U.S. patent application number 12/498691 was filed with the patent office on 2010-08-05 for bcl-2-modifying factor (bmf) sequences and their use in modulating apoptosis.
This patent application is currently assigned to The Walter And Eliza Hall Institute Of Medical Research. Invention is credited to Jennifer BEAUMONT, Leigh COULTAS, David Ching Siang HUANG, Lorraine Ann O'REILLY, Hamsa PUTHALAKATH, Andreas STRASSER, Andreas VILLUNGER.
Application Number | 20100199368 12/498691 |
Document ID | / |
Family ID | 3829329 |
Filed Date | 2010-08-05 |
United States Patent
Application |
20100199368 |
Kind Code |
A1 |
STRASSER; Andreas ; et
al. |
August 5, 2010 |
BCL-2-MODIFYING FACTOR (BMF) SEQUENCES AND THEIR USE IN MODULATING
APOPTOSIS
Abstract
The present invention relates generally to novel molecules
capable of, inter alia, modulating apoptosis in mammalian cells and
to genetic sequences encoding same. More particularly, the present
invention relates to a novel member of the Bcl-2 family of
proteins, referred to herein as "Bmf", and to genetic sequences
encoding same and to regulatory sequences such as a promoter
sequence directing expression of Bmf. Bmf comprises a BH3 domain
which facilitates interaction to pro-survival Bcl-2 family members
thereby triggering apoptosis. Bmf is regarded, therefore, as a
BH3-only molecule. The molecules of the present invention are
useful, for example, in therapy, diagnosis, antibody generation and
as a screening tool for therapeutic agents capable of modulating
physiological cell death or survival and/or modulating cell cycle
entry. The present invention further contemplates genetically
modified animals in which one or both alleles of Bmf are mutated or
partially or wholly deleted alone or in combination with a mutation
in one or both alleles of another Bcl-2-type molecule such as but
not limited to Bim. The genetically modified animals are useful
inter alia in screening for agents which ameliorate the symptoms of
diseases caused by defects in apoptosis or which specifically
promote apoptosis of target cells.
Inventors: |
STRASSER; Andreas; (Ascot
Vale, AU) ; PUTHALAKATH; Hamsa; (Keilor East, AU)
; VILLUNGER; Andreas; (Williamstown, AU) ;
COULTAS; Leigh; (Brunswick East, AU) ; BEAUMONT;
Jennifer; (Dianella, AU) ; O'REILLY; Lorraine
Ann; (Cheltenham, AU) ; HUANG; David Ching Siang;
(North Fitzroy, AU) |
Correspondence
Address: |
SCULLY SCOTT MURPHY & PRESSER, PC
400 GARDEN CITY PLAZA, SUITE 300
GARDEN CITY
NY
11530
US
|
Assignee: |
The Walter And Eliza Hall Institute
Of Medical Research
Parkville
AU
|
Family ID: |
3829329 |
Appl. No.: |
12/498691 |
Filed: |
July 7, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10479307 |
Jun 30, 2004 |
7572900 |
|
|
PCT/AU02/00693 |
May 30, 2002 |
|
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12498691 |
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Current U.S.
Class: |
800/18 ; 514/1.1;
530/350; 530/388.1; 536/23.5; 800/13; 800/14; 800/17 |
Current CPC
Class: |
A61P 43/00 20180101;
A61P 31/12 20180101; A61K 38/00 20130101; A01K 2217/05 20130101;
C07K 14/4747 20130101; A61P 35/00 20180101; A61P 37/00
20180101 |
Class at
Publication: |
800/18 ;
536/23.5; 530/350; 514/12; 530/388.1; 800/13; 800/14; 800/17 |
International
Class: |
A01K 67/00 20060101
A01K067/00; C07H 21/04 20060101 C07H021/04; C07K 14/435 20060101
C07K014/435; A61K 38/16 20060101 A61K038/16; C07K 16/00 20060101
C07K016/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 30, 2001 |
AU |
PR5351/01 |
Claims
1. An isolated nucleic acid molecule comprising a nucleotide
sequence encoding or complementary to a sequence encoding an amino
acid sequence substantially as set forth in one of SEQ ID NO:2 or
SEQ ID NO:6 or SEQ ID NO:8 or a derivative or homolog thereof or
having at least about 45% or greater similarity to one or more of
SEQ ID NO:2 or SEQ ID NO:6 or SEQ ID NO:8 or a derivative or
homolog thereof, wherein the derivative comprises a BH3 domain and
binds a member of the pro-survival Bcl-2.
2-5. (canceled)
6. The nucleic acid molecule of claim 1 comprising a nucleotide
sequence set forth in SEQ ID NO:1 or a nucleotide sequence having
at least about 45% similarity thereto or a nucleotide sequence
capable of hybridizing to SEQ ID NO:1 or its complementary form
under low stringency conditions.
7. (canceled)
8. The nucleic acid molecule of claim 1 comprising a nucleotide
sequence set forth in SEQ ID NO:5 or a nucleotide sequence having
at least about 45% similarity thereto or a nucleotide sequence
capable of hybridizing to SEQ ID NO:5 or its complementary form
under low stringency conditions.
9. The nucleic acid molecule of claim 1 comprising a nucleotide
sequence set forth in SEQ ID NO:7 or a nucleotide sequence having
at least about 45% similarity thereto or a nucleotide sequence
capable of hybridizing to SEQ ID NO:7 or its complementary form
under low stringency conditions.
10-13. (canceled)
14. An isolated protein comprising an amino acid sequence encoded
by a nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:5 or
SEQ ID NO:7 or a nucleotide sequence having at least about 45%
similarity to the nucleotide sequence set forth in SEQ ID NO: 1 or
SEQ ID NO:5 or SEQ ID NO:7 or a nucleotide sequence capable of
hybridizing to SEQ ID NO:1 or SEQ ID NO:5 or SEQ ID NO:7 or a
complement thereof under low stringency conditions.
15. The isolated protein of claim 14 comprising an amino acid
sequence encoded by the nucleotide sequence set forth in SEQ ID
NO:11.
16-18. (canceled)
19. The isolated protein of claim 14 comprising an amino acid
sequence as set forth in SEQ ID NO:2 or having at least 45%
similarity thereto.
20. (canceled)
21. The isolated protein of claim 14 comprising an amino acid
sequence as set forth in SEQ ID NO:6 or having at least 45%
similarity thereto.
22. The isolated protein of claim 14 comprising an amino acid
sequence as set forth in SEQ ID NO:8 or having at least 45%
similarity thereto.
23-26. (canceled)
27. A variant of an isolated bmf nucleic acid molecule comprising
at least one nucleotide mutation in said nucleic acid molecule
resulting in at least one amino acid addition, substitution and/or
deletion to the polypeptide encoded by said variant wherein said
polypeptide cannot bind, couple or otherwise associate with a
dynein light chain, such as DLC2.
28. The variant of claim 27 wherein the mutation results in an
altered amino acid sequence in the region which binds to the dynein
light chain.
29. A variant of an isolated Bmf polypeptide comprising at least
one amino acid addition, substitution and/or deletion wherein said
polypeptide cannot bind, couple or otherwise associate with the
dynein light chain.
30. A method of modulating activity of Bmf in a mammal, said method
comprising administering to said mammal a modulating effective
amount of an agent for a time and under conditions sufficient to
increase or decrease Bmf activity.
31. A method of modulating apoptosis in a mammal, said method
comprising administering to said mammal an effective amount of an
agent for a time and under conditions sufficient to modulate the
expression of a nucleotide sequence encoding bmf.
32. A method of modulating apoptosis in a mammal, said method
comprising administering to said mammal an effective amount of an
agent for a time and under conditions sufficient to modulate the
activity of Bmf.
33-34. (canceled)
35. The method of claim 30 or 31 or 32 wherein the mammal is a
human.
36. A pharmaceutical composition comprising bmf, Bmf or derivative
thereof or an agent capable of modulating bmf expression or Bmf
activity together with one or more pharmaceutically acceptable
carriers and/or diluents, bmf, Bmf or said agent are referred to as
the active ingredients.
37. A monoclonal antibody having specificity for Bmf or bmf or
derivative thereof.
38-39. (canceled)
40. A genetically modified animal in which one or both alleles of
bmf are mutated alone or in combination with another mutation in
one or both alleles for another Bcl-2 molecule such as but not
limited to genes encoding Blk, Bad, Bik, Hrk, Bid, Bim, Noxa and/or
Puma.
41. The genetically modified animal of claim 40 wherein said animal
is a mouse, rat or pig.
42-47. (canceled)
Description
FIELD OF THE INVENTION
[0001] The present invention relates generally to novel molecules
capable of, inter alia, modulating apoptosis in mammalian cells and
to genetic sequences encoding same. More particularly, the present
invention relates to a novel member of the Bcl-2 family of
proteins, referred to herein as "Bmf", and to genetic sequences
encoding same and to regulatory sequences such as a promoter
sequence directing expression of Bmf. Rolf comprises a BH3 domain
which facilitates interaction to pro-survival Bcl-2 family members
thereby triggering apoptosis. Bird is regarded, therefore, as a
BH3-only molecule. The molecules of the present invention are
useful, for example, in therapy, diagnosis, antibody generation and
as a screening tool for therapeutic agents capable of modulating
physiological cell death or survival and/or modulating cell cycle
entry. The present invention further contemplates genetically
modified animals in which one or both alleles of Bmf are mutated or
partially or wholly deleted alone or in combination with a mutation
in one or both alleles of another Bcl-2-type molecule such as but
not limited to Bim. The genetically modified animals are useful
inter alia in screening for agents which ameliorate the symptoms of
diseases caused by defects in apoptosis or which specifically
promote apoptosis of target cells.
BACKGROUND OF THE INVENTION
[0002] Reference to any prior art in this specification is not, and
should not be taken as, an acknowledgment or any form of suggestion
that this prior art forms part of the common general knowledge in
any country.
[0003] Apoptosis, the physiologic and genetically modulated process
of cell death, is of central importance for modelling tissues, and
maintaining homeostasis in multicellular organisms (Kerr et al.,
Br. J. Cancer 26: 239-257, 1972; Jacobson et al., Cell 88: 347-354,
1997). Great progress is being made towards understanding the
biochemistry underlying this intrinsic suicide program. The
cellular apoptotic effector molecules include a set of cysteine
proteinases, termed caspases, that degrade critical cellular
substrates (Nicholson et al., Trends Biochem. Sci. 22: 299-306,
1997). The regulatory machinery that governs the activation of the
caspases is loss well understood. However, a family of proteins of
which Bcl-2 is the prototypic molecule (and is referred to as the
Bcl-2 family of proteins) plays a central role (Jacobson, Curr.
Biol. 7: R277-R281, 1997; Reed, Nature 387: 773-776, 1997; Kroemer,
Nature Med. 3: 614-620, 1997; Adams and Cory, Science 281:
1322-1326, 1998).
[0004] Bcl-2 was the first intracellular regulator of apoptosis to
be identified (Vaux et al., Nature 335: 440-442, 1988) and high
levels enhance cell survival under diverse cytotoxic conditions.
Other cellular homologs, such as Bcl-x.sub.L. (Boise et al., Cell
74: 597-608, 1993) and Bcl-w (Gibson et al., Oncogene 665-675,
1996), also enhance cell survival, as do more distantly related
viral homologs, such as the adenovirus E1B 19K protein (White et
al., Mol. Cell. Biol. 12: 2570-2580, 1992) and Epstein-Barr virus
BHRF-1 (Henderson et al., Proc. Natl. Acad. USA 90: 8479-8483,
1993).
[0005] Pro-apoptotic BH3-only members of the Bcl-2 family are
essential for initiation of apoptosis in species as distantly
related as mice and C. elegans (Huang and Strasser, Cell 103: 839,
2000). EGL-1, the so far only recognized BH3-only protein in C.
elegans, is required for all developmentally programmed cell deaths
in, this organism. In contrast, a number of BH3-only proteins have
already been identified in mammals: Blk, Bad, Bile, Hrk, Bid, Bim,
Noxa and Puma. Experiments with knock-out mice have shown that
different apoptosis stimuli require distinct BH3-only proteins for
their initiation. (Huang and Strasser, 2000, supra). For example,
Bim is essential for apoptosis induced by cytokine withdrawal or
antigen receptor stimulation, but is dispensable for cell death
induced by glucocorticoids (Bouillet et al., Science 286: 1735,
1999; Bouillet et al., Nature 415, 922, 2002). In contrast, Bid is
involved in Fas-induced killing of hepatocytes (Yin et al., Nature
400: 886, 1999). Moreover, different cell types may require
distinct BH3-only proteins for their developmentally programmed
death. Consistent with this idea, Bim-deficient mice have an
abnormal accumulation of lymphoid and myeloid cells but
erythropoiesis appears normal (Bouillet et al., 1999, supra). These
results indicate that individual mammalian BH13-only proteins have
specific functions.
[0006] The pro-apoptotic activity of BH3-only proteins is subject
to stringent control. In C. elegans, EGL-1. is regulated by the
transcriptional represser TRA-1A in a group of neurons that is
required for egg-laying (Conradt and Horvitz, Cell 93: 519,1998).
Some mammalian BH3-only proteins are also subject to
transcriptional regulation. For example, Noxa was discovered as a
p53-inducible gene and is therefore a prime candidate for mediating
DNA damage-induced apoptosis (Oda et al., Science 288: 1053, 2000).
Several mammalian BH3-only proteins can also be regulated
post-translationally (Huang and Strasser, 2000, supra). In growth
factor-stimulated cells, Bad is phosphorylated and sequestered away
from pro-survival Bcl-2 family members by binding to 14-3-3
scaffold proteins (Zha et al., Cell 87: 619, 1996). In healthy
cells, Bim is sequestered to the microtubular dynein motor complex
by binding to dynein light chain, DLC1/LC8 (Puthalakath et al.,
Mol. Cell. 3: 287, 1999). Certain apoptotic stimuli, such as
UV-radiation or treatment with taxol, free aim (still bound to
DLC1) and allow it to translocate to, bind and inactivate
pro-survival Bcl-2 family members. This process occurs
independently of the cell death executioner cysteine proteases
(caspases) and therefore constitutes an upstream signalling event
in apoptosis (Puthalakath et al., 1999, supra). In contrast, the
pro-apoptotic activity of Bid is unleashed upon cleavage by a
variety of caspases (e.g. caspase-8) or by the serine protease
granzyme B (Li et al., Cell 94: 491-501, 1998; Luc et al., Cell 94:
481-490, 1998), indicating that it functions as part of an
amplification mechanism rather than as an initiator of apoptosis.
These observations demonstrate that through sequestration to
specific sites in the cell, different BH3-only proteins function as
sensors for distinct forms of intra-cellular stress.
[0007] In work leading to the present invention, the inventors
sought novel BH3-only proteins which played a role in
embryogenesis. In accordance with the present invention, the
inventors cloned "Bmf" (Bcl-2 modifying factor) which was
identified through yeast 2-hybrid screening of a day 17 mouse
embryonic library using Mcl-1. as bait. Bad is proposed to induce
cell death and act as a "death-ligand" for certain. or all members
of the pro-survival Bcl-2 family. The identification of this new
gene permits the identification and rational design of a range of
products for use in therapy, diagnosis, antibody generation and
involving modulation of physiological cell death. These therapeutic
molecules may act as either antagonists or agonists of Bmf's
function and will be useful in cancer, autoimmune or degenerative
disease therapy.
SUMMARY OF THE INVENTION
[0008] Throughout this specification, unless the context requires
otherwise, the word "comprise", Or variations such as "comprises"
or "comprising", will be understood to imply the inclusion of a
stated element or integer or group of elements or integers but not
the exclusion of any other element or integer or group of elements
or integers.
[0009] Nucleotide and amino acid sequences are referred to by a
sequence identifier number (SEQ ID NO:). The SEQ ID NOs: correspond
numerically to the sequence identifiers <400>1 (SEQ ID NO:1),
<400>2 (SEQ ID NO:2), etc. A sequence listing is provided
after the claims.
[0010] Specific mutations in an amino acid sequence are represented
herein as "X.sub.1nX.sub.2" where X.sub.1 is the original amino
acid residue before mutation, n is the residue number and X.sub.2
is the mutant amino acid. Reference to Xn is a reference to a
particular amino acid in an amino acid sequence where X is the
amino acid and n is the residue number. The abbreviation X may be
to the three letter or single letter amino acid code.
[0011] The present invention is predicated in part on the
identification of a novel member of the pro-survival Bcl-2 family.
This protein is referred to herein as "Bcl-2 modifying factor" or
"Bmf", The protein was identified by yeast 2-hybrid screening of a
mouse embryonic library using Mcl-1 as bait. Bmf is an
apoptosis-inducing BH3-only protein and is activated by
anoikis.
[0012] Accordingly, one aspect of the present invention provides a
nucleic acid molecule comprising a nucleotide sequence encoding a
polypeptide having one or more of the identifying characteristics
of Bmf or a derivative or homolog thereof.
[0013] Another aspect of the present invention provides a nucleic
acid molecule comprising a nucleotide sequence encoding or
complementary to a sequence encoding an amino acid sequence
substantially as set forth in one of SEQ ID NO:2 or SEQ ID NO:4 or
SEQ ID NO:6 or SEQ ID NO:8 or a derivative or homolog thereof or
having at least about 45% or greater similarity to one or more of
SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 or a
derivative or homolog thereof.
[0014] Yet another aspect of the present invention contemplates a
nucleic acid molecule comprising a nucleotide sequence
substantially as set forth in one of SEQ ID NO:1 or SEQ ID NO:3 or
SEQ ID NO:5 or SEQ ID NO:7 or a derivative or homolog thereof
capable of hybridising to one of SEQ ID NO:1 or SEQ ID NO:3 ar SEQ
ID NO:5 or SEQ ID NO:7 under low stringency conditions and which
encodes an amino acid sequence corresponding to an amino acid
sequence set forth in one of SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID
NO:6 or SEQ ID NO:8 or a sequence having at least about 45%
similarity to one or more of SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID
NO:6 or 8.
[0015] Still yet another aspect of the present invention
contemplates a nucleic acid molecule comprising a sequence of
nucleotides substantially as set forth in SEQ ID NOS:1 or SEQ ID
NO:3 or SEQ m NO:5 or SEQ ID NO:7.
