U.S. patent application number 10/870197 was filed with the patent office on 2004-12-16 for mammalian pro-apoptotic bok genes and their uses.
This patent application is currently assigned to The Board of Trustees of the Leland Stanford Junior University. Invention is credited to Hsu, Sheau Yu, Hsueh, Aaron J.W..
Application Number | 20040253629 10/870197 |
Document ID | / |
Family ID | 22059282 |
Filed Date | 2004-12-16 |
United States Patent
Application |
20040253629 |
Kind Code |
A1 |
Hsueh, Aaron J.W. ; et
al. |
December 16, 2004 |
Mammalian pro-apoptotic bok genes and their uses
Abstract
Nucleic acid compositions encoding a pro-apoptotic protein, Bok
(Bcl-2-related ovarian killer) are identified. Bok has conserved
Bcl-2 homology domains 1, 2 and 3 and a C-terminal transmembrane
region present in other Bcl-2 related proteins, but lacks the BH4
domain found only in anti-apoptotic Bcl-2 proteins. Over-expression
of Bok induces apoptosis. Cell killing induced by Bok is suppressed
by co-expression with selective anti-apoptotic Bcl-2 proteins. Bok
is highly expressed in the ovary, testis and uterus, particularly
in granulosa cells, the cell type that undergoes apoptosis during
follicle atresia. Identification of Bok as a new pro-apoptotic
protein with wide tissue distribution and hetero-dimerization
properties facilitates elucidation of apoptosis mechanisms in
reproductive and other tissues, and provides a means for
manipulating apoptosis.
Inventors: |
Hsueh, Aaron J.W.;
(Stanford, CA) ; Hsu, Sheau Yu; (Menlo Park,
CA) |
Correspondence
Address: |
BOZICEVIC, FIELD & FRANCIS LLP
1900 UNIVERSITY AVE
SUITE 200
EAST PALO ALTO
CA
94303
US
|
Assignee: |
The Board of Trustees of the Leland
Stanford Junior University
|
Family ID: |
22059282 |
Appl. No.: |
10/870197 |
Filed: |
June 16, 2004 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10870197 |
Jun 16, 2004 |
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09682667 |
Oct 4, 2001 |
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6780604 |
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09682667 |
Oct 4, 2001 |
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09517358 |
Mar 2, 2000 |
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6437097 |
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09517358 |
Mar 2, 2000 |
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09186250 |
Nov 4, 1998 |
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6043055 |
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60064943 |
Nov 7, 1997 |
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Current U.S.
Class: |
435/6.13 ;
435/226; 435/320.1; 435/325; 435/69.1; 514/16.5; 514/18.9; 514/9.8;
536/23.2 |
Current CPC
Class: |
C07K 14/4747 20130101;
A01K 2217/075 20130101; C07K 14/4702 20130101 |
Class at
Publication: |
435/006 ;
514/012; 435/069.1; 435/226; 435/320.1; 435/325; 536/023.2 |
International
Class: |
C12Q 001/68; C12N
009/64; G01N 033/574 |
Goverment Interests
[0002] This invention was made with Government support under Grant
no. HD-31566, awarded by the National Institutes of Health. The
Government may have certain rights in this invention.
Claims
What is claimed is:
1. An isolated nucleic acid encoding a mammalian Bok protein.
2. The isolated nucleic acid according to claim 1, wherein said Bok
protein comprises the amino acid sequence as set forth in SEQ ID
NO:2, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:8.
3. The isolated nucleic acid according to claim 1, wherein said Bok
protein is a BH3.sup.i variant protein.
4. An isolated nucleic acid comprising at least 18 contiguous
nucleotides of the sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID
NO:5 or SEQ ID NO:7.
5. An isolated nucleic acid that hybridizes under stringent
conditions to the nucleic acid sequence of SEQ ID NO:1, SEQ ID
NO:3, SEQ ID NO:5 or SEQ ID NO:7.
6. An isolated nucleic acid encoding a BH3i variant of a
pro-apoptotic Bok related protein.
7. The isolated nucleic acid of claim 6, wherein said pro-apoptotic
Bok related protein is Bak or Bax.
8. An expression cassette comprising a transcriptional initiation
region functional in an expression host and operably linked to a
nucleic acid having a sequence of the isolated nucleic acid
according to claim 1.
9. A cell comprising an expression cassette according to claim 8 as
part of an extrachromosomal element or integrated into the genome
of a host cell as a result of introduction of said expression
cassette into said host cell, and the cellular progeny of said host
cell.
10. A method for producing pro-apoptotic protein, said method
comprising: growing a cell according to claim 9, whereby said
protein is expressed; and isolating said protein free of other
proteins.
11. A purified polypeptide composition comprising at least 50
weight % of the protein present as a Bok protein or a fragment
thereof.
12. A purified polypeptide composition comprising at least 50
weight % of the protein present as a BH3.sup.i variant of a
pro-apoptotic Bok related protein.
13. A monoclonal antibody binding specifically to a Bok
protein.
14. A non-human transgenic animal model for Bok gene function
wherein said transgenic animal comprises an introduced alteration
in a Bok gene.
15. A method of inducing apoptosis in a susceptible cell
population, the method comprising: upregulating expression of Bok
or a BH3.sup.i variant of a pro-apoptotic Bok related protein in
said cell population; wherein apoptosis is induced.
16. The method of claim 15, wherein said susceptible cell
population comprises reproductive tissue.
17. The method of claim 15, wherein said upregulating step
comprises induction of expression of an endogenous Bok gene.
18. The method of claim 15, wherein said upregulating step
comprises introduction and expression of an exogenous Bok coding
sequence.
19. The method of claim 15, wherein said upregulating step
comprises introduction and expression of an exogenous coding
sequence for a BH3.sup.i variant of a pro-apoptotic Bok related
protein.
Description
CROSS REFERENCE
[0001] This application is a Continuation of U.S. application Ser.
No. 09/517,358 filed Mar. 2, 2000, which is a Divisional of U.S.
application Ser. No. 09/186,250 filed Nov. 4, 1998, now U.S. Pat.
No. 6,043,055 issued on Mar. 28, 2000, which claims benefit of U.S.
Provisional Application Ser. No. 60,064,943 filed Nov. 7, 1997.
INTRODUCTION
BACKGROUND
[0003] Apoptosis or programmed cell death is important during
embryonic development, metamorphosis, tissue renewal,
hormone-induced tissue atrophy and many pathological conditions. In
multi-cellular organisms, apoptosis ensures the elimination of
superfluous cells including those that are generated in excess,
have already completed their specific functions or are harmful to
the whole organism. In reproductive tissues that are characterized
by cyclic functional changes, massive cell death occurs under the
control of hormonal signals. A growing body of evidence suggests
that the intracellular "death program" activated during apoptosis
is similar in different cell types and conserved during
evolution.
[0004] Apoptosis involves two essential steps. The Bcl-2 family of
proteins that consists of different anti- and pro-apoptotic members
is important in the "decision" step of apoptosis. In contrast, the
"execution" phase of apoptosis is mediated by the activation of
caspases, cysteine proteases homologous to the C. elegans protease
ced-3, that induce cell death via the proteolytic cleavage of
substrates vital for cellular homeostasis. Bcl-2-related proteins
act upstream from caspases in the cell death pathway and recent
studies demonstrated that another C. elegans gene, ced-4, or its
mammalian homolog Apaf-1 can bridge between Bcl-2/ced-9 family
members and caspases.
[0005] The proto-oncogene Bcl-2 was originally isolated at the
breakpoint of the t(14,18) chromosomal translocation associated
with follicular B-cell lymphoma. Over-expression of Bcl-2
suppresses apoptosis induced by a variety of agents both in vitro
and in vivo. Subsequent studies identified a family of
Bcl-2-related proteins possessing several conserved BH (Bcl-2
homology) domains important for homo- or hetero-dimerization
between family members. In addition, a C-terminal transmembrane
region for membrane anchoring is also conserved in most members.
Based on their differential roles in regulating apoptosis, the
Bcl-2-related proteins can be separated into anti-apoptotic (Bcl-2,
Bcl-xL, Mcl-1, Bcl-w and Bfl-1/A1) and pro-apoptotic members (Bax,
BAD, Bak, Bik, Hrk and BID). Through hetero-dimerization, the
balance between pro- and anti-apoptotic proteins presumably
determines cell fate. The anti-apoptotic effect of Bcl-2 is not
universal, however, because Bcl-2 over-expression is not effective
in blocking Fas-mediated apoptosis and the apoptosis of
auto-reactive thymocytes during negative selection. Recent
identification of multiple Bcl-2-related proteins suggests that
selective Bcl-2 members may act in a tissue- and
dimerization-specific manner.
[0006] References
[0007] Bcl related genes are discussed in Yin et al. (1994) Nature
369:321-323; Chittenden et al. (1995) EMBO J. 14:5589-5596; and
White (1996) Genes Dev. 10:1-15.
[0008] Sequences of exemplary bcl-related genes may be accessed in
Genbank. The human hrk gene has the accession no. U76376 and is
described in Inohara et al. (1997) EMBO J. 16:1686-1694. The human
bcl-w gene has the accession no. U59747 and is described in Gibson
et al. (1996) Oncogene 13:665-675. Human A1 gene has the accession
no. U29680, and is described in Karsan et al. (1996) Blood
87:3089-3096. The human Bak gene has the accession no. U23765, and
is described in Chittenden et al. (1995) Nature 374:733-736. The
human Bak-2 gene has the accession no. U16812, and is described in
Kiefer et al. (1995) Nature 374:736-739. The human Bik gene has the
accession no. U34584, and is described in Boyd et al. (1995)
Oncogene 11:1921-1928. The human Bfl-1 gene has the accession no.
U27467, and is described in Choi et al. (1995) Oncogene 11:
1693-1698. The human bcl-2 gene has the accession no. M13995, and
is described in Tsujimoto and Croce (1986) P.N.A.S. 83:5214-5218.
The human Bax genes have the accession nos. L22475, L22474 and
L22473, and are described in Oltvai et al. (1993) Cell 74:609-619.
The EBV BHRF1 gene has the accession no. A22899, and is described
in WO 9311267. The human mcl-1 gene is described in Kozopas et al.
(1993) P.N.A.S. 90:3516-3520, and OMIM 159552.
[0009] The EST fragment, Genbank accession no. AA103989, contains
partial sequence of the 5' end of the mouse Bok gene.
SUMMARY OF THE INVENTION
[0010] Isolated nucleotide compositions and sequences are provided
for Bok genes. The provided nucleic acids include splice variants
encoding long forms of the protein, as well as short forms having a
truncation that deletes all or a part of the BH3 domain. The short
form of Bok and other related pro-apoptotic proteins may be
naturally occurring or synthetic. These short forms induce cell
killing without heterodimerization with antiapoptotic proteins.
[0011] The Bok nucleic acid compositions find use in identifying
homologous or related genes; in producing compositions that
modulate the expression or function of its encoded protein; for
gene therapy; mapping functional regions of the protein; and in
studying associated physiological pathways. In addition, modulation
of the gene activity in vivo is used for prophylactic and
therapeutic purposes, such as treatment of cancer and other
proliferative disorders, identification of cell type based on
expression, and the like.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 is a graph showing quantitative analysis of cell
killing by Bok and the inhibitory effects of P35. The number of
.beta.-gal-expressing cells (mean.+-.SEM, n=3) was determined at 36
h after transfection. Data from cells transfected with three
independent clones (1, 2 and 3) encoding Bok are presented. CHO
cells were transfected with a total of 2.1 .mu.g plasmid DNA
including 2.0 .mu.g of pcDNA3 expression constructs and 0.1 .mu.g
of the pCMV-.beta.-gal reporter. In cells transfected with two
different pcDNA3 expression plasmids, 1.0 .mu.g each was used.
Similar results were-obtained in three separate experiments.
[0013] FIG. 2 is a graph showing suppression of Bok-induced
apoptosis by selective anti-apoptotic Bcl-2 members in CHO cells.
Cell killing by Bok and the antagonistic effects of Mcl-1 and BHRF1
were analyzed. Cell transfection and estimation of apoptosis were
as described for FIG. 1. Co-expression of Bcl-2 was ineffective in
suppressing Bok-induced apoptosis.
[0014] FIG. 3 is a schematic representation of wild type Bax and
Bak together with Bax-S and Bak-S constructs with BH3-BH1 deletions
similar to that found in Bok-S. The BH domains are boxed and the
junctional sequences derived from the fusion of BH3 and BH1 domains
(BH3/1) in the mutants are also shown. Numbering of amino acid in
the BH1, BH3 and BH3/1 domains are indicated at the bottom of amino
acid residues.
DETAILED DESCRIPTION OF THE INVENTION
[0015] Nucleic acid compositions encoding Bok, a pro-apoptotic
member of the bcl-2 protein family, are provided. Included are
splice variants encoding long forms of the protein, as well as
short forms having a truncation that deletes all or a part of the
BH3 domain. Also provided are truncated forms of pro-apoptotic
proteins related to Bok, e.g. Bax, Bak, etc. These short forms may
be naturally occurring or synthetic. The long forms associate with
anti-apoptotic proteins to form heterodimers, while the short forms
induce cell killing without such heterodimerization.
[0016] As used herein, the term "Bok" is intended to generically
refer to the polypeptide or nucleic acids as set forth in the
Seqlist attached herewith, homologs thereof, and sequences having
substantial similarity and function. Bok occurs naturally in a long
form (herein Bok-L), as exemplified by the amino acid sequences
provided in SEQ ID NO:2 and SEQ ID NO:6, which are rat and human,
respectively. A short form (herein Bok-S) also occurs naturally, as
exemplified by SEQ ID NO:4 and SEQ ID NO:8, in which there is a
deletion, leading to the fusion of the N-terminal half of the BH3
domain to the C-terminal half of the BH1 domain (herein,
BOK-BH3.sup.inactive).
[0017] The term "BH3.sup.inactive", or "BH3.sup.i" is intended to
generically refer to naturally occurring splice variants and
synthetic variants of Bok or pro-apoptotic Bok-related proteins,
e.g. Bax, Bak, etc., in which deletions or amino acid substitutions
made in the BH3 domain substantially inactivate or abrogate the
heterodimerization activity of the protein. These variants may also
be referred to as "channel only" proteins, because they retain the
ability to form channels in the mitochondria that promote
apoptosis.
