Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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BRCA2 TRANSCRIPTIONAL ACTIVATOR
DOMAIN AND USES THEREOF
- The present invention provides polypeptides, nucleic
acid encoding the polypeptides, substances which interact
with the polypeptides, oligonucleotide probes and primers,
and various methods and uses thereof. In particular it
relates to transcriptional activator polypeptides and
substances which modulate their activity. It is founded on
the surprising discovery, supported by clear experimental
data, that portions of the polypeptide encoded by the tumour
suppressor gene, BRCA2 (Tavtigian, S.V., Simard, J., Rommens,
J., Couch, F. Shattuck-Eidens, D., et al. (1996), Nat. Genet.
12, 333-337), have transcriptional activation capacity, and
that these portions are flanked in full-length polypeptide by
regions which interact with inhibitor molecules which inhibit
transcriptional activation. Experimental evidence is also
provided showing that a mutation that is associated with
familial breast cancer and is within the transcriptional
activator domain severely reduces activation of
transcription. Furthermore, molecules (polypeptide domains)
which interact with this region of the BRCA2 polypeptide have
been identified experimentally and may be used to modulate
transcriptional activation.
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Figure 1 shows that sequences within exon 3 of HRCA2
show sequence similarity to the activation domain of c-jun.
Below the alignment is the exon structure of the BRCA2
protein. Various portions of BRCA2 are shown below, along
with relative figures for transcription activation when fused
to the GAL4 DNA binding domain (1-147), compared with the
activity of the GAL4 DNA binding domain alone. The results
represent an average of several independent experiments, of
which details are provided below.
Figure 2 shows the protein and DNA sequence of residues
1-197 of BRCA2, including the various domains and regions
employed in aspects and embodiments of the present invention.
A mutation of tyr to cys at residue 42 is indicated, such
mutation being associated with familial breast cancers (3).
Nordling et al.(10) have also reported (after the priority
date of the present invention) that a large deletion which
disrupts the exon 3 transcription activator domain of BRCA2
is the disease-causing mutation in a Swedish breast/ovarian
cancer family.
Figure 3 shows the amino acid and DNA sequence of a
portion of the protein named "BBP1" (BRCA2 Binding Protein-1)
found to interact with the BRCA2 TAD and to modulate its
transcriptional activation.
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Figure 4 shows a portion of the BBP1 protein and
encoding DNA expressed and demonstrated to interact with
BRCA2 TAD, modulating its transcriptional activation.
According to a first aspect of the present invention
there is provided a polypeptide which has the amino acid
sequence of a fragment of BRCA2 protein and which is able to
act as transcriptional activator. Further aspects provide
use of such a polypeptide in activating transcription and
methods of activating transcription which employ such a
polypeptide.
A fragment of BRCA2 according to the present invention
may in some embodiments have less than about 300 amino acids,
less than about 200 amino acids, less than about 150 amino
acids, less than about 120 amino acids, less than about 100
amino acids, or less than about 70 amino acids.
Generally in order for a polypeptide to have
transcriptional activator function requires a DNA binding
domain which recognises a site within a promoter sequence.
~ 20 Thus, polypeptides according to this aspect of the present
invention may include or be fused or otherwise operably
linked to a DNA binding domain, which may be heterologous or
foreign to BRCA2, e.g. being of a polypeptide such as GAL4,
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or LexA or any suitable example, of which many are known and
in standard use in the art. See e.g. "Gene regulation" by
David Latchman, published by Unwin Hyman Ltd (1990). ,
There are many examples of transcription activation
domains being fused to DNA binding domains of polypeptides
such as GAL4, in order to study and/or manipulate
transcriptional activation. For instance, Martin et a1.
(Nature (1995) 375: 691-694) fused activation domain portions
of E2F1 to the DNA binding domain of GAL4, to study
interaction with MDM2, while Brown et a1. (The FN~O J. (1995)
14 (1): 124-131) fused portions of c-Fos protein to the DNA
binding domain of GAL4, to investigate transcriptional
activation and particularly silencing of such activation on
inclusion of inhibitory domains within the fusion protein.
A polypeptide according to the present invention may
include or consist essentially of amino acids 18-105 or 23-
105 of the human BRCA2 polypeptide (residue 23 is the
boundary of exon 3), the sequences of which are shown in
Figure 2, or an amino acid sequence which is a fragment,
mutant, variant, allele or derivative thereof. For instance, .
particular embodiments of the present invention may make
individual use of the two fragments of the exon 3 activation
domain demonstrated experimentally as described below to have
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the ability to activate transcription, i.e. the fragment
which is amino acids 18 to 60 (shown to be a primary
activating region) and the fragment which is amino acids 60
to 105 (shown to be an auxiliary activating region). Other
5 fragments within the fragment of BRCA2 residues 18-105 may be
employed as transcription activators provided they retain the
ability to so function. Alanine scanning and other
techniques of systematic alteration and/or fragmentation of a
polypeptide may be used to identify regions and/or residues
functionally involved or required.
Instead of using a wild-type BRCA2 fragment, the
polypeptide may include an amino acid sequence which differs
by one or more amino acid residues from the wild-type amino
acid sequence, by one or more of addition, insertion,
deletion and substitution of one or more amino acids. Thus,
variants, derivatives, alleles, mutants and homologues, e.g.
from other organisms, are included.
Preferably, the amino acid sequence shares homology with
the sequence or Figure 2, preferably at least about 30%, or
40%, or 50%, or 60%, or 70%, or 75%, or BO%, or 85% homology,
or at least about 90% or 95% homology.
As is well-understood, homology at the amino acid level
is generally in terms of amino acid similarity or identity.
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Similarity allows for "conservative variation", i.e.
substitution of one hydrophobic residue such as isoleucine,
valine, leucine or methionine for another, or the .
substitution of one polar residue for another, such as
S arginine for lysine, glutamic for aspartic acid, or glutamine
for asparagine. Similarity may be as defined and determined
by the TBLASTN program, of Altschul et al. (1990) J. Mol.
Biol. 215: 403-10, which is in standard use in the art, or
more preferably using the algorithm GAP (Genetics Computer
Group, Madison, WI). GAP uses the Needleman and Wunsch
algorithm to align two complete sequences that maximizes the
number of matches and minimizes the number of gaps.
Generally, the default parameters are used, with a gap
creation penalty = 12 and gap extension penalty = 4. Use of
either of the terms "homology" and "homologous" herein does
not imply any necessary evolutionary relationship between
compared sequences, in keeping for example with standard use
of terms such as "homologous recombination" which merely
requires that two nucleotide sequences are sufficiently
similar to recombine under the appropriate conditions.
Homology may be over the full-length of the relevant
polypeptide or may more preferably be over a contiguous
sequence of about 15, 20, 25, 30, 40, 50 or more amino acids,
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compared with the relevant wild-type amino acid sequence.
-Preferred polypeptides may include a transcriptional
activation domain with at least about 50%, 60%, 70%, 80%,
85%, 90% or 95% identity with that of BRCA2 exon 3, as shown
in Figure 2, or the fragment which is amino acids 18 to 60,
or the fragment which is amino acids 60 to 105.
At the nucleic acid level sequence identity may be
assessed by means of hybridization of molecules under
stringent conditions. The present invention extends to
nucleic acid that hybridizes with any one or more of the
specific sequences disclosed herein under s~~ringent
conditions. Suitable conditions include, e.g. for detection
of sequences that are about 80-90% identical, hybridization
overnight at 42°C in 0.25M Na2HP04, pH 7.2, 6.5% SDS, 10%
1S dextran sulfate and a final wash at 55°C in O.1X SSC, 0.1%
SDS. For detection of sequences that are greater than about
90% identical, suitable conditions include hybridization
overnight at 65°C in 0.25M Na2HP04, pH 7.2, 6.5% SDS, 10%
dextran sulfate and a final wash at 60°C in O.1X SSC, 0.1%
' 20 SDS.
For convenience herein, the residues 18-105 fragment of
BRCA2 polypeptide is referred to as the BRCA2 transcription
activation domain (~~TAD'~), the residues 18-60 fragment as the
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BRCA2 primary activating region or "PAR", and the residues
60-105 as the BRCA2 auxiliary activating region or "AAR",
bearing in mind that mutants, alleles, variants, derivatives -
and smaller fragments may be employed in the present
invention and are therefore generally encomr~assed by use of
such terms in the description unless e.g. context requires
otherwise.
One or more additional portions of the BRCA2 polypeptide
may be included, if desired. One such embodiment includes
the portion from residues 126 to 197, or a fragment, mutant,
allele, variant or derivative thereof. In further
embodiments of various aspects of the present invention, a
polypeptide includes or consists essentially of the fragment
of BRCA2 at residues 126 to 197, which may be used for
instance in looking for and/or obtaining substances which
interact with it and which may have an effect on a BRCA2
function.
Further experimental evidence included below indicates
that within the BRCA2 polypeptide the transcription
activation domain of the invention, the TAD including the PAR
and the AAR, is flanked by two "inhibitory regions" which are .
bound by molecules which inhibit activation of transcription
by the TAD, as demonstrated experimentally and described
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below. The position of the two inhibitory regions is shown
_in Figure 1 ("IRl" and "IR2"), with sequence information
' being given in Figure 2.
Further polypeptides according to the present invention
include IR1 and/or IR2 in addition to the TAD. Of course,
mutant, allele, derivative or variant sequences, or
fragments, may be used instead of the wild-type sequence
shown. As discussed below, a polypeptide which includes IR1
and/or IR2 with the BRCA2 TAD may be used in the
identification and isolation of molecules which interact with
or bind the inhibitory regions to inhibit transcriptional
activation by the TAD, and substances which interfere with
such interaction or binding and/or inhibition of
transcriptional activation. Also, peptides including or
consisting essentially of all or part of the IR1 and IR2
sequences may be used similarly in the identification and
isolation of molecules which interact or bind, preferably
which inhibit transcriptional activation by the TAD upon
interaction or binding, and in the identification and
' 20 isolation of molecules which interfere with such interaction
. or binding to modulate inhibition of activation of
transcription by the TAD.
Inhibitor domains have been shown to be present in a
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number of transcription factors (references 4, 5, 6 below).
_ In c-Fos and others there is evidence that the inhibitor is
functioning by binding a protein or proteins to mediate
repression of transcriptional activation. This evidence
5 comes from "squelching" experiments in which the inhibitor
domain was added in excess to compete away any potential
repressor protein (4). Similar experiments may be performed
using polypeptides and peptides of the present invention.
Addition of BRCA2 IR1 and/or IR2 peptides in excess may
10 increase the activation capacity of inactive BRCA2 fragment
residues 1-125. There are other instances where an inhibitor
domain functions via intra-molecular interaction which
"masks" the activation domain. As discussed more fully
below, assay methods and means, for example using a high
throughput screen, may be used to find molecules which
activate BRCA2 transcriptional activation, which may do this
by binding the inhibitor domain and preventing an intra-
molecular interaction.
A peptide according to a further aspect of the invention
may include or consist essentially of the IR1 and/or IR2
sequences shown in Figure 2. Where additional amino acids
are included, which amino acids may be from BRCA2 (so that
the peptide is a larger fragment of BRCA2) or may be
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heterologous or foreign to BRCA2, the peptide may be about
-20, 25, 30 or 35 amino acids in length. A peptide according
to this aspect may be included within a larger fusion
protein, particularly where the peptide is fused to a non-
BRCA2 (i.e. heterologous or foreign) sequence, such as a
polypeptide or protein domain.
Further similar aspects relate in the same or similar
terms to peptides including or consisting essentially of
fragments of BBP1 (see below).
Various screening and assay formats employing
polypeptides and peptides according to the present invention
are discussed below.
A convenient way of producing a polypeptide or peptide
according to the present invention is to express nucleic acid
encoding it, by use of nucleic acid in an expression system.
Accordingly the present invention also provides in
various aspects nucleic acid encoding the polypeptides and
peptides of the invention. (This includes "BBP1" (BRCA2
Binding Protein 1) discussed further below.)
Generally, nucleic acid according to the present
invention is provided as an isolate, in isolated and/or
purified form, or free or substantially free of material with
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which it is naturally associated, such as free or
_substantially free of nucleic acid flanking the gene in the
human genome, except possibly one or more regulatory '
sequences) for expression. Nucleic acid may be wholly or
partially synthetic and may include genomic DNA, cDNA or RNA.
