WO2001075099A1

WO2001075099A1 - Nucleic acid molecule encoding a uterine estrogen agonist-inducible protein

Info

Publication number: WO2001075099A1
Application number: PCT/AU2001/000379
Authority: WO
Inventors: The Hung Huynh
Original assignee: HUGHES EDWARD JOHN LANGFO; National Cancer Centre of Singapore Pte Ltd
Current assignee: HUGHES EDWARD JOHN LANGFO; National Cancer Centre of Singapore Pte Ltd
Priority date: 2000-04-04
Filing date: 2001-04-04
Publication date: 2001-10-11
Anticipated expiration: 2002-10-04

Abstract

The present invention relates generally to a nucleic acid molecule comprising a nucleotide sequence corresponding to an estrogen agonist-inducible genetic sequence in cells derived from the uterus. The nucleic acid molecule of the present invention encodes a protein which is proposed to mediate the effects of estrogenic hormones and other estrogen agonists in inducing epithelial proliferation and differentiation in uterine and ovarian tissues. The present invention provides, therefore, a means of monitoring uterine and ovarian growth and development as well as the level of estrogenic activity in tissues including cancer tissue by, for example, measuring expression of the subject nucleic acid molecule and/or detecting the level of the estrogen agonist-mediating polypeptide encoded by said nucleic acid molecule. The present invention further provides compositions comprising the subject polypeptide or its derivatives, homologs, analogs or agonists. The present invention further contemplates a method of modulating the growth of uterine and/or ovarian tissue including uterus- or ovary-derived cancer tissue by the administration of an agonist or antagonist of expression of the subject nucleic acid molecule or activity of the polypeptide encoded by same. The instant nucleic acid molecule also provides directly or indirectly via its expression product a means for diagnosing, detecting or otherwise monitoring uterine cancers and/or ovarian cancers. The present invention further provides a promoter and/or other regulatory regions associated with the expression of the subject nucleic acid molecule.

Description

NUCLEIC ACID MOLECULE ENCODING A UTERINE ESTROGEN AGONIST-INDUCIBLE PROTEIN

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

Bibliographic details of the publications numerically referred to in this specification are collected at the end of the description.

Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other country.

The female genital tract, including the uterus, exhibits considerable control over the ability of a conceptus to develop (1, 2). The uterus expresses and secretes a number of growth factors (3-8) and other regulatory polypeptides (7, 9) in response to ovarian steroid hormones. These polypeptides are thought to play a part in directing or limiting the growth and development of the uterus. Estrogens promote the growth, differentiation and remodelling of the uterus during the estrus cycle and pregnancy (10-12). They modulate the expression of genes involved in the regulation of cell growth and differentiation including EGF, IGF-I and their receptors (5, 13-20). Preovulatory ovarian estrogen secretion is important for uterine cellular proliferation and epithelial differentiation during early stages of pregnancy (21).

The CUB domain is an extracellular domain of approximately 110 residues which is found in functionally diverse (mostly developmental) proteins. Proteins that contain the CUB domain include mammalian complement subcomponents Cls/CIR (22, 23); hamster serine protease, Casp (24, 25); and mammalian complement-activating component of Ra-reactive factor (RARF) (26); vertebrate bone morphogenic protein 1 (BMP-1) (27-31); neuropilin (A5 antigen) (32-39); mammalian hyaluronate-binding protein TSG-6, a serum- and growth factor-induced protein possibly involved in cell-cell and cell-matrix interactions during inflammation and tumorigenesis (40); and mammalian spermadhesins (24, 41, 42). Bovine acidic seminal fluid contains a CUB domain and belongs to the spermadhesin family. It functions both as a mitogen and growth factor in vitro (43) and a stimulator of progesterone secretion in cultured ovarian cells (43).

The anti-estrogen drug tamoxifen improves the survival of women with breast cancer and has proved to be clinically useful for the treatment of metastic estrogen receptor-positive tumours (44). However, long-term administration of tamoxifen has been reported to be associated with an increased risk of endometrial cancer in post-menopausal women (45). Tamoxifen causes endometrial thickening in some postmenopausal women. Over 40% of women on tamoxifen had an endometrium >8 mm thick compared with only 5% of control women of placebo (46). It has been suggested that the estrogenic effect of tamoxifen on the atrophic postmenopausal endometrium causes hyperplasia that may progress to atypia and cancer in a manner similar to that seen with estrogen replacement therapy.

In work leading up to the present invention, the inventors sought to identify tamoxifen- and estradiol-regulated genes in the uterus using differential display to examine the transcript expression profile of the ovariectomized uterus under conditions of tamoxifen supplementation. In accordance with the present invention, a novel tamoxifen-inducible nucleic acid molecule has been isolated. By virtue of its activation by growth hormone, estradiol and tamoxifen, tissue-specific expression and localization on the cell membrane, the instant nucleic acid molecule is proposed to be important in normal and neoplastic uterine and ovarian growth. It is also proposed that the nucleic acid molecule and/or its expression product will serve as a biomarker for uterine and ovarian cancer and aid in their diagnosis and treatment.

SUMMARY OF THE INVENTION

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO:). The SEQ ID NOs: correspond numerically to the sequence identifiers <400>1, <400>2, etc. A sequence listing is provided after the claims.

One aspect of the present invention provides a nucleic acid molecule comprising a sequence of nucleotides corresponding to a uterine estrogen agonist-inducible genetic sequence.

Another aspect of the present invention contemplates a nucleic acid molecule comprising a sequence of nucleotides corresponding to a uterine estrogen agonist-inducible genetic sequence which is expressed in estrogen agonist treated ovariectomized uterus cells or tissue but substantially not or in very low amounts in non-estrogen agonist treated ovariectomized uterine tissue.

A further aspect of the present invention provides a nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:l or a nucleotide sequence having at least 50% similarity thereto or a nucleotide sequence capable of hybridizing to SEQ ID NO: 1 or a complementary form thereof under low stringency conditions.

Still another aspect of the present invention is directed to a nucleic acid molecule a comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:l or a nucleotide sequence having at least 50% similarity thereto or a nucleotide sequence capable of hybridizing to SEQ ID NO:l or a complementary form thereof under low stringency conditions wherein said nucleic acid molecule corresponds to a uterine estrogen agonist-inducible genetic sequence.

Still a further aspect of the present invention provides a nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:3 or a nucleotide sequence having at least 50% similarity thereto or a nucleotide sequence capable of hybridizing to SEQ ID NO: 3 or a complementary form thereof under low stringency conditions.

Yet another aspect of the present invention is directed to a nucleic acid molecule a comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:3 or a nucleotide sequence having at least 50% similarity thereto or a nucleotide sequence capable of hybridizing to SEQ ID NO:3 or a complementary form thereof under low stringency conditions wherein said nucleic acid molecule corresponds to a uterine estrogen agonist-inducible genetic sequence.

Even yet another aspect of the present invention relates to a genetic construct comprising a nucleotide sequence corresponding to a uterine estrogen agonist-inducible genetic sequence.

Even still another aspect of the present invention contemplates a method for producing a uterine estrogen agonist-mediating polypeptide said method comprising introducing into a cell a nucleic acid molecule comprising a nucleotide sequence corresponding to a uterine estrogen agonist-inducible genetic sequence and subjecting said cell to conditions sufficient to permit expression of the nucleotide sequence and then recovering the polypeptide or membrane portions comprising same.

Another aspect of the present invention provides a polypeptide comprising a sequence of amino acids corresponding to a uterine estrogen agonist-mediating polypeptide. A further aspect of the present invention is directed to a polypeptide comprising sequence of amino acids encoded by the nucleotide sequence substantially as set forth in SEQ ID NO:l or a nucleotide sequence having at least 50% similarity thereto or a nucleotide sequence capable of hybridizing SEQ ID NO:l under low stringency conditions.

Still another aspect of the present invention provides a polypeptide comprising a sequence of amino acids substantially as set forth in SEQ ID NO:2 or an amino acid sequence having at least about 50%ι similarity thereto.

Still a further aspect the present invention provides a polypeptide comprising a sequence of amino acids substantially as set forth in SEQ ID NO:2 or an amino acid sequence having at least about 50% similarity thereto and which polypeptide corresponds to a uterine estrogen agonist-mediating polypeptide or a mutant, derivative, homologue or analogue of said polypeptide.

Yet another aspect of the present invention is directed to a polypeptide comprising sequence of amino acids encoded by the nucleotide sequence substantially as set forth in SEQ ID NO: 3 or a nucleotide sequence having at least 50% similarity thereto or a nucleotide sequence capable of hybridizing SEQ ID NO: 3 under low stringency conditions.

Still another aspect of the present invention provides a polypeptide comprising a sequence of amino acids substantially as set forth in SEQ ID NO:4 or an amino acid sequence having at least about 50% similarity thereto.

Still a further aspect the present invention provides a polypeptide comprising a sequence of amino acids substantially as set forth in SEQ ID NO:4 or an amino acid sequence having at least about 50% similarity thereto and which polypeptide corresponds to a uterine estrogen agonist-mediating polypeptide or a mutant, derivative, homologue or analogue of said polypeptide. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a photographic representation showing differentially expressed mRNA bands in uterine tissues of control ONX and ONX-tamoxifen-treated rats. Total RΝA was isolated from uteri of ONX and ONX-tamoxifen-treated rats was subjected to differential display. The band representing mRΝA that was induced during tamoxifen treatment is marked by an arrowhead. Northern blot analysis of total RNA from uteri of ONX and ONX-tamoxifen- treated rats was used to confirmed the presence of a differentially expressed mRΝA in the ONX-tamoxifen-treated uteri. Blots were hybridized with a ³²P-labelled cDΝA fragment that was isolated from the above differential gel (A) or GAPDH cDΝA (B).