[0018] The BH3.sup.i variants will usually have at least less than
about 50% of the anti-apoptotic protein binding activity of the
parent "long" form, more usually less than about 75% of the
anti-apoptotic protein binding activity, and preferably less than
about 95% of anti-apoptotic protein binding activity. Examples are
provided herein of BH3.sup.i variants, including but limited to:
alanine substitutions at the highly conserved Bok glycine 75
residue, truncations of Bax and Bak in the BH3 domain, splice
variants of Bok where there is a deletion of the amino acids
76-118; and a glycine substitution was made for leucine 71 to
leucine 74 (BokGGGG: 71 LLRL 74 to 71 GGGG 74).
[0019] The BH3 domain has the consensus motif sequence: [SEQ ID
NO:11} LRRAGDEFE.RYRR, and generally corresponds to the region of
amino acids 71-82 in Bok (SEQ ID NO:9 and SEQ ID NO:10). A
substitution at the conserved gly75 residue is shown to be
sufficient for inactivation.
[0020] Modulation of pro-apoptotic gene activity, which may include
Bok or other pro-apoptotic BH3.sup.i variants, in vivo is used for
prophylactic and therapeutic purposes where it is desirable induce
cell death in specific populations. The specificity of Bok for
reproductive tissues is particularly useful in this respect.
Diseases where there is hyperproliferation of reproductive tissue,
e.g. uterine, testicular and ovarian carcinomas, endometriosis,
squamous and glandular epithelial carcinomas of the cervix, etc.
are reduced in cell number by upregulating Bok expression to cause
apoptosis in susceptible cells. Expression can be regulated by
introduction of exogenous Bok genes, or by inducing expression of
the native gene. Introduction of exogenous Bok gene and its channel
domain only variants into tumor cells following direct injection or
using tumor-specific carriers can serve as effective therapies.
[0021] The isolated Bok genes are useful for in vitro, i.e. cell
culture or cell-free assays, investigation of apoptosis pathways,
identification of cell type based on expression, and the like. The
protein is useful as an immunogen for producing specific
antibodies, in screening for biologically active agents that act to
regulate Bok gene expression, or that directly mimic, agonize or
antagonize Bok protein function.
[0022] Bok is expressed mainly in mammalian reproductive tissues,
including ovary, testis and uterus. Bok is also expressed in
diverse other tissues, albeit at lower levels. It forms a
heterodimer with specific anti-apoptotic Bcl-2 proteins, including
mcl-1, BHRF1 and Bfl-1. The protein-protein interaction is mediated
by the conserved BH1, 2 and 3 domain regions of Bok, particularly
by the BH3 domain. Over-expression of Bok induces apoptosis in
certain cells, particularly reproductive cells. The rat cDNA
sequence is provided as SEQ ID NO:1, the encoded polypeptide
product as SEQ ID NO:2. The gene encodes a 213 amino acid
polypeptide. The rat short form is provided as SEQ ID NO:3, the
encoded polypeptide as SEQ ID NO:4. The nucleotide sequences of the
human long and short forms are provided as SEQ ID NO:5 and 7; the
encoded polypeptides as SEQ ID NO:6 and 8.
[0023] Many members of the bcl-2 gene family have been identified
and characterized, as previously indicated. Other proteins in the
pathway have also been identified, including caspases, and Apaf-1.
The availability of isolated genes and gene products in this
pathway allows the in vitro reconstruction of the pathway and its
regulation, using native or genetically altered molecules, or a
combination thereof. Also of interest is the use of the genomic
region 5' to Bok or related genes, particularly those members that
are hormonally regulated, in order to investigate the role of
particular transcription factors in regulating expression.
Identification of Bok Sequences
[0024] Homologs of Bok are identified by any of a number of
methods. A fragment of the provided cDNA may be used as a
hybridization probe against a cDNA library from the target organism
of interest, where low stringency conditions are used. The probe
may be a large fragment, or one or more short degenerate
primers.
[0025] Nucleic acids having sequence similarity are detected by
hybridization under low stringency conditions, for example, at
50.degree. C. and 10.times.SSC (0.9 M NaCl/0.09 M sodium citrate)
and remain bound when subjected to washing at 55.degree. C. in
1.times.SSC. Sequence identity may be determined by hybridization
under stringent conditions, for example, at 50.degree. C. or higher
and 0.1.times.SSC (9 mM NaCl/0.9 mM sodium citrate). Nucleic acids
that are substantially identical to the provided Bok sequences,
e.g. allelic variants, genetically altered versions of the gene,
etc., bind to the provided Bok sequences under stringent
hybridization conditions. By using probes, particularly labeled
probes of DNA sequences, one can isolate homologous or related
genes. The source of homologous genes may be any species, e.g.
primate species, particularly human; rodents, such as rats and
mice, canines, felines, bovines, ovines, equines, yeast, nematodes,
etc.
[0026] Between mammalian species, e.g. human and mouse, homologs
have substantial sequence similarity, i.e. at least 75% sequence
identity between nucleotide sequences. Sequence similarity is
calculated based on a reference sequence, which may be a subset of
a larger sequence, such as a conserved motif, coding region,
flanking region, etc. A reference sequence will usually be at least
about 18 nt long, more usually at least about 30 nt long, and may
extend to the complete sequence that is being compared. Algorithms
for sequence analysis are known in the art, such as BLAST,
described in Altschul et al. (1990) J Mol Biol 215:403-10. The
sequences provided herein are essential for recognizing Bok related
and homologous proteins in database searches.
Bok Nucleic Acid Compositions
[0027] Nucleic acids encoding Bok may be cDNA or genomic DNA or a
fragment thereof. The term "Bok gene" shall be intended to mean the
open reading frame encoding specific Bok polypeptides, e.g. splice
variants; introns; as well as adjacent 5' and 3' non-coding
nucleotide sequences involved in the regulation of expression, up
to about 20 kb beyond the coding region, but possibly further in
either direction. The gene may be introduced into an appropriate
vector for extrachromosomal maintenance or for integration into a
host genome.
[0028] The term "cDNA" as used herein is intended to include all
nucleic acids that share the arrangement of sequence elements found
in native mature mRNA species, where sequence elements are exons
and 3' and 5' non-coding regions. Normally mRNA species have
contiguous exons, with the intervening introns, when present,
removed by nuclear RNA splicing, to create a continuous open
reading frame encoding a Bok protein.
[0029] A genomic sequence of interest comprises the nucleic acid
present between the initiation codon and the stop codon, as defined
in the listed sequences, including all of the introns that are
normally present in a native chromosome. It may further include the
3' and 5' untranslated regions found in the mature mRNA. It may
further include specific transcriptional and translational
regulatory sequences, such as promoters, enhancers, etc., including
about 1 kb, but possibly more, of flanking genomic DNA at either
the 5' or 3' end of the transcribed region. The genomic DNA may be
isolated as a fragment of 100 kbp or smaller; and substantially
free of flanking chromosomal sequence. The genomic DNA flanking the
coding region, either 3' or 5', or internal regulatory sequences as
sometimes found in introns, contains sequences required for proper
tissue and stage specific expression.
[0030] The sequence of the 5' flanking region may be utilized for
promoter elements, including enhancer binding sites, that provide
for developmental regulation in tissues where Bok is expressed. The
tissue specific expression is useful for determining the pattern of
expression, for providing promoters that mimic the native pattern
of expression, and for determination of transcription factors that
regulate expression. Naturally occurring polymorphisms in the
promoter region are useful for determining natural variations in
expression, particularly those that may be associated with
disease.
[0031] Alternatively, mutations may be introduced into the promoter
region to determine the effect of altering expression in
experimentally defined systems. Methods for the identification of
specific DNA motifs involved in the binding of transcriptional
factors are known in the art, e.g. sequence similarity to known
binding motifs, gel retardation studies, etc. For examples, see
Blackwell et al. (1995) Mol Med 1: 194-205; Mortlock et al. (1996)
Genome Res. 6: 327-33; and Joulin and Richard-Foy (1995) Eur J
Biochem 232: 620-626.
[0032] The regulatory sequences may be used to identify cis acting
sequences required for transcriptional or translational regulation
of Bok expression, especially in different tissues or stages of
development, and to identify cis acting sequences and trans acting
factors that regulate or mediate Bok expression. Such transcription
or translational control regions may be operably linked to a Bok
gene in order to promote expression of wild type or altered Bok or
other proteins of interest in cultured cells, or in embryonic,
fetal or adult tissues, and for gene therapy. Expression of Bok may
be regulated through hormonal control.
[0033] The nucleic acid compositions of the subject invention may
encode all or a part of the subject polypeptides. Double or single
stranded fragments may be obtained of the DNA sequence by
chemically synthesizing oligonucleotides in accordance with
conventional methods, by restriction enzyme digestion, by PCR
amplification, etc. For the most part, DNA fragments will be of at
least 15 nt, usually at least 18 nt or 25 nt, and may be at least
about 50 nt. Such small DNA fragments are useful as primers for
PCR, hybridization screening probes, etc. Larger DNA fragments,
i.e. greater than 100 nt are useful for production of the encoded
polypeptide. For use in amplification reactions, such as PCR, a
pair of primers will be used. The exact composition of the primer
sequences is not critical to the invention, but for most
applications the primers will hybridize to the subject sequence
under stringent conditions, as known in the art. It is preferable
to choose a pair of primers that will generate an amplification
product of at least about 50 nt, preferably at least about 100 nt.
Algorithms for the selection of primer sequences are generally
known, and are available in commercial software packages.
Amplification primers hybridize to complementary strands of DNA,
and will prime towards each other.
[0034] The Bok genes are isolated and obtained in substantial
purity, generally as other than an intact chromosome. Usually, the
DNA will be obtained substantially free of other nucleic acid
sequences that do not include a Bok sequence or fragment thereof,
generally being at least about 50%, usually at least about 90% pure
and are typically "recombinant", i.e. flanked by one or more
nucleotides with which it is not normally associated on a naturally
occurring chromosome.
[0035] The DNA may also be used to identify expression of the gene
in a biological specimen. The manner in which one probes cells for
the presence of particular nucleotide sequences, as genomic DNA or
RNA, is well established in the literature and does not require
elaboration here. DNA or mRNA is isolated from a cell sample. The
mRNA may be amplified by RT-PCR, using reverse transcriptase to
form a complementary DNA strand, followed by polymerase chain
reaction amplification using primers specific for the subject DNA
sequences. Alternatively, the mRNA sample is separated by gel
electrophoresis, transferred to a suitable support, e.g.
nitrocellulose, nylon, etc., and then probed with a fragment of the
subject DNA as a probe. Other techniques, such as oligonucleotide
ligation assays, in situ hybridizations, and hybridization to DNA
probes arrayed on a solid chip may also find use. Detection of mRNA
hybridizing to the subject sequence is indicative of Bok gene
expression in the sample.
[0036] The sequence of a Bok gene, including flanking promoter
regions and coding regions, may be mutated in various ways known in
the art to generate targeted changes in promoter strength, sequence
of the encoded protein, etc. The DNA sequence or protein product of
such a mutation will usually be substantially similar to the
sequences provided herein, i.e. will differ by at least one
nucleotide or amino acid, respectively, and may differ by at least
two but not more than about ten nucleotides or amino acids. The
sequence changes may be substitutions, insertions or deletions.
Deletions may further include larger changes, such as deletions of
a domain or exon. Of particular interest is the creation of
BH3.sup.i variants. Other modifications of interest include epitope
tagging, e.g. with the FLAG system, HA, etc. For studies of
subcellular localization, fusion proteins with green fluorescent
proteins (GFP) may be used.
[0037] Techniques for in vitro mutagenesis of cloned genes are
known. Examples of protocols for site specific mutagenesis may be
found in Gustin et al., Biotechniques 14:22 (1993); Barany, Gene
37:111-23 (1985); Colicelli et al., Mol Gen Genet 199:537-9 (1985);
and Prentki et al., Gene 29:303-13 (1984). Methods for site
specific mutagenesis can be found in Sambrook et al., Molecular
Cloning: A Laboratory Manual, CSH Press 1989, pp. 15.3-15.108;
Weiner et al., Gene 126:35-41 (1993); Sayers et al., Biotechniques
13:592-6 (1992); Jones and Winistorfer, Biotechniques 12:528-30
(1992); Barton et al., Nucleic Acids Res 18:7349-55 (1990); Marotti
and Tomich, Gene Anal Tech 6:67-70 (1989); and Zhu, Anal Biochem
177:120-4 (1989). Such mutated genes may be used to study
structure-function relationships of Bok, or to alter properties of
the protein that affect its function or regulation.
[0038] Other nucleic acids of the invention include pro-apoptotic
BH3.sup.i variants. These may be synthesized by using techniques of
in vitro mutagenesis and genetic engineering to inactivate the BH3
domain of Bok related genes. The wild-type sequence of these genes
are known and publically available, e.g. in Genbank the human Bak
gene has the accession no. U23765; human Bak-2 gene has the
accession no. U16812; human Bik gene has the accession no. U34584;
human Bax genes have the accession nos. L22475, L22474 and L22473.
One of skill in the art can generate the physical nucleic acid from
the database sequence by various means, e.g. synthesis of primers
and PCR amplification, screening cDNA libraries, etc.
Bok Polypeptides
[0039] The subject nucleic acids may be employed for producing all
or portions of Bok polypeptides or BH3.sup.i variants of
pro-apoptotic Bok related polypeptides. For expression, an
expression cassette may be employed. The expression vector will
provide a transcriptional and translational initiation region,
which may be inducible or constitutive, where the coding region is
operably linked under the transcriptional control of the
transcriptional initiation region, and a transcriptional and
translational termination region. These control regions may be
native to a Bok gene, or may be derived from exogenous sources.
[0040] The peptide may be expressed in prokaryotes or eukaryotes in
accordance with conventional ways, depending upon the purpose for
expression. For large scale production of the protein, a
unicellular organism, such as E. coli, B. subtilis, S. cerevisiae,
insect cells in combination with baculovirus vectors, or cells of a
higher organism such as vertebrates, particularly mammals, e.g. COS
7 cells, may be used as the expression host cells. In some
situations, it is desirable to express the Bok gene in eukaryotic
cells, where the Bok protein will benefit from native folding and
post-translational modifications. Small peptides can also be
synthesized in the laboratory. Peptides that are subsets of the
complete Bok sequence, e.g. peptides of at least about 4 amino
acids, usually at least about 8 amino acids, more usually at least
about 16 amino acids, up to and including functional domains, and
the complete Bok polypeptide, may be used to identify and
investigate parts of the protein important for function, or to
raise antibodies directed against these regions.