Where nucleic acid according to the invention includes RNA,
reference to the sequence shown should be construed as
encompassing the RNA equivalent, with U substituted for T.
Nucleic acid sequences encoding a polypeptide in
accordance with the present invention can be readily prepared
by the skilled person using the information and references
contained herein and techniques known in the art (for
example, see Sambrook, Fritsch and Maniatis, "Molecular
Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory
Press, 1989, and Ausubel et al, Short Protocols in Molecular
Biology, John Wiley and Sons, 1992), given the nucleic acid
sequence and clones available (e. g. Tavtigian, S.V., Simard,
J., Rommens, J., Couch, F. Shattuck-Eidens, D., et al.
(1996), Nat. Genet. 12, 333-337). These techniques include
(i) the use of the polymerase chain reaction (PCR) to amplify
samples of such nucleic acid, e.g. from genomic sources, (ii)
chemical synthesis, or (iii) preparing cDNA sequences.
Modifications to the BRCA2 sequences can be made, e.g. using
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site directed mutagenesis, to lead to the expression of
_ modified BRCA2 polypeptide or to take account of codon
preference in the host cells used to express the nucleic
acid.
In order to obtain expression of the nucleic acid
sequences, the sequences can be incorporated in a vector
having one or more control sequences operably linked to the
nucleic acid to control its expression. The vectors may
include other sequences such as promoters or enhancers to
drive the expression of the inserted nucleic acid, nucleic
acid sequences so that the polypeptide or peptide is produced
as a fusion and/or nucleic acid encoding secretion signals so
that the polypeptide produced in the host call is secreted
from the cell. Polypeptide can then be obtained by
transforming the vectors into host cells in which the vector
is functional, culturing the host cells so that the
polypeptide is produced and recovering the polypeptide from
the host cells or the surrounding medium. Prokaryotic and
eukaryotic cells are used for this purpose in the art,
including strains of E. coli, yeast, and eukaryotic cells
such as COS or CHO cells.
Thus, the present invention also encompasses a method of
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making a polypeptide or peptide (as disclosed), the method
-including expression from nucleic acid encoding the
polypeptide or peptide (generally nucleic acid according to
the invention). This may conveniently be achieved by growing
a host cell in culture, containing such a vector, under
appropriate conditions which cause or allow expression of the
polypeptide. Polypeptides and peptides may also be expressed
in in vitro systems, such as reticulocyte lysate.
Systems for cloning and expression of a polypeptide in a
variety of different host cells are well known. Suitable
host cells include bacteria, eukaryotic cells such as
mammalian and yeast, and baculovirus systems. Mammalian cell
lines-available in the art for expression of a heterologous
polypeptide include Chinese hamster ovary cells, HeLa cells,
baby hamster kidney cells, COS cells and many others. A
common, preferred bacterial host is E. coli.
Suitable vectors can be chosen or constructed,
containing appropriate regulatory sequences, including
promoter sequences, terminator fragments, polyadenylation
sequences, enhancer sequences, marker genes and other
sequences as appropriate. Vectors may be plasmids, viral
e.g. 'phage, or phagemid, as appropriate. For further
details see, for example, Molecular Cloning: a Laboratory
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Manual: 2nd edition, Sambrook et al., 1989, Cold Spring
Harbor Laboratory Press. Many known techniques and protocols
for manipulation of nucleic acid, for example in preparation
of nucleic acid constructs, mutagenesis, sequencing,
5 introduction of DNA into cells and gene expression, and
analysis of proteins, are described in detail in Current
Protocols in Molecular Biology, Ausubel et al. eds., John
Wiley & Sons, 1992.
Thus, a further aspect of the present invention provides
10 a host cell containing heterologous nucleic acid as disclosed
herein.
The nucleic acid of the invention may be integrated into
the genome (e. g. chromosome) of the host cell. Integration
may be promoted by inclusion of sequences which promote
15 recombination with the genome, in accordance with standard
techniques. The nucleic acid may be on an extra-chromosomal
vector within the cell, or otherwise identifiably
heterologous or foreign to the cell.
A still further aspect provides a method which includes
' 20 introducing the nucleic acid into a host cell. The
introduction, which may (particularly for in vitro
introduction) be generally referred to without limitation as
"transformation", may employ any available technique. For
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eukaryotic cells, suitable techniques may include calcium
_phosphate transfection, DEAE-Dextran, electroporation,
liposome-mediated transfection and transduction using
retrovirus or other virus, e.g. vaccinia or, for insect
cells, baculovirus. For bacterial cells, suitable techniques
may include calcium chloride transformation, electroporation
and transfection using bacteriophage. As an alternative,
direct injection of the nucleic acid could be employed.
Marker genes such as antibiotic resistance or
sensitivity genes may be used in identifying clones
containing nucleic acid of interest, as is well known in the
art.
The introduction may be followed by causing or allowing
expression from the nucleic acid, e.g. by culturing host
cells (which may include cells actually transformed although
more likely the cells will be descendants of the transformed
cells) under conditions for expression of the gene, so that
the encoded polypeptide (or peptide) is produced. If the
polypeptide is expressed coupled to an appropriate signal
leader peptide it may be secreted from the cell into the
culture medium. Following production by expression, a
polypeptide or peptide may be isolated and/or purified from
the host cell and/or culture medium, as the case may be, and
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subsequently used as desired, e.g. in the formulation of a
composition which may include one or more additional
components, such as a pharmaceutical composition which
includes one or more pharmaceutically acceptable excipients,
vehicles or carriers (e. g. see below).
Introduction of nucleic acid may take place in vivo by
way of gene therapy, as discussed below.
A host cell containing nucleic acid according to the
present invention, e.g. as a result of introduction of the
nucleic acid into the cell or into an ancestor of the cell
and/or genetic alteration of the sequence endogenous to the
cell or ancestor (which introduction or alteration may take
place in vivo or ex vivo), may be comprised (e.g. in the
soma) within an organism which is an animal, particularly a
mammal, which may be human or non-human, such as rabbit,
guinea pig, rat, mouse or other rodent, cat, dog, pig, sheep,
goat, cattle or horse, or which is a bird, such as a chicken.
Genetically modified or transgenic animals or birds
comprising such a cell are also provided as further aspects
of the present invention.
This may have a therapeutic aim. (Gene therapy is
discussed below.) Also, the presence of a mutant, allele,
derivative or variant sequence within cells of an organism,
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particularly when.in place of a homologous endogenous
_sequence, may allow the organism to be used as a model in
testing and/or studying substances which modulate activity of
the encoded polypeptide in vitro or are otherwise indicated
to be of therapeutic potential. Conveniently, however,
assays for such substances may be carried out in vitro,
within host cells or in cell-free systems.
A polypeptide according to the present invention may be
used in screening for molecules which affect or modulate its
activity or function. Such molecules may be useful in a
therapeutic (possibly including prophylactic) context.
Combinatorial library technology provides an efficient way of
testing a potentially vast number of different substances for
ability to modulate bind to and/or activity of a polypeptide.
Such libraries and their use are known in the art. The use
of peptide libraries may be preferred in certain
circumstances.
In various further aspects the present invention relates
to screening and assay methods and means, and substances
identified thereby. _
Thus, further aspects of the present invention provide
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the use of a polypeptide or peptide of the invention as
_disclosed, and/or encoding nucleic acid therefor, in
' screening or searching for and/or obtaining/identifying a
substance, e.g. peptide or chemical compound, which interacts
and/or binds with the polypeptide or peptide and/or
interferes with its function or activity or that of another
substance, e.g. polypeptide or peptide, which interacts
and/or binds with the polypeptide or peptide of the
invention. For instance, a method according to one aspect of
the invention includes providing a polypeptide or peptide of
the invention and bringing it into contact with a substance,
which contact may result in binding between the polypeptide
or peptide and the substance. Binding may be determined by
any of a number of techniques available in the art, both
qualitative and quantitative.
In various aspects the present invention is concerned
with provision of assays for substances which inhibit
interaction between the BRCA2 IR1 and IR2 peptide motifs
(discussed above) and the inhibitory molecule or molecules
which bind these regions to inhibit transcriptional
activation by the TAD.
Further assays are for substances which interact with or
bind the TAD and/or modulate its ability to activate
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transcription.
A protein termed "BBP1" (BRCA2 Binding Protein-1) has
been found to interact with the BRCA2 TAD, specifically the
PAR. A domain of BBP1 was expressed (see experimental
5 section) and shown to repress activity of the BRCA2 TAD.
Assays of the invention may therefore use the BRCA2 TAD, or
suitable fragment thereof, and BBP1, or suitable fragment
thereof, in obtaining substances able to interfere with their
interaction and/or modulate the effect of Bi3P1 on BRCA2
10 transcription activation.
As noted, the IR1 and IR2 peptides of the invention and
polypeptides including one or more of these may be used in
screening for and obtaining the inhibitory molecules which
interact or bind IR1 and/or IR2 within BRCA2 in vivo to
15 inhibit transcriptional activation.
A substance which binds the TAD may inhibit
transcriptional activation, or may stimulate and/or enhance
it. For instance, it is known that c-Jun transcriptional
activator function is stimulated by phosphorylation of the
20 active site by a kinase (Jun N-terminal kinase, Jnk). A
phosphorylase removes the phosphate to deactivate the
transcriptional activator function. A molecule that inhibits
or prevents the dephosphorylation may be used to enhance
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transcriptional activation. Such a molecule may include a
binding portion of the kinase. As discussed elsewhere
herein, the BRCA2 TAD has significant sequence homology with
the TAD of Jun and a kinase is able to bind it. Thus, a
kinase which operates on the BRCA2 TAD, or a HRCA2 TAD
binding portion of the kinase, has been obtained and used to
modulate transcriptional activation.
Phosphorylation may be determined for example by
immobilising a BRCA2 fragment, mutant, variant or derivative
thereof, e.g. on a bead or plate, and detecting
phosphorylation using an antibody or other binding molecule
which binds the relevant site of phosphorylation with a
different affinity when the site is phosphorylated from when
the site is not phosphorylated. Such antibodies may be
obtained by means of any standard technique as discussed
elsewhere herein, e.g. using a phosphorylated peptide (such
as a fragment of BRCA2). Binding of a binding molecule which
discriminates between the phosphorylated and non-
phosphorylated form of BRCA2 or relevant fragment, mutant,
variant or derivative thereof may be assessed using any
technique available to those skilled in the art, which may
involve determination of the presence of a suitable label,
such as fluorescence. Phosphorylation may be determined by
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immobilisation of BRCA2 or a fragment, mutant, variant or
derivative thereof, on a suitable substrate such as a bead or
plate, wherein the substrate is impregnated with scintillant,
such as in a standard scintillation proximetry assay, with
phosphorylation being determined via measurement of the
incorporation of radioactive phosphate. Phosphate
incorporation into BRCA2 or a fragment, mutant, variant or
derivative thereof, may be determined by precipitation with
acid, such as trichloroacetic acid, and collection of the
precipitate on a suitable material such as nitrocellulose
filter paper, followed by measurement of incorporation of
radiolabeled phosphate. SDS-PAGE separation of substrate may
be employed followed by detection of radiolabel.
As described below, the present inventors have obtained
in their experimental work substances, in particular
polypeptide domains, which interact with BRCA2 polypeptide
fragments in accordance with the present invention.
Another aspect of the present invention provides an
assay (A) which includes:
(a) bringing into contact a polypeptide or peptide according
to the invention including BRCA2 IR1 and/or IR2 as
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disclosed, and a putative binding molecule or other test
substance; and
(b) determining interaction or binding between the
polypeptide or peptide and the test substance.
A substance which interacts with or binds to the BRCA2
TAD, IR1 and/or IR2 may be isolated and/or purified,
manufactured and/or used to modulate transcriptional
activation as discussed.
In another aspect, the present invention provides an
assay (B) which includes:
(a) bringing into contact a polypeptide according to
the present invention, including a BRCA2 TAD and optionally a
IR1 and/or IR2 and a DNA binding domain capable of binding a
nucleotide sequence within a promoter, and a putative
inhibitor compound under conditions where the polypeptide, in
the absence of inhibitor, is capable of binding the
nucleotide sequence within the promoter to activate
transcription;
(b) providing a nucleic acid molecule which includes a
promoter which includes the nucleotide sequence to which the
polypeptide is capable of binding to activate transcription
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of a sequence operably linked to the promoter; and
_ (c) measuring the degree of modulation or alteration of
transcriptional activation caused by said inhibitor compound.