[0016] Still another aspect of the present invention is directed to
an isolated nucleic acid molecule encoding bmf or a derivative
thereof; said nucleic acid molecule selected from the list
consisting of:-- [0017] (i) a nucleic acid molecule comprising a
nucleotide sequence encoding the amino acid sequence set forth in
one of SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 or
a derivative or homolog thereof or having at least about 45%
similarity to one or more of SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID
NO:6 or SEQ ID NO:8; [0018] (ii) a nucleic acid molecule comprising
a nucleotide sequence substantially as set forth in one of SEQ ID
NO:1 or SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:7 or a derivative
or homolog thereof; [0019] (iii) a nucleic acid. molecule capable
of hybridizing under low stringency conditions to the nucleotide
sequence substantially as set forth in one of SEQ ID NO:1 or SEQ ID
NO:3 or SEQ ID NO:5 or SEQ ID NO:7 a derivative or homolog and
encoding an amino acid sequence corresponding to an amino acid
sequence as set forth in one of SEQ ID NO:SEQ ID NO:2 or SEQ ID
NO:4 or SEQ ID NO:6 or SEQ ID NO:8 a derivative or homolog or a
sequence having at least about 45% similarity to one or more of SEQ
ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8; [0020] (iv) a
nucleic acid molecule capable of hybridizing to the nucleic acid
molecule of paragraphs (i) or (ii) or (iii) under low stringency
conditions and encoding an amino acid sequence having at least
about 45% similarity to one or more of SEQ ID NO:2 or SEQ ID NO:4
or SEQ ID NO:6 or SEQ ID NO:8; and [0021] (v) a derivative or
mammalian homolog of the nucleic acid molecule of paragraphs (i) or
(ii) or (iii) or (iv).
[0022] A further aspect of the present invention is directed to an
isolated polypeptide selected from the list consisting of:-- [0023]
(i) a polypeptide having an amino acid sequence substantially as
set forth in one of SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or
SEQ TO NO:8 or derivative or homolog thereof or a sequence having
at least about 45% similarity to one or more of SEQ ID NO:2 or SEQ
NO:4 or SEQ ID NO:6 or SEQ ID NO:8; [0024] (ii) a polypeptide
encoded by a nucleotide sequence substantially as set forth in one
of SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:7 or
derivative or homolog thereof or a sequence encoding an amino acid
sequence having at least about 45% similarity to one or more of SEQ
ID NO:2 or SEQ M NO:4 or SEQ ID NO:6 or SEQ ID NO:8; [0025] (iii) a
polypeptide encoded by a nucleic acid molecule capable of
hybridizing to the nucleotide sequence as set forth in one of SEQ
ID NO:1 or SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:7 or derivative
or homolog thereof under low stringency conditions and which
encodes an amino acid sequence substantially as set forth in SEQ ID
NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 or derivative or
homolog thereof or an amino acid sequence having at least about 45%
similarity to one or more of SEQ NO:2 or SEQ ID NO:4 or SEQ ID
NO:60 or SEQ ID NO:8; [0026] (iv) a polypeptide as defined in
paragraphs (i) or (ii) or (iii) in homodimeric form; and [0027] (v)
a polypeptide as defined in paragraphs (i) or (ii) or (iii) in
heterodimeric form.
[0028] Yet another aspect of the present invention provides a
method of producing a genetically modified non-human animal, said.
method comprising introducing into embryonic stem cells of an
animal a genetic construct comprising a bmf nucleotide sequence
carrying a single or multiple nucleotide substitution, addition
and/or deletion or inversion or insertion wherein there is
sufficient bmf nucleotide sequences to promote homologous
recombination with a bmf gene within the genome of said embryonic
stem cells selecting for said homologous recombination and
selecting embryonic stem cells which carry a mutated bmf gene and
then generating a genetically modified animal from said embryonic
stem cell.
BRIEF DESCRIPTION OF THE FIGURE
[0029] FIG. 1 is a representation showing Buff, a novel mammalian
BH3-only protein. (A) Predicted amino acid sequence of mouse and
human Bmf. The nine amino acids that are conserved with the dynein
light chain-binding motif of Bim are indicated by a box marked with
a single asterisk (*). The short BH3 region, identified by hidden
Markov modeling (Krogh et al., J. Mol. Biol. 235: 1501, 1994), is
indicated by a box marked with two asterisks (**). (B) Alignment of
the BH3 region of Bmf with other pro-apoptotic Bcl-2 family
members. Black boxes indicate identical amino acids and grey boxes
indicate similar residues. (C) Wild-type Bmf, but not a BH3 mutant,
binds pro-survival Bcl-2 and Bcl-w. Co-immunoprecipitation
experiments were carried out as previously described (Puthalakath
et al., 1999, supra). Briefly, 293T cells were transiently
co-transfected with expression constructs for FLAG-tagged Bcl-2 (or
Bcl-w) and EE(Glu-Glu)-tagged Bmf or L138A mutant Bmf. Cells were
metabolically labeled with .sup.35S-methionine 24 hours after
transfection and harvested after overnight culture. Volumes of cell
lysates with equivalent trichloroacetic acid (TCA)-precipitable
.sup.35S counts were used for immunoprecipitations with mAbs to the
FLAG or EE epitope tags. (D) Interaction of endogenous Bmf with
Bcl-2 MCF-7 cells. Lysates from 10.sup.7 MCF-7 cells, prepared in
lysis buffer containing 1% v/v Triton X-100, were
immunoprecipitated either with Bcl-2-100 (anti-human Bcl-2) mAb or
an isotype matched control mAb coupled to sepharose. Bound proteins
were eluted. from the beads by boiling in Laemmli buffer (non
reducing), size fractionated on SDS-PAGE and transferred onto
nitrocellulose filters. Western blotting was performed with a rat
anti-Bmf mAb (9G10). The asterisk (*) indicates the light chain of
the mAb used for immunoprecipitation. (E) Wild-type Bmf but not a
BH3 mutant, kills L929 fibroblasts. L929 fibroblasts were
transfected with empty vector, expression constructs for hygromycin
resistance alone, or with wild-type Bmf a BH3 mutant (L138A) of Bmf
or Bmf lacking its BH3 domain. Transfected cells were plated in
medium, containing hygromycin and resulting drug-resistant colonies
counted after 10-14 days. Values are means (+/-SD) of three
independent experiments. (F and G) Expression of Bmf in cell lines
and tissues. For Northern blot analysis (F), 4 .mu.g of poly
A.sup.+ RNA from various cell lines or from mouse embryos
(embryonic day 9 to 1-day after birth) were electrophoresed,
blotted and probed with a mouse bmf cDNA probe. Probing with a
gapdh cDNA clone was used as the loading control. For Western blot
analysis (G), 50 .mu.g of total protein from various mouse tissues
was size-fractionated by SDS-PAGE, electroblotted onto
nitrocellulose filters and probed with affinity purified rabbit
polyclonal antibodies to Bird. Probing with a ;monoclonal antibody
to HSP70 served as the loading control.
[0030] FIG. 2 is a representation showing Bmf is regulated by
interaction with DLC2. (A) Expression of bmf mRNA in thymocytes
treated with various apoptotic stimuli. Total RNA was isolated from
thymocytes (freshly isolated) or at the indicated time points after
culture in the absence of cytokines or treatment with dexamethasone
(1 .mu.M), .gamma.-radiation (10 Gy) or ionomycin (1 .mu.g/mL).
These conditions all induce substantial apoptosis and, hence, no
RNA could be harvested after 7 hours of treatment. Then 2 .mu.g RNA
was reverse transcribed using AMV reverse transcriptase. Five fold
dilutions of the cDNA were subjected to PCR analysis using bmf
specific primers. After transfer of the PCR products,
nitrocellulose filters were probed with a .sup.32P-labeled internal
bmf oligonucleotide probe. (B) Bmf binds to DLC2 through its dynein
light chain binding region. Co-immunoprecipitation experiments were
performed as described in the legend to FIG. 1C, from lysates of
293T cells transiently expressing FLAG-tagged DLC2 and EE-tagged wt
Bmf, a BH3 mutant (L138A) of Bmf or DLC binding region mutants of
Bmf (A69P or AAA), Bid or Bax. The asterisk (*) indicates the light
chain of the mAb used for immunoprecipitation. (C) Interaction with
DLC2 regulates the pro-apoptotic potency of Bmf. FDC-P1 cells
stably expressing Bcl-2 plus FE-tagged wt Bmf, a BH3 mutant (L138A)
of Bmf or DLC binding region mutants of Bmf (A69P or AAA) were
deprived of IL-3 for 1-6 days. Cell viability was assessed by
propidium iodide staining and flow cytometric analysis. Values are
means (+/-SD) of three independent experiments done with four
independent clones of each genotype.
[0031] FIG. 3 is a photographic representation showing that Bmf
associates with the actin-based myosin V motor complex through
DLC2. (A) Lysates from 10.sup.7 MCF-7 cells were separated into P1,
P2 and S fractions. Proteins from each fraction were then
size-fractionated by SDS-PAGE, transferred onto nitrocellulose and
probed with mAbs specific to Bmf, Bim.sub.L (O'Reilly et al.,
Biotechniques 25: 824, 1998), myosin V (Espreafico et al., J. Cell
Biol. 119: 1541, 1992) or dynein intermediate chain IC74 (Sigma).
(B) MCF-7 cells were treated for 3 hours with either cytochalasin D
(10 .mu.M) or toxin B (10 ng/mL), then fractionated and processed
as described under (A). (C) Characterization of novel in Abs that
recognize both DLC1/LC8 and DLC2, or just DLC1/LC8. Extracts from
293T cells transiently expressing FLAG-tagged DLC1 or DLC2 were run
on SDS-PAGE gels, electroblotted onto nitrocellulose membranes and
probed with rat monoclonal antibodies 11F7 (which recognizes both
DLC1 and DLC2) or 10D6 (which recognizes only DLC1). The faint
bands of lower molecular weight marked by arrows indicate
endogenous DLC1. (D) Myosin V is associated mostly with DLC2
whereas dynein predominantly associates with DLC1/LC8. Cytoplasmic
dynein was enriched from MCF-7 cells (Paschal et al., Methods
Enzymol. 196: 181, 1991) and myoSin V was purified from mouse
spleen (m) or chicken brain (c) (Cheney, Methods Enzymol. 298: 3,
1998). These enriched fractions were analyzed by Western blotting
using rat mAbs 11F7 (recognizes DLC1/LC8 and DLC2) or 10D6
(recognizes only DLC1/LC8). Nitrocellulose membranes were probed
with antibodies to myosin V or IC74 (Sigma) to demonstrate purity
of the myosin and dynein motor fractions. (E) Extracts from mouse
spleen cells (200 .mu.g protein) were incubated for 3 hours at
4.degree. C. with recombinant GST or CTST-tagged FADD, Bmf or
Bim.sub.L proteins, and the bound proteins recovered on glutathione
sepharose beads. Bound proteins were eluted from the beads by
boiling in Laemmli buffer (non-reducing), size-fractionated by
SDS-PAGE and electro-blotted onto nitrocellulose membranes, which
were probed with an antibody to myosin V (Espreafico et al., 1992,
supra,). The nitrocellulose membrane was stained with amido black
(bottom to document that comparable amounts of proteins were used
in the pull down experiments. (F) Lysates from 10.sup.7 MCF-7 cells
were fractionated through a 5-20% w/v sucrose gradient. The pellet
and soluble fractions were analyzed by Western blotting for the
presence of Bmf, Bim, DLC1/LC8 or DLC2.
[0032] FIG. 4 is a photographic representation showing that Bmf and
Bim are released from their sequestration sites in response to
distinct apoptotic stimuli. (A) MCF-7 cells were cultured in the
presence of the broad-spectrum caspase inhibitor zVAD-fmk (50
.mu.M). Lysates from control (untreated) cells were compared with
those from cells subjected to various apoptotic stimuli, including
anoikis (culturing cells for 24 hours in suspension on poly-hema
coated bacterial Petri dishes), UV-irradiation (100 J/m.sup.2),
paclitaxel (taxol 1 .mu.M). Lysates of 10.sup.7 cells were
fractionated through sucrose gradients. The pellet and soluble
fractions were collected and analyzed by Western blotting for Bmf
and Bim using specific monoclonal antibodies. (B) During anoikis,
Bmf translocates to mitochondria and binds to Bcl-2. Mitochondria
were purified as previously described from 2.times.10.sup.8 healthy
MCF-7 cells or cells subjected to anoikis. Mitochondrial proteins
were extracted in lysis buffer containing 1% v/v Triton X-100
(Puthalakath et al., 1999, supra). Immmunoprecipitations were
performed with anti-human Bcl-2 mAb (Bcl 2-100) bound to sepharose
beads. Bound proteins were eluted by boiling the beads in Laemmli
buffer (non-reducing), size-fractionated by SDS-PAGE,
electroblotted onto nitrocellulose membranes and probed with mAbs
to Bcl-2, Brill or dynein light chains.
[0033] FIG. 54. is a diagrammatic representation showing the
genomic organization of the bmf gene locus of the mouse.
[0034] FIG. 5B is a diagrammatic representation of a bmf targeting
construct in NEB193neo or NEB193hygro for use in generating
knock-out mice.
[0035] Single and three letter abbreviations used throughout the
specification are defined below.
TABLE-US-00001 SINGLE AND THREE LETTER AMINO ACID ABBREVIATIONS
THREE-LETTER ONE-LETTER AMINO ACID ABBREVIATION SYMBOL Alanine Ala
A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys
C Glutamine Gln Q Glutamic acid Glu E Glycine Gly G Histidine His H
Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M
Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T
Tryptophan Trp W Tyrosine Tyr Y Valine Val V Any residue Xaa X
[0036] A summary of sequence identifiers is provided below:--
TABLE-US-00002 SUMMARY OF SEQENCE IDENTIFIERS SEQ ID NO:
DESCRIPTION 1 Nucleotide sequence of mouse bmf 2 Amino acid
sequence of mouse Bmf 3 Nucleotide sequence of human bmf 4 Amino
acid sequence of human Bmf 5 Nucleotide sequence of BH3 domain of
mouse bmf 6 Amino acid sequence of BH3 domain of mouse Bmf 7
Nucleotide sequence of BH3 domain of human bmf 8 Amino acid
sequence of BH3 domain of human bmf 9 Nucleotide sequence of mouse
bmf promoter 10 Nucleotide sequence of human bmf promoter 11 5'
sense primer 12 3' antisense primer 13 internal bmf primer 14 5'
sense primer 15 3' antisense primer 16 internal primer 17 predicted
amino acid sequence of mouse Bmf 18 predicted amino acid sequence
of human Bmf 19 partial amino acid sequence of Bmf 20 partial amino
acid sequence of Bim 21 partial amino acid sequence of EGL-1 22
partial amino acid sequence of Bak 23 partial amino acid sequence
of Bax 24 partial amino acid sequence of Bid 25 partial amino acid
sequence of Bik 26 partial amino acid sequence of Hrk 27 partial
amino acid sequence of Bad
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0037] The present invention is predicated in part on the
identification of a novel member of the Bcl-2 family of proteins.
The protein is called "Bmf" for "Bcl-2 modifying factor". It is
proposed that in healthy cells, Bmf is sequestered to the actin
based myosin. V motor complex by binding to a dynein light chain
and in particular dynein light chain 2 (DLC2). It is further
proposed that certain apoptotic stimuli, such as anoikis, release
Bmf from the myosin V motor complex allowing it to translocate and
bind to Bcl-2. Consequently, Bmf functions as a sensor of
intracellular damage by sequestration to motor complexes on
distinct cytoskeletal structures.
[0038] Accordingly, one aspect of the present invention provides a
nucleic acid molecule comprising a nucleotide sequence encoding or
complementary to a sequence encoding an amino acid sequence
substantially as set forth in one of SEQ ID NO:2 or SEQ ID NO:4 or
SEQ ID NO:6 or SEQ ID NO:8 or a derivative or homolog thereof or
having at least about 45% or mater similarity to one or more of SEQ
ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 or a
derivative or homolog thereof.
[0039] The term "similarity" as used herein includes exact identity
between compared sequences at the nucleotide or amino acid level.
Where there is non-identity at the nucleotide level, "similarity"
includes differences between sequences which result in different
amino acids that are nevertheless related to each other at the
structural, functional, biochemical and/or conformational levels.
Where there is non-identity at the amino acid level, "similarity"
includes amino acids that are nevertheless related to each other at
the structural, functional, biochemical and/or conformational
levels. In a particularly preferred embodiment, nucleotide and
sequence comparisons are made at the level of identity rather than
similarity.
[0040] Terms used to describe sequence relationships between. two
or more polynucleotides or polypeptides include "reference
sequence", "comparison window", "sequence similarity", "sequence
identity", "percentage of sequence similarity", "percentage of
sequence identity", "substantially similar" and "substantial
identity". A "reference sequence" is at least 12 but frequently 15
to 18 and often at least 25 or above, such as 30 monomer units,
inclusive of nucleotides and amino acid residues, in length.
Because two polynucleotides may each comprise (1) a sequence (i.e.
only a portion of the complete polynucleotide sequence) that is
similar between the two polynucleotides, and (2) a sequence that is
divergent between the two polynucleotides, sequence comparisons
between two (or more) polynucleotides are typically performed by
comparing sequences of the two polynucleotides over a "comparison
window" to identify and compare local regions of sequence
similarity. A "comparison window" refers to a conceptual segment of
typically 12 contiguous residues that is compared to a reference
sequence. The comparison window may comprise additions or deletions
(i,e. gaps) of about 20% or less as compared to the reference
sequence (which does not comprise additions or deletions) for
optimal alignment of the two sequences. Optimal alignment of
sequences for aligning a comparison window may be conducted by
computerised implementations of algorithms (GAP, BESTFIT, FASTA,
and TPASTA in the Wisconsin Genetics Software Package Release 7.0,
Genetics Computer Group, 575 Science Drive Madison, Wis., USA) or
by inspection and the best alignment (i.e. resulting in the highest
percentage homology over the comparison window) generated by any of
the various methods selected. Reference also may be made to the
BLAST family of programs as, for example, disclosed by Altschul et
al. (Nucl. Acids. Res. 25: 3389, 1997). A detailed discussion of
sequence analysis can be found in Unit 19.3 of Ausubel et al.
("Current Protocols in Molecular Biology", John Wiley & Sons
inc., 1994-1998, Chapter 15).
[0041] The terms "sequence similarity" and "sequence identity" as
used herein refers to the extent that sequences are identical or
functionally or structurally similar on a nucleotide-by-nucleotide
basis or an amino acid-by-amino acid basis over a window of
comparison. Thus, a "percentage of sequence identity", for example,
is calculated by comparing two optimally aligned sequences over the
window of comparison, determining the number of positions at which
the identical nucleic acid base (e.g. A, T, C, G, I) or the
identical amino acid residue (e.g. Ala, Pro, Ser, Thr, Gly, Val,
Leu, Ile, Phe, Tyr, Tip, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and
Met) occurs in both sequences to yield the number of matched
positions, dividing the number of matched positions by the total
number of positions in the window of comparison (i.e., the window
size), and multiplying the result by 100 to yield the percentage of
sequence identity. For the purposes of the present invention,
"sequence identity" will be understood to mean the "match
percentage" calculated by the DNASIS computer program (Version 2.5
for windows; available from Hitachi Software engineering Co., Ltd.,
South San Francisco, Calif., USA) using standard defaults as used
in the reference manual accompanying the software. Similar comments
apply in relation to sequence similarity.