Figure 2 is a photographic representation showing Northern blot analysis of UO-44 gene expression in female adult rat tissues. Total RNA derived from various tissues of an 80-day old female rat was subjected to Northern blot analysis. Blots were hybridized with rat UO-44 (A) and GAPDH (B) cDNAs. Tissues are: Mg: mammary gland; Fa: abdominal fat; Mu: red muscle; Ov: ovary; He: heart; Lu: lung; Li: liver; Sto: stomach; Int: small intestine; Spl: spleen; Pi: pituitary; Br: brain; Ki: kidney; and Ut: uterus.

Figure 3 is a representation showing ovarian-steroid dependent UO-44 gene expression. Female rats were underwent ovariectomy. The uteri were collected at the indicated times after ovariectomy. Total RNA derived from uteri was subjected to Northern blotting. Blots were hybridised with GAPDH (A, D) and rat UO-44 (B, E) cDNAs. Time-dependent tamoxifen- induced UO-44 gene expression in uteri of ONX-rats is shown in (E). Densitometric scanning of the UO-44 band is shown in (C, F). Bars with different letters are significantly different from one another at (pθ.01). Data are expressed as the mean ± SEM.

Figure 4 is a representation showing effects of tamoxifen treatment on UO-44 expression in the uteri of ONX rats. Ovariectomized (OVX) rats were treated with indicated amount of tamoxifen (TAM), 1.2 μg/day estradiol (E₂) or 5 mg tamoxifen plus 1 mg ICI 182780 (T M + ICI) for 3 weeks. Total RΝA derived from uteri was subjected to Northern blotting. Blots were hybridized with GAPDH (A) and rat UO-44 (B) cDNAs. Densitometric scanning of the UO-44 band (C) and the effect of each treatment on uterine weight (D) are shown. Uteri of ovary intact (I) serves as a positive control. Bars with different letters are significantly different from one another at (pO.Ol). Data are expressed as the mean + SEM.

Figure 5 is a representation showing effects of tamoxifen and ICI 182780 on UO-44 gene expression and uterine weight of ovary-intact rats. Ovary-intact rats were treated with indicated concentrations of tamoxifen (TAM, A), and ICI 182780 (ICI, B) for 3 weeks. Total RNA derived from uteri was subjected to Northern blot analysis. Blots were hybridized with GAPDH and rat UO-44 cDNAs. Densitometric scanning of the UO-44 band and the effect of the treatments on uterine weight are shown. Bars with different letters are significantly different from one another at (pθ.01). Data are expressed as the mean ± SEM.

Figure 6 is a representation showing effects of growth hormone on UO-44 gene expression in the uterus of hypophysectomized rats. Hypophysectomized rats were treated with vehicle, or indicated concentration of human growth hormone (GH) per gram body weight for 3 weeks. Total RNA derived from uteri was analyzed by Northern blotting. Blots were hybridized with rat UO-44 or GAPDH cDNAs (A). Densitometric scanning of the UO-44 band (B) and the effect of GH on uterine weight (C) are shown. Bars with different letters are significantly different from one another at (pO.Ol). Data are expressed as the mean ± SEM.

Figure 7 is a photographic representation showing effects of tamoxifen, ICI 182780, growth hormone, and estradiol on UO-44 gene expression in the ovaries of hypophysectomized rats. Hypophysectomized rats were treated with vehicle (C), 1 μg human growth hormone per gram body weight (GH), 1.0 mg ICI 182780 per kg BW per week (ICI), 1.2 μg 17-β estradiol per day (E₂) and 5 mg tamoxifen per kg BW per day (TAM). Total RNA derived from ovaries was analyzed by Northern blotting. Blots were hybridized with rat UO-44 (A) or GAPDH cDNAs (B). Hypophysectomy had no effect on UO-44 gene expression in the ovaries

Figure 8 is a photographic representation showing detection of UO-44 mRNA in the tamoxifen-treated uteri. In situ hybridization with antisense RNA probe for UO-44 gene expression in the uteri of ONX-tamoxifen-treated uteri at low (A) and high magnification (B). (C) Sense control UO-44 probe showing no background staining in uterus of tamoxifen- treated rats. In situ hybridization with antisense RΝA probe for UO-44 gene expression in the ONX-tamoxifen-ICI-treated uteri (D). UO-44 mRΝA was localized in the luminal secretory epithelial cells and glandular epithelial cells. ICI abolished tamoxifen-induced UO-44 expression in the uteri of ONX rats.

Figure 9 is a photographic representation showing in situ hybridization with antisense RΝA probe for UO-44 expression in rat ovaries. Low- (A) and high- (B) magnification showing the non-uniform UO-44 mRΝA distribution in the follicles and granulosa cells. Abundant levels of UO-44 expression were detected in the granulosa cells of medium size follicles. Moderate levels UO-44 mRΝA were detected in granulosa cells of large and small follicles. (C) A sense control UO-44 probe showed no background staining in the ovarian tissue. (D) ICI 182780- treated ovary hybridized with anti-sense UO-44 showed very faint staining signal.

Figure 10 is a photograpic representation showing Subcellular localization of UO-44 protein. Human breast cancer MCF-7 cells were transfected with mammalian expression vector containing full length UO-44 cDΝA (UO-44 pcDΝA3.1/His) or control ρcDNA3.1/His vector as described in Example 6. Plasma membrane-enriched subcellular fractions and cytosolic proteins were isolated and Western blot analysis was performed as described in Example 6. Blots were incubated with mouse anti 6-Histidine antibody and horseradish peroxidase-conjugated donkey anti-mouse secondary antibody. Blots were visualized with a chemiluminescent detection system. Molecular weights of irnmunoreactive bands are shown. Clones 1 and 2 are mock transfectants; and UO-44- 12 and UO-44- 15 are UO-44 expressing clones. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is predicted in part on the identification, cloning and characterization of a nucleic acid molecule in the form of a genetic sequence from cells of uterine origin, the expression of which, is induced by a group of molecules referred to herein as "estrogen agonists". Cells of uterine origin are deemed herein to include ovarian cells. For the purposes of the present invention, an "estrogen agonist" is deemed to include estrogen and its functional derivatives, homologs and analogs as well as estradiol, tamoxifen, diethylstilbestrol and growth hormone. Tamoxifen acts as an anti-estrogen in breast tissue but exhibits estrogen-like activity in the uterus (47). Expression of the genetic sequence, however, is not induced by the anti-estrogen ICI 182780 which is a 7 alpha-alkylsulfϊnyl analogue of estradiol, ICI 182780 is an estrogen antagonist.

It is proposed, in accordance with the present invention, that the subject genetic sequence, and more particularly its expression product, is involved in or otherwise associated with uterine growth mediated by an estrogen agonist. Again, reference herein to uterine growth includes ovarian growth. For brevity, the subject genetic sequence is referred to herein as a "uterine estrogen agonist-inducible genetic sequence". The corresponding expression product is referred to herein as a "uterine estrogen agonist-mediating polypeptide".

Accordingly, one aspect of the present invention provides a nucleic acid molecule comprising a sequence of nucleotides corresponding to a uterine estrogen agonist-inducible genetic sequence.

As stated above, an estrogen agonist includes but is not limited to estrogen, estradiol, tamoxifen, diethylstilbestrol and growth hormone.

Reference herein to a "genetic sequence" includes reference to a gene as well as a cDNA sequence or a mRNA sequence or hybrid forms thereof. It is also encompassed by the term "nucleic acid molecule". The nucleic acid molecule may, therefore, be DNA or RNA such as but not limited to single or double stranded DNA or RNA. The term "gene" is used in its broadest sense and includes cDNA corresponding to the exons of a gene. Accordingly, reference herein to a "gene" is to be taken to include:-

(i) a classical genomic gene consisting of transcriptional and/or translational regulatory sequences and/or a coding region and or non-translated sequences (i.e. introns, 5'- and 3'- untranslated sequences); or

(ii) mRNA or cDNA corresponding to the coding regions . (i.e. exons) and 5'- and 3'- untranslated sequences of the gene.

The term "gene" is also used to describe synthetic or fusion molecules encoding all or part of an expression product, hi particular embodiments, the term "nucleic acid molecule", "genetic sequence" and "gene" may be used interchangeably.

The term "corresponding" means that the nucleotide sequences of the nucleic acid molecule has complete or partial identity or at least similarity with the uterus estrogen agonist-inducible genetic sequence in the genome or corresponds to the coding regions of this genetic sequence including any or all of its splice variants and polymorphisms. Where the corresponding sequences are not identical, it may differ by one or more nucleotide substitutions, additions and/or deletions whether or not such charges are degenerate.

The uterus estrogen agonist-inducible genetic sequence is conveniently identified following differential display.

Accordingly, another aspect of the present invention contemplates a nucleic acid molecule comprising a sequence of nucleotides corresponding to a uterine estrogen agonist-inducible genetic sequence which is expressed in estrogen agonist treated ovariectomized uterus cells or tissue but substantially not or in very low amounts in non-estrogen agonist treated ovariectomized uterine tissue. The term "low amounts" includes basal levels of expression product or at least 20% less than the amount in estrogen agonist treated ovariectomized uterus cells. The term "low amounts" also includes not detecting any activity using the procedures currently available.