A compound which increases the level of transcriptional
activation, particularly, when IR1 and IR2 are not included
may be useful in enhancing transcriptional activation by
BRCA2 TAD, which has therapeutic potential given BRCA2's role
as a tumour suppressor and the experimental evidence
described below on the effect of a mutation within the BRCA2
TAD which is associated with familial breast cancer on
decreasing BRCA2 TAD transcriptional activation.
A compound which binds IR1 and/or IR2 may inhibit
transcriptional activation by the polypeptide. Thus,
inhibition of transcriptional activation allows for
identification of molecules which bind IR1 and/or IR2,
including a natural ligand.
A molecule which binds IR1 and/or IR2 in vivo, e.g. a
molecule naturally present in a mammalian, e.g. human, tumour
(e.g. breast tumour) or non-tumour cell, and preferably a
molecule which inhibits transcriptional activation by the
BRCA2 TAD, obtainable using assay (A) or assay (B) may be
isolated and may be manufactured, and may be subsequently
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used to assay for.substances which interfere with its binding
_to IR1 and/or IR2 within BRCA2 polypeptide and/or ability to
' inhibit BRCA2 TAD transcriptional activation.
5 Thus, a further aspect of the invention employs a
peptide or polypeptide which includes BRCA2 IR1 and/or IR2
(bearing in mind these terms always, unless context requires
otherwise, allow for the use of a mutant, variant,
derivative, allele or fragment) and a molecule obtainable by
10 assay (A) or assay (B) which binds IR1 or IR2 in an assay for
substances which interfere with such binding and may
therefore inhibit inhibition of the transcriptional
activation capacity of the BRCA2 TAD.
Such an assay may include bringing into contact a
15 peptide or polypeptide including a IR1 or IR2 sequence and a
binding molecule for the IR1 or IR2 sequence (such as
obtainable by means of assay (A) or assay (B)) in the
presence of a test substance, wherein the test conditions are
such that in the absence of a substance able to interfere
20 with binding between the IR1 and/or IR2 sequence and the
binding molecule such binding occurs, and determining binding
between the IR1 and/or IR2. This may be followed by
isolation and/or manufacture and/or use of a substance which
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tests positive for ability to interefere with the binding of
interest.
It is not necessary to use the entire proteins for
assays of the invention which test for binding between two
molecules. Fragments may be generated and used in any
suitable way known to those of skill in the art. Suitable
ways of generating fragments include, but are not limited to,
recombinant expression of a fragment from encoding DNA. Such
fragments may be generated by taking encoding DNA,
identifying suitable restriction enzyme recognition sites
either side of the portion to be expressed, and cutting out
said portion from the DNA. The portion may then be operably
linked to a suitable promoter in a standard commercially
available expression system. Another recombinant approach is
to amplify the relevant portion of the DNA with suitable PCR
primers. Small fragments (up to about 20 or 30 amino acids)
may also be generated using peptide synthesis methods which
are well known in the art.
For example, in a preferred embodiment of the invention
a polypeptide may be fused to a heterologous DNA binding
domain such as that of the yeast transcription factor GAL 4.
The GAL 4 transcription factor includes two functional
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domains. These domains are the DNA binding domain (GAL4DBD)
_and the GAL4 transcriptional activation domain (GAL4TAD). By
' fusing one polypeptide or peptide to one of those domains and
another polypeptide or peptide to the respective counterpart,
a functional GAL 4 transcription factor is restored only when
two polypeptides or peptides of interest interact. Thus,
interaction of the polypeptides or peptides may be measured
by the use of a reporter gene probably linked to a GAL 4 DNA
binding site which is capable of activating transcription of
said reporter gene. This assay format is described by Fields
and Song, 1989, Nature ~Q; 245-246. This type of assay
format can be used in both mammalian cells and in yeast.
Other combinations of DNA binding domain and transcriptional
activation domain are available in the art .end may be
preferred, such as the LexA DNA binding domain and the VP60
transcriptional activation domain.
The precise format of the assay of the invention may be
varied by those of skill in the art using routine skill and
knowledge. For example, the interaction between the
polypeptides may be studied in vitro by labelling one with a
detectable label and bringing it into contact with the other
which has been immobilised on a solid support. Suitable
detectable labels include 35S-methionine which may be
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incorporated into recombinantly produced peptides and
_polypeptides. Recombinantly produced peptides and
polypeptides may also be expressed as a fusion protein
containing an epitope which can be labelled with an antibody.
The protein which is immobilized on a solid support may
be immobilized using an antibody against that protein bound
to a solid support or via other technologies which are known
per se. A preferred in vitro interaction may utilise a
fusion protein including glutathione-S-transferase (GST).
This may be immobilized on glutathione agarose beads. In an
in vitro assay format of the type described above a test
compound can be assayed by determining its ability to
diminish the amount of labelled peptide or polypeptide which
binds to the immobilized GST-fusion polypeptide. This may be
determined by fractionating the glutathione-agarose beads by
SDS-polyacrylamide gel electrophoresis. Alternatively, the
beads may be rinsed to remove unbound protein and the amount
of protein which has bound can be determined by counting the
amount of label present in, for example, a suitable
scintillation counter.
An assay according to the present invention may also
take the form of an in vivo assay. The in vivo assay may be
performed in a cell line such as a yeast strain in which the
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relevant polypeptides or peptides are expressed from one or
_more vectors introduced into the cell.
A further assay according to the present invention tests
for ability of a substance to modulate transcriptional
activation by HRCA2 TAD, e.g. by inhibiting interaction of
IR1 and/or IR2 with a binding molecule or respective binding
molecules {such as one or more ligands obtainable by assay
(A) or assay (B)) which inhibit such transcriptional
activation.
Such an assay may involve:
(a) bringing into contact a polypeptide according to
the present invention, including a BRCA2 TAD and a IR1 and/or
IR2 and a DNA binding domain capable of binding a nucleotide
sequence within a promoter, a binding molecule or molecules
for IR1 and/or IR2 which inhibits) transcriptional
activation by the BRCA2 TAD, and a test compound, under
conditions in which the binding molecule or molecules) for
IR1 and/or IR2 inhibits) transcriptional activation of the
promoter by the BRCA2 TAD,;
(b) providing a nucleic acid molecule which includes a
promoter which includes the nucleotide sequence to which the
polypeptide is capable of binding to activate transcription
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of a sequence operably linked to the promoter when the
polypeptide is not bound by the IRl and/or IR2 binding
molecule or molecules; and
(c) measuring the degree of modulation or alteration of
5 transcriptional activation caused by said test compound.
A reporter gene construct including a promoter which
includes a nucleotide sequence to which the DNA binding
domain binds, operably linked to a reporter gene, may be
10 introduced into an expression system such as a cell together
with one or more expression vectors encoding polypeptide or
peptide components of an assay according to the present
invention. Two or more binding sites (for example 3, 4 or 5)
may be present in the nucleic acid construct and this may
15 enhance sensitivity of the assay.
By "promoter" is meant a sequence of nucleotides from
which transcription may be initiated of DNA operably linked
downstream (i.e. in the 3' direction on the sense strand of
double-stranded DNA).
20 "Operably linked" in the context of a sequence of
interest and a promoter means joined as part of the same
nucleic acid molecule, suitably positioned and oriented for
transcription to be initiated from the promoter. DNA
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operably linked to,a promoter is "under transcriptional
-initiation regulation" of the promoter.
' "Promoter activity" is used to refer to ability to
initiate transcription. The level of promoter activity is
quantifiable for instance by assessment of the amount of mRNA
produced by transcription from the promoter or by assessment
of the amount of protein product produced by translation of
mRNA produced by transcription from the promoter. The amount
of a specific mRNA present in an expression system may be
determined for example using specific oligonucleotides which
are able to hybridise with the mRNA and which are labelled or
may be used in a specific amplification reaction such as the
polymerase chain reaction. Use of a reporter gene
facilitates determination of promoter activity by reference
to protein production.
Generally, the gene may be transcribed into mRNA which
may be translated into a peptide or polypeptide product which
may be detected and preferably quantitated following
expression. A gene whose encoded product may be assayed
following expression is termed a "reporter gene", i.e. a gene
which "reports" on promoter activity.
A reporter gene preferably encodes an enzyme which
catalyses a reaction which produces a detectable signal,
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preferably a visually detectable signal, such as a coloured
-product. Many examples are known, including (3-galactosidase
and luciferase. (3-galactosidase activity may be assayed by
production of blue colour on substrate, the assay being by
eye or by use of a spectrophotometer to measure absorbance.
Fluorescence, for example that produced as a result of
luciferase activity, may be quantitated using a
spectrophotometer. Radioactive assays may be used, for
instance using chloramphenicol acetyltransferase, which may
also be used in non-radioactive assays. The presence and/or
amount of gene product resulting from expression from the
reporter gene may be determined using a molecule able to bind
the product, such as an antibody or fragment thereof. The
binding molecule may be labelled directly or indirectly using
any standard technique.
Those skilled in the art are well aware of a multitude
of possible reporter genes and assay techniques which may be
used to determine gene activity. Any suitable reporter/assay
may be used and it should be appreciated that no particular
choice is essential to or a limitation of the present
invention.
A method of screening for ability of a substance to
modulate activity of a promoter may include contacting an
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expression system,. such as a host cell, containing assay
_components as herein disclosed with a test or candidate
' substance and determining expression of the heterologous
gene.
The level of expression in the presence of the test
substance may be compared with the level of expression in the
absence of the .test substance. A difference in expression in
the presence of the test substance indicates ability of the
substance to modulate gene expression. An increase in
expression of the gene compared with expression of another
gene not linked to a promoter as disclosed herein indicates
specificity of the substance for modulation of the promoter.
A promoter construct may be introduced into a cell line
using any technique previously described to produce a stable
cell line containing the reporter construct integrated into
the genome. The cells may be grown and incubated with test
compounds for varying times. The cells may be grown in 96
well plates to facilitate the analysis of large numbers of
compounds. The cells may then be washed and the reporter
gene expression analysed. For some reporters, such as
luciferase the cells will be lysed then analysed.
Following identification of a substance which modulates
or affects promoter activity, the substance may be
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investigated further. Furthermore, it may be manufactured
_ and/or used in preparation, i.e. manufacture or formulation,
of a composition such as a medicament, pharmaceutical
composition or drug. These may be administered to
individuals.
The amount of test substance or compound which may be
added to an assay of the invention will normally be
determined by trial and error depending upon the type of
compound used. Typically, from about 0.01 ~0 100 nM
concentrations of putative inhibitor compound may be used,
for example from 0.1 to 10 nM. Greater concentrations may be
used when a peptide is the test substance.
Compounds which may be used may be natural or synthetic
chemical compounds used in drug screening programmes.
Extracts of plants which contain several characterised or
uncharacterised components may also be used. A further class
of putative inhibitor compounds can be derived from the BRCA2
polypeptide and/or a ligand which binds IR1 and/or IR2.
Peptide fragments of from 5 to 40 amino acids, for example
from 6 to 10 amino acids from the region of the relevant
polypeptide responsible for interaction, may be tested for
their ability to disrupt such interaction.
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The skilled person can use the techniques described
- herein and others well known in the art to produce large
amounts of peptides, for instance by expression from encoding
nucleic acid.
5 Peptides can also be generated wholly or partly by
chemical synthesis. The compounds of the present invention
can be readily prepared according to well-established,
standard liquid or, preferably, solid-phase peptide synthesis
methods, general descriptions of which are broadly available
10 (see, for example, in J.M. Stewart and J.D. Young, Solid
Phase Peptide Synthesis, 2nd edition, Pierce Chemical
Company, Rockford, Illinois (1984), in M. Bodanzsky and A.
Bodanzsky, The Practice of Peptide Synthesis, Springer
Verlag, New York (1984); and Applied Biosystems 430A Users
15 Manual, ABI Inc., Foster City, California), or they may be
prepared in solution, by the liquid phase method or by any
combination of solid-phase, liquid phase and solution
chemistry, e.g. by first completing the respective peptide
portion and then, if desired and appropriate, after removal
20 of any protecting groups being present, by introduction of
the residue X by reaction of the respective carbonic or
sulfonic acid or a reactive derivative thereof.
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Antibodies. directed to the site of interaction in either
- protein form a further class of putative inhibitor compounds.
