment whose 3' end was at a Eag I site 308 bp
downstream of the transcription start site was then cloned into a
promoterless luciferase reporter vector (FIG. 2A). This fragment
was completely sequenced by primer walking. Subclones were then
generated by restriction endonuclease digestion. Luciferase
activity was determined after co-transfection with expression
vectors encoding wt or H175R mutant p53. For in vitro p53 binding
experiments, oligonucleotides containing two copies of the
predicted p53-binding site (FIG. 2A) were subcloned into a modified
pBR322 vector, excised as a .about.260 bp restriction fragment, and
end-labeled. Immunochemical assays were performed as described
previously (28).
[0056] Flow cytometry and other assays. Cells were collected with
the aid of trypsin and incubated with CM-H2DCF-DA or NAO (Molecular
Probes, Eugene, OR) at concentrations of 10 and 0.4 .mu.M,
respectively, for 20 minutes at 37.degree. C. prior to analysis by
flow cytometry (14, 15). To determine the fraction of apoptotic
cells after various treatments, cells were stained with the
DNA-binding dye H33258 and evaluated by fluorescence microscopy or
flow cytometry as described (1). Superoxide production was assessed
with lucigenin (29). In brief, 4-5.times.10.sup.6 cells were
collected with rubber policeman and resuspended in 1 ml of Earle's
Balanced Salt Solution (Gibco BRL 14015-069, Life Technologies).
Dark-adapted lucigenin (bis-N-methulacridinium nitrate, Sigma
M8010) was added to the samples to a final concentration of 20
.mu.M. Light emission was detected using a Berthold LB 9505C
luminometer for 60 minutes at 37.degree. C. Glutathione
concentrations were measured using an assay kit purchased from
Oxford Biomedical Res. Inc. according to the manufacturer's
instructions. Caspase activation was assessed by cleavage of PARP
(polyADP-ribose polymerase). Lysates from cells infected with
Ad-p53 were Western blotted with an anti-PARP antibody, and the
cleavage fragments were quantitated by densitometry (4).
EXAMPLE 1
[0057] To evaluate the patterns of gene expression following p53
expression, we employed SAGE, a technique which allows the
quantitative evaluation of cellular mRNA populations (3). In brief,
the method revolves around short sequence "tags" (11 bp), generated
from defined positions within each mRNA molecule. Expression
patterns are deduced from the abundance of individual tags. To
induce apoptosis, the colorectal cancer line DLD-1, containing an
inactive endogenous p53 gene, was infected with a replication
defective adenovirus encoding p53 (Ad-p53). As previously shown,
DLD-1 cells are among the .about.50% of CRC lines that undergo
apoptosis in response to p53 (4). RNA was purified from cells 16
hours after infection, at least 8 hours before the onset of
morphological signs of apoptosis.
[0058] A total of 101,694 tags were analyzed, approximately half
from cells infected with Ad-p53 and half from cells infected with a
control virus (Ad-lacZ) encoding .beta.-galactosidase. These tags
corresponded to 7,202 different transcripts. Comparison of the two
SAGE libraries indicated a remarkable similarity in expression
profiles (FIG. 1A). Of the 7,202 transcripts detected, only 14
(0.19%) were expressed at levels more than 10-fold greater in
p53-expressing than in control cells; conversely, only 20
transcripts were expressed at levels less than 10-fold lower in the
p53-expressing cells.
[0059] As previous data indicated p53-mediated transcriptional
activation as the likely basis of p53 action (5), we concentrated
on the 14 tags appearing at higher levels in the p53-expressing
cells. The mRNA transcripts corresponding to 13 of these tags were
successfully identified (Table 1). In each case, the induction was
confirmed by Northern blot analysis (examples in FIG. 1B). Only two
of these genes (called PIGs, for p53-induced genes) had been
implicated as targets of p53-transcriptional activation (1, 2, 5,
6) and seven had not previously been described at all. Other genes
previously implicated in p53-mediated responses were induced to
lower levels (e.g., MDM2, thrombospondin) or not at all (e.g., bax
and cyclin G1) in the human CRC cells studied here (4).
EXAMPLE 2
[0060] PIGs were induced at relatively short times after p53
expression, at least 12 hours prior to any morphological or
biochemical signs of apoptosis (FIG. 1B). This time course
suggested that PIGs were directly induced by the transcriptional
activation properties of p53. To formally test this conjecture in a
representative case, we evaluated the genomic structure and
sequence of PIG3. By screening a bacterial artificial chromosome
(BAC) library, a genomic clone was identified that contained all
PIG3 coding sequences. The gene was localized to chromosome 2p (see
Methods), and the intron-exon structure and sequence of the
promoter region were determined.
[0061] A 6.1 kb ApaLI fragment of genomic DNA containing the
presumptive promoter was then cloned upstream of a luciferase
reporter gene (FIG. 2A). The resulting construct was transfected
into three different human cell lines together with wild type (wt)
or mutant p53.
[0062] As shown in FIG. 2B, wt p53 induced substantial activity
through the PIG3 promoter in all three lines. Mutant p53 had no
transcriptional activation capacity. Analysis of a truncated
promoter showed that the p53-responsive elements lay within a
fragment containing only 862 bp of sequence upstream of the PIG3
transcription start site (FIG. 2A). Determination of the sequence
of this 6.1 kb Apa L1 fragment revealed a single 20 bp sequence
predicted to bind p53, located at 308 nt upstream of the
transcription start site p53. A DNA fragment containing two copies
of this sequence, but not a derivative of this fragment altered at
critical residues, was found to bind strongly to p53 in vitro (FIG.
2C).
EXAMPLE 3
[0063] As a further test of the p53-dependency of PIG3 induction,
we determined whether PIG3 could be induced by endogenous p53
rather than through the exogenous Ad-p53 source. Six CRC cancer
cell lines were each treated with adriamycin, a DNA-damaging and
apoptotic-inducing agent known to increase endogenous p53 levels.
PIG3, like p21, was found to be strongly induced in the three lines
with wild-type p53 genes, but not in the three lines with mutant
p53 genes.
EXAMPLE 4
[0064] The sequences of the PIGs provided important clues to their
potential functions (Table 1). In particular, several were
predicted to encode proteins with activities related to the redox
status of cells. PIG12 is a novel member of the microsomal
glutathione transferase family of genes (FIG. 3A). PIG8 is the
human homologue of a mouse gene (Ei24) whose expression is induced
in a p53-dependent manner by etoposide, a quinone known to generate
reactive oxygen species (ROS) (6) (FIG. 3C). PIG6 is a homolog of
proline oxidoreductase (FIG. 3D), a mitochondrial enzyme that
catalyzes the first step in the conversion of proline to glutamate
(7). Glutamate is one of the three amino acids required for
formation of glutathione, a major regulator of cellular redox
status. The p21 gene, which can also be considered a PIG, can be
induced by ROS, independently of p53 (8). PIG4 encodes a serum
amyloid protein that can be induced by oxidative stress (9). PIG1
belongs to the galectin family, members of which can stimulate
superoxide production (10). PIG7 has been shown to be induced by
TNF-.beta., a known inducer of oxidative stress. PIG3 is a novel
gene that is highly related to TED2, a plant NADPH oxidoreductase
(11) (FIG. 3B). Interestingly, TED2 is one of the few genes
implicated in the apoptotic process necessary for the formation of
plant meristems (11). The closest relative of PIG3 in mammals is an
NADPH-quinone oxidoreductase which has been shown to be a potent
generator of ROS (12).
[0065] Previous studies have shown that ROS are powerful inducers
of apoptosis (13). The SAGE-based characterization of p53-induced
genes suggested that p53 might induce apoptosis by stimulating the
production of ROS. To test this hypothesis, the production of ROS
was measured in p53-expressing cells using carboxy-methyl
dichlorofluorescein (DCF-diacetate (CM-DCF-DA) and flow cytometry
(14). This analysis showed that ROS were induced following Ad-p53
infection and that ROS continued to increase as apoptosis
progressed (FIG. 4A). The magnitude of the increase in ROS, as
assessed by DCF fluorescence, was similar in p53-expressing cells
to that observed in cells treated with the powerful oxidant
menadione (FIG. 4A). No change in DCF fluorescence was observed
following infection with a control adenovirus (FIG. 4A).
[0066] As an assay for the functional consequences of ROS
production, we examined the cellular content of cardiolipin, a
major component of the mitochondrial membrane which is especially
sensitive to cellular oxidation (15). Using nonyl-acridine orange
(NAO) as a probe, cardiolipin was found to decrease soon after
p53-induced ROS was detected (FIG. 4A), demonstrating significant
injury to a major mitochondrial component.
EXAMPLE 5
[0067] To determine the specificity of PIG expression for the
p53-dependent apoptotic process, we performed experiments with
other inducers of ROS or apoptosis. We found that PIGs were not
expressed simply as a result of ROS production, as none were
induced following treatment with menadione and only p21 was induced
by hydrogen peroxide in DLD-1 cells. Similarly, the specificity of
PIG induction for p53-dependent apoptosis was confirmed by the
demonstration that other inducers of apoptosis (indomethacin or
ceramide) did not result in the expression of any PIG, despite
extensive cell death.
EXAMPLE 6
[0068] To clarify the relationship between p53 expression, PIG
activation, ROS production, and apoptosis, we carried out more
detailed time course experiments. PIG induction began within six
hours after Ad-53 infection (FIG. 1B and FIG. 4B), while
intracellular ROS production, as assessed with lucigenin
chemiluminescence, could first be observed at 18 hours (FIG. 4B).
This ROS production led to oxidative stress, as evidenced by a
48+/-12% decrease in intracellular glutathione concentration at 21
hours. Mitochondrial lipid degradation (NAO) was not observed until
three to six hours after the onset of a measurable ROS increase and
was accompanied by morphologic (chromatin condensation and
fragmentation) and biochemical (caspase-mediated degradation of
PARP) signs of apoptosis (FIG. 4B). These observations are
consistent with previous studies showing that mitochondrial damage
is rapidly followed by classic signs of programmed cell death
(13).
[0069] The time courses illustrated in FIG. 4B suggest a cascade
wherein p53 transcriptionally induces redox-controlling genes
resulting in the production of ROS, in turn leading to oxidative
damage to mitochondria and apoptosis. To determine whether these
steps were causally associated, we inhibited each step with
specific pharmacologic agents and determined the effect of this
inhibition on other components of the pathway.