In a particularly preferred embodiment, the nucleic acid molecule is referred to herein as "UO-44". The murine form of UO-44, such as from rat (i.e. rUO-44) comprises the nucleotide sequence substantially as set forth in SEQ ID NO:l. The human form of UO-44 (hUO-44) comprises the nucleotide sequence set forth in SEQ ID NO:3. The terms "UO- 44", "rUO-44", "hUO-44" or equivalent forms also encompass derivatives, homologs and analogs of UO-44.

Accordingly, another aspect of the present invention provides a nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:l or a nucleotide sequence having at least 50% similarity thereto or a nucleotide sequence capable of hybridizing to SEQ ID NO:l or a complementary form thereof under low stringency conditions.

More particularly, the present invention is directed to a nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:l or a nucleotide sequence having at least 50% similarity thereto or a nucleotide sequence capable of hybridizing to SEQ H) NO:l or a complementary form thereof under low stringency conditions wherein said nucleic acid molecule corresponds to a uterine estrogen agonist-inducible genetic sequence.

In a related embodiment, the present invention provides a nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:3 or a nucleotide sequence having at least 50% similarity thereto or a nucleotide sequence capable of hybridizing to SEQ ID NO:3 or a complementary form thereof under low stringency conditions. More particularly, the present invention is directed to a nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO: 3 or a nucleotide sequence having at least 50% similarity thereto or a nucleotide sequence capable of hybridizing to SEQ ID NO:3 or a complementary form thereof under low stringency conditions wherein said nucleic acid molecule corresponds to a uterine estrogen agonist-inducible genetic sequence.

Alternative percentage similarities contemplated and encompassed by the present invention include at least about 60%, at least about 70%, at least about 80%, at least about 90% and at least about 95% or above such as 96%, 97%, 98% or 99%.

Generally, the percentage similarity is measured with respect to the entire nucleotide sequence of SEQ ID NO:l or SEQ ID NO:3. However, the percentage similarity may also be relative to at least about 30 contiguous nucleotides of SEQ ID NO:l or SEQ ID NO:3 and preferably at least 30 contiguous nucleotides within a region characteristic of said uterine estrogen agonist-inducible genetic sequence. An example of the latter is a region encoding a CUB domain, a hydrophobic transmembrane domain, an anchor domain and/or a zona pellucida domain.

The term "characteristic" is not to imply that the sequence is unique to the uterine estrogen agonist-inducible genetic sequence, although such an aspect is encompassed by the present invention. The "characteristic" may need only be present in the genetic sequence.

The term "similarity" as used herein includes exact identity between compared sequences at the nucleotide or amino acid level. Where there is non-identity at the nucleotide level, "similarity" includes differences between sequences which result in different amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. Where there is non-identity at the amino acid level, "similarity" includes amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels, hi a particularly preferred embodiment, nucleotide and sequence comparisons are made at the level of identity rather than similarity.

Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include "reference sequence", "comparison window", "sequence similarity", "sequence identity", "percentage of sequence similarity", "percentage of sequence identity", "substantially similar" and "substantial identity". A "reference sequence" is at least 12 but frequently 15 to 18 and often at least 25 or above, such as 30 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two polynucleotides may each comprise (1) a sequence (i.e. only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of typically 12 contiguous residues that is compared to a reference sequence. The comparison window may comprise additions or deletions (i.e. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as, for example, disclosed by Altschul et al. (1997) (57). A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al. (1998) (58).

The terms "sequence similarity" and "sequence identity" as used herein refers to the extent that sequences are identical or functionally or structurally similar on a nucleotide-by- nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity", for example, is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g. A, T, C, G, I) or the identical amino acid residue (e.g. Ala, Pro, Ser, Thr, Gly, Nal, Leu, He, Phe, Tyr, Tip, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present invention, "sequence identity" will be understood to mean the "match percentage" calculated by the DΝASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software. Similar comments apply in relation to sequence similarity.

Reference herein to a low stringency includes and encompasses from at least about 0 to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization, and at least about 1 M to at least about 2 M salt for washing conditions. Generally, low stringency is at from about 25-30°C to about 42°C. The temperature may be altered and higher temperatures used to replace formamide and/or to give alternative stringency conditions. Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization, and at least about 0.5 M to at least about 0.9 M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt for hybridization, and at least about 0.01M to at least about 0.15M salt for washing conditions. In general, washing is carried out T_m = 69.3 + 0.41 (G+C)% (59). However, the T_m of a duplex DΝA decreases by 1°C with every increase of 1% in the number of mismatch base pairs (60). Formamide is optional in these hybridization conditions. Accordingly, particularly preferred levels of stringency are defined as follows: low stringency is 6 x SSC buffer, 0.1% w/v SDS at 25-42°C; a moderate stringency is 2 x SSC buffer, 0.1% w/v SDS at a temperature in the range 20°C to 65°C; high stringency is 0.1 x SSC buffer, 0.1% w/v SDS at a temperature of at least 651°C.

The nucleic acid molecule of the present invention and in particular UO-44, is generally in isolated form. This means that it has undergone at least one purification or isolation step from a biological sample. The nucleic acid molecule of the present invention may, therefore, be referred to as an "isolated nucleic acid molecule", a "biologically pure nucleic acid molecule" and/or an "isolated, purified nucleic acid molecule" amongst other suitable terms. An isolation step includes centrifugation, precipitation, chromatographic separation as well as electrophoretic separation.

As stated above, the subject nucleic acid molecule and in particular UO-44 may correspond to the genomic form of the uterine estrogen agonist-inducible genetic sequence or it may comprise one or more nucleotide substitutions, additions and/or deletions. These may be naturally occurring alterations in the nucleotide sequence or they may be artificially induced by any number of techniques. Furthermore, the nucleic acid molecule may comprise a fusion of heterologous sequences such as from different homologs from different individuals or different mammalian sources. One particularly useful form of the nucleic acid molecule is a "humanized" form of a non-human derived nucleic acid molecule. Conveniently, all such nucleotide modifications are encompassed by nucleic acid molecules being capable of hybridizing to SEQ ID NO:l or SEQ ID NO:3 or its complementary form under low stringency conditions.

The nucleic acid molecule of the present invention may be in single stranded or double stranded form and may be alone or in combination with a vector.

Accordingly, another aspect of the present invention relates to a genetic construct comprising a nucleotide sequence corresponding to a uterine estrogen agonist-inducible genetic sequence. Preferably, the nucleotide sequence is operably linked to a promoter sequence. The promoter sequence may be naturally associated with the uterine estrogen agonist-inducible sequence or it may be another promoter. Other nucleotide sequences may be located on the genetic construct such as those encoding a reporter molecule or selectable molecule and/or those involved in regulation of the subject nucleotide sequence.

In a particularly preferred embodiment, the genetic construct comprises UO-44 or a derivative or homologue thereof.

A "derivative" includes a mutant, part, portion or fragment of UO-44.

The nucleic acid molecule of the present invention and particularly UO-44 or its derivatives or homologs is conveniently used to produce a recombinant polypeptide, i.e. a uterine estrogen agonist-mediating polypeptide.

Accordingly, another aspect of the present invention contemplates a method for producing a uterine estrogen agonist-mediating polypeptide said method comprising introducing into a cell a nucleic acid molecule comprising a nucleotide sequence corresponding to a uterine estrogen agonist-inducible genetic sequence and subjecting said cell to conditions sufficient to permit expression of the nucleotide sequence and then recovering the polypeptide or membrane portions comprising same.

This method may be practiced using the cell into which the nucleic acid molecule has been introduced or progeny of the cell. The cell may be a prokaryotic or eukaryotic cell. Examples of the latter include insect cells, mammalian cells, uterine cell lines and yeast cells.

Preferably, the nucleotide sequence is UO-44 or its derivatives or homologs. Accordingly, another aspect of the present invention provides a polypeptide comprising a sequence of amino acids corresponding to a uterine estrogen agonist-mediating polypeptide.

Reference herein to a "polypeptide" includes a peptide and protein.

Preferably, the uterine estrogen agonist-mediating polypeptide is present in estrogen agonist treated ovariectomized uterus cells or tissue but substantially not or in low amounts in non-estrogen agonist treated ovariectomized uterine tissue.

Preferably, the polypeptide is encoded by UO-44 or its derivative or homologue.

Accordingly, another aspect of the present invention is directed to a polypeptide comprising sequence of amino acids encoded by the nucleotide sequence substantially as set forth in SEQ ID NO:l or a nucleotide sequence having at least 50% similarity thereto or a nucleotide sequence capable of hybridizing SEQ ID NO:l under low stringency conditions.

More particularly, the present invention provides a polypeptide comprising a sequence of amino acids substantially as set forth in SEQ ID NO:2 or an amino acid sequence having at least about 50% similarity thereto.

Even more particularly, the present invention provides a polypeptide comprising a sequence of amino acids substantially as set forth in SEQ ID NO:2 or an amino acid sequence having at least about 50% similarity thereto and which polypeptide corresponds to a uterine estrogen agonist-mediating polypeptide or a mutant, derivative, homologue or analogue of said polypeptide.

In a related embodiment, the present invention is directed to a polypeptide comprising sequence of amino acids encoded by the nucleotide sequence substantially as set forth in

SEQ ID NO: 3 or a nucleotide sequence having at least 50% similarity thereto or a nucleotide sequence capable of hybridizing SEQ ID NO: 3 under low stringency conditions.

More particularly, the present invention provides a polypeptide comprising a sequence of amino acids substantially as set forth in SEQ ID NO:4 or an amino acid sequence having at least about 50% similarity thereto.

Even more particularly, the present invention provides a polypeptide comprising a sequence of amino acids substantially as set forth in SEQ ID NO:4 or an amino acid sequence having at least about 50% similarity thereto and which polypeptide corresponds to a uterine estrogen agonist-mediating polypeptide or a mutant, derivative, homologue or analogue of said polypeptide.