Candidate inhibitor antibodies may be characterised and their
binding regions determined to provide single chain antibodies
and fragments thereof which are responsible for disrupting
the interaction. Antibodies in accordance with the present
invention which discriminate between BRCA2 which is wild-type
and BRCA2 which is mutated in the region of interest, are
useful in screening procedures as discussed further below.
Antibodies may be obtained using techniques which are
standard in the art. Methods of producing antibodies include
immunising a mammal (e. g. mouse, rat, rabbit, horse, goat,
sheep or monkey) with the protein or a fragment thereof.
Antibodies may be obtained from immunised animals using any
of a variety of techniques known in the art, and screened,
preferably using binding of antibody to antigen of interest.
For instance, Western blotting techniques or
immunoprecipitation may be used (Armitage et al., 1992,
Nature 357: 80-82). Isolation of antibodies and/or antibody-
producing cells from an animal may be accompanied by a step
of sacrificing the animal.
As an alternative or supplement to immunising a mammal
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with a peptide, an antibody specific for a protein may be
- obtained from a recombinantly produced library of expressed
immunogiobulin variable domains, e.g. using lambda
bacteriophage or filamentous bacteriophage which display
functional immunoglobulin binding domains on their surfaces;
for instance see W092/01047. The library may be naive, that
is constructed from sequences obtained from an organism which
has not been immunised with any of the proteins (or
fragments), or may be one constructed using sequences
obtained from an organism which has been exposed to the
antigen of interest.
Antibodies according to the present invention may be
modified in a number of ways. Indeed the term "antibody"
should be construed as covering any binding substance having
a binding domain with the required specificity. Thus the
invention covers antibody fragments, derivatives, functional
equivalents and homologues of antibodies, including synthetic
molecules and molecules whose shape mimics that of an
antibody enabling it to bind an antigen or epitope.
Example antibody fragments, capable of binding an
antigen or other binding partner are the Fab fragment
consisting of the VL, VH, Cl and CH1 domains; the Fd fragment
consisting of the VH and CH1 domains; the Fv fragment
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consisting of the.VL and VH domains of a single arm of an
- antibody; the dAb fragment which consists of a VH domain;
isolated CDR regions and F(ab~)2 fragments, a bivalent
fragment including two Fab fragments linked by a disulphide
bridge at the hinge region. Single chain Fv fragments are
also included.
A hybridoma producing a monoclonal antibody according to
the present invention may be subject to genetic mutation or
other changes. It will further be understood by those
skilled in the art that a monoclonal antibody can be
subjected to the techniques of recombinant DNA technology to
produce other antibodies or chimeric molecules which retain
the specificity of the original antibody. Such techniques
may involve introducing DNA encoding the immunoglobulin
variable region, or the complementarity determining regions
(CDRs), of an antibody to the constant regions, or constant
regions plus framework regions, of a different
immunoglobulin. See, for instance, EP184187A, GB 2188638A or
EP-A-0239400. Cloning and expression of chimeric antibodies
are described in EP-A-0120694 and EP-A-0125023.
Hybridomas capable of producing antibody with desired
binding characteristics are within the scope of the present
invention, as are host cells, eukaryotic or prokaryotic,
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containing nucleic acid encoding antibodies (including
_ antibody fragments) and capable of their expression. The
invention also provides methods of production of the
antibodies including growing a cell capable of producing the
antibody under conditions in which the antibody is produced,
and preferably secreted.
The reactivities of antibodies on a sample (e.g, the
subject of a diagnostic test) may be determined by any
appropriate means. Tagging with individual reporter
molecules is one possibility. The reporter molecules may
directly or indirectly generate detectable, and preferably
measurable, signals. The linkage of reporter molecules may
be directly or indirectly, covalently, e.g. via a peptide
bond or non-covalently. Linkage via a peptide bond may be as
a result of recombinant expression of a gene fusion encoding
antibody and reporter molecule.
One favoured mode is by covalent linkage of each
antibody with an individual fluorochrome, phosphor or laser
dye with spectrally isolated absorption or emission
characteristics. Suitable fluorochromes include fluorescein,
rhodamine, phycoerythrin and Texas Red. Suitable chromogenic
dyes include diaminobenzidine.
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Other reporters include macromolecular colloidal
-particles or particulate material such as latex beads that
are coloured, magnetic or paramagnetic, and biologically or
chemically active agents that can directly or indirectly
5 cause detectable signals to be visually observed,
electronically detected or otherwise recorded. These
molecules may be enzymes which catalyse reactions that
develop or change colours or cause changes in electrical
properties, for example. They may be molecularly excitable,
10 such that electronic transitions between energy states result
in characteristic spectral absorptions or emissions. They
may include chemical entities used in conjunction with
biosensors. Biotin/avidin or biotin/streptavidin and
alkaline phosphatase detection systems may be employed.
15 The mode of determining binding is not a feature of the
present invention and those skilled in the art are able to
choose a suitable mode according to their preference and
general knowledge.
20 Antibodies in accordance with the present invention may
be used in screening for the presence of a particular
polypeptide, for example in a test sample containing cells or
cell lysate as discussed, such as a BRCA2 polypeptide
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including a mutation in the TAD, IR1 and/or IR2, where such
_mutation is reflected in an alteration in one or more
' epitopes allowing discrimination between the mutant and wild-
type regions of BRCA2. Screening methods using antibodies
are discussed further below.
Antibodies may also be used in purifying and/or
isolating a polypeptide according to the present invention,
for instance following production of the polypeptide by
expression from encoding nucleic acid therefor. Antibodies
may modulate the activity of the polypeptide to which they
bind and so, if that polypeptide has a deleterious effect in
an individual, may be useful in a therapeutic context (which
may include prophylaxis).
An antibody may be provided in a kit, which may include
instructions for uae of the antibody, e.g. in determining the
presence of a particular substance in a test sample. One or
more other reagents may be included, such as labelling
molecules, buffer solutions, elutants and so on. Reagents
may be provided within containers which protect them from the
external environment, such as a sealed vial.
Other candidate inhibitor compounds may be based on
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modelling the 3-dimensional structure of a polypeptide or
_ peptide fragment and using rational drug design to provide
potential inhibitor compounds with particular molecular
shape, size and charge characteristics.
In a further aspect, the invention provides compounds
obtainable by an assay according to the present invention,
for example peptide compounds based on portions of the BRCA2
polypeptide.
A compound found to have the ability to modulate
transcriptional activity of HRCA2 TAD, with therapeutic
potential in anti-tumour treatment, may be used in
combination with any other anti-tumour compound. In such a
case, the assay of the invention, when conducted in vivo,
need not measure the degree of inhibition of binding or
transcriptional activation caused by the inhibitor compound
being tested. Instead the effect on tumorigenicity may be
measured. It may be that such a modified assay is run in
parallel or subsequent to the main assay of the invention in
order to confirm that any effect on tumorigenicity is as a
result of the inhibition of binding or transcriptional
activation caused by said inhibitor compound and not merely a
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general toxic ef fe~ct .
The present invention extends in various aspects not
only to a substance identified as a modulator of BRCA2
transcriptional activity, in accordance with what is
disclosed herein, but also a pharmaceutical composition,
medicament, drug or other composition including such a
substance, a method including administration of such a
composition to a patient, e.g. for anti-tumour or other anti-
proliferative treatment, which may include preventative
treatment, use of such a substance in manufacture of a
composition for administration, e.g. for anti-tumour or other
anti-proliferative treatment, and a method of making a
pharmaceutical composition including admixing such a
substance with a pharmaceutically acceptable excipient,
vehicle or carrier, and optionally other ingredients.
Also encompassed within the scope of the present
invention are functional mimetics of peptide fragments of
BRCA2 polypeptide TAD, IR1 or IR2, or the binding ligand or
ligands for IR1 and/or IR2, which interfere with interaction
between these polypeptides, and/or transcriptional activation
by BRCA2 TAD. The term "functional mimetic" means a
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substance which may not contain an active portion of the
- relevant amino acid sequence, and probably is not a peptide
at all, but which retains the relevant interfering activity.
The design and screening of candidate mimetics is described
in detail below.
A substance identified using the present invention may
be peptide or non-peptide in nature. Non-peptide "small
molecules" are often preferred for many in vivo
pharmaceutical uses. Accordingly, a mimetic or mimick of the
substance (particularly if a peptide) may be designed for
pharmaceutical use.
The designing of mimetics to a known pharmaceutically
active compound is a known approach to the development of
pharmaceuticals based on a "lead" compound. This might be
desirable where the active compound is difficult or expensive
to synthesise or where it is unsuitable for a particular
method of administration, e.g. peptides are not well suited
as active agents for oral compositions as they tend to be
quickly degraded by proteases in the alimentary canal.
Mimetic design, synthesis and testing may be used to avoid
randomly screening large number of molecules for a target
property.
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There are several steps commonly taken in the design of
-a mimetic from a compound having a given target property.
Firstly, the particular parts of the compound that are
critical and/or important in determining the target property
5 are determined. In the case of a peptide, this can be done
by systematically varying the amino acid residues in the
peptide, e.g. by substituting each residue in turn. These
parts or residues constituting the active region of the
compound are known as its "pharmacophore".
10 Once the pharmacophore has been found, its structure is
modelled to according its physical properties, e.g.
stereochemistry, bonding, size and/or charge, using data from
a range of sources, e.g. spectroscopic techniques, X-ray
diffraction data and NMR. Computational analysis, similarity
15 mapping (which models the charge and/or volume of a
pharmacophore, rather than the bonding between atoms) and
other techniques can be used in this modelling process.
In a variant of this approach, the three-dimensional
structure of the ligand and its binding partner are modelled.
20 This can be especially useful where the ligand and/or binding
. partner change conformation on binding, allowing the model to
take account of this the design of the mimetic.
A template molecule is then selected onto which chemical
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groups which mimic the pharmacophore can be grafted. The
-template molecule and the chemical groups grafted on to it
can conveniently be selected so that the mimetic is easy to
synthesise, is likely to be pharmacologically acceptable, and
does not degrade in vivo, while retaining the biological
activity of the lead compound. The mimetic or mimetics found
by this approach can then be screened to see whether they
have the target property, or to what extent they exhibit it.
Further optimisation or modification can then be carried out
to arrive at one or more final mimetics for in vivo or
clinical testing.
Whether it is a polypeptide, antibody, peptide, nucleic
acid molecule, small molecule, mimetic or other
pharmaceutically useful compound according to the present
invention that is to be given to an individual,
administration is preferably in a "prophylactically effective
amount" or a "therapeutically effective amount" (as the case
may be, although prophylaxis may be considered therapy), this
being sufficient to show benefit to the individual. The
actual amount administered, and rate and time-course of
administration, will depend on the nature and severity of
what is being treated. Prescription of treatment, e.g.
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decisions on dosage etc, is within the responsibility of
-general practitioners and other medical doctors.
A composition may be administered alone or in
combination with other treatments, either simultaneously or
sequentially dependent upon the condition to be treated.
Pharmaceutical compositions according to the present
invention, and for use in accordance with the present
invention, may include, in addition to active ingredient, a
pharmaceutically acceptable excipient, carrier, buffer,
stabiliser or other materials well known to those skilled in
the art. Such materials should be non-toxic and should not
interfere with the efficacy of the active ingredient. The
precise nature of the carrier or other material will depend
on the route of administration, which may be oral, or by
injection, e.g. cutaneous, subcutaneous or intravenous.
Pharmaceutical compositions for oral administration may
be in tablet, capsule, powder or liquid form. A tablet may
include a solid carrier such as gelatin or an adjuvant.
Liquid pharmaceutical compositions generally include a liquid
carrier such as water, petroleum, animal or vegetable oils,
mineral oil or synthetic oil. Physiological saline solution,
dextrose or other saccharide solution or glycols such as
ethylene glycol, propylene glycol or polyethylene glycol may
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be included.
- For intravenous, cutaneous or subcutaneous injection, or
injection at the site of affliction, the active ingredient
will be in the form of a parenterally acceptable aqueous
solution which is pyrogen-free and has suitable pH,
isotonicity and stability. Those of relevant skill in the
art are well able to prepare suitable solutions using, for
example, isotonic vehicles such as Sodium Chloride Injection,
Ringer's Injection, Lactated Ringer's Injection.
Preservatives, stabilisers, buffers, antioxidants and/or
other additives may be included, as required.
A polypeptide, peptide or other substance able to
interfere with the interaction of the relevant polypeptide,
peptide or other substance as disclosed herein may be
provided in a kit, e.g. sealed in a suitable container which
protects its contents from the external environment. Such a
kit may include instructions for use.