[0041] With the availability of the protein or fragments thereof in
large amounts, by employing an expression host, the protein may be
isolated and purified in accordance with conventional ways. A
lysate may be prepared of the expression host and the lysate
purified using HPLC, exclusion chromatography, gel electrophoresis,
affinity chromatography, or other purification technique. The
purified protein will generally be at least about 80% pure,
preferably at least about 90% pure, and may be up to and including
100% pure. Pure is intended to mean free of other proteins, as well
as cellular debris.
[0042] The expressed Bok polypeptides are useful for the production
of antibodies, where short fragments provide for antibodies
specific for the particular polypeptide, and larger fragments or
the entire protein allow for the production of antibodies over the
surface of the polypeptide. Epitopes for immunization may comprise
one or more of the conserved BH domains. Antibodies may be raised
to the wild-type or variant forms of Bok. Antibodies may be raised
to isolated peptides corresponding to these domains, or to the
native protein.
[0043] Antibodies are prepared in accordance with conventional
ways, where the expressed polypeptide or protein is used as an
immunogen, by itself or conjugated to known immunogenic carriers,
e.g. KLH, pre-S HBsAg, other viral or eukaryotic proteins, or the
like. Various adjuvants may be employed, with a series of
injections, as appropriate. For monoclonal antibodies, after one or
more booster injections, the spleen is isolated, the lymphocytes
immortalized by cell fusion, and then screened for high affinity
antibody binding. The immortalized cells, i.e. hybridomas,
producing the desired antibodies may then be expanded. For further
description, see Monoclonal Antibodies: A Laboratory Manual, Harlow
and Lane eds., Cold Spring Harbor Laboratories, Cold Spring Harbor,
N.Y., 1988. If desired, the mRNA encoding the heavy and light
chains may be isolated and mutagenized by cloning in E. coli, and
the heavy and light chains mixed to further enhance the affinity of
the antibody. Alternatives to in vivo immunization as a method of
raising antibodies include binding to phage "display" libraries,
usually in conjunction with in vitro affinity maturation.
Modulation of Gene Expression
[0044] The Bok genes, gene fragments, or the encoded protein or
protein fragments, including BH3.sup.i variants of related
pro-apoptotic sequence, are useful in gene therapy to treat
disorders associated with a deficit in pro-apoptosis proteins,
including different states of tumorgenesis. This approach is also
useful in treating proliferative conditions of reproductive cells,
such as uterine cell hyperplasia, leiomyoma and tumorigenesis.
Expression vectors may be used to introduce the coding sequence
into a cell. Such vectors generally have convenient restriction
sites located near the promoter sequence to provide for the
insertion of nucleic acid sequences. Transcription cassettes may be
prepared comprising a transcription initiation region, the target
gene or fragment thereof, and a transcriptional termination region.
The transcription cassettes may be introduced into a variety of
vectors, e.g. plasmid; retrovirus, e.g. lentivirus; adenovirus; and
the like, where the vectors are able to transiently or stably be
maintained in the cells, usually for a period of at least about one
day, more usually for a period of at least about several days to
several weeks.
[0045] The exogenous coding sequence or protein may be introduced
into tissues or host cells by any number of routes, including viral
infection, microinjection, or fusion of vesicles. Methods that
localize the agent to the particular targeted tissues are of
interest.
[0046] Antisense molecules can be used to down-regulate expression
of Bok in cells. The anti-sense reagent may be antisense
oligonucleotides (ODN), particularly synthetic ODN having chemical
modifications from native nucleic acids, or nucleic acid constructs
that express such anti-sense molecules as RNA. The antisense
sequence is complementary to the mRNA of the targeted gene, and
inhibits expression of the targeted gene products. Antisense
molecules inhibit gene expression through various mechanisms, e.g.
by reducing the amount of mRNA available for translation, through
activation of RNAse H, or steric hindrance. One or a combination of
antisense molecules may be administered, where a combination may
comprise multiple different sequences.
[0047] Antisense molecules may be produced by expression of all or
a part of the target gene sequence in an appropriate vector, where
the transcriptional initiation is oriented such that an antisense
strand is produced as an RNA molecule. Alternatively, the antisense
molecule is a synthetic oligonucleotide. Antisense oligonucleotides
will generally be at least about 7, usually at least about 12, more
usually at least about 20 nucleotides in length, and not more than
about 500, usually not more than about 50, more usually not more
than about 35 nucleotides in length, where the length is governed
by efficiency of inhibition, specificity, including absence of
cross-reactivity, and the like. It has been found that short
oligonucleotides, of from 7 to 8 bases in length, can be strong and
selective inhibitors of gene expression (see Wagner et al. (1996)
Nature Biotechnology 14:840-844).
[0048] A specific region or regions of the endogenous sense strand
mRNA sequence is chosen to be complemented by the antisense
sequence. Selection of a specific sequence for the oligonucleotide
may use an empirical method, where several candidate sequences are
assayed for inhibition of expression of the target gene in an in
vitro or animal model. A combination of sequences may also be used,
where several regions of the mRNA sequence are selected for
antisense complementation.
[0049] Antisense oligonucleotides may be chemically synthesized by
methods known in the art (see Wagner et al. (1993) supra. and
Milligan et al., supra.) Preferred oligonucleotides are chemically
modified from the native phosphodiester structure, in order to
increase their intracellular stability and binding affinity. A
number of such modifications have been described in the literature,
which alter the chemistry of the backbone, sugars or heterocyclic
bases.
[0050] Among useful changes in the backbone chemistry are
phosphorothioates; phosphorodithioates, where both of the
non-bridging oxygens are substituted with sulfur;
phosphoroamidites; alkyl phosphotriesters and boranophosphates.
Achiral phosphate derivatives include 3'-O'-5'-S-phosphorothioate,
3'-S-5'-O-phosphorothioate, 3'-CH2-5'-O-phosphonate and
3'-NH-5'-O-phosphoroamidate. Peptide nucleic acids replace the
entire ribose phosphodiester backbone with a peptide linkage. Sugar
modifications are also used to enhance stability and affinity. The
.alpha.-anomer of deoxyribose may be used, where the base is
inverted with respect to the natural .beta.-anomer. The 2'-OH of
the ribose sugar may be altered to form 2'-O-methyl or 2'-O-allyl
sugars, which provides resistance to degradation without comprising
affinity. Modification of the heterocyclic bases must maintain
proper base pairing. Some useful substitutions include deoxyuridine
for deoxythymidine; 5-methyl-2'-deoxycytidine and
5-bromo-2'-deoxycytidine for deoxycytidine.
5-propynyl-2'-deoxyuridine and 5-propynyl-2'-deoxycytidine have
been shown to increase affinity and biological activity when
substituted for deoxythymidine and deoxycytidine, respectively.
Genetically Altered Cell or Animal Models for Bok Function
[0051] The subject nucleic acids can be used to generate transgenic
animals or site specific gene modifications in cell lines.
Transgenic animals may be made through homologous recombination,
where the normal Bok locus is altered. Alternatively, a nucleic
acid construct is randomly integrated into the genome. Vectors for
stable integration include plasmids, retroviruses and other animal
viruses, YACs, and the like.
[0052] The modified cells or animals are useful in the study of
pro-apoptotic gene function and regulation. For example, a series
of small deletions and/or substitutions may be made in the Bok gene
to determine the role of different exons in oncogenesis, signal
transduction, etc. Of interest are the use of Bok to construct
transgenic animal models for proliferative disorders, e.g.
endometriosis, where expression of Bok is specifically reduced or
absent. Specific constructs of interest include anti-sense Bok,
which will block Bok expression, expression of dominant negative
Bok mutations. A detectable marker, such as lac Z may be introduced
into the Bok locus, where upregulation of Bok expression will
result in an easily detected change in phenotype.
[0053] One may also provide for expression of the Bok gene or
variants thereof in cells or tissues where it is not normally
expressed or at abnormal times of development. By providing
expression of Bok protein in cells in which it is not normally
produced, one can induce changes in cell behavior.
[0054] DNA constructs for homologous recombination will comprise at
least a portion of the Bok gene with the desired genetic
modification, and will include regions of homology to the target
locus. Conveniently, markers for positive and negative selection
are included. Methods for generating cells having targeted gene
modifications through homologous recombination are known in the
art. For various techniques for transfecting mammalian cells, see
Keown et al. (1990) Methods in Enzymology 185:527-537.
[0055] For embryonic stem (ES) cells, an ES cell line may be
employed, or embryonic cells may be obtained freshly from a host,
e.g. mouse, rat, guinea pig, etc. Such cells are grown on an
appropriate fibroblast-feeder layer or grown in the presence of
leukemia inhibiting factor (LIF). When ES or embryonic cells have
been transformed, they may be used to produce transgenic animals.
After transformation, the cells are plated onto a feeder layer in
an appropriate medium. Cells containing the construct may be
detected by employing a selective medium. After sufficient time for
colonies to grow, they are picked and analyzed for the occurrence
of homologous recombination or integration of the construct. Those
colonies that are positive may then be used for embryo manipulation
and blastocyst injection. Blastocysts are obtained from 4 to 6 week
old superovulated females. The ES cells are trypsinized, and the
modified cells are injected into the blastocoel of the blastocyst.
After injection, the blastocysts are returned to each uterine horn
of pseudopregnant females. Females are then allowed to go to term
and the resulting offspring screened for the construct. By
providing for a different phenotype of the blastocyst and the
genetically modified cells, chimeric progeny can be readily
detected.
[0056] The chimeric animals are screened for the presence of the
modified gene and males and females having the modification are
mated to produce homozygous progeny. If the gene alterations cause
lethality at some point in development, tissues or organs can be
maintained as allogeneic or congenic grafts or transplants, or in
in vitro culture. The transgenic animals may be any non-human
mammal, such as laboratory animals, domestic animals, etc. The
transgenic animals may be used in functional studies, drug
screening, etc.
In vitro Models for Bok Function
[0057] The availability of a number of members in the bcl-2 gene
family, as previously described, allows in vitro reconstruction of
the apoptosis pathway. Two or more of the components may be
combined in vitro, and the behavior assessed in terms of activation
of transcription of specific target sequences; modification of
protein components, e.g. proteolytic processing, phosphorylation,
methylation, etc.; ability of different protein components to bind
to each other, etc. The components may be modified by sequence
deletion, substitution, etc. to determine the functional role of
specific domains.
[0058] Drug screening may be performed using an in vitro model, a
genetically altered cell or animal, or purified protein. One can
identify ligands or substrates that bind to, modulate or mimic the
action of Bok and other pro-apoptotic proteins. Areas of
investigation include the development of cancer treatments, agents
that modulate Bok expression, etc.
[0059] Drug screening identifies agents that provide a replacement
for Bok function in abnormal cells. Conversely, agents that reverse
Bok function may stimulate controlled growth and healing. Of
particular interest are screening assays for agents that have a low
toxicity for human cells. A wide variety of assays may be used for
this purpose, including labeled in vitro protein-protein binding
assays, electrophoretic mobility shift assays, immunoassays for
protein binding, and the like. The purified protein may also be
used for determination of three-dimensional crystal structure,
which can be used for modeling intermolecular interactions.
[0060] The term "agent" as used herein describes any molecule, e.g.
protein or pharmaceutical, with the capability of altering or
mimicking the physiological function of Bok. Generally a plurality
of assay mixtures are run in parallel with different agent
concentrations to obtain a differential response to the various
concentrations. Typically, one of these concentrations serves as a
negative control, i.e. at zero concentration or below the level of
detection.
[0061] Candidate agents encompass numerous chemical classes, though
typically they are organic molecules, preferably small organic
compounds having a molecular weight of more than 50 and less than
about 2,500 daltons. Candidate agents comprise functional groups
necessary for structural interaction with proteins, particularly
hydrogen bonding, and typically include at least an amine,
carbonyl, hydroxyl or carboxyl group, preferably at least two of
the functional chemical groups. The candidate agents often comprise
cyclical carbon or heterocyclic structures and/or aromatic or
polyaromatic structures substituted with one or more of the above
functional groups. Candidate agents are also found among
biomolecules including peptides, saccharides, fatty acids,
steroids, purines, pyrimidines, derivatives, structural analogs or
combinations thereof.
[0062] Candidate agents are obtained from a wide variety of sources
including libraries of synthetic or natural compounds. For example,
numerous means are available for random and directed synthesis of a
wide variety of organic compounds and biomolecules, including
expression of randomized oligonucleotides and oligopeptides.
Alternatively, libraries of natural compounds in the form of
bacterial, fungal, plant and animal extracts are available or
readily produced. Additionally, natural or synthetically produced
libraries and compounds are readily modified through conventional
chemical, physical and biochemical means, and may be used to
produce combinatorial libraries. Known pharmacological agents may
be subjected to directed or random chemical modifications, such as
acylation, alkylation, esterification, amidification, etc. to
produce structural analogs.
[0063] Where the screening assay is a binding assay, one or more of
the molecules may be joined to a label, where the label can
directly or indirectly provide a detectable signal. Various labels
include radioisotopes, fluorescers, chemiluminescers, enzymes,
specific binding molecules, particles, e.g. magnetic particles, and
the like. Specific binding molecules include pairs, such as biotin
and streptavidin, digoxin and antidigoxin etc. For the specific
binding members, the complementary member would normally be labeled
with a molecule that provides for detection, in accordance with
known procedures.
[0064] A variety of other reagents may be included in the screening
assay. These include reagents like salts, neutral proteins, e.g.
albumin, detergents, etc that are used to facilitate optimal
protein-protein binding and/or reduce non-specific or background
interactions. Reagents that improve the efficiency of the assay,
such as protease inhibitors, nuclease inhibitors, anti-microbial
agents, etc. may be used. The mixture of components are added in
any order that provides for the requisite binding. Incubations are
performed at any suitable temperature, typically between 4 and
40.degree. C. Incubation periods are selected for optimum activity,
but may also be optimized to facilitate rapid high-throughput
screening. Typically between 0.1 and 1 hours will be
sufficient.