The present invention encompasses a range of derivatives of the uterine estrogen agonist- mediating polypeptide. These are all encompassed by the term "UO-44" or reference to the UO-44 polypeptide. Derivatives include fragments, parts, portions, mutants, homologs and analogs of the subject polypeptide. Derivatives also include single or multiple amino acid substitutions, deletions and/or additions to the subject polypeptide. "Additions" to amino acid sequences or nucleotide sequences include fusions with other peptides, polypeptides or proteins or fusions to nucleotide sequences. Reference herein to the "uterine estrogen agonist-mediating polypeptide" or reference to the "subject polypeptide" includes reference to all derivatives thereof including functional and non-functional derivatives.

Analogs of the subject polypeptide contemplated herein include, but are not limited to, modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecule or their analogs.

Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH₄; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH .

The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitization, for example, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4- chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2- chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation with N- bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate. Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3- hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. A list of unnatural amino acid, contemplated herein is shown in Table 1.

Crosslinkers can be used, for example, to stabilize 3D conformations, using homo- bifunctional crosslinkers such as the bifunctional imido esters having (CH₂)_n spacer groups with n=l to n=6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N-hydroxysuccinimide and another group specific-reactive moiety such as maleimido or dithio moiety (SH) or carbodiimide (COOH). In addition, peptides can be conformationally constrained by, for example, incorporation of C_α and N_crmethylamino acids, introduction of double bonds between C_a and Cβ atoms of amino acids and the formation of cyclic peptides or analogs by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.

The present invention further contemplates chemical analogs of the subject polypeptide capable of acting as antagonists or agonists of the polypeptide or which can act as functional analogs of polypeptide. Chemical analogs may not necessarily be derived from the instant polypeptide but may share certain conformational similarities. Alternatively, chemical analogs may be specifically designed to mimic certain physiochemical properties of the subject polypeptide. Chemical analogs may be chemically synthesised or may be detected following, for example, natural product screening. The latter refers to molecules identified from various environmental sources such as river beds, coral, plants, microorganisms and insects.

These types of modifications may be important to stabilize the subject polypeptide if administered to an individual or for use as a diagnostic reagent. Other derivatives contemplated by the present invention include a range of glycosylation variants from a completely unglycosylated molecule to a modified glycosylated molecule. Altered glycosylation patterns may result from expression of recombinant molecules in different host cells.

TABLE 1

Non-conventional Code Non-conventional Code amino acid amino acid

c.-aminobutyric acid Abu L-N-methylalanine Nmala α-amino-c_-methylbutyrate Mgabu L-N-methylarginine Nmarg aminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgln carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa L-Nmethylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucine Nmile D-alanine Dal L-N-methylleucine Nmleu

D-arginine Darg L-N-methyllysine Nmlys

D-aspartic acid Dasp L-N-methylmethionine Nmmet

D-cysteine Dcys L-N-methylnorleucine Nmnle

D-glutamine Dgln L-N-methylnorvaline Nmnva D-glutamic acid Dglu L-N-methylorni thine N orn

D-histidine Dhis L-N-methylphenylalanine Nmphe

D-isoleucine Dile L-N-methylproline Nmpro

D-leucine Dleu L-N-methylserine Nmser

D-lysine Dlys L-N-methylthreonine Nmthr .D-methionine Dmet L-N-methyltryptophan Nmtrp

D-ornithine Dorn L-N-methyltyrosine Nmtyr

D-phenylalanine Dphe L-N-methylvaline Nmval

D-proline Dpro L-N-methylethylglycine Nmetg

D-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine Dthr L-norleucine Nle

D-tryptophan Dtrp L-norvaline Nva D-tyrosine Dtyr α-methyl-aminoisobutyrate Maib

D-valine Dval C--methyl-γ-aminobutyrate Mgabu

D-α-methylalanine Dmala α-methylcyclohexylalanine Mchexa

D-α-methylarginine Dmarg α-methylcylcopentylalanine Mcpen D-α-methylasparagine Dmasn α-methyl-α-napthylalanine Manap

D-α-methylaspartate Dmasp c-methylpenicillamine Mpen

D-α-methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu

D-α-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg

D-α-methylhistidine Dmhis N-(3-aminopropyl)glycine Norn D-α-methylisoleucine Dmile N-amino-ce-methylbutyrate Nmaabu

D-α-methylleucine Dmleu α-napthylalanine Anap

D-c.-methyllysine Dmlys N-benzylglycine Nphe

D-c-methylmethionine Dmmet N-(2-carbamylethyl)glycine Ngln

D-α-methylornithine Dmorn N-(carbamylmethyl)glycine Nasn D-α-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine Nglu

D-α-methylproline Dmpro N-(carboxymethyl)glycine Nasp

D-α-methylserine Dmser N-cyclobutylglycine Ncbut

D-α-methylthreonine Dmthr N-cycloheptylglycine Nchep

D-c--methyltryptophan Dmtrp N-cyclohexylglycine Nchex D-C--methyltyrosine Dmty N-cyclodecylglycine Ncdec

D-C--methylvaline Dmval N-cylcododecylglycine Ncdod

D-N-methylalanine Dnmala N-cyclooctylglycine Ncoct

D-N-methylarginine Dnmarg N-cyclopropylglycine Ncpro

D-N-methylasparagine Dnmasn N-cycloundecylglycine Ncund D-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine Nbhm

D-N-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine Nbhe

D-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine Narg

D-N-methylglutamate Dnmglu N-(l-hydroxyethyl)glycine Nthr

D-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine Nser D-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine Nhis D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine Nhtrp

D-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate Nmgabu

N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnrnmet

D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen N-methylglycine Nala D-N-methylphenylalanine Dnmphe

N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro

N-(l-methylpropyl)glycine Nile D-N-methylserine Dnmser

N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr

D-N-methyltryptophan Dnmtrp N-(l-methylethyl)glycine Nval D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap

D-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acid Gabu N-(p-hydroxyphenyl)glycine Nhtyr

L-t-butylglycine Tbug N-(thiomethyl)glycine Ncys

L-ethylglycine Etg penicillamine Pen L-homophenylalanine Hphe L-α-methylalanine Mala

L-o.-methylarginine Marg L-α-methylasparagine Masn

L-α-methylaspartate Masp L-α-methyl-t-butylglycine Mtbug

L-c-methylcysteine Mcys L-methylethylglycine Metg

L-α-methylglutamine Mgln L-α-methylglutamate Mglu L-α-methylhistidine Mhis L-α-methylhomophenylalanine Mhphe

L-α-methylisoleucine Mile N-(2-methylthioethyl)glycine Nmet

L-α-methylleucine Mleu L-α-methyllysine Mlys

L-α-methylmethionine Mmet L-o.-methylnorleucine Mnle

L-α-methylnorvaline Mnva L-c.-methylornithine Morn L-o;-methylphenylalanine Mphe L-α-methylproline Mpro

L-α-methylserine Mser L-α-methylthreonine Mthr

L-α-methyltryptophan Mtrp L-C--methyltyrosine Mtyr

L-c.-methylvaline Mval L-N-methylhomophenylalanine Nmhphe

N-(N-(2,2-diρhenylethyl) Nnbhm N-(N-(3,3-diρhenylρropyl) Nnbhe carbamylmethyl)glycine carbamylmethyl)glycine 1 -carboxy- 1 -(2,2-diphenyl- Nmbc ethylamino)cyclopropane

The identification of the subject nucleic acid molecule and its corresponding polypeptide permits the generation of a range of therapeutic molecules capable of modulating estrogen agonist-mediating cell growth and proliferation in the uterus including ovaries. Modulators contemplated by the present invention includes agonists and antagonists of gene expression or polypeptide activity. Antagonists include antisense molecules, ribozymes, co- suppression molecules, antibodies inhibitor peptide fragments and soluble receptors. Agonists include molecules which increase promoter activity or interfere with negative regulatory mechanisms.

Another embodiment of the present invention contemplates a method for modulating expression of a uterine estrogen agonist-inducible genetic sequence in a mammal such as a human, primate or laboratory test animal, said method comprising contacting a gene corresponding to said genetic sequence with an effective amount of a modulator of gene expression for a time and under conditions sufficient to up-regulate or down-regulate or otherwise modulate expression of the uterine estrogen agonist inducible genetic sequence.

Another aspect of the present invention contemplates a method of modulating activity of the uterus estrogen agonist-mediating polypeptide in a mammal such as a human, primate or laboratory test animal, said method comprising administering to said mammal a modulating effective amount of a molecule for a time and under conditions sufficient to increase or decrease activity of the uterine estrogen agonist-inducible genetic sequence. The molecule may be a proteinaceous molecule or a chemical entity and may also be a derivative of the subject polypeptide or its ligand or a chemical analogue or truncation mutant of said polypeptide or its ligand. Modulating levels of the instant polypeptide or its genetic sequence may be important in modulating uterine growth and development as well as in the treatment of uterine and ovarian cancers.

Accordingly, the present invention contemplates a composition comprising a uterine estrogen agonist-mediating polypeptide and one or more pharmaceutically acceptable carriers and/or diluents. These components are referred to as the "active ingredients".

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) and sterile powders for the extemporaneous preparation of sterile injectable solutions. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as licithin. The preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient. When the active ingredients are suitably protected they may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions in such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.01 μg and about 2000 mg of active compound per subject. Alternative amounts include between about 1.0 peg and about 1500 ng, between about 1 μg and about 1000 mg and between about 10 μg and about 500 mg per subject. Equivalent doses may alsobe expressed per kilogram of body weight.