[0070] First, cells were treated with the transcriptional inhibitor
5,6-dichlorobenimidizole riboside (DRB) at 8 hours following Ad-p53
infection (16). Though p53 expression was already near maximal at
this time, DRB was found to block apoptosis at 24 hours by 83+/-3%
as well as to inhibit the expression of PIGs. The translational
inhibitor cycloheximide, when given up to 8 hours following Ad-p53
infection, was found to similarly block apoptosis (by 79% at 24
hours). Thus both transcription and translation were required for
p53-induced apoptosis in CRC cells, as observed in some other
systems (2, 5) and as expected for classic programmed cell death
(2, 5).
[0071] Second, p53-expressing cells were treated with pyrrolidine
dithiocarbamate (PDTC), an anti-oxidant which has been shown to
block ROS-associated apoptosis (17). PDTC was indeed able to block
the apoptosis elicited by p53. However, PDTC inhibits many enzymes,
and its specificity is questionable (17). We therefore treated
cells with diphenyleneiodonium chloride (DPI), a specific inhibitor
of flavin-dependent oxidoreductases which has been used to block
production of ROS in a variety of systems (18). Cells were treated
with DPI 12 hours after Ad-p53 infection, when PIG production was
already underway. PIG3 expression, apoptosis, and ROS production
were measured 12 hours later. DPI (25 .mu.M) did not inhibit PIG3
production but did inhibit ROS production by 71-85% and inhibited
apoptosis by 73-77% in three independent experiments.
[0072] Finally, we treated cells with bongkrekic acid (BA), a
specific inhibitor of mitochondrial ATP translocase which can block
the mitochondrial permeability transition pore opening thought to
be required for ROS-dependent forms of apoptosis (13). When cells
were treated 12 hours after Ad-p53 infection, BA was found to
inhibit neither PIG3 expression nor ROS production, but inhibited
subsequent apoptosis by 86-93%. BA was non-toxic at the dose used
(100 .mu.M). While BA inhibited the p53-apoptotic process dependent
on ROS production, it had no effect on the p53-mediated growth
arrest dependent on p21 as assessed by flow cytometry.
[0073] The gene expression profile, time courses, and pharmacologic
inhibition studies reported above strongly support a three step
model underlying p53's induction of apoptosis. We propose that p53
transcriptionally activates a specific subset of genes, including
oxidoreductases, long before any morphological or biochemical
evidence of cell death (Table 1 and FIG. 4B). The proteins encoded
by these genes then collectively increase the content of ROS, which
in turn damage mitochondria. Leakage of calcium and proteinaceous
components from damaged mitochondria then stimulate the caspases
that are ubiquitously activated during the apoptotic process.
(19-22).
[0074] Data from several experimental systems are consistent with
this model. For example, apoptosis induced by irradiation, which is
dependent on p53 in certain cell types, has been suggested to
proceed through a process involving ROS and mitochondrial damage
(23). Additionally, an SV40 large T antigen mutant, which binds p53
only at the permissive temperature, was shown to induce apoptosis
at the non-permissive temperature through a ROS-related mechanism
(24). More recently, it was shown that p53-induced apoptosis in
smooth muscle cells is ROS-dependent (25). Though the basis for ROS
production and the involvement of mitochondria were not
investigated in these previous studies, they suggest that the
events we observed in CRC cells are unlikely to be cell-type or
species specific and may often underlie p53-associated apoptotic
processes. The fact that one of the PIGs is highly related to Ted2,
an oxidoreductase implicated in plant cell apoptosis (11), and that
apoptosis in plants may also proceed through a ROS-directed pathway
(11), adds further interest to this model.
[0075] Though observations by us and others are consistent with
this model, they raise several unanswered questions. For example,
we do not yet know which of the PIGs, are primarily responsible for
the induction of ROS. We suspect that their combination, rather
than any single one, is necessary for ROS generation. This
conjecture is supported by preliminary experiments which
demonstrate that PIG3 alone does not induce apoptosis when
overexpressed. Though we have concentrated on the most highly
induced PIGs, the SAGE analysis revealed at least 26 other genes
which were induced by p53 to significant but lower levels than p21
and PIG1 -PIG13. Some of these genes may play a role in redox
regulation.
[0076] It is also not known why some cells enter into apoptosis
following p53 expression while others undergo a prolonged growth
arrest (4). The possibility that PIGs are only induced in the
former has been excluded by examination of PIG expression in such
lines; most PIGs were induced by p53 in each of ten CRC lines
tested, regardless of whether the cells underwent apoptosis or
growth arrest. A more likely possibility is that different cells
have different capacities to cope with generators of oxidative
stress and that cells with a low capacity succumb to apoptosis.
This possibility is supported by numerous studies which show that
the response to ROS varies significantly with cell type and growth
conditions (13). Hopefully, the experiments and genes reported here
will open a new window into the p53 apoptotic process that will
facilitate inquiry into these issues.
References
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necessary for the p53-mediated G1 arrest in human cancer cells.
Cancer Res. 55, 5187-5190 (1995).
[0078] 2. Oren, M. Relationship of p53 to the control of apoptotic
cell death. Semin. Cancer Biol. 5, 221-227 (1994).
[0079] 3. Velculescu, V. E., Zhang, L., Vogelstein, B. &
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1TABLE 1.sup.1 SEQ ID ACCES- NO: SAGE TAG SION DESCRIPTION 4
CCCGCCTCTT D38112 mitochondrial 16S rRNA 4 CCCGCCTCTT T10098 seq816
human cDNA clone b4HB3MA-COT8-HAP-Ft 4 CCCGCCTCTT T10208 seq907
human cDNA clone b4HB3MA-COT8-HAP-Ft 4 CCCGCCTCTT T26521 AB291H2F
human cDNA clone LLAB291H2 3'. 4 CCCGCCTCTT W27281 28g3 human
retina cDNA randomly primed sublibrary 4 CCCGCCTCTT T17062 NIB250
human cDNA 3'end similar to human mitochondrial mRNA 5 AATCTGCGCC
M13755 human interferon-induced 17-kDa/15-kDa protein mRNA* 5
AATCTGCGCC M21786 human interferon-induced 15-Kd protein (ISG)
gene* 6 GTGACCACGG K03432 18S rRNA 7 TTTCCTCTCA X57348 human mRNA
(clone 9112). 8 TGCCTGCACC X61683 human gene for cystatin C exon 3
8 TGCCTGCACC X05607 human mRNA for cysteine proteinase inhibitor
precursor 9 TCACCCACAC R01174 ye77b03.s1 human cDNA clone 123725 3'
9 TCACCCACAC N95827 zb66e05.s1 human cDNA clone 308576 3' 10
TAAACCTGCT U06643 PIG1, human keratinocyte lectin 14 (HKL-14) mRNA*
10 TAAACCTGCT L07769 PIG1, human galectin-7 mRNA, complete CDs.* 11
CCCAAGCTAG X54079 human mRNA for heat shock protein HSP27 12
AGCCCGCCGC AF001294 human IPL (IPL) mRNA 12 AGCCCGCCGC N29541
yw89f12.s1 human cDNA clone 259439 3' 13 GACATCAAGT Y00503 human
mRNA for keratin 19. 13 GACATCAAGT J03607 human 40-kDa keratin
inter- mediate filament precursor 14 TGTCCTGGTT U03106 human
wild-type p53 activated fragment-1 (WAF1)* 14 TGTCCTGGTT U09579
human melanoma differentiation associated (mda-6)* 14 TGTCCTGGTT
L26165 human DNA synthesis inhibitor mRNA, complete CDs.* 14
TGTCCTGGTT L25610 human cyclin-dependent kinase inhibitor mRNA* 15
AGCTCACTCC AF010314 PIG10, homologous to none* 16 AGGCTGTCCA
AF010315 PIG11, homologous to none* 17 TGAGTCCCTG AF010316 PIG12,
microsomal GST homolog* 18 CCCTCCTCCG F19653 PIG2, human EST
sequence (011-X4-27) from skeletal muscle* 18 CCCTCCTCCG Z49878
PIG2, Guanidinoacetate N-methyltransferase* 19 GAGGCCAACA AF010309
PIG3, quinone oxidoreductase homologue 19 GAGGCCAACA H42923
yo10e11.s1 human cDNA clone 177548 3'. 19 GAGGCCAACA W07320
za94c09.r1 Soares fetal lung NbHL19W human 20 TGGGGCCGCA U33271
PIG5, human normal keratinocyte mRNA, clone B4, partial* 21
TCCTTGGACC AF010311 PIG6, homologous to Drosophila PUT1, partial*
22 CTGGGCCTGA AF010312 PIG7* 23 AGCTGGTTTCC AF010313 PIG8, human
homolog of mouse EI24* 24 GAGGTGCCGG J00277 human (genomic clones
J00206 lambda-[SK2-T2, HS578T]; J00276 cDNA clones RS-[3,4, 6])
K00954 c-Ha-ras1 proto-oncogene, complete coding sequence 24
GAGGTGCCGG W25059 zb67e08.r1 Soares fetal lung NbHL19W human 25
ACAACGTCCA T16546 NIB1466 human cDNA 3'end 25 ACAACGTCCA D85815
human DNA for rhoHP1 26 GTGCGGAGGA X56653 PIG4, human SAA2 alpha
gene, exon 3 and exon 4* 26 GTGCGGAGGA X51439 PIG4, human mRNA for
serum amyloid A (SAA) protein partial* 26 GTGCGGAGGA X51441 PIG4,
human mRNA for serum amyloid A (SAA) protein partial* 26 GTGCGGAGGA
X51442 PIG4, human mRNA for serum amyloid A (SAA) protein partial*
26 GTGCGGAGGA X51445 PIG4, human mRNA for serum amyloid A (SAA)
protein partial* 26 GTGCGGAGGA M23698 PIG4, human serum amyloid A1
(SAA1) mRNA, complete* 26 GTGCGGAGGA M23699 PIG4, human serum
amyloid A2-alpha (SAA2) mRNA* 26 GTGCGGAGGA M26152 PIG4, human
serum amyloid A (SAA) mRNA, complete* 26 GTGCGGAGGA M10906 PIG4,
human serum amyloid A (SAA) mRNA* 26 GTGCGGAGGA H45773 PIG4,
yp23c09.r1 human cDNA clone 188272 5' simil* 26 GTGCGGAGGA T28677
PIG4, EST51616 human cDNA 5' end similar to serum* 27 CGTCCCGGAG
U33822 PIG9, human tax1-binding protein TXBP181 mRNA, complete* 27
CGTCCCGGAG D52048 PIG9, human fetal brain cDNA 5'-end GEN-064D09*
28 GTGCTCATTC AB000584 human mRNA for TGF-beta superfamily protein
29 GCTGACTCAG M99425 human thrombospondin mRNA, 3' end. 30
AGATGCTGCA PIG13 31 CTCAGACAGT AA046881 EST homologous to 40S
ribosomal protein 32 TCCGGCCGCG NO MATCH 33 AGCCACTGCA Alu repeat
34 GCTTTTAAGG L06498 human ribosomal protein S20 (RPS20) mRNA 35
GGGCCAATAA D29121 human keratinocyte cDNA, clone 142 35 GGGCCAATAA
AA178918 human cDNA clone 612020 36 AAGGGCTCTT M20560 human
lipocortin-III mRNA 36 AAGGGCTCTT M63310 human 1,2-cyclic-inositol-
phosphate phosphodiesterase (ANX3) mRNA .sup.1Gene assignments were
based on the following list of GenBank sequences (GenBank Release
94). In each case, tentative assignments were based on the
identification of a 10 bp SAGE tag adjacent to a NlaIII site. The
final assignment was further refined by using an 11 bp SAGE tag and
elimination of non 3' end NlaIII sites and genomic sequences. In
some cases, the assignment was confirmed by Northern blot analysis
as indicated by the asterisk following #the description. In other
cases, a single assignment could not be made, and more than one
gene is listed.
