AU764590B2 - Mammary-specific transcription factor BrEts - Google Patents

Description

1 MAMMARY-SPECIFIC TRANSCRIPTION FACTOR BrEts FIELD OF THE INVENTION The present invention relates generally to transcription factors and specifically to a new member of the Ets family of proteins which is denoted BrEts.

BACKGROUND OF THE INVENTION The Ets family of proteins, which currently includes about 30 related proteins in species ranging from flies to humans, are characterised by their possession of the Ets DNA binding domain, which specifically interacts with sequences containing the common core trinucleotide

GGA.

The Ets family comprises a group of transcription regulators which are increasingly being recognised for their involvement in early embryonic development and late tissue maturation as well as directed stage-specific and tissue-restricted programs of target gene expression. The Ets family of proteins includes Ets 1 which, in humans, is expressed in the endothelia and developing vessels of the embryo, and in blood vessels in the adult when angiogenesis resumes, for example, during the vascularization of tumours. Ets 1 in chicken embryos is expressed in a number 25 of cells during blood vessel formations. In Xenopus, the family member Ets 2 appears to be required for oocyte maturation. Evidence is also accumulating for a role for Ets family members in Drosophila development, lymphocyte differentiation and viral infectious cycles, (Wasylyk B.; Hahn S.L. and Giovane The Ets family of transcription factors. Eur. J. Biochem, 211, 7-18 (1993)).

The applicant has now identified a novel transcription factor designated BrEts which appears to play a significant role in the transcriptional regulation of milk protein synthesis and/or development of the mammary gland.

H:\RBell\Keep\54549.99.doc 13/01/03 la It is broadly to this transcription factor that the present invention is directed.

All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does onot constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

S 15 For the purposes of this specification it will be clearly understood that the word "comprising" means "including but not limited to", and that the word "comprises" has a corresponding meaning.

SUMMARY OF THE INVENTION Accordingly, in one aspect, the present invention may broadly be said to consist of an isolated transcription factor BrEts polypeptide which has the amino acid sequence set out in Figure 2, or a variant thereof having 25 substantially equivalent activity thereto.

gee H:\RBe11\Keep\s4549.99.doc 13/01/03 WO 00/12552 PCT/NZ99/00144 -2- The transcription factor BrEts polypeptide may be expressed almost exclusively in the pregnant or lactating mammary gland and be induced to express in early pregnancy, continuing throughout lactation and terminating during days 2-3 of involution.

Conveniently, the transcription factor polypeptide of the invention may be obtained by expression of a DNA sequence coding therefore in a host cell or organism or by isolation from .pregnant or lactating mammary tissue or mammary cells in culture.

In a further aspect, the present invention provides an isolated nucleic acid molecule encoding a transcription factor polypeptide of the invention or functional variant thereof.

This nucleic acid molecule can be an RNA, DNA or cDNA molecules but is preferably a DNA molecule.

The nucleic acid molecule may be isolated from a mammary gland cell, and in particular, from a mammary gland cell selected from the group consisting of cow, sheep, mouse, rat, goat and human.

The present invention also provides the promoter region of the BrEts gene. This promoter is mammary specific, and controls the expression of the BrEts gene via known transcription factors, switching it on during pregnancy and lactation and switching it off during days 2-3 of involution.

Also provided by the present invention are recombinant expression vectors which contain a DNA molecule of the invention or functional variant thereof, and hosts transformed with the vector of the invention capable of expressing a polypeptide of the invention. The DNA molecule may or may not comprise the promoter region of the BrEts gene.

In a still further aspect, the invention provides a method of producing a transcription factor polypeptide of the invention comprising the steps of: culturing a host cell which has been transformed or transfected with a vector as defined above to express the encoded polypeptide; and recovering the expressed polypeptide.

SUBSTITUTE SHEET (Rule 26) WO 00/12552 PCT/NZ99/00144 -3- An additional aspect of the present invention provides a ligand that binds to a transcription factor polypeptide of the invention. Most usually, the ligand is, an antibody or antibody binding fragment which is immunoreactive with the transcription factor BrEts. Such ligands also form a part of this invention.

In further aspects, the present invention provides methods of assaying samples for the presence of ligands; test kits suitable for use in such assays; and methods of modulating the development of the mammary gland by administering selected products of the invention to a mammal.

In a further embodiment, the invention provides a method of manipulating milk protein synthesis in cells in culture and/or transgenic animals to enhance the level of production, alter the composition of milk and/or alter the timing of milk protein synthesis.