The tablets, troches, pills, capsules and the like may also contain the components as listed hereafter: a binder such as gum, acacia, corn starch or gelatin; xcipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetemng agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound(s) may be incorporated into sustained-release preparations and formulations. Pharmaceutically acceptable carriers and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

The principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form. A unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.01 μg to about 2000 mg. Expressed in proportions, the active compound is generally present in from about 0.5 μg to about 2000 mg/ml of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients. Alternatively, amounts administered may be represented in terms of amounts/kg body weight, hi this case, amounts range from about 0.001 μg to about 1000 mg/kg body weight may be administered 500 mg/kg body weight or about 10.01 μg to about or above 0.1 μg to about 250 mg/kg body weight are contemplated by the present invention.

The pharmaceutical composition may also comprise genetic molecules such as a vector capable of transfecting target cells where the vector carries a nucleic acid molecule capable of modulating expression of the uterus estrogen agonist-inducible genetic sequence. The vector may, for example, be a viral vector.

Still another aspect of the present invention is directed to antibodies to the uterine estsrogen agonist-mediating polypeptide and its derivatives. Such antibodies may be monoclonal or polyclonal and may be selected from naturally occurring antibodies to the polypeptide or may be specifically raised to the polypeptide or its derivatives thereof. The antibodies and/or recombinant polypeptide or its derivatives of the present invention are particularly useful as therapeutic or diagnostic agents.

For example, a recombinant polypeptide and its derivatives can be used to screen for naturally occurring antibodies to estrogen agonist-mediating polypeptides. These may occur, for example in some autoimmune diseases. Alternatively, specific antibodies can be used to screen for the presence of levels of estrogen agonist-mediating polypeptides. Techniques for such assays are well known in the art and include, for example, sandwich assays and ELISA. Knowledge of levels of subject polypeptides may be important for diagnosis of certain cancers or a predisposition to cancers or for monitoring certain therapeutic protocols.

Antibodies to the subject polypeptides of the present invention may be monoclonal or polyclonal. Alternatively, fragments of antibodies may be used such as Fab fragments. Furthermore, the present invention extends to recombinant and synthetic antibodies and to antibody hybrids. A "synthetic antibody" is considered herein to include fragments and hybrids of antibodies. The antibodies of this aspect of the present invention are particularly useful for immunotherapy and may also be used as a diagnostic tool for assessing apoptosis, cancer, tissue regeneration or development, muscle development or health of neural tissue or for monitoring the program of a therapeutic regimin.

For example, specific antibodies can be used to screen for the subject polypeptides. The latter would be important, for example, as a means for screening for levels of a polypeptide in a cell extract or other biological fluid or purifying a polypeptide made by recombinant means from culture supernatant fluid. Techniques for the assays contemplated herein are known in the art and include, for example, sandwich assays and ELISA.

It is within the scope of this invention to include any second antibodies (monoclonal, polyclonal or fragments of antibodies or synthetic antibodies) directed to the first mentioned antibodies discussed above. Both the first and second antibodies may be used in detection assays or a first antibody may be used with a commercially available anti- immunoglobulin antibody. An antibody as contemplated herein includes any antibody specific to any region of the polypeptide.

Both polyclonal and monoclonal antibodies are obtainable by immunization with the enzyme or protein and either type is utihzable for immunoassays. The methods of obtaining both types of sera are well known in the art. Polyclonal sera are less preferred but are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of subject polypeptide, or antigenic parts thereof, collecting serum from the animal, and isolating specific sera by any of the known immunoadsorbent techniques. Although antibodies produced by this method are utihzable in virtually any type of immunoassay, they are generally less favoured because of the potential heterogeneity of the product.

The use of monoclonal antibodies in an immunoassay is particularly preferred because of the ability to produce them in large quantities and the homogeneity of the product. The preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art.

Another aspect of the present invention contemplates a method for detecting a uterus estrogen agonist-mediating polypeptide in a biological sample from a subject said method comprising contacting said biological sample with an antibody specific for said uterine estrogen agonist-inducible genetic sequence or its derivatives or homologs for a time and under conditions sufficient for an antibody-polypeptide complex to form, and then detecting said complex.

The presence of the instant polypeptide may be accomplished in a number of ways such as by Western blotting and ELISA procedures. A wide range of immunoassay techniques are available as can be seen by reference to U.S. Patent Nos. 4,016,043, 4, 424,279 and 4,018,653. Sandwich assays are among the most useful and commonly used assays and are favoured for use in the present invention. A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay, an unlabelled antibody is immobilized on a solid substrate and the sample to be tested brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-antigen complex, a second antibody specific to the antigen, labelled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of another complex of antibody-antigen-labelled antibody. Any unreacted material is washed away, and the presence of the antigen is determined by observation of a signal produced by the reporter molecule. The results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of hapten. Variations on the forward assay include a simultaneous assay, in which both sample and labelled antibody are added simultaneously to the bound antibody. These techniques are well known to those skilled in the art, including any minor variations as will be readily apparent. In accordance with the present invention the sample is one which might contain a subject polypeptide including membrane preparation or tissue biopsy. The sample is, therefore, generally a biological sample comprising biological fluid but also extends to preparations containing membranes.

In the typical forward sandwich assay, a first antibody having specificity for the instant polypeptide or antigenic parts thereof, is either covalently or passively bound to a solid surface. The solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid supports may be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay. The binding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing, the polymer-antibody complex is washed in preparation for the test sample. An aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient (e.g. 2-40 minutes or where more convenient, overnight) and under suitable conditions (e.g. for about 20°C to about 40°C) to allow binding of any subunit present in the antibody. Following the incubation period, the antibody subunit solid phase is washed and dried and incubated with a second antibody specific for a portion of the hapten. The second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to the hapten.

An alternative method involves immobilizing the target molecules in the biological sample and then exposing the immobilized target to specific antibody which may or may not be labelled with a reporter molecule. Depending on the amount of target and the strength of the reporter molecule signal, a bound target may be detectable by direct labelling with the antibody. Alternatively, a second labelled antibody, specific to the first antibody is exposed to the target-first antibody complex to form a target-first antibody-second antibody tertiary complex. The complex is detected by the signal emitted by the reporter molecule.

By "reporter molecule" as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative. The most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes) and chemiluminescent molecules, hi the case of an enzyme immunoassay, an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan. Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta-galactosidase and alkaline phosphatase, amongst others. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable colour change. Examples of suitable enzymes include alkaline phosphatase and peroxidase. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. In all cases, the enzyme-labelled antibody is added to the first antibody hapten complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the complex of antibody-antigen- antibody. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of hapten which was present in the sample. "Reporter molecule" also extends to use of cell agglutination or inhibition of agglutination such as red blood cells on latex beads, and the like.

Alternately, fluorescent compounds, such as fluorescein and rhodamine, may be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope. As in the EIA, the fluorescent labelled antibody is allowed to bind to the first antibody- hapten complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to the light of the appropriate wavelength the fluorescence observed indicates the presence of the hapten of interest. Immunofluorescene and EIA techniques are both very well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed.

The present invention also contemplates genetic assays such as involving PCR analysis to detect a uterus estrogen agonist-inducible genetic sequence its derivatives. Alternative methods or methods which may be used in conjunction include direct nucleotide sequencing or mutation scanning such as single stranded conformation polymorphoms analysis (SSCP) as well as specific oligonucleotide hybridization.

The isolation and cloning of UO-44 permits the monitoring of uterine growth and development and also provides a marker for uterine and ovarian cancers.

The UO-44 has been cloned from rat and, in particular, Rattus norvegirus (i.e. rUO-44) as well as from humans (i.e. hUO-44). This is done, however, with the understanding that the present invention extends to UO-44 homologs from any species but in particular from primate and livestock animals.

Accordingly, the present invention extends to a UO-44 homologue from a primate or livestock animal, said homologue comprising a nucleotide sequence capable of hybridizing to SEQ ID NO:l and/or SEQ ID NO: 3 or their complementary forms under stringency conditions.

The present invention further extends to promoter and other regulatory regions associated with the UO-44 gene as well as genomic clones. Genomic clones are particularly useful in gene therapy. The present invention further extends to genetically modified animals such as mice, rats, rabbits, sheep or non-human primates which are unable to express UO-44 or which have modified levels of expression of UO-44 genetic material. Such animals and in particular mice and rats are excellent models for screening, inter alia, for antagonists and agonists.

The present invention is further described by the following non-limiting Examples.