The experimental evidence included below demonstrates
that a mutation within the BRCA2 TAD which is associated with
familial breast cancer severely reduces the ability of the
TAD to activate transcription.
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Given such experimental evidence, oligonucleotides
-designed to hybridise to a region within the TAD or IR1 or
IR2 may be used in diagnostic and prognostic screening.
Oligonucleotide probes or primers, as well as the full-
length BRCA2 TAD (optionally including IR1 and/or IR2)
sequence (and mutants, alleles, variants and derivatives) are
useful in screening a test sample containing nucleic acid for
the presence of alleles, mutants and variants, especially
those that confer susceptibility or predisposition to
proliferative disorders, including cancers, the probes
hybridising with a target sequence from a sample obtained
from the individual being tested. The conditions of the
hybridisation can be controlled to minimise non-specific
binding, and preferably stringent to moderately stringent
hybridisation conditions are preferred. The skilled person
is readily able to design such probes, label them and devise
suitable conditions for the hybridisation reactions, assisted
by textbooks such as Sambrook et al (1989) and Ausubel et al
(1992) .
Nucleic acid isolated and/or purified from one or more
cells (e. g. human) or a nucleic acid library derived from
nucleic acid isolated and/or purified from cells (e. g. a cDNA
library derived from mRNA isolated from the cells), may be
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probed under conditrions for selective hybridisation and/or
-subjected to a specific nucleic acid amplification reaction
such as the polymerase chain reaction (PCR).
A method may include hybridisation of one or more (e. g.
5 two) probes or primers to target nucleic acid. Where the
nucleic acid is double-stranded DNA, hybridisation will
generally be preceded by denaturation to produce single-
stranded DNA. The hybridisation may be as part of a PCR
procedure, or as part of a probing procedure not involving
10 PCR. An example procedure would be a combination of PCR and
low stringency hybridisation. A screening procedure, chosen
from the many available to those skilled in the art, is used
to identify successful hybridisation events and isolated
hybridised nucleic acid.
I5 Binding of a probe to target nucleic acid (e.g. DNA) may
be measured using any of a variety of techniques at the
disposal of those skilled in the art. For instance, probes
may be radioactively, fluorescently or enzymatically
labelled. Other methods not employing labelling of probe
20 include examination of restriction fragment length
polymorphisms, amplification using PCR, RNAase cleavage and
allele specific oligonucleotide probing.
Probing may employ the standard Southern blotting
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technique. For instance DNA may be extracted from cells and
-digested with different restriction enzymes. Restriction
fragments may then be separated by electrophoresis on an
agarose gel, before denaturation and transfer to a
nitrocellulose filter. Labelled probe may be hybridised to
the DNA fragments on the filter and binding determined. DNA
for probing may be prepared from RNA preparations from cells.
Those skilled in the art are well able to employ
suitable conditions of the desired stringency for selective
l0 hybridisation, taking into account factors such as
oligonucleotide length and base composition, temperature and
so on.
PCR techniques for the amplification of nucleic acid are
described in US Patent No. 4,683,195. In general, such
techniques require that sequence information from the ends of
the target sequence is known to allow suitable forward and
reverse oligonucieotide primers to be designed to be
identical or similar to the polynucleotide sequence that is
the target for the amplification. PCR includes steps of
denaturation of template nucleic acid (if double-stranded),
a annealing of primer to target, and polymerisation. The
nucleic acid probed or used as template in the amplification
reaction may be genomic DNA, cDNA or RNA. PCR can be used to
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amplify specific sequences from genomic DNA, specific RNA
-sequences and cDNA transcribed from mRNA, bacteriophage or
plasmid sequences. References for the general use of PCR
techniques include Mullis et al, Cold Spring Harbor Symp.
Quant. Biol., 51:263, (1987), Ehrlich (ed), PCR technology,
Stockton Press, NY, 1989, Ehrlich et al, Science, 252:1643-
1650, (1991), "PCR protocols; A Guide to Methods and
Applications", Eds. Innis et al, Academic Press, New York,
(1990).
On the basis of amino acid sequence information, -
oligonucleotide probes or primers may be designed, taking
into account the degeneracy of the genetic code, and, vuhere
appropriate, cadon usage of the organism from the candidate
nucleic acid is derived. An oligonucleotide for use in
nucleic acid amplification may have about 10 or fewer codons
(e.g. 6, 7 or 8), i.e. be about 30 or fewer nucleotides in
length (e.g. 18, 21 or 24). Generally specific primers are
upwards of 14 nucleotides in length, but generally need not
be more than 18-20. Those skilled in the art are well versed
in the design of primers for use processes such as PCR.
A further aspect of the present invention provides an
oligonucleotide or polynucleotide fragment of the nucleotide
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sequence shown for .the BRCA2 TAD, IR1 and IR2 regions in
Figure 2 or a complementary sequence, in particular for use
in a method of obtaining and/or screening nucleic acid. The
sequences referred to above may be modified by addition,
substitution, insertion or deletion of one or more
nucleotides, but preferably without abolition of ability to
hybridise selectively with nucleic acid with the sequence
shown for the BRCA2 region in Figure 2, that is wherein the
degree of homology of the oligonucleotide or polynucleotide
with the sequence given is sufficiently high.
In some preferred embodiments, oligonucleotides
according to the present invention that are fragments of the
sequences shown for regions of BRCA2 in Figure 2, or any
allele associated with susceptibility to cancer or other
disorder of cell proliferation, are at least about 10
nucleotides in length, more preferably at least about 15
nucleotides in length, more preferably at least about 20
nucleotides in length. Such fragments themselves
individually represent aspects of the present invention.
Fragments and other oligonucleotides may be used as primers
or probes as discussed but may also be generated (e.g. by
PCR) in methods concerned with determining the presence in a
test sample of a sequence indicative of susceptibility to
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cancer or other disorder of cell-cycle regulation.
- Preferred probes or primers according to certain
embodiments of this aspect of the present invention are
designed to hybridise with and/or amplify a fragment of the
BRCA2 TAD, shown in Figure 2, including the codon for residue
42, mutation at which is associated with cancer
susceptibility and is demonstrated herein to severely reduce
transcriptional activation by the BRCA2 TAD. This is as a
result of a change in the coding nucleotide sequence from TAT
to TGT.
Thus, suitable oligonucleotides for probing or priming
around codon 42 or other codon associated with disease
susceptibility in the coding sequence shown in Figure 2 may
be about 20 nucleotides in length, or may include a
contiguous sequence of about 20 nucleotides of the coding
sequence shown (either sense strand or anti-sense strand,
also known as coding and non-coding strands respectively).
The oligonucleotide sequence may be designed so that it
anneals to the sense or anti-sense strand with the codon of
interest (e. g. 42) towards the middle of the oligonucleotide,
or at or adjacent the 3' or 5' end, different possibilities
being preferred for different purposes.
Exemplary sequences of oligonucleotides designed to
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anneal around codon.42 of the wild-type sequence shown in
Figure 2 thus include:
5'-TATAATTCTGAACCTGCAGA-3'
(corresponding to a portion of the sense strand, and thus
5 complementary to the anti-sense strand with codon 42 at the
5' end)
5'-ATAATTCTGAACCTGCAGAT-3'
(corresponding to a portion of the sense strand, and thus
complementary to the anti-sense strand with the nucleotide
10 (A) that is changed (to T) in the mutation discussed herein
at the 5' end)
5'-ATAGGGTGGAGCTTCTGAAG-3'
(corresponding to a portion of the anti-sense strand, and
thus complementary to the sense strand with the anti-codon
15 for codon 42 at the 5' end of the oligonucleotide)
5'-TAGGGTGGAGCTTCTGAAGA-3'
(corresponding to a portion of the anti-sense strand, and
thus complementary to the sense strand with the nucleotide
complementary to the A that is changed in the mutation
20 discussed herein at the 5' end of the oligonucleotide)
- 5'-GCTCCACCCTATAATTCTG-3'
5'-CAGAATTATAGGGTGGAGC-3
(respectively complementary to the anti-sense and sense
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strands having codon 42 (or its complement) towards the
_middle of the oligonucleotides).
These oligonucleotides will anneal without mismatch to
the wild-type sequence but will include a mismatch where the
TAT at codon 42 is mutated to TGT.
Oligonucleotides that will anneal to the mutated
sequence including TGT at codon 42 without mismatch, and the
wild-type sequence with mismatch, include
5'-TGTAATTCTGAACCTGCAGA-3'
5'-ACAATTCTGAACCTGCAGAT-3'
5'-GTAGGGTGGAGCTTCTGAAG-3'
5'-CAGGGTGGAGCTTCTGAAGA-3'
5'-GCTCCACCCTGTAATTCTG-3'
5'-CAGAATTACAGGGTGGAGC-3
(corresponding to the oligonucleotides given above which
anneal without mismatch to the wild-type sequence, but with
the requisite change at the position corresponding to codon
42) .
Similarly designed oligonucleotides may be employed for
any mutation or other sequence alteration within a BRCA2
fragment according to the present invention.
Detection of mismatches and other techniques useful for
screening of mutations are discussed further below.
i
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Other oligonucleotides useful in accordance with various
-aspects of the present invention, such as methods involving
amplification of sequences encoding particular BRCA2
fragments, include:
5'-ATGCCTATTGGATCCAAAGA-3'
(complementary to the 3' end of the antisense strand of the
coding sequence shown in Figure 2)
5'-GAACTTGACCAAGACATATC-3'
(complementary to the 3' end of the sense strand of the
coding sequence shown in Figure 2)
5'-ATCCATTTTAGTTTTCACTG-3'
(complementary to the sense strand of the coding sequence at
the 3' end of the region encoding IR2, ending with codon 125
in Figure 2)
5'-TAAGTCTAATTTGAATTTAT-3'
(complementary to the sense strand of the coding sequence at
the 3' end of the region encoding the TAD AAR, ending with
codon 105)
5'-AACCTATTTAAACTCCACAA-3'
(complementary to the anti-sense strand of the coding
sequence at its 3' end corresponding to the 5' end of the
region of the sense strand encoding the TAD AAR, beginning
with codon 60)
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5'-GTTTGGTTCGTAATTGTTGT-3'
(complementary to the sense strand of the coding sequence at
the 3' end of the region encoding the TAD PAR, ending with
codon 6 0 )
5'-CGCTGCAACAAAGCAGATTT-3'
(complementary to the anti-sense strand of the coding
sequence at its 3' end corresponding to the 5' end of the
region of the sense strand encoding the TAD PAR, beginning at
codon 18 ) .
These and other oligonucleotides may be used in various
combinations to amplify particular regions of the BRCA2
fragment, including the whole fragment for which the sequence
is shown in Figure 2, the TAD, the TAD AAR, the TAD PAR, and
the TAD plus IR1 and/or IR2.
Methods involving use of nucleic acid in diagnostic
and/or prognostic contexts, for instance in determining
susceptibility to, e.g., cancer, and other methods concerned
with determining the presence of sequences indicative of,
e.g., cancer susceptibility
A number of methods are known in the art for analysing
biological samples from individuals to determine whether the
individual carries an BRCA2 allele with a mutation within the
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region for which the sequence is shown in Figure 2
-predisposing them to disease. The purpose of such analysis
may be used for diagnosis or prognosis, and serve to detect
the presence of, e.g., an existing cancer, to help identify
the type of cancer, to assist a physician in determining the
severity or likely course of the cancer and/or to optimise
treatment of it. The methods may be used to detect alleles
that are statistically associated with a susceptibility to
cancer or other proliferative disorder in the future, e.g.
early onset cancer, identifying individuals who would benefit
from regular screening to provide early diagnosis of cancer.
Broadly, the methods divide into those screening for the
presence of nucleic acid sequences and those that rely on
detecting the presence or absence of polypeptide. The
methods make use of biological samples from individuals that
are suspected of contain the nucleic acid sequences or
polypeptide. Examples of biological samples include blood,
plasma, serum, tissue samples, tumour samples, saliva and
urine.