[0106]
2TABLE 2.sup.2 SEQ ACCES- ID SION NO: SAGE TAG NUMBER DESCRIPTION
37 GTAAGTGTAC J01415 12S rRNA 38 TGTACCTGTA K00558 human
alpha-tubulin mRNA 39 AACGACCTCG V00599 human mRNA fragment
encoding beta-tubulin 40 AGTTTGTTAG M33011 human
carcinoma-associated antigen GA733-2 mRNA 41 GACTCGCCCA M98326
human P1-Cdc46 mRNA 42 GGGCCAATAA D29121 human keratinocyte cDNA,
clone 142 42 GGGCCAATAA AA178918 human cDNA clone 612020 43
GGGTTTTTAT L28809 human dbpB-like protein mRNA 44 AGAAATACCA
AA455253 human cDNA clone 814816 3' 45 TACCATCAAT J02642 human
glyceraldehyde 3-phos- phate dehydrogenase mRNA 46 GGATTGTCTG
M34081 human small nuclear ribonucleo- protein particle SmB mRNA 47
TACTAGTCCT X15183 human mRNA for 90-kDa heat-shock protein 48
AATATTGAGA U62962 human Int-6 mRNA, complete CDs 49 GAGGGAGTTT
U14968 human ribosomal protein L27a mRNA 50 AAGGGCGCGG M20560 human
lipocortin-III mRNA 50 AAGGGCGCGG M63310 human 1,2-cyclic-inositol-
phosphate phosphodiesterase (ANX3) mRNA 51 TTCACAAAGG X61970 human
mRNA for macropain subunit zeta 52 CTGCACTTAC D28480 human mRNA for
hMCM2 53 GATCCCAACT V00594 human mRNA for metallothionein from
cadmium-treated cells 54 GGGAAGCAGA X77770 mitochondrial mRNA 55
GCTTTCTCAC K00365 human mitochondrial Ser-tRNA 56 TTCATTATAA M26708
human prothymosin alpha mRNA 57 TAAGGAGCTG X77770 human RPS26 mRNA
58 TGAGGGAATA M10036 human triosephosphate isomerase mRNA 59
GGGATGGCAG M98326 human transfer valyl-tRNA synthetase mRNA 60
TCTTCTCTG NO MATCH 61 GCACCTTATT NO MATCH 62 ACTTTAAACT NO MATCH 63
CCATTCCACT NO MATCH 64 TCAAATGCAT M16342 human small nuclear
ribonucleo- protein (hnRNP) C protein mRNA 65 GAAAAATGGT X61156
human mRNA for laminin- binding protein 66 ACTAACACCC U18810 human
PACAP type-e/VIP type-2 receptor mRNA 67 TTGGGGTTTC M12937 human
ferritin heavy subunit mRNA .sup.2Gene assignments were based on
the following list of GenBank sequences (GenBank Release 94). In
each case, tentative assignments were based on the identification
of a 10 bp SAGE tag adjacent to a NlaIII site. The final assignment
was further refined by using an 11 bp SAGE tag and elimination of
non 3' end NlaIII sites and genomic sequences.
[0107]
Sequence CWU 1
1
87 1 20 DNA Homo sapiens 1 cagcttgccc acccatgctc 20 2 18 DNA Homo
sapiens 2 ggccaggagt aagtaact 18 3 18 DNA Homo sapiens 3 gccctggtct
gccgcgga 18 4 10 DNA Homo sapiens 4 cccgcctctt 10 5 10 DNA Homo
sapiens 5 aatctgcgcc 10 6 10 DNA Homo sapiens 6 gtgaccacgg 10 7 10
DNA Homo sapiens 7 tttcctctca 10 8 10 DNA Homo sapiens 8 tgcctgcacc
10 9 10 DNA Homo sapiens 9 tcacccacac 10 10 10 DNA Homo sapiens 10
taaacctgct 10 11 10 DNA Homo sapiens 11 cccaagctag 10 12 10 DNA
Homo sapiens 12 agcccgccgc 10 13 10 DNA Homo sapiens 13 gacatcaagt
10 14 10 DNA Homo sapiens 14 tgtcctggtt 10 15 10 DNA Homo sapiens
15 agctcactcc 10 16 10 DNA Homo sapiens 16 aggctgtcca 10 17 10 DNA
Homo sapiens 17 tgagtccctg 10 18 10 DNA Homo sapiens 18 ccctcctccg
10 19 10 DNA Homo sapiens 19 gaggccaaca 10 20 10 DNA Homo sapiens
20 tggggccgca 10 21 10 DNA Homo sapiens 21 tccttggacc 10 22 10 DNA
Homo sapiens 22 ctgggcctga 10 23 11 DNA Homo sapiens 23 agctggtttc
c 11 24 10 DNA Homo sapiens 24 gaggtgccgg 10 25 10 DNA Homo sapiens
25 acaacgtcca 10 26 10 DNA Homo sapiens 26 gtgcggagga 10 27 10 DNA
Homo sapiens 27 cgtcccggag 10 28 10 DNA Homo sapiens 28 gtgctcattc
10 29 10 DNA Homo sapiens 29 gctgactcag 10 30 10 DNA Homo sapiens
30 agatgctgca 10 31 10 DNA Homo sapiens 31 ctcagacagt 10 32 10 DNA
Homo sapiens 32 tccggccgcg 10 33 10 DNA Homo sapiens 33 agccactgca
10 34 10 DNA Homo sapiens 34 gcttttaagg 10 35 10 DNA Homo sapiens
35 gggccaataa 10 36 10 DNA Homo sapiens 36 aagggctctt 10 37 10 DNA
Homo sapiens 37 gtaagtgtac 10 38 10 DNA Homo sapiens 38 tgtacctgta
10 39 10 DNA Homo sapiens 39 aacgacctcg 10 40 10 DNA Homo sapiens
40 agtttgttag 10 41 10 DNA Homo sapiens 41 gactcgccca 10 42 10 DNA
Homo sapiens 42 gggccaataa 10 43 10 DNA Homo sapiens 43 gggtttttat
10 44 10 DNA Homo sapiens 44 agaaatacca 10 45 10 DNA Homo sapiens
45 taccatcaat 10 46 10 DNA Homo sapiens 46 ggattgtctg 10 47 10 DNA
Homo sapiens 47 tactagtcct 10 48 10 DNA Homo sapiens 48 aatattgaga
10 49 10 DNA Homo sapiens 49 gagggagttt 10 50 10 DNA Homo sapiens
50 aagggcgcgg 10 51 10 DNA Homo sapiens 51 ttcacaaagg 10 52 10 DNA
Homo sapiens 52 ctgcacttac 10 53 10 DNA Homo sapiens 53 gatcccaact
10 54 10 DNA Homo sapiens 54 gggaagcaga 10 55 10 DNA Homo sapiens
55 gctttctcac 10 56 10 DNA Homo sapiens 56 ttcattataa 10 57 10 DNA
Homo sapiens 57 taaggagctg 10 58 10 DNA Homo sapiens 58 tgagggaata
10 59 10 DNA Homo sapiens 59 gggatggcag 10 60 9 DNA Homo sapiens 60
tcttctctg 9 61 10 DNA Homo sapiens 61 gcaccttatt 10 62 10 DNA Homo
sapiens 62 actttaaact 10 63 10 DNA Homo sapiens 63 ccattccact 10 64
10 DNA Homo sapiens 64 tcaaatgcat 10 65 10 DNA Homo sapiens 65
gaaaaatggt 10 66 10 DNA Homo sapiens 66 actaacaccc 10 67 10 DNA
Homo sapiens 67 ttggggtttc 10 68 498 DNA Homo sapiens 68 ttaaagcaaa
gaattccccg gtcccagcca tgtccaacgt cccccacaag tcctcgctgc 60
ccgagggcat ccgccctggc acggtgctga gaattcgcgg cttggttcct cccaatgcca
120 gcaggttcca tgtaaacctg ctgtgcgggg aggagcaggg ctccgatgcc
gccctgcatt 180 tcaacccccg gctggacacg tcggaggtgg tcttcaacag
caaggagcaa ggctcctggg 240 gccgcgagga gcgcgggccg ggcgttcctt
tccagcgcgg gcagcccttc gaggtgctca 300 tcatcgcgtc agacgacggc
ttcaaggccg tggttgggga cgcccagtac caccacttcc 360 gccaccgcct
gccgctggcg cgcgtgcgcc tggtggaggt gggcggggac gtgcagctgg 420
actccgtgag gatcttctga gcagaagccc aggcggcccg gggccttggc tggcaaataa
480 agcgttagcc cgcagcgc 498 69 993 DNA Homo sapiens 69 cggcggcgcg
cgatcgaggt cgggtcgccg tccagcctgc agcatgagcg cccccagcgc 60
gacccccatc ttcgcgcccg gcgagaactg cagccccgcg tggggggcgg cgcccgcggc
120 ctacgacgca gcggacacgc acctgcgcat cctgggcaag ccggtgatgg
agcgctggga 180 gaccccctat atgcacgcgc tggccgccgc cgcctcctcc
aaagggggcc gggtcctgga 240 ggtgggcttt ggcatggcca tcgcagcgtc
aaaggtgcag gaggcgccca ttgatgagca 300 ttggatcatc gagtgcaatg
acggcgtctt ccagcggctc cgggactggg ccccacggca 360 gacacacaag
gtcatcccct tgaaaggcct gtgggaggat gtggcaccca ccctgcctga 420
cggtcacttt gatgggatcc tgtacgacac gtacccactc tcggaggaga cctggcacac
480 acaccagttc aacttcatca agaaccacgc ctttcgcctg ctgaagccgg
ggggcgtcct 540 cacctactgc aacctcacct cctgggggga gctgatgaag
tccaagtact cagacatcac 600 catcatgttt gaggagacgc aggtgcccgc
