WO1998048013A1 - Brca2 transcriptional activator domain and uses thereof - Google Patents

Abstract

Exon 3 of BRCA2 encodes a transcriptional activator domain ('TAD'), including a primary activating region ('PAR') and an auxiliary activating region ('AAR'), flanked by two inhibitory regions ('IR1' and 'IR2') which are bound by molecules which inhibit activation of transcription by the TAD. Appropriate fragments of BRCA2 polypeptide are useful in activating transcription and in assays for substances able to modulate transcriptional activation by BRCA2.

Description

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. Figure 1 shows that sequences within exon 3 of BRCA2

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 GA 4 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 "BBPl" (BRCA2 Binding Protein-1) found to interact with the BRCA2 TAD and to modulate its transcriptional activation. Figure 4 shows a portion of the BBPl 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. 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 , 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 al .

(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 al . { The EMBO 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 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

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 80%, 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. 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

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, I) . GAP uses the Needleman and unsch

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, 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 stringent

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%

dextran sulfate and a final wash at 55°C in 0. IX 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 0. IX SSC, 0.1% 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 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 encompassed 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 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 IRl 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 IRl 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 IRl 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

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 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

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 IRl and/or IR2 peptides in excess may

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 intramolecular interaction.

A peptide according to a further aspect of the invention

0 may include or consist essentially of the IRl 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 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 BBPl (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 "BBPl" (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 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

sequence (s) 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 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 cell 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 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 Manual: 2nd edition, Sambrook et al . , 1989, Cold Spring

JHarbor Laboratory Press. Many known techniques and protocols

for manipulation of nucleic acid, for example in preparation

of nucleic acid constructs, mutagenesis, sequencing,

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

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

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 introducing the nucleic acid into a host cell. The introduction, which may (particularly for in vi tro

introduction) be generally referred to without limitation as "transformation", may employ any available technique. For 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 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, 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 vi tro,

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 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 IRl 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 transcription.

A protein termed "BBPl" (BRCA2 Binding Protein-1) has

been found to interact with the BRCA2 TAD, specifically the

PAR. A domain of BBPl was expressed (see experimental

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 BBPl, or suitable fragment

thereof, in obtaining substances able to interfere with their

interaction and/or modulate the effect of BBPl on BRCA2

transcription activation.

As noted, the IRl 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 IRl and/or IR2 within BRCA2 in vivo to

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

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 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 BRCA2 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 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 IRl and/or IR2 as 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, IRl 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

IRl 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 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 IRl 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 IRl and/or IR2 may inhibit transcriptional activation by the polypeptide. Thus,

inhibition of transcriptional activation allows for

identification of molecules which bind IRl and/or IR2 ,

including a natural ligand.

A molecule which binds IRl 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 used to assay for substances which interfere with its binding _ to IRl and/or IR2 within BRCA2 polypeptide and/or ability to inhibit BRCA2 TAD transcriptional activation.

Thus, a further aspect of the invention employs a

peptide or polypeptide which includes BRCA2 IRl 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

assay (A) or assay (B) which binds IRl 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

peptide or polypeptide including a IRl or IR2 sequence and a

binding molecule for the IRl 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

with binding between the IRl and/or IR2 sequence and the

binding molecule such binding occurs, and determining binding between the IRl and/or IR2. This may be followed by

isolation and/or manufacture and/or use of a substance which 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 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 340: 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 and 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 vi tro 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 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 vi tro interaction may utilise a

fusion protein including glutathione-S-transferase (GST) .

This may be immobilized on glutathione agarose beads. In an

in vi tro 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 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 BRCA2 TAD, e.g. by inhibiting interaction of

IRl 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 IRl and/or

IR2 and a DNA binding domain capable of binding a nucleotide

sequence within a promoter, a binding molecule or molecules

for IRl and/or IR2 which inhibit (s) transcriptional

activation by the BRCA2 TAD, and a test compound, under

conditions in which the binding molecule or molecule (s) for

IRl and/or IR2 inhibit (s) 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 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 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

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

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) .

"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 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 rnRNA 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, preferably a visually detectable signal, such as a coloured

-product. Many examples are known, including β-galactosidase and luciferase. β-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 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 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 to 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 IRl 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. 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. 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

(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 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 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. 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 with a peptide, an antibody specific for a protein may be

- obtained from a recombinantly produced library of expressed immunoglobulin variable domains, e.g. using lambda

bacteriophage or filamentous bacteriophage which display

functional immunoglobulin binding domains on their surfaces;

for instance see WO92/01047. 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.

0 Example antibody fragments, capable of binding an antigen or other binding partner are the Fab fragment consisting of the VL, VH, Cl and CHI domains; the Fd fragment consisting of the VH and CHI domains; the Fv fragment consisting of the NL 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 D A technology to

produce other antibodies or chimeric molecules which retain

the specificity of the original antibody. Such techniques may involve introducing DΝA 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, 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 . 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

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,

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.

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.

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 including a mutation in the TAD, IRl 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 use 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 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 BRCA2 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 0 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 general toxic effect.