Exemplary approaches for detecting nucleic acid or
polypeptides include:
(a) comparing the sequence of nucleic acid in the sample
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with an BRCA2 nucleic acid sequence to determine whether the
_ sample from the patient contains one or more mutations in a
region for which the sequence is shown in Figure 2; or,
(b) determining the presence in a sample from a patient
5 of the polypeptide encoded by BRCA2 and, if present,
determining whether the polypeptide is includes a region
corresponding to that shown in Figure 2 (e. g. including TAD,
IR1 and/or IR2), and/or is mutated in such a region; or,
(c) using DNA fingerprinting to compare the restriction
10 pattern produced when a restriction enzyme cuts a sample_of
nucleic acid from the patient With the restriction pattern
obtained from a region corresponding to that shown in Figure
2 for normal gene or from known mutations thereof; or,
(d) using a specific binding member capable of binding
15 to a nucleic acid sequence (either a normal sequence or a
known mutated sequence) encoding a fragment of BRCA2
corresponding to a region for which the sequence is shown in
Figure 2 (e. g. including TAD, IR1 and/or IR2), the specific
binding member including nucleic acid hybridisable with the
20 BRCA2 sequence, or substances including an antibody domain
with specificity f or a native or mutated BRCA2 fragment
nucleic acid sequence or the polypeptide encoded by it, the
specific binding member being labelled so that binding of the
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specific binding member to its binding partner is detectable;
-or ,
(e) using PCR involving one or more primers based on
normal or mutated BRCA2 gene sequence to screen for normal or
mutant sequences within the region of BRCA2 corresponding to
a region for which the sequence is shown in Figure 2 (e. g.
including TAD, IR1 and/or IR2) in a sample from a patient.
A "specific binding pair" includes a specific binding
member (sbm) and a binding partner (bp) which have a
particular specificity for each other and which in normal
conditions bind to each other in preference to other
molecules. Examples of specific binding pairs are antigens
and antibodies (see above), molecules and receptors and
complementary nucleotide sequences. The skilled person will
be able to think of many other examples and they do not need
to be listed here. Further, the term "specific binding pair"
is also applicable where either or both of the specific
binding member and the binding partner include a part of a
larger molecule. In embodiments in which the specific
binding pair are nucleic acid sequences, they will be of a
length to hybridise to each other under the conditions of the
assay, preferably greater than 10 nucleotides long, more
preferably greater than 15 or 20 nucleotides long.
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In most embodiments for screening for susceptibility
alleles, the BRCA2 nucleic acid in the sample will initially
be amplified, e.g. using PCR, to increase the amount of the
analyte as compared to other sequences present in the sample.
This allows the target sequences to be detected with a high
degree of sensitivity if they are present in the sample.
This initial step may be avoided by using highly sensitive
array techniques that are becoming increasingly important in
the art.
To reiterate in further detail, the identification of a
significant region of the BRCA2 gene and its implication with
disorders of cell proliferation paves the way for aspects of
the present invention to provide the use of materials and
methods, such as are disclosed and discussed above, for
establishing the presence or absence in a test sample of an
variant form of the gene, in particular an allele or variant
specifically associated with cancer. This may be for
diagnosing a predisposition of an individual to cancer. It
may be for diagnosing cancer of a patient with the disease as
being associated with the gene.
This allows for planning of appropriate therapeutic
and/or prophylactic treatment, permitting stream-lining of
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treatment by targeting those most likely to benefit.
_ A variant form of the gene may contain one or more
insertions, deletions, substitutions and/or additions of one
or more nucleotides compared with the wild-type sequence
which may or may not disrupt the transcriptional activation
function of the region examined herein. Differences at the
nucleic acid level are not necessarily reflected by a
difference in the amino acid sequence of the encoded
poiypeptide. However, a mutation or other difference in a
gene may result in a frame-shift or stop codon, which could
seriously affect the nature of the polypeptide produced, or a
point mutation or gross mutational change to the encoded
polypeptide, including insertion, deletion, substitution
and/or addition of one or more amino acids or regions in the
polypeptide, which may affect transcriptional activation.
There are various methods for determining the presence
or absence in a test sample of a particular nucleic acid
sequence, such as the sequence shown for a BRCA2 fragment in
Figure 2 or a mutant, variant or allele thereof.
Tests may be carried out on preparations containing
genomic DNA, cDNA and/or mRNA. Testing cDNA or mRNA has the
advantage of the complexity of the nucleic acid being reduced
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by the absence of intron sequences, but the possible
disadvantage of extra time and effort being required in
making the preparations. RNA is more difficult to manipulate
than DNA because of the wide-spread occurrence of RN'ases.
Nucleic acid in a test sample may be sequenced and the
sequence compared with the sequence shown in Figure 2 to
determine whether or not a difference is present. If so, the
difference can be compared with known susceptibility alleles
to determine whether the test nucleic acid contains one or
more of the variations indicated, or the difference can be
investigated for association with cancer.
Since it will not generally be time- or labour-efficient
to sequence all nucleic acid in a test sample or even the
whole BRCA2 gene fragment corresponding to the region shown
in Figure 2, a specific amplification reaction such as PCR
using one or more pairs of primers may be employed to amplify
the region of interest in the nucleic acid. The amplified
nucleic acid may then be sequenced as above, and/or tested in
any other way to determine the presence or absence of a
particular feature. Nucleic acid for testing may be prepared
from nucleic acid removed from cells or in a library using a
variety of other techniques such as restriction enzyme digest
and electrophoresis.
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Nucleic acid may be screened using a variant- or allele-
specific probe. Such a probe corresponds in sequence to a
region of the BRCA2 gene, or its complement, containing a
sequence alteration known to be associated with
5 susceptibility to cancer or other proliferative disorder.
Under suitably stringent conditions, specific hybridisation
of such a probe to test nucleic acid is indicative of the
presence of the sequence alteration in the test nucleic acid.
For efficient screening purposes, more than one probe may be
10 used on the same test sample.
Allele- or variant-specific oligonucleotides may
similarly be used in PCR to specifically amplify particular
sequences if present in a test sample. Assessment of whether
a PCR band contains a gene variant may be carried out in a
15 number of ways familiar to those skilled in the art. The PCR
product may for instance be treated in a way that enables one
to display the mutation or polymorphism on a denaturing
polyacrylamide DNA sequencing gel, with specific bands that
are linked to the gene variants being selected.
20 An alternative or supplement to looking for the presence
of variant sequences in a test sample is to look for the
presence of the normal sequence, e.g. using a suitably
specific oligonucleotide probe or primer.
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Use of oligonucleotide probes and primers has been
discussed in more detail above.
Approaches which rely on hybridisation between a probe
and test nucleic acid and subsequent detection of a mismatch
may be employed. Under appropriate conditions (temperature,
pH etc.), an oligonucleotide probe will hybridise with a
sequence which is not entirely complementary. The degree of
base-pairing between the two molecules will be sufficient for
them to anneal despite a mis-match. Various approaches are
well known in the art for detecting the presence of a mis-
match between two annealing nucleic acid molecules.
For instance, RN'ase A cleaves at the site of a mis-
match. Cleavage can be detected by electrophoresing test
nucleic acid to which the relevant probe or probe has
annealed and looking for smaller molecules (i.e. molecules
with higher electrophoretic mobility) than the full length
probe/test hybrid. Other approaches rely on the use of
enzymes such as resolvases or endonucleases.
Thus, an oligonucleotide probe that has the sequence of
a region of the normal BRCA2 gene (either sense or anti-sense
strand) corresponding to the fragment shown in Figure 2 in
which at least one mutation associated with, e.g., cancer
susceptibility is known to occur, may be annealed to test
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nucleic acid and the presence or absence of a mis-match
determined. Detection of the presence of a mis-match may
indicate the presence in the test nucleic acid of a mutation
associated with, e.g., cancer susceptibility. On the other
hand, an oligonucleotide probe that has the sequence of a
region of the BRCA2 gene including a mutation associated
with, e.g., cancer susceptibility may be annealed to test
nucleic acid and the presence or absence of a mis-match
determined. The presence of a mis-match may indicate that
the nucleic acid in the test sample has the normal sequence.
In either case, a battery of probes to different regions of
the gene may be employed.
The presence of differences in sequence of nucleic acid
molecules may be detected by means of restriction enzyme
digestion, such as in a method of DNA fingerprinting where
the restriction pattern produced when one or more restriction
enzymes are used to cut a sample of nucleic acid is compared
with the pattern obtained when a sample containing the normal
gene or a variant or allele is digested with the same enzyme
or enzymes.
A test sample of nucleic acid may be provided for
example by extracting nucleic acid from cells, e.g. in saliva
or preferably blood, or for pre-natal testing from the
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amnion, placenta or foetus itself.
Nucleic acid according to the present invention, such as
a full-length coding sequence or oligonucleotide probe or
primer, may be provided as part of a kit, e.g. in a suitable
container such as a vial in which the contents are protected
from the external environment. The kit may include
instructions for use of the nucleic acid, e.g. in PCR and/or
a method for determining the presence of nucleic acid of
interest in a test sample. A kit wherein the nucleic acid is
intended for use in PCR may include one or more other
reagents required for the reaction, such as polymerase,
nucleosides, buffer solution etc. The nucleic acid may be
labelled. A kit for use in determining the presence or
absence of nucleic acid of interest may include one or more
articles and/or reagents for performance of the method, such
as means for providing the test sample itself, e.g. a swab
for removing cells from the buccal cavity or a syringe for
removing a blood sample (such components generally being
sterile). In a further aspect, the present invention
provides an apparatus for screening for BRCA2 TAD, IR1 and/or
IR2 nucleic acid, the apparatus including storage means
including the BRCA2 nucleic acid sequence as set out in
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Figure 2, or a fragment thereof, the stored sequence being
_used to compare the sequence of the test nucleic acid to
determine the presence of mutations.
There are various methods for determining the presence
or absence in a test sample of a particular polypeptide, such
as a polypeptide including a fragment of BRCA2 corresponding
to a region for which the amino acid sequence is shown in
Figure 2 or an amino acid sequence mutant, variant or allele
thereof.
A sample may be tested for the presence of a binding
partner for a specific binding member such as an antibody (or
mixture of antibodies), specific for one or more particular
variants of the BRCA2 polypeptide shown in the figures, or a
mutant, variant or allele thereof.
A sample may be tested for the presence of a binding
partner for a specific binding member such as an antibody (or
mixture of antibodies), specific for the BRCA2 polypeptide
shown in the figures.
In such cases, the sample may be tested by being
contacted with a specific binding member such as an antibody
under appropriate conditions for specific binding, before
binding is determined, for instance using a reporter system
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as discussed. Where a panel of antibodies is used, different
_reporting labels may be employed for each antibody so that
binding of each can be determined.
A specific binding member such as an antibody may be
5 used to isolate and/or purify its binding partner polypeptide
from a test sample, to allow for sequence and/or biochemical
analysis of the polypeptide to determine whether it has the
sequence and/or properties of the BRCA2 polypeptide whose
sequence is shown in the figures, or if it is a mutant or
10 variant form. Amino acid sequence is routine in the art
using automated sequencing machines.
Nucleic acid according to the present invention,
encoding a polypeptide functional as a transcriptional
15 activator, may be used in methods of gene therapy, for
instance in treatment of individuals with the aim of
preventing or curing (wholly or partially) cancer or other
disorder involving loss of proper regulation of the cell-
cycle and/or cell growth.
20 Nucleic acid encoding an authentic biologically active
BRCA2 fragment polypeptide, i.e. with ability to activate
transcription, may be used in a method of gene therapy, to
treat a patient who is unable to synthesize the active
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polypeptide or unable to synthesize it at the normal level,
thereby providing the effect provided by wild-type and
suppressing the occurrence of cancer and/or reduce the size
or extent of existing defects in cell-cycle and/or growth
regulation in the target cells.
Vectors such as viral vectors have been used in the
prior art to introduce genes into a wide variety of different
target cells. Typically the vectors are exposed to the
target cells so that transfection can take place in a
sufficient proportion of the cells to provide a useful
therapeutic or prophylactic effect from the expression of the
desired polypeptide. The transfected nucleic acid maybe
permanently incorporated into the genome of each of the
targeted tumour cells, providing long lasting effect, or
alternatively the treatment may have to be repeated
periodically.
A variety of vectors, both viral vectors and plasmid
vectors, are known in the art, see US Patent No. 5,252,479
and WO 93/07282. In particular, a number of viruses have
been used as gene transfer vectors, including papovaviruses,
such as SV40, vaccinia virus, herpesviruses, including HSV
and EBV, and retroviruses. Many gene therapy protocols in
the prior art have used disabled murine retroviruses.