gctgctggag gccggcttcc ggagggagaa 660 catccgtacg gaggtgatgg
cgctggtccc accggccgac tgccgctact acgccttccc 720 acagatgatc
acgcccctgg tgaccaaagg ctgagccccc accccggccc ggccacaccc 780
atgccctccg ccgtgccttc ctggccggga gtccagggtg tcgcaccagc cctgggctga
840 tcccagctgt gtgtcaccag aagctttccc ggcttctctg tgaggggtcc
caccagccca 900 gggctgatcc cagctgtgtg tcaccagcag ctttcccagc
ttgctctgtg agggtcactg 960 ctgcccactg cagggtgccc tgaggtgaag ccg 993
70 1670 DNA Homo sapiens 70 ccagccgtcc attccggtgg aggcagaggc
agtcctgggg ctctggggct cgggctttgt 60 caccgggacc cgcagagcca
gaaccactcg gcgccgctgg tgcatgggag gggagccggg 120 ccaggagtaa
gtaactcata cgggcgccgg ggacccgggt cggctggggg cttccaactc 180
agagggagtg tgatttgcct gatcctcttc ggcgttgtcc tgctctgccg catccagccc
240 tgtaccgcca tcccacttcc cgccgttccc atctgtgttc cgggtgggat
cggtctggag 300 gcggccgagg acttcccagg caggagctcg gggcggaggc
gggtccgcgg cagaccaggg 360 cagcgaggcg ctggccggca gggggcgctg
cggtgccagc ctgaggctgg ctgctccgcg 420 aggatacagc ggcccctgcc
ctgtcctgtc ctgccctgcc ctgtcctgtc ctgccctgcc 480 ctgccctgtc
ctgtcctgcc ctgccctgcc ctgtgtcctc agacaatatg ttagccgtgc 540
actttgacaa gccgggagga ccggaaaacc tctacgtgaa ggaggtggcc aagccgagcc
600 cgggggaggg tgaagtcctc ctgaaggtgg cggccagcgc cctgaaccgg
gcggacttaa 660 tgcagagaca aggccagtat gacccacctc caggagccag
caacattttg ggacttgagg 720 catctggaca tgtggcagag ctggggcctg
gctgccaggg acactggaag atcggggaca 780 cagccatggc tctgctcccc
ggtgggggcc aggctcagta cgtcactgtc cccgaagggc 840 tcctcatgcc
tatcccagag ggattgaccc tgacccaggc tgcagccatc ccagaggcct 900
ggctcaccgc cttccagctg ttacatcttg tgggaaatgt tcaggctgga gactatgtgc
960 taatccatgc aggactgagt ggtgtgggca cagctgctat ccaactcacc
cggatggctg 1020 gagctattcc tctggtcaca gctggctccc agaagaagct
tcaaatggca gaaaagcttg 1080 gagcagctgc tggattcaat tacaaaaaag
aggatttctc tgaagcaacg ctgaaattca 1140 ccaaaggtgc tggagttaat
cttattctag actgcatagg cggatcctac tgggagaaga 1200 acgtcaactg
cctggctctt gatggtcgat gggttctcta tggtctgatg ggaggaggtg 1260
acatcaatgg gcccctgttt tcaaagctac tttttaagcg aggaagtctg atcaccagtt
1320 tgctgaggtc tagggacaat aagtacaagc aaatgctggt gaatgctttc
acggagcaaa 1380 ttctgcctca cttctccacg gagggccccc aacgtctgct
gccggttctg gacagaatct 1440 acccagtgac cgaaatccag gaggcccata
gtacatggag gccaacaaga acataggcaa 1500 gatcgtcctg gaactgcccc
agtgaaggag gatgggggca ggacaggacg cggccacccc 1560 aggcctttcc
agagcaaacc tggagaagat tcacaataga caggccaaga aacccggtgc 1620
ttcctccaga gccgtttaaa gctgatatga ggaaataaag agtgaactgg 1670 71 526
DNA Homo sapiens 71 cagctacagc acagatcagc accatgaagc ttctcacggg
cctggttttc tgctccttgg 60 tcctgagtgt cagcagccga agcttctttt
cgttccttgg cgaggctttt gatggggctc 120 gggacatgtg gagagcctac
tctgacatga gagaagccaa ttacatcggc tcagacaaat 180 acttccatgc
tcgggggaac tatgatgctg ccaaaagggg acctgggggt gcctgggccg 240
cagaagtgat cagcaatgcc agagagaata tccagagact cacaggccat ggtgcggagg
300 actcgctggc cgatcaggct gccaataaat ggggcaggag tggcagagac
cccaatcact 360 tccgacctgc tggcctgcct gagaaatact gagcttcctc
ttcactctgc tctcaggaga 420 cctggctatg agccctcggg gcagggattc
aaagttagtg aggtctatgt ccagagaagc 480 tgagatatgg catataatag
gcatctaata aatgcttaag aggtgg 526 72 842 DNA Homo sapiens 72
gcctcaaggg ctacgtcaac cacagcctgt ccgtcttcca caccaaggac ttccaggacc
60 ctgatgggat tgagggctca gaaaacgtga ctctgtgcag atacagggac
taccgcaatc 120 ccccgattac aacttctccg agcagttctg gttcctcctg
gccatccgcc tggccttcgt 180 catcctcttt gagcacgtgg ccttgtgcat
caagctcatc gccgcctggt tcgtgcccga 240 catccctcag tcggtgaaga
acaaggttct ggaggtgaag taccagaggc tgcgtgagaa 300 gatgtggatg
gaaggcagag gctgggtggg gtgggggctg gctctcggcc cccaatgcct 360
gcccatccca ccccagcatc catcttcagt gccaggagca cagacgtgta gggccagagc
420 ccgtccagag gccaccagga gctgagacag tgccaccacc agcacctccc
acaaacccac 480 cctgtgcgtg ttgaggggtg ctgtgagaag gctgtgccca
tgtggggccg caggaatccc 540 ctgtatgttc agggctgtga gctgccaccc
tattccgcct gctccgtctt tgtggggctc 600 tcaggcttgg cacagccctg
acttgaactc tgggtgagcc tgggcaccca cagaactggg 660 agtgagggct
cctcaggcag ccacaaggca ggaaaactgg cgcaaatttc ctgggcctcc 720
ctctgacttc tgggcgccag atcctgccgt gccccctacc tggctgttgg gggtgtcctg
780 agcccacctc gctggcctgt tcccttcagc caacccgttt ctgcagtaaa
attaagcctg 840 tc 842 73 901 DNA Homo sapiens 73 ggcgcatacc
tggcccagga gcgagcccgt gcgcagatcg gctatgagga ccccatcaac 60
cccacgtacg aggccaccaa cgccatgtac cacaggtgcc tggactacgt gttggaggag
120 ctgaagcaca acgccaaggc caaggtgatg gtggcctccc acaatgagga
cacagtgcgc 180 ttcgcactgc gcaggatgga ggagctgggc ctgcatcctg
ctgaccacca ggtgtacttt 240 ggacagctgc taggcatgtg tgaccagatc
agcttcccgc tgggccacgg ctggctaccc 300 cgtgtacaag tacgtgccct
atggccccgt gatggaggtg ctgccctact tgtccccgcc 360 gtgccctgga
agaacagcag cctcatgaag ggcacccatt cgggagcggc actggctgtg 420
gctggagctc ttgaagcggc tccgaactgg caacctcttc catcgccctg cctagcaccc
480 gccagcacac cctctagcct tccagcaccc cccgccccct gctccaggcc
attcaaccaa 540 caagctgcaa gccaaacccc aatccttcaa cacagattca
ccttttttca ccccaccact 600 ttgcagagct tgcttggagg tgaggtcagg
tgcctcccag cccttgccca gagtatgggc 660 actcaggtgt gggccgaacc
tgatacctgc ctgggacagc cactggaaac ttttgggaac 720 tctcctctga
aatgtgtggg cccaaggccc ccacctctgt gacccccatg tccttggacc 780
tagaggattg tccaccttct gccaaggcca gcccacacag cccgagcccc ttggggagca
840 gtggccgggc tggggaggcc tgcctggtca ataaaccact gttcctgcaa
aaaaaaaaaa 900 a 901 74 1677 DNA Homo sapiens 74 cacgcgcagc
atagcagagt cgacactaga ggcatccaaa gaataccggc acgagcaggc 60
ggcgcgggcg gcggttaaaa tgtcggttcc aggaccttac caggcggcca ctgggccttc
120 ctccgcacca tccgcacctc catcctatga agagacagtg gctgttaaca
gttattaccc 180 cactcctcca gctcccatgc ctgggccaac tacggggctt
gtgacggggc ctgatgggaa 240 gggcatgaat cctccttcgt attataccca
gccagcgccc atccccaata acaatccaat 300 taccgtgcag acggtctacg
tgcagcaccc catcaccttt ttggaccgcc ctatccaaat 360 gtgttgtcct
tcctgcaaca agatgatcgt gagtcagctg tcctataacg ccggtgctct 420
gacctggctg tcctgcggga gcctgtgcct gctgggggtg catagcggcc tgctgcttca
480 tccccttctg cgtggatgcc ctgcaggacg tggaccatta ctgtcccaac
tgcagagctc 540 tcctgggcac ctacaagcgt ttgtaggact cagccagacg
tggagggagc cgggtgccgc 600 aggaagtcct ttccacctct catccagctt
cacgcctggt ggaggttctg ccctggtggt 660 ctcacctctc cagggggccc
accttcatgt cttcttttgg ggggaatacg tcgcaaaact 720 aacaaatctc
caaaccccag aaattgctgc ttggagtcgt gcataggact tgcaaagaca 780
ttccccttga gtgtcagttc cacggtttcc tgcctccctg agaccctgag tcctgccatc
840 taactgttga tcattgccct atccgaatat tttcctgtcg accccgggcc