[0042] Another aspect of the present invention contemplates a
nucleic acid molecule comprising a nucleotide sequence
substantially as set forth in one of SEQ ID NO:1 or SEQ ID NO:3 or
SEQ ID NO:5 or SEQ ID NO:7 or a derivative or homolog thereof
capable of hybridizing to one of SEQ NO:1 or SEQ ID NO:3 or SEQ ID
NO:5 or SEQ ID NO:7 under low stringency conditions and which
encodes an amino acid sequence corresponding to an amino acid
sequence set forth in one of SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID
NO:6 or SEQ ID NO:8 or a sequence having at least about 45%
similarity to one or more of SEQ ID NO:2 or SEQ DD NO:4 or SEQ ID
140:6 or SEQ ID NO:8.
[0043] More particularly, the present invention contemplates a
nucleic acid molecule comprising a sequence of nucleotides
substantially as set forth in SEQ ID NO:1 or SEQ 73 NO:3 or SEQ ID
NO:5 or SEQ ID NO:7.
[0044] Preferably, the subject nucleic acid molecules encode a
polypeptide having the identifying characteristics of Bmf or its
homologs or derivatives including functional derivatives.
[0045] Reference herein to a low stringency includes and
encompasses from at least about 0 to at least about 15% v/v
formamide and from at least about 1 M to at least about 2 M salt
for hybridization, and at least about 1 M to at least about 2 M
salt for washing conditions. Generally, low stringency is at from
about 25-30.degree. C. to about 42.degree. C. The temperature may
be altered and higher temperatures used to replace formamide and/or
to give alternative stringency conditions. Alternative stringency
conditions may be applied where necessary, such as medium
stringency, which includes and encompasses from at least about 16%
v/v to at least about 30% v/v formamide and from at least about 0.5
M to at least about 0.9 M salt for hybridization, and at least
about 0.5 M to at least about 0.9 M salt for washing conditions, or
high stringency, which includes and encompasses from at least about
31% v/v to at least about 50% v/v formamide and from at least about
0.01 M to at least about 0.15 M salt for hybridization, and at
least about 0.01 M to at least about 0.15 M salt for washing
conditions. In general, washing is carried out T.sub.m=69.3+0.41
(G+C) % (Mamamur and Doty, J. Mol. Biol. 5: 109, 1962). However,
the T.sub.m of a duplex DNA decreases by 1.degree. C. with every
increase of 1% in the number of mismatch base pairs (Bonner and
Lasky, Eur. J. Biochem. 46: 83, 1974). Formamide is optional in
these hybridization conditions. Accordingly, particularly preferred
levels of stringency are defined as follows: low stringency is
6.times.SSC buffer, 0.1% w/v SDS at 25-42.degree. C.; a moderate
stringency is 2.times.SSC buffer, 0.1% w/v SDS at a temperature in
the range 20.degree. C. to 65.degree. C.; high stringency is
0.1.times.SSC buffer, 0.1% w/v SDS at a temperature of at least
65.degree. C.
[0046] The nucleic acid molecule according to this aspect of the
present invention corresponds herein to "bmf". This gene has been
determined in accordance with the present invention to induce
apoptosis. The product of the bin/. gene is referred to herein as
"Bmf" without limiting this invention in any way, human bmf has
been mapped to human chromosome location 15q14. Bmf is known as a
"BH3-only" protein since the only Bcl-2 homology region which it
contains is BH3. It thereby forms a novel member of a Bcl-2 related
BH3-only pro-apoptotic group which also comprises, for example,
Bik/Nbk, Bid, Bim and Hrk.
[0047] The nucleic acid molecule encoding bmf is preferably a
sequence of deoxyribonucleic acids such as cDNA sequence, an mRNA
sequence or a genomic sequence. A genomic sequence may also
comprise exons and introns. A genomic sequence may also include a
promoter region or other regulatory region. The bmf genetic
sequence includes splice variants.
[0048] Reference hereinafter to "Bmf" and "bmf" should be
understood as a reference to all forms of Bmf and bmf,
respectively, including, by way of example, polypeptide and cDNA
isoforms of bmf which may be identified as arising from alternative
splicing of bmf mRNA. Reference hereinafter to Bmf and bmf in the
absence of a reference to its derivatives should be understood to
include reference to its derivatives thereof including any splice
variants.
[0049] The protein and/or gene is preferably from a human, primate,
livestock animal (e.g. sheep pig, cow, horse, donkey) laboratory
test animal (e.g. mouse, rat, rabbit, guinea pig) companion animal
(e.g. dog, cat), captive wild animal (e.g. fox, kangaroo, koala,
deer), ayes (e.g. chicken, geese, duck, emu, ostrich), reptile or
fish.
[0050] Derivatives include fragments (such as peptides), parts,
portions, chemical equivalents, mutants, homologs or mimetics from
natural, synthetic or recombinant sources including fusion
proteins. Derivatives may be derived from insertion, deletion or
substitution of amino acids. Amino acid insertional derivatives
include amino and/or carboxylic terminal fusions as well as
intrasequence insertions of single or multiple amino acids.
insertional amino acid sequence variants are those in which one or
more amino acid residues are introduced into a predetermined site
in the protein although random insertion is also possible with
suitable screening of the resulting product. Deletional variants
are characterized by the removal of one or more amino acids from
the sequence. Substitutional amino acid variants are those in which
at least one residue in the sequence has been removed and a
different residue inserted in its place. Additions to amino acid
sequences including fusions with other peptides, polypeptides or
proteins. Mutants should be understood to include, but is not
limited to, the specific Bmf or bmf mutant molecules described
herein. Derivatives include, for example, peptides derived from the
BH3 region, from the dynein binding region or from a site of
phosphorylation. Peptides include, for example, molecules
comprising at least 4 contiguous amino acids corresponding to at
least 4 contiguous amino acids of Bmf as herein defined. Use of the
term "polypeptides" herein should be understood to encompass
peptides, polypeptides and proteins.
[0051] The derivatives of Bmf include fragments having particular
epitopes or parts of the entire Bmf protein fused to peptides,
polypeptides or other proteinaceous or non-proteinaceous molecules.
For example, Bmf or derivative thereof may be fused to a molecule
to facilitate its entry into a cell. Analogues of Bmf contemplated
herein include, but are not limited to, modification to side
chains, incorporating of unnatural amino acids and/or their
derivatives during peptide, polypeptide or protein synthesis and
the use of crosslinkers and other methods which impose
conformational constraints on the proteinaceous molecules or their
analogues. Derivatives of nucleic acid sequences may similarly be
derived from single or multiple nucleotide substitutions, deletions
and/or additions including fusion with other nucleic acid
molecules. The derivatives of the nucleic acid molecules of the
present invention include oligonucleotides, PCR primers, antisense
molecules, molecules suitable for use in co-suppression and fusion
of nucleic acid molecules.
[0052] Examples of side chain modifications contemplated by the
present invention include modifications of amino groups such as by
reductive alkylation by reaction with an aldehyde followed by
reduction with NaBH.sub.4; amidination with methylacetimidate;
acylation with acetic anhydride; carbamoylation of amino groups
with cyanate; trinitrobenzylation of amino groups with
2,4,6-tinitrobenzene sulphonic acid (TNBS); acylation of amino
groups with succinic anhydride and tetrahydrophthalic anhydride;
and pyridoxylation of lysine with pyridoxal-5-phosphate followed by
reduction with NaBH.sub.4.
[0053] The guanidine group of arginine residues may be modified by
the formation of heterocyclic condensation products with reagents
such as 2,3-butanedione, phenylglyoxal and glyoxal.
[0054] The carboxyl group may be modified by carbodiimide
activation via O-acylisourea formation followed by subsequent
derivitisation, for example, to a corresponding amide.
[0055] Sulphydryl groups may be modified by methods such as
carboxymethylation with iodoacetic acid or iodoacetamide; performic
acid oxidation to cysteic acid; formation of a mixed disulphides
with other thiol compounds; reaction with maleimide, maleic
anhydride or other substituted maleimide; formation of mercurial
derivatives using 4-chloromercuribenzoate,
4-chloromercuriphenylsulphonic acid, phenylmercury chloride,
2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation
with cyanate at alkaline pH.
[0056] Tryptophan residues may be modified by for example,
oxidation with N-bromosuccinimide or alkylation of the indole ring
with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine
residues on the other hand, may be altered. by nitration. with
tetranitromethane to form a 3-nitrotyrosine derivative.
[0057] Modification of the imidazole ring of a histidine residue
may be accomplished by alkylation with iodoacetic acid derivatives
or N-carbethoxylation with diethylpyrocarbonate.
[0058] Examples of incorporating unnatural amino acids and
derivatives during peptide synthesis include, but are not limited
to, use of norleucine, 4-amino butyric acid,
4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid,
t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine,
4-amino-3-hydroxy-G-methylheptanoic acid, 2-thienyl alanine and/or
D-isomers of amino acids. A list of unnatural amino acid,
contemplated herein is shown in Table 1.
TABLE-US-00003 TABLE 1 Non-conventional Non-conventional amino acid
Code amino acid Code .alpha.-aminobutyric acid Abu
L-N-methylalanine Nmala .alpha.-amino-.alpha.-methylbutyrate Mgabu
L-N-methylarginine Nmarg aminocyclopropane- Cpro
L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid
Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys
aminonorbornyl- Norb L-N-methylglutamine Nmgln carboxylate
L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa
L-Nmethylhistidine Nmhis cyclopentylalanine Cpen
L-N-methylisolleucine Nmile D-alanine Dal L-N-methylleucine Nmleu
D-arginine Darg L-N-methyllysine Nmlys D-aspartic acid Dasp
L-N-methylmethionine Nmmet D-cysteine Dcys L-N-methylnorleucine
Nmnle D-glutamine Dgln L-N-methylnorvaline Nmnva D-glutamic acid
Dglu L-N-methylornithine Nmorn D-histidine Dhis
L-N-methylphenylalanine Nmphe D-isoleucine Dile L-N-methylproline
Nmpro D-leucine Dleu L-N-methylserine Nmser D-lysine Dlys
L-N-methylthreonine Nmthr D-methionine Dmet L-N-methyltryptophan
Nmtrp D-ornithine Dorn L-N-methyltyrosine Nmtyr D-phenylalanine
Dphe L-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycine
Nmetg D-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine
Dthr L-norleucine Nle D-tryptophan Dtrp L-norvaline Nva D-tyrosine
Dtyr .alpha.-methyl-aminoisobutyrate Maib D-valine Dval
.alpha.-methyl-.gamma.-aminobutyrate Mgabu D-.alpha.-methylalanine
Dmala .alpha.-methylcyclohexylalanine Mchexa
D-.alpha.-methylarginine Dmarg .alpha.-methylcylcopentylalanine
Mcpen D-.alpha.-methylasparagine Dmasn
.alpha.-methyl-.alpha.-napthylalanine Manap
D-.alpha.-methylaspartate Dmasp .alpha.-methylpenicillamine Mpen
D-.alpha.-methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu
D-.alpha.-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg
D-.alpha.-methylhistidine Dmhis N-(3-aminopropyl)glycine Norn
D-.alpha.-methylisoleucine Dmile N-amino-.alpha.-methylbutyrate
Nmaabu D-.alpha.-methylleucine Dmleu .alpha.-napthylalanine Anap
D-.alpha.-methyllysine Dmlys N-benzylglycine Nphe
D-.alpha.-methylmethionine Dmmet N-(2-carbamylethyl)glycine Ngln
D-.alpha.-methylornithine Dmorn N-(carbamylmethyl)glycine Nasn
D-.alpha.-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine Nglu
D-.alpha.-methylproline Dmpro N-(carboxymethyl)glycine Nasp
D-.alpha.-methylserine Dmser N-cyclobutylglycine Ncbut
D-.alpha.-methylthreonine Dmthr N-cycloheptylglycine Nchep
D-.alpha.-methyltryptophan Dmtrp N-cyclohexylglycine Nchex
D-.alpha.-methyltyrosine Dmty N-cyclodecylglycine Ncdec
D-.alpha.-methylvaline Dmval N-cylcododecylglycine Ncdod
D-N-methylalanine Dnmala N-cyclooctylglycine Ncoct
D-N-methylarginine Dnmarg N-cyclopropylglycine Ncpro
D-N-methylasparagine Dnmasn N-cycloundecylglycine Ncund
D-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine Nbhm
D-N-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine Nbhe
D-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine Narg
D-N-methylglutamate Dnmglu N-(1-hydroxyethyl)glycine Nthr
D-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine Nser
D-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine Nhis
D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine Nhtrp
D-N-methyllysine Dnmlys N-methyl-.gamma.-aminobutyrate Nmgabu
N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet
D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen
N-methylglycine Nala D-N-methylphenylalanine Dnmphe
N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro
N-(1-methylpropyl)glycine Nile D-N-methylserine Dnmser
N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr
D-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine Nval
D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap
D-N-methylvaline Dnmval N-methylpenicillamine Nmpen
.gamma.-aminobutyric acid Gabu N-(p-hydroxyphenyl)glycine Nhtyr
L-t-butylglycine Tbug N-(thiomethyl)glycine Ncys L-ethylglycine Etg
penicillamine Pen L-homophenylalanine Hphe L-.alpha.-methylalanine
Mala L-.alpha.-methylarginine Marg L-.alpha.-methylasparagine Masn
L-.alpha.-methylaspartate Masp L-.alpha.-methyl-t-butylglycine
Mtbug L-.alpha.-methylcysteine Mcys L-methylethylglycine Metg
L-.alpha.-methylglutamine Mgln L-.alpha.-methylglutamate Mglu
L-.alpha.-methylhistidine Mhis L-.alpha.-methylhomophenylalanine
Mhphe L-.alpha.-methylisoleucine Mile N-(2-methylthioethyl)glycine
Nmet L-.alpha.-methylleucine Mleu L-.alpha.-methyllysine Mlys
L-.alpha.-methylmethionine Mmet L-.alpha.-methylnorleucine Mnle
L-.alpha.-methylnorvaline Mnva L-.alpha.-methylornithine Morn
L-.alpha.-methylphenylalanine Mphe L-.alpha.-methylproline Mpro
L-.alpha.-methylserine Mser L-.alpha.-methylthreonine Mthr
L-.alpha.-methyltryptophan Mtrp L-.alpha.-methyltyrosine Mtyr
L-.alpha.-methylvaline Mval L-N-methylhomophenylalanine Nmhphe
N-(N-(2,2-diphenylethyl) Nnbhm N-(N-(3,3-diphenylpropyl) Nnbhe
carbamylmethyl)glycine carbamylmethyl)glycine
1-carboxy-1-(2,2-diphenyl- Nmbc ethylamino)cyclopropane
[0059] Crosslinkers can be used, for example, to stabilize 3D
conformations, using home-bifunctional crosslinkers such as the
bifunctional imido esters having (CH.sub.2).sub.n spacer groups
with n=1 to n=6, glutaraldehyde, N-hydroxysuccinimide esters and
hetero-bifunctional reagents which usually contain an
amino-reactive moiety such as N-hydroxysuccinimide and another
group specific-reactive moiety such as maleimido or dithio moiety
(SH) or carbodiimide (COOH). In addition, peptides can be
conformationally constrained by, for example, incorporation of
C.sub..alpha. and N.sub..alpha.-methylamino acids, introduction of
double bonds between C.sub..alpha. and C.sub..beta. atoms of amino
acids and the formation of cyclic peptides or analogues by
introducing covalent bonds such as forming an amide bond between
the N and C termini, between two side chains or between a side
chain and the N or C terminus.
[0060] The nucleic acid molecule of the present invention is
preferably in isolated folio. or ligated to a vector, such as an
expression vector. By "isolated" is meant a nucleic acid molecule
having undergone at least one purification step and this is
conveniently defined, for example, by a composition comprising at
least about 10% subject nucleic acid molecule, preferably at least
about 20%, more preferably at least about 30%, still more
preferably at least about 40-50%, even still more preferably at
least about 60-70%, yet even still more preferably 80-90% or
greater of subject nucleic acid molecule relative to other
components as determined by molecular weight, encoding activity,
nucleotide sequence, base composition or other convenient means.
The nucleic acid molecule of the present invention may also be
considered, in a preferred embodiment, to be biologically pure.
[0061] In a particularly preferred embodiment, the nucleotide
sequence corresponding to bmf is a cDNA sequence comprising a
sequence of nucleotides as set forth in one of SEQ ID NO:1 or SEQ
NO:3 or SEQ ID NO:5 or SEQ ID NO:7 or is a derivative or homolog
thereof including a nucleotide sequence having similarity to one of
SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:7 and which
encodes an amino acid sequence corresponding to an amino acid
sequence as set forth in one of SEQ 3D NO:2 or SEQ ID NO:4 or SEQ
3D NO:6 or SEQ NO:8 or a sequence having at least about 45%
similarity to one or more of SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID
NO:6 or SEQ ID NO:8.
[0062] A derivative of the nucleic acid molecule of the present
invention also includes nucleic acid molecules capable of
hybridizing to the nucleotide sequences as set forth in one of SEQ
ID NO:1 or SEQ M NO:3 or SEQ JD NO:5 or SEQ NO:7 under low
stringency conditions. Preferably, said low stringency is at
42.degree. C.
[0063] in another embodiment, the present invention is directed to
an isolated nucleic acid molecule encoding 1:nif or a derivative
thereof, said nucleic acid molecule selected from the list
consisting of:-- [0064] (i) a nucleic acid molecule comprising a
nucleotide sequence encoding the amino acid sequence set forth in
one of SEQ ID NO:2 or SEQ ID NO:4 or SEQ M NO:6 or SEQ. JD NO:8 or
a derivative or homolog thereof or having at least about 45%
similarity to one or more of SEQ ID NO:2 or SEQ ID NO:4 or SEQ NO:6
or SEQ ID NO:8; [0065] (ii) a nucleic acid molecule comprising a
nucleotide sequence substantially as set forth in one of SEQ ID
NO:1 or SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:7 or a derivative
or homolog thereof; [0066] (iii) a nucleic acid molecule capable of
hybridizing under low stringency conditions to the nucleotide
sequence substantially as set forth in one of SEQ ID NO:1 or SEQ ID
NO:3 or SEQ ID NO:5 or SEQ ID NO:7 a derivative or homolog and
encoding an amino acid sequence corresponding to an amino acid.
sequence as set forth in one of SEQ ID NO:2 or SEQ ID NO:4 or SEQ
ID NO:6 or SEQ ID NO:8 or a derivative or homolog or a sequence
having at least about 45% similarity to one or more of SEQ ID NO:2
or SEQ TD NO:4 or SEQ 3D NO:6 or SEQ ID NO:8; [0067] (iv) a nucleic
acid molecule capable of hybridizing to the nucleic acid molecule
of paragraphs (i) or (iii) under low stringency conditions and
encoding an amino acid sequence having at least about 45%
similarity to one or more of SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID
NO:6 or SEQ ID NO:8; and [0068] (v) a derivative or mammalian
homolog of the nucleic acid molecule of paragraphs (i) or (ii) or
(iii) or (iv).
[0069] Reference here to an ability to hybridize to a particular
sequence (e.g. SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID
NO:7) also includes, in the alternative, an ability to hybridize to
its complementary form. In other words, nucleic acid molecules are
encompassed which hybridize to SEQ ID NO:1 or SEQ DD NO:3 or SEQ ID
NO:5 or SEQ BD NO:7 or their complementary forms.
[0070] The nucleic acid molecule may be ligated to an expression
vector capable of expression in a prokaryotic cell (e.g. E. coli)
or a eukaryotic cell (e.g. yeast cells, fungal cells, insect cells,
mammalian cells or plant cells). The nucleic acid molecule may be
ligated or fused or otherwise associated with a nucleic acid
molecule encoding another entity such as, for example, a signal
peptide, a cytokine or other member of the Bcl-2 family.
[0071] The present invention extends to the promoter for bmf from
murine or other mammalian species. Nucleotide sequences comprising
the murine and human bmf promoters are shown in SEQ DD NO:9 and SEQ
ID NO:10, respectively. The present invention extends to mutants
and derivatives of these promoters and their use in genetic
con-tracts, gene therapy and in generating genetically modified
animals. A mutant or derivative of a promoter includes one which
comprises a nucleotide sequence having at least 70% similarity to
SEQ ID NOS:9 or 10 or which is capable of hybridizing to SEQ ID
NO:9 or SEQ SD NO:10 or their complementary ferns under low
stringency conditions.
[0072] The present invention extends to the expression product of
the nucleic acid molecule hereinbefore cleaned.
[0073] The expression product is Bmf having an amino acid sequence
set forth in one of SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or
SEQ ID NO:8 or is a derivative or homolog thereof as defined above
or is a mammalian homolog having an amino acid sequence of at least
about 45% similarity to the amino acid sequence set forth in one of
SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 or
derivative or homolog thereof.
[0074] Another aspect of the present invention is directed to an
isolated polypeptide selected from the list consisting of:-- [0075]
(i) a polypeptide having an amino acid sequence substantially as
set forth in one of SEQ ID NO:2 or SEQ m NO:4 or SEQ ID NO:6 or SEQ
ID NO:8 or derivative or homolog thereof or a sequence having at
least about 45% similarity to one or more of SEQ ID NO:2 or SEQ ID
NO:4 or SEQ ID NO:6 or SEQ ID NO:8; [0076] (ii) a polypeptide
encoded by a nucleotide sequence substantially as set forth in one
of SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:7 or
derivative or homolog thereof or a sequence encoding an amino acid
sequence having at least about 45% similarity to one or more of SEQ
ID NO:2 or SEQ 7D NO:4 or SEQ ID NO:6 or SEQ ID NO:8; [0077] (iii)
a polypeptide encoded by a nucleic acid molecule capable of
hybridizing to the nucleotide sequence as set forth in one of SEQ
II) NO:1 or SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:7 or derivative
or homolog thereof under low stringency conditions and which
encodes an amino acid sequence substantially as set forth in SEQ ID
NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 or derivative or
homolog thereof or an amino acid sequence having at least about 45%
similarity to one or more of SEQ ID NO:2 or SEQ NO:4 SEQ NO:6 or
SEQ ID NO:8; [0078] (iv) a polypeptide as defined in paragraphs (i)
or (ii) (iii) in homodimeric form; and [0079] (v) a polypeptide as
defined in paragraphs (i) or (ii) or (iii) in heterodimeric
form.
[0080] As defined earlier, the present invention extends to
peptides or derivatives thereof of &nil Preferably, said
peptide comprises at least 5 contiguous amino acids of the
polypeptide defined in SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or
SEQ ID NO:8. The present invention also extends to nucleic acid
molecules encoding the peptides of the present invention.
[0081] Another aspect of the present invention provides a nucleic
acid molecule comprising a nucleotide sequence encoding a
polypeptide having one or more of the identifying characteristics
of Bmf or a derivative or homolog thereof.
[0082] Reference herein to "identifying characteristics" of Bmf
includes one or more of the following features:-- [0083] (i) a
polypeptide which induces apoptosis; [0084] (ii) a polypeptide
having an amino acid sequence substantially as set forth in SEQ ID
NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 or a derivative
or homolog thereof; [0085] (iii) a polypeptide having an amino
acid. sequence of at least 45% similarity to SEQ TD NO:2 or SEQ ID
NO:4 SEQ NO:6 or SEQ ID NO:8; [0086] (iv) a polypeptide as defined
in paragraph (ii) or (iii) which induces apoptosis; [0087] (v) a
polypeptide encoded by a nucleic acid sequence substantially as set
forth in SEQ ID NO:1 or SEQ ID NO:3 or SEQ ID NO:5 or SEQ ID NO:7
or derivative or homolog thereof; [0088] (vi) a polypeptide encoded
by a nucleic acid molecule capable of hybridizing to the nucleotide
sequence as set forth in one of SEQ ID NO:1 or SEQ ID NO:3 or SEQ
ID NO:5 or SEQ ID NO:7 under low stringency conditions; [0089]
(vii) a polypeptide as defined in paragraph (v) or (vi) which
induces apoptosis; and [0090] (viii) a non apoptosis inducing
derivative of the polypeptide defined in paragraphs (i) to
(vii).
[0091] The present invention should be understood to extend to the
expression product of the nucleic acid molecule according to this
aspect of the present invention.
[0092] Although not intending to limit the invention to any one
theory or mode of action, the BH3 region is responsible for some of
the cytotoxic actions of Bmf. The BH3 region forms an amphipathic
.alpha.-helix that interacts with the elongated hydrophobic cleft
formed by the BH1, BH2 and BH3 regions of pro-survival molecules
such as, for example, Bcl-x.sub.L. The pro-apoptotic action of Bmf
reflects its ability to bind to the anti-apoptotic members of the
Bcl-2 family.
[0093] Still without limiting the invention to any one theory or
mode of action, the pro-apoptotic activity of Bmf is thought to be
regulated both at the transcriptional level and at the
post-translational level. Sequence analysis of the non-coding 5'
region of Bmf has revealed a number of putative binding sites for
transcription factors such as AP1. Bmf is proposed to interact via
a dynein light chain such as DLC2. A dynein light chain is a highly
conserved protein which is a component of the myosin V motor
complex.
[0094] The interaction of Bmf with the myosin V motor complex
regulates the pro-apoptotic activity of Bmf. Single or multiple
amino acid mutations in Bmf which abolish binding to the dynein
light chain are encompassed by the present invention.
[0095] Accordingly, a related aspect of the present invention is
directed to a variant of an isolated bmf nucleic acid molecule
comprising one or more nucleotide mutations in said nucleic acid
molecule resulting in at least one amino acid addition,
substitution and/or deletion to the polypeptide encoded by said
variant wherein said polypeptide cannot bind, couple or otherwise
associate with a dynein light chain, such as DLC2.
[0096] Preferably, the mutation results in an altered amino acid
sequence in the region which binds the dynein light chain. The
present invention should be understood to extend to variants of Bmf
comprising a mutation resulting in an amino acid addition,
substitution and/or deletion in a region functionally equivalent to
the regions hereinbefore defined.
[0097] Accordingly, the present invention is more particularly
directed to a variant of an isolated bmf nucleic acid molecule
comprising one or more nucleotide mutations in said nucleic acid
molecule resulting in at least one amino acid addition,
substitution and/or deletion in the region of the polypeptide
encoded by said variant which binds the dynein light chain, wherein
said polypeptide cannot bind, couple or otherwise associate with a
dynein light chain.
[0098] Mutations contemplated by the present invention which occur
in combination with one or more mutations in another location are
also contemplated by the present invention.
[0099] The present invention extends to the expression products of
the nucleic acid molecule variants defined according to this aspect
of the present invention.
[0100] Accordingly, the present invention is directed to a variant
of an isolated Bmf polypeptide comprising at least one amino acid
addition, substitution and/or deletion wherein said polypeptide
cannot bind, couple or otherwise associate with the dyncin light
chain.
[0101] The present invention extends to derivatives of the nucleic
acid molecules and polypeptides according to this aspect of the
present invention. The term "derivatives" should be understood as
previously defined.
[0102] As hereinbefore defined, reference to "Bmf" and "bmf" should
be understood to include reference to the variant molecules defined
according to this aspect of the present invention.
[0103] The Bmf of the present invention may be in multimeric form
meaning that two or more molecules are associated together. Where
the same Bra molecules are associated together, the complex is a
homomultimer. An example of a homomultimer is a homodimer. Where at
least one Bmf is associated with at least one non-Bmf molecule,
then the complex is a heteromultimer such as a heterodimer. A
heteromultimer may include a molecule of another member of the
Bcl-2 family or other molecule capable of modulating apoptosis.
Furthermore, the present invention contemplates fusion, or hybrids
or heteromeric dimers between Bmf and other molecules such as
Bim.
[0104] The present invention contemplates, therefore, a method for
modulating expression of bmf in a mammal, said method comprising
administering to said mammal a modulating effective amount of an
agent for a time and under conditions sufficient to up-regulate or
down-regulate or otherwise modulate expression of bmf. For example,
bmf antisense sequences such as oligonucleotides may be introduced
into a cell to enhance the ability of that cell to survive.
Conversely, a nucleic acid molecule encoding Bmf or a derivative
thereof may be introduced to decrease the survival capacity of any
cell expressing the endogenous bmf gene. Modulation of the
expression of bmf should be understood to extend to modulating
transcriptional and translation events such as the splicing pattern
of Bmf RNA.
[0105] Another aspect of the present invention contemplates a
method of modulating activity of Buff in a mammal, said method
comprising administering to said mammal a modulating effective
amount of an agent for a time and under conditions sufficient to
increase or decrease Bmf activity.
[0106] Modulation of said activity by the administration of an
agent to a mammal can be achieved by one of several techniques,
including but in no way limited to introducing into said mammal a
proteinaceous or non-proteinaceous molecule which:
(i) modulates expression of bmf; (ii) functions as an antagonist of
Bmf; and (iii) functions as an agonist of Bim.
[0107] Said proteinaceous molecule may be derived from natural or
recombinant sources including fusion proteins or following, for
example, natural product screening. Said non-proteinaceous molecule
may be, for example, a nucleic acid molecule or may be derived from
natural sources, such as for example natural product screening or
may be chemically synthesized. The present invention contemplates
chemical analogues of Bmf capable of acting as agonists or
antagonists of Bmf. Chemical agonists may not necessarily be
derived from Bmf but may share certain conformational similarities.
Alternatively, chemical agonists may be specifically designed to
mimic certain physiochemical properties of Bmf Antagonists may be
any compound capable of blocking, inhibiting or otherwise
preventing Bmf from carrying out its normal or pathological
biological functions. Antagonists include, but are not limited to
parts of Bmf or peptides thereof, monoclonal antibodies specific
for Bmf or parts of Bmf, and antisense nucleic acids or
oligonucleotides which prevent transcription or translation of bit
genes or mRNA in mammalian cells. Agonists of Bmf and bmf include,
for example, the derivative or variant molecules or peptides
hereinbefore defined which interact with anti-apoptotic molecules
such as Bcl-2, to prevent their functional activity thereby
promoting apoptosis. Agonists may also include molecules capable of
disrupting or preventing binding of Bmf to the dynein light chain
or the interaction of dynein light chain with dynein intermediate
chain.
[0108] Said proteinaceous or non.-proteinaceous molecule may act
either directly or indirectly to modulate the expression of bmf or
the activity of Bmf. Said molecule acts directly if it associates
with Bmf or Bmf to modulate the expression or activity of bmf or
Bmf. Said molecule acts indirectly if it associates with a molecule
other than bmf or Bmf which other molecule either directly or
indirectly modulates the expression or activity of bmf or Bmf.
[0109] Accordingly, the method of the present invention encompasses
the regulation of bmf or Bmf expression or activity via the
induction of a cascade of regulatory steps which lead to the
regulation of bmf or Bmf expression or activity.
[0110] Increased bmf expression or Bmf activity is useful, for
example, for treatment or prophylaxis in conditions such as cancer
and deletion of autoreactive lymphocytes in autoimmune disease.
Decreased bmf expression or Bmf activity is useful in regulating
inhibition or prevention of cell death or degeneration such as
under cytotoxic conditions during, for example, .gamma.-irradiation
and chemotherapy or during HIV/AIDS or other viral infections,
ischaemia or myocardial infarction, Since Bmf is expressed in germ
cells, modulating bmf expression or Bmf activity is useful, for
example, as a contraceptive or method of sterilisation by
preventing generation of fertile sperm.
[0111] Another aspect of the present invention contemplates a
method of modulating apoptosis in a mammal, said method comprising
administering to said mammal an effective amount of an agent for a
time and under conditions sufficient to modulate the expression of
a nucleotide sequence encoding bmf.
[0112] Yet another aspect of the present invention contemplates a
method of modulating apoptosis in a mammal, said method comprising
administering to said mammal an effective amount of an agent for a
tisane and under conditions sufficient to modulate the activity of
Bmf.
[0113] Still another aspect of the present invention contemplates a
method of modulating apoptosis in a mammal, said method comprising
administering to said mammal an effective amount of Bmf or bmf or
derivative thereof.
[0114] The Brat bmf or derivative thereof or agent used may also be
linked to a targeting means such as a monoclonal antibody, which
provides specific delivery of the Bmf, bmf or agent to the target
cells.
[0115] In a preferred embodiment of the present invention, the Bmf,
bmf or agent used in the method is linked to an antibody specific
for said target cells to enable specific delivery to these
cells.
[0116] Modulation of Bmf or bmf may be accompanied simultaneously
or sequentially with the modulation of other molecules or genes
such as but not limited to bim or Bim.
[0117] Administration of the Bmf, bmf or agent, in the form of a
pharmaceutical composition, may be performed by any convenient
means. Bmf, bmf or agent of the pharmaceutical composition are
contemplated to exhibit therapeutic activity when administered in
an amount which depends on the particular case. The variation
depends, for example, on the human or animal and the Bmf, bmf or
agent chosen. A broad range of doses may be applicable. Considering
a patient, for example, from about 0.01 mg to about 10 mg of Bmf or
agent may be administered per kilogram of body weight per day.
Dosage regimes may be adjusted to provide the optimum therapeutic
response. For example, several divided doses may be administered
daily, weekly, monthly or other suitable time intervals or the dose
may be proportionally reduced as indicated by the exigencies of the
situation, The Bmf or agent may be administered in a convenient
manner such as by the oral, intravenous (where water soluble),
intranasal, intraperitoneal, intramuscular, subcutaneous,
intradermal or suppository routes or implanting (e.g. using slow
release molecules). With particular reference to use of Bmf or
agent, these peptides may be administered in the form of
pharmaceutically acceptable nontoxic salts, such as acid addition
salts or metal complexes, e.g. with zinc, iron or the like (which
are considered as salts for purposes of this application),
illustrative of such acid addition salts are hydrochloride,
hydrobromide, sulphate, phosphate, maleate, acetate, citrate,
benzoate, succinate, malate, ascorbate, tartrate and the like. If
the active ingredient is to be administered in tablet form, the
tablet may contain a binder, such as tragacanth, corn starch or
gelatin; a disintegrating agent, such as alginic acid; and a
lubricant, such as magnesium stearate.
[0118] A further aspect of the present invention relates to the use
of the invention in relation to mammalian disease conditions. For
example, the present invention is particularly applicable to, but
in no way limited to, use in therapy or prophylaxis in relation to
cancer, degenerative diseases, autoimmune disorders, viral
infections or for germ cell regulation.
[0119] Accordingly, another aspect of the present invention relates
to a method of treating a mammal, said method comprising
administering to said mammal an effective amount of an agent for a
time and under conditions sufficient to modulate the expression of
bmf wherein said modulation results in modulation of apoptosis.
[0120] In another aspect, the present invention relates to a method
of treating a mammal, said method comprising administering to said
mammal an effective amount of an agent for a time and under
conditions sufficient to modulate the activity of Bmf wherein said
modulation results in modulation of apoptosis.
[0121] In another aspect, the present invention relates to a method
of treating a mammal, said method comprising administering to said
mammal an effective amount of Bmf or derivative thereof for a time
and under conditions sufficient to modulate apoptosis.
[0122] Yet another aspect of the present invention relates to a
method of treating a mammal, said method comprising administering
to said mammal an effective amount of bmf or derivative thereof for
a time and under conditions sufficient to modulate apoptosis.
[0123] In yet another aspect, the present invention relates to the
use of an agent capable of modulating the expression of bmf or
derivative thereof in the manufacture of a medicament for the
modulation of apoptosis.
[0124] Another aspect of the present invention relates to the use
of an agent capable of modulating the expression of Bmf or
derivative thereof in the manufacture of a medicament for the
modulation of apoptosis.
[0125] A further aspect of the present inventions relates to the
use of Bmf or bmf or derivative thereof in the manufacture of a
medicament for the modulation of apoptosis.
[0126] Still yet another aspect of the present invention relates to
agents for use in modulating bmf expression wherein modulating
expression of said bmf modulates apoptosis.
[0127] A further aspect of the present invention relates to agents
for use in modulating Bmf expression wherein modulating expression
of said Bmf modulates apoptosis.
[0128] Another aspect of the present invention relates to Bmf or
bmf or derivative thereof for use in modulating apoptosis.
[0129] In a related aspect of the present invention, the mammal
undergoing treatment may be human or an animal in need of
therapeutic of prophylactic treatment.
[0130] In yet another further aspect, the present invention
contemplates a pharmaceutical composition comprising bmf, Bmf or
derivative thereof or an agent capable of modulating bmf expression
or Bmf activity together with one or more pharmaceutically
acceptable carriers and/or diluents. bmf, Bmf or said agent are
referred to as the active ingredients.
[0131] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions (where water soluble) and sterile powders
for the extemporaneous preparation of sterile injectable solutions.
It must be stable under the conditions of manufacture and storage
and must be preserved against the contaminating action of
microorganisms such as bacteria and fungi. The carrier can be a
solvent or dilution medium comprising, for example, water, ethanol,
polyol (for example, glycerol, propylene glycol and liquid
polyethylene glycol, and the like), suitable mixtures thereof and
vegetable oils. The proper fluidity can be maintained, for example,
by the use of surfactants. The preventions of the action of
microorganisms can be brought about by various anti-bacterial and
anti-fungal agents, for example, parabens, chlorobutanol, phenol,
sorbic acid, thirmerosal and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars or
sodium chloride. Prolonged absorption of the injectable
compositions can be brought about by the use in the compositions of
agents delaying absorption, for example, aluminium monostearate and
gelatin.
[0132] Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate
solvent with the active ingredient and optionally other active
ingredients as required, followed by filtered sterilization or
other appropriate means of sterilization. In the case of sterile
powders for the preparation of sterile injectable solutions,
suitable methods of preparation include vacuum drying and the
freeze-drying technique which yield a powder of active ingredient
plus any additionally desired ingredient.
[0133] When bmf, Bmf and/or Bad. modulators are suitably protected,
they may be orally administered, for example, with an inert diluent
or with an assimilable edible carrier, or it may be enclosed in
hard or soft shell gelatin capsule, or it may be compressed into
tablets, or it may be incorporated directly with the food of the
diet or administered via breast milk. For oral therapeutic
administration, the active ingredient may be incorporated with
excipients and used in the form of ingestible tablets, buccal
tablets, troches, capsules, elixirs, suspensions, syrups, wafers
and the like. Such compositions and preparations should contain at
least 1% by weight of active compound. The percentage of the
compositions and preparations may, of course, be varied and may
conveniently be between about 5 to about 80% of the weight of the
unit. The amount of active compound in such therapeutically useful
compositions is such that a suitable dosage will be obtained.
Preferred compositions or preparations according to the present
invention are prepared so that an oral dosage unit form contains
between about 0.1 .mu.g and 200 mg of active compound. Alternative
dosage amounts include from about 1 .mu.g to about 1000 mg and from
about 10 .mu.g to about 500 mg. These dosages may be per individual
or per kg body weight. Administration may be per hour, day, week,
month or year.
[0134] The tablets, troches, pills, capsules, creams and the like
may also contain the components as listed hereafter. A binder such
as gun, acacia, corn starch or gelatin; excipients such as
dicalcium phosphate; a disintegrating agent such as corn starch,
potato starch, alginic acid and the like; a lubricant such as
magnesium stearate; and a sweetening agent such as sucrose, lactose
or saccharin may be added or a flavouring agent such as peppermint,
oil of wintergreen or cherry flavouring. When the dosage unit form
is a capsule, it may contain, in addition to materials of the above
type, a liquid carrier. Various other materials may be present as
coatings or to otherwise modify the physical form of the dosage
unit. For instance, tablets, pills or capsules may be coated with
shellac, sugar or both. A syrup or elixir may contain the active
compound, sucrose as a sweetening agent, methyl and propylparabens
as preservatives, a dye and flavouring such as cherry or orange
flavour. Of course, any material used in preparing any dosage unit
form should be pharmaceutically pure and substantially non-toxic in
the amounts employed. In addition, the active compound(s) may be
incorporated into sustained-release preparations and
formulations.
[0135] Pharmaceutically acceptable carriers and/or diluents include
any and all solvents, dispersion media, coatings, anti-bacterial
and anti-fungal agents, isotonic and absorption delaying agents and
the like. The use of such media and agents for pharmaceutical
active substances is well known in the art and except insofar as
any conventional media or agent is incompatible with the active
ingredient, their use in the therapeutic compositions is
contemplated. Supplementary active ingredients can also be
incorporated into the compositions.
[0136] It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the
mammalian subjects to be treated; each unit containing a
predetermined quantity of active material calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the novel dosage unit
forms of the invention are dictated by and directly dependent on
(a) the unique characteristics of the active material and the
particular therapeutic effect to be achieved, and (b) the
limitations inherent in the art of compounding such an active
material for the treatment of disease in living subjects having a
diseased condition in which bodily health is impaired as herein
disclosed in detail.
[0137] The principal active ingredient is compounded for convenient
and effective administration in effective amounts with a suitable
pharmaceutically acceptable carrier in dosage unit form as
hereinbefore disclosed. A unit dosage form can, for example,
contain the principal active compound in amounts ranging from 0.5
.mu.g to about 2000 mg. Expressed in proportions, the active
compound is generally present in from about 0.5 .mu.g to about 2000
mg/ml of carrier. In the case of compositions containing
supplementary active ingredients, the dosages are determined by
reference to the usual dose and manner of administration of the
said ingredients.
[0138] The pharmaceutical composition may also comprise genetic
molecules such as a vector capable of transfecting target cells
where the vector carries a nucleic acid molecule capable of
modulating bmf expression or Bmf activity. The vector may, for
example, be a viral vector.
[0139] Conditions requiring modulation of physiological cell death
include enhancing survival of cells utilising, for example,
antisense sequence in patients with neurodegenerative diseases,
myocardial infarction, muscular degenerative disease, hypoxia,
ischaemia, HIV infection or for prolonging the survival of cells
being transplanted for treatment of disease. Alternatively, the
molecules of the present invention are useful for, for example,
reducing the survival capacity of tumour cells or autoreactive
lymphocytes. The antisense sequence may also be used for modifying
in vitro behaviour of cells, for example, as part of a protocol to
develop novel lines from cell types having unidentified growth
factor requirements; for facilitating isolation of hybridoma cells
producing monoclonal antibodies, as described below; and for
enhancing survival of cells from primary explants while they are
being genetically modified.
[0140] Still another aspect of the present invention is directed to
an immunointeractive molecule comprising an antigen binding portion
having specificity for Bmf or bmf or derivative thereof.
[0141] Reference to "immunointeractive molecule" should be
understood as a reference to any molecule comprising an antigen
binding portion or a derivative of said molecule. Examples of
molecules contemplated by this aspect of the present invention
include, but are not limited to, monoclonal and polyclonal
antibodies (including synthetic antibodies, hybrid antibodies,
humanized antibodies, catalytic antibodies) and T cell antigen
receptor binding molecules. Preferably, said immunoreactive
molecule is a monoclonal antibody.
[0142] According to this preferred embodiment, there is provided a
monoclonal antibody having specificity for Bmf or bmf or derivative
thereof.
[0143] Reference to a molecule "having specificity for Bmf or bmf"
should be understood as a reference to a molecule, such as a
monoclonal antibody, having specificity for any one or more
epitopes of Bmf or bmf. These epitopes may be conformational
epitopes, linear epitopes or a combination of conformational and
linear epitopes of either the native Bmf or bmf molecule or the
denatured molecule.
[0144] More preferably, there is provided a monoclonal antibody
having specificity for Bmf
[0145] The immunointeractive molecules of the present invention may
be naturally occurring, synthetic or recombinantly produced. For
example, monoclonal or polyclonal antibodies may be selected from
naturally occurring antibodies to Bmf or bmf or may be specifically
raised to Bmf or bmf in the case of the latter, Burl or bmf may
first need to be associated with a carrier molecule. The antibodies
and/or recombinant Bmf of the present invention axe particularly
useful as therapeutic or diagnostic agents. Alternatively,
fragments of antibodies may be used such as Fab fragments,
Furthermore, the present invention extends to recombinant and
synthetic antibodies, to antibody hybrids and to antibodies raised
against non-Bmf antigens but which are cross-reactive with any one
or more Bmf epitopes, A "synthetic antibody" is considered herein
to include fragments and hybrids of antibodies. The antibodies of
this aspect of the present invention are particularly useful for
immunotherapy and may also be used as a diagnostic tool for
assessing apoptosis or monitoring the program of a therapeutic
regime.
[0146] For example, Bmf and bmf can be used to screen for naturally
occurring antibodies to Bmf and bmf, respectively. These may occur,
for example in some degenerative disorders.
[0147] For example, specific antibodies can be used to screen for
tuff proteins. The latter would be important, for example, as a
means for screening for levels of Bmf in a cell extract or other
biological fluid or purifying Bmf made by recombinant means from
culture supernatant fluid. Techniques for the assays contemplated
herein are known in the art and include, for example, sandwich
assays, ELISA and flow cytometry.
[0148] It is within the scope of this invention to include any
second antibodies (monoclonal, polyclonal or fragments of
antibodies) directed to the first mentioned antibodies discussed
above. Both the first and second antibodies may be used in
detection assays or a first antibody may be used with a
commercially available anti-immuno globulin antibody. An antibody
as contemplated herein includes any antibody specific to any region
of Bmf.
[0149] Both polyclonal and monoclonal antibodies are obtainable by
immunization with the protein or peptide derivatives and either
type is utilizable for immunoassays. The methods of obtaining both
types of sera are well known in the art, Polyclonal sera are less
preferred but are relatively easily prepared by injection of a
suitable laboratory animal with an effective amount of Bmf, or
antigenic parts thereof, collecting serum from the animal, and
isolating specific sera by any of the known immunoadsorbent
techniques. Although antibodies produced by this method are
utilizable in virtually any type of immunoassay, they are generally
less favoured because of the potential heterogeneity of the
product.
[0150] The use of monoclonal antibodies in an immunoassay is
particularly preferred because of the ability to produce them in
large quantities and the homogeneity of the product. The
preparation of hybridoma cell lines for monoclonal antibody
production derived by fusing an immortal cell line and lymphocytes
sensitized against the immunogenic preparation can be done by
techniques which are well known to those who are skilled in the
art. (See, for example, Douillard and Hoffman, Basic Pacts about
Hybridomas, in Compendium of Immunology Vol. II, ed. by Schwartz,
1981; Kohler and Milstein, Nature 256: 495-499, 1975; Kohler and
Milstein, European Journal of Immunology 6: 511-519, 1976).
[0151] Screening for immunointeractive molecules, such as
antibodies, can be a time consuming and labour intensive process.
However, the inventors have developed a rapid and efficient flow
cytometric screening procedure for the identification of
immunointereactive molecules, and in particular antibodies,
directed to low abundance cytoplasmic proteins such as, but not
limited to, Bmf.
[0152] The method according to this aspect of the present invention
is based on the analysis of a population of cells, following the
incubation of these cells with the antibody of interest together
with or separately to a reporter molecule, said population of cells
comprising both cells expressing the protein of interest and cells
which do not express the protein of interest. This analysis is
preferably flow cytometric analysis and the cells expressing the
protein of interest are preferably transfected with a nucleic acid
molecule encoding the protein of interest to thereby express high
levels of said protein. Where the protein is a cytoplasmic protein
the cells are permeabalized prior to incubation with the antibody
of interest, By screening a population of cells comprising both
cells which do not express and cells which do express the protein
of interest, determination of which antibodies bind to the protein
of interest is simplified since where the subject antibody is
directed to the protein of interest, a double fluorescence peak is
observed. The lower intensity peak represents background staining
while the higher fluorescence intensity peak is the result of
specific staining. Where the antibody being screened according to
this method is not directed to the protein of interest, a single
peak of low fluorescence intensity is observed. Antibodies not
specific to the protein of interest but bound to some unknown
epitope present in both populations of cells produces a single peak
with high fluorescence intensity. This technique provides a rapid
and accurate method of screening for immunointeractive molecules
directed to low abundance intracytoplasmic molecules (O'Reilly,
1998, supra).
[0153] Accordingly, another aspect of the present invention
provides a method of detecting an immunointeractive molecule, in a
sample, specific for a protein of interest produced by a cell, said
method comprising contacting the sample to be tested with a
population of cells comprising a defined ratio of cells producing
the protein of interest and cells not producing the protein of
interest for a time and under conditions sufficient for
immunointeractive molecules, if present in said sample, to interact
with said protein of interest and the subjecting said
immunointeractive molecule-protein complex to detecting means.
[0154] Preferably said immunointeractive molecule is an
antibody.
[0155] More preferably, said detecting means comprises an
anti-immunoglobulin antibody labelled with a reporter molecule
capable of giving a detectable signal. Even more preferably said
reporter molecule is fluorochrome.
[0156] Reference to "sample" should be understood as a reference to
any sample potentially comprising an immunointeractive molecule,
such as an antibody. Said immunointeractive molecule may be
produced by natural, recombinant or synthetic means.
[0157] The method of the present invention is predicated on
subjecting the cells incubated with the sample of the present
invention to flow cytometric analysis to produce a fluorescent
signal wherein a differential fluorescent signal is indicative of
antibody binding to the target protein expressed by said cells.
[0158] The method exemplified herein is directed, but not limited
to, screening for immunointeractive molecules comprising an antigen
binding site directed to epitopes of Bmf. The promyelomoncytic cell
line FDC-P1 is transfected with a Bcl-2 expression construct and an
BE (Glu-Glu) epitope-tagged Brat. construct. A 1:1 ratio of Bcl-2
transfected cells to Bmf transfected cells are fixed, permeabilized
and contacted with the immunointeractive molecule of interest, such
as a hybridoma supernatant. Visualization of antibodies bound
intracellular molecules can be achieved via a number of techniques
known to those skilled in the art, including, for example, the use
of fluorescently labelled reporter molecules. Where the antibody of
interest is directed to Bmf, a double fluorescence peak is
observed, the lower intensity peak representing background staining
of the Bcl-2 transfected negative control cells.
[0159] In another aspect of the present invention, the molecules of
the present invention are also useful as screening targets for use
in applications such as the diagnosis of disorders which are
regulated by Bmf, For example, screening for the levels of Bmf or
bmf in tissue as an indicator of a predisposition to or the
development or, cancer, a degenerative disease or infertility. The
screening of this aspect of the present invention may also be
directed to detecting mutations in Bmf or bmf.
[0160] Accordingly, another aspect of the present invention
contemplates a method for detecting Bmf in a biological sample from
a subject, said method comprising contacting said biological sample
with an immunointeractive molecule as hereinbefore defined specific
for Bmf or its derivatives thereof for a time and under conditions
sufficient for an immunointeractive molecule-Bmf complex to form,
and then detecting said complex.
[0161] Preferably said immunointeractive molecule is an antibody.
Even more preferably, said antibody is a monoclonal antibody.
[0162] Reference to biological sample according to this aspect of
the present invention should be understood as a reference to any
sample comprising tissue from a subject, said "tissue" should be
understood in its broadest sense to include biological fluid,
biopsy samples or any other fowl of tissue or fluid or extracts
therefrom such as DNA or RNA properties.
[0163] Still another aspect of the present invention contemplates a
method for detecting bmf in a biological sample from a subject,
said method comprising contacting said biological sample with an
immunointeractive molecule as hereinbefore defined specific for bmf
or its derivatives thereof for a time and under conditions
sufficient for an immunointeractive molecule-bmf complex to form,
and then detecting said complex.
[0164] Reference to an "immunointeractive" molecule should be
understood as a reference to any molecule which couples, binds or
otherwise associates with bmf or Bmf or derivative thereof. For
example, said interactive molecule may be a nucleic acid molecule
or an anti-nuclear antibody.
[0165] The presence of Bmf may be determined in a number of ways
such as by Western blotting, ELISA or flow cytomety procedures. Bmf
mRNA or DNA may be detected, for example, by in situ hybridization
or Northern blotting or Southern blotting. These, of course,
include both single-site and two-site or "sandwich" assays of the
non-competitive types, as well as in the traditional competitive
binding assays. These assays also include direct binding of a
labelled antibody to a target.
[0166] Sandwich assays are among the most useful and commonly used
assays and are favoured for use in the present invention. A number
of variations of the sandwich assay technique exist, and all are
intended to be encompassed by the present invention. Briefly, in a
typical forward assay, an unlabelled antibody is immobilized on a
solid substrate and the sample to be tested brought into contact
with the bound molecule. After a suitable period of incubation, for
a period of time sufficient to allow formation of an
antibody-antigen complex, a second antibody specific to the
antigen, labelled with a reporter molecule capable of producing a
detectable signal is then added and incubated, allowing time
sufficient for the formation of another complex of
antibody-antigen-labelled antibody. Any unreacted material is
washed away, and the presence of the antigen is determined by
observation of a signal produced by the reporter molecule. The
results may either be qualitative, by simple observation of the
visible signal, or may be quantitated by comparing with a control
sample containing known, amounts of hapten. Variations on the
forward assay include a simultaneous assay, in which both sample
and labelled antibody are added simultaneously to the bound
antibody. These techniques are well known to those skilled in the
art, including any minor variations as will be readily apparent. In
accordance with the present invention the sample is one which might
contain Bmf including cell extract, tissue biopsy or possibly
serum, saliva, mucosal secretions, lymph, tissue fluid and
respiratory fluid. The sample is, therefore, generally a biological
sample comprising biological fluid but also extends to fermentation
fluid and supernatant fluid such as from a cell culture.
[0167] In the typical forward sandwich assay, a first antibody
having specificity for the Bmf or antigenic parts thereof, is
either covalently or passively bound to a solid surface. The solid
surface is typically glass or a polymer, the most commonly used
polymers being cellulose, polyacrylamide, nylon, polystyrene,
polyvinyl chloride or polypropylene. The solid supports may be in
the form of tubes, beads, discs of microplates, or any other
surface suitable for conducting an immunoassay. The binding
processes are well-known in the art and generally consist of
cross-linking covalently binding or physically adsorbing, the
polymer-antibody complex is washed in preparation for the test
sample. An aliquot of the sample to be tested is then added to the
solid phase complex and incubated for a period of time sufficient
(e.g. 2-40 minutes or overnight if more convenient) and under
suitable conditions (e.g. from room temperature to about 40.degree.
C. such as 25.degree. C.) to allow binding of any subunit present
in the antibody. Following the incubation period, the antibody
subunit solid phase is washed and dried and incubated with a second
antibody specific for a portion of the hapten. The second antibody
is linked to a reporter molecule which is used to indicate the
binding of the second antibody to the hapten.
[0168] An alternative method involves immobilizing the target
molecules in the biological sample and then exposing the
immobilized target to specific antibody which may or may not be
labelled with a reporter molecule. Depending on the amount of
target and the strength of the reporter molecule signal, a bound
target may be detectable by direct labelling with the antibody.
Alternatively, a second labelled antibody, specific to the first
antibody is exposed to the target-first antibody complex to form a
target-first antibody-second antibody tertiary complex. The complex
is detected by the signal emitted by the reporter molecule.
[0169] By "reporter molecule" as used in the present specification,
is meant a molecule which, by its chemical nature, provides an
analytically identifiable signal which allows the detection of
antigen-bound antibody. Detection may be either qualitative or
quantitative. The most commonly used reporter molecules in this
type of assay are either enzymes, fluorophores or radionuclide
containing molecules (i.e. radioisotopes) and chemiluminescent
molecules.
[0170] In the case of an enzyme immunoassay, an enzyme is
conjugated to the second antibody, generally by means of
glutaraldehyde or periodate. As will be readily recognized,
however, a wide variety of different conjugation techniques exist,
which are readily available to the skilled artisan. Commonly used
enzymes include horseradish peroxidase, glucose oxidase,
.beta.-galactosidase and alkaline phosphatase, amongst others. The
substrates to be used with the specific enzymes are generally
chosen for the production, upon hydrolysis by the corresponding
enzyme, of a detectable color change. Examples of suitable enzymes
include alkaline phosphatase and peroxidase. It is also possible to
employ fluorogenic substrates, which yield a fluorescent product
rather than the chromogenic substrates noted above. In all cases,
the enzyme-labelled antibody is added to the fast antibody hapten
complex, allowed to bind, and then the excess reagent is washed
away. A solution containing the appropriate substrate is then added
to the complex of antibody-antigen-antibody. The substrate will
react with the enzyme linked to the second antibody, giving a
qualitative visual signal, which may be further quantitated,
usually spectrophotometrically, to give an indication of the amount
of hapten which was present in the sample. "Reporter molecule" also
extends to use of cell agglutination or inhibition of agglutination
such as red blood cells on latex beads, and the like.
[0171] Alternately, fluorescent compounds, such as fluorescein and
rhodamine, may be chemically coupled to antibodies without altering
their binding capacity. When activated by illumination with light
of a particular wavelength, the fluorochrome-labelled antibody
adsorbs the light energy, inducing a state to excitability in the
molecule, followed by emission of the light at a characteristic
color visually detectable with a light microscope. As in the EIA,
the fluorescent labelled antibody is allowed to bind to the first
antibody-hapten. complex. After washing off the unbound reagent,
the remaining tertiary complex is then exposed to the light of the
appropriate wavelength the fluorescence observed indicates the
presence of the hapten of interest. Immunofluorescence and ETA
techniques are both very well established in the art and are
particularly preferred for the present method.
[0172] However, other reporter molecules, such as radioisotope,
chemiluminescent or bioluminescent molecules, may also be
employed.
[0173] The present invention also contemplates genetic assays such
as involving PCP, analysis to detect bmf or its derivatives.
[0174] The present invention further provides genetically modified
animals in which one or both alleles of bmf are mutated alone or in
combination with, another mutation in one or both alleles for
another Bcl-2 molecule snob as but not limited to genes encoding
Blk, Bad, Bik, Hrk, Bid, Bim, Noxa, blx3 and/or Puma. The animals
may also have mutations in other genes or alleles of genes.
Preferably, the genetically modified animals are laboratory test
animals such as murine species (e.g. mice, rats), rabbits, guinea
pigs or hamsters, livestock animals such as sheep, pigs, horses or
cows or non-human mammals such as primates, Conveniently, and
preferably, the genetically modified animal is a murine species
such as a mouse or rat.
[0175] The genetic modification is generally in the form of a
mutation such as a single or multiple nucleotide substitution,
deletion and/or addition or inversion or insertion. Generally, such
a genetically modified animal is referred to as a "knock-out"
animal.
[0176] Genetically modified animals and in particular knock-out
murine animals may be prepared by any number of means. In one
method, a targeting DNA construct is prepared comprising a
nucleotide sequence which is sufficiently homologous to a target
sequence such a bmf or bim to permit homologous recombination. The
bmf or him targeting sequence may be isogenic or non-isogenic to
the target Bmf or Bim sequence. The targeting DNA construct
generally comprises a selectable marker within the targeting
sequence such that by homologous recombination, the target bmf or
bim gene is disrupted by an insertional mutation. The targeting DNA
construct is generally introduced into an embryonic stem cell or
embryonic stern cell line. One suitable targeting vector is shown
in FIG. 5A.
[0177] As an alternative to using a selectable marker, a mutation
may be introduced which induces a phenotypic change which may then
be selected or detected.
[0178] Accordingly, another aspect of the present invention
provides a method of producing a genetically modified non-human
animal, said method comprising introducing into embryonic stem
cells of an animal a genetic construct comprising a bmf nucleotide
sequence carrying a single or multiple nucleotide substitution,
addition and/or deletion or inversion or insertion wherein there is
sufficient bmf nucleotide sequences to promote homologous
recombination with a bmf gene within the genome of said embryonic
stern cells selecting for said homologous recombination and
selecting embryonic stem cells which carry a mutated bmf gene and
then generating a genetically modified animal from said embryonic
stem cell.
[0179] Preferably, the genetically modified animal is a murine
species such as a mouse or rat,
[0180] The Bmf nucleotide sequence may be isogenic or non-isogenic
to the bmf gene in the embryonic stem cell.
[0181] The term "isogenic" means that the bmf nucleotide sequence
in the construct is derived from the same animal strain from which
the embryonic stem cell has been derived.
[0182] The present invention further contemplates
non-homologous-mediated integration of the target DNA sequence.
[0183] A range of selectable markers may be employed and reference
may be made to U.S. Pat. No. 5,789,215 for general
methodologies.
[0184] The above method may be similarly adopted for introducing a
plurality of mutations into different genes such as, in addition to
bmf, other Bcl-2 genes (e.g. those encoding Bim, Blk, Bad, Bid,
Hrk, Noxa or Puma) and/or other structural or regulatory genes.
[0185] Breeding protocols may also be adopted to introduce
mutations or other genetic modifications into Bra. In one approach,
an EMS or other mutagenized mouse is crossed with a non-mutagenized
mouse to produce a G1 generation. The G1 generation may then be
crossed with an index strain to produce GIFT kindreds which are
then screened phenotypically for mutation in bmf. Mutations in bmf
may be dominant or recessive and mutations may be detected directly
on bmf or by its effect on another gene or on its effect in
alleviating the effects of a first mutation. on another gene.
[0186] genetically modified animals including bock-out mice
carrying mutations in one or both bmf alleles alone or in
combination with mutations in other genes such as other Bcl-2
family genes are encompassed by the present invention.
[0187] The present invention is further described by the following
Examples.
Example 1
Identification and Cloning of Bmf
[0188] The inventors sought novel BH3-only proteins that played a
role in embryogenesis. Since Mcl-1-deficient mice have the most
severe developmental defect of all knock-out mice lacking
pro-survival Bcl-2 family members, Mcl-1 was used as bait. Bmf
modifying factor) was identified through yeast 2-hybrid screening
of a day 17 mouse embryonic library using Mcl-1 as bait. The method
used is as follows.
[0189] The cDNA libraries from day 17 mouse embryos or from mouse
embryos from embryonic day 9 to one day post-partum were prepared
in pAD-GAL4-2.1 (HybriZAP-2.1 kit, Stratagene). The bait vector was
made by cloning mouse mcl-1 lacking the sequences encoding its
hydrophobic C-terminus into pGBT-9 (Clontech). Yeast transformation
and plasmid rescue were performed as previously described
(Puthalakath et al., 1999, supra). 7.times.10.sup.5 clones were
screened and one positive clone was obtained. Interaction between
Mcl-1 and the novel protein was confirmed by .beta.-galactosidase
staining (Puthalakath et al., 1999, supra). Sequence analysis
revealed that the clone was a partial one lacking the 5' end. This
partial clone was used as the probe to isolate full-length clones
by screening a cDNA library derived from the p53.sup.-/- KO52DA20
thymoma cell line (Strasser et al., Cell 79: 329, 1994). Human bmf
was isolated by screening a human activated T cell cDNA library
using mouse bmf as probe. To screen for Bmf-interacting proteins,
mouse bmf was sub cloned into a pGBT-9 derivative harboring the
gene for chloramphenicol acetyltransferase as the selection marker.
Out of 5.times.10.sup.6 clones screened, 60 positive clones were
initially selected, of which 6 were later found to be false
positives.
[0190] Detailed sequence analysis (Krogh et al., 1994, supra)
revealed that Bmf harbors a BH3 domain most similar to that found
in Bim, Bik and EGL-1 (FIGS. 1A and B). In. the yeast 2-hybrid
system, Bmf interacted with Mc1-1 and other pro-survival Bcl-2
proteins (Bcl-2, Bcl-x.sub.L and Bcl-w) but not with the
pro-apoptotic family members tested (Bax, Bid and Bad). When
transiently overexpressed in 293T cells, Bmf could be
co-immunoprecipitated with pro-survival Bcl-2 fatally members Bcl-2
and Bcl-w (FIG. 1C), as well as Bcl-x.sub.L and Mcl-1, but did not
bind pro-apoptotic Bax or the BH3-only protein Bim. The interaction
of Bmf with Bcl-2 or Bcl-w was ,greatly diminished by mutating the
invariant leucine (L138A) within its BH3 domain (FIG. 1C).
Furthermore, mutations of conserved residues within the BH1 (G145E)
or BH2 (W188A) domain of Bcl-2, which abolish its binding to Bim
(O'Connor et al., EMBO J. 17: 384, 1998) or Bax (Yin et al., Nature
369: 321, 1994), also disrupt its binding to Bmf Significantly,
endogenous Bmf could be co-immunoprecipitated with endogenous Bcl-2
from detergent lysed MCF-7 human breast carcinoma cells (PIG. 1D),
excluding the possibility that these proteins associate only when
overexpressed.
[0191] The biological activity of Bmf was investigated by
transiently overexpressing it in Jurkat human T lymphoma cells, as
well as in stably transfected L929 mouse fibroblasts (FIG. 1E) or
in IL-3-dependent FDC-P1 mouse promyelocytic cells (FIG. 2C).
Expression of Bmf triggered apoptosis in .about.80% of Jurkat cells
within 24 hours and reduced formation of L929 fibroblast colonies
by about 65% (FIG. 1E). Bmf-induced apoptosis in Jurkat cells could
be blocked by the caspase inhibitor baculovirus p35, or by
co-expression of Bcl-2 or its homologs (Bcl-x.sub.L, Bcl-w, Mcl-1)
but not by BH1 (G145E) or BH2 (W188A) domain mutants of Bcl-2.
Consistent with its pro-apoptotic activity, high levels of Bmf
could be expressed stably in FDC-P1 cells only when Bcl-2 (or one
of its homologs) was also expressed. Such Bmf/Bcl-2 co-expressing
FDC-P1 cells died more rapidly than. Bcl-2 expressing cells in
response to cytokine withdrawal (FIG. 2C), .gamma.-irradiation or
treatment with etoposide. In all the cell death assays performed,
Bmf mutants that lack the BH3 domain or have the L138A. mutation in
it were inert (FIGS. 1E and 2C). These results establish that Bmf
is a BH3-only protein that binds pro-survival Bcl-2 family members
to initiate apoptosis.
Example 2
Expression Patterns of Bmf
[0192] The expression pattern of Bmf was investigated by Northern
blotting, RT-PCR and Western blotting. bmf mRNA was found in many
cell lines of B and T lymphoid, myeloid or fibroblastoid origin and
in mouse embryos at all developmental stages from E9 to birth (FIG.
1F). Western blotting of cell lysates using affinity purified
rabbit polyclonal antibodies or rat monoclonal antibodies
(described below) detected a single band corresponding to Bmf in
many organs, with prominent levels found in pancreas, liver, kidney
and hematopoietic tissues (FIG. 1G). Thus, Bmf is expressed during
embryogenesis and in many adult tissues.
[0193] Monoclonal rat antibodies to dynein light chains and Bmf
were generated using a previously published protocol (O'Reilly et
al., 1998, supra). In brief; Wistar rats were immunized with
purified recombinant mouse DLC1/LC8 or mouse Bmf. Spleen cells from
immunized rats were fused with Sp2/0 myeloma cells. The resulting
hybridoma clones were screened for production of specific
antibodies by immunofluorescent staining and flow cytometric
analyses. Hybridomas were cloned twice and antibodies were purified
either on a protein-G column (Amersham Pharmacia) or on a sepharose
column conjugated with MAR 18.5 (monoclonal mouse anti-rat
Ig.kappa.) antibodies. Monoclonal antibody 11F7 (rat IgG
2a/.kappa.) recognizes mouse and human DLC1/LC8 and DLC2 whereas
10D6 (rat .mu./.kappa.) detects mouse and human DLC1/LC8 but not
DLC2. Monoclonal antibodies 9610 and 12E10 (both rat
.gamma.2a/.kappa.) detect endogenous mouse and human Bmf by Western
blotting and immunoprecipitation. To generate polyclonal anti-Bmf
antibodies, New Zealand White rabbits were immunized with 500 .mu.g
of recombinant mouse Bmf. Booster immunisations were given at
intervals of three weeks. Serum was collected after 12 days and
purified over a sepharose, column conjugated with recombinant mouse
Bmf protein.
Example 3
Apoptotic Structure
[0194] To assess whether bmf expression was induced by apoptotic
stimuli, RT-PCR analyses were performed of mRNA from thymocytes
exposed to various forms of stress, including cytokine deprivation,
.gamma.-irradiation or treatment with dexamethasone or ionomycin
(described below). None of these stimuli had any impact on bmf
expression (FIG. 2A), prompting the inventors to investigate
whether Bmf is regulated post-translationally, perhaps by
interacting with other proteins. A yeast 2-hybrid screen of a mouse
embryo cDNA library with Bmf as bait isolated 14 independent clones
of Mcl-1 and, surprisingly, more than 40 clones encoding dynein
light chain (DLC). In a previous screen, Bim had isolated
exclusively DLC1/LC8 (Puthalakath et al., 1999, supra). In
contrast, most dynein light chain clones interacting with Bmf
encoded the closely related protein. DLC2 (Naisbitt et al., J.
Neurosci, 20: 4524, 2000). Co-immunoprecipitation experiments in
transiently transfected 293T cells confirmed the interaction of Bmf
with DLC2 (FIG. 2B). Sequence comparison revealed that Bmf has, in
addition to the BH3 domain, a short region (aa67-DKATQTLSP) that
closely resembles one in Dim (aa51-DKSTQLESP) that mediates its
binding to DLC1/LC8 (FIG. 1A). This is the DLC-binding motif of
Brat because mutations within it (A69P or D67K.sub.68A69>AAA,
hereafter referred to as AAA mutation) abrogated the interaction of
Bmf with. DLC2 in yeast and in mammalian cells (FIG. 2B). Moreover,
upon IL-3 deprivation or .gamma.-irradiation, FDC-P1 cells
co-expressing Bcl-2 and non-DLC2-binding mutants of Bmf died much
more rapidly than those co-expressing Bcl-2 and wild-type Bad (FIG.
2C). These Bmf mutants also suppressed the formation of L929
fibroblast colonies more potently than wild-type Bmf. Hence,
interaction with DLC2 negatively regulates the pro-apoptotic
activity of Bmf.
[0195] RT-PCR. analysis of bmf mRNA expression was performed using
the following primers: 5' (sense) primer 5'CCGGATGGATCACCAGGAATG3'
[SEQ ID NO:11], 3' (antisense) primer 5'CAGAGCTGACAAAGGCACAG3' [SEQ
ID NO:12]. Detection of the PCR products on Southern blots was
performed using the internal bmf primer 5'CCACTTCCTGGAGAACATCA3'
[SEQ ID NO:13]. For analysis of GAPDH expression, the following
primers were used: 5' (sense) primer 5'TGATGACATCAAGAAGGTGGTGAAG3'
[SEQ ID NO:14], 3' (antisense) primer 5'TCCTTGGAGGCCATGTAGGCCAT3'
[SEQ ID NO:15] and the internal primer 5'CCCGOCATCGAAGGTGGAAGAG3'
[SEQ ID NO:16].
Example 4
Functional Model
[0196] The question considered by the inventors is why Bmf is
controlled by binding to highly related partners, DLC-1 or DLC-2.
It is proposed that Bmf is sequestered to sites within the cell in
order to sense distinct stress stimuli, Separation of cellular
proteins into the filamentous actin and the paclitaxel
(taxol)-polymerizable microtubular fractions revealed that,
consistent with previous results (Puthalakath et al., 1999, supra),
Bim and dynein intermediate chain (IC74) largely co-migrated with
microtubular components (P2), whereas Bmf and myosin V were
confined to the filamentous actin-containing P1 fraction (FIG. 3A).
Furthermore, treating cells with actin depolymerizing agents, such
as cytochalasin ID or C. difficcili toxin B, released Bmf from the
filamentous actin-containing P1 fraction whereas the fractionation
of Bim was unaffected (FIG. 3B).
[0197] For subcellular fractionation, 5.times.10.sup.6 MCF-7 cells
were lysed in 500 .mu.l extraction buffer containing 1%
Triton-X-100. Cell debris and nuclei were removed by centrifugation
at 2000 g. The supernatant was then incubated for 13 minutes at
37.degree. C. with 100 .mu.M paclitaxel (taxol) and 5 units of
apyrase (Sigma). This mixture was then loaded on top of a 0.5 mL
cushion of 7.5% sucrose (made in the extraction buffer) and
centrifuged at 140,000g for 30 minutes at 30.degree. C. The pellet
was saved as the microtubular P2,fraction and the supernatant as
the S fraction. To obtain the actin-enriched P1 fraction without
contamination by microtubular constituents, MCF-7 cells were
cultured for 2 hrs in the presence of 2 .mu.g/mL colchicine and 1
.mu.g/mL nocodazole prior to lysis. These lysates were then cleared
of cell debris and nuclei (described above) and subsequently
centrifuged for 60 minutes at 4.degree. C. at 140,000g to obtain
the pellet (P1) fraction. For fractionation of extracts on sucrose
gradients, 10.sup.7 cells were lysed in 500 .mu.L extraction
buffer. After removing cellular debris and nuclei, the supernatants
were treated with 100 .mu.M paclitaxel (taxol) plus 5 units of
apyrase and incubated at 37.degree. C. for 13 minutes before
loading onto a 5-20% sucrose gradient (prepared in extraction
buffer containing 1% Triton X-100) and centrifuging for 18 hours at
15.degree. C. at 140,000g.
[0198] The distinct localization of Bmf and Bim may be determined
largely by their preferred dynein light chain partners. Contrary to
a previous report (Benashski et al., J. Biol. Chem. 272: 20929,
1997), by using monoclonal antibodies that either recognize only
DLC1/LC8 or both. DLC1/LC8 and DLC2 (FIG. 3C), the inventors showed
that purified myosin V motor complexes contained DLC2 but not
DLC1/LC8 (FIG. 30). This observation indicated that Bmf, by being
preferentially bound to DLC2, might be complexed with myosin V on
filamentous actin rather than forming part of the dynein motor
complex. Consistent with this notion, incubation of extracts from
mouse spleen cells with recombinant Bmf and Bim confirmed that only
Bmf associated with myosin V (FIG. 3E). Furthermore, Bmf and Bim
showed distinct migration patterns after subcellular fractionation
of lysates from MCF-7 cells on sucrose gradients (FIG. 3F). Since
DLC1/LC8 forms homodimers avidly and since it binds Bim and IC74
through the same region (Lo el at., J. Biol. Chem. 276: 14059,
2001), one partner of a DLC1/LC8 homodimer probably interacts with
IC74 whilst the other binds Bim, thereby sequestering it to the
microtubular dynein motor complex. It is likely that DLC2
homodimers sequester Bmf to filamentous actin by binding with one
arm to Bmf and with the other to myosin V.
[0199] The inventors next investigated whether Bmf and Bim are
activated by distinct apoptotic stimuli using cells that express
both proteins endogenously. Consistent with our previous results
(Puthalakath et al., 1999, supra), UV-irradiation of MCF-7 cells
released Bim from the pellet fraction where the dynein motor
complex resided. When lysates of healthy or damaged MCF-7 cells
were compared by sucrose gradient centrifugation, it became
apparent that Bmf also translocated from denser to lighter
fractions in response to UV-irradiation (FIG. 4A). Treatment with
paclitaxel (taxol), a chemotherapeutic drug known to polymerize
microtubules, released Bim but not Bmf (FIG. 4A). Consistent with a
critical role for Bim in this pathway to apoptosis, Bim-deficient
thymocytes are abnormally resistant to the cytotoxic effects of
paclitaxel (Frisch and Ruoslahti, Science 286(5445): 1735-1738,
1999). On the other hand, anoikis (absence of cell attachment and
integrin signaling), an apoptotic stimulus that affects the actin
cytoskeleton (Frisch and Ruoslahti, 1997, supra), resulted in the
selective release of Bmf but not Bim (FIG. 4A). Since these
experiments were conducted in the presence of the broad-spectrum
caspase inhibitor zVAD-fink at a concentration (50 .mu.M)
sufficient to block caspase activation, the release of Bmf and/or
Bim are likely to represent initiating events in apoptosis
signaling rather than being a consequence of apoptotic changes.
Importantly, the inventors showed that endogenous Bmf (together
with DLC2) released during anoikis could be co-immunoprecipitated
with endogenous Bcl-2 isolated from mitochondria (FIG. 4B). In
contrast, negligible Bmf was found complexed with Bcl-2 isolated
from mitochondria of healthy cells.
[0200] Collectively, the inventors' data demonstrate that Bmf and
Bim represent two pro-apoptotic BH3-only proteins that transduce
distinct death signals caused by different forms of cell stress.
They seem to represent sentinels mounted on the main cytoskeletal
structures to monitor the well-being of the cell. For example,
disturbance of the microtubules by paclitaxel activates Bim but not
Bmf whereas anoikis, which affects the actin cytoskeleton,
activates Bud. but not Bim. Since deregulated expression of
anti-apoptosis Bcl-2 can promote tumorigenesis (Strasser et al.,
Nature 348: 331, 1990), it is possible that abnormalities in
pro-apoptosis BH3-only proteins can also cause cancer, The gene for
human bmf is located On. chromosome 15q14, identified as the site
of a candidate tumor suppressor gene lost in many metastastic but
not primary carcinomas (Wick et al., Oncogene 12: 973, 1996).
Anoikis has been implicated as a barrier against metastatic tumor
growth (Ruoslahti and Reed, Cell 77: 477, 1994). Metastatic tumors
harboring 15q14 mutations may, therefore, have abnormalities in
their expression or function of Bmf.
Example 5
Generation of Bmf Knock-Out Mice
[0201] Mice are selected with a C57BL/6 background which are back
crossed into C57BL/6. Offspring are genotyped using PCR using
primers specific for wild-type or mutant bmf genes.
[0202] A bmf targeting vector is generated as shown in FIG. 5A. A
neomycin or hygromycin sequence is used as the selectable marker.
The construct is introduced into embryonic stem cells and
transformed cells selected using neomycin or hygromycin. The
transformed embryonic stem cells are then used to generate
genetically modified mice.
Example 6
Genomic Organization of Bmf and Identification of Promoter
Regions
[0203] The genomic organization of the murine bmf gene is shown in
FIG. 5A. Upstream of this region comprises a promoter region as
outlined in SEQ ID NO:9. A corresponding promoter from the human
bmf gene is outlined in SEQ ID NO:10.
[0204] Those skilled in the art will appreciate that the invention
described herein is susceptible to variations and modifications
other than those specifically described. It is to be understood
that the invention includes all such variations and modifications.
The invention also includes all of the steps, features,
compositions and compounds referred to or indicated in this
specification, individually or collectively, and any and all
combinations of any two or more of said steps or features.
Sequence CWU 1
1
2714688DNAMus musculus 1cggcacgagc ggagcgggcg tattttggaa acaataccgc
gcggtgtgcc gtggcctcct 60cccgcgccag ctcgcgcctg cagcagtcgc tgccgcagcc
cgcgccaccg cctcccaccg 120cagcccgctg gagtttgccc ccttcttccc
aatcgagtgt gggcaccaag ccccccgagt 180gttcttcacc ctggaccctg
gcgcagagcc ctggcatcac aactcggagg ctgagacgct 240gtcctggagt
cacccaggag agatggagcc acctcagtgt gtggaggagc tagaagatga
300tgtgttccag tcagaggatg gggagccagg gacacagcct gggggcttgc
tctctgctga 360cctgtttgcc cagagccagc tggactgtcc cctcagtcga
ctccagctct tccctctcac 420ccactgctgt ggtcccggac tccggcccat
aagccaggaa gacaaggcca ctcagaccct 480cagtccagct tccccaagcc
agggtgtcat gctgccttgt ggggtgacag aggaacccca 540gagactcttt
tacggcaacg ctggctacag gcttcctctc cctgccagtt tccctgcagg
600ctcacccctt ggggagcagc cccctgaagg acagttcctt cagcaccgag
cagaggtgca 660gatcgccaga aagcttcagt gtattgcaga ccagttccat
cggcttcata cgcaacaaca 720ccagcagaac cgagaccgtg cgtggtggca
ggtcttcctc ttccttcaaa acctcgccct 780gaacagacaa gaaaacaggg
aaggggtggg gccctggtga ggctggaccg ccctggccgg 840atggatcacc
aggaatgcag tctgggagga cagatactgt cttgttaagt tttgttatcc
900gtaactttct atccatgtgt acatattaca tgccccagtg ggatcttctt
tccccgttca 960gaacctccac tgaggatggg ggcctttgtc aaacactgtt
gaaggagagg cagctgtgtc 1020tgctggtaga gttcctaagg ctctgaagat
gaccagttgg tgatgttctc caggaagtgg 1080actgagactt gctaccggag
ctggttaagt caggttaggc tcccagtacc atcaaacatg 1140tcagccttcc
ttcgtgcctg atggatcatg gctttaaccc accaggaccc tgtctgggag
1200cctcctggct aagatcaact gtgccttggc tagcccgatt cacgtctggg
ttcccacttg 1260gcacagccag cgcccaccgc atgggctcag ggattcttac
tcagacgctg tcaacttccg 1320gaagacctca gggagggtcg gacttctcca
agaacccctc aaatttgcca caaaatgaat 1380caaacttgag aggcttcaag
tctgactcgg tcctacatgg aagccccttg attggtcccc 1440aagggtgcaa
actactgcca cctttgctgg agccaagtag ccagtcacca tgccgttgta
1500cccagcggcc tgtgcctttg tcagctctgt tttcagaaga ccctgccaat
gactggactg 1560gatctgtctg catccatctc agcagaagca cttggagatg
gggtgggctt gctgggggcg 1620tgggaaccca gcagccatcc cactgataag
ttggtcccgg gggaatccta gacctcttgg 1680tttccttgga aacgtgtacc
tcctccccct tacccccatt ccttttctgt acctagcggg 1740atacaggaag
aagctaggac tgaggctttg tcagctgaaa agtgactttg gaagtaacag
1800acaatgttta gaccatggaa actgcagagc tgacacatct tgaatctccc
tttagctttc 1860agctaggcca gaaaggagag ttctagaacc cagcaggacc
ttagctgcct gggagctcct 1920tccttctctc cccgctgcag tgctcaggga
gggaacgaaa agcagctgat cttagaaatt 1980aagtcctaag tgttatgtat
gtaaggaaag acattggtgc tataatgaag acgagccaag 2040gctcaaagaa
agatggcttc cctagacaaa gaagaacaca ggattattca aggactttgg
2100aggagaaact cagagtagac tcaaattgaa caagcccagt gactccccat
tctgcagaag 2160tggggggggg gggcggattt cagtaccata aaagggcacc
ggatctcctg ggtgtccagt 2220tgggcaggtt tctccctgcc aaggctaccg
acagaggggg ctcatagagg ctgaggtcca 2280gcctgtgccc tgctctctag
gctgcacatc ctgatgtggg cagggtctgc ccagagtaag 2340gaatgycttt
cctaattygc acaaaggtac tgtgtgcact ctcagtggtc ctgaggagga
2400ccttaaggca tgtgtataag cctttgtgtt tttctgctca gatgacctgg
agatattacc 2460tcttcttggc cacaatcatt tttctcaggg tctataccat
agttcgtctg ctaagaaagt 2520gggtttctgt ttgcaaggag aggggaccca
agtaatgaat atgaagactg gttctgaggg 2580tgggcttgat tatttgtgaa
gcccagcctt cctacatccc accctccctg cagtttagat 2640gtgtgtggga
catatgtggt cttccccttt cctggatcaa ggctatgaca ggcaggcttc
2700tggtatgtgc ttggtacatg tgctgcaata catgtgcttg atggtacatg
cgtgcccctc 2760cccatacaga accttgtact tcagttatct ttaacataac
ttagtagact ttccatctgt 2820cctaaggcat gactgtcagc ttcagcaacc
ttgctaggtg caggctgggt accctgtggc 2880ctgagaactg agcccagagt
ttgacctgaa cattgagcct ggaccaggca gaagctggtc 2940agaatgtact
gtctccattt tgggccaagc cgggaccaaa cttactaact caggcaagcc
3000agggtgaaac ttaaaccctc ttttatcttt aacctgaatc caggctggag
ttgaagctca 3060atgataaagg gcttgcctgt cggtcaagag accctcggat
ggatcccagg actgtaaaaa 3120aaaaataaat aaaaatatgc cccctctctc
caacctcttg gaggcagctg gcagcccagc 3180tcaaggaggc aagaggttcc
ccctggctgc ttgctttaat tggccggtga tctctgcctg 3240gtgaattggt
tttatagtct ctgagtccag cccctggaca accccycycc ctttgcctct
3300ccagaactcc aggagcttgg ctgtctctag tgaagagtgg agattcagat
tctgagggag 3360ggagggaggg tctgacctgt gtggtgacct gtaaccagct
gaaggcaaca ggcttttgtg 3420gtgtgctgag gatggggggt gggagaagag
ctggaaaact ctgtagaaat gccctagagg 3480aacagaagcc ctgtattcag
gggccaaact gctctactaa ttcacaatcc ccaggaggca 3540tttctaaaag
ccagtgaaca gatctggaga ggaacatccc ccagtcccgt cactgactcc
3600cccagggaga ctggggtggt acgcaggaga gtggggggag ggtgggcact
ttgcctggcc 3660ctgtctctct gtttcaaagt ttggctgtgc tgttcaaatg
tccccaaagg ccttgtcttg 3720ctcaatcaga ggtggaaaaa gatcacctcc
cggtgacctc cttcaggccc caggggatgg 3780gctgtgtcca gagctatgtg
gcagagcttg tttccagagc ccagagaggc cccgctggcc 3840tgccttgctg
ccccagtggc gaaacgcaag cccaacactg agcatggcag ggagtgggtg
3900ttggggacag tctagccaag gcccctgtct gtcccaagct ttccaggaac
taagcagtga 3960ggtcactgtg acagtcctgc ctggtcagcg aggagaatat
tctgagcttt cccgctctgg 4020gacttactgg ggagggggcg ggtaccagtg
tatggagtaa caagacatca gccttcgtgg 4080ctctgcagga tttgggggaa
tcagtacagt ggccactgtg gctgctatga agataaggcc 4140ttttggagac
aggctatcct ggcctgatac cctggttgga cctagaggga tacagggtgg
4200agccacagtc atttcaagtt ccctggggtt cctgtgggct gcagacctag
gggacagagc 4260ctcaaggaag acagcagaac aaagctgagc tcttccatat
atgccagctt tggccaaggt 4320gctatgaggt ctgtgacccc aggcttgcac
tgcccctttc tctacagttg tctgatgtcc 4380accccatctc tgggttcaca
gaaactgaca cggggagacc cagcgagatg tagcatttcc 4440ttcatcccca
gtctggttgg cccggggtct gggcatcctg ccttaaggaa aggaatgggg
4500accattattt tttaacctgt atataatatt tgaccttttt ttggttcttt
atatgtatac 4560atgtgtaaat atcctctcat ccatcaagtg gtacagtttt
taataaaacc atttaaagca 4620aaaaaaaaaa aaaaaaactc gagagtactt
ctagagcggc cgcgggccca tcgattttcc 4680acccggaa 46882185PRTMus
musculus 2Met Glu Pro Pro Gln Cys Val Glu Glu Leu Glu Asp Asp Val
Phe Gln1 5 10 15Ser Glu Asp Gly Glu Pro Gly Thr Gln Pro Gly Gly Leu
Leu Ser Ala 20 25 30Asp Leu Phe Ala Gln Ser Gln Leu Asp Cys Pro Leu
Ser Arg Leu Gln 35 40 45Leu Phe Pro Leu Thr His Cys Cys Gly Pro Gly
Leu Arg Pro Ile Ser 50 55 60Gln Glu Asp Lys Ala Thr Gln Thr Leu Ser
Pro Ala Ser Pro Ser Gln65 70 75 80Gly Val Met Leu Pro Cys Gly Val
Thr Glu Glu Pro Gln Arg Leu Phe 85 90 95Tyr Gly Asn Ala Gly Tyr Arg
Leu Pro Leu Pro Ala Ser Phe Pro Ala 100 105 110Gly Ser Pro Leu Gly
Glu Gln Pro Pro Glu Gly Gln Phe Leu Gln His 115 120 125Arg Ala Glu
Val Gln Ile Ala Arg Lys Leu Gln Cys Ile Ala Asp Gln 130 135 140Phe
His Arg Leu His Thr Gln Gln His Gln Gln Asn Arg Asp Arg Ala145 150
155 160Trp Trp Gln Val Phe Leu Phe Leu Gln Asn Leu Ala Leu Asn Arg
Gln 165 170 175Glu Asn Arg Glu Gly Val Gly Pro Trp 180
18534959DNAHomo sapiens 3gaacgatact agtggaccca aagaattcgg
cacgagctcg tgttttaatt cggcacgagc 60cgcaccgtgc ggagtggcct cctcccgccc
cggcctgtgc ccgccgccgc cgccgcccct 120gcctgcgcct cccgcctcct
gccgcagccc gctgggcttt ttccctcctt cccaatcgag 180tctgggcgtc
cagcccccga gtgctcgtca cgctggaccc tggcgcggag ccctggcatc
240acgactcgga ggccgagact ctctcctgga gtcacccagg agagatggag
ccatctcagt 300gtgtggagga gctggaggat gatgtgttcc aaccagagga
tggggagccg gtgacccaac 360ccgggagctt gctctctgct gacctgtttg
cccagagcct actggactgc cccctcagcc 420gacttcagct cttccctctc
acccactgct gtggccctgg ccttcgaccc accagccagg 480aagacaaagc
tacccagact ctcagcccag cctcccccag ccaaggtgtc atgctgcctt
540gtggggtgac tgaggaaccc cagcgactct tttatggcaa tgctggctat
cggcttcctc 600tccctgccag tttcccagca gtcttgccca ttggggagca
gccccccgaa gggcagtggc 660aacatcaagc agaggtacag attgcccgaa
agcttcagtg cattgcagac cagttccacc 720ggcttcatgt gcagcaacac
cagcagaacc aaaatcgtgt gtggtggcag atcctcctct 780tcctgcacaa
ccttgctttg aatggagaag agaacaggaa cggggcaggc cctaggtgag
840ggtgggctgc cctcttcaca tggggcacca ggaacaccgt ctggaacagg
aaggacatcg 900ggcaggactg acactgtgtc ttgtgaaatt gtttttttgt
tgttattttg tgttttaatt 960ttttttaatt tctctctgag tgtacataca
acatactcaa gcgggacctt ctttctctgt 1020caggcccttg acctggaatg
ggggcctgtg tcaaacactg ttgaaggaga ggctgatgtg 1080tctgtgatgg
tgagaattcc caarggctct gacaagtaga ttcttcgact gaggaatcta
1140ccagttgycg aagatgatcc gttagtgatg ttctctggga agtggactgt
ggtttttcca 1200gaggaactca gttaagaaat cgagagtgga ttagactccc
cagttccacc aaacctatga 1260gccttccact gtggatgggg gccgtgatcc
tgatggtcac attgctttaa cccagcaggg 1320cttcggccag gggctttcca
cttgaggata gcagcttcac taggctggcc ggccagctcc 1380acatctgact
gggttcttac ttctcagcca gtacctgccc catgggctca aggattcctg
1440gccagctcct gccacctcca gcagacctca gggagggttg ggtttctcta
aggacccctc 1500aaacatgtcg caaaatgaac caaacttctg gctaggcctc
aaaactgact tggtcccact 1560tggaggcccc aggattggtc ctgaggtaca
gagccactgc caccactggc ggcctgggac 1620cagctgggtg tcagccacgg
atgagccgaa tagccagtca gcatgttgct gctggcagcc 1680tgtgcctttg
tcagctcttc tttcaaaaga ccccaccgac agaccgcatt ccacccccaa
1740catcggcact gagggacatc gggggcaagt ttgcagtggg gccggaaaat
atggtggcca 1800ccctaccatg agagtcagcc gtaggggacc ccagacccct
tggtttcctt ggaaacaaca 1860tacctcttcc cccttatccc cagtcctttt
tcatacctag tgggatacag aagaagccag 1920gacagtggct ttgccagcta
agtgactttt agagtaacag ataacgatta gagtggggaa 1980ccgtcaaagc
tgggtacaca tttcctatct tcctccagct tccagctagg ccagaagggc
2040atgctctgga acccagcagg atcacagctg cctgtgcacg tcttcccttc
tctccctgct 2100gcggcactta tggagaggat ctaaagcagc cggtgtggca
gctccgatag caggcacagg 2160gaatctgtta accagacgct agaaactaaa
ttataacttt tgcatatgtg agaaaagaca 2220acattggtgc taacagtgaa
gtaaggccca aggaaagacg gcttccctgg acaaagaaga 2280cctcagggct
acccaaggaa ataggaggag tctagagtag actcacaaat ctgaacaagc
2340ccaagtcttc cagttctgag gagaggaggt cttcagtact attgaaggag
acattgatct 2400tctggatgta cagttgtgca ggttgttcac tgcacaaggg
cacctgcagc taatggaggc 2460tggaattcag cttgtgttct gctcactaag
ctgtgtgcgt gccctggtgt ggggctactt 2520ctcccaagaa gaaggggtgc
ctttcctaat ttgcaaaggt gccatatggg ctcacagcac 2580cctggaggag
aagggcctca aactgtgcat gtaagccttt gttttgttct gctcagatat
2640tctggatatt acctcttctt ggtcagaatt ctattctcag ggtgtgtacc
atgatttgtc 2700tgctaagaag acgggttcct gtttttgcag agaaggagcc
ggggaccagc ttgaagactg 2760gctctgggac acactgacca ttgtgaaatt
cagccttccc tgtgggtctc cataccattt 2820tatgatgtgt atgggacata
cgttgttctc ccctctctgg atcaaggtgg tgacaggcag 2880cctgctgccg
tatgcttcag tggcacacac caaaaaaacc gggattattt acagcagcca
2940ctattcaccc cttttggcag actctccacc tgagttcagt gagagagaaa
atgatttaga 3000tcttggtgct ggggcaaggc catcagcttc agagaccttg
ccaggcccag gctgggtgcc 3060ctgtggcctg agaactgagc ccagactttg
acaaacccac ctcaacatca agcctggggc 3120aggtggaagg tggtctgact
gcactgtctc catcttatgc aaagccagga actcactcac 3180tgtcttaagt
gagccagggc aaaatttaca cccctcacgt ttccctctcc cttcttcccc
3240accaacaaaa cccaggaggt agctggcaga gtagctgtca ggaaggagaa
aggttctttc 3300tggctggttg tttttaattg gcttagtgat ctaaactggc
cccttcctcc tctgcctggt 3360gagttggcct aaacattcct caagtctagc
ttcaggagac ctgcccctcc cccccgacct 3420ccaccccctc agactccatc
ccccaccccc aggagcctgg ctgtctctag cgaggggtgg 3480agaatcagat
ttagagggag ggagagtctg acctggatcc tgacctgtaa ccagctgaag
3540acggtggagg ctttgtggct tgctgagggt gggggttggg agagggactg
gaaaccttcc 3600tcctcgggaa agaaatgcct gggaggaagg gaagcctgat
attcagggtc aaaacagccc 3660ttctaattca caaccccaaa gcaggggttt
ctagaagttg gtcaacagat ctggtgaggg 3720aagtccccag gccagacgct
ggactcctgc aatgagggtg gaggactcag cctggcctct 3780gcctggcctg
tttcaaagtc tgggccactg gcagccttct cttgctcaat ccgaggtgga
3840aaaagatcac cctcccagtg acctcccctc agcccccagg ggagtaggtc
tgtgtccgaa 3900gctatgtggc aggcttgttt gaaggaccca gaggggccca
gctgacctgt ctcactgctc 3960ctggcagccc agccccatcg gcaggtggcc
cctgcctggg ggttattcgg gacggttcag 4020ccagggcctc ccaggaagag
tgctgtggag ccacggtgac tgtcctggac agcaaggagc 4080atgctacccc
agtgagcact tctttttgag ggacttgatg gggaggtggg gtaggcagaa
4140gggacgtggc agaagcggga agactttggt caccatggct ctgcaggcct
tctgcaaatc 4200agtgctggcc tgggcttgga accagttctt gtgtgtgaag
gtgaggactc ttggaagcag 4260gccatcctgg ccagacaccc gtagcagaag
gggctcacct gtcaccctta ggcagcctga 4320gggctggtgg ggacttttga
gtcttgaggg gatagcggaa aaagctgagc tcataggtgc 4380ccagccagcc
tcccagctaa aggtgctcag agccccmctg ccccctctct ctgtgcaggt
4440ggccagtgcc cctccctgct ctgggagcat tgctagcctt ctaccccatc
cctggatcca 4500caggggctat cgaggagacc cagtgagaat gtagcatttt
gttcatcccc agggtagctg 4560ccctggggtc tgggcactct gcctctggga
gagaggaaga agaaaggggc cccatttttt 4620aaaaaactgt acagagcctt
tggctttatg tgtttatgtt cttcacatgc atatgtgtgt 4680atgtgtgtat
atctttcccc ccatcaattg gtacaatttt taataaaatc atttaaagca
4740aaaaaaaaaa aaaaaaactm ragaawamwt ctagagcggc cgcgggccca
tcgattttcc 4800acccgggtrg ggtaccaggt tagtgtaccc aattcgccct
atagtgagtc gtattacaat 4860tcactggccg tcgtttacaa cgtcgtgact
gggaaaacct ggcgttacca acttaatcgc 4920cttgagcaca tcccctttcg
ccagctggcg taatagcga 49594184PRTHomo sapiens 4Met Glu Pro Ser Gln
Cys Val Glu Glu Leu Glu Asp Asp Val Phe Gln1 5 10 15Pro Glu Asp Gly
Glu Pro Val Thr Gln Pro Gly Ser Leu Leu Ser Ala 20 25 30Asp Leu Phe
Ala Gln Ser Leu Leu Asp Cys Pro Leu Ser Arg Leu Gln 35 40 45Leu Phe
Pro Leu Thr His Cys Cys Gly Pro Gly Leu Arg Pro Thr Ser 50 55 60Gln
Glu Asp Lys Ala Thr Gln Thr Leu Ser Pro Ala Ser Pro Ser Gln65 70 75
80Gly Val Met Leu Pro Cys Gly Val Thr Glu Glu Pro Gln Arg Leu Phe
85 90 95Tyr Gly Asn Ala Gly Tyr Arg Leu Pro Leu Pro Ala Ser Phe Pro
Ala 100 105 110Val Leu Pro Ile Gly Glu Gln Pro Pro Glu Gly Gln Trp
Gln His Gln 115 120 125Ala Glu Val Gln Ile Ala Arg Lys Leu Gln Cys
Ile Ala Asp Gln Phe 130 135 140His Arg Leu His Val Gln Gln His Gln
Gln Asn Gln Asn Arg Val Trp145 150 155 160Trp Gln Ile Leu Leu Phe
Leu His Asn Leu Ala Leu Asn Gly Glu Glu 165 170 175Asn Arg Asn Gly
Ala Gly Pro Arg 180554DNAMus musculus 5cagatcgcca gaaagcttca
gtgtattgca gaccagttcc atcggcttca tacg 54618PRTMus musculus 6Gln Ile
Ala Arg Lys Leu Gln Cys Ile Ala Asp Gln Phe His Arg Leu1 5 10 15His
Thr754DNAHomo sapiens 7cagattgccc gaaagcttca gtgcattgca gaccagttcc
accggcttca tgtg 54818PRTMus musculus 8Gln Ile Ala Arg Lys Leu Gln
Cys Ile Ala Asp Gln Phe His Arg Leu1 5 10 15His Val92000DNAMus
musculus 9actctgacca gctcagacat gcacttttgg ccaagcctct gggtaaaata
atatacctcc 60ttttggctta gtgccttttg ctggaaatct atcaatttcg ggggactgtg
ctactggcta 120accttgactc cccactcttt cccttttccc tctttgtcct
ttggagacag tatttttcta 180ttctagcaac cacagccctc aatatggctg
gttcattctg tttcaggtga cttctgaggg 240agcaaaggaa agaatgatgg
gaaaaaggta caaggagact tcaggctcta ttgggctgaa 300ggtcccagct
ccactgaact taaatctaag tttacaaccc agagagcagc tcggtggtca
360gagcactgct gagccctact gcccctggtg ggtcagggtt gttgcaggac
tggctccctc 420tcactgctct ctggactcat cattttatct ttcctaaaac
tcaggcttgg tcaagtcact 480catctccaat aaaatctttg ctgcctcccc
tttataaaat acagattctt cagtctggca 540ttccagggac acctctgtag
tctgacctga acccacctgc ccatgcctac cgttcgtaaa 600tctcagtgtg
cacccaatga tccagtcacc aagcctccct ctgctccctg cacaggtaag
660ctctccagct cagcacttca gtctgctacc cttaggcctg ggctattctt
ttttatttta 720ttttattttt atttttttga gacggagtct cattgtcgcc
caggctggag tgcggtggcg 780ctatctccgc ccactgcaac cttcacctcc
taggttcaag caattctcct acctcagcct 840cccaagtagc tgggattaca
ggcatgcgcc accacgcccg cctgattttt gtatttttag 900tagagacagg
gtttcccgtg ttggccaggc tggtctccaa ccactgagct caagtgaacc
960acccgcctcg gcctcccaaa gtgttgggat tacaggcgtg agacatgtgc
atctggtttt 1020ccttattgaa aaatatttcc cattcaattt aaaagctgtc
tccctgaggc cttctccagc 1080ctttgtattc tgaatttttc tgcattcctc
aatagtcatt atccttggaa tgaatacaat 1140tccaataact tttcactatt
ttcagggcta cctctcctag tagactgtaa gctccttgag 1200ggcagacgcc
aggtttctgg cactcagccc aatatctggc acagagtagg tgctcggtaa
1260atgcctgtga agtagcccat agactccctt acgcggtctg caggacatgc
tgctctcctg 1320gcagcaccag cacagtctct aaatgctgcc atatgcgaga
tatgtgtcaa ccgctcaagc 1380agccccggcc ttcttgagcg ctccgcttct
cagccaggtg cttattttgc cagtgcccac 1440gcccagtcaa gaagaggacc
taagggctcc cctggatgtg tttgtttcaa acacaccttc 1500agctttggag
ctgcagtttt cttccgacct gctcggcagg cgggagggag ctttcccctg
1560aggctgggat cccataggga cccgcaccct gcaccctgca cccactggac
gcaccagcct 1620cataaaaaaa ctccccgcct ccttcccccc tccctttgtg
gacgcgcagc aattattctg 1680cccattgccg tgaaaaagaa gacaaaagtt
actttggcgc cccctctccc acacctaata 1740cagaggattc agggactctc
tggcgcttcc agagcctgtg ttagggacag aatccgcact 1800ggcgacggcg
ctccgactgc gcttctggcg acggtcggaa ttttgctcgg ccccttgcaa
1860tgtttccatg ggaaggttcg tacattcgtg accgtccctg gcagcggccc
agcccgggac 1920ttggcgcttc actcgccatt ggtcagtcct cggcgtgacg
cgcagggggg cggggcctca 1980tcagctgttt gcgggatgcc 2000101833DNAHomo
sapiens 10ctagtgtttg gttctcagca cccacatggt ggctcacacc catccttaac
tccaatttca 60ggagattcca ccccctctta tagtcccctt gggcatcagg cacacacctg
gtacacacac 120atgcatgaag gcaaaacacc cacacgaata aagtaaaaat
aaatatatct aaaaacaatt 180aaaaaagaaa aacagaaaga aagaaaccaa
agtaaataaa taaaaatttt aaaacaaaaa 240atagccaaag acaattgagt
ctgggagccc cagatccagc cagcttgtat cttagagcag 300actgtaagta
gagactgtaa ctaagcccag cttctctgct ttgcccgtgt ctctattgca
360gctcatttag gttgtggcag ggctctgttc ctttcactga tttctgcctc
cccactcagt 420tttccccaaa attcatcttc tgtaaaatct ttgctaccag
tcctctataa agtaattcct 480tggggctgaa gagatggctc aagtgggtaa
aagcactggt tgctcttcca gaggacccag
540gttcaattcc aagaactcac acggcagttc atagctgtct gtaactctgg
ttccggatga 600cacgctgtca caaagacata catacaggcg aaacactaac
gcacataaaa taaaaattaa 660caaattactt ttaaaaaata cagactcctc
agcctggtat tccagggggc acctctgcaa 720tctgacctga atctacctgc
ccataccctc acatcctaaa tcatagtgaa gatgccagag 780agcttgatgg
gggggagggg ggacgggggg ggcgtgcagc tgatcttgct ttccagacat
840gttaagtact ttattctagc tccctgacct ctctgggtca ggactgtttt
atttggtttt 900tatttagtga aatattcccc attcttcaag gcccgactca
aaaatctttg cttattatcc 960ctaaaatagc accattcaag ctggtttcca
tcattcacat gtgaagtatc ggttcattga 1020ggtcaggcac cagaagtcta
gcacttagac caggacctgg cacatagtag gtgttcaaaa 1080aaatgtggag
tcatccctag attggccata gtacacgaga tgcgttttgc tttcttagca
1140gtccaagaac agccttctgc tgctgccata gatagggctg tccgagctac
ccctttgatt 1200tcccctctgc tagagtcctt ttttcaacag tgatcttcca
cagtctagaa gagaggacat 1260ggcctttctt ttgcacttga tggatccgaa
tgtcatttgg ttctagatgc agacacacgt 1320gttctcctcc agctttcctc
agacacgccc aacaggaaag agggcagttt tttgttttgg 1380ttttttattt
gttttgcttt ttttttatta gggaccagca gctcggtttt tttttttttc
1440cgctatatat tacaaggcgc gtctgaaaac tcccagtctc ctcctgagcc
ggctttttgg 1500aagtcctgca ataactcagc ccatttccaa gtagacaaaa
gtcacttagg cgtcttgtcc 1560ccccactacc tagtagaaaa cctagggact
ctctgtcgtt tctggggctt gcattaggaa 1620ccgaatcctc accctctaca
gcgctccaat tgcgcttctg acggaggtca gaaatgcgcc 1680cggcccctta
taatgtttcc aagggaaggt ttgtacattg gtgattgtcc ctggcagctg
1740cccagcttga gacttggacc tccacttgcc attggtcagt ggttggagtg
acgcaaagag 1800gggcggggcc tcatcagctg tttgcgggat gcc
18331121DNAArtificial SequenceSynthetic oligonucleotide primer
11ccggatggat caccaggaat g 211219DNAArtificial SequenceSynthetic
oligonucleotide primer 12cagagctgac aaaggcaag 191320DNAArtificial
SequenceSynthetic oligonucleotide primer 13ccacttcctg gagaacatca
201425DNAArtificial SequenceSynthetic oligonucleotide primer
14tgatgacatc aagaaggtgg tgaag 251523DNAArtificial SequenceSynthetic
oligonucleotide primer 15tccttggagg ccatgtaggc cat
231622DNAArtificial SequenceSynthetic oligonucleotide primer
16cccggcatcg aaggtggaag ag 2217185PRTMus musculus 17Met Glu Pro Pro
Gln Cys Val Glu Glu Leu Glu Asp Asp Val Phe Gln1 5 10 15Ser Glu Asp
Gly Glu Pro Gly Thr Gln Pro Gly Gly Leu Leu Ser Ala 20 25 30Asp Leu
Phe Ala Gln Ser Gln Leu Asp Cys Pro Leu Ser Arg Leu Gln 35 40 45Leu
Phe Pro Leu Thr His Cys Cys Gly Pro Gly Leu Arg Pro Ile Ser 50 55
60Gln Glu Asp Lys Ala Thr Gln Thr Leu Ser Pro Ala Ser Pro Ser Gln65
70 75 80Gly Val Met Leu Pro Cys Gly Val Thr Glu Glu Pro Gln Arg Leu
Phe 85 90 95Tyr Gly Asn Ala Gly Tyr Arg Leu Pro Leu Pro Ala Ser Phe
Pro Ala 100 105 110Gly Ser Pro Leu Gly Glu Gln Pro Pro Glu Gly Gln
Phe Leu Gln His 115 120 125Arg Ala Glu Val Gln Ile Ala Arg Lys Leu
Gln Cys Ile Ala Asp Gln 130 135 140Phe His Arg Leu His Thr Gln Gln
His Gln Gln Asn Arg Asp Arg Ala145 150 155 160Trp Trp Gln Val Phe
Leu Phe Leu Gln Asn Leu Ala Leu Asn Arg Gln 165 170 175Glu Asn Arg
Glu Gly Val Gly Pro Trp 180 18518184PRTHomo sapiens 18Met Glu Pro
Ser Gln Cys Val Glu Glu Leu Glu Asp Asp Val Phe Gln1 5 10 15Pro Glu
Asp Gly Glu Pro Val Thr Gln Pro Gly Ser Leu Leu Ser Ala 20 25 30Asp
Leu Phe Ala Gln Ser Leu Leu Asp Cys Pro Leu Ser Arg Leu Gln 35 40
45Leu Phe Pro Leu Thr His Cys Cys Gly Pro Gly Leu Arg Pro Thr Ser
50 55 60Gln Glu Asp Lys Ala Thr Gln Thr Leu Ser Pro Ala Ser Pro Ser
Gln65 70 75 80Gly Val Met Leu Pro Cys Gly Val Thr Glu Glu Pro Gln
Arg Leu Phe 85 90 95Tyr Gly Asn Ala Gly Tyr Arg Leu Pro Leu Pro Ala
Ser Phe Pro Ala 100 105 110Val Leu Pro Ile Gly Glu Gln Pro Pro Glu
Gly Gln Trp Gln His Gln 115 120 125Ala Glu Val Gln Ile Ala Arg Lys
Leu Gln Cys Ile Ala Asp Gln Phe 130 135 140His Arg Leu His Val Gln
Gln His Gln Gln Asn Gln Asn Arg Val Trp145 150 155 160Trp Gln Ile
Leu Leu Phe Leu His Asn Leu Ala Leu Asn Gly Glu Glu 165 170 175Asn
Arg Asn Gly Ala Gly Pro Arg 1801915PRTArtificial SequenceSynthetic
peptide; partial amino acid sequence of Bmf 19Ile Ala Arg Lys Leu
Gln Cys Ile Ala Asp Gln Phe His Arg Leu1 5 10 152015PRTArtificial
SequenceSynthetic peptide; partial amino acid sequence of Bim 20Ile
Ala Gln Glu Leu Arg Arg Ile Gly Asp Glu Phe Asn Ala Tyr1 5 10
152115PRTArtificial SequenceSynthetic peptide; partial amino acid
sequence of EGL-1 21Ile Gly Ser Lys Leu Ala Ala Met Cys Asp Asp Phe
Asp Ala Gln1 5 10 152215PRTArtificial SequenceSynthetic peptide;
partial amino acid sequence of Bak 22Val Gly Arg Gln Leu Ala Ile
Ile Gly Asp Asp Ile Asn Arg Arg1 5 10 152315PRTArtificial
SequenceSynthetic peptide; partial amino acid sequence of Bax 23Leu
Ser Glu Cys Leu Lys Arg Ile Gly Asp Glu Leu Asp Ser Asn1 5 10
152415PRTArtificial SequenceSynthetic peptide; partial amino acid
sequence of Bid 24Leu Ala Leu Arg Leu Ala Cys Ile Gly Asp Glu Met
Asp Val Ser1 5 10 152515PRTArtificial SequenceSynthetic peptide;
partial amino acid sequence of Bik 25Leu Ala Leu Arg Leu Ala Cys
Ile Gly Asp Glu Met Asp Val Ser1 5 10 152615PRTArtificial
SequenceSynthetic peptide; partial amino acid sequence of Hrk 26Thr
Ala Ala Arg Leu Lys Ala Leu Gly Asp Glu Leu His Gln Arg1 5 10
152715PRTArtificial SequenceSynthetic peptide; partial amino acid
sequence of Bad 27Tyr Gly Arg Glu Leu Arg Arg Met Ser Asp Glu Phe
Val Asp Ser1 5 10 15
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