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As an alternative to the use of viral vectors other
_known methods of introducing nucleic acid into cells includes
electroporation, calcium phosphate co-precipitation,
mechanical techniques such as microinjection, transfer
mediated by liposomes and direct DNA uptake and receptor-
mediated DNA transfer.
As mentioned above, the aim of gene therapy using
nucleic acid encoding an BRCA2 polypeptide, or an active
portion thereof, is to increase the amount of the expression
product of the nucleic acid in cells in which the level of -
the wild-type polypeptide is absent or present only at
reduced levels. Such treatment may be therapeutic in the
treatment of cells which are already cancerous or pre-
cancerous or prophylactic in the treatment of individuals
known through screening to have an BRCA2 susceptibility
allele and hence a predisposition to cancer.
Receptor-mediated gene transfer, in which the nucleic
acid is linked to a protein ligand via polylysine, with the
ligand being specific for a receptor present on the surface
of the target cells, is an example of a technique for
specifically targeting nucleic acid to particular cells.
As noted, a polypeptide termed "BBP1" (BRCA2 Binding
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Protein 1) has been found to interact with the PAR of BRCA2
_and a portion of it able to repress transcriptional
activation by BRCA2 TAD.
The isolated polypeptide, isolated nucleic acid encoding
it, vectors and host cells including the nucleic acid, and
methods of making the polypeptide all represent further
aspects of the present invention.
Thus in one aspect the present invention provides a
polypeptide including the amino acid sequence shown in Figure
3 or the amino acid sequence shown in Figure 4. In one
embodiment a polypeptide according to the present invention
may be approximately up to about 2000 amino acids, or as
encoded by the approximately 7.5kb mRNA discussed below. In
one embodiment a polypeptide according to the invention
includes the amino acid sequence of Figure 3 or Figure 4, is
able to bind BRCA2 TAD and is obtainable from human ovary or
testis cells. Antibodies directed against the amino acid
sequence of Figure 3 or Figure 4, or a suitable fragment
thereof, may be used in isolation and/or purification of the
polypeptide from ovary or testis cells. The polypeptide may
be provided in isolated form and may be formulated into a
composition containing at least one additional component,
such as a pharmaceutically acceptable excipient.
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Nucleic acid encoding the BBP1 polypeptide is also
provided as an aspect of the present invention. The
invention provides a nucleic acid molecule including the
encoding nucleotide sequence shown in Figure 3 or Figure 4.
The nucleic acid molecule may be RNA of approximately 7.5 kb
including said encoding sequence (the RNA equivalent) or a
cDNA copy of such RNA and obtainable by probing a cDNA
library or RNA generated from human ovary or testes.
Nucleic acid according to this aspect of the present
invention is obtainable using one or more oligonucleotide
probes or primers designed to hybridise with one or more
fragments of the nucleic acid sequence shown in Figure 3 or
Figure 4, particularly fragments of relatively rare sequence,
based on codon usage or statistical analysis. A primer
designed to hybridise with a fragment of the nucleic acid
sequence shown in Figure 3 or Figure 4 may be used in
conjunction with one or more oligonucleotides designed to
hybridise to a sequence in a cloning vector within which
target nucleic acid has been cloned, or in so-called "RACE"
(rapid amplification of cDNA ends) in which cDNA's in a
library are ligated to an oligonucleotide linker and PCR is
performed using a primer which hybridises with the sequence
shown in Figure 3 or Figure 4 and a primer which hybridises
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to the oligonucleotide linker.
In the context of cloning, it may be necessary for one
or more gene fragments to be ligated to generate a full-
length coding sequence. Also, where a full-length encoding
5 nucleic acid molecule has not been obtained, a smaller
molecule representing part of the full molecule, may be used
to obtain full-length clones. Inserts may be prepared from
partial cDNA clones and used to screen cDNA libraries. The
full-length clones isolated may be subcloned into expression
10 vectors and activity assayed by transfectio.l into suitable
host cells, e.g. with a reporter plasmid.
A method may include hybridisation of one or more (e. g.
two) probes or primers to target nucleic acid. Where the
nucleic acid is double-stranded DNA, hybridisation will
15 generally be preceded by denaturation to produce single-
stranded DNA. The hybridisation may be as part of a PCR
procedure, or as part of a probing procedure not involving
PCR. An example procedure would be a combination of PCR and
low stringency hybridisation. A screening procedure, chosen
20 from the many available to those skilled in the art, is used
to identify successful hybridisation events and isolated
hybridised nucleic acid.
Binding of a probe to target nucleic acid (e.g. DNA) may
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be measured using any of a variety of techniques at the
disposal of those skilled in the art. For instance, probes
may be radioactively, fluorescently or enzymatically
labelled. Other methods not employing labelling of probe
include examination of restriction fragment length
polymorphisms, amplification using PCR, RNAase cleavage and
allele specific oligonucleotide probing.
Probing may employ the standard Southern blotting
technique. For instance DNA may be extracted from cells and
digested with different restriction enzymes. Restriction
fragments may then be separated by electrophoresis on an
agarose gel, before denaturation and transfer to a
nitrocellulose filter. Labelled probe may be hybridised to
the DNA fragments on the filter and binding determined. DNA
for probing may be prepared from RNA preparations from cells.
Preliminary experiments may be performed by hybridising
under low stringency conditions various probes to Southern
blots of DNA digested with restriction enzymes. Suitable
conditions would be achieved when a large number of
hybridising fragments were obtained while the background
hybridisation was low. Using these conditions nucleic acid
libraries, e.g. cDNA libraries representative of expressed
sequences, may be searched.
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Those skilled, in the art are well able to employ
_suitable conditions of the desired stringency for selective
hybridisation, taking into account factors such as
oligonucleotide length and base composition, temperature and
so on.
The BBP1 polypeptide, fragments, mutants, variants and
derivatives thereof, and encoding nucleic acid may be used in
the same or similar terms as fragments of BRCA2 as discussed
above, e.g. in assays. Peptide fragments may be used to
modulate transcriptional activation by BRCA2.
Further aspects and embodiments will be apparent to
those of ordinary skill in the art upon consideration of the
above disclosure and the following experimental report,
presented by way of illustration of embodiments of the
present invention and without limitation.
All documents mentioned in this specification are hereby
incorporated herein by reference.
' 20
EXPERIMENTAL EXEMPLIFICATION
The BRCA2 gene encodes a large 3418 residue protein of
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unknown function which is found mutated in 45°s of familial
breast cancers (1). The present inventors have scrutinised
the BRCA2 protein sequence for any similarity to proteins of
known function. It has surprisingly been found that exon 3
sequences at the N-terminus of BRCA2 (within a region highly
conserved between human and mouse) show sequence similarity
to the activation domain of c-jun (Figure 1). The homology
overlaps the delta region of c-jun which contains the binding
site f or the Jun N-terminal Kinase (JNK, (2)). In view of
the similarity of exon 3 sequences to a transcriptional
activation domain, the inventors investigated whether this
region of BRCA2 has transcriptional activation capacity.
Surprisingly, it has been found that BRCA2 sequences
spanning exon 3 (23-105) are able to activate transcription
in yeast, when linked to the lexA DNA binding domain. In
contrast, all the other highly conserved domains of BRCA2
{919-1171, 1500-1589, 2034-2223 and 3200-3326) do not show
any activation potential in this assay. These various
domains were fused to the lexA DNA binding domain and used to
drive the activity of a promoter including a lexA binding
site, which was linked to the B-gal gene. The results are
shown in Table 1.
BRCA2 exon 3 sequences (18-105) also have potent
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activation potential in two different mammalian cell lines,
'U20S (Figure 1) and NMuMG (data not shown) when these
sequences are linked to the GAL4 DNA binding domain.
Fusions of portions of the BRCA2 protein and the DNA
binding domain of GAL4 (1-147) were co-transfected into U20S
cells along with a target promoter 5GAL4EIBCAT and CAT
activity was then measured 24 h following transfection. The
activity shown represents the relative value compared to the
activity of the GAL4 DNA binding domain alone. The results
represent an average of several independent experiments.- The
expression levels of the different constructs were
established by western blotting using a GAL4 specific
antibody.
The c-jun homology region (60-105) contributes to this
activation potential but has relatively little independent
activity, whereas the adjacent region (18-60) remains
significant, although reduced activation capacity. Residues
18-60 therefore represent a primary activating region (PAR)
whereas residues (60-105) represent an auxiliary activating
region (AAR).
' Within the PAR lies a tyr residue at position 42 which
is found mutated to cys in familial breast cancers (3) (shown
in Figure 2). Introduction of this tyr to cys mutation into
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PAR severely compromises its activation potential.
Further characterisation of this region reveals that the
activation potential within exon 3 is under negative control
of inhibitory regions (IR1 and IR2) present directly N- and
5 C-terminal to exon 3. Together these small regions
completely mask the activation potential of BRCA2. This type
of regulation by inhibitor regions has been shown to be
operational in a number of transcription factors (4,5,6) and
in the case of c-Fos, inhibitor function has been
10 demonstrated in the context of the intact protein assayed on
its natural binding site (4).
These results provide the first insight into the
potential function and regulation of the BRCA2 protein. They
provide indication that (a) BRCA2 has the ability to
15 stimulate transcription, (b) its activation potential is
under negative control by inhibitory sequences and (c) signal
transduction pathways culminating in the stimulation of JNK-
like kinases may regulate BRCA2 activity.
To date two genes, BRAC1 and BRCA2, have been implicated
20 in the generation of familial breast cancers. Although these
two proteins have no apparent sequence similarity,
circumstantial evidence relating mainly to their size and
overlapping expression patterns, has raised the possibility
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that their functions may be related (7). Our finding that
-BRCA2, like BRAC1 (8,9) has transcriptional activation
potential provides the first functional evidence to support
this. Indeed, the fact that the mutations found naturally in
breast cancers disrupt the activation potential of both BRAC1
or BRCA2, argues that compromising this activity may be an
important step in the generation of familial breast cancers.
This is further supported by the Nordling et al. finding
noted above, i.e. that a large deletion disrupting the exon 3
transcription activator domain of BRCA2 is the disease-
causing mutation in a Swedish breast/ovarian cancer family.
Using two hybrid screens, polypeptide regions which
interact with BRCA2 fragments according to the present
invention are identified. Polypeptides or peptides including
such regions may be included in screens and assays as
discussed above and may be used to modulate BRCA2
transcriptional activation. Furthermore, they may themselves
be built into screens and assays such as two hybrid assays to
' 20 obtain and/or identify molecules which modulate or interfere
with their interaction with BRCA2 and/or their action on
BRCA2 transcriptional activation.
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Identification of ,a protein which binds the BRCA2 PAR and
_ modulates i is transcriptional activi ty, and cloning of
encoding nucleic acid
In two hybrid screens (see below) using the BRCA2 PAR a
protein was identified which binds the PAR.
The protein has been termed "BBP1" - BRCA2 Binding
Protein 1.
An initial partial encoding sequence was obtained and is
shown in Figure 4.
More encoding sequence of 2.2kb was obtained. Northerns
showed that the BBP1 message is about 7.Skb, and expressed
highly in ovary and testes, where BRCA2 is also highly
expressed (comparisons with other tissues).
Demonstration that BBPI modulates transcription activity of
the BRCA2 TAD
A portion of the BHP1 protein as shown in Figure 4 was
expressed in mammalian cells as above and shown to repress
activity of the BRCA2 activation domain.
The BRCA2 TAD was linked to the GAL4 DNA binding domain
and a reporter gene under control of a promoter including
GAL4 sites included in the cells.
Titration in of increasing concentrations of the BHP1
a i
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expression vector gave increasing repression of expression of
-the reporter construct.
Mapping of interaction site for BBP1 in BRCA2
Overlapping fragments of BRCA2 TAD were used to map the
BBP1 binding site to within amino acids 18-46 of BRCA2. The
overlap is a highly conserved region in BRCA2 between human
and mouse and is where the noted mutation associated with
breast cancer lies (residue 42).
This provides ample basis for screens and assays for
substances which interfere with interaction between BHP1 and
BRCA2 and have an effect on BRCA2 transcriptional activation.
The following describes two hybrid assay techniques for
yeast. When performing a two hybrid assay to look for
substances which interfere with the interaction between two
polypeptides or peptides it may be preferred to use mammalian
cells instead of yeast cells. The same principles apply and
similar methods are well known to the person skilled in the
art.