accagtggct 900 cttttttcct gcttccatgg gcctttctgg tggcagtctc
aaactgagga agccacagtt 960 gcctcatttt tgaggctgtt ctccccagga
gcttcggctg gaaccaggcc tttaggtggc 1020 cttaccattt atctctatat
ccggctcttt cccgttccct ggatggacaa aaatcttgcc 1080 cttgacagga
ctttaacagg gcttgggctt tgagattctg ttaacccgca ggacttcatt 1140
aggcacacaa gattcacctt aatttctcta aatttttttt tttttaaaat accaagggaa
1200 gggggctaat taacaaccca gtacaggaca tatccacaag ggtcggtaaa
tggcatgcta 1260 ggaaaaatag gggccttgga tcttattcac tggccctgtc
ttccccttgg tttctcttgt 1320 ggccagatct ttcagttgcc ccttttccat
aacaggggat tttttttctt cataggagtt 1380 aattattatg ggaacagttt
tttatggacc tcccttttgg tctggaaata ccttttcgaa 1440 cagaatttct
tttttttaaa aaaaaacaga gatggggtct tactatgttg cccaggctgg 1500
tgtcgaactc ctgggctcaa gcgatccttc tgccttggcc tcccgaagtg ctgggattgc
1560 aggcataagc ttaccatgct gggcctgaac ataatttcaa gaggaggatt
tataaaacca 1620 ttttctgtaa tcaaatgatt ggtgtcattt tcccatttgc
acaatgtagt ctcactt 1677 75 2608 DNA Homo sapiens 75 agctcgccgg
cctttggtct ccaggacttg tcccagcagc ccctcgaact gagaattaca 60
ccatcggacc cctggctctg aggccttcag acttggactg tgtcacactg ccaggcttcc
120 agggctccaa cttgcagacg gcctgttgtg ggacagtctc tgtaatcgcg
aaagcaacca 180 tggaagacct gggggaaaac accatggttt tatccaccct
gagatctttg aacaacttca 240 tctctcagcg tgtggaggga ggctctggac
tggatatttc tacctcggcc ccaggttctc 300 tgcagatgca gtaccagcag
agcatgcagc tggaggaaag agcagagcag atccgttcga 360 agtcccacct
catccaggtg gagcgggaga aaatgcagat ggagctgagt cacaagaggg 420
ctcgagtgga gctggagaga gcagccagca ccagtgccag gaactacgag cgtgaggtcg
480 accgcaacca ggagctcctg acgcgcatcc ggcagcttca ggagcgggag
gccggggcgg 540 aggagaagat gcaggagcag ctggagcgca acaggcagtg
tcagcagaac ttggatgctg 600 ccagcaagag gctgcgtgag aaagaggaca
gtctggccca ggctggcgag accatcaacg 660 cactgaaggg gaggatctcg
gaactgcagt ggagcgtgat ggaccaggag atgcgggtga 720 agcgcctgga
gtcggagaag caggacgtgc aggagcagct ggacctgcaa cacaaaaaat 780
gccaggaagc caatcagaaa atccaggaac tccaggccag ccaagaagca agagcagacc
840 acgagcagca gattaaggat ctggagcaga agctgtccct gcaagagcag
gatgcagcga 900 ttgtgaagaa catgaagtct gagctggtac ggctccctag
gctggaacgg gagctggagc 960 agctgcggga ggagagcgca ctgcgggaga
tgagagagac caacgggctg ctccaggaag 1020 agctggaagg gctgcagagg
aagctggggc gccaggagaa gatgcaggag acgctggttg 1080 gcttggagct
ggagaacgag aggctgctgg ccaagctgca aagctgggag agactggacc 1140
agaccatggg cctgagcatc aggactccag aagacctttc cagattcgtg gttgagctgc
1200 agcagaggga gcttgccttg aaggacaaga acagcgccgt caccagcagc
gcccgggggc 1260 tggagaaggc caggcagcag ctgcaggagg agctccggca
ggtcagcggc cagctgttgg 1320 aggagaggaa gaagcgcgag acccacgagg
cgctggcccg gaggctccag aaacgggtcc 1380 tgctgctcac caaggagcgg
gacggtatgc gggccatcct ggggtcctac gacagcgagc 1440 tgaccccggc
cgagtactca ccccagctga cgcggcgcat gcgggaggct gaggatatgg 1500
tgcagaaggt gcacagccac agcgccgaga tggaggctca gctgtcgcag gccctggagg
1560 agctgggagg ccagaaacaa agagcagaca tgctggagat ggagctgaag
atgctgaagt 1620 ctcagtccag ctctgccgaa cagagcttcc tgttctccag
ggaggaggcg gacacgctca 1680 ggttgaaggt cgaggagctg gaaggcgagc
ggagtcggct ggaggaggaa aagaggatgc 1740 tggaggcaca gctggagcgg
cgagctctgc agggtgacta tgaccagagc aggaccaaag 1800 tgctgcacat
gagcctgaac cccaccagtg tggccaggca gcgcctgcgc gaggaccaca 1860
gccagctgca ggcggagtgc gagcgactgc gcgggctcct gcgcgccatg gagagaggag
1920 gcaccgtccc agccgacctt
gaggctgccg ccgcgagtct gccatcgtcc aaggaggtgg 1980 cagagctgaa
gaagcaggtg gagagtgccg agctgaagaa ccagcggctc aaggaggttt 2040
tccagaccaa gatccaggag ttccgcaagg cctgctacac gctcaccggc taccagatcg
2100 acatcaccac ggagaaccag taccggctga cctcgctgta cgccgagcac
ccaggcgact 2160 gctcatcttc aaggccacca gcccctcggg ttccaagatg
cagctactgg agacagagtt 2220 ctcacacacc gtgggcgagc tcatcgaggt
gcacctgcgg cgccaggaca gcatccctgc 2280 cttcctcagc tcgctcaccc
tcgagctctt cagccgccag accgtggcgt agcctgcagg 2340 ctcgggggca
tagccggagc cactctgctt ggcctgacct gcaggtcccc tgccccgcca 2400
gccacaggct gggtgcacgt cctgcctctc cagccccaca gggcagcagc atgactgaca
2460 gacacgctgg gacctacgtc gggcttcctg ctggggcggc cagcaccctc
tccacgtgca 2520 gaccccatgc gtcccggagc ctggtgtgtg ggcgtcggcc
accagcctgg gttcctcacc 2580 ttgtgaaata aaatcttctc ccctaaaa 2608 76
2326 DNA Homo sapiens 76 aggccggaga ggaggcggtg cggcggtggc
cgtgcggaga cccggtccag acgcctggcg 60 gccgccggca cacaaggcgc
tttctagctc cctcccccga gcgcacagcc cgcctccttc 120 cgcggcgcct
gcagtggcac ggattgctct gccctaccgt gacgcgctcc ggagacgctc 180
tgcgggtcct ggacaccggg tccggcggcg tggggacgac agacggaggc gaacgcatcc
240 ggtagccggt ccgcgagcca tcgttcgggg cgcagtcctc tccccggctg
gccctccttt 300 ctccggggca ttcgccaccg cttccctggg gctgagacga
ccggttcgtc gcctccttgc 360 ccgtgaccgt cgctagaact cagttgtgcg
ttgcggccag tcgccactgc tgagtggaag 420 caaaatgtca gtcagtgtgc
atgagaaccg caagtccagg gccagcagcg gctccattaa 480 catctatctg
tttcacaagt cctcctacgc tgacagcgtc ctcactcacc tgaatctttt 540
acgccagcag cgtctcttca ctgacgtcct tctccatgcc ggaaatagga ccttcccttg
600 ccaccgggca gtgctggctg catgcagtcg ctactttgag gccatgttca
gtggtggcct 660 gaaagagagc caggacagtg aggtcaactt tgacaattcc
atccacccag aagtcttgga 720 gctgctgctt gactatgcgt actcctcccg
ggtcattcat caattggaag gaaaatgcag 780 aaattcgctc ctgggaagct
tggtgacatg ctggagtttc aaggacatcc gggatgcatg 840 tgcagagttc
ctggaaaaga acctgcatcc caccaactgc ctgggcatgc tgctgctgtc 900
tgatgcacac cagtgcacca agctgtacga actatcttgg agaatgtgtc tcagcaactt
960 ccaaaccatc aggaagaatg aagatttcct ccagctgccc caggacatgg
tagtgcaact 1020 cttgtccagt gaagagctgg agacagagga tgaaaggctt
gtgtacgagt ctgcaattaa 1080 ctggatcagc tatgacctga agaagcgcta
ttgctacctc ccagaactgt tgcagacagt 1140 aacgcgggca cttctgccag
ccatctatct catggagaat gtggccatgg aggaactcat 1200 caccaagcag
agaaagagta aggaaattgt ggaagaggcc atcaggtgca aactaaaaat 1260
cctgcagaat gacggtgtgg taaccagcct ctgtgcccga cctcggaaaa ctggccatgc
1320 cctcttcctt ctgggaggac agactttcat gtgtgacaag ttgtatctgg
tagaccagaa 1380 ggccaaagaa atcattccca aggctgacat tcccagccca
agaaaagagt ttagtgcatg 1440 tgcgattggc tgcaaagtgt acattactgg
ggggcggggg tctgaaaatg gggtctcgaa 1500 agatgtctgg gtttatgata
ccctgcacga ggagtggtcc aaggctgccc ccatgctggt 1560 ggccaggttt
ggccatggct ctgctgaact gaagcactgc ctgtatgtgg ttggggggca 1620
cacggccgca actggctgcc tcccggcctc cccctcagtc tctctaaagc aggtagaaca
1680 ttatgacccc acaatcaaca aatggaccat ggcggcccca cgtccgagaa
ggcgttacaa 1740 ctgcgcacag gtagtgagtg ccaaacttaa gttatttgct
ttcggaggta ccagtgtcag 1800 tcatgacaag ctccccaaag ttcagtgtta
cgatcagtgt gaaaacaggt ggactgtacc 1860 ggccacctgt ccccagccct