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, IRl or IR2 , or the binding ligand or ligands for IRl and/or IR2 , which interfere with interaction between these polypeptides, and/or transcriptional activation by BRCA2 TAD. The term "functional mimetic" means a 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 0 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. 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

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" . 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

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. 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 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. 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 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. Given such experimental evidence, oligonucleotides

-designed to hybridise to a region within the TAD or IRl or

IR2 may be used in diagnostic and prognostic screening.

Oligonucleotide probes or primers, as well as the full- length BRCA2 TAD (optionally including IRl 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 probed under conditions 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.

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

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.

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

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.

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.

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 oligonucleotide 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) ,

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 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, where

appropriate, codon 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 sequence shown for the BRCA2 TAD, IRl 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 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 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

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

(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

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

discussed herein at the 5' end of the oligonucleotide) 5 ' -GCTCCACCCTATAATTCTG-3 ^■ 5 ' -CAGAATTATAGGGTGGAGC-3 (respectively complementary to the anti-sense and sense 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. 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) 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 60)

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 IRl 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 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 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

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,

IRl and/or IR2 ) , and/or is mutated in such a region; or,

(c) using DNA fingerprinting to compare the restriction

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

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, IRl and/or IR2) , the specific

binding member including nucleic acid hybridisable with the

0 BRCA2 sequence, or substances including an antibody domain

with specificity for 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 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, IRl 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. 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 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

polypeptide. 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 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 . 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

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

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

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. 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. 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 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 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, IRl and/or IR2 nucleic acid, the apparatus including storage means including the BRCA2 nucleic acid sequence as set out in 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 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

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

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

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.

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 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 may be

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. 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 "BBPl" (BRCA2 Binding 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. Nucleic acid encoding the BBPl 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 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

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

vectors and activity assayed by transfection 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

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

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 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. 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 BBPl 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.

EXPERIMENTAL EXEMPLIFICATION

The BRCA2 gene encodes a large 3418 residue protein of unknown function which is found mutated in 45% 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 for 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 activation potential in two different mammalian cell lines,

TJ20S (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 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 (IRl and IR2) present directly N- and

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

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

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, BRACl and BRCA2 , have been implicated

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 that their functions may be related (7) . Our finding that

-BRCA2 , like BRACl (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 BRACl

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 obtain and/or identify molecules which modulate or interfere with their interaction with BRCA2 and/or their action on BRCA2 transcriptional activation. Identification of a protein which binds the BRCA2 PAR and ^ modulates i ts 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 "BBPl" - 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 BBPl message is about 7.5kb, and expressed highly in ovary and testes, where BRCA2 is also highly

expressed (comparisons with other tissues) .

De-Tions ra ion that BBPl modulates transcription activi ty of

the BRCA2 TAD

A portion of the BBPl 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 BBPl expression vector gave increasing repression of expression of

-the reporter construct.

Mapping of interaction si te for BBPl in BRCA2 Overlapping fragments of BRCA2 TAD were used to map the

BBPl 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 BBPl 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 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 β-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 on pBTM116. It contains the coding sequence for the full

JLength 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 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

sequences (depending on whether the aim is to obtain a

naturally occurring ligand for the BRCA2 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 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 TAD or for IRl 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 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 β-

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 β-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 pVP16 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 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 URA3 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 Transformation

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 are 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 μg of plasmid DNA together with 100 μg of sonicated sheared salmon sperm DNA is mixed

with 100 μl of the yeast suspension. After a further addition of a buffer of 700 μl 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 μl 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 μl 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.

Larσe Scale Library 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 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 μg 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 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 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 μl on plates

made from -THULL media. If the bait alone can activate the

β-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, 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-Cl (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 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, IRl 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 Using residues 18-141 of BRCA2 as an affinity column a

kinase has been purified from HeLa nuclear extracts which phosphorylates within amino acids 60-105 of BRCA2 ,

specifically the region of BRCA2 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: 238-239.

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. 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 β-galactosidase liquid assay

using various fragments of BRCA2 fused to the DNA binding

domain of lexA.

TABLE 1

bait activity TUl

18-105 112±2

919-1171 (BRC1) 0

1500-1589 (BRC4) 0

2034-2223 (BRC8) 0

3200-3326 0

Claims

1. A fragment of BRCA2 which is able to act as

transcriptional activator when operably linked to a heterologous DNA binding domain.

2. A fragment according to claim 1 which is of less than

about 200 amino acids.

3. A fragment according to claim 1 or claim 2 which includes amino acids 23-105 of the human BRCA2 polypeptide

the sequence of which is shown in Figure 2.

4. A fragment according to claim 3 which includes amino acids 18-105 of the human BRCA2 polypeptide the sequence of

which is shown in Figure 2.

5. A fragment according to claim 1 or claim 2 which includes amino acids 18 to 60 of the human BRCA2 polypeptide the sequence of which is shown in Figure 2.

6. A fragment according to claim 1 or claim 2 which includes amino acids 60 to 105 of the human BRCA2 polypeptide the sequence of which is shown in Figure 2.

7. A fragment according to any of claims 1 to 6 including

amino acids 1-17 and/or amino acids 106-125 of the human BRCA2 polypeptide the sequence of which is shown in Figure 2.