EXAMPLE 1 Animals and drug administration

Animal experiments were approved by the local Animal Care Committee. Intact, hypophysectomized (Hypox) or ovariectomized (OVX) female Sprague-Dawley rats, 50 days old at the beginning of the experiments, were obtained from Charles River, Quebec. Except for ovarian-dependent and time dependent tamoxifen induced UO-44 gene expression studies, OVX and Hypox animals were used in these experiments 2 weeks after ovariectomy and hypophysectomy, respectively. To study the effect of growth hormone on UO-44 gene expression, groups of Hypox rats (n=15) were daily injected with 0.5, 1, 1.5 and 2 μg recombinant human growth hormone (GH) (Genentech) per gram body weight for 21 days. To study the effect of estradiol on UO-44 gene expression, groups of ovariectomized rats (n=15) were implanted with 0.5 cm silastic tubes (0.04 in. ID, Dow Corning, Michigan) containing 17-β estradiol (Sigma) on the back of their necks. Control rats experienced the same surgical implantation with empty silastic tubes. Based on previous published work (49), the released rate of 17 β-estradiol from silastic implants was documented to be 2.4 μg/cm day. Tamoxifen (Sigma) was dissolved in castor oil at a concentration of 10 mg/ml. To study the effects of tamoxifen on UO-44 gene expression, groups of ovariectomized rats (n=15) daily received 1, 2, 3, 4 and 5 mg tamoxifen per kg body weight via subcutaneous injections for 3 weeks. To study the effects of ovariectomy on UO-44 gene expression in the uterus, groups of rats (n=15) were underwent ovariectomy and the uteri were collected at 6, 24, 48, 72, 96, 120 and 144 hr post-oavriectomy. To study the time dependent tamoxifen- induced UO-44 gene expression, groups of OVX-rats (n=15) were injected with 5 mg tamoxifen and the uteri were collected at 0, 6, 12, 18 and 24 h post-injection. Preformulated ICI 182780 (AstraZeneca Pharmaceuticals) was supplied at a concentration of 50 mg/ml in castor oil solution. To study the effects of ICI 182780 on UO-44 expression in ovary intact rats, groups of female Sprague-Dawley rats (n=15) received weekly subcutaneous injections of castor oil alone, 1, 1.5 or 2 mg of ICI 182780 per kg BW for 3 weeks. The pilot study showed that maximal reduction of uterine weight could be achieved at a dose of 1.5 mg ICI per kg body weight per week. At the end of the experiments, animals were sacrificed by carbon dioxide exposure. The uteri or ovaries were excised, trimmed, weighed and snap- frozen in liquid nitrogen and stored at -70°C for RNA extraction. Parts of the uterus and one ovary were embedded in OCT for in situ hybridization studies.

EXAMPLE 2 mRNA differential display

Ovariectomized rats were either untreated (OVX) or treated with 5 mgkg BW tamoxifen per day (OVX-TAM) for 14 days. Total RNA was isolated from uteri using RNAzol prernix solution and RNAzol B method (Tel-Test, Friendswood, TX) as previously described (49). Differential display was performed using RNA derived from uteri of OVX and OVX-TAM rats according to the protocol supplied with the RNAmap (trademark) kit (GenHunter Corp., Nashville, TN). Briefly, 5 μg of DNase I-treated total RNA were reverse transcribed with 300 units of Moloney murine leukemia virus reverse transcriptase (Pharmacia) in the presence of 1 niM T₁₂MG, T₁₂MA, T₁₂MT, or T₁₂MC primer (GenHunter) where M is a mixture containing dG, dA, and dC. Two tenth of this reaction was used in the PCR amplification reaction containing 2.5 niM each of dNTPs, 10 mCi of [α-³³P]dATP (NEN), and two primers: 1 mM T₁ oligonucleotide and 2 mM of one of the five arbitrary primers, AP-1 (5 - AGCCAGCGAA-3' [SEQ ID NO:5]); AP-2 (5'-GACCGCTTGT-3' [SEQ ID NO:6]); AP-3 (5'-AGGTGACCGT-3' [SEQ ID NO:7]); AP-4 (5'-GGTACTCCAC-3' [SEQ ID NO:8]); and AP-5 (5'-GTTGCGATCC-3' [SEQ ID NO:9]). These reactions contained 1 unit of AmpHTag DNA polymerase (Perkin Elmer). The cycling parameters for PCR were: 94°C for 30 s, 42°C for 120 s, 72°C for 45 s for 45 cycles. After PCR amplification, the PCR-amplified fragments were separated on 6% w/v denaturing polyacrylamide gel. The gel was dried and exposed to Kodak XAR film with mtensifying screens at -70°C, and cDNA representing differentially expressed mRNAs were excised from the dried gels for elution and reamphfication as described by GenHunter Corporation. Eluted DNA samples were reamplified by PCR using corresponding pair of primers under the same conditions as described above. Reamplified cDNA fragments were separated in 2.% v/v low melting agarose and used as probes in Northern blots to verify their differential expression in uteri. Desired fragments were subcloned into TA vector ( ivitrogen) and subjected to nucleotide sequence. EXAMPLE 3 cDNA library construction

Total RNA was isolated from tamoxifen-treated uteri (pool of 5 rats) using RNAzol premix solution and RNAzol B method (Tel-Test, Friendswood, TX) as described above. Poly A⁺ RNA was isolated from total RNA using Oligotex mRNA Kits (Qiagen) according to the manufacturer's protocol. Ten μg of poly A⁺ RNA derived from uteri of OVX-TAM rats were used to construct a unidirectional cDNA library in the vector pcDNA3.0 (hivitrogen, Carlsbad, CA) designed for expression in mammalian cells using the CMV promoter. cDNA was primed using the unidirectional Notl "T" primer so giving inserts in the correct orientation for expression. Double stranded cDΝA was size enriched and transformed into TOPI OF' cells after ligation into the vector. The 320 bp DΝA probe of rat UO-44 cDΝA was used to screen this rat uterine cDΝA library as described (50). Clones identified by this probe were isolated and sequenced by the Sanger dideoxy chain termination method and their nucleotide sequences were compared with those deposited in the GenBank and EMBL databases.

EXAMPLE 4 In situ hybridization

For mRΝA in situ hybridization, recombinant plasmid pcDΝA3.0 containing a 500-bp UO-44 fragment (nucleotide 1780 to 2280 of the UO-44 sequence, GenBank Accession No. AF022147) was linearized to generate sense and antisense digoxigenin-labeled RNA probes using Dig RNA Labeling kit (Boehringer Mannheim). Serial 7-8 μm OCT-frozen sections from either uterus or ovary were heated for 2 min at 50°C and dried for 30 min. Prehybridization, hybridization, posthybridization and immunological detection were performed according to the manufacturer's protocol and these sections were subsequently counterstained with haematoxylin. EXAMPLE 5 Northern blotting

Total RNA was isolated from indicated tissues of female rats as described (51). Northern blots were performed on total RNA and blots were hybridized with rat UO-44 or human GAPDH (ATCC) cDNAs as previously described (51). mRNA levels were determined by densitometric scanning of autoradiographs.

EXAMPLE 6 Stably infected MCF- 7 cell lines

The entire coding region of UO-44 cDNA was cloned into the mammalian expression vector pcDNA3.1 His (Invitrogen, Carlsbad, CA) to create the UO-44-pcDNA3.1/His expression vector. The UO-44-pcDNA3.1/His sequence was confirmed by sequencing. MCF-7 cells were seeded at a density of 2 x 10⁵ in 100 mm culture dishes in 90% α-MEM (Life Technologies, Inc.) containing 10% w/v FCS with Garamycin 24 hr prior to transfection. Cells were transfected with 5 μg of UO-44-pDNA3.1/His DNA or pDNA3.1/His control plasmid DNA and 28 μl of Lipofectamine reagent (Life Technologies) following recommendations of the manufacturer. Forty-eight hours following transfection, cells were subcultured at 1:10 and replaced with growth medium containing 800 μg/ml G418 (Calbiochem, La Jolla, CA). After 4 weeks, clones were isolated, expanded and assayed for UO-44 expression by Western blot analysis.

EXAMPLE 7 Western analysis

To localize the UO-44 protein, controls and UO-transfected MCF-7 cells were grown to 90% confluence. Plasma membrane-enriched subcellular fractions and cytosol were prepared by differential centrifugation as described previously (52). Plasma membrane and cytosolic proteins were subjected to Western blot analysis as described (53). Blots were incubated with mouse anti 6-Histidine antibody (Epitope Tagging) Ab-1 (NeoMarkers, Union City, CA) (1:500 dilution) and horseradish peroxidase-conjugated donkey anti-mouse secondary antibody (1:7500). Blots were visualized with a chemiluminescent detection system (ECL, Amersham) and exposed to film for 10 sec to 45 sec.

EXAMPLE 8

Isolation and sequence analysis of UO-44 cDNA

Using differential display methodology, several differentially displayed bands representing cDNA corresponding to gene whose expression was up-regulated during tamoxifen treatment were isolated. One of the bands, which was present in the uteri of tamoxifen-treated rats but disappeared in uteri derived from ovariectomized rats, was selected for further characterization (Figure 1 A). This 320 bp DNA fragment was used to probe Northern blots of mRNA obtained from uteri of OVX and OVX-tamoxifen-treated rats. A strong signal corresponding to an approximate molecular weight of 2.2 kb emerged in RNA isolated from ONX-tamoxifen-treated uteri, while no detectable signal was observed in the RΝA derived from uteri of ONX rats (Figure IB). The results suggested that the gene isolated was upregulated by tamoxifen. This 320 bp D A fragment was then subcloned into a TA vector and subjected to nucleotide sequence analysis.

Using this 320 bp DΝA probe to screen the cDΝA library prepared from tamoxifen-treated ovariectomized rat uterus resulted in the isolation of 14 clones. The longest clone contained a 2.2 kb insert. Comparison of the nucleotide sequence of the cDΝA against the non-redundant nucleotide database of GenBank established that this cDΝA (GenBank Accession No. AF022147) shared 99% with the estrogen regulated gene 1 (ERG1) cDNA (54).