ggcgtataca cagccaagca agctgtcctg ggggaaccca 1920 ggatttttta
ttatgggggg tgatacagaa tttctctgcc tgcttctgct tataaattcg 1980
caacagtgag acttaccagt ggaccaaagg tgggagatgt gacagcaaag cgcatgagct
2040 gccatgctgt tggcctctgg aaacaaactc ttacgtggtt ggaggatact
ttgggcattc 2100 agcgatgcaa gactttggac tgctacgatc caacattaga
cgtgtggaac agcatcacca 2160 ctgtcccgta ctcgctgatt cctactgcat
tttgtcagca cctggaaaca tctgccttct 2220 taaatgcagt acattctaaa
gagaagatga gcatgagctc actccatcac tcgatgagat 2280 aatatgagat
ttctacttcg gagaggccaa gtctaatgaa gagaaa 2326 77 2302 DNA Homo
sapiens 77 ctaaatcaag ctggagtcat gagggtagtg ggctaagtcg agggtccagc
ctcttctgcc 60 aggaagccct tcttgctttt gagagagggc tgtgaccacc
ccccatcctt ctccctacac 120 tcccagccaa cctagtgccc aagcagctaa
acttggcttc cttctaatcc tggaaaaccc 180 tgtacccctc ctcctcaatc
tggccctctc cacatgcaca ccctgagaac acacacagac 240 acacaacaca
cacacataca cacccctgaa cacacacaca gacacacata cacccatgat 300
gtgagcaaac acacacacgt gcgccttcat agcccagcca aggcatcgca ggcagggtgt
360 gctgcctgag atggcacctc cctttcagcc attcttcaag aatgggccac
acacagctag 420 aagtcctctc ccagctagaa gtcctgtccc actctcctgg
cctgacaaga tgagctctcc 480 tgggaccttg ctctagggca ctctgcctct
accctaggac actggaatgc cctgggagcc 540 ccctccctgc aaccagcctg
agttcagccc cacggacaaa gggacacaca gcccccaatg 600 gagaccattg
taagtggtgg ggctgggaga ggaggaacag aaggaaagcc atagcgctct 660
cttgcccctt ggcatgtacc ccaaggcctg atggccactg ggctcagcct gtcccccact
720 cctgcctgct tcccggtgag ctgcccccga cacgtgcagc ccgggctgcc
tccagggtct 780 ggctgagtgg gatcaggtgg ccctccaact cagcacagga
aataagtaga aacatttcag 840 caggccacct cccctcatct tccccgccct
gtccagcgcc ctggcaaagg ctgacaactg 900 gctgtcttgg ggccgaacag
ccctgcctgc tctgagggcc acagcctgtg ctgcataccc 960 accgcccagc
ttctccctga gggcccacca gcctgtgctg catacccacc acccagcttc 1020
tccctgaggg cccaccagcc tgtgctgtac accccgttag tccctgatcc caaccttctc
1080 cctcctgcca gcacaccgat gcacacaccg gaagtggcga gcccaagccc
tggggacagg 1140 tgtagggaga aaagcagccc caggcctcag actcgctctc
ccatcactgg catagagtgg 1200 gaggatggct ggagggtgtc tataggtaca
gcccgctctg gctgctgcca ggtgggcccc 1260 tgccaggggt cctcacccct
gtccaccctg tgcctggctg tccctgcacc cagatacagc 1320 aacatggcct
gtacccagca gagtggtggc aaccaccatg gttacagcgg atgccccgag 1380
actctgcttg gtaaacgtgg cagagcagaa tgggaggctg ggaccctgag gaagggcccc
1440 tctcctggca tctgtctctt gctacctaag cctgtgcctc tccctaaaga
gctgcctccc 1500 tgctgccgag ccctggtctg gccacgagcc actactgcct
cccacaggca ccactgcctc 1560 ccgctgctgc ccacaggtgg tgccgccaat
gggcagtgcc tccaggccga agccttcaat 1620 cccccatctt gagccagggc
ctaaatcctc ttaatagtga tggttggttt tgtcctccca 1680 ttaactgcag
gtgggatttc cacctggggg aatgaggctt gcgttgttcg ggcgtctgct 1740
ggccctgaga catccagtct tccacactca actgtgggat gggagggtgg cgtggcttta
1800 ccccatggag gctgttccag ggctctgggc acacagctgt gctcacacaa
aatactgggt 1860 ggcttggttt agagctaatt gtagtggaag cctgcaaggt
tgaggggtga aggggagggg 1920 gcttgcaagg tccaggtaaa gatctggaaa
gacagaacgt acagcttgga gggcaagggg 1980 gactctaaag tgcaaggaga
tttacagttg ggaaaggagg cagtggcaga ggggttgagg 2040 gacaggggcc
cttaagtcca gcgaggaaag ctcggtgtgg ggcccgctct acgctccgtt 2100
tggggtgacc tggaacgcct cttctcccag ctccctccag ccatcagcag cctcttgtca
2160 agcttctgcc tcgccccagt ctatccccaa ccccaaatca agaccacctt
tcttcaacgg 2220 tcactattta ttctttgttc ctttttcttt tgtgtaagaa
acattcacaa aaaccagtgc 2280 caaaaccatc aaaaaaaaaa aa 2302 78 1729
DNA Homo sapiens 78 tggccagaga tgcctgccca cagcctggtg atgagcagcc
cggccctccc ggccttcctg 60 ctctgcagca cgctgctggt catcaagatg
tacgtggtgg ccatcatcac gggccaagtg 120 aggctgcgga agaaggcctt
tgccaacccc gaggatgccc tgagacacgg aggaggcccc 180 cagtattgca
ggagcgaccc cgacgtggaa cgctgcctca gggcccaccg gaacgacatg 240
gagaccatct accccttcct tttcctgggc ttcgtctact cctttctggg tcctaaccct
300 tttgtcgcct ggatgcactt cctggtcttc ctcgtgggcc gtgtggcaca
caccgtggcc 360 tacctgggga agctgcgggc acccatccgc tccgtgacct
acaccctggc ccagctcccc 420 tgcgcctcca tggctctgca gatcctctgg
gaagcggccc gccacctgtg accagcagct 480 gatgcctcct tggccaccag
accatgggcc aagagccgcc gtggctatac ctggggactt 540 gatgttcctt
ccagattgtg gtgtgggccc tgagtcctgg tttcctggca gcctgctgcg 600
cgtgtgggtc tctgggcaca gtgggcctgt gtgtgtgccc gtgtgtgtgt atgtgtgtgt
660 gtatgtttct tagccccttg gattcctgca cgaagtggct gatgggaacc
atttcaagac 720 agattgtgaa gattgataga aaatccttca gctaaagtaa
cagagcatca aaaacatcac 780 tccctctccc tccctaacag tgaaaagaga
gaagggagac tctatttaag attcccaaac 840 ctaatgatca tctgaatccc
gggctaagaa tgcagacttt tcagactgac cccagaaatt 900 ctggcccagc
caatctagag gcaagcctgg ccatctgtat tttttttttc caagacagag 960
tcttgctctc gttgcccaag ctggagtgaa gtggtacaat ctggctcact gcagcctccg
1020 cctcccgggt tcaagcgatt ctcccgcctc agcctcctga gtagctggga
ttacaggcgc 1080 gtatcaccat acccagctaa tttttgtatt tttagtagag
acgggttcac catgttgccc 1140 aggagggtct cgaactcctg gcctcaagtg
atccacgcct cggcctccca aagtgctggg 1200 atgacaggca tgaatcactg
tgctcagcca ccatctggag tttaaaagga cctcccatgt 1260 gagtccctgt
gtggccaggc cagggacccc tgccagttct atgtggaagc aaggctgggg 1320
tcttgggttc ctgtatggtg gaagctgggt gagccaagga cagggctggc tcctctgccc
1380 ccgctgacgc ttcccttgcc gttggctttg gatgtctttg ctgcagtctt
ctctctggct 1440 caggtgtggg tgggaggggc ccacaggaag ctcagccttc
tcctcccaag gtttgagtcc 1500 ctccaaaggg cagtgggtgg aggaccggga
gctttgggtg accagccact caaaggaact 1560 ttctggtccc ttcagtatct
tcaaggtttg gaaactgcaa atgtcccctg atggggaatc 1620 ctgtgtgtgt
gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgttt tctcctagac 1680
ccgtgacctg agatgtgtga tttttagtca ttaaatggaa gtgtctgcc 1729 79 136
PRT Homo sapiens 79 Met Ser Asn Val Pro His Lys Ser Ser Leu Pro Glu
Gly Ile Arg Pro 1 5 10 15 Gly Thr Val Leu Arg Ile Arg Gly Leu Val
Pro Pro Asn Ala Ser Arg 20 25 30 Phe His Val Asn Leu Leu Cys Gly
Glu Glu Gln Gly Ser Asp Ala Ala 35 40 45 Leu His Phe Asn Pro Arg
Leu Asp Thr Ser Glu Val Val Phe Asn Ser 50 55 60 Lys Glu Gln Gly
Ser Trp Gly Arg Glu Glu Arg Gly Pro Gly Val Pro 65 70 75 80 Phe Gln
Arg Gly Gln Pro Phe Glu Val Leu Ile Ile Ala Ser Asp Asp 85 90 95
Gly Phe Lys Ala Val Val Gly Asp Ala Gln Tyr His His Phe Arg His 100
105 110 Arg Leu Pro Leu Ala Arg Val Arg Leu Val Glu Val Gly Gly Asp
Val 115 120 125 Gln Leu Asp Ser Val Arg Ile Phe 130 135 80 236 PRT
Homo sapiens 80 Met Ser Ala Pro Ser Ala Thr Pro Ile Phe Ala Pro Gly
Glu Asn Cys 1 5 10 15 Ser Pro Ala Trp Gly Ala Ala Pro Ala Ala Tyr
Asp Ala Ala Asp Thr 20 25 30 His Leu Arg Ile Leu Gly Lys Pro Val
Met Glu Arg Trp Glu Thr Pro 35 40 45 Tyr Met His Ala Leu Ala Ala