[0065] Other assays of interest detect agents that mimic Bok
function. For example, an expression construct comprising a Bok
gene may be introduced into a cell line under conditions that allow
expression. The level of Bok activity is determined by a functional
assay. In one screening assay, candidate agents are added, and the
ability to down-regulate its activity is detected. In another
assay, the ability of candidate agents to enhance Bok function is
determined. Alternatively, candidate agents are added to a cell
that lacks functional Bok, and screened for the ability to
reproduce Bok in a functional assay.
[0066] The compounds having the desired pharmacological activity
may be administered in a physiologically acceptable carrier to a
host for treatment of proliferative diseases, etc. The compounds
may also be used to enhance Bok function. The inhibitory agents may
be administered in a variety of ways, orally, topically,
parenterally e.g. subcutaneously, intraperitoneally, by viral
infection, intravascularly, etc. Topical treatments are of
particular interest. Depending upon the manner of introduction, the
compounds may be formulated in a variety of ways. The concentration
of therapeutically active compound in the formulation may vary from
about 0.1-100 wt. %.
[0067] The pharmaceutical compositions can be prepared in various
forms, such as granules, tablets, pills, suppositories, capsules,
suspensions, salves, lotions and the like. Pharmaceutical grade
organic or inorganic carriers and/or diluents suitable for oral and
topical use can be used to make up compositions containing the
therapeutically-active compounds. Diluents known to the art include
aqueous media, vegetable and animal oils and fats. Stabilizing
agents, wetting and emulsifying agents, salts for varying the
osmotic pressure or buffers for securing an adequate pH value, and
skin penetration enhancers can be used as auxiliary agents.
Diagnostic Uses
[0068] The subject nucleic acid and/or polypeptide compositions may
be used to analyze a patient sample for the presence of
polymorphisms associated with a disease state or genetic
predisposition to a disease state. Biochemical studies may be
performed to determine whether a sequence polymorphism in a Bok
coding region or control regions is associated with disease.
Disease associated polymorphisms may include deletion or truncation
of the gene, mutations that alter expression level, etc.
[0069] Changes in the promoter or enhancer sequence that may affect
expression levels of Bok can be compared to expression levels of
the normal allele by various methods known in the art. Methods for
determining promoter or enhancer strength include quantitation of
the expressed natural protein; insertion of the variant control
element into a vector with a reporter gene such as
.beta.-galactosidase, luciferase, chloramphenicol
acetyltransferase, etc. that provides for convenient quantitation;
and the like.
[0070] A number of methods are available for analyzing nucleic
acids for the presence of a specific sequence, e.g. a disease
associated polymorphism. Where large amounts of DNA are available,
genomic DNA is used directly. Alternatively, the region of interest
is cloned into a suitable vector and grown in sufficient quantity
for analysis. Cells that express Bok may be used as a source of
mRNA, which may be assayed directly or reverse transcribed into
cDNA for analysis. The nucleic acid may be amplified by
conventional techniques, such as the polymerase chain reaction
(PCR), to provide sufficient amounts for analysis. The use of the
polymerase chain reaction is described in Saiki, et al. (1985)
Science 239:487, and a review of techniques may be found in
Sambrook, et al. Molecular Cloning: A Laboratory Manual, CSH Press
1989, pp.14.2-14.33. Alternatively, various methods are known in
the art that utilize oligonucleotide ligation as a means of
detecting polymorphisms, for examples see Riley et al. (1990)
N.A.R. 18:2887-2890; and Delahunty et al. (1996) Am. J. Hum. Genet.
58:1239-1246.
[0071] A detectable label may be included in an amplification
reaction. Suitable labels include fluorochromes, e.g. fluorescein
isothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin,
allophycocyanin, 6-carboxyfluorescein (6-FAM),
2',7'-dimethoxy-4',5'-dichloro-6-carboxyflu- orescein (JOE),
6-carboxy-X-rhodamine (ROX), 6-carboxy-2',4',7',4,7-hexach-
lorofluorescein (HEX), 5-carboxyfluorescein (5-FAM) or
N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), radioactive
labels, e.g. .sup.32P, .sup.35S, .sup.3H; etc. The label may be a
two stage system, where the amplified DNA is conjugated to biotin,
haptens, etc. having a high affinity binding partner, e.g. avidin,
specific antibodies, etc., where the binding partner is conjugated
to a detectable label. The label may be conjugated to one or both
of the primers. Alternatively, the pool of nucleotides used in the
amplification is labeled, so as to incorporate the label into the
amplification product.
[0072] The sample nucleic acid, e.g. amplified or cloned fragment,
is analyzed by one of a number of methods known in the art. The
nucleic acid may be sequenced by dideoxy or other methods, and the
sequence of bases compared to a wild-type Bok sequence.
Hybridization with the variant sequence may also be used to
determine its presence, by Southern blots, dot blots, etc. The
hybridization pattern of a control and variant sequence to an array
of oligonucleotide probes immobilised on a solid support, as
described in U.S. Pat. No. 5,445,934, or in WO95/35505, may also be
used as a means of detecting the presence of variant sequences.
Single strand conformational polymorphism (SSCP) analysis,
denaturing gradient gel electrophoresis (DGGE), and heteroduplex
analysis in gel matrices are used to detect conformational changes
created by DNA sequence variation as alterations in electrophoretic
mobility. Alternatively, where a polymorphism creates or destroys a
recognition site for a restriction endonuclease, the sample is
digested with that endonuclease, and the products size fractionated
to determine whether the fragment was digested. Fractionation is
performed by gel or capillary electrophoresis, particularly
acrylamide or agarose gels.
[0073] Screening for mutations in Bok may be based on the
functional or antigenic characteristics of the protein. Protein
truncation assays are useful in detecting deletions that may affect
the biological activity of the protein. Various immunoassays
designed to detect polymorphisms in Bok proteins may be used in
screening. Where many diverse genetic mutations lead to a
particular disease phenotype, functional protein assays have proven
to be effective screening tools. The activity of the encoded Bok
protein may be determined by comparison with the wild-type
protein.
[0074] Antibodies specific for a Bok may be used in staining or in
immunoassays. Samples, as used herein, include biological fluids
such as semen, blood, cerebrospinal fluid, tears, saliva, lymph,
dialysis fluid and the like; organ or tissue culture derived
fluids; and fluids extracted from physiological tissues. Also
included in the term are derivatives and fractions of such fluids.
The cells may be dissociated, in the case of solid tissues, or
tissue sections may be analyzed. Alternatively a lysate of the
cells may be prepared.
[0075] Diagnosis may be performed by a number of methods to
determine the absence or presence or altered amounts of normal or
abnormal Bok in patient cells. For example, detection may utilize
staining of cells or histological sections, performed in accordance
with conventional methods. Cells are permeabilized to stain
cytoplasmic molecules. The antibodies of interest are added to the
cell sample, and incubated for a period of time sufficient to allow
binding to the epitope, usually at least about 10 minutes. The
antibody may be labeled with radioisotopes, enzymes, fluorescers,
chemiluminescers, or other labels for direct detection.
Alternatively, a second stage antibody or reagent is used to
amplify the signal. Such reagents are well known in the art. For
example, the primary antibody may be conjugated to biotin, with
horseradish peroxidase-conjugated avidin added as a second stage
reagent. Alternatively, the secondary antibody conjugated to a
flourescent compound, e.g. flourescein, rhodamine, Texas red, etc.
Final detection uses a substrate that undergoes a color change in
the presence of the peroxidase. The absence or presence of antibody
binding may be determined by various methods, including flow
cytometry of dissociated cells, microscopy, radiography,
scintillation counting, etc.
[0076] Diagnostic screening may also be performed for polymorphisms
that are genetically linked to a disease predisposition,
particularly through the use of microsatellite markers or single
nucleotide polymorphisms. Frequently the microsatellite
polymorphism itself is not phenotypically expressed, but is linked
to sequences that result in a disease predisposition. However, in
some cases the microsatellite sequence itself may affect gene
expression. Microsatellite linkage analysis may be performed alone,
or in combination with direct detection of polymorphisms, as
described above. The use of microsatellite markers for genotyping
is well documented. For examples, see Mansfield et al. (1994)
Genomics 24:225-233; Ziegle et al. (1992) Genomics 14:1026-1031;
Dib et al., supra.
[0077] It is to be understood that this invention is not limited to
the particular methodology, protocols, formulations and reagents
described, as such may, of course, vary. It is also to be
understood that the terminology used herein is for the purpose of
describing particular embodiments only, and is not intended to
limit the scope of the present invention which will be limited only
by the appended claims.
[0078] It must be noted that as used herein and in the appended
claims, the singular forms "a", "and", and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a complex" includes a plurality of such
complexes and reference to "the formulation" includes reference to
one or more formulations and equivalents thereof known to those
skilled in the art, and so forth.
[0079] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood to one of
ordinary skill in the art to which this invention belongs. Although
any methods, devices and materials similar or equivalent to those
described herein can be used in the practice or testing of the
invention, the preferred methods, devices and materials are now
described.
[0080] All publications mentioned herein are incorporated herein by
reference for the purpose of describing and disclosing, for
example, the methods, ligands, and methodologies that are described
in the publications which might be used in connection with the
presently described invention. The publications discussed above and
throughout the text are provided solely for their disclosure prior
to the filing date of the present application. Nothing herein is to
be construed as an admission that the inventors are not entitled to
antedate such disclosure by virtue of prior invention.
[0081] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the subject invention, and are
not intended to limit the scope of what is regarded as the
invention. Efforts have been made to ensure accuracy with respect
to the numbers used (e.g. amounts, temperature, concentrations,
etc.) but some experimental errors and deviations should be allowed
for. Unless otherwise indicated, parts are parts by weight,
molecular weight is average molecular weight, and pressure is at or
near atmospheric.
Experimental
EXAMPLE 1
Isolation and Characterization of Bok cDNA
[0082] Materials and Methods
[0083] Two-hybrid screening. The full-length open reading frame
(ORF) of rat Mcl-1 cDNA was fused in frame with the GAL4-binding
domain (GAL4-BD) into the pGBT-9 yeast shuttle vector (Clontech,
Palo Alto, Calif.). This vector was used to identify Mcl-1
interacting proteins by screening 1.5 million transformants from a
GAL4-activation domain (AD)-tagged ovarian fusion Matchmaker cDNA
library. The ovarian cDNAs were prepared from 27-day-old
Sprague-Dawley rats primed for 36 h with 10 IU equine gonadotropin.
Yeast cells were first transformed with pGBT9-Mcl-1 and colonies
selected in plates deficient for tryptophan. In the second step,
cells were transformed with cDNAs from the ovarian library before
selection of clones in plates lacking tryptophan, leucine and
histidine. Positive transformants were further selected for growth
in media containing 5 mM 3-aminotriazole. Individual AD-fusion
cDNAs were retrieved following transformation of E. coli cells.
[0084] A total of 40 potential Mcl-1-interacting clones were
re-screened against the empty vector or vector encoding different
Bcl-2 proteins to eliminate false positives. Three clones of Bok
cDNAs were isolated based on their ability to interact with Mcl-1
in an HF7c yeast reporter strain (Fields & Song (1989) Nature
340, 245-247). DNA sequence analysis and comparison with known
genes using the BLASTX algorithm revealed that the positive clones
encode a polypeptide sharing high homology with Bcl-2 proteins.
Further analysis of expressed sequence tags (EST) in the GenBank
revealed that EST accession number AA103989 has greater than 98%
identity with the 5'-sequence of cloned cDNA and contains extra
5'-sequence of the murine Bok homolog. Full-length ORF and
5'-untranslated sequence of rat Bok were obtained by PCR using the
GALA-AD-tagged ovarian Matchmaker cDNA library as the template and
an upstream primer based on murine EST. Complementary DNA fragments
with an identical ORF were also obtained in separate PCR using a
rat brain cDNA library (Stratagene, La Jolla, Calif.) as the
template. Interactions between Bok and different Bcl-2 members were
assessed in the yeast two-hybrid system using pGBT9 GAL4-BD and
pGADGH GALA-AD vectors (Bartel et al. (1993) in Cellular
Interaction in Development: A Practical Approach, ed. Hartley, D.
A. (Oxford University Press, Oxford), pp. 153-179). Specific
binding of different protein pairs was evaluated based on the
activation of GAL1-HIS3 and GAL4-lacZ reporter genes.
[0085] Cell culture and transfection with plasmids. For the
expression of Bcl-2 proteins in eukaryotic cells, PCR-generated ORF
of different cDNAs were subcloned into the pcDNA3 vector
(Invitrogen, Inc., San Diego, Calif.). Following transfection of
cDNAs, cell death was monitored (Kumar et al. (1994) Genes Dev. 8,
1613-1626). CHO cells (2.times.10.sup.5/well) were cultured in
Dulbecco's modified Eagle's medium (DMEM)/F12 supplemented with 10%
fetal bovine serum, 100 U/ml penicillin, 100 .mu.g/ml streptomycin
and 2 mM glutamine. One day later, cells were transfected using the
lipofectamine procedure (Life Technologies, Gaithersburg, Md.) with
the empty pcDNA3 expression vector or the same vector containing
different cDNAs, together with {fraction (1/10)}-{fraction (1/20)},
fractions of an indicator plasmid pCMV-.beta.-gal to allow the
identification of transfected cells. Inclusion of 10- to 20-fold
excess of expression vectors as compared to the pCMV-.beta.-gal
reporter plasmid ensured that most of the .beta.-galactosidase
expressing cells also expressed the protein(s) under investigation.
Cells were incubated with plasmids in a serum-free medium for 4 h,
followed by adding fetal bovine serum to a final concentration of
5% and further incubation for 14 h. After an additional culture in
fresh medium for 18 h, cells were fixed by 0.25% glutaraldehyde and
stained with X-gal (0.4 mg/ml) to detect .beta.-galactosidase
expression. The number of blue cells was counted by microscopic
examination (Kumar et al. (1994) Genes Dev. 8, 1613-1626). To
verify the nature of cell death, total cellular DNA was extracted
for 3'-end labeling of DNA ends at 18 h after transfection with
.sup.32P-ddATP before gel fractionation to identify
internucleosomal DNA fragmentation (Hsu et al. (1996) Endocrinology
137, 4837-4843). Statistical differences among treatment groups
were analyzed using one-way ANOVA and Scheffe F-test.
[0086] Northern and Southern blots and in situ analyses For
Northern blot analysis of Bok expression, tissues were collected
from 27-day-old Sprague-Dawley rats (Simonsen Lab, Gilroy, Calif.).