The full-length UO-44 cDNA contains 2282 bp of nucleotide sequence. An initiator ATG codon (position 253) is followed by a single open reading frame of 607 amino acids with a calculated molecular weight of 68639 Da. The ATG initiation site is contained in the sequence for initiation by eukaryotic ribosomes described by Kozak (55). The open reading frame ends in a TGA teiniinator codon at position 2074 followed by 208 nucleotides in the 3' untranslated region. Within the first 265 amino acids of UO-44, two regions were identified that bear homology to the CUB motifs (complement subcomponents Clr/Cls, Uegf protein and bone morphogenetic protein) (56). The first CUB domain began at Cys-32 and the second at Cys-154. UO-44 also contained a zona pellucida domain at amino acids 276-523. The UO-44 amino acid sequence predicted a membrane protein with two transmembrane helices. The hydrophobic transmembrane region was 13 amino acids in length and was located between amino acids 5 and 17, while the anchor transmembrane region was 19 amino acids in length and is located between amino acids 571 and 589. There was a putative transmembrane domain near the carboxyl terminus. UO-44 teiminated in a short 19 amino acid polypeptide presumably positioned within the cytoplasm.

EXAMPLE 9 Tissue distribution of UO-44

To determine the expression of rat UO-44 gene in various tissues, total RNA obtained from tissues of mature female rats was analyzed by Northern blotting. Figure 2 shows that transcription of the UO-44 gene was observed only in uterus and ovary. UO-44 mRNA levels in adipose tissue, mammary gland, liver, kidney, muscle, heart, stomach, small intestine, spleen, brain, pituitary, and muscle were undetectable, suggesting that the UO-44 gene may be expressed at a very low level or not at all in these tissues. Since this gene is highly expressed in the uterus and ovary, it has been designated Uterine-Ovarian specific gene 44 or UO-44.

EXAMPLE 10

Hormonal regulation of UO-44

To detennine if UO-44 gene expression is steroid-hormone dependent, uteri were collected from rats at different times following ovariectomy. As shown in Figure 3, UO-44 mRNA rapidly disappeared following ovariectomy. Six hours post-ovariectomy, UO-44 mRNA levels dropped to 30% of controls and no UO-44 transcripts were detected at 144 hr post- ovariectomy (Figure 3). Re-expression of UO-44 gene could be achieved by tamoxifen treatment. Tamoxifen-induced UO-44 gene expression was observed as early as 6 hr post- tamoxifen injection (Figure 3). Treatment of ONX rats with various doses of tamoxifen resulted in a dose dependent up-regulation of UO-44 gene expression (Figure 4). ICI 182780 potently attenuated tamoxifen-induced UO-44 gene expression (Figure 4). Estradiol was more potent than tamoxifen to induce UO-44 gene expression. Progesterone was unable to restore UO-44 gene expression. In all cases, the changes in expression of UO-44 gene was positively correlated with the changes in uterine weight (Figure 4C).

To further examine the estrogenic effects of tamoxifen on the uterine growth and expression UO-44 in the uterus, ovary intact rats were treated with various doses of tamoxifen and a pure antiestrogen ICI 182780. Figure 5 shows that treatment of rats with tamoxifen for 3 weeks resulted in a significant increase in basal UO-44 expression. At the dose of 2 mg and 5 mg per kg body weight per day, tamoxifen stimulated UO-44 gene expression by 2- and 3-fold respectively. ICI 182780, on the other hand, was a very potent inhibitor of UO-44 gene expression, completely abolishing UO-44 gene expression in the uterus at the dose of 1 mg per kg body weight per week as compared with the ovary-intact uterus (Figure 5B). There is a positive correlation between UO-44 gene expression and uterine weight.

Uterine weight is known to increase by the influence of growth hormone (49), and this led the inventors to investigate whether UO-44 gene expression in the uterus was under the regulation of growth hormone. Northern blot analysis was performed using uteri and ovaries obtained from hypophysectomized (Hypox) rats treated with vehicle or various doses of recombinant human growth hormone. Comparing to uterine weight of ovary intact rats (Figure 5), hypophysectomy caused a sigmficant reduction in uterine weight which was coincident with the disappearance of UO-44 mRNA (Figure 6). Upon administration of growth hormone, UO-44 gene expression in the uterus was restored. Hypophysectomy, on the other hand, had very little or no effect on UO-44 gene expression in the ovary (Figure 7). However, blockage of estrogenic activity by aόhninistration of a pure antiestrogen ICI 182780 led to a dramatic reduction in UO-44 mRNA in the ovaries of hypophysectomized rats (Figure 7B).^' EXAMPLE 11 Localization of UO-44 in the uterus and ovary

Figure 5 showed that UO-44 mRNA was undetectable in uteri derived from ovariectomized rats and was expressed followed tamoxifen treatment. To detemiine the cell-type specific expression of UO-44 in the uterus following tamoxifen, in situ hybridization was performed on sections of uteri derived from OVX-tamoxifen-treated rats using an antisense RNA probe specific for rat UO-44. High levels of UO-44 were detected in the luminal epithelial cells and glandular population of the uteri following tamoxifen treatment (Figures 8A and 8B). The effects of tamoxifen on UO-44 expression was abolished by ICI treatment (Figure 8D) suggesting that tamoxifen acts as an estrogen to induce UO-44 expression. No staining was seen in smooth muscle cells. Hybridization with the sense UO-44 RNA probe showed no background staining (Figure 9C).

In the ovaries, varying amounts of UO-44 mRNA was detected in granulosa cells of a mixed population of follicles (Figure 10A). High levels of UO-44 expression were observed in the granulosa cells of medium size follicles (Figure 9A). Low to moderate UO-44 expression was detected in granulosa cells of small and large follicles (Figure 9A). Furthermore, lack of uniform UO-44 mRNA among granulosa cells within the same follicle was noted (Figure 9B). A control sense UO-44 probe produced no background staining in ovarian tissue (Figure 9C). UO-44 staining signals were almost lost in sections of ovaries from rats treated with ICI 182780 (Figure 9D).

EXAMPLE 12

Subcellular localization of UO-44 protein

To further demonstrate the subcellular localization of UO-44 protein, human breast cancer

MCF-7 cells were transfected with a mammalian expression vector containing full length UO- 44 cDNA (UO-44 ρcDNA3.1/His) or control pcDNA3.1 His vector. As shown in Figure 10,

6-Histidine antibody recognized a protein of approximately 68-69 kDa in plasma membrane- enriched subcellular fractions of UO-44 transfectants but not in the cytosol. No protein of identical size was detected in mock-transfected cells.

EXAMPLE 11 Identification and cloning of human UO-44 (hUO-44) homolog

The murine UO-44 cDNA was used to identify a human homolog. Using the murine coding region as a probe in a screening procedure, a full length human UO-44 (hUO-44) cDNA from a human uterus library was identified, cloned and sequenced to its entirety. The full length hUO-44 possess an open reading frame (ORF) of 357 amino acids with a calculated molecular weight of 40.17 kDa and a pi of 8.19. The nucleotide and corresponding amino acid sequence is set forth in SEQ ID NO:3 and SEQ ID NO:4, respectively.

A transcription and translational study (TnT) of the appropriate hUO-44 cDNA cloned into pBSIISK vector produced a translated product of about 40 kDa which was in accordance with that predicted from the ORF of the full length hUO-44 cDNA.

Further characterization of the full length hUO-44 showed that at the amino acid level, the hUO-44 possesses a ZP domain but no CUB domains that were present in the murine homolog. Further efforts, i.e. 5' RACE targeted at the isolation of a probable 5' end of the hUO-44 did not reveal the existence of any other cDNA fragments. Therefore, the UO-44 cDNA is deemed to be full length. A transmembrane domain present in the murine molecule was also present in the hUO-44.

A northern analysis conducted employing the hUO-44 probe without the poly 'A' tail using three commercially available poly 'A' mRNA Northern blots revealed the presence of two transcripts (-2.1 kb and 3.0 kb). A very much weaker signal was detected at about 5.0 kb.

The hUO-44 gene is also present in a human placenta library. Data indicate that the hUO- 44 gene is approximately 7-8 kb inclusive of the promoter regions. There are also six exons encoding the full length hUO-44 protein and these exons were also found to comply with the consensus exon-intron junctions.

BLAST data from the human genome sequence database from NCBI revealed that the hUO-44 gene is aligned to a specific contig clone from chromosome 10 with six BLAST hits, all with a homology of greater than 97% confidence. These six BLAST hits coincide well with the exons of hUO-44.

The contig clone where hUO-44 gene resides is known as NT008720. Within this genomic contig clone, there exists a gene known a the DMBTl gene (deleted in malignant brian tumor gene 1). There is evidence fro an involvement of the inactivation of tumor suppressor genes at chromosome lOq in the carcinogenesis of brain tumors, melanomas and carcinomas of the lung, the prostate, the pancreas and the endometrium.

The locality of this gene and that of hUO-44 appears to be a distinct locus for potential tumor suppressor genes owing to recent LOH studies.

The DMBTl gene has at least 54 exons which span a genomic region of about 80 kb. This organisation of the domain structure of the DMBTl protein reveals certain degrees of similarity to that of the hUO-44 protein with the existence of the ZP and transmembrane domains. The presence of the CUB domains is also reminiscent to that of the murine homolog which possess two CUB domains.

Thus, hUO-44 may also function as a tumor suppressor gene.