Ala Ala Ser Ser Lys Gly Gly Arg Val 50 55 60 Leu Glu Val Gly Phe
Gly Met Ala Ile Ala Ala Ser Lys Val Gln Glu 65 70 75 80 Ala Pro Ile
Asp Glu His Trp Ile Ile Glu Cys Asn Asp Gly Val Phe 85 90 95 Gln
Arg Leu Arg Asp Trp Ala Pro Arg Gln Thr His Lys Val Ile Pro 100 105
110 Leu Lys Gly Leu Trp Glu Asp Val Ala Pro Thr Leu Pro Asp Gly His
115 120 125 Phe Asp Gly Ile Leu Tyr Asp Thr Tyr Pro Leu Ser Glu Glu
Thr Trp 130 135 140 His Thr His Gln Phe Asn Phe Ile Lys Asn His Ala
Phe Arg Leu Leu 145 150 155 160 Lys Pro Gly Gly Val Leu Thr Tyr Cys
Asn Leu Thr Ser Trp Gly Glu 165 170 175 Leu Met Lys Ser Lys Tyr Ser
Asp Ile Thr Ile Met Phe Glu Glu Thr 180 185 190 Gln Val Pro Ala Leu
Leu Glu Ala Gly Phe Arg Arg Glu Asn Ile Arg 195 200 205 Thr Glu Val
Met Ala Leu Val Pro Pro Ala Asp Cys Arg Tyr Tyr Ala 210 215 220 Phe
Pro Gln Met Ile Thr Pro Leu Val Thr Lys Gly 225 230 235 81 322 PRT
Homo sapiens 81 Met Leu Ala Val His Phe Asp Lys Pro Gly Gly Pro Glu
Asn Leu Tyr 1 5 10 15 Val Lys Glu Val Ala Lys Pro Ser Pro Gly Glu
Gly Glu Val Leu Leu 20 25 30 Lys Val Ala Ala Ser Ala Leu Asn Arg
Ala Asp Leu Met Gln Arg Gln 35 40 45 Gly Gln Tyr Asp Pro Pro Pro
Gly Ala Ser Asn Ile Leu Gly Leu Glu 50 55 60 Ala Ser Gly His Val
Ala Glu Leu Gly Pro Gly Cys Gln Gly His Trp 65 70 75 80 Lys Ile Gly
Asp Thr Ala Met Ala Leu Leu Pro Gly Gly Gly Gln Ala 85 90 95 Gln
Tyr Val Thr Val Pro Glu Gly Leu Leu Met Pro Ile Pro Glu Gly 100 105
110 Leu Thr Leu Thr Gln Ala Ala Ala Ile Pro Glu Ala Trp Leu Thr Ala
115 120 125 Phe Gln Leu Leu His Leu Val Gly Asn Val Gln Ala Gly Asp
Tyr Val 130 135 140 Leu Ile His Ala Gly Leu Ser Gly Val Gly Thr Ala
Ala Ile Gln Leu 145 150 155 160 Thr Arg Met Ala Gly Ala Ile Pro Leu
Val Thr Ala Gly Ser Gln Lys 165 170 175 Lys Leu Gln Met Ala Glu Lys
Leu Gly Ala Ala Ala Gly Phe Asn Tyr 180 185 190 Lys Lys Glu Asp Phe
Ser Glu Ala Thr Leu Lys Phe Thr Lys Gly Ala 195 200 205 Gly Val Asn
Leu Ile Leu Asp Cys Ile Gly Gly Ser Tyr Trp Glu Lys 210 215 220 Asn
Val Asn Cys Leu Ala Leu Asp Gly Arg Trp Val Leu Tyr Gly Leu 225 230
235 240 Met Gly Gly Gly Asp Ile Asn Gly Pro Leu Phe Ser Lys Leu Leu
Phe 245 250 255 Lys Arg Gly Ser Leu Ile Thr Ser Leu Leu Arg Ser Arg
Asp Asn Lys 260 265 270 Tyr Lys Gln Met Leu Val Asn Ala Phe Thr Glu
Gln Ile Leu Pro His 275 280 285 Phe Ser Thr Glu Gly Pro Gln Arg Leu
Leu Pro Val Leu Asp Arg Ile 290 295 300 Tyr Pro Val Thr Glu Ile Gln
Glu Ala His Ser Thr Trp Arg Pro Thr 305 310 315 320 Arg Thr 82 122
PRT Homo sapiens 82 Met Lys Leu Leu Thr Gly Leu Val Phe Cys Ser Leu
Val Leu Ser Val 1 5 10 15 Ser Ser Arg Ser Phe Phe Ser Phe Leu Gly
Glu Ala Phe Asp Gly Ala 20 25 30 Arg Asp Met Trp Arg Ala Tyr Ser
Asp Met Arg Glu Ala Asn Tyr Ile 35 40 45 Gly Ser Asp Lys Tyr Phe
His Ala Arg Gly Asn Tyr Asp Ala Ala Lys 50 55 60 Arg Gly Pro Gly
Gly Ala Trp Ala Ala Glu Val Ile Ser Asn Ala Arg 65 70 75 80 Glu Asn
Ile Gln Arg Leu Thr Gly His Gly Ala Glu Asp Ser Leu Ala 85 90 95
Asp Gln Ala Ala Asn Lys Trp Gly Arg Ser Gly Arg Asp Pro Asn His 100
105 110 Phe Arg Pro Ala Gly Leu Pro Glu Lys Tyr 115 120 83 253 PRT
Homo sapiens 83 Gly Ala Tyr Leu Ala Gln Glu Arg Ala Arg Ala Gln Ile
Gly Tyr Glu 1 5 10 15 Asp Pro Ile Asn Pro Thr Tyr Glu Ala Thr Asn
Ala Met Tyr His Arg 20 25 30 Cys Leu Asp Tyr Val Leu Glu Glu Leu
Lys His Asn Ala Lys Ala Lys 35 40 45 Val Met Val Ala Ser His Asn
Glu Asp Thr Val Arg Phe Ala Leu Arg 50 55 60 Arg Met Glu Glu Leu
Gly Leu His Pro Ala Asp His Gln Val Tyr Phe 65 70 75 80 Gly Gln Leu
Leu Gly Met Cys Asp Gln Ile Ser Phe Pro Leu Gly His 85 90 95 Gly
Trp Leu Pro Arg Val Gln Val Arg Ala Leu Trp Pro Arg Asp Gly 100 105
110 Gly Ala Ala Leu Leu Val Pro Ala Val Pro Trp Lys Asn Ser Ser Leu
115 120 125 Met Lys Gly Thr His Ser Gly Ala Ala Leu Ala Val Ala Gly
Ala Leu 130 135 140 Glu Ala Ala Pro Asn Trp Gln Pro Leu Pro Ser Pro
Cys Leu Ala Pro 145 150 155 160 Ala Ser Thr Pro Ser Ser Leu Pro Ala
Pro Pro Ala Pro Cys Ser Arg 165 170 175 Pro Phe Asn Gln Gln Ala Ala
Ser Gln Thr Pro Ile Leu Gln His Arg 180 185 190 Phe Thr Phe Phe His
Pro Thr Thr Leu Gln Ser Leu Leu Gly Gly Glu 195 200 205 Val Arg Cys
Leu Pro Ala Leu Ala Gln Ser Met Gly Thr Gln Val Trp 210 215 220 Ala
Glu Pro Asp Thr Cys Leu Gly Gln Pro Leu Glu Thr Phe Gly Asn 225 230
235 240 Ser Pro Leu Lys Cys Val Gly Pro Arg Pro Pro Pro Leu 245 250
84 228 PRT Homo sapiens 84 Met Ser Val Pro Gly Pro Tyr Gln Ala Ala
Thr Gly Pro Ser Ser Ala 1 5 10 15 Pro Ser Ala Pro Pro Ser Tyr Glu
Glu Thr Val Ala Val Asn Ser Tyr 20 25 30 Tyr Pro Thr Pro Pro Ala
Pro Met Pro Gly Pro Thr Thr Gly Leu Val 35 40 45 Thr Gly Pro Asp
Gly Lys Gly Met Asn Pro Pro Ser Tyr Tyr Thr Gln 50 55 60 Pro Ala
Pro Ile Pro Asn Asn Asn Pro Ile Thr Val Gln Thr Val Tyr 65 70 75 80
Val Gln His Pro Ile Thr Phe Leu Asp Arg Pro Ile Gln Met Cys Cys 85
90 95 Pro Ser Cys Asn Lys Met Ile Val Ser Gln Leu Ser Tyr Asn Ala
Gly 100 105 110 Ala Leu Thr Trp Leu Ser Cys Gly Ser Leu Cys Leu Leu
Gly Val His 115 120 125 Ser Gly Leu Leu Leu His Pro Leu Leu Arg Gly
Cys Pro Ala Gly Arg 130 135 140 Gly Pro Leu Leu Ser Gln Leu Gln Ser
Ser Pro Gly His Leu Gln Ala 145 150 155 160 Phe Val Gly Leu Ser Gln
Thr Trp Arg Glu Pro Gly Ala Ala Gly Ser 165 170 175 Pro Phe His Leu
Ser Ser Ser Phe Thr Pro Gly Gly Gly Ser Ala Leu 180
185 190 Val Val Ser Pro Leu Gln Gly Ala His Leu His Val Phe Phe Trp
Gly 195 200 205 Glu Tyr Val Ala Lys Leu Thr Asn Leu Gln Thr Pro Glu
Ile Ala Ala 210 215 220 Trp Ser Arg Ala 225 85 803 PRT Homo sapiens
85 Met Glu Asp Leu Gly Glu Asn Thr Met Val Leu Ser Thr Leu Arg Ser
1 5 10 15 Leu Asn Asn Phe Ile Ser Gln Arg Val Glu Gly Gly Ser Gly
Leu Asp 20 25 30 Ile Ser Thr Ser Ala Pro Gly Ser Leu Gln Met Gln
Tyr Gln Gln Ser 35 40 45 Met Gln Leu Glu Glu Arg Ala Glu Gln Ile
Arg Ser Lys Ser His Leu 50 55 60 Ile Gln Val Glu Arg Glu Lys Met
Gln Met Glu Leu Ser His Lys Arg 65 70 75 80 Ala Arg Val Glu Leu Glu
Arg Ala Ala Ser Thr Ser Ala Arg Asn Tyr 85 90 95 Glu Arg Glu Val
Asp Arg Asn Gln Glu Leu Leu Thr Arg Ile Arg Gln 100 105 110 Leu Gln
Glu Arg Glu Ala Gly Ala Glu Glu Lys Met Gln Glu Gln Leu 115 120 125
Glu Arg Asn Arg Gln Cys Gln Gln Asn Leu Asp Ala Ala Ser Lys Arg 130
135 140 Leu Arg Glu Lys Glu Asp Ser Leu Ala Gln Ala Gly Glu Thr Ile
Asn 145 150 155 160 Ala Leu Lys Gly Arg Ile Ser Glu Leu Gln Trp Ser
Val Met Asp Gln 165 170 175 Glu Met Arg Val Lys Arg Leu Glu Ser Glu