In another embodiment, the invention provides a genetic marker for DNA assisted selection for milk production or animal performance in dairy cattle.

In yet another embodiment, the invention provides a method of treatment and/or prophylaxis of breast cancer comprising administering agents that block or down regulate the level of the polypeptide or variant thereof, in a suitable pharmaceutically acceptable form to a patient in need thereof.

In a further embodiment, the invention provides a method of diagnosis of breast cancer comprising monitoring the level of expression or number of copies of the gene for the polypeptide of the invention or variant thereof and thereby detecting the presence of abnormal expression or number of copies of the gene and correlating this abnormally with the possible presence of cancerous cells. Such diagnostic techniques may be supplemented with histological examination of mammary tissue biopsy samples, for example.

In yet another embodiment, the invention provides pharmaceutical compositions comprising the polypeptide of the invention or variants thereof.

In yet another embodiment, the invention provides the use of a polypeptide of the invention or variants thereof in the methods of the invention.

SUBSTITUTE SHEET (Rule 26) WO 00/12552 PCT/NZ99/00144 -4- While the invention is broadly as defined above, it will be appreciated by those persons skilled in the art that it is not limited thereto and that it also includes embodiments of which the following description gives examples.

DESCRIPTION OF THE FIGURES In particular preferred aspects of the invention will be described in relation to the accompanying drawings in which: Figure 1 shows the nucleotide sequence alignment of two murine M BrEts 1, M BrEts 2 and a bovine B BrEts cDNA. Dashes denote gaps introduced in order to maximise alignment.

Figure 2 shows the predicted amino acid sequences of murine M BrEts and bovine B BrEts. Differences between the two sequences are highlighted and the underlined amino acids represent the characteristic conserved Ets domain.

Figure 3 shows the Ets DNA binding domain sequences of other members of the Ets family of proteins reproduced from Wasylyk et al, Eur.J.Biochem 211, 7-18, 1993.

Figure 4 shows a Northern blot showing expression of BrEts in different tissues from mice.

Figure 5 shows the expression pattern of BrEts in the mammary gland of mice from gestation through to lactation and involution.

Figure 6 shows BrEts expression in lactating human mammary gland.

Figure 7 shows an increased BrEts expression in rat mammary gland tumour.

Figure 8 shows over expression of BrEts in human breast cancer cell line T47-D.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a substantially pure transcription factor BrEts polypeptide which has the amino acid sequence(s) set out in Figure 2, or a variant thereof having substantially equivalent activity thereto.

SUBSTITUTE SHEET (Rule 26) WO 00/12552 PCTINZ99/00144 The term "substantially pure" means BrEts substantially free of contaminating proteins, lipids, carbohydrate or any other material. A person skilled in the art can purify BrEts using standard techniques for protein purification which will form a single band on a SDS polyacrylamide gel. BrEts purity may also be determined from amino acid analysis using known techniques. All peptides of BrEts having functional activity are included in the invention.

The term "Ets" means a transformation specific protein produced by the gene ets (Wasylyk et al, Eur.J.Biochem 211, 7-18, 1993).

The term "variant" as used herein refers to a polypeptide wherein the amino acid sequence exhibits substantially 60% or greater homology with the amino acid sequence set out in Figure 2, preferably 75% homology and most preferably 90-95% homology to the sequence set out in Figure 2. The variant may be arrived at by modification of the native amino acid sequence by such modifications as insertion, substitution or deletion of one or more amino acids. The term "variant" also includes homologous sequence which hybridise to the sequence set out in Figure 2 under standard hybridisation conditions defined as 2 x SSC at 65 0 C or under reduced stringency hybridisation conditions defined as 6 x SSC at 55 0

C.

In addition, peptides having substantial identity to the above-mentioned peptides can also be employed in preferred embodiments. Here "substantial sequence identity" means that two peptide sequences, when optimally aligned such as by the programs GAP or BESTFIT using default gap weights, share at least 60%, preferably 75% and most preferably 90-95% sequence identity.

The reader will appreciate that modifications of the polypeptides and peptides of the invention are possible. The production of peptide fragments is also well within the capabilities of an art skilled worker.

The polypeptide and peptides of the invention can be prepared in a variety of ways. For example, they can be produced by isolation from natural source, by synthesis using any suitable known techniques (such as by stepwise, solid phase, synthesis described by Merryfield (1963), J.Amer.Chem.soc. Vol 85:2149-2156) or as preferred, through employing recombinant DNA techniques.