5 8. A mutant, variant or derivative of a BRCA2 fragment

according to any of claims 1 to 7 , which mutant, variant or

derivative is able to act as transcriptional activator when

operably linked to a heterologous DNA binding domain and has

at least 80% sequence similarity with said fragment.

10

9. A fragment according to any of claims 1 to 7 or a

mutant, variant or derivative thereof according to claim 8 fused to a sequence of amino acids heterologous to BRCA2.

15 10. A substance including a fragment according to any of claims 1 to 7 or a mutant, variant or derivative thereof according to claim 8 operably linked to a heterologous DNA

binding domain.

20 11. An isolated nucleic acid molecule encoding a fragment

according to any of claims 1 to 7 or a mutant, variant or derivative thereof according to claim 8.

12. Nucleic acid according to claim 11 wherein said

fragment, mutant, variant or derivative is fused to a

sequence of amino acids heterologous to BRCA2.

13. Nucleic acid according to claim 11 or claim 12 operably linked to regulatory sequences for expression of the encoded

product .

14. A host cell transformed with nucleic acid according to

claim 13.

15. A method for production of a fragment according to any

of claims 1 to 7 or a mutant, variant or derivative thereof according to claim 8, the method including causing expression

from nucleic acid according to claim 13.

16. A method according to claim 15 including culturing a

host cell transformed with said nucleic acid under conditions

for expression of the encoded product.

17. A method according to claim 15 or claim 16 wherein said

fragment, mutant, variant or derivative is isolated and/or

purified.

18. A method according to claim 17 including formulating said fragment, mutant, variant or derivative into a

composition including at least one additional component.

19. A method of producing a transcription factor, the method

including operably linking a fragment according to any of claims 1 to 7 or a mutant, variant or derivative according to

claim 8 to a DNA binding domain to form a transcription

factor.

20. A method of activating transcription from a promoter

including a motif for a DNA binding domain, the method

including bringing into contact the promoter and a substance

according to claim 10 wherein said heterologous DNA binding

domain is able to bind said motif, under conditions wherein the DNA binding domain binds said motif and transcription from the promoter is activated.

21. An assay method which includes: (a) bringing into contact a substance including a

fragment according to any of claims 1 to 6 or a mutant, variant or derivative thereof according to claim 8 and a test compound; and (b) determining interaction between said substance and

said test compound.

22. An assay method which includes:

(a) bringing into contact a substance including a fragment according to any of claims 1 to 5 or a mutant,

variant or derivative thereof according to claim 8 , or a

fragment of BRCA2 including amino acids 18-46 as shown in

Figure 2, a substance including a fragment of BBPl of which

the amino acid sequence is shown in Figure 3 or Figure 4, or

a mutant, variant or derivative thereof which is able to bind

BRCA2; and a test compound, under conditions in which in the

absence of the test compound being an inhibitor, the two said

substances interact ;

(b) determining interaction between said substance.

23. An assay method which includes:

(a) bringing into contact a substance according to

claim 10 including a DNA binding domain able to bind a motif within a promoter, the method including bringing into contact

a substance according to claim 10 and a putative inhibitor compound under conditions where the substance, 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 motif to which said DNA binding

domain is capable of binding to activate transcription 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.

24. An assay method which includes:

(a) bringing into contact a substance including a

fragment according to any of claims 1 to 6 or a mutant,

variant or derivative thereof according to claim 8 and a test

compound in the presence of a kinase under conditions in

which the kinase normally phosphorylates said fragment,

mutant, variant or derivative; and

(b) determining phosphorylation of said fragment,

mutant, variant or derivative.

25. An assay method which includes:

(a) bringing into contact a substance including amino

acids 1-17 and/or 106-125, and a putative binding molecule or

other test compound; and

(b) determining interaction or binding between the substance and the test compound.

26. An assay method according to any of claims 21 to 25

wherein the test compound is a peptide fragment of BRCA2 or BBPl or a mimetic thereof.

27. A method wherein a test compound identified as testing

positive in a method according to any of claims 21 to 26 is

formulated into a composition including at least one

additional component.

28. Use of a fragment according to any of claims 1 to 7 or a

mutant, variant or derivative thereof according to claim 8 in an assay to identify a compound which is able to modulate transcriptional activation by BRCA2.

29. A peptide which is a fragment of a fragment according to

any of claims 1 to 7 or a mutant, variant or derivative thereof according to claim 8 or a fragment of BBPl the amino acid sequence of which is shown in Figure 3 or Figure 4, or a

non-peptidyl mimetic of said peptide, which peptide or mimetic thereof is able to modulate transcriptional activation by BRCA2.

30. A peptide according to claim 29 including residues 18-46

of the amino acid sequence of human BRCA2 polypeptide which

is shown in Figure 2.

31. A peptide which includes amino acids 1-17 of the human

BRCA2 polypeptide the sequence of which is shown in Figure 2.

32. A peptide which includes amino acids 106-125 of the

human BRCA2 polypeptide the sequence of which is shown in

Figure 2.