For Bok expression in ovarian cells, ovaries were obtained from
26-day-old rats implanted for two days with a diethylstilbestrol
capsule to stimulate development of multiple early antral follicles
(Bicsak et al. (1986) Endocrinology 119, 2711-2719). Granulosa
cells were prepared by needle puncture. For the extraction of total
RNA, tissues were homogenized in Tri-Reagent solution (Molecular
Research Center, Inc., Cincinnati, Ohio) and at least two pools
from each treatment group were used. In addition, poly (A+) RNA was
isolated using the Oligotex oligo-dT resin (Qiagen, Inc.,
Chatsworth, Calif.). Aliquots of each sample were denatured and
fractionated in 1% agarose gels containing formaldehyde before
northern blotting analysis. Membranes were pre-hybridized for 4 h
at 65.degree. C. in a solution containing 50% formamide, 5.times.
sodium phosphate buffer (SSPE), 5.times. Denhardt's solution, 0.5%
SDS and 500 .mu.g/ml yeast tRNA. This was followed by overnight
hybridization in the same conditions but with 1.times.10.sup.6
cpm/ml of .sup.32P-labeled Bok or GAPDH cRNA probe. After
hybridization, the membranes were washed twice in 2.times.SSC, 1%
SDS at room temperature, followed by two washes in 0.1.times.SSC,
1% SDS at 65.degree. C. before exposure to Kodak RX films (Eastman
Kodak, Rochester, N.Y.). For studies on the conservation of the Bok
gene during evolution, the Zoo blot (Clontech) containing genomic
DNA from different vertebrate species was hybridized with a
.sup.32P-labeled rat Bok cDNA probe under stringent conditions.
[0087] For in situ hybridization analysis of Bok mRNA expression,
ovaries from 26-day-old rats were isolated and fixed at 4.degree.
C. for 4 h in 4% paraformaldehyde in PBS (pH 7.4), followed by
overnight dehydration in 0.5M sucrose. Tissue blocks were embedded
in Tissue-Tek solution (Sakura Finetek USA Inc., Torrence, Calif.)
and snapped frozen in liquid nitrogen. Twelve .mu.m thick
cryo-sections were mounted on charged microscopic slides (Fischer
Scientific, Pittsburgh, Pa.), post-fixed in 4% paraformaldehyde and
stored at -70C for up to 1 month. Hybridization and washes of
cryosections were as previously described (Hsu et al, supra.).
After two weeks of exposure under NTB2 emulsion (Kodak), the slides
were developed, counter-stained and mounted with Permount (Fisher
Scientific, Fair Lawn, N.J.) for observation and photography using
a Nikon Optiphot microscope.
[0088] Results
[0089] Using the anti-apoptotic protein Mcl-1 as bait, we screened
an ovarian fusion cDNA library and isolated three Bok clones.
Subsequent DNA sequencing and identification of homologous murine
ESTs allowed isolation of full-length Bok cDNAs following PCR of
ovarian and brain cDNA libraries. The ORF of Bok encoded a protein
of 213 amino acids showing no identity with any known gene. The
novel protein has a predicted molecular weight of 23.5 kD and a pI
of 9.1. The methionine initiation codon conformed to the consensus
Kozak sequence and hydrophobicity analysis predicted the presence
of a C-terminal transmembrane domain. In addition, two potential
phosphorylation sites were found near the N-terminal region.
Comparison of DNA sequences among different Bcl-2 proteins
indicated that Bok was a novel member of this family showing
conserved BH 1, 2 and 3 domains. However, the BH4 domain, known to
be important for the anti-apoptotic function of mammalian Bcl-2
proteins, was missing in Bok. Closer comparison indicated the core
BH1 domain of Bok (TWGK) was less conserved as compared with other
Bcl-2 proteins (NWGR). For the BH3 domain found in pro-apoptotic
members, the core sequence (GDE) was conserved in Bok but the
flanking sequences were different. Furthermore, analysis of the
phylogenetic relatedness of the different Bcl-2 members suggests
that, during evolution, Bok diverged early from other Bcl-2
proteins.
[0090] We investigated interactions between Bok and different anti-
and pro-apoptotic Bcl-2 proteins. Bok only interacts with selective
anti-apoptotic Bcl-2 proteins in the yeast two-hybrid system. Yeast
cells were grown in the selective media containing 5 mM
3-aminotriazole and without Trp, Leu and His. Prominent growth of
yeast colonies expressing Bok fused to the GAL4 activation domain
together with Mcl-1, BHRF1 or Bfl-1 fused to the GAL4 binding
domain could be seen. Minimal growth of yeast colonies was found in
cells that express the same Bok expressing vector together with
Bcl-2, Bcl-xL, Bcl-w, BAD, Bax, Bak or Bik fused to the GAL4
binding domain. In addition, prominent growth of colonies
expressing Bcl-xL and different pro-apoptotic Bcl-2 proteins
indicated that the lack of growth in yeast cells expressing Bok and
different pro-apoptotic family members was not due to suppression
of cell growth by these pro-apoptotic proteins.
[0091] Of interest, Bok did not interact with any pro-apoptotic
members tested. To demonstrate that the lack of interactions
between Bok and pro-apoptotic Bcl-2 proteins was not due to the
killing of yeast cells by these apoptosis agonists, we also tested
the growth of yeast cells co-transformed with Bcl-xL and different
pro-apoptotic proteins. Although Bcl-xL showed negligible
interaction with Bok, it interacted strongly with all the
pro-apoptotic members tested.
[0092] To further study the restricted dimerization property of Bok
with selective anti-apoptotic proteins, we tested the growth of
yeast cells that were co-transformed with different pairs of pro-
and anti-apoptotic Bcl-2 proteins. Several pro-apoptotic proteins
(Bak, Bik and Bax), unlike Bok, all interacted strongly with
diverse anti-apoptotic proteins tested, data shown in Table 1,
suggesting the restricted hetero-dimerization property of Bok was
unique.
1 TABLE 1 pGBT-9 BHRF1 Mcl-1 Bfl-1 Bcl-2 Bcl-xL Bcl-w Bok -- ++ ++
+ -- -- -- Bax -- ++ ++ ++ ++ ++ ++ Bak -- ++ ++ ++ ++ ++ ++ Bik --
++ ++ ++ ++ ++ ++ Summary of protein-protein interactions between
pairs of pro-apoptotic proteins # (Bok, Bak, Bik and Bax) and
different anti-apoptotic Bcl-2 members. The positive # signs
indicate prominent (++) or moderate (+) yeast cell growth whereas #
the negative signs (-) indicate the absence of reporter gene
expression.
[0093] The ability of Bok to regulate apoptosis in mammalian cells
was investigated. In CHO cells, transfection with expression
vectors encoding Bok for 36 h induced cell death. The pro-apoptotic
effect of Bok was specific because transfection of the empty
plasmid or the same plasmid containing Bok cDNA in reverse
orientation did not affect cell survival. Furthermore,
co-expression of P35, a cysteine protease inhibitor derived from
the baculovirus (Bump et al. (1995) Science 269, 1885-1888),
prevented Bok-induced cell killing as indicated by increases in the
number of viable cells.
[0094] Normal cell morphology was found in cells transiently
transfected with the empty pcDNA3 expression vector (2.1 .mu.g
DNA/35 mm dish) or the vector containing Bok cDNA in reverse
orientation. Cells were also transfected with the Bok expression
vector without or with an equal amount of the P35-expressing
construct.
[0095] The 3'-end labeling of genomic DNA fragments at an earlier
time point (18 h) further demonstrated the induction of
internucleosomal DNA fragmentation following Bok over-expression,
confirming the induction of apoptosis. The observed DNA
fragmentation was blocked by co-expression with P35. CHO cells were
treated as described in above. At 18 h after transfection, cellular
DNA was extracted for analysis of DNA fragmentation using a 3'-end
labeling method.
[0096] Quantitative analysis also indicated that Bok
over-expression decreased viable cell number by 75% whereas
co-expression of P35 completely reversed Bok killing (FIG. 1),
substantiating the involvement of caspases in Bok action.
[0097] We further tested if the restricted hetero-dimerization of
Bok with selective anti-apoptotic Bcl-2 members found in the yeast
two-hybrid system could be substantiated in mammalian cells. Bok
was co-expressed with Mcl-1, BHRF1 or Bcl-2 in CHO cells. As shown
in FIG. 2, Bok-induced apoptosis was attenuated following
co-expression with Mcl-1 or BHRF1; but co-expression with Bcl-2 was
ineffective in blocking Bok action. Transfection of the same Bcl-2
expression vector was, however, capable of blocking apoptosis
induced by staurosporin.
[0098] The expression of Bok mRNA in diverse rat tissues was
examined. High levels of Bok transcript of .about.1.5 kb in size
were abundant in the ovary, testis and uterus, less abundant in the
brain, heart and intestine and negligible in other tissues
examined. Further analysis of Bok mRNA in isolated granulosa cells
demonstrate high levels of expression in these cells that undergo
apoptosis during follicle degeneration. In situ hybridization
analyses further confirmed high levels of the Bok transcript in the
granulosa cells of antral and preantral follicles, with minimal
signals in theca and interstitial cells
[0099] For northern blot analysis, poly (A)+-selected RNA from
different tissues of rats at 27 days of age or from isolated
granulosa cells of estrogen-treated rats were hybridized with a
.sup.32P-labeled Bok cRNA probe. After washing, the blots were
exposed to X-ray films at -70C for five days. Subsequent
hybridization with a GAPDH cRNA probe was performed to estimate
nucleic acid loading (8 h exposure). For in situ hybridization
analysis, ovaries from immature eCG-treated rats were probed with
the anti-sense Bok cRNA. Positive signals were found in granulosa
cells of preantral follicles and an antral follicle. No signal was
found in a section hybridized with the sense Bok probe.
[0100] Conservation of the Bok gene in diverse vertebrates was
tested using Southern blot hybridization of genomic DNA from
different species. DNA was digested with the EcoRI enzyme and
probed with a Bok cDNA probe. Following hybridization at 68C, the
membrane was washed under high stringency conditions (1% SDS,
0.1.times.SSC at 65C) before exposure. Under high stringency
washing conditions, the rat cDNA cross-hybridized strongly with
rat, human and monkey genomic DNA and weakly with dog, cow and
rabbit DNA. Negligible hybridization signals were found for chicken
DNA.
[0101] A cDNA encoding the human bok gene was isolated from a human
ovary cDNA library. A partial sequence of the human protein is
provided in SEQ ID NO:3.
[0102] Discussion
[0103] A new pro-apoptotic Bcl-2-related protein Bok has been
identified based on its binding to an ovarian anti-apoptosis
protein Mcl-1. In addition to its elevated expression in several
reproductive tissues, Bok was found in diverse other tissues. In
addition, Bok shows a selective hetero-dimerization property by
interacting with some (Mcl-1, BHRF1 and Bfl-1) but not other
(Bcl-2, Bcl-xL and Bcl-w) anti-apoptotic proteins. Coupled with
findings that Bok-induced apoptosis could only be antagonized by
selective anti-apoptotic proteins, the present data suggest that
different pro- and anti-apoptotic Bcl-2 protein pairs may play
tissue-specific roles in the regulation of apoptosis. Due to the
higher expression of Bok to ovarian granulosa cells and several
reproductive tissues characterized by hormonally regulated cyclic
cell turnover, further analyses of Bok action in the gonads and
uterus could provide unique models to study the hormonal regulation
of apoptosis. Because most of the Bcl-2-related proteins have been
identified in the lymphoid system, the present yeast two-hybrid
screen provides an experimental paradigm to isolate novel Bcl-2
homologs essential for apoptosis regulation in other tissues.
[0104] Although the mechanism by which the Bcl-2 proteins
participates in the "decision" step of apoptosis is not clear, the
ratio of anti- and pro-apoptotic Bcl-2 members and their hetero-
and homo-dimerization are believed to determine whether a cell will
respond to an apoptotic signal. Among the Bcl-2 family of proteins,
several homology domains have been found to be essential for their
function. Bok contains conserved BH1, 2 and 3 domains but lacks the
BH4 domain found in most anti-apoptotic members. In addition, the
conserved NH1 region important for the survival function of several
anti-apoptotic Bcl-2 proteins is also absent in Bok. Consistent
with its structural features, over-expression of Bok in CHO cells
induces apoptosis based on observed cell morphology and
internucleosomal DNA fragmentation. Bok-induced cell killing, like
that induced by Bax and BAD, is mediated by caspases as
demonstrated by the suppressive actions of the baculoviral P35
protein.
[0105] Among the pro-apoptotic Bcl-2 proteins, Bok is most similar
to Bax and Bak in having the BH1, 2 and 3 domains plus the
C-terminal transmembrane sequence. Studies on Bax and Bak with
truncation in different BH domains suggested that these
pro-apoptotic proteins might exert their effects by
hetero-dimerizing with Bcl-2 or Bcl-xL. Competitive dimerization
between selective pairs of anti- and pro-apoptotic Bcl-2 proteins
is believed to be involved in the "decision" step of apoptosis.
Furthermore, interactions among the Bcl-2 family of proteins appear
to exhibit a defined selectivity and hierarchy. For example, the
anti-apoptotic E1B protein shows preferential binding to
pro-apoptotic Bcl-2 proteins, whereas the pro-apoptotic Hrk binds
only to the anti-apoptotic family member. Thus, the pro-apoptotic
protein Bok may regulate apoptosis through similar mechanisms by
forming hetero-dimers with selective anti-apoptotic proteins.
[0106] Analysis of the relatedness of amino acid sequences of
different Bcl-2 proteins indicated that Bok is not closely related
to any particular Bcl-2 member and probably diverged early during
evolution. The less conserved BH1 domain of Bok may determine its
unique hetero-dimerization property. In both yeast and mammalian
cells, Bok interacts with some but not other anti-apoptotic
proteins, suggesting the possible evolution of selective pairs of
death agonists and antagonists with restricted hetero-dimerization
properties to confer tissue specificity of the death program.