SUBSTITUTE SHEET (Rule 26) WO 00/12552 PCT/NZ99/00144 -6- The variants of both the polypeptide and peptides can similarly be made by any of those techniques known in the art. For example, variants can be prepared by site-directed mutagenesis of the DNA encoding the native amino acid sequence as described by Adelman et al. DNA 2:183 (1983).

Where it is preferred, recombinant techniques used to produce the polypeptide or peptide of the invention, the first step is to obtain DNA encoding the desired product. Such DNA comprises a still further aspect of this invention.

The DNA of the invention may encode a native or modified polypeptide or peptide of the invention or an active fragment thereof. In its preferred forms, the DNA comprises at least nucleotides of the sequence of Figure 1.

The DNA can be isolated from any appropriate natural source or can be produced as intron free cDNA using conventional techniques. DNA can also be produced in the form of synthetic oligonucleotides where the size of the active fragment to be produced permits.

By way of example, the Triester method of Matteucci et al. J. Am.Chem.Soc. Vol 103:3185-3191 (1981) may be employed.

Where desirable, the DNA of the invention can also code for a fusion protein comprising the polypeptide or peptide of the invention and a carrier protein. This carrier protein will generally be cleavable from the polypeptide, peptide or fragment under controlled conditions. Examples of commonly employed carrier proteins are P-galactosidase and glutathione-S-transferase.

As indicated above, the invention also contemplates variants of the polypeptide or peptide which differ from the native amino acid sequence by insertion, substitution or deletion of one or more amino acids. Where such a variant is desired, the nucleotide sequence of the native DNA is altered appropriately. This alteration can be made through elective synthesis of the DNA or by modification of the native DNA by, for example, site-specific or cassette mutagenesis. Preferably, where portions of cDNA or genomic DNA require sequence modifications, site-specific primer directed mutagenesis is employed using techniques standard in the art. Any such variants and fragments of the present invention include all those which hybridise to the sequence of Fig 1 under stringent conditions.

SUBSTITUTE SHEET (Rule 26) WO 00/12552 PCT/NZ99/00144 -7- In a further aspect, the present invention consists in replicable transfer vectors suitable for use in preparing a polypeptide or peptide of the invention. These vectors may be constructed according to techniques well known in the art, or may be selected from cloning vectors available in the art.

The cloning vector may be selected according to the host or host cell to be used. Useful vectors will generally have the following characteristics: the ability to self-replicate; the possession of a single target for any particular restriction endonuclease; and desirably, carry genes for a readily selectable marker such as antibiotic resistance.

Two major types of vector possessing these characteristics are plasmids and bacterial viruses (bacteriophages or phages). Preferred vectors are bacterial or mammalian expression vectors such as the pET and pcDNA series available from Novagen and Invitrogen.

The DNA molecules of the invention may be expressed by placing them in operable linkage with suitable control sequences in a replicable expression vector. Control sequences may include origins of replication, a promoter, enhancer and transcriptional terminator sequences amongst others. The selection of the control sequence to be included in the expression vector is dependent on the type of host or host cell intended to be used for expressing the DNA.

Generally, prokaryotic, yeast or mammalian cells are useful hosts. Also included within the term hosts are plasmid vectors. Suitable prokaryotic hosts include E. coli, Bacillus species and various species of Pseudomonas. Commonly used promoters such as Plactamase (penicillinase), lactose (lac) promoter systems and inducable promoters such as IPTG are all well known in the art. Any available promoter system compatible with the host of choice can be used. Vectors used in yeast are also available and well known.

A suitable example is the 2 micron origin of replication plasmid.

Similarly, vectors for use in mammalian cells are also well known. Such vectors include well known derivatives of SV-40, adenovirus, retrovirus-derived DNA sequences and vectors derived from a combination of plasmid and phage DNA.

SUBSTITUTE SHEET (Rule 26) WO 00/12552 PCT/NZ99/00144 -8- Further eukaryotic expression vectors are known in the art P.J. Southern and P.Berg, J. Mol. Appl. Genet. 1 327-341 (1982); S. Subramani et al., Mol.Cell.Biol. 1, 854-864 (1981); R J. Kaufmann and P.A. Sharp, "Amplification and Expression of Sequences Cotransfected with a Modular Dihydrofolate Reducase Complementary DNA Gene, J AMol. Biol. 159, 601-621 (1982); R J. Kaufmann and P.A. Sharp, Mol.Cell.Biol. 159, 601- 664(1982); S.I. Scahill et al., "Expressions And Characterization Of The Product Of A Human Immune Interferon DNA Gene In Chinese Hamster Ovary Cells," Proc. Natl.