Lys Gln Asp Val Gln Glu 180 185 190 Gln Leu Asp Leu Gln His Lys Lys
Cys Gln Glu Ala Asn Gln Lys Ile 195 200 205 Gln Glu Leu Gln Ala Ser
Gln Glu Ala Arg Ala Asp His Glu Gln Gln 210 215 220 Ile Lys Asp Leu
Glu Gln Lys Leu Ser Leu Gln Glu Gln Asp Ala Ala 225 230 235 240 Ile
Val Lys Asn Met Lys Ser Glu Leu Val Arg Leu Pro Arg Leu Glu 245 250
255 Arg Glu Leu Glu Gln Leu Arg Glu Glu Ser Ala Leu Arg Glu Met Arg
260 265 270 Glu Thr Asn Gly Leu Leu Gln Glu Glu Leu Glu Gly Leu Gln
Arg Lys 275 280 285 Leu Gly Arg Gln Glu Lys Met Gln Glu Thr Leu Val
Gly Leu Glu Leu 290 295 300 Glu Asn Glu Arg Leu Leu Ala Lys Leu Gln
Ser Trp Glu Arg Leu Asp 305 310 315 320 Gln Thr Met Gly Leu Ser Ile
Arg Thr Pro Glu Asp Leu Ser Arg Phe 325 330 335 Val Val Glu Leu Gln
Gln Arg Glu Leu Ala Leu Lys Asp Lys Asn Ser 340 345 350 Ala Val Thr
Ser Ser Ala Arg Gly Leu Glu Lys Ala Arg Gln Gln Leu 355 360 365 Gln
Glu Glu Leu Arg Gln Val Ser Gly Gln Leu Leu Glu Glu Arg Lys 370 375
380 Lys Arg Glu Thr His Glu Ala Leu Ala Arg Arg Leu Gln Lys Arg Val
385 390 395 400 Leu Leu Leu Thr Lys Glu Arg Asp Gly Met Arg Ala Ile
Leu Gly Ser 405 410 415 Tyr Asp Ser Glu Leu Thr Pro Ala Glu Tyr Ser
Pro Gln Leu Thr Arg 420 425 430 Arg Met Arg Glu Ala Glu Asp Met Val
Gln Lys Val His Ser His Ser 435 440 445 Ala Glu Met Glu Ala Gln Leu
Ser Gln Ala Leu Glu Glu Leu Gly Gly 450 455 460 Gln Lys Gln Arg Ala
Asp Met Leu Glu Met Glu Leu Lys Met Leu Lys 465 470 475 480 Ser Gln
Ser Ser Ser Ala Glu Gln Ser Phe Leu Phe Ser Arg Glu Glu 485 490 495
Ala Asp Thr Leu Arg Leu Lys Val Glu Glu Leu Glu Gly Glu Arg Ser 500
505 510 Arg Leu Glu Glu Glu Lys Arg Met Leu Glu Ala Gln Leu Glu Arg
Arg 515 520 525 Ala Leu Gln Gly Asp Tyr Asp Gln Ser Arg Thr Lys Val
Leu His Met 530 535 540 Ser Leu Asn Pro Thr Ser Val Ala Arg Gln Arg
Leu Arg Glu Asp His 545 550 555 560 Ser Gln Leu Gln Ala Glu Cys Glu
Arg Leu Arg Gly Leu Leu Arg Ala 565 570 575 Met Glu Arg Gly Gly Thr
Val Pro Ala Asp Leu Glu Ala Ala Ala Ala 580 585 590 Ser Leu Pro Ser
Ser Lys Glu Val Ala Glu Leu Lys Lys Gln Val Glu 595 600 605 Ser Ala
Glu Leu Lys Asn Gln Arg Leu Lys Glu Val Phe Gln Thr Lys 610 615 620
Ile Gln Glu Phe Arg Lys Ala Cys Tyr Thr Leu Thr Gly Tyr Gln Ile 625
630 635 640 Asp Ile Thr Thr Glu Asn Gln Tyr Arg Leu Thr Ser Leu Tyr
Ala Glu 645 650 655 His Pro Gly Asp Cys Ser Ser Ser Arg Pro Pro Ala
Pro Arg Val Pro 660 665 670 Arg Cys Ser Tyr Trp Arg Gln Ser Ser His
Thr Pro Trp Ala Ser Ser 675 680 685 Ser Arg Cys Thr Cys Gly Ala Arg
Thr Ala Ser Leu Pro Ser Ser Ala 690 695 700 Arg Ser Pro Ser Ser Ser
Ser Ala Ala Arg Pro Trp Arg Ser Leu Gln 705 710 715 720 Ala Arg Gly
His Ser Arg Ser His Ser Ala Trp Pro Asp Leu Gln Val 725 730 735 Pro
Cys Pro Ala Ser His Arg Leu Gly Ala Arg Pro Ala Ser Pro Ala 740 745
750 Pro Gln Gly Ser Ser Met Thr Asp Arg His Ala Gly Thr Tyr Val Gly
755 760 765 Leu Pro Ala Gly Ala Ala Ser Thr Leu Ser Thr Cys Arg Pro
His Ala 770 775 780 Ser Arg Ser Leu Val Cys Gly Arg Arg Pro Pro Ala
Trp Val Pro His 785 790 795 800 Leu Val Lys 86 516 PRT Homo sapiens
86 Met Ser Val Ser Val His Glu Asn Arg Lys Ser Arg Ala Ser Ser Gly
1 5 10 15 Ser Ile Asn Ile Tyr Leu Phe His Lys Ser Ser Tyr Ala Asp
Ser Val 20 25 30 Leu Thr His Leu Asn Leu Leu Arg Gln Gln Arg Leu
Phe Thr Asp Val 35 40 45 Leu Leu His Ala Gly Asn Arg Thr Phe Pro
Cys His Arg Ala Val Leu 50 55 60 Ala Ala Cys Ser Arg Tyr Phe Glu
Ala Met Phe Ser Gly Gly Leu Lys 65 70 75 80 Glu Ser Gln Asp Ser Glu
Val Asn Phe Asp Asn Ser Ile His Pro Glu 85 90 95 Val Leu Glu Leu
Leu Leu Asp Tyr Ala Tyr Ser Ser Arg Val Ile His 100 105 110 Gln Leu
Glu Gly Lys Cys Arg Asn Ser Leu Leu Gly Ser Leu Val Thr 115 120 125
Cys Trp Ser Phe Lys Asp Ile Arg Asp Ala Cys Ala Glu Phe Leu Glu 130
135 140 Lys Asn Leu His Pro Thr Asn Cys Leu Gly Met Leu Leu Leu Ser
Asp 145 150 155 160 Ala His Gln Cys Thr Lys Leu Tyr Glu Leu Ser Trp
Arg Met Cys Leu 165 170 175 Ser Asn Phe Gln Thr Ile Arg Lys Asn Glu
Asp Phe Leu Gln Leu Pro 180 185 190 Gln Asp Met Val Val Gln Leu Leu
Ser Ser Glu Glu Leu Glu Thr Glu 195 200 205 Asp Glu Arg Leu Val Tyr
Glu Ser Ala Ile Asn Trp Ile Ser Tyr Asp 210 215 220 Leu Lys Lys Arg
Tyr Cys Tyr Leu Pro Glu Leu Leu Gln Thr Val Thr 225 230 235 240 Arg
Ala Leu Leu Pro Ala Ile Tyr Leu Met Glu Asn Val Ala Met Glu 245 250
255 Glu Leu Ile Thr Lys Gln Arg Lys Ser Lys Glu Ile Val Glu Glu Ala
260 265 270 Ile Arg Cys Lys Leu Lys Ile Leu Gln Asn Asp Gly Val Val
Thr Ser 275 280 285 Leu Cys Ala Arg Pro Arg Lys Thr Gly His Ala Leu
Phe Leu Leu Gly 290 295 300 Gly Gln Thr Phe Met Cys Asp Lys Leu Tyr
Leu Val Asp Gln Lys Ala 305 310 315 320 Lys Glu Ile Ile Pro Lys Ala
Asp Ile Pro Ser Pro Arg Lys Glu Phe 325 330 335 Ser Ala Cys Ala Ile
Gly Cys Lys Val Tyr Ile Thr Gly Gly Arg Gly 340 345 350 Ser Glu Asn
Gly Val Ser Lys Asp Val Trp Val Tyr Asp Thr Leu His 355 360 365 Glu
Glu Trp Ser Lys Ala Ala Pro Met Leu Val Ala Arg Phe Gly His 370 375
380 Gly Ser Ala Glu Leu Lys His Cys Leu Tyr Val Val Gly Gly His Thr
385 390 395 400 Ala Ala Thr Gly Cys Leu Pro Ala Ser Pro Ser Val Ser
Leu Lys Gln 405 410 415 Val Glu His Tyr Asp Pro Thr Ile Asn Lys Trp
Thr Met Ala Ala Pro 420 425 430 Arg Pro Arg Arg Arg Tyr Asn Cys Ala
Gln Val Val Ser Ala Lys Leu 435 440 445 Lys Leu Phe Ala Phe Gly Gly
Thr Ser Val Ser His Asp Lys Leu Pro 450 455 460 Lys Val Gln Cys Tyr
Asp Gln Cys Glu Asn Arg Trp Thr Val Pro Ala 465 470 475 480 Thr Cys
Pro Gln Pro Trp Arg Ile His Ser Gln Ala Ser Cys Pro Gly 485 490 495
Gly Thr Gln Asp Phe Leu Leu Trp Gly Val Ile Gln Asn Phe Ser Ala 500
505 510 Cys Phe Cys Leu 515 87 153 PRT Homo sapiens 87 Met Pro Ala
His Ser Leu Val Met Ser Ser Pro Ala Leu Pro Ala Phe 1 5 10 15 Leu
Leu Cys Ser Thr Leu Leu Val Ile Lys Met Tyr Val Val Ala Ile 20 25
30 Ile Thr Gly Gln Val Arg Leu Arg Lys Lys Ala Phe Ala Asn Pro Glu
35 40 45 Asp Ala Leu Arg His Gly Gly Gly Pro Gln Tyr Cys Arg Ser
Asp Pro 50 55 60 Asp Val Glu Arg Cys Leu Arg Ala His Arg Asn Asp
Met Glu Thr Ile 65 70 75 80 Tyr Pro Phe Leu Phe Leu Gly Phe Val Tyr
Ser Phe Leu Gly Pro Asn 85 90 95 Pro Phe Val Ala Trp Met His Phe
Leu Val Phe Leu Val Gly Arg Val 100 105 110 Ala His Thr Val Ala Tyr
Leu Gly Lys Leu Arg Ala Pro Ile Arg Ser 115 120 125 Val Thr Tyr Thr
Leu Ala Gln Leu Pro Cys Ala Ser Met Ala Leu Gln 130 135 140 Ile Leu
Trp Glu Ala Ala Arg His Leu 145 150
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