Apoptosis induced by Bok in transfected CHO cells could be mediated
through inhibition of the protection afforded by Mcl-1 or other Bok
partners. It is likely that Bok may interact with its dimerization
partner(s) including Mcl-1 and Bfl-1 in reproductive tissues to
regulate apoptosis. Of interest, our recent data indicated that
Mcl-1, but not Bcl-2, is highly expressed in ovarian cells. Based
on the suppression of Bok-induced apoptosis by BHRF1, it is
possible that reproductive tissues expressing Bok are potential
targets for this anti-apoptotic protein encoded by the Epstein-Barr
virus. Recent studies have suggested that anti-apoptotic proteins
may bind to ced-4/Apaf-1 homologs, which, in turn, activate
downstream caspases. Elucidation of the hetero-dimerization
partner(s) for Bok in gonads and uterus would allow
characterization of the putative ced-4 homologs in these
tissues.
[0107] Recent crystallographic analyses of complexes formed between
the anti-apoptotic protein Bcl-xL and the BH3 domain of the
pro-apoptotic Bak indicated that the .alpha.-helix in the BH3
domain of different Bcl-2 proteins plays a central role in defining
the binding specificity to Bcl-xL. Because Bok does not interact
with Bcl-xL in the yeast two-hybrid system, further studies on the
BH3 region of Bok and related proteins could define the specificity
of hetero-dimerization among different pro- and anti-apoptotic
protein pairs and their role in apoptosis regulation.
[0108] Although over-expression of Bax and Bak induces yeast cell
death, the present Bok fusion protein did not affect yeast cell
survival. In addition, lack of interactions between Bok and
different pro-apoptotic Bcl-2 proteins in the two-hybrid assay are
not due to detrimental effects of these death agonists on yeast
cells because co-transformation of these apoptosis agonists with
Bcl-xL led to activation of the reporter genes. It is likely that
moderate expression of these death agonists using the present
expression vector may not significantly affect yeast cell survival,
thus allowing studies on interactions between different Bcl-2
proteins.
[0109] The majority of ovarian follicles and about 50% of
testicular germ cells undergo apoptosis under normal physiological
conditions, whereas the menstruation involves monthly apoptosis of
uterine endometrial cells. The restricted expression of Bok in the
gonads and uterus suggests its potential role in the regulation of
apoptosis in these tissues. It is likely that selective pairs of
Bcl-2 agonists/antagonists may play tissue-specific roles in the
regulation of apoptosis. Indeed, mutant mice deficient in Bcl-2 or
Bax showed abnormality in apoptosis regulation only in distinct
cell lineage. Although Bax-deficient mice showed an accumulation of
granulosa cells in atretic follicles, these cells were still
apoptotic, suggesting the involvement of additional pro-apoptotic
factors during ovarian follicle atresia. Because the pro-apoptotic
protein Bax has been suggested to function as a tumor suppressor
gene in colon adenocarcinomas and because inactivation of Bax in
transgenic mice leads to enhanced tumorigenesis, it would also be
interesting to investigate changes in Bok function during gonadal
and uterine tumorigenesis. Because cyclic variations in
reproductive hormones are essential in the regulation of apoptosis
in gonadal and uterine tissues, future investigations on the
hormonal regulation of the Bok and its dimerization partner(s) in
these reproductive tissues would allow the design of novel
strategies to modulate reproductive functions. These studies could
also provide understanding on the role of Bok in gonadal and
uterine diseases associated with aberrant regulation of
apoptosis.
EXAMPLE 2
Characterization of a Bok Splicing Variant with a Truncated BH3
Domain, which Induces Apoptosis but does not Dimerize with
Anti-Apoptotic Bcl-2 Proteins in vitro
[0110] A Bok splicing variant is identified in which the region
encoded by exon three is absent, creating a truncated short form
(Bok-S) of the full-length Bok protein (Bok-L). The skipping of
exon three maintains the original reading frame and retains the BH2
and the C-terminal membrane anchoring domains; however, parts of
the BH3 and BH1 domains were deleted. Functional analysis indicated
that Bok-S is still capable of inducing apoptosis. The truncated
Bok has lost its ability to heterodimerize with Mcl-1, BHRF-1 and
Bfl-1, suggesting that the proapoptotic activity of this variant is
not mediated by its binding to antiapoptotic Bcl-2 proteins.
[0111] Materials and Methods
[0112] Reverse transcription-PCR of the Bok-S transcript. Total RNA
from different tissues was isolated from 27-day-old Sprague-Dawley
rats using an anion exchange resin chromatographic column (Qiagen,
Chatsworth, Calif.) before reverse transcription with oligo (dT) 18
as primer in a reaction containing RNase H-free reverse
transcriptase from Moloney murine leukemia virus (Clontech, Palo
Alto, Calif.). For PCR amplification of Bok cDNAs, aliquots of DNA
equivalent to 0.5 .mu.g total RNA were used in each reaction (50
.mu.l). To minimize contamination during PCR, control reactions
containing a single primer or RNA without reverse transcriptase
were routinely performed. All PCR was carried out under high
stringency conditions (94.degree. C., 45 s, 68.degree. C. 45 s,
72.degree. C. 4 min) for 30 cycles.
[0113] Isolation of Bok genomic DNA, Southern blot hybridization
and genomic analysis. A genomic DNA fragment was isolated from a
mouse BAC genomic DNA library (Genome Systems Inc., St. Louis, Mo.)
using the full-length Bok cDNA probe. The Bok genomic fragment was
first analyzed by restriction enzyme mapping, followed by
subcloning into the pUC18 vector for dideoxy sequencing analysis of
both DNA strands. Overlapping clones were isolated to define the
direction of individual clones and to facilitate assignment of
intron-exon junctions. For Southern blot hybridization analysis,
genomic DNA (10 .mu.g) was digested with indicated restriction
enzymes, separated by electrophoresis on a 0.8% Agarose gel, and
then transferred onto a Nylon membrane (Hybond-N, Amersham Corp.,
Arlington Heights, Ill.). Hybridization was performed in the
QuickHyb buffer (Clontech) at 60 C with .sup.32P-labeled cDNA
probes. The filters were washed with 0.1.times.SSC and 0.5% SDS at
65 C before exposure.
[0114] Generation of mutant constructs. Specific mutations in the
BH3 domain of Bok-L were generated by a two-step PCR mutagenesis
method using a Bok cDNA template as previously described (Hsu et
al. (1997) Mol Endocrinol 11: 1858-1867). The resulting PCR
products were evaluated for correct size on a 1% Agarose gel,
purified, and subcloned into the EcoR1 site of the pcDNA3 vector
for mammalian cell expression (Invitrogen, Inc., San Diego,
Calif.). Truncated Bax and Bak constructs (Bax-S and Bak-S) with
homologous deletion of the BH3-BH1 region found in Bok-S were also
generated using two-step overlapping PCR, and subcloned into the
EcoRV site of the pcDNA3 vector for eukaryotic cell expression (Hsu
et al. supra.) For the yeast two-hybrid assay, mutant Bok-L cDNAs
were subcloned into the pGADGH expression vector. Restriction
mapping and dideoxy sequencing confirmed proper orientation and the
authenticity of the inserts.
[0115] Yeast two-hybrid assay. To study dimerization between
different Bcl-2 family proteins and variants or mutants of Bok,
cDNAs for Bok-L, Bok-S or Bok mutants were fused to the activation
domain (AD) of GAL4 in a yeast shuttle vector pGADGH. Complementary
DNAs encoding different Bcl-2 proteins were fused to the
GAL4-binding domain (BD) of pGBT9. After transformation of yeast
cells, colonies containing different protein pairs were selected on
plates lacking tryptophan and leucine. To test for specific
protein-protein interactions, positive transformants were further
selected for growth in media deficient for tryptophan, leucine and
histidine but containing 5-30 mM 3-aminotriazole to inhibit
endogenous histidine production. A minimum of five independent
transformants containing each pair of fusion cDNAs was routinely
analyzed.
[0116] Analysis of apoptosis in transfected CHO cells. Apoptosis
was monitored following transfection of different cDNAs as
previously described (Hsu et al. (1997) Proc. Natl. Acad. Sci. USA
94:12401-12406). CHO cells (2.times.10.sup.5 cells/well) were
cultured in Dulbecco's modified Eagle's medium (DMEM)/F12
supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100
g/ml streptomycin and 2 mM glutamine. One day later, cells were
transfected using the lipofectamine procedure (Life Technologies,
Gaithersburg, Md.) with the pcDNA3 expression vector with or
without different cDNA inserts, together with {fraction (1/10)} to
{fraction (1/20)} amounts of an indicator plasmid pCMV-.beta.-gal
to allow the identification of transfected cells. Inclusion of a
10-fold excess of expression vectors as compared with the
pCMV-.beta.-gal reporter plasmid ensured that most of the
.beta.-galactosidase-expressing cells also expressed the protein(s)
under investigation. Cells were incubated with plasmids in a
serum-free medium for 4 h, followed by the addition of fetal bovine
serum to a final concentration of 5% and further incubation for 14
h. After an additional culture in fresh medium for 18 h, cells were
fixed by using 0.25% glutaraldehyde and stained with X-gal to
detect .beta.-galactosidase expression. The number of blue cells
was counted by microscopic examination. Data are expressed as the
percentage (mean.+-.SEM) of viable cells as compared to the control
group.
[0117] In vitro direct protein-binding assay. To further
demonstrate the specificity of interactions between Bok variants
and antiapoptotic proteins, direct protein-protein interactions
were studied using recombinant Bok and FLAG-tagged BHRF-1
translated in vitro. .sup.35S-methionine labeled or nonlabeled
proteins were generated using the TNT coupled reticulocyte lysate
system (Promega, Madison, Wis.). Pairs of proteins were incubated
in the binding buffer (PBS, 0.2% NP-40 and protease inhibitor
cocktail; Sigma, St. Louis, Mo.) for 2 h at 4.degree. C. followed
by incubation with 1.5 .mu.g of M2 antibody against the FLAG tag
(Kodak, Rochester, N.Y.) under gentle agitation. The complexes
formed between the antibody and recombinant proteins were
precipitated with Protein A Sepharose (Pharmacia Biotech, Uppsala,
Sweden) and resolved using 12-15% SDS PAGE. Following fixation,
gels were treated with Amplify fluorographic agents (Amersham Life
Science Inc., England) before exposure to x-ray films.
[0118] Statistical analyses and sequence analysis. One-way analysis
of variance followed by Scheffe's F-test was used to determine the
statistical significance of cell viability employing the STATVIEW
software (Abacus Concepts, Inc., Berkeley, Calif.). The
hydropathicity of protein sequence was analyzed using Biology
Workbench version 2.0 (http://biology.ncsa.uiuc.edu/BW/BW.cgi).
[0119] Results
[0120] Existence of long and short Bok splicing variants in
reproductive tissues. We amplified Bok cDNA from a rat ovarian cDNA
library using primers flanking the open reading frame (ORF) of Bok.
A 513 bp PCR product was obtained in addition to the predicted 642
bp band. DNA sequencing of the lower molecular weight product
indicated it was identical to that of the Bok cDNA except that
nucleotides encoding amino acid 76-118 were missing. This short
transcript (Bok-S) encoded a 170 amino acid polypeptide and the
deletion of 43 amino acids from the full-length 213 amino acid
protein (Bok-L) led to the fusion of the N-terminal half of the BH3
domain to the C-terminal half of the BH1 domain. To confirm the
authenticity of this variant, reverse transcription-PCR was
performed using total RNA from different tissues. Electrophoresis
analysis confirmed the presence of a PCR product of 642 bp in the
ovary, uterus and testis, tissues known to express the Bok
transcript. In addition, a lower band of 513 bp was found in the
ovary, less in the uterus and absent from the testis. Negative
control reactions using only a single primer or RNA without reverse
transcription did not generate any products. Subsequent subcloning
and sequencing confirmed that the 642 bp and 513 bp bands encode
the expected Bok-L and Bok-S transcripts, respectively.
[0121] The deduced amino acid sequence of Bok-S showed that a
presumptive alternative splicing led to the disruption of both BH1
and BH3 domains of Bok-L, changing the original BH3 sequence [SEQ
ID NO:9] 71 LLRLGDELEQIR 82 to [SEQ ID NO:10] 71 LLRLGITWGKVV 82.
However, Kyte-Doolittle hydropathicity analysis suggested that the
hydropathicity profile of the BH3/BH1 fusion region found in Bok-S
did not differ substantially from that of the original BH1 domain
in Bok-L. Furthermore, the 5 and 6 regions predicted, based on
their homology to similar regions in Bax and Bak, were unaltered in
the truncated Bok-S. These regions have been postulated to be
important for channel formation in the mitochondria by different
Bcl-2 proteins.
[0122] Bok gene arrangement and the derivation of alternative
splicing variants. To elucidate the mechanism by which two Bok
isoforms were generated, the Bok gene and its exon/intron junctions
were analyzed. Following the screening of a bacterial artificial
chromosome-based mouse genomic DNA library using a mouse Bok cDNA
fragment, one genomic clone for Bok was isolated. The amino acid
sequence of the coding region for the mouse clone was found to be
identical to its rat counterpart. Southern blot hybridization of
mouse genomic DNA digested with different restriction enzymes using
cDNA probes corresponding to two different regions of the Bok gene
demonstrated the presence of a single Bok gene in the mouse.
Further characterization of the genomic clone indicated that the
entire Bok gene spanned 11 kb and consisted of 5 exons. The Bok ORF
was encoded by sequences in exons II to V whereas exon I contained
only untranslated sequences. Comparison of the ORF of Bok-S with
genomic sequences indicated that Bok-S was derived following the
splicing out of exon III.
[0123] Bok-S promotes cell death in transfected cells. To study the
role of Bok-S in apoptosis regulation, expression vectors
containing Bok-S in either sense or antisense orientation were
constructed. Transfection of CHO cells with either Bok-S or Bok-L,
but not the reverse construct, significantly reduced the number of
transfected cells, demonstrating that Bok-S retained its ability to
induce apoptosis despite the loss of the BH3 sequence. In addition,
cell killing induced by either Bok-S or Bok-L was antagonized by
cotransfection with P35, a baculoviral-derived caspase inhibitor.
Bok-S does not heterodimerize with antiapoptotic Bcl-2 proteins.