Acad. Sci. USA. 80, 4654-4659 (1983); G. Urlaub and L.A. Chasi, Proc. Natl. Acad. Sci.

USA. 77, 4216-4220, (1980).

The expression vectors useful in the present invention contain at least one expression control sequence that is operatively linked to the DNA sequence or fragment to be expressed. The control sequence is inserted in the vector in order to control and to regulate the expression of the cloned DNA sequence. Examples of useful expression control sequences are the lac system, the rp system, the tac system, the trc system, major operator and promoter regions of phage lambda, the glycolytic promoters of yeast acid phosphatase, e.g. Pho5, the promoters of the yeast alpha-mating factors, and promoters derived from polyoma, adenovirus, retrovirus, and simian virus, and cytomegalovirus e.g.

the early and late promoters of SV40, and other sequences known to control the expression of genes of prokaryotic and eukaryotic cells and their viruses or combinations thereof.

In the construction of a vector it is also an advantage to be able to distinguish the vector incorporating the foreign DNA from unmodified vectors by a convenient and rapid assay.

Such assays include measurable colour changes, antibiotic resistance and the like. In one preferred vector, the P-galactosidase gene is used, which gene is detectable by clones exhibiting a blue phenotype on X-gal plates. This facilitates selection. Once selected, the vectors may be isolated from the culture using routine procedures such as freeze-thaw extraction followed by purification.

For expression, vectors containing the DNA of the invention to be expressed and control signals are inserted or transformed into a host or host cell. Some useful expression host cells include well-known prokaryotic and eukaryotic cells. Some suitable prokaryotic hosts include, for example, E.oli, such as E. coli S G-936, E. coli HB 101, E. coli W3110, E.coli X1776, E. coli X2282, E. coli DHT, E. coli MR01 and F. coli XL1 blue MRF, Pseudomonas. Bacillus. such as Bacillus subtilis. and Streptomyces. Suitable eukaryotic SUBSTITUTE SHEET (Rule 26) WO 00/12552 PCT/NZ99/00144 -9cells include yeast and other fungi, insect, animal calls, such as COS cells and CHO cells, HC 11 cells, human cells and plant cells in tissue culture.

Depending on the host used, transformation is performed according to standard techniques appropriate to such cells. For prokaryotes or other cells that contain substantial cell walls, the calcium treatment process (Cohen, S N Proceedings, National Academy of Science, USA .62 2110 (1972)) may be employed. For mammalian cells without such cell walls the calcium phosphate precipitation method of Graeme and Van Der Eb, Virology 52:546 (1978) or liposomal reagents are preferred. Transformations into plants may be carried out using Arobacterium tumefaciens (Shaw et al., Gene 23:315 (1983) or into yeast according to the method of Van Solingen et al. J.Bact. 130: 946 (1977) and Hsiao et al.

Proceedings, National Academy of Science, 76: 3829 (1979).

Upon transformation of the selected host with an appropriate vector the polypeptide or peptide encoded can be produced, often in the form of fusion protein, by culturing the host cells. The polypeptide or peptide of the invention may be detected by rapid assays as indicated above. The polypeptide or peptide is then recovered and purified as necessary.

Recovery and purification can be achieved using any of those procedures known in the art, for example by absorption onto and elution from an anion exchange resin. This method of producing a polypeptide or peptide of the invention constitutes a further aspect of the present invention.

Host cells transformed with the vectors of the invention also form a further aspect of the present invention.

In addition, a further aspect of the present invention provides a ligand that binds to a polypeptide or peptide of the invention.

Ligands may be of two functional types. The first functional type of ligand is a molecule which binds to the transcriptional factor BrEts and stimulates it in performing its normal function (an agonist ligand). The second functional type of ligand is a molecule which binds to the transcriptional factor BrEts and inhibits or prevents it performing its normal function (an antagonist ligand).

SUBSTITUTE SHEET (Rule 26) WO 00/12552 PCT/NZ99/00144 In one embodiment the ligand may be an antibody or antibody binding fragment raised against the polypeptide or peptide. Such antibodies may be polyclonal, but are preferably monoclonal.

Polyclonal antibodies may be produced according to the method used by Koelle el al.; Cell 67:59-77, 1991 incorporated herein by reference. Useful antibody production protocols are outlined in US Patent 5,514,578. Monoclonal antibodies may be produced by methods known in the art. These methods include the immunological method described by Kohler and Milstein in Nature 256:495-497 (1975) as well as by the recombinant DNA method described by Huse et al. Science 246:1275-1281 (1989).

An understanding of the tertiary structure and spatial interactions between the transcriptional factor BrEts (especially ligand-binding domains) and its ligand will provide ways to select highly specific ligands which may be bound only by a modification of a natural receptor ligand-binding domain. Also, this knowledge will provide directions for new designs using the combination of transcriptional factor BrEts with ligands and methods to design and select peptide mimetics of ligands with high specificity by techniques such as phage differential display.

It is likely that BrEts acts as a signalling molecule. This signalling pathway is probably stimulated through some form of receptor (via external stimuli), leading to activation and translocation to the nucleus where it binds DNA and activates transcription of target genes.

In another embodiment the ligand, therefore, may comprise molecules that bind to the polypeptide or peptide of the invention which are derived from natural sources. Such molecules may include protein transcription factors which bind to DNA in conjunction or association with BrEts to initiate transcription. Such proteins may include STAT5a and Accordingly, in a further aspect, the present invention provides a method of assaying samples for the presence of ligands. Assaying processes using polypeptides or peptides as a ligand binding agent or probe are well within the capacity of the art skilled worker.

The selection of the segment to be used as a probe will allow particular functionally associated SUBSTITUTE SHEET (Rule 26) WO 00/12552 PCT/NZ99/00144 11 segments to be isolated. For example, if a segment of the DNA binding domain is used as a probe, other nucleic acid segments encoding other DNA binding domains will be isolated.

It will also be appreciated that the selection of probes highly specific for BrEts, such as the N-terminal region of the polypeptide, will provide an opportunity to assay samples in a rapid and highly specific manner by detecting the presence of BrEts expression.

Samples of material to be screened may be prepared in the form of substrate solutions, then exposed to the ligand binding agent or probe. The presence of a ligand binding agent/ligand complex may be detected according to methods also known in the art.

Examples of such methods include agglutination, radio immunoassay, fluorescence or enzyme immunoassay techniques as well as nucleic acid hybridisation techniques. A suitable screening test is an ELISA assay. In this method of the invention it is presently preferred that the DNA binding domain be used as the ligand binding agent.

It will be appreciated by the reader that a further aspect of the invention therefore contemplates the use of the polypeptides or peptides of the invention in the preparation of probes for the detection of other transcriptional factors of the Ets family of ligands.

In a further aspect the present invention provides test kits suitable for use in such assays.

An example of such a test kit is an ELISA assay test kit including a ligand binding agent of the invention.

As seen from figures 4 and 5, BrEts expression is specific to the mammary gland and, moreover, the expression is switched on during pregnancy and lactation suggesting that BrEts may be linked to milk protein synthesis.

In a further embodiment, the invention therefore provides a method of manipulating milk protein synthesis in cells in culture and/or transgenic animals to enhance the level of production, alter the composition of milk and/or alter the timing of milk protein synthesis.

By overexpression of BrEts the timing and level of expression of specific milk protein genes may be altered in cultured cells or transgenic animals, for example BrEts could be inserted into a gene cassette under the control of a mammary specific promoter such as P-casein or P-lactoglobulin or a promoter that expresses in all cell types (constittive expression) or the BrEts promoter. This cassette would also contain 3' flanking DNA that could stabilise the mRNA and may contain downstream regulatory sequences. This DNA cassette could be introduced into the genome of mammals of micro injection of the DNA SUBSTITUTE SHEET (Rule 26) WO 00/12552 PCT/NZ99/00144 12into the pronuclei of eggs (as described in L'Huillier et al PNAS 93; 6698-6703) which are subsequently transferred back to recipient animals and allowed to develop to term.

This technique for the production of transgenic animals is described by Hogan et al (1996; In: manipulating the mouse embryo, Cold Spring Habor Lab. Press). Another way to produce transgenic animals would involve transfection of cells in culture that are derived from an embryo, or foetal or adult tissues followed by nuclear transfer and embryo transfer to recipient animals, or the gene cassette may be bound to mammalian sperm and delivered to the egg via in vitro or in vivo fertilisation to produce a non-human transgenic animal. Manipulation of the developmental regulation or the level of expression of BrEts could be used to alter the level of milk protein synthesis or production or the composition of specific proteins in the milk.

Transgenic mammals may also be produced by gene therapy techniques whereby said gene cassette is injected into the mammary gland or appropriate tissue of an animal or human in vivo or by transfection of the mammary gland or appropriate tissue using chemically- mediated transfection techniques such as liposome reagents which bind to the DNA and facilitate cellular uptake of the gene cassette.

In another embodiment, the invention provides a genetic marker for DNA assisted selection for milk production or animal performance in dairy cattle.

As seen from figures 7 and 8, BrEts is over expressed in tumour cells suggesting a role for BrEts in the etiology of breast cancer.

In yet another embodiment, the invention therefore provides a method of diagnosis of breast cancer comprising detection of the presence of diseased cells by analysis of the expression level of BrEts RNA or protein in milk and/or tissue biopsy samples.

In yet another embodiment, the invention provides a method of treatment and/or prophylaxis of breast cancer, comprising administering an effective dose of antisense RNA to the nucleic sequence of the transcription factor of the invention, or antibody to the transcription factor of the invention in a suitable pharmaceutically acceptable form to a patient in need thereof and in turn reducing the level of expression of BrEts or reducing the level of free active BrEts.

In yet a further embodiment there is provided methods of manipulating milk protein synthesis in mammals.

SUBSTITUTE SHEET (Rule 26) WO 00/12552 PCT/NZ99/00144 13- In a first method it is desirable to bring about the over expression of the BtEts transcriptional factor in mammals, particularly cows, by the introduction of the BrEts DNA to increase transcriptional regulation of milk protein synthesis or by the introduction of BrEts gene into mammalian cells in vivo.

Milk protein synthesis in mammals, particularly cows, may also be switched on prematurely, by induction of the BrEts gene or by introduction of the transcription factor gene into the mammary cells in vivo of immature mammals.

One way in which the milk protein synthesis may be disrupted or delayed is by introducing the "anti-sense" RNA of the transcriptional factor BrEts into a mammal. This anti-sense RNA binds to the sense transcriptional factor RNA produced by the mammary cells, thereby blocking the availability of the transcriptional factor BrEts to induce transcription of the milk protein genes which in turn reduces milk protein synthesis or the composition of proteins in the milk.

Delivery of the transcriptional factor BrEts gene or anti-sense RNA to the mammal may be achieved by way of vectors as is known in the art.

Another powerful way to deliver the transcriptional factor BrEts gene or anti-sense RNA to a mammal is by germline transformation by methods known in the art.

Non-limiting examples illustrating the invention will now be provided. It will be appreciated that the above description is provided by way of example only and variations in both the materials and techniques used which are known to those persons skilled in the art are contemplated.

PROTOCOL

The general strategy of cloning the transcriptional factor BrEts was based on reverse transcription (RT) using an oligo dT primer followed by PCR with the aforementioned primer and a 5'-specific oligonucleotide with redundancy. The PCR product generated was then used to screen a cDNA library. Positive phase identified in this first screen were purified through two rounds of plaque purification and excised from the phage. The resulting double-stranded DNA was used as a probe to screen a murine mammary gland cDNA library. cDNA clones in the bluescript SK plasmid were identified and verified by automated sequencing.

SUBSTITUTE SHEET (Rule 26) WO 00/12552 PCT/NZ99/001 44 -14- Isolation and Characterisation of Bovine and Murine BrEts The murine forms of the gene for transcription factor BrEts have been isolated from a mammary gland cDNA library. The bovine clone was isolated first using a redundant oligonucleotide and oligo dT primer in PCR, after reverse transcription of mammary RNA. Subsequently, the mouse cDNA clone was isolated with the bovine cDNA or PCR product as the probe. Both genes have been sequenced and the inferred amino acid sequences determined (Figures 1 and The amino acid sequence for the murine and bovine clones are highly homologous (see figure The gene has been assigned to the Ets family of proteins because it contains a highly conserved region known as the Ets DNA binding domain. Figure 3 shows the Ets DNA binding domain sequence of BrEts and other members of the Ets protein family. The BrEts gene, is however, unique from the other Ets family members at the 5' end of the gene, and bears little resemblance to any genes currently in Genebank and other nucleic acid databases.