Two Hybrid Screens for Interacting Molecules
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The following, method is used to isolate molecules which
_interact with a BRCA2 polypeptide or peptide in accordance
with the present invention.
The yeast two hybrid system is based on a protein
interaction assay in yeast (Fields and Song. 1989. Nature
340, 245-246). The following protocol contains several
modifications of the original Fields strategy and facilitates
large scale library screens. It has been designed and
optimised and was first used by Ann Vojtek to isolate c-raf
and a-raf clones (Vojtek et al. 1993. Cell 74, 205-214): The
method described below is essentially identical to the one
outlined in Vojtek et al. It uses the same set of vectors
(the different bait constructs are described below) and yeast
strains. The Two Hybrid System is based on an in vivo yeast
protein interaction assay.
In general yeast are transformed with a reporter gene
construction which expresses a selective marker protein (e. g.
encoding (3-galactosidase). The promoter of that gene has
been designed such that it contains binding site for the LexA
DNA-binding protein. Gene expression from that plasmid is
usually very low. Two more expression vectors are
transformed into the yeast containing the selectable marker
expression plasmid. The first of those two vectors is based
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on pBTM116. It contains the coding sequence for the full
length LexA gene linked to a multiple cloning site. This
multiple cloning site is used to clone a gene of interest,
i.e. encoding a BRCA2 polypeptide or peptide in accordance
5 with the present invention, in frame on to the LexA coding
region. The second yeast expression vector contains the
activation domain of the herpes simplex transactivator VP16
fused to random sequences of a cDNA library or a library of
sequences encoding peptides with diverse e.g. random
10 sequences (depending on whether the aim is to obtain a
naturally occurring ligand for the HRCA2 polypeptide or
peptide or to screen for peptides which interact). Those two
plasmids facilitate expression from the reporter construct
containing the selectable marker only when the LexA fusion
15 construct (bait) interacts with a polypeptide or peptide
sequence derived from the cDNA or peptide library.
A modification of this when looking for peptides which
interfere with interaction between a BRCA2 polypeptide or
peptide and a ligand or other binding molecule, e.g. for the
20 TAD or for IR1 and/or IR2, employs the BRCA2 polypeptide or
peptide as a fusion with the LexA DNA binding domain, and the
binding molecule as a fusion with VP60, and involves a third
expression cassette, which may be on a separate expression
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vector, from which a peptide or a library of peptides of
diverse and/or random sequence may be expressed. A reduction
in reporter gene expression (e.g. in the case of (3-
galactosidase a weakening of the blue colour) results from
the presence of a peptide which disrupts the interaction
between the BRCA1 polypeptide or peptide and the binding
molecule, which interaction is required for transcriptional
activation of the (3-galactosidase gene.
As noted, instead of using LexA and VP60, other similar
combinations of proteins which together form a functional
transcriptional activator may be used, such as the GAL4 DNA
binding domain and the GAL4 transcriptional activation
domain.
Two Hybrid Plasmids
Amino Acids 1-197 of a human BRCA2 clone are amplified
by PCR and cloned as in frame with the LexA gene in pBTM116,
which contains the TRP1 gene which allows selection of
transformed yeast on tryptophan negative plates.
The pVPl6 library vector carries the LEU2 gene which
allows selection on Leucine negative plates. A human cDNA
library cloned next to the activation domain of VP16 is
generated by random primed synthesis of poly A+ RNA. The
i
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vast majority of inserts have a length of 400 - 600
nucleotides.
Two reporter constructs are in use and both are provided
by the yeast strain L40. The first construct has a
selectable marker, the LYS2 gene, which allows growth on
Lysine negative plates. It contains the coding region for
the histidine gene under the control of a promoter containing
four binding sites for the LexA operator. The second
reporter gene has a UR.A3 gene as selectable marker which
allows growth on uracil negative plates. It contains the
coding region for the lacZ gene controlled by a promoter
containing eight binding sites for the lexA protein.
Yeast Transformation
Small Scale Transforms ion
10 ml of YPAD are inoculated with a colony of L40 and
incubated overnight at 30°C. Thereafter, the culture is
diluted to an OD600 of around 0.4 in 50 ml YPAD and grown for
an additional 2-4 hours. Cells are then pelleted at 2500 rpm
at room temperature and re-suspended in 40 ml TE. Yeast axe
then repelleted at 2500 rpm and resuspended in 2 ml of 100 mM
LiAc in 0.5 x TE. This yeast suspension is incubated at room
temperature for 10 minutes. 1 ~g of plasmid DNA together
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with 100 ug of sonicated sheared salmon sperm DNA is mixed
with 100 ~,1 of the yeast suspension. After a further
addition of a buffer of 700 ~C1 containing 100 mM (LiAc), 40~
PEG-3350 in 1 x TE, the solution is mixed well and incubated
at 30°C for 30 minutes. To stop the transformation process
88 ~.1 DMSO is added and the mixture heat- shocked at 40°C for
7 minutes. Cells are pelletted in a microfuge for 10 seconds
and re-suspended in 1 ml TE. Cells are then re-washed in 1
ml TE and re-suspended in 50-100 ~,l TE and plated on
selective plates. Plates are incubated at 30°C and colonies
picked after 2-3 days.
Small scale transformation may be used to test the
induction of the beta-galactosidase activity by the
LexA/BRCA2 fragment fusion plasmid. The beta-galactosidase
filter assay is described below.
~3e Scale Librarv Transformation
The LexA/BRCA2 fragment fusion plasmid is introduced
into L40 by selecting for growth on tryptophan minus plates
after a small scale transformation. The resulting strain is
used to grow a 2 ml overnight culture in yeast selective
medium minus tryptophan and minus uracil. Thereafter, the
culture is diluted with 100 ml of the same medium. The next
i
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day the mid log phase culture is used to inoculate 1 litre of
YPAD medium (pre-warmed to 30°C). The optical density at 600
nm should be about 0.3. This culture is grown at 30°C for 3
hours. During this time the cells should roughly double in
number. Yeast are pelletted at 2500 rpm for 5 minutes at
room temperature and re-suspended in 500 ml of TE. After a
re-spin the cells are taken up in 10 ml of 100 mM Li Ac in
0.5 x TE. To this a mixture of 0.5 ml of 10 mg/ml denatured
salmon sperm DNA and 200 ~g of library plasmid is added. The
suspension is mixed well. After this 70 ml of a solution
containing 100 mM LiAc, 40% PEG-3350 in 1 x TBE is added and
mixed well. This mixture is incubated for 30 minutes at
30°C. The transformation mixture is then transferred to a
sterile 2 litre beaker and 8.8 ml of DMSO was added. After
mixing the suspension is heat shocked at 42°C in a water bath
for 6 minutes. Thereafter, the suspension is diluted with
200 ml of YPA and rapidly cooled to room temperature in a
water bath. Cells are then pelletted at 2500 rpm for 5
minutes at room temperature, washed with 250 ml YPA medium
and re-suspended in 1 litre of pre-warmed YPAD medium. At
this stage incubation at 30°C is allowed for 1 hour with
gentle shaking. The culture is then pelletted at 2500 rpm
for 5 minutes at room temperature and re-suspended in 500 ml
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of selective medium omitting uracil, tryptophan leucine (-
UTL). After a further respin cells are resuspended in 1
litre of pre-warmed -UTL medium with shaking at 30°C for
about 4 hours. Thereafter, cells are pelletted at 2500 rpm
5 for 5 minutes at room temperature and washed twice with
selective medium omitting tryptophan, histidine, uracil and
leucine (-THULL). The final pellet is resuspended in 10 ml
of -THULL medium and plated in aliquots of 100 ~.1 on plates
made from -THULL media. If the bait alone can activate the
10 ~i-galactosidase gene, this is suppressed by the inclusion of
3 amino 124-triazole at an appropriate concentration (e. g.
50mM). After 2-3 days colonies are picked to a grid. A
nitrocellulose filter lift is used in a beta-galactosidase
filter assay for analysis of lacZ induction.
Beta-galactosidase Filter Assay
Filters are removed from the plates and immersed for 3-5
seconds in liquid nitrogen. Filters are then placed, colony
side up, at room temperature until thawed. The beta-
galactosidase assay is performed in the lid of a petri dish.
3 ml of Z-buffer (60 mM Na2HP04, 40 mM NaH2P04, 10 mM KC1, 1
mM MgS04, pH7.0) containing 30 ml of 50 mg/ml X-gal.
Circularised Whatman filters (#1) are placed into the buffer,
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followed by the nitrocellulose filters, colonies facing up.
The lid is then covered with the bottom of the petri dish and
placed at 30°C. Interactions are detectable by the
appearance of a blue colour after 20 to 40 minutes.
Recovering of Plasmids from Yeast and Shuttling into E.coli
Viable cells are recovered from colonies and grown in a
50 ml overnight culture with the appropriate selection. The
next morning cells are pelletted at 2500 rpm for 5 minutes at
room temperature. Pellets are resuspended in 0.3 ml of lysis
buffer (2.5 M LiCl, 50 mM Tris-C1 (pH 8.0), 4~ Triton X-100,
62.5 mM EDTA). At this stage solution is transferred to 1.5
ml tubes and 150 ml of glass beads (0.45 - 0.50 mm) together
with 0.3 ml phenol/chloroform are added. After vigorous
shaking for 1 minute samples are centrifuged for 1 minute and
the aqueous phase transferred to a new tube. DNA is
precipitated twice with ethanol and resuspended in 25 ml TE
followed by electroporation of DNA into E.coli.
Verification of Interacting Partners
Recovered library plasmids from positive yeast colonies
are retransformed into the L40 strain containing the
LexA/BRCA2 fragment bait vector using the small scale
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transformation procedure. Using the beta-galactosidase
filter assay positive colonies are identified with the
LexA/BRCA2 fragment bait. No induction of beta-galactosidase
activity is detected in colonies transformed with a LexA
instead of a LexA/BRCA 2 fragment clone. The underlying
protein interactions of the positive colonies are significant
and consequently further analysed.
Identification of a Full Length cDNA of A BRCA2 Binding
Molecule
In order to isolate a full length cDNA clone of a
molecule which binds BRCA2 TAD, IR1 and/or IR2, a plasmid
cDNA library is plated on ampicillin resistant plates.
Nitrocellulose filter lifts of those plates are hybridised to
DNA sequences obtained through the yeast two hybrid screen.
Washes are done at high stringency and positive signals are
identified. Isolated DNA is sequenced and if necessary
partial clones are ligated to provide a full length coding
sequence. A full length sequence may be verified as such by
primer walking sequencing in both directions.
Identification of a kinase able to bind the activation domain
of BRCA2
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Using residues 18-141 of BRCA2 as an affinity column a
kinase has been purified from HeLa nuclear extracts which
phosphoryiates within amino acids 60-105 of BRCA2,
specifically the region of HRCA2 which is homologous to the
JNK binding site in c-jun. This was demonstrated by deletion
of residues 80-107 in BRCA2, which abolished binding of the
kinase. This mutation also reduced the activation capacity
of BRCA2. The region of BRCA2 residues 60-105 is
phosphorylated in vivo.
The kinase was found not to be stimulated by uv and is
therefore not JNK. This was confirmed by experiments in
which recombinant JNK was found not to bind the region of
BRCA2.
REFERENCES
1. Wooster, R. et al. (1994).Science, 265, 2088-2090.
2. Derijard, B. et al. (1994) . Cell, 76, 1025-1037.
3. Friend, S. and the Breast Cancer Information Core
Steering Committee (1995).Breast cancer information on
the web. Nature Genetics 11: 23B-239.
4. Brown, H.J. et al. (1995).EMBO J., 14, 124-131.
5. Dennig, J. et al. (1996).EMBO J., 15, 5659-5667.
6. Dubendorff, J.W.,
et al. (1992). Genes
& Development,
6, 2524-2535.
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7. Thakur, S. et al. (1997). Mol & Cell. Biol. 17, 444-
452.
8. Chapman, M.S. and Verma, I.M. (1996). Nature, 382, 678-
679.
9. Varo, A. et al. PNAS, 93, 13595-13599.
10. Nordling, M., et al., Cancer Research, 58, 1372-1375,
April 1, 1998
TABLE 1 shows the results of a (3-galactosidase liquid assay
using various fragments of BRCA2 fused to the DNA binding
domain of lexA.
bait ~etivity ~U1
18-105 112~2
919-1171 (BRC1) 0
1500-1589 (BRC4) 0
2034-2223 (BRC8) 0
3200-3326 0