Examination of the putative promoter using the Matlnspector N2.2 progam reveals potential regulatory elements, i.e. TATA box, CEBPB, API STAT, CREB, etc. A ΝfkappaB element is also present and this has been strongly implicated in immune regulated genes. BIBLIOGRAPHY

1. Pope, W. F., et al. Theriogenology 23: 91-105, 1985.

2. Wilmut, L., et al. Theriogenology, 23: 107-119, 1985.

3. Simmen, R. M., et al. J.CellBiol, 103: 154a, 1986.

4. J-keda, T., et al. J. Biol Chem., 259: 4049-4064, 1984.

5. DiAugustine, R.P., et al. Endocrinol, 122:2355-2363, 1988.

6. Murphy, L. J., et al Rev., ϋ:443-453, 1990.

7. Huynh, H., et al. Cancer Res., 54: 3115-3119, 1994.

8. Tang, X. -M., et al. Endocrinol, 135: 450-459, 1994.

9. Roberts, R. M., et al. J. Reprod. Fert, 82: 875-892, 1988.

10. Jensen, EN., et al. Annu. Rev. Biochem., 41: 203-230, 1972.

11. Anderson, J. Ν., et al. Endocrinol, 96: 160-167. 1975.

12. Katzenellenbogen, B. S., et al Biochemical Actions of Hormones, vol. 3. New York: Academics Press, 1975.

13. Katzenellenbogen, B. S., et al. Mol.Endocrinol, 1: 445-450, 1987.

14. Travers, M. T., et al. FEBSLett., 211:21-30, 1987.

15. Murphy, L. J., et al. Endrocrinol, 120: 1882-1888, 1987.

16. Weisz, A., et al. Mol. Endocrinol, 2: 816-824, 1988.

17. Loose-Mitchell, D. S., et al. Mol. Endocrinol, 2: 946-951, 1988.

18. Ghahary, A., et al Endocrinol, 125: 597-604. 1989.

19. Chippetta, C, et al. Journal of Steriod Biochemistry & Molecular Biology, 41: 113- 123, 1992.

20. Nelson, K.G., et al. Endocrinol, 131: 1657-1665, 1992.

21. Yoshinaga, K., et al. Endocrinol, 85: 103-112, 1969.

22. Brenner. S., Nature, 554:528-530, 1988.

23. Rawlins, N.D., et al. Methods Enzymol., 244: 19-61, 1994.

24. Bork, P., et al. J.Mol. Biol, 231: 539-545, 1993.

25. Kinoshita, H., et al. FEBSLett, 250: 411-415, 1989.

26. Takashi, A., et al Biochem, Biophys. Res. Commun., 190: 681-687, 1993.

27. Murphy, G. J., et al FEBS Lett, 289: 4-7, 1991. 28. Rawlings, N. D., et al. Methods Enzymol, 248: 183-228, 1995.

29. Woessner, J. F. Jr. FASEB J., 5: 2145-2154, 1991.

30. Hits, L. A., et al. Biol, Chem Hoppe Seyler, 373: 381-385, 1992.

31. Montecucco, C, et al. Trends Biochem. Sci, 18: 324-327, 1993.

32. Kane, W. H., et al. Proc. Natl Acad. Sci. U.S.A. 83: 6800-6804, 1986.

33. Johnson, J. D., et al. Proc. Natl. Acad. Sci. U.S.A., 90: 5677-5681, 1993.

34. Couto, J.R., et al. DNA Cell Biol, 15: 281-286, 1996.

35. Kane, W. H., et al. Blood, 71: 539-555, 1988.

36. Ortel, T.L., et al. J. Biol. Chem., 269: 15898-15905, 1994.

37. Xue, J., et al. Biochem., 32: 5917-5923, 1993.

38. Mc Mullen, B.A., et al.Protein Sci, 4: 740-746, 1995.

39. Hvarregaard, J., et al. Eur. J. Biochem., 240: 628-636, 1996.

40. Le,. T. H., et al. J. Cell Biol, 116: 545-557, 1992.

41. Calvete, J. J., et al. Eur. J. Biochem., 218: 719-725, 1993.

42. Kwok, S. C, et al DNA Cell Biol, 12: 605-610, 1993.

43. Einspanier, R., et al. Biochem. Biophys. Res. Commun., 179: 1006-1010, 1991.

44. Jordan, V.C., et al. Endocr. Rev., 20: 253-278, 1999. >

45. Fornander, T., et al. Lancet, 117-120, 1989.

46. Katzenellenbegen., et al. Breast Cancer Research and Treatment. 44: 23-38, 1997.

47. Powles, T. J., et al Endocrine-Related Cancer, 4: 135-139, 1997.

48. Robaire, B., et al. Biol. Repro., 21 : 445-463, 1979.

49. Huynh F. and Pollak, M., Cancer Research 53: 5585-5588, 1993.

50. Ausubel, E. F., et al. Current protocols, 1993.

51. Huynh, H., et al. Cancer Res., 53: 1727-1730, 1993.

52. Pollak, M., et al. Cancer Lett, 38: 223-230, 1987.

53. Huynh, H., etal. Cancer Res., 55: 2225-2231, 1995.

54. Chen, D. et al, Journal of Biological Chemnistry 274: 32215-32224, 1999.

55. Kozak, M., Cell 44: 283-292, 1986.

56. Bork, P. and Beckmann, G., Journal of Molecular Biology 231: 539-545, 1993.

57. Altschul et al. Nucl. Acids Res. 25: 3389. 1997. 58. Ausubel et al. "Current Protocols in Molecular Biology", John Wiley & Sons fric, 1994-1998, Chapter 15.

59. Marmur and Doty J. Mol. Biol. 5: 109, 1962.

60. Bonner and Laskey Eur. J. Biochem. 46: 83, 1974.

Claims

1. An isolated nucleic acid molecule comprising a sequence of nucleotides corresponding to a uterine estrogen agonist-inducible genetic sequence in a mammal.

2. An isolated nucleic acid molecule according to Claim 1 wherein the uterine estrogen agonist-inducible genetic sequence is expressed in estrogen agonist treated ovariectomized uterus cells or tissue but substantially not or in very low amounts in non- estrogen agonist treated ovariectomized uterine tissue.

3. An isolated nucleic acid molecule according to Claim 1 or 2 comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:l or a nucleotide sequence having at least 50%> similarity thereto or a nucleotide sequence capable of hybridizing to SEQ ID NO:l or a complementary form thereof under low stringency conditions.

4. An isolated nucleic acid molecule according to Claim 1 or 2 comprising a sequence of nucleotides encoding an amino acid sequence substantially as set forth in SEQ ID NO:2 or an amino acid sequence having at least 50% similarity thereto.

5. An isolated nucleic acid molecule according to Claim 1 or 2 comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:3 or a nucleotide sequence having at least 50% similarity thereto or a nucleotide sequence capable of hybridizing to SEQ ID NO:3 or a complementary form thereof under low stringency conditions.

6. An isolated nucleic acid molecule according to Claim 1 or 2 comprising a sequence of nucleotides encoding an amino acid sequence substantially as set forth in SEQ ID NO:4 or an amino acid sequence having at least 50% similarity thereto.

7. An isolated nucleic acid molecule according to Claim 3 comprising the nucleotide sequence set forth in SEQ ID NO:l.

8. An isolated nucleic acid molecule according to Claim 4 comprising the nucleotide sequence set forth in SEQ ID NO:3.

9. A genetic construct comprising a nucleic acid molecule having a sequence of nucleotides corresponding to a uterine estrogen agonist-inducible genetic sequence in a mammal.

10. A genetic construct according to Claim 9 wherein the uterine estrogen agonist-inducible genetic sequence is expressed in estrogen agonist treated ovariectomized uterus cells or tissue but substantially not or in very low amounts in non-estrogen agonist treated ovariectomized uterine tissue.

11. A genetic construct according to Claim 9 or 10 comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:l or a nucleotide sequence having at least 50% similarity thereto or a nucleotide sequence capable of hybridizing to SEQ ID NO:l or a complementary form thereof under low stringency conditions.

12. A genetic construct according to Claim 9 or 10 comprising a sequence of nucleotides encoding an amino acid sequence substantially as set forth in SEQ ID NO:2 or an amino acid sequence having at least 50% similarity thereto.

13. A genetic construct according to Claim 9 or 10 comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:3 or a nucleotide sequence having at least 50% similarity thereto or a nucleotide sequence capable of hybridizing to SEQ ID NO: 3 or a complementary form thereof under low stringency conditions.

14. A genetic construct according to Claim 9 or 10 comprising a sequence of nucleotides encoding an amino acid sequence substantially as set forth in SEQ ID NO:4 or an amino acid sequence having at least 50% similarity thereto.

15. A genetic construct according to Claim 14 comprising the nucleotide sequence set forth in SEQ ID NO:l.

16. A genetic construct according to Claim 15 comprising the nucleotide sequence set forth in SEQ ID NO:3.

17. A genetic construct according to any one of Claims 9 to 16 wherein the nucleic acid molecule is operably linked to a promoterΛ

18. An isolated polypeptide comprising a sequence of amino acids encoded by a uterine estrogen agonist-inducible genetic sequence.

19. An isolated polypeptide according to Claim 18 wherein the uterine estrogen agonist-inducible genetic sequence is expressed in estrogen agonist treated ovariectomized uterus cells or tissue but substantially not or in very low amounts in non-estrogen agonist treated ovariectomized uterine tissue.

20. An isolated polypeptide according to Claim 18 or 19 comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:l or a nucleotide sequence having at least 50% similarity thereto or a nucleotide sequence capable of hybridizing to SEQ ID NO:l or a complementary form thereof under low stringency conditions.

21. An isolated polynucleotide according to Claim 18 or 19 comprising a sequence of nucleotides encoding an amino acid sequence substantially as set forth in SEQ JD NO:2 or an amino acid sequence having at least 50% similarity thereto.

22. An isolated polynucleotide according to Claim 18 or 19 comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:3 or a nucleotide sequence having at least 50% similarity thereto or a nucleotide sequence capable of hybridizing to SEQ ID NO: 3 or a complementary form thereof under low stringency conditions.

23. An isolated polynucleