U.S. patent application number 10/443982 was filed with the patent office on 2003-09-25 for anti-proliferation domain of human bcl-2 and dna encoding the same.
This patent application is currently assigned to ST. LOUIS UNIVERSITY. Invention is credited to Chinnadurai, Govindaswamy.
Application Number | 20030180791 10/443982 |
Document ID | / |
Family ID | 24616105 |
Filed Date | 2003-09-25 |
United States Patent
Application |
20030180791 |
Kind Code |
A1 |
Chinnadurai, Govindaswamy |
September 25, 2003 |
Anti-proliferation domain of human Bcl-2 and DNA encoding the
same
Abstract
A domain of Bcl-2 that suppresses apoptosis by allowing cell
survival permits cell proliferation when mutated. The wild type
domain includes amino acid residues 51 to 97 (SEQ ID NO:13) of
Bcl-2. Peptides including the domain and nucleotides encoding the
domain are useful in molecular screening of human tumors for the
presence of mutations that allow proliferation of cells that were
otherwise marked for apoptosis. The peptides are also useful to
screen for proteins that play a role in the modulation of cellular
proliferation.
Inventors: |
Chinnadurai, Govindaswamy;
(St. Louis, MO) |
Correspondence
Address: |
SUGHRUE MION, PLLC
2100 PENNSYLVANIA AVENUE, N.W.
WASHINGTON
DC
20037
US
|
Assignee: |
ST. LOUIS UNIVERSITY
|
Family ID: |
24616105 |
Appl. No.: |
10/443982 |
Filed: |
May 23, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10443982 |
May 23, 2003 |
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09759136 |
Jan 16, 2001 |
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09759136 |
Jan 16, 2001 |
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09274647 |
Mar 23, 1999 |
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6207452 |
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09274647 |
Mar 23, 1999 |
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09079186 |
May 15, 1998 |
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5952179 |
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09079186 |
May 15, 1998 |
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08652245 |
May 23, 1996 |
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5821082 |
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Current U.S.
Class: |
435/6.11 ;
435/320.1; 435/325; 435/6.12; 435/69.3; 435/7.23; 530/350;
536/23.5 |
Current CPC
Class: |
C07K 14/4747 20130101;
C07K 2319/00 20130101; C07K 16/18 20130101 |
Class at
Publication: |
435/6 ; 435/7.23;
435/69.3; 435/320.1; 435/325; 530/350; 536/23.5 |
International
Class: |
C12Q 001/68; G01N
033/574; C07H 021/04; C12P 021/02; C12N 005/06; C07K 014/47 |
Goverment Interests
[0001] This invention was made with government support under grants
CA-33616 and CA-31719 from the National Cancer Institute. The
government has certain rights in the invention.
Claims
What is claimed:
1. A truncated bcl-2 gene comprising nucleotides 151 to any one of
nucleotides 255-291 (SEQ ID NOS:14-50), and fragments of the
truncated bcl-2 gene that contain at least one codon encoding an
amino acid needed to maintain full or partial anti-proliferation
activity of the AP domain.
2. The truncated bcl-2 gene of claim 1, wherein the fragments
contain at least one of nucleotides 151-153, nucleotides 169-171,
nucleotides 184-186, nucleotides 204-210, nucleotides 220-222, and
nucleotides 223-225.
3. The truncated bcl-2 gene of claim 1, which comprises nucleotides
151 to any one of nucleotides 255-291 (SEQ ID NO: 14-50).
4. The truncated bcl-2 gene of claim 1, which comprises nucleotides
151-255. (SEQ ID NO:14).
5. The truncated bcl-2 gene of claim 1, which consists of
nucleotides 151 to any one of nucleotides 255-291 (SEQ ID
NO:14-50).
6. The truncated bcl-2 gene of claim 1, which consists of
nucleotides 151-255 (SEQ ID NO:14).
7. RNA complementary to the truncated bcl-2 gene of anyone of
claims 1-6.
8. A truncated Bcl-2 protein comprising residues 51 to any of 85-97
(SEQ ID NOS: 1-13) and fragments of the truncated Bcl-2 protein
that contain at least one amino acid needed to maintain full or
partial anti-proliferation activity of the AP domain.
9. The truncated Bcl-2 protein of claim 8, wherein the fragments
contain at least one of Ser residue 51, Pro residue 57, Ser residue
62, Thr and Ser residues 69 and 70, Thr residue 74, and Pro residue
75.
10. The truncated Bcl-2 protein of claim 8 comprising residues
51-85 (SEQ ID NO:1) and fragments of the truncated protein
comprising residues 51-85 that contain at least one of Ser residue
51, Pro residue 57, Ser residue 62,.Thr and Ser residues 69 and 70,
Thr residue 74, and Pro residue 75.
11. The truncated Bcl-2 protein of claim 8 which consists of
residues 51 to any of 85-97 (SEQ ID NOS:1-13).
12. The truncated Bcl-2 protein of claim 8 which consists of
residues 51-85 (SEQ ID NO:1).
13. A method of screening for mutations in the AP domain of Bcl-2,
said method comprising: (a) isolating genomic DNA, cDNA or mRNA
from a specimen to be screened; (b) amplifying DNA fragments
encoding the AP domain or portions thereof from the genomic DNA,
cDNA or mRNA (c) denaturing the amplified product; (d) subjecting
the denatured product to electrophoresis; and (e) detecting
mutations by comparing the mobility of the denatured amplified
product to a control DNA encoding the AP domain or portions thereof
corresponding positionally to the DNA fragments amplified in step
(b); wherein said control DNA encoding the AP domain or portions
thereof is selected from the truncated bcl-2 gene and fragments
thereof claimed in claim 1.
14. The method of claim 13, wherein truncated bcl-2 gene extends
from nucleotide no. 151--nucleotide no. 255 (SEQ ID NO:14).
15. A method of screening for mutations in the AP domain of Bcl-2,
said method comprising: (a) isolating genomic DNA, cDNA or mRNA
from a specimen to be screened; (b) amplifying DNA fragments
encoding the AP domain or portions thereof from the genomic DNA,
cDNA or mRNA; (c) mixing the amplified product with labeled PCR
product from the corresponding position in a control AP domain or
portion thereof; (d) denaturing and annealing the mixed PCR
products; and (e) analyzing for mismatched nucleotides by
electrophoresis following chemical modification; wherein the
control DNA encoding the AP domain or portion thereof is selected
from the truncated bcl-2 gene and fragments thereof claimed in
claim 1.
16. The method of claim 15, wherein truncated bcl-2 gene extends
from nucleotide no. 151--nucleotide no. 255 (SEQ ID NO:14).
17. A method of screening for mutations in the AP domain of Bcl-2,
said method comprising: (a) isolating genomic DNA, cDNA or mRNA
from a specimen to be screened; (b) amplifying DNA fragments
encoding the AP domain or portions thereof from the genomic DNA,
cDNA or mRNA; and (c) sequencing the amplified DNA product.
18. An isolated cDNA comprising a sequence that encodes a
polypeptide that binds in a double transformation to a truncated
Bcl-2 protein defined by residues 51 to any of residues 85-97 (SEQ
ID NOS:1-13) and fragments thereof that contain at least one amino
acid needed to maintain full or partial anti-proliferation activity
of the AP domain.
19. The polypeptide encoded by the isolated cDNA of claim 18.
20. A method for screening for a polypeptide that binds the AP
domain of Bcl-2 protein, said method comprising: (a) conducting a
double transformation wherein one vector expresses a fusion protein
comprising the AP domain or a fragment thereof and a reporter
molecule and the other vector expresses a fusion protein comprising
a complementary protein for the reporter molecule and said
polypeptide to be screened; (b) monitoring for activation of the
reporter molecule; and (c) isolating cDNA that encodes the protein
that binds to said AP domain or said fragment thereof, wherein said
AP domain or fragment thereof is a truncated Bcl-2 protein
comprising residues 51 to any of 85-97 (SEQ ID NOS:1-13) or a
fragment thereof that contains at least one amino acid needed to
maintain full or partial anti-proliferation activity of the AP
domain.
21. An isolated cDNA comprising a sequence that encodes a
polypeptide that binds wt bcl-2 in a double transformation and does
not bind Bcl-2.DELTA.51 to 85-97 or deletions of a fragment of a
bcl-2 gene that contains at least one codon encoding an amino acid
needed to maintain full or partial anti-proliferation activity of
the AP domain.
22. A polypeptide encoded by the isolated cDNA of claim 21.
23. A method of screening for a polypeptide that binds the AP
domain of Bcl-2 protein, said method comprising: (a) conducting a
first double transformation wherein one vector expresses a fusion
protein comprising the AP domain and a reporter molecule and the
other vector expresses a fusion protein comprising a complementary
protein for the reporter molecule and said polypeptide to be
screened; (b) conducting a second double transformation wherein one
vector expresses a fusion protein comprising Bcl-2 with the AP
domain or fragments of Bcl-2 that contain at least one amino acid
needed to maintain full or partial anti-proliferation activity of
the AP domain deleted and a reporter molecule and the other vector
expresses a fusion protein comprising a complementary protein for
the reporter molecule and said polypeptide to be screened; (c)
monitoring for activation of the reporter molecule in both double
transformations; and (d) isolating cDNA that encodes a polypeptide
that binds in step (a) but not in step (b).
24. A method for screening for a polypeptide that interacts with
the AP domain of Bcl-2 protein, the method comprising: (a)
expressing cDNA that encodes a polypeptide to be screened; (b)
immobilizing the expressed polypeptide; and (c) detecting
interaction with a polypeptide comprising the AP domain or fragment
thereof wherein the AP domain or fragment thereof is a truncated
Bcl-2 protein comprising residues 51 to any of residues 85-97 (SEQ
ID NOS:1-13) or a fragment thereof that contains at least one amino
acid needed to maintain full or partial anti-proliferation activity
of the AP domain.
25. A method for screening for a polypeptide that interacts with
the AP domain of Bcl-2 protein, the method comprising: (a)
immobilizing a polypeptide comprising the AP domain or fragment of
the AP domain; (b) contacting the immobilized polypeptide with
putative interacting protein; and (c) identifying interacting
protein; wherein the AP domain or fragment thereof is a truncated
Bcl-2 protein comprising residues 51 to any of residues 85-97 (SEQ
ID NOS:1-13) or a fragment thereof that contains at least one amino
acid needed to maintain full or partial anti-proliferation activity
of the AP domain.
26. An isolated antibody that binds to the AP domain of Bcl-2 or
fragments of Bcl-2 that contain at least one amino acid needed to
maintain full or partial anti-proliferation activity of the AP
domain.
27. The isolated antibody of claim 27, wherein the AP domain
consists of Bcl-2 residue 51 to any of residues 85-97 (SEQ ID
NO:1-13) or fragments of the AP domain that contain at least one of
Ser residue 51, Pro residue 57, Ser residue 62, Thr and Ser
residues 69 and 70, Thr residue 74, and Pro residue 75.
28. The isolated antibody of claim 26, wherein the AP domain
consists of Bcl-2 residues 51-85 (SEQ ID NO:1).
29. The isolated antibody of any one of claims 26, 27 or 28,
wherein the antibody is a monoclonal antibody that specifically
binds to the AP domain.
30. A hybridoma that makes the monoclonal antibody of claim 29.
31. A method of producing isolated AP domain or fragments thereof,
said method comprising: (a) constructing a vector comprising DNA
encoding the AP domain or fragments thereof containing at least one
codon encoding an amino acid needed to maintain full or partial
anti-proliferation activity of the AP domain; (b) transforming a
suitable host cell with said vector of step (a); (c) culturing said
host cell under conditions that allow expression of said domain or
fragments thereof by said host cell; and (d) isolating said domain
or fragment thereof expressed by said host cell of step (c).
32. The method of claim 31, wherein said host cell is a mammalian
cell.
Description
FIELD OF THE INVENTION
[0002] The present invention relates generally to the field of cell
physiology, and more particularly to tumorigenesis and to
apoptosis, i.e. programmed cell death. The novel peptides and
nucleotides of the invention are useful in molecular screening of
human tumors for the presence of mutations that allow the
proliferation of cells that were otherwise marked for apoptosis.
The novel peptides and nucleotides are also useful to screen for
proteins that play a role in the modulation of cellular
proliferation.
BACKGROUND OF THE INVENTION
[0003] The bcl-2 gene was discovered as typically involved in the
t(14;18) chromosomal translocations observed in human follicular
lymphoma (1-3). This chromosomal rearrangement results in
deregulated high-level expression of the bcl-2 gene. In addition,
Bcl-2 is also expressed at elevated levels in a variety of other
tumors (4-6). The Bcl-2 protein suppresses apoptosis induced by a
multitude of stimuli (7,8). Suppression of apoptosis by Bcl-2,
while allowing cell survival, is characterized by growth arrest
associated with Bcl-2 activity (40). Although bcl-2 was discovered
as a candidate oncogene, conventional transformation assays
indicate that it does not possess dominant oncogenic activity (9).
It is therefore believed that unlike other oncogenes, bcl-2
contributes to oncogenesis primarily by extending cell viability,
thereby perturbing the homeostatic mechanisms that control cell
number and by providing an environment for other genetic changes
(10).
[0004] In spite of a lack of detectable autonomous transforming
activity, bcl-2 has been shown to synergize with c-myc in the
generation of malignant cells (11). Since constitutive expression
of c-myc induces apoptosis under certain conditions (12-14) that
can be suppressed by Bcl-2 (14-16), it appears that the
c-myc-cooperating oncogenic activity of bcl-2 may be related to its
anti-apoptosis activity. In addition, Bcl-2 can also efficiently
suppress apoptosis induced by tumor suppressor proteins such as p53
(17-21). This suggests that Bcl-2 may contribute to oncogenesis by
suppressing apoptosis induced by oncogenes and tumor suppressor
genes.
[0005] Although mutations within the Bcl-2 protein that permit
proliferation of cells that would otherwise undergo total apoptosis
could play a more direct role (as opposed to deregulated
expression) in oncogenesis, thus far no such mutants have been
identified in naturally arising tumors or under experimental
conditions.
SUMMARY OF THE INVENTION
[0006] The present inventor here describes the identification and
characterization of a hitherto unrecognized domain within human
Bcl-2, which the inventor has designated the "anti-proliferation
(AP) domain", that is required for the proliferation-restraining
activity of Bcl-2. Mutants in this domain of Bcl-2 are described
that retain the ability to suppress apoptosis induced by the p53
tumor suppressor protein and Myc onco-protein, while allowing
concomitant cell proliferation.
[0007] More specifically, the present inventor has identified a
deletion mutant of Bcl-2 that has a novel activity. The deletion
mutant, designated Bcl2.DELTA.51-85, not only suppresses apoptosis
induced by the tumor suppressor protein p53 and the Myc
onco-protein, but unlike wt Bcl-2, permits continued cell
proliferation. These results may have important implications for
oncogenesis involving Bcl-2. Unlike other oncogenes, the bcl-2
proto-oncogene promotes cell survival without significant cell
proliferation.
[0008] These results suggest that certain mutations can inactivate
a proliferation-restraining activity. Further, the observed effect
against oncogene/anti-oncogene-induced apoptosis may potentially
prove to be of considerable significance in oncogenic events
involving Bcl-2. Such inactivating mutations within the
non-conserved region of Bcl-2 may enhance tumorigenesis by
antagonizing the apoptotic activities of p53 and Myc as well as by
permitting continued cell proliferation.
[0009] The molecular basis for the loss of
proliferation-restraining activity in the Bcl-2 mutant has been
partially elucidated as described in Example 3. The results suggest
that the loss of activity does not correlate with the ability of
Bcl-2 to interact with several proteins. However, the interaction
between the Bcl-2 mutant and the death-promoting protein Bax
appears to be enhanced compared to the interaction of Bax with wild
type Bcl-2. It is not clear whether this enhancement is due to an
increased affinity of the Bcl-2 mutant for Bax or increased
stability of the Bcl-2/Bax complex. The importance of the Bcl-2/Bax
interaction to the proliferation-restraining function of Bcl-2 is
unknown.
[0010] Also, the region deleted in Bcl2.DELTA.51-85 contains
several Ser and Thr residues. It has been reported that Bcl-2
activity can be modulated by phosphorylation (34, 46, 47, and 49).
Analysis of the activity of several Bcl-2 mutants containing amino
acid substitutions at Ser or Thr residues, as described in Example
4, suggests that modulation of the proliferation-restraining
activity by phosphorylation is possible. Alternative explanations
to account for the mutant phenotype are also possible. The deleted
region is rich in Ala and Pro residues. Substitution of Pro
residues in two positions within the AP domain resulted in Bcl-2
mutants that permit enhanced cell proliferation. The possibility
that these residues play some negative regulatory role in Bcl-2
activity remains to be investigated.
[0011] In one aspect then, the invention provides isolated
oligonucleotides that encode the Bcl-2 AP domain or fragments of
the domain. The oligonucleotides and short segments thereof are
useful for screening for mutations in the Bcl-2 AP domain by
methods known in the art, such as single strand conformational
polymorphism (SSCP) and PCR mismatch analysis.
[0012] In another aspect, the present invention is directed to
identifying protein/protein interactions between the Bcl-2 AP
domain and known or as yet unidentified cellular proteins. The
Bcl-2 AP domain is also useful in the identification and cloning of
genes whose protein products interact with this domain in Bcl-2.
The interacting proteins may play a role in modulation of cellular
proliferation.
[0013] The present invention also relates to an isolated
polypeptide that is the Bcl-2 AP domain and fragments of the
domain. The domain may be a target for allosteric regulators of
Bcl-2 function, such as protein kinases and/or phosphatases.
Accordingly, peptides derived from this domain, prepared
synthetically or as bacterially expressed fusion proteins, can be
used as substrates to identify and characterize potential
regulatory kinases and/or phosphatases.
[0014] The invention further provides screening methods to identify
molecules that modulate the proliferation-restraining activity of
the AP domain. In one aspect, such screening methods involve the
effect of a putative modulating molecule on the short term or long
term proliferation of cells in culture expressing the AP domain. In
another aspect, putative modulating molecules can be identified by
screening for agents that disrupt necessary protein/protein
interactions mediated by the AP domain, using in vitro binding
assays.
[0015] In yet another aspect, the invention provides for expression
vectors containing genetic sequences, hosts transformed with such
expression vectors, and methods for producing the AP domain and
fragments of the domain that hinder or completely block
proliferation.
[0016] In additional aspects, the present invention relates to
antibodies that specifically bind to the AP domain and fragments of
the domain that hinder or completely block cell proliferation.
Peptides comprising the domain are useful for producing antibodies
thereto. Such antibodies are useful for detecting and isolating
proteins comprising the AP domain in biological specimens
including, for example, cells from all human tissues including
heart tissue, lung tissue, tumor cells, brain tissue, placenta,
liver, skeletal muscle, kidney, and pancreas, as well as for
modulating the proliferation-restraining activity of proteins
comprising the AP domain, in and from such biological specimens,
and constitute additional aspects of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 shows the domain structure of Bcl-2. The various
conserved domains (BH1-4) are indicated. BH1-3 are conserved among
both survival-promoting and death-promoting members of the Bcl-2
family of proteins. BH1 and 2 are described in ref. 33. BH3 is
described in ref. 32. BH4 is conserved among survival-promoting
members and corresponds to box A described by Reed and coworkers
(ref. 38). TM indicates transmembrane domain. NH-1 indicates the
E1B nineteen K homology domain (21). The amino acid sequence (SEQ
ID NO:1) deleted in mutant Bcl2.DELTA.51085 is indicated.
[0018] FIGS. 2A to 2E illustrate suppression of p53-induced
apoptosis by Bcl-2. FIG. 2A shows an immunoprecipitation analysis
of Bcl-2 and Bcl2.DELTA.51-85 expression in BRK-p53val135-E1A
cells. FIG. 2B is a graph showing survival/proliferation of
BRK-p53val135-E1A cells at 32.5.degree. C. .circle-solid., pRcCMV
vector; .box-solid., wt Bcl-2; .tangle-solidup., Bcl2.DELTA.51-85.
FIGS. 2C-E show the growth of colonies of cells transfected with
vectors carrying various Bcl-2 genes. The Figures illustrate the
long-term proliferation of BRK-p53val135-E1A cells. FIG. 2C, pRcCmV
vector; FIG. 2D, wt Bcl-2; FIG. 2E, Bcl2.DELTA.51-85.
[0019] FIGS. 3A and 3B show suppression of Myc-induced apoptosis by
Bcl-2. FIG. 3A shows the results of an immunoprecipitation analysis
of Bcl-2 and Bcl2.DELTA.51-85 expression in Rat1MycER-Hygro cells.
FIG. 3B is a graph showing survival/proliferation of RatMycER-Hygro
cells. .circle-solid., pRcCMV vector; .box-solid., wt Bcl-2;
.tangle-solidup., Bcl2.DELTA.51-85.
[0020] FIGS. 4A and B show the interaction of Bax with Bcl-2 and
Bcl2.DELTA.51-85. BSC40 cells were transfected with pTM-HA Bax and
pTM-Bcl-2 or pTM-Bcl2.DELTA.51-85 and infected with vaccinia virus
vTF7-3. .sup.35S-labeled proteins were immunoprecipitated either
with HA mouse monoclonal antibody (FIG. 4A) or Bcl-2 rabbit
polyclonal antibody (FIG. 4B) and analyzed on 13%
SDS-polyacrylamide gels. FIG. 5 is a graph showing survival of
BRK-p53val153-EIA cells expressing point mutants of Bcl-2. The
number of viable cells was determined at various times after
shifting to 32.5.degree. C. by trypan blue exclusion and is plotted
as a percentage of the number of live cells at the start of the
experiment.
DETAILED DESCRIPTION OF THE INVENTION
[0021] Isolated, in the context of the invention, indicates that
some intervention occurs that increases the level of purity of a
molecule over that found in nature.
[0022] As mentioned above, the present invention is based upon the
discovery of a heretofore unidentified domain of the human Bcl-2
protein. This "anti-proliferation" or "AP" domain is required for
modulation of cell proliferation. More specifically, the AP domain
completely blocks cell proliferation.
[0023] The nucleotide sequence of human Bcl-2 according to this
invention is based on those described in reference 41, Genbank
Accession #X06487 and in reference 42, Genbank Accession #M13994.
The Bcl-2 nucleotide sequence described in reference 41 has a G at
position 189 and an A at position 287. The Bcl-2 nucleotide
sequence described in reference 42 contains an alternate nucleotide
(C in place of G) at position 189 and an alternate nucleotide (G in
place of A) at position 287 resulting in an amino acid change at
residue 96 of Thr to Ala. The nucleotide sequence for Bcl-2
reported by Cleary et al (51), Genbank Accession #M14745, contains
an alternate nucleotide at position 175 (A in place of C),
resulting in an amino acid change at residue 59 of Pro to Thr.
[0024] As used herein, the phrase "anti-proliferation (or AP)
domain" means a truncated human Bcl-2 protein comprising amino acid
residue 51 to any of amino acid residues 85-97. (SEQ ID
NOS:1-13).
[0025] Thus, in addition to the core residues, i.e. residues 51 to
any of amino acid residues 85-97, the AP domain can include
stretches of 1 or more amino acids in the amino-terminal direction
from residue 85 and/or 1 or more amino acids in the
carboxyl-terminal direction from residue 97, provided that the
protein is truncated. That is, the AP domain of the present
invention is not intended to include the full-length Bcl-2 protein.
For example, the AP domain can include the core residues, i.e.,
residue 51 to any of amino acid residues 85-97, and/or 5, 10, 15,
20 residues, and so on, in increments of 5 to the amino-terminus of
residue 51 and/or carboxyl-terminus of residue 97.
[0026] The sequences of the polypeptide that make up the core
residues, i.e. SEQ ID NOS:1-13, are set forth in the section
following the examples. The sequences of any additional stretches
upstream or downstream of the core residues may be ascertained from
the literature (E.G. 41, 42, 51) and protein databases such as
EMBL.
[0027] Further, the data in Example 5 herein indicates that certain
amino acids are needed to maintain full or partial
anti-proliferation activity of the AP domain. For example, any one
of Ser at position 51, Pro at position 57, Ser at position 62,
Thr-Ser at positions 69 and 70, Thr at position 74, and Pro at
position 75 may contribute to the anti-proliferation function of
the AP domain which is lost when residues 51-85 are deleted. Thus,
fragments of the AP domain that include any one or more of the
residues that fully or partially restores anti-proliferation
activity are within the present invention.
[0028] Functional equivalents of the polypeptide that make up the
core residues as defined by SEQ ID NOS: 1-13 are also within the
present invention. By "functional equivalent" is meant a peptide
possessing a biological activity or immunological characteristic
substantially similar to that of the polypeptides that make up the
core residues, and is intended to include "variants", "analogs",
"homologs", or "chemical derivatives" possessing such activity or
characteristics. Functional equivalents of the polypeptides that
make up the core residues, then, may not share an identical amino
acid sequence, and conservative or non-conservative amino acid
substitutions of conventional or unconventional amino acids are
possible. However, in the present invention any of Ser at position
51, Pro at position 57, Ser at position 62, Thr and Ser at
positions 69 and 70, Thr at position 74, or Pro at position 75, may
not be Ala.
[0029] Reference herein to "conservative" amino acid substitution
is intended to mean the interchangeability of amino acid residues
having similar side chains. For example, glycine, alanine, valine,
leucine and isoleucine make up a group of amino acids having
aliphatic side chains; serine and threonine are amino acids having
aliphatic-hydroxyl side chains; asparagine and glutamine are amino
acids having amide-containing side chains; phenylalanine, tyrosine
and tryptophan are amino acids having aromatic side chains; lysine,
arginine and histidine are amino acids having basic side chains;
and cysteine and methionine are amino acids having
sulfur-containing side chains. Interchanging one amino acid from a
given group with another amino acid from that same group would be
considered a conservative substitution. Preferred conservative
substitution groups include asparagine-glutamine, alanine-valine,
lysine-arginine, phenylalanine-tyrosine and
valine-leucine-isoleucine.
[0030] Functional equivalents that possess immunological
characteristics substantially similar to that of the polypeptides
that make up the core residues are useful, for example, as an
antigen for raising antibodies against the AP domain or fragments
thereof or for detection or purification of antibodies against the
AP domain or fragments thereof.
[0031] The nucleotide sequences that encode the AP domain as
defined herein are also within the present invention. The nucleic
acid compositions of the invention will generally be in RNA or DNA
forms, mixed polymeric forms, or any synthetic nucleotide structure
capable of binding in a base-specific manner to a complementary
strand of nucleic acid. The described nucleic acid embodiment is
typically derived from genomic DNA or cDNA, prepared by synthesis,
or derived from combinations thereof, including polymerase chain
reaction (PCR) products.
[0032] The oligonucleotides that encode the core amino acids are
those bounded by nucleotide 151 to any of nucleotides 255-291 (SEQ
ID NO: 14-50), where nucleotide 1 is the first nucleotide of the
codon encoding the first amino acid of Bcl-2. In these sequences
the nucleotide at position 175 can be C or A, the nucleotide at
position 189 can be C or G, and the nucleotide at position 287 can
be A or G.
[0033] The oligonucleotide sequences that encode the core residues,
i.e. SEQ ID NOS: 14-50, are set forth in the section following the
examples. The cDNA sequences toward the 5' end of nucleotide 151
and toward the 3' end of nucleotide 291 may be ascertained from the
literature (E.G. 22, 42, 51) as well as from sequence databases
such as Genbank.
[0034] Oligonucleotide fragments of oligonucleotide sequences that
encode the AP domain are also included within the present invention
and include fragments that contain at least one codon encoding an
amino acid needed to maintain full or partial anti-proliferation
activity of the AP domain. Examples are fragments that retain any
one of the codons defined by nucleotides 151-153 (coding for Ser
51), 169-171 (coding for Pro 57), nucleotides 184-186 (coding for
Ser 62), 204-210 (coding for Thr 69 and Ser 70), 220-222 (coding
for Thr 74), and 223-225 (coding for Pro 75).
[0035] The instant oligonucleotides and polypeptides may be
obtained as described herein, such as by recombinant means. For
example, nucleotide sequences encoding the AP domain polypeptides
or fragments thereof of the invention may be inserted into a
suitable DNA vector, such as a plasmid, and the vector used to
transform a suitable host. The recombinant AP polypeptide or
fragment is produced in the host by expression. The transformed
host may be a prokaryotic or eukaryotic cell, including a mammalian
cell. The instant oligonucleotides and polypeptides may also be
used to obtain homologous nucleic acids and proteins by
hybridization, for example, an instant nucleic acid can be used as
a probe of a gene bank to identify clones with suitable homology
therewith. Also, within the confines of available technology, the
oligonucleotides may be synthesized in vitro using, for example,
solid phase oligonucleotide and oligopeptide synthetic methods
known in the art.
[0036] The present invention also includes fusion polypeptides
between the AP domain, or fragments thereof, or truncated wt Bcl-2
polypeptides including the AP domain, and other proteins or
polypeptides. For example, fusions may include proteins that serve
as purification targets, such as, but not limited to glutathione
S-transferase (GST) (43) and the FLAG epitope tag (Eastman Kodak).
In addition, fusions may include polypeptides that may have amino
acid residues that have been or can be chemically modified by
phosphorylation, biotinylation, acylation, or other moieties, using
methods known in the art. Fusion polypeptides will typically be
made by using either synthetic polypeptide or recombinant nucleic
acid methods known in the art.
[0037] The functional importance of the AP domain is related to its
ability to regulate cell proliferation. This regulation may be
mediated by one or more protein/protein interactions between the
domain and known Bcl-2 interacting proteins such as Bax (44),
Nip1-3 (29), Bik (32), Bak (31), R-ras (52), BAG-1 (45) and c-raf-1
(30) (see also Example 4) or as yet unidentified cellular proteins.
The polypeptides of the present invention are useful to screen for
proteins that interact with the AP domain, and these proteins and
cDNA's encoding these proteins are also part of the invention. Such
molecules are useful as agents for modulation of tumorigenesis and
apoptotic activity of cells.
[0038] Methods for screening for proteins that interact with the AP
domain are well known in the art and include the yeast two-hybrid
system (39, 28) and expression cloning strategies using recombinant
fusion proteins. (53, 54)
[0039] The in vivo genetic strategy designated `two hybrid` cloning
(39, 28) permits rapid genetic screening in yeast of molecules that
associate, and the method has been used to isolate from expression
libraries cDNA clones that code for proteins interacting with
several known proteins.
[0040] Briefly, the method relies on the double transformation of
yeast hosts with plasmids that encode fusion proteins. One plasmid
carries partial sequences for a reporter molecule, for example, the
GAL4 DNA binding domain, at the amino terminus of the fusion
protein and sequences for the known protein, to which a ligand is
sought, also known as the "bait" at the carboxyl-terminus. For
example, the bait can be the AP domain polypeptide.
[0041] The second plasmid comprises sequences encoding a
complementary protein for the reporter molecule, in the above case,
required by the GAL4 DNA binding domain, such as the GAL4
activation domain, at the amino terminus and expressed products of
individual cDNA from a bank at the carboxyl-terminus. A suitable
host is used to enable the selection planned. In the scenario
discussed, the host would be one wherein the expression of
.beta.-galactosidase is under the control of the GALL promoter.
[0042] Selection of double transformants are those that express
.beta.-galactosidase, hence would be blue colonies on an X-gal
plate because the bait protein encoded by the cDNA of the second
plasmid bind and that interaction juxtaposes the two GAL4
regulatory elements required for .beta.-galactosidase
expression.
[0043] An additional related strategy is to isolate positive clones
from the two hybrid assay that interact with GAL4 DNA-binding
domain-Bcl-2 (wt) fusion but not with a GAL4 DNA-binding
domain-Bcl-2.DELTA.51-85 fusion. Such interacting proteins may
require the identified domain for their interaction.
[0044] Thus, the present invention provides a method for screening
for a polypeptide that binds the AP domain of Bcl-2 protein, the
method comprising:
[0045] (a) conducting a double transformation wherein one vector
expresses a fusion protein comprising the AP domain or a fragment
thereof and a reporter molecule and the other vector expresses a
fusion protein comprising a complementary protein for the reporter
molecule and the polypeptide to be screened;
[0046] (b) monitoring for activation of the reporter molecule;
and
[0047] (c) isolating cDNA that encodes the protein that binds to
the AP domain or the fragment thereof,
[0048] wherein the AP domain or fragment thereof is a truncated
Bcl-2 protein comprising residues 51 to any of residues 85-97 (SEQ
ID NOS:1-13) or a fragment thereof that contains at least one amino
acid needed to maintain full or partial anti-proliferation activity
of the AP domain.
[0049] In a related embodiment, the present invention also provides
a method of screening for a polypeptide that binds the AP domain of
Bcl-2 protein, the method comprising:
[0050] (a) conducting a first double transformation wherein one
vector expresses a fusion protein comprising the AP domain and a
reporter molecule and the other vector expresses a fusion protein
comprising a complementary protein for the reporter molecule and
the polypeptide to be screened;
[0051] (b) conducting a second double transformation wherein one
vector expresses a fusion protein comprising Bcl-2 with the AP
domain or a fragment of Bcl-2 that contains at least one amino acid
needed to maintain full or partial anti-proliferation activity of
the AP domain deleted and a reporter molecule and the other vector
expresses a fusion protein comprising a complementary protein for
the reporter molecule and the polypeptide to be screened;
[0052] (c) monitoring for activation of the reporter molecule in
both double transformations; and
[0053] (d) isolating cDNA that encodes a polypeptide that binds in
step (a) but not in step (b).
[0054] In a second example of methods of screening for proteins
that interact with the AP domain, a cDNA encoding Bcl-2 residues
51-85 is cloned into an E.coli expression vector that will encode a
glutathione S-transferase (GST)-Bcl-2 domain fusion protein.
[0055] The fusion protein is isolated following expression in
bacteria and radiolabeled for use as a probe to screen for cDNA of
proteins capable of interacting with the AP domain from a human
cell .lambda.-phage expression library. (53, 54) Briefly, a
.lambda.-phage expression library (e.g. .lambda.-ZAP, Stratagene)
is plated on E.coli and resulting plaques are transferred to
isopropyl-.beta.-D-thogalactoside (IPTG)-impregnated nitrocellulose
filters to induce protein expression. .sup.32P-radiolabeled GST-AP
domain fusion proteins or unlabelled GST-AP domain fusion proteins
that can be detected with an anti-GST antibody, are used as a probe
to screen for expressed proteins capable of interacting with the AP
domain. Positive clones can be isolated and the gene encoding a
protein capable of interacting with the AP domain can be sequenced
and characterized.
[0056] Thus, the present invention provides a method for screening
for a polypeptide that interacts with the AP domain of Bcl-2
protein, the method comprising:
[0057] (a) expressing cDNA that encodes a polypeptide to be
screened;
[0058] (b) immobilizing the expressed polypeptide; and
[0059] (c) detecting interaction with a polypeptide comprising the
AP domain or fragment thereof;
[0060] wherein the AP domain or fragment thereof is a truncated
Bcl-2 protein comprising residues 51 to any of residues 85-97 (SEQ
ID NOS:1-13) or a fragment thereof that contains at least one amino
acid needed to maintain full or partial anti-proliferation activity
of the AP domain.
[0061] Alternatively, the biochemical isolation of interacting
molecules is also possible using isolated polypeptides comprising
the Bcl-2 AP domain. For example, GST-AP domain fusion proteins can
be immobilized on glutathione (GSH)-agarose columns to capture
interacting proteins from cell lysates. Cell lysates from
BRK-p53val135-E1A cells are passed over the column. Following
washing to remove non-binding proteins, interacting proteins can be
eluted using GSH or other conditions known to disrupt
protein/protein interactions such as salt, pH, guanidine HCl, or
detergent gradients. Eluted proteins can be identified, for
example, by SDS-PAGE and microsequencing. If necessary,
oligonucleotide probes based on the protein sequence can be used to
clone the corresponding gene from an appropriate cDNA library.
[0062] Thus, the present invention provides a method for screening
for a polypeptide that interacts with the AP domain of Bcl-2
protein, the method comprising:
[0063] (a) immobilizing a polypeptide comprising the AP domain or
fragment of the AP domain;
[0064] (b) contacting the immobilized polypeptide with putative
interacting protein; and
[0065] (c) identifying interacting protein;
[0066] wherein the AP domain or fragment thereof is a truncated
Bcl-2 protein comprising residues 51 to any of residues 85-97 (SEQ
ID NOS:1-13) or a fragment thereof that contains at least one amino
acid needed to maintain full or partial anti-proliferation activity
of the AP domain.
[0067] The present invention includes the use of the AP domain or
fragments for the identification of agents that modulate AP domain
mediated functions. Such agents may include peptides comprising the
AP domain or mutants of the AP domain or comprising an AP domain. A
"mutant" as used herein refers to a peptide having an amino acid
sequence that differs from the amino acid sequence of the naturally
occurring peptide or protein by at least one amino acid. Mutants
may have the same biological and immununological activity as the
naturally occurring AP domain. However, the biological or
immunological activity of mutants may differ or be lacking.
Identification of such agents can be accomplished by the screening
of peptide or compound libraries, or other information banks, in
assays for agonists or antagonists that enhance or inhibit AP
domain function, e.g. survival-promoting and
proliferation-restraining activity, as well as protein binding.
[0068] For example, BRK-p53val135-E 1 A cells expressing Bcl-2 or a
truncated version of Bcl-2 comprising the AP domain can be used to
screen for agents that inhibit the proliferation-restraining
activity the AP domain detected by increased proliferation in the
short term assay and/or allowing colony formation in the long term
assay.
[0069] In another example, agents can be identified that modulate
the proliferation-restraining activity of the AP domain by
screening for compounds that influence protein/protein interactions
mediated by the AP domain using an in vitro binding assay. In such
as an assay, a GST fusion protein comprising the AP domain is
immobilized to GSH-agarose. Binding of a radiolabaled-interacting
protein in the presence of one or more compounds to be tested would
be quantitated by scintillation counting. Inhibitors of the
interaction would result in a decrease in associated interacting
protein. For rapid-throughput screening, the GST/AP-domain fusion
protein and biotinylated interacting protein are used in a
multi-well plate format. Biotinylated proteins can be expressed and
isolated from E.coli using PinPoint vectors (Promega) by known
methods. The purified biotinylataed protein is immobilized on a
neutravidin-coated plate and binding of the GST/Ap-domain fusion
protein in the presence of test compounds is detected by ELISA
using an anti-GST monoclonal antibody. Inhibitors of the
interaction would score as a decreased ELISA signal.
[0070] A high speed screen using immobilized or "tagged"
combinatorial libraries can be used to identify agents that bind
directly to the AP domain. Such agents are candidates to be tested
for their ability to enhance or inhibit the
proliferation-restraining activity of Bcl-2.
[0071] The AP domain may be a target for allosteric regulators of
Bcl-2 function such as protein kinases and/or phosphatases.
Phosphorylation of Bcl-2 has been reported (46-49) and it has been
suggested that phosphorylation/dephosphorylation may play a role in
the regulation of Bcl-2 function. The identified domain of Bcl-2
contains several potential phosphorylation sites. Thus, the
polypeptides of the present invention comprising the AP domain can
be used as substrates to measure an enzymatic activity, such as
kinase or phosphatase. In this aspect, in vitro kinase assays are
carried out by incubating cell lysates, such as derived from
BRK-p53val135-E1A cells, with AP domain polypeptides, prepared
synthetically or as bacterially expressed fusion proteins, in the
presence of .sup.23P-labeled ATP in 10 mM Tris buffer containing 10
mM MgCl.sub.2 and 1 .mu.M unlabeled ATP. Phosphatase activity is
detected by incubating cell lysates with phosphorylated AP domain
polypeptide, derived from in vitro kinase assays described above or
isolated from cells, and following the release of radiolabeled
phosphate from the AP domain. Purification and sequencing of the
protein responsible for this activity can be accomplished by
standard methods such as those described in "Protein Purification:
Principles and Practice," by Robert Scopes (Ed: C. Cantor, Springer
Verlag, Heidelberg, 1982).
[0072] Synthetic peptides or fusion proteins containing this domain
can be used for immunizing animals in the production of polyclonal
or monoclonal antibodies that bind to this domain in Bcl-2. Such
antibodies would be useful as reagents for studying the function of
this domain. For example, microinjection of anti-domain antibodies
may alter the cell cycle arrest activity of Bcl-2. Such antibodies
may also prove to be useful in screening for mutations in this
domain of Bcl-2 that cause alterations in antibody binding. These
mutations may correlate with alterations in Bcl-2 function.
[0073] The AP polypeptides of the invention also may be used for
the detection of Bcl-2 by means of standard assays including
radioimmunoassays and enzyme immunoassays.
[0074] The polypeptides of the present invention or fusion proteins
thereof are also useful to make antibodies for detection or
determination of proteins comprising the AP domain, for example, in
fractions from tissue/organ excisions, by means of immunochemical
or other techniques in view of the antigenic properties
thereof.
[0075] Immunization of animals with polypeptides comprising the AP
domain alone or in conjunction with adjuvants by known methods can
produce antibodies specific for the AP domain polypeptide.
Antiserum obtained by conventional procedures may be utilized for
this purpose. For example, a mammal, such as a rabbit, may be
immunized with a peptide comprising the AP domain, thereby inducing
the formation of polyclonal antibodies thereagainst. Monoclonal
antibodies also may be generated using known procedures.
[0076] If the target molecule is poorly immunogenic, known methods
for enhancing immunogenicity, such as, use of adjuvants, use of
fragments of the target molecule as antigen, conjugating the target
molecule or fragments thereof to a known carrier, such as albumin
or keyhole limpet hemocyanin, immunizing immune cells in vitro and
the like, as known in the art can be used.
[0077] Antibodies against the AP domain polypeptides or fragments
thereof of the invention may be used to screen cDNA expression
libraries for identifying clones containing cDNA inserts encoding
structurally related, immunocrossreactive proteins that may be
members of an AP domain family of proteins. Screening of cDNA and
mRNA expression libraries is known in the art. Similarly,
antibodies against AP domain polypeptides or fragments thereof can
be used to identify or purify immunocrossreactive proteins related
to this domain, or to detect or determine the amount of proteins
containing the AP domain in a cell or cell population, for example,
in tissue or cells, such as lymphocytes, obtained from a patient.
Known methods for such measurements include immunopreciptiation of
cell extracts followed by PAGE, in situ detection by
immunohistochemical methods, and ELISA methods, all of which are
well know in the art. In addition, antibodies against the AP domain
or fragments thereof may be used to modulate the
proliferation-restraining activity of proteins comprising the AP
domain.
[0078] Accordingly, the present invention also provides an isolated
antibody that binds to the AP domain of Bcl-2 and a hybridoma that
makes monoclonal antibody that specifically binds to the AP
domain.
[0079] The cDNA of the present invention may be used for screening
for mutations in the AP domain in, for example, human tumors.
Indeed, mutations within this domain associated with non-Hodgkin's
lymphomas have been reported including a change in the nucleotide
(T in place of C) at position 175 resulting in a substitution of
Pro 59 with Ser (50).
[0080] Methods for screening for such mutations have been
described, and include single strand conformational polymorphism
(SSCP) of polymerase chain reaction-amplified DNA fragments
(SSPC-PCR) (55, 56) and PCR-mismatch analysis (50, 51).
[0081] In SSCP-PCR, oligonucleotide primers are used to amplify the
segment of the Bcl-2 gene encoding the AP domain from DNA or mRNA
isolated from a test sample or from cDNA made from the test sample.
The PCR product is then heat denatured, subjected to
electrophoresis on polyacrylamide gels and transferred to a nylon
membrane. The fragment can be detected by a chemiluminescence
detection system and the relative mobility of the test fragment
with a control fragment from wt Bcl-2 is determined. A single base
change can be detected by this method.
[0082] Accordingly, the present invention provides a method of
screening for mutations in the AP domain of Bcl-2, the method
comprising:
[0083] (a) isolating genomic DNA, cDNA or mRNA from a specimen to
be screened;
[0084] (b) amplifying DNA fragments encoding the AP domain or
portions thereof from the genomic DNA, cDNA or mRNA;
[0085] (c) denaturing the amplified product;
[0086] (d) subjecting the denatured product to electrophoresis;
and
[0087] (e) detecting mutations by comparing the mobility of the
denatured amplified product to a control DNA encoding the AP domain
or portions thereof corresponding positionally to the DNA fragments
amplified in step (b);
[0088] wherein the control DNA encoding the AP domain or portions
thereof is from the truncated cDNA encoding the bcl-2 gene and
fragments of the truncated cDNA.
[0089] Alternatively, in PCR-mismatch analysis, PCR products from
the test sample are mixed with radiolabeled PCR products from the
wild type Bcl-2 AP domain. The mixed PCR material is denatured and
then annealed. Chemical modification and cleavage of heteroduplexes
containing mismatched nucleotides is analyzed by gel
electrophoresis. PCR-generated DNAs containing mutations are then
subcloned and sequenced to identify the precise nature of the
mutation.
[0090] Thus, the present invention provides a method of screening
for mutations in the AP domain of Bcl-2, said method
comprising:
[0091] (a) isolating genomic DNA, cDNA or mRNA from a specimen to
be screened;
[0092] (b) amplifying DNA fragments encoding the AP domain or
portions thereof from the genomic DNA, cDNA or mRNA;
[0093] (c) mixing the amplified product with labeled PCR product
from the corresponding position in a control AP domain or portion
thereof;
[0094] (d) denaturing and annealing the mixed PCR products; and
[0095] (e) analyzing for mismatched nucleotides by electrophoresis
following chemical modification;
[0096] wherein the control DNA encoding the AP domain or portions
thereof is selected from the truncated cDNA encoding the bcl-2 gene
and fragments of the truncated cDNA.
[0097] In a related embodiment, the present invention also provides
a method of screening for mutations in the AP domain of Bcl-2, said
method comprising:
[0098] (a) isolating genomic DNA, cDNA or mRNA from a specimen to
be screened;
[0099] (b) amplifying DNA fragments encoding the AP domain or
portions thereof from the genomic DNA, cDNA or mRNA; and
[0100] (c) sequencing the amplified DNA product.
[0101] Of course, the polynucleotide sequences of the invention may
be used in the PCR method to detect the presence of mRNA encoding
AP domain polypeptides in for, example, cells from all human
tissues including heart tissue, lung tissue, tumor cells, brain
tissue, placenta, liver, skeletal muscle, kidney, and pancreas.
EXAMPLES
[0102] The invention will now be described by means of working
examples that are not intended to be limiting.
[0103] Materials and Methods
[0104] Plasmids. Plasmid pRcCMV-Bcl-2 was constructed by cloning
the human bcl-2 gene (22) into the HindIII and XbaI sites of the
mammalian expression vector pRcCMV (Invitrogen). Mutant
Bcl2.DELTA.51-85 was constructed by PCR mutagenesis using a
mutagenic oligonucleotide primer
5'-GGA-CCA-CAG-GTG-GCA-CCG-GGC-TGA-GGC-TAG-CGG-AGA-AGA-AGC-CCG-GTG-CGG-GG-
G-CG-3' (SEQ ID NO:51) and two other primers complementary to the
5' and 3' ends of bcl-2. This mutagenesis introduces an NheI site,
and substitutes an alanine and a serine residue in the deleted
region. The PCR product was cloned into the HindIII and XbaI sites
of pRcCMV to generate pRcCMV-Bcl2.DELTA.51-85. pTM1-based plasmids
expressing wt Bcl-2 and mutant Bcl2.DELTA.51-85 were constructed by
cloning the respective genes into the NcoI and SalI sites of the
vector pTM1 (23).
[0105] Cell lines. The BRK-p53val135-E1A cell line has been
described (21) and was maintained at 38.5.degree. C. in Dulbecco's
modified Eagle medium (DMEM) supplemented with 10% fetal calf
serum. BRK-p53val135-E1A cells stably expressing Bcl-2 were
generated by transfection of various pRcCMV-based Bcl-2 expression
plasmids and selection with G418 (250 .mu.g/ml)(GIBCO/BRL). Rat1a
and Rat1MycER-Hygro cells have been described (14,24). Cells
expressing ER fusion proteins were maintained in DMEM media without
phenol red and 10% fetal calf serum (certified low estrogen
content, GIBCO/BRL). Rat1MycER-Hygro cells expressing Bcl-2 were
selected by transfection with pRcCMV-Bcl2 or
pRcCMV-Bcl2.DELTA.51-85 and selection with 400 .mu.g/ml G418. DNA
transfections were carried out by the standard calcium phosphate
method.
[0106] Cell death assays. BRK-p53val135-E1A cells were plated at
5.times.10.sup.5 cells/35 mm dish. After 12 hours at 38.5.degree.
C., the dishes were shifted to 32.5.degree. C., and at various
intervals cells were trypsinized in triplicates, stained with 0.2%
trypan blue and viable cells were counted. Similarly
5.times.10.sup.5 Rat1-Hygro cells were plated in 35 mm dishes,
incubated for 12 hours at 37.degree. C., washed three times in
serum-free DMEM and maintained in fresh media containing 0.1% fetal
calf serum and 1 .mu.M .beta.-estradiol. Viable cell number was
determined at various intervals.
[0107] Immunoprecipitation. Bcl-2 or Bcl2.DELTA.51-85 proteins were
co-expressed with HA epitope-tagged Bax using the vaccinia virus/T7
coupled expression system as previously described (29). BSC40 cells
were transfected with pTM1 expression plasmids using LipofectAMINE
(GIBCO/BRL) and infected with the recombinant vaccinia virus vTF7-3
(23) expressing the T7 RNA polymerase. Sixteen hours
post-infection, cells were metabolically labeled with 500 .mu.Ci of
.sup.35S-methionine and -cysteine mixture for two hours and lysed
in isotonic buffer (29) containing protease inhibitors (0.04 mg/ml
aprotinin, 0.2 mg/ml leupeptin, 200 .mu.M phenylmethylsulfonyl
fluoride). Lysates were precleared with protein A-Sepharose for 1
hour, which was removed by centrifugation. The proteins were
immunoprecipitated with a rabbit polyclonal antibody specific for
human Bcl-2 or with HA monoclonal antibody (12CA5; Boehringer
Mannheim). The proteins were analyzed by electrophoresis on 13% SDS
polyacrylamide gels and detected by fluorography.
EXAMPLE 1
Effect on p53-Induced Apoptosis
[0108] A non-conserved region located between residues 51 and 85
was examined (FIG. 1) with the rationale that such sequences may
regulate the activity of Bcl-2.
[0109] Deletion of this region of Bcl-2 (Bcl2.DELTA.51-85) did not
significantly alter the level of expression of the mutant protein
(FIGS. 2A; 3A). The effect of Bcl-2 wt and mutant Bcl2.DELTA.51-85
on apoptosis induced by the tumor suppressor protein p53 (25) was
tested. Baby rat kidney (BRK) cells transformed with adenovirus E1A
and a ts mutant of p53 (p53val135) (26) express very high levels of
mutant p53 at the non-permissive (38.5.degree. C.) temperature and
undergo rapid apoptosis after the p53 protein assumes wt
conformation at 32.5.degree. C. (27). This apoptosis can be
efficiently suppressed by Bcl-2 (20). BRK-p53val135-E1A cells were
transfected with pRcCMV vector or pRcCMV-Bcl-2 or
pRcCMV-Bcl2.DELTA.51-85 and G418 resistant colonies were selected
at 38.5.degree. C. As expected, wt Bcl-2 efficiently suppressed
cell death compared to cells transfected with pRcCMV vector (FIG.
2A). Cells expressing Bcl2.DELTA.51-85 did not lose cell viability
significantly at 32.5.degree. C. Surprisingly, however, these cells
also proliferated efficiently at this temperature in contrast to
cells expressing wt Bcl-2 (FIG. 2B). Deletion of additional
residues, form 51-98, resulted in a mutant, Bcl-2.DELTA.51-98 that
was unable to suppress cell death in this assay (FIG. 5, Table 1)
suggesting that residues between 85 and 98 may be critical for
Bcl-2 survival function.
[0110] The effect of mutant Bcl2.DELTA.51-85 on long term
proliferation was also determined. Pooled cell lines transfected
with Bcl-2 wt or Bcl2.DELTA.51-85 or pRcCMV vector were plated at
low cell density, maintained at 32.5.degree. C. for three weeks and
stained with Giemsa (FIGS. 2C-2E). Cells tranfected with pRcCMV
died rapidly without forming any detectable colonies. Cells
tranfected with wt Bcl-2 survived for an extended period, but
formed very few proliferating colonies. Consistent with their
behavior in short term cell survival/proliferation assays (FIG.
2B), cells transfected with mutant Bcl2.DELTA.51-85 formed numerous
proliferating colonies. These results indicate that the mutant
Bcl2.DELTA.51-85 facilitates long term proliferation of cells under
conditions that otherwise result in apoptosis.
EXAMPLE 2
Effect on Myc-Induced Apoptosis
[0111] The effect of Bcl2.DELTA.51-85 on Myc-induced apoptosis was
also tested. Rat1 cells expressing the c-myc gene fused to the
human estrogen receptor (c-mycER-Hygro) undergo apoptosis after Myc
expression is activated by addition of .beta.-estradiol and cells
are deprived of serum (13,14). The c-mycER-Hygro cells were
tranfected with pRcCMV vector or pRcCMV-based plasmids expressing
wt Bcl-2 or mutant Bcl2.DELTA.51-85 and pooled G418-resistant cell
lines were established. Immunoprecipitation (FIG. 3A) and
protein-blot (not shown) analyses revealed that the various Rat1
cell lines expressed comparable levels of wt or the mutant Bcl-2
proteins. The effect of Bcl-2 expression on Myc-induced apoptosis
was then determined by treating the cells with 1 .mu.M f-estradiol
in media containing 0.1% fetal calf serum.
[0112] Deregulated Myc expression induced significant cell death.
Expression of wt Bcl-2 resulted in about 60% cell survival. As in
the case of BRK/p53val135-E1 A cells, the expression of
Bcl2.DELTA.51-85 mutant not only suppressed cell death but also
induced significant proliferation on mycER-Hygro cells in low serum
after a lag period of about one day.
EXAMPLE 3
Interaction of Cellular Proteins with Bcl2.DELTA.51-85
[0113] In order to determine if deletion of the amino acid region
encompassing residues 51-85 affected interaction of various
cellular proteins, the interaction of several cellular proteins
that have been previously reported to interact with Bcl1-2 either
by two-hybrid interaction studies in yeast (28) or by
co-immunoprecipitation analyses was examined. In these studies, no
major difference was observed in the patterns of interaction of
Nip1-3 (29), c-Raf-1 (30), R-ras (52), Bak (31), and Bik (32) (not
shown). In contrast, the level of interaction between Bax (33) and
Bcl2.DELTA.51-85 appeared to be significantly enhanced in
comparison to wt bcl-2 in co-immunoprecipitation assays (FIGS. 4A
and 4B). This enhanced interaction appears to be significant
considering that the total level of Bax was similar in cells
expressing either Bcl2.DELTA.51-85 or wt Bcl-2.
EXAMPLE 4
Characterization of Critical Residues Within the Bcl-2 Residue
51-85 Domain
[0114] In an effort to characterize critical residues within the
Bcl-2 residue 51-85 domain, several Bcl-2 mutants encoding single
amino acid substitutions were constructed and tested for their
effect on cell survival and proliferation. In the short term
survival assay (FIG. 5 and Table 1), none of the point mutants gave
an enhanced proliferation activity comparable to the Bcl-2D51-85
mutant, though two mutants, P75A and S51 A, had some effect. While
most of the point mutations resulted in Bcl-2 molecules that
retained at least significant survival function, substitution of
serine at position 62 with alanine completely abolished survival
activity. This result demonstrates that this region has substantial
influence on the survival function of Bcl-2 as well as modulation
of proliferation. In the long term assay (Table 1), several of the
point mutants permitted significant colony formation, suggesting
that these residues may contribute to the proliferation-restraining
activity of Bcl-2. One substitution mutant, S51A, had a
hyperprotective effect that was apparent in the long term assay.
With wild type Bcl-2, the BRK-p53val135-E1 A cells eventually die
when subjected to the prolonged exposure at 32.5.degree. C. used in
the long term assay. In contrast, cells expressing Bcl-2 S51A
survived for the duration of the assay, though no significant
colony formation was observed.
1TABLE 1 Comparison of survival activity and long term colony
formation for Bcl-2 mutants. Survival activity and long-term
proliferation (colony formation) was measured in BRK-p53val135-E1a
cells at 32.5.degree. C. as described for FIG. 2. .DELTA. indicates
deleted residues, substitution mutations (such as S51A) are
indicated by the amino acid changed followed by the position number
and the substituted amino acid. For survival activity, + is normal
+\- is partial, ++ and +++ are above normal. For colony formation,
+ is small, ++ is medium, +++ is large colonies. Mat indicates that
cells were present without obvious colony formation. -, indicates
no cells remaining. Survival colony Bcl-2 mutant Activity formation
vector - - wild type + - .DELTA.51-85 ++ +++ .DELTA.51-98 - - S51A
+++ mat T56A +.backslash.- - P57A +.backslash.- + S62A - -
TS69-70AA +.backslash.- + T74A +.backslash.- ++ P75A ++ +
[0115] A straightforward interpretation of these results is that
the effect of the Bcl-2.DELTA.51-85 mutant is a sum of the
hyperproliferative function of the S51A mutant and the
proliferative effects of the P57A, TS69-70AA, T74A, and P75A
mutants.
Sequences
[0116] Polypeptide and nucleotide sequences referred to herein by
SEQ ID NOS. are listed below.
[0117] In the polypeptide sequences (Pro/Thr) and (Thr/Ala) means
that the amino acid at that position can be Pro or Thr and Thr or
Ala, respectively.
[0118] In the nucleic acid sequences M represents A or C, S
represents C or G, and R represents A or G.
2 Ser Gln Pro Gly His Thr Pro His (Pro/Thr) Ala Ala Ser Arg Asp Pro
Val Ala Arg Thr Ser (SEQ ID NO:1) Pro Leu Gln Thr Pro Ala Ala Pro
Gly Ala Ala Ala Gly Pro Ala. Ser Gln Pro Gly His Thr Pro His
(Pro/Thr) Ala Ala Ser Arg Asp Pro Val Ala Arg Thr Ser (SEQ ID NO:2)
Pro Leu Gln Thr Pro Ala Ala Pro Gly Ala Ala Ala Gly Pro Ala Leu.
Ser Gln Pro Gly His Thr Pro His (Pro/Thr) Ala Ala Ser Arg Asp Pro
Val Ala Arg Thr Ser (SEQ ID NO:3) Pro Leu Gln Thr Pro Ala Ala Pro
Gly Ala Ala Ala Gly Pro Ala Leu Ser. Ser Gln Pro Gly His Thr Pro
His (Pro/Thr) Ala Ala Ser Arg Asp Pro Val Ala Arg Thr Ser (SEQ ID
NO:4) Pro Leu Gln Thr Pro Ala Ala Pro Gly Ala Ala Ala Gly Pro Ala
Leu Ser Pro. Ser Gln Pro Gly His Thr Pro His (Pro/Thr) Ala Ala Ser
Arg Asp Pro Val Ala Arg Thr Ser (SEQ ID NO:5) Pro Leu Gln Thr Pro
Ala Ala Pro Gly Ala Ala Ala Gly Pro Ala Leu Ser Pro Val. Ser Gln
Pro Gly His Thr Pro His (Pro/Thr) Ala Ala Ser Arg Asp Pro Val Ala
Arg Thr Ser (SEQ ID NO:6) Pro Leu Gln Thr Pro Ala Ala Pro Gly Ala
Ala Ala Gly Pro Ala Leu Ser Pro Val Pro. Ser Gln Pro Gly His Thr
Pro His (Pro/Thr) Ala Ala Ser Arg Asp Pro Val Ala Arg Thr Ser (SEQ
ID NO:7) Pro Leu Gln Thr Pro Ala Ala Pro Gly Ala Ala Ala Gly Pro
Ala Leu Ser Pro Val Pro Pro. Ser Gln Pro Gly His Thr Pro His
(Pro/Thr) Ala Ala Ser Arg Asp Pro Val Ala Arg Thr Ser (SEQ ID NO:8)
Pro Leu Gln Thr Pro Ala Ala Pro Gly Ala Ala Ala Gly Pro Ala Leu Ser
Pro Val Pro Pro Val. Ser Gln Pro Gly His Thr Pro His (Pro/Thr) Ala
Ala Ser Arg Asp Pro Val Ala Arg Thr Ser (SEQ ID NO:9) Pro Leu Gln
Thr Pro Ala Ala Pro Gly Ala Ala Ala Gly Pro Ala Leu Ser Pro Val Pro
Pro Val Val. Ser Gln Pro Gly His Thr Pro His (Pro/Thr) Ala Ala Ser
Arg Asp Pro Val Ala Arg Thr Ser (SEQ ID NO:10) Pro Leu Gln Thr Pro
Ala Ala Pro Gly Ala Ala Ala Gly Pro Ala Leu Ser Pro Val Pro Pro Val
Val His. Ser Gln Pro Gly His Thr Pro His (Pro/Thr) Ala Ala Ser Arg
Asp Pro Val Ala Arg Thr Ser (SEQ ID NO:11) Pro Leu Gln Thr Pro Ala
Ala Pro Gly Ala Ala Ala Gly Pro Ala Leu Ser Pro Val Pro Pro Val Val
His Leu. Ser Gln Pro Gly His Thr Pro His (Pro/Thr) Ala Ala Ser Arg
Asp Pro Val Ala Arg Thr Ser (SEQ ID NO:12) Pro Leu Gln Thr Pro Ala
Ala Pro Gly Ala Ala Ala Gly Pro Ala Leu Ser Pro Val Pro Pro Val Val
His Leu (Thr/Ala). Ser Gln Pro Gly His Thr Pro His (Pro/Thr) Ala
Ala Ser Arg Asp Pro Val Ala Arg Thr Ser (SEQ ID NO:13) Pro Leu Gln
Thr Pro Ala Ala Pro Gly Ala Ala Ala Gly Pro Ala Leu Ser Pro Val Pro
Pro Val Val His Leu (Thr/Ala) Leu. TCCCAGCCCG GGCACACGCC CCATMCAGCC
GCATCCCGSG ACCCGGTCGC (SEQ ID NO:14) CAGGACCTCG CCGCTGCAGA
CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGC TCCCAGCCCG GGCACACGCC
CCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQ ID NO:15) CAGGACCTCG
CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCG TCCCAGCCCG
GGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQ ID NO:16)
CAGGACCTCG CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGC
TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQ ID
NO:17) CAGGACCTCG CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC
CTGCGCT TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQ
ID NO:18) CAGGACCTCG CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC
CTGCGCTC TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC
(SEQ ID NO:19) CAGGACCTCG CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC
GCCGCGGGGC CTGCGCTCA TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG
ACCCGGTCGC (SEQ ID NO:20) CAGGACCTCG CCGCTGCAGA CCCCGGCTGC
CCCCGGCGCC GCCGCGGGGC CTGCGCTCAG TCCCAGCCCG GGCACACGCC CCATMCAGCC
GCATCCCGSG ACCCGGTCGC (SEQ ID NO:21) CAGGACCTCG CCGCTGCAGA
CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTCAG C TCCCAGCCCG GGCACACGCC
CCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQ ID NO:22) CAGGACCTCG
CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CC
TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQ ID
NO:23) CAGGACCTCG CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC
CTGCGCTCAG CCC TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG
ACCCGGTCGC (SEQ ID NO:24) CAGGACCTCG CCGCTGCAGA CCCCGGCTGC
CCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CCCG TCCCAGCCCG GGCACACGCC
CCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQ ID NO:25) CAGGACCTCG
CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CCCGG
TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQ ID
NO:26) CAGGACCTCG CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC
CTGCGCTCAG CCCGGT TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG
ACCCGGTCGC (SEQ ID NO:27) CAGGACCTCG CCGCTGCAGA CCCCGGCTGC
CCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CCCGGTG TCCCAGCCCG GGCACACGCC
CCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQ ID NO:28) CAGGACCTCG
CCGCTGGAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CCCGGTGC
TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQ ID
NO:29) CAGGACCTCG CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC
CTGCGCTCAG CCCGGTGCC TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG
ACCCGGTCGC (SEQ ID NO:30) CAGGACCTCG CCGCTGCAGA CCCCGGCTGC
CCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CCCGGTGCCA TCCCAGCCCG GGCACACGCC
CCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQ ID NO:31) CAGGACCTCG
CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CCCGGTGCCA C
TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQ ID
NO:32) CAGGACCTCG CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC
CTGCGCTCAG CCCGGTGCCA CC TCCCAGCCCG GGCACACGCC CCATMCAGCC
GCATCCCGSG ACCCGGTCGC (SEQ ID NO:33) CAGGACCTCG CCGCTGCAGA
CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CCCGGTGCCA CCT
TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQ ID
NO:34) CAGGACCTCG CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC
CTGCGCTCAG CCCGGTGCCA CCTG TCCCAGCCCG GGCACACGCC CCATMCAGCC
GCATCCCGSG ACCCGGTCGC (SEQ ID NO:35) CAGGACCTCG CCGCTGCAGA
CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CCCGGTGCCA CCTGT
TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQ ID
NO:36) CAGGACCTCG CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC
CTGCGCTCAG CCCGGTGCCA CCTGTG TCCCAGCCCG GGCACACGCC CCATMCAGCC
GCATCCCGSG ACCCGGTCGC (SEQ ID NO:37) CAGGACCTCG CCGCTGCAGA
CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CCCGGTGCCA CCTGTGGT
TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQ ID
NO:38) CAGGACCTCG CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC
CTGCGCTCAG CCCGGTGCCA CCTGTGGTC TCCCAGCCCG GGCACACGCC CCATMCAGCC
GCATCCCGSG ACCCGGTCGC (SEQ ID NO:39) CAGGACCTCG CCGCTGCAGA
CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CCCGGTGCCA CCTGTGGTCC
TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQ ID
NO:40) CAGGACCTCG CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC
CTGCGCTCAG CCCGGTGCCA CCTGTGGTCC A TCCCAGCCCG GGCACACGCC CCATMCAGCC
GCATCCCGSG ACCCGGTCGC (SEQ ID NO:41) CAGGACCTCG CCGCTGCAGA
CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CCCGGTGCCA CCTGTGGTCC
AC TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQ ID
NO:42) CAGGACCTCG CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC
CTGCGCTCAG CCCGGTGCCA CCTGTGGTCC ACC TCCCAGCCCG GGCACACGCC
CCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQ ID NO:43) CAGGACCTCG
CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CCCGGTGCCA
CCTGTGGTCC ACCT TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG
ACCCGGTCGC (SEQ ID NO:44) CAGGACCTCG CCGCTGCAGA CCCCGGCTGC
CCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CCCGGTGCCA CCTGTGGTCC ACCTG
TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQ ID
NO:45) CAGGACCTCG CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC
CTGCGCTCAG CCCGGTGCCA CCTGTGGTCC ACCTGR TCCCAGCCCG GGCACACGCC
CCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQ ID NO:46) CAGGACCTCG
CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CCCGGTGCCA
CCTGTGGTCC ACCTGRC TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG
ACCCGGTCGC (SEQ ID NO:47) CAGGACCTCG CCGCTGCAGA CCCCGGCTGC
CCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CCCGGTGCCA CCTGTGGTCC ACCTGRCC
TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQ ID
NO:48) CAGGACCTCG CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC
CTGCGCTCAG CCCGGTGCCA CCTGTGGTCC ACCTGRCCC TCCCAGCCCG GGCACACGCC
CCATMCAGCC GCATCCCGSG ACCCGGTCGC (SEQ ID NO:49) CAGGACCTCG
CCGCTGCAGA CCCCGGCTGC CCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CCCGGTGCCA
CCTGTGGTCC ACCTGRCCCT TCCCAGCCCG GGCACACGCC CCATMCAGCC GCATCCCGSG
ACCCGGTCGC (SEQ ID NO:50) CAGGACCTCG CCGCTGCAGA CCCCGGCTGC
CCCCGGCGCC GCCGCGGGGC CTGCGCTCAG CCCGGTGCCA CCTGTGGTCC ACCTGRCCCT
C
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[0176]
Sequence CWU 1
1
* * * * *