The tissue specificity of BrEts gene expression was tested using known methods such as Northern analysis. Figure 4 shows that expression of the BrEts gene is almost exclusively confined to the mammary gland. Very low levels of expression can be seen in the spleen, lung and salivary glands but levels 100-1000 fold below that observed in the mammary gland.

The expression pattern of BrEts in mammary gland of mice was investigated using known methods Northern analysis. Figure 5 shows that BrEts expression is switched on at day 4-6 of gestation and continues throughout pregnancy and lactation until day 2 of involution, at which point transcription is turned off.

This pattern of expression correlates with the transcription of milk protein genes such as P-casein (Harris et al 1988, Nucl. Ac. Res 16.10379), which indicates that BrEts may be an important switch for milk protein gene expression. However, its role in this process is not yet defined. The high degree of sequence conservation between the murine and bovine clones of BrEts (figures 1 and 2) suggest that BrEts may play a critical role in mammary development or milk protein gene expression in these and other mammals.

DNA sequences encoding BrEts can be expressed in vitro by DNA transfer into a suitable host cell using known methods of transfection. Figure 5 shows steady-state RNA for murine BrEts gene in CHO cells using a plasmid pTracer-BrEts.

SUBSTITUTE SHEET (Rule 26) WO 00/12552 PCT/NZ99/00144 Other members of the Ets family of proteins have been implicated in tumour development (Wasylyk et al 1993 J. Biochem 211:7-18) and BrEts is upregulated in that mammary gland tumours (see figure The neu oncogene (also called ErbB, and HER2) is overexpressed in 20-30% of breast cancers and some members of the Ets family of proteins are activated as downstream molecules in this cellular cascade (Chang et al 1997, Oncogen, in press).

The overexpression of BrEts in human breast cancer cell line T47-D (and in mammary tumours in rats) is shown in Figure 8 indicating that BrEts may indeed be involved in the etiology of human breast cancer. This experiment was carried out using known techniques of cell culture, RNA extraction and Northern analysis.

INDUSTRIAL APPLICATION Potential applications related to the gene/protein/antibody related to BrEts or functional fragments or homologues thereof include the following: i. Manipulation of milk protein synthesis in cells in culture and/or transgenic animals to enhance the level of production, composition of milk or timing of milk protein synthesis.

ii. As a marker for DNA assisted selection for milk production or animal performance in dairy cattle.

d5* iii. Associated with breast cancer as a reagent in assay/detection systems a marker for the onset of breast cancer as a point of intervention to halt the progression tumour development by genetic means iv. Manipulation of the development of the mammary gland in transgenic or nontransgenic animals.

It will be appreciated that it is not intended to limit the invention to the aforementioned examples only, many variations, such as might readily occur to a person skilled in the art being possible, without departing from the scope thereof.

SUBSTITUTE SHEET (Rule 26)

Claims (29)

1. An isolated transcription factor BrEts polypeptide which has the amino acid sequence set out in Figure 2, or a variant thereof having substantially equivalent activity thereto.

2. A polypeptide of claim 1, which is detectably labelled.

3. An isolated nucleic acid molecule encoding a transcription factor BrEts polypeptide according to claim 1, or variant thereof having substantially equivalent activity the polypeptide of claim 1.

4. A nucleic acid molecule according to claim 3, which is selected from the group of RNA, DNA or cDNA molecules.

5. A nucleic acid molecule according to claim 4, which is a DNA molecule.

6. A nucleic acid molecule according to any one of claims 3 to 5, isolated from a mammalian cell.

7. A nucleic acid molecule according to claim 6, wherein the mammalian cell is a mammary gland cell.

8. A nucleic acid molecule according to claim 7, 30 wherein the mammary gland cell is selected from the group consisting of cow, sheep, mouse, rat, goat and human mammary gland cell.

9. A recombinant expression vector or gene construct 35 comprising a nucleic acid molecule according to any one of claims 3 to 8. H:\lauraw\Keep\Speci\54549.99.doc 24/06/03 17 A host transformed with a vector or gene construct according to claim 9.

11. A host according to claim 10 which is a mammalian cell.

12. A host according to claim 11, selected from a mammary cell in culture or mammary cell in vivo.

13. A method of producing a transcription factor BrEts polypeptide according to claim 1, comprising the steps of: culturing a host cell which has been transformed or transfected with a vector according to claim 9 to express the encoded polypeptide; and recovering the expressed polypeptide.