Because Bok was isolated based on its ability to bind Mcl-1 and the
dimerization between pro- and antiapoptotic Bcl-2 proteins has been
suggested to be important in apoptosis, we analyzed whether Bok-S
that maintained its cell killing ability could still dimerize with
antiapoptotic Bcl-2 proteins. In the two-hybrid assay, interactions
between Bok-S and different Bcl-2 family members were tested. Bok-S
did not interact with any Bcl-2 proteins tested whereas Bok-L
interacted strongly with Mcl-1, Bfl-1 and BHRF-1, as previously
reported. To further confirm findings in yeasts, a direct
protein-protein interaction assay was performed using in vitro
translated recombinant Bok variants and the antiapoptotic protein
BHRF-1 that exhibited strongest interaction with Bok-L in yeast.
BHRF-1 interacted strongly with Bok-L in vitro but showed
negligible interaction with Bok-S. Thus, heterodimerization of
Bok-S with antiapoptotic Bcl-2 proteins is probably not needed for
apoptosis induction.
[0124] BH3 mutants of Bok defective in heterodimerization still
retain proapoptotic activity. Because Bok-S lacking a BH3 domain
still retained its cell killing potential, we hypothesized that the
BH3 domain might be dispensable for the proapoptotic activity of
Bok-L. Bok-L cDNAs with alanine or glycine substitutions in the BH3
domain were constructed and the ability of these mutants to promote
apoptosis was studied. The mutants included alanine substitutions
at the highly conserved glycine 75 or glycine 75 plus flanking
aspartic acid 76 and glutamic acid 77 (BokADE: G 75 A and BokAAA:
75 AAA 77). In addition, a glycine substitution was made for
leucine 71 to leucine 74 (BokGGGG: 71 LLRL 74 to 71 GGGG 74).
Transfection of these Bok-L mutants reduced the number of viable
CHO cells as compared to the group with cells transfected with the
pcDNA3 vector without an insert. In contrast, constructs with
mutant cDNAs in reverse orientation had no effect on cell survival.
These data suggested that the BH3 domain in Bok-L is dispensable
for apoptosis induction. We further tested the ability of these BH3
domain mutants of Bok-L to dimerize with antiapoptotic Bcl-2
proteins in the yeast two-hybrid assay. Substitution of residues in
the BH3 domain of Bok-L abolished its interaction with Mcl-1 or
Bfl-1. In addition, the ability of Bok-L to interact with BHRF-1
was also abated by glycine substitution at residues 71-74 of Bok-L.
Similar to findings using the two-hybrid assay, in vitro translated
BokGGGG mutant also lost its ability to interact with Bok-L
effectively in the direct protein-protein interaction test. These
data suggested that the cell killing ability of these BH3 mutants
is not correlated to their ability to dimerize with antiapoptotic
Bcl-2 proteins.
[0125] Mutants of Bax and Bak with deletion of their BH3 domain
resembling Bok-S also retain proapoptotic activity. Because Bok-L
is similar in structure to two other proapoptotic proteins Bax and
Bak, deletion mutants with truncation of the BH3-BH1 region similar
to that found in Bok-S were constructed for these proteins and
named as Bax-S and Bak-S (FIG. 3). Full-length Bak and Bax, like
Bok-L and Bok-S, effectively reduced cell viability in the CHO cell
transfection assay. Of interest, overexpression of Bax-S or Bak-S
also significantly reduced the viability of transfected cells,
suggesting that the BH3-BH1 regions deleted in these two
proapoptotic proteins are not essential for apoptosis
induction.
[0126] Discussion
[0127] A naturally occurring variant of Bok with proapoptotic
activity but exhibiting negligible dimerization with antiapoptotic
Bcl-2 members is identified. Bok-S with a 43-amino acid deletion
between the BH3 and BH1 domains was likely the result of
alternative mRNA splicing, leading to the skipping of exon three
during post-transcriptional modification. Analysis of Bok variants
and Bok mutants with alterations in the BH3 domain indicated that
the BH3 domain of Bok-L is critical for heterodimerization but
dispensable for apoptosis induction. Likewise, similar deletions
between BH3 and BH1 domains of the homologous proapoptotic proteins
Bax and Bak also retained cell killing ability. Thus, Bok-L could
promote apoptosis independent of heterodimerization and Bok-S
represents a novel proapoptotic Bcl-2 member capable of inducing
cell death without binding to or interference by antiapoptotic
Bcl-2 partners. This functional Bok variant with retention of the
region spanning BH1 and BH2 domains and the TM sequence provides a
unique model for further studies of apoptosis mechanisms regulated
by Bcl-2 family proteins.
[0128] The bifunctional antiapoptotic Bcl-2 proteins play a pivotal
role in the decision step of apoptosis. These proteins, represented
by Bcl-xL, maintain a channel structure important in the control of
mitochondrial membrane potential and volume homeostasis. Regulation
of these channels controls the release of cytochrome C essential
for the activation of Apaf-1 and caspases important for apoptosis
execution. The antiapoptotic Bcl-2 proteins also function as
docking proteins for proapoptotic Bcl-2 members. Because several
mutants of Bcl-2 and Bcl-xL simultaneously lost antiapoptotic
activity and the ability to bind proapoptotic Bcl-2 proteins, it is
believed that dimerization of Bcl-2 protein pairs mediated by the
consensus BH domains is important in apoptosis regulation.
Crystallographic studies and computer modeling showed that the
conserved BH1, BH2 and BH3 domains of Bcl-xL and related proteins
constitute an elongated hydrophobic cleft capable of interaction
with the amphipathic helix formed by BH3 domains of proapoptotic
partners. Upon heterodimerization, anti- and proapoptotic Bcl-2
partners antagonize the actions of the other. It is likely that one
of the mechanisms by which Bok-L exerts its proapoptotic action is
through dimerization with antiapoptotic partners.
[0129] Mammalian proapoptotic Bcl-2 proteins can be divided into
two subgroups based on domain arrangement. Together with Bax and
Bak, Bok-L belongs to the first subgroup showing the conserved BH1,
BH2, BH3 and TM domains. In contrast, members of the second
subgroup (BAD, BID, Hrk/DP5, Bik/Nbk and Bim) possess only the BH3
domain, with or without the TM region. Earlier studies suggested
that proapoptotic proteins function by antagonizing the action of
antiapoptotic proteins mediated by BH3 domains. Mutations in the
BH3 domain of proapoptotic proteins abolished their dimerization
with antiapoptotic partners and cell killing activity. In addition,
polypeptides containing minimal BH3 domain sequences bind
antiapoptotic proteins and induce apoptosis in transfected cells or
cell-free systems. More recent studies, however, demonstrated that
Bax, like Bcl-xL and Bcl-2, also shows intrinsic ion channel
activity in the artificial membrane. In addition, mutations in the
BH1, 2 or 3 domains of Bax do not affect its ability to promote
apoptosis. Likewise, Bak mutants accelerate chemotherapy-induced
apoptosis independent of its heterodimerization property. These
data suggest that the first subgroup of proapoptotic proteins,
including Bax, Bak and Bok, could induce apoptosis through channel
formation in addition to their role as ligands for antiapoptotic
Bcl-2 proteins. Because the second BH3-only subgroup members lack
the region spanning BH1 and BH2 domains important for pore
formation and mainly reside in the cytoplasm, they are believed to
serve as ligands or facilitators of the pore forming Bcl-2
proteins.
[0130] Our findings that substitution of conserved residues in the
BH3 domain of Bok-L abates its ability to dimerize with
antiapoptotic proteins are in accord with studies on the BH3 domain
of its proapoptotic homologues. Similarly, truncation of the
conserved BH3 domain in the naturally-occurring Bok-S variant also
disrupted heterodimerization but retained cell killing ability,
indicating the BH3 domain is dispensable for apoptosis induction.
Thus, Bok-S represents a new form of proapoptotic protein
consisting of only minimal functional modules and manifesting
proapoptotic action without direct interactions with antiapoptotic
proteins. As shown in the above data, truncation of the region
between BH3 and BH1 from Bok-L does not affect the homologous
.alpha.5 and .alpha.6 regions proposed to be important for channel
formation in Bax. In addition, the hydropathicity property between
the 5'-end of the BH1 region and the C-terminal TM domain is not
altered by the truncation found in Bok-S. It is likely that the
BH3/1, BH2 and TM domains found in Bok-S comprise a module
sufficient for mediating apoptosis through a
heterodimerization-independent pathway. Future studies on the
channel-forming property of the naturally-occurring Bok-S and other
channel-forming Bcl-2 proteins are important for understanding the
mechanisms of apoptosis. The channel-forming hypothesis is further
supported by the finding that Bax-S and Bak-S with truncation at
the BH3-BH1 regions homologous to that of Bok-S also retain
proapoptotic activity. Recent studies also indicated that, during
apoptosis, activated caspases cleave the N-terminal BH4 domain of
antiapoptotic proteins Bcl-2 and Bcl-xL to yield truncated
molecules resembling the proapoptotic Bax, Bak or Bok in terms of
the BH domain arrangement. Of interest, deletion of the BH4 domain
from these antiapoptotic proteins confers proapoptotic activity and
mitochondrial release of cytochrome C, presumably mediated through
the C-terminal channel-forming region.
[0131] Like Bok, splicing variants have been reported for Bcl-2,
Bcl-x and Bax genes. The Bcl-xL gene encodes three different
variants, each with a distinct function; the long form of Bcl-x (L)
exhibits antiapoptotic activity whereas Bcl-x-short and Bcl-x-are
proapoptotic. Also, Bcl-2 variants lacking the TM domain show
decreased antiapoptotic activity. The proapoptotic Bax gene also
encodes a number of splicing variants with unknown function.
[0132] At least three mechanisms could be proposed for the action
of proapoptotic Bcl-2 proteins: 1) The subgroup of proapoptotic
proteins with only the BH3 domain (e.g. the soluble BAD protein)
heterodimerizes with membrane-bound antiapoptotic proteins to
regulate apoptosis; 2) The subgroup of membrane-bound proapoptotic
proteins with BH1, BH2, BH3 and TM domains, represented by Bok-L,
heterodimerizes with antiapoptotic proteins (Mcl-1/Bfl-1) or
functions as mitochondrial channels to regulate apoptosis; and 3)
The unique Bok-S variant does not dimerize with antiapoptotic
proteins but probably forms mitochondrial channels to regulate
apoptosis. Because Bok-S does not interact with antiapoptotic
proteins, apoptosis mediated through Bok-S may be important in
situations when unwanted cells need to be eliminated quickly
despite the presence of antiapoptotic proteins in the same cell. In
the ovary and uterus known to express high levels of Bok
transcripts, Bok-S expression could provide a short circuit to
promote cell demise in hormone-dependent cell populations that
express abundant antiapoptotic proteins (such as Mcl-1) but have to
be removed swiftly due to cyclic cell turnover during reproductive
cycles. The search for novel death promoters that interact
specifically with proapoptotic Bcl-2 proteins could also be
simplified based on the lack of interaction between Bok-S and other
Bcl-2 proteins. Further characterization of this unique
proapoptotic protein would allow a better understanding of
intracell ular mechanism of apoptosis, particularly for
hormone-regulated cell death.
Sequence CWU 1
1
18 1 642 DNA r. rattus 1 atggaggtgc tgcggcgctc ttctgtcttc
gctgcggaga tcatggacgc ctttgatcgc 60 tcgcccacag acaaggagct
ggtggcccag gctaaagcac taggccggga gtacgtgcac 120 gcgcggcttt
tgcgcgccgg cctctcctgg agcgctccag agcgtgcctc gcctgcccct 180
ggaggacgcc tggcagaggt gtgcaccgtg ctgctgcgct tgggagatga gctggagcag
240 atccgtccca gcgtatatcg gaatgtggcc cggcagctgc acatccccct
gcagtctgag 300 cctgtggtga ctgatgcctt cctcgctgtg gccggccaca
tcttctcagc aggtatcaca 360 tggggcaagg tagtgtccct gtactcggtg
gctgcgggac tagcggtgga ctgcgtccgg 420 caagctcagc cagccatggt
tcatgccctg gttgactgcc tgggggaatt tgtacgcaag 480 ccctggcca
cctggcttcg gaggcgtggt ggatggacgg acgtcctcaa gtgtgtggtc 540
gcacagacc ctggcttccg ctcccactgg ctcgtggcca cactctgcag ctttggccgc
600 tcctgaagg ctgcattctt cctcctgttg ccagagagat ga 642 2 213 PRT r.
rattus 2 Met Glu Val Leu Arg Arg Ser Ser Val Phe Ala Ala Glu Ile
Met Asp 1 5 10 15 Ala Phe Asp Arg Ser Pro Thr Asp Lys Glu Leu Val
Ala Gln Ala Lys 20 25 30 Ala Leu Gly Arg Glu Tyr Val His Ala Arg
Leu Leu Arg Ala Gly Leu 35 40 45 Ser Trp Ser Ala Pro Glu Arg Ala
Ser Pro Ala Pro Gly Gly Arg Leu 50 55 60 Ala Glu Val Cys Thr Val
Leu Leu Arg Leu Gly Asp Glu Leu Glu Gln 65 70 75 80 Ile Arg Pro Ser
Val Tyr Arg Asn Val Ala Arg Gln Leu His Ile Pro 85 90 95 Leu Gln
Ser Glu Pro Val Val Thr Asp Ala Phe Leu Ala Val Ala Gly 100 105 110
His Ile Phe Ser Ala Gly Ile Thr Trp Gly Lys Val Val Ser Leu Tyr 115
120 125 Ser Val Ala Ala Gly Leu Ala Val Asp Cys Val Arg Gln Ala Gln
Pro 130 135 140 Ala Met Val His Ala Leu Val Asp Cys Leu Gly Glu Phe
Val Arg Lys 145 150 155 160 Thr Leu Ala Thr Trp Leu Arg Arg Arg Gly
Gly Trp Thr Asp Val Leu 165 170 175 Lys Cys Val Val Ser Thr Asp Pro
Gly Phe Arg Ser His Trp Leu Val 180 185 190 Ala Thr Leu Cys Ser Phe
Gly Arg Phe Leu Lys Ala Ala Phe Phe Leu 195 200 205 Leu Leu Pro Glu
Arg 210 3 513 DNA r.rattus CDS (1)...(513) 3 atg gag gtg ctg cgg
cgc tct tct gtc ttc gct gcg gag atc atg gac 48 Met Glu Val Leu Arg
Arg Ser Ser Val Phe Ala Ala Glu Ile Met Asp 1 5 10 15 gcc ttt gat
cgc tcg ccc aca gac aag gag ctg gtg gcc cag gct aaa 96 Ala Phe Asp
Arg Ser Pro Thr Asp Lys Glu Leu Val Ala Gln Ala Lys 20 25 30 gca
cta ggc cgg gag tac gtg cac gcg cgg ctt ttg cgc gcc ggc ctc 144 Ala
Leu Gly Arg Glu Tyr Val His Ala Arg Leu Leu Arg Ala Gly Leu 35 40
45 tcc tgg agc gct cca gag cgt gcc tcg cct gcc cct gga gga cgc ctg
192 Ser Trp Ser Ala Pro Glu Arg Ala Ser Pro Ala Pro Gly Gly Arg Leu
50 55 60 gca gag gtg tgc acc gtg ctg ctg cgc ttg gga atc aca tgg
ggc aag 240 Ala Glu Val Cys Thr Val Leu Leu Arg Leu Gly Ile Thr Trp
Gly Lys 65 70 75 80 gta gtg tcc ctg tac tcg gtg gct gcg gga cta gcg
gtg gac tgc gtc 288 Val Val Ser Leu Tyr Ser Val Ala Ala Gly Leu Ala
Val Asp Cys Val 85 90 95 cgg caa gct cag cca gcc atg gtt cat gcc
ctg gtt gac tgc ctg ggg 336 Arg Gln Ala Gln Pro Ala Met Val His Ala
Leu Val Asp Cys Leu Gly 100 105 110 gaa ttt gta cgc aag acc ctg gcc
acc tgg ctt cgg agg cgt ggt gga 384 Glu Phe Val Arg Lys Thr Leu Ala
Thr Trp Leu Arg Arg Arg Gly Gly 115 120 125 tgg acg gac gtc ctc aag
tgt gtg gtc agc aca gac cct ggc ttc cgc 432 Trp Thr Asp Val Leu Lys
Cys Val Val Ser Thr Asp Pro Gly Phe Arg 130 135 140 tcc cac tgg ctc
gtg gcc aca ctc tgc agc ttt ggc cgc ttc ctg aag 480 Ser His Trp Leu
Val Ala Thr Leu Cys Ser Phe Gly Arg Phe Leu Lys 145 150 155 160 gct
gca ttc ttc ctc ctg ttg cca gag aga tga 513 Ala Ala Phe Phe Leu Leu
Leu Pro Glu Arg * 165 170 4 170 PRT r.rattus 4 Met Glu Val Leu Arg
Arg Ser Ser Val Phe Ala Ala Glu Ile Met Asp 1 5 10 15 Ala Phe Asp
Arg Ser Pro Thr Asp Lys Glu Leu Val Ala Gln Ala Lys 20 25 30 Ala
Leu Gly Arg Glu Tyr Val His Ala Arg Leu Leu Arg Ala Gly Leu 35 40
45 Ser Trp Ser Ala Pro Glu Arg Ala Ser Pro Ala Pro Gly Gly Arg Leu
50 55 60 Ala Glu Val Cys Thr Val Leu Leu Arg Leu Gly Ile Thr Trp
Gly Lys 65 70 75 80 Val Val Ser Leu Tyr Ser Val Ala Ala Gly Leu Ala
Val Asp Cys Val 85 90 95 Arg Gln Ala Gln Pro Ala Met Val His Ala
Leu Val Asp Cys Leu Gly 100 105 110 Glu Phe Val Arg Lys Thr Leu Ala
Thr Trp Leu Arg Arg Arg Gly Gly 115 120 125 Trp Thr Asp Val Leu Lys
Cys Val Val Ser Thr Asp Pro Gly Phe Arg 130 135 140 Ser His Trp Leu
Val Ala Thr Leu Cys Ser Phe Gly Arg Phe Leu Lys 145 150 155 160 Ala
Ala Phe Phe Leu Leu Leu Pro Glu Arg 165 170 5 642 DNA H.sapiens CDS
(1)...(642) 5 atg gag gtg ctg cgg cgc tct tcg gtc ttc gct gcg gag
atc atg gac 48 Met Glu Val Leu Arg Arg Ser Ser Val Phe Ala Ala Glu
Ile Met Asp 1 5 10 15 gcc ttt gat cgc tgg ccc aca gac aag gag ctg
gtg gcc cag gct aaa 96 Ala Phe Asp Arg Trp Pro Thr Asp Lys Glu Leu
Val Ala Gln Ala Lys 20 25 30 gca cta ggc cgg gag tac gtg cac gcg
cgg ctt ttg cgc gcc ggc ctc 144 Ala Leu Gly Arg Glu Tyr Val His Ala
Arg Leu Leu Arg Ala Gly Leu 35 40 45 tcc tgg agc gct cca gag cgt
gcc tcg cct gcc cct gga gga cgc ctg 192 Ser Trp Ser Ala Pro Glu Arg
Ala Ser Pro Ala Pro Gly Gly Arg Leu 50 55 60 gca gag gtg tgc acc
gtg ctg ctg cgc ttg gga gat gag ctg gag cag 240 Ala Glu Val Cys Thr
Val Leu Leu Arg Leu Gly Asp Glu Leu Glu Gln 65 70 75 80 atc cgt ccc
agc gta tat cgg aat gtg gcc cgg cag ctg cac atc cct 288 Ile Arg Pro
Ser Val Tyr Arg Asn Val Ala Arg Gln Leu His Ile Pro 85 90 95 ctg
cag tct gag cct gtg gtg act gat gcc ttc ctc gct gtg gcc ggc 336 Leu
Gln Ser Glu Pro Val Val Thr Asp Ala Phe Leu Ala Val Ala Gly 100 105
110 cac atc ttc tca gca ggt atc aca tgg ggc aag gta gtg tcc ctg tac
384 His Ile Phe Ser Ala Gly Ile Thr Trp Gly Lys Val Val Ser Leu Tyr
115 120 125 tcg gcg gct gcg gga cta gcg gtg gac tgc gtc cgg caa gct
cag cca 432 Ser Ala Ala Ala Gly Leu Ala Val Asp Cys Val Arg Gln Ala
Gln Pro 130 135 140 gcc atg gtt cat gcc ctg gtt gac tgc ctg ggg gaa
ttt gta cgc aag 480 Ala Met Val His Ala Leu Val Asp Cys Leu Gly Glu
Phe Val Arg Lys 145 150 155 160 acc ttg gct acc tgg ctt cgg agg cgt
ggt gga tgg acg gac gtc ctc 528 Thr Leu Ala Thr Trp Leu Arg Arg Arg
Gly Gly Trp Thr Asp Val Leu 165 170 175 aag tgt gtg gtc agc aca aaa
cct ggc ttc cgc tcc cac tgg ctc gtg 576 Lys Cys Val Val Ser Thr Lys
Pro Gly Phe Arg Ser His Trp Leu Val 180 185 190 gcc aca ctc tgc agc
ttt ggc cgc ttc ctg aag gct gca ttc ttc ctc 624 Ala Thr Leu Cys Ser
Phe Gly Arg Phe Leu Lys Ala Ala Phe Phe Leu 195 200 205 ctg ttg cca
gag aga tga 642 Leu Leu Pro Glu Arg * 210 6 213 PRT H.sapiens 6 Met
Glu Val Leu Arg Arg Ser Ser Val Phe Ala Ala Glu Ile Met Asp 1 5 10
15 Ala Phe Asp Arg Trp Pro Thr Asp Lys Glu Leu Val Ala Gln Ala Lys
20 25 30 Ala Leu Gly Arg Glu Tyr Val His Ala Arg Leu Leu Arg Ala
Gly Leu 35 40 45 Ser Trp Ser Ala Pro Glu Arg Ala Ser Pro Ala Pro
Gly Gly Arg Leu 50 55 60 Ala Glu Val Cys Thr Val Leu Leu Arg Leu
Gly Asp Glu Leu Glu Gln 65 70 75 80 Ile Arg Pro Ser Val Tyr Arg Asn
Val Ala Arg Gln Leu His Ile Pro 85 90 95 Leu Gln Ser Glu Pro Val
Val Thr Asp Ala Phe Leu Ala Val Ala Gly 100 105 110 His Ile Phe Ser
Ala Gly Ile Thr Trp Gly Lys Val Val Ser Leu Tyr 115 120 125 Ser Ala
Ala Ala Gly Leu Ala Val Asp Cys Val Arg Gln Ala Gln Pro 130 135 140
Ala Met Val His Ala Leu Val Asp Cys Leu Gly Glu Phe Val Arg Lys 145
150 155 160 Thr Leu Ala Thr Trp Leu Arg Arg Arg Gly Gly Trp Thr Asp
Val Leu 165 170 175 Lys Cys Val Val Ser Thr Lys Pro Gly Phe Arg Ser
His Trp Leu Val 180 185 190 Ala Thr Leu Cys Ser Phe Gly Arg Phe Leu
Lys Ala Ala Phe Phe Leu 195 200 205 Leu Leu Pro Glu Arg 210 7 513
DNA H. sapiens CDS (1)...(513) 7 atg gag gtg ctg cgg cgc tct tcg
gtc ttc gct gcg gag atc atg gac 48 Met Glu Val Leu Arg Arg Ser Ser
Val Phe Ala Ala Glu Ile Met Asp 1 5 10 15 gcc ttt gat cgc tgg ccc
aca gac aag gag ctg gtg gcc cag gct aaa 96 Ala Phe Asp Arg Trp Pro
Thr Asp Lys Glu Leu Val Ala Gln Ala Lys 20 25 30 gca cta ggc cgg
gag tac gtg cac gcg cgg ctt ttg cgc gcc ggc ctc 144 Ala Leu Gly Arg
Glu Tyr Val His Ala Arg Leu Leu Arg Ala Gly Leu 35 40 45 tcc tgg
agc gct cca gag cgt gcc tcg cct gcc cct gga gga cgc ctg 192 Ser Trp
Ser Ala Pro Glu Arg Ala Ser Pro Ala Pro Gly Gly Arg Leu 50 55 60
gca gag gtg tgc acc gtg ctg ctg cgc ttg gga atc aca tgg ggc aag 240
Ala Glu Val Cys Thr Val Leu Leu Arg Leu Gly Ile Thr Trp Gly Lys 65
70 75 80 gta gtg tcc ctg tac tcg gcg gct gcg gga cta gcg gtg gac
tgc gtc 288 Val Val Ser Leu Tyr Ser Ala Ala Ala Gly Leu Ala Val Asp
Cys Val 85 90 95 cgg caa gct cag cca gcc atg gtt cat gcc ctg gtt
gac tgc ctg ggg 336 Arg Gln Ala Gln Pro Ala Met Val His Ala Leu Val
Asp Cys Leu Gly 100 105 110 gaa ttt gta cgc aag acc ttg gct acc tgg
ctt cgg agg cgt ggt gga 384 Glu Phe Val Arg Lys Thr Leu Ala Thr Trp
Leu Arg Arg Arg Gly Gly 115 120 125 tgg acg gac gtc ctc aag tgt gtg
gtc agc aca aaa cct ggc ttc cgc 432 Trp Thr Asp Val Leu Lys Cys Val
Val Ser Thr Lys Pro Gly Phe Arg 130 135 140 tcc cac tgg ctc gtg gcc
aca ctc tgc agc ttt ggc cgc ttc ctg aag 480 Ser His Trp Leu Val Ala
Thr Leu Cys Ser Phe Gly Arg Phe Leu Lys 145 150 155 160 gct gca ttc
ttc ctc ctg ttg cca gag aga tga 513 Ala Ala Phe Phe Leu Leu Leu Pro
Glu Arg * 165 170 8 170 PRT H. sapiens 8 Met Glu Val Leu Arg Arg
Ser Ser Val Phe Ala Ala Glu Ile Met Asp 1 5 10 15 Ala Phe Asp Arg
Trp Pro Thr Asp Lys Glu Leu Val Ala Gln Ala Lys 20 25 30 Ala Leu
Gly Arg Glu Tyr Val His Ala Arg Leu Leu Arg Ala Gly Leu 35 40 45
Ser Trp Ser Ala Pro Glu Arg Ala Ser Pro Ala Pro Gly Gly Arg Leu 50
55 60 Ala Glu Val Cys Thr Val Leu Leu Arg Leu Gly Ile Thr Trp Gly
Lys 65 70 75 80 Val Val Ser Leu Tyr Ser Ala Ala Ala Gly Leu Ala Val
Asp Cys Val 85 90 95 Arg Gln Ala Gln Pro Ala Met Val His Ala Leu
Val Asp Cys Leu Gly 100 105 110 Glu Phe Val Arg Lys Thr Leu Ala Thr
Trp Leu Arg Arg Arg Gly Gly 115 120 125 Trp Thr Asp Val Leu Lys Cys
Val Val Ser Thr Lys Pro Gly Phe Arg 130 135 140 Ser His Trp Leu Val
Ala Thr Leu Cys Ser Phe Gly Arg Phe Leu Lys 145 150 155 160 Ala Ala
Phe Phe Leu Leu Leu Pro Glu Arg 165 170 9 12 PRT R. rattus 9 Leu
Leu Arg Leu Gly Asp Glu Leu Glu Gln Ile Arg 1 5 10 10 12 PRT H.
sapiens 10 Leu Leu Arg Leu Gly Ile Thr Trp Gly Lys Val Val 1 5 10
11 13 PRT Artificial Sequence Consensus motif 11 Leu Arg Arg Ala
Gly Asp Glu Phe Glu Arg Tyr Arg Arg 1 5 10 12 4 PRT H. sapiens 12
Thr Trp Gly Lys 1 13 4 PRT H. sapiens 13 Asn Trp Gly Arg 1 14 3 PRT
H. sapiens 14 Gly Asp Glu 1 15 24 PRT H. sapiens 15 Leu Arg Arg Ile
Gly Asp Glu Leu Asp Ser Asn Ala Asp Gly Asn Phe 1 5 10 15 Asn Trp
Gly Arg Val Val Ala Leu 20 16 24 PRT H. sapiens 16 Leu Ala Ile Ile
Gly Asp Asp Ile Asn Arg Arg Phe Glu Ser Gly Ile 1 5 10 15 Asn Trp
Gly Arg Val Val Ala Leu 20 17 13 PRT H. sapiens 17 Leu Arg Arg Ile
Gly Asn Phe Asn Trp Gly Arg Val Val 1 5 10 18 12 PRT H. sapiens 18
Leu Ala Ile Ile Gly Ile Asn Trp Gly Arg Val Val 1 5 10
* * * * *
References