14. A ligand which binds to a transcription factor BrEts polypeptide according to claim 1, wherein the ligand is an antibody or fragment thereof, a phage display molecule, Stat5a or

15. A ligand according to claim 14, which is an agonist ligand.

16. A ligand according to claim 14, which is an antagonist ligand.

17. A ligand according to claim 14, which is selected 30 from an antibody or antibody binding fragment which is immunoreactive with the transcription factor BrEts.

18. A method of assaying a sample for the presence of a ligand according to any one of claims 14 to 17, the 35 method comprising the steps of: contacting a sample with a polypeptide (or variant) according to claim 2; and H:\lauraw\Keep\Speci\54549.99.doc 24/06/03 18 detecting the presence of any bound labelled polypeptides.

19. A test kit for use in a method according to claim 18, comprising a polypeptide according to claim 2. A test kit according to claim 19, comprising an ELISA assay test kit including a ligand according to any one of claims 14 to 17.

21. A method of modulating the development of the mammary gland of a mammal, said method comprising either: administering a polypeptide or variant thereof according to claim 1 to said mammal; (ii) developing a transgenic non-human mammal by the insertion of a vector or gene construct comprising the coding sequence for a nucleic acid molecule according to any one of claims 3 to 8 attached to a mammary-specific promoter and delivering said vector or gene construct into a one-cell embryo followed by embryo transfer to produce a transgenic non-human mammal; (iii) gene therapy techniques whereby a vector or gene construct according to claim 9, is injected into the mammary gland or appropriate tissue of an animal or human 25 in vivo; or (iv) transfection of the mammary gland or appropriate tissue with a vector or gene construct according to claim 9 using chemically mediated techniques. 30 22. A method according to claim 21, wherein the vector or gene construct in alternative (ii) is bound to mammalian sperm and delivered to the egg via in vitro or in vivo fertilisation to produce a non-human transgenic mammal.

23. A method according to claim 20, wherein the transfection of alternative (iv) is carried out using H:\lauraw\Keep\Speci\5449.99.doc 24/06/03 19 liposome reagents which bind to the DNA and facilitate cellular uptake of the gene construct.

24. A method of manipulating milk protein synthesis in cells in culture or in a transgenic or non-transgenic mammal to enhance the level of production, alter the composition of milk and/or alter the timing of milk protein synthesis, said method comprising transforming or transfecting a mammary cell in culture or in a transgenic or non-transgenic mammal with a vector or gene construct according to claim 9, said transgenic mammal being produced by the methods defined in alternatives (ii) to (iv) in claim 21.

25. A genetic marker for DNA assisted selection for milk production or animal performance in dairy cattle comprising a nucleic acid molecule which hybridises to a nucleic acid molecule according to any one of claims 2 to 8.

26. A method of treatment and/or prophylaxis of breast cancer comprising administering agents that block or down regulate the level of a polypeptide or variant thereof according to claim 1, in a suitable pharmaceutically S. 25 acceptable form to a patient in need thereof.

27. A method of diagnosis of breast cancer comprising monitoring the level of expression or number of copies of the gene for a polypeptide or variant thereof according to 30 claim 1, and thereby detecting the presence of abnormal S expression or number of copies of the gene and correlating this abnormality with the possible presence of cancerous cells. 35 28. A method according to claim 27, further comprising histological examination of mammary tissue biopsy samples. H;\lauraw\Keep\Speci\54549.99.doc 24/06/03 20

29. A pharmaceutical composition comprising a polypeptide or variant thereof according to claim 1, together with a pharmaceutically acceptance carrier. Use of a polypeptide or variant thereof according to claim 1, in a method according to any one of claims 13, 18, 21 to 24, 26 and 27.

31. Use of a polypeptide or variant thereof according to claim 1, in the manufacture of a composition for modulating the development of the mammary gland of a mammal.

32. Use of a polypeptide or variant thereof according to claim 1, in the manufacture of a composition for the treatment and/or prophylaxis of breast cancer.

33. An isolated transcription factor BrEts polypeptide according to claim 1, substantially as described herein with reference to any example thereof.

34. An isolated nucleic acid molecule according to claim 3, substantially as described herein with reference 25 to any example thereof. Dated this 2 4 th day of June 2003 AGRESEARCH LIMITED By their Patent Attorneys 30 GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia H.\1auraw\Kee\Speci\54549.99.doc 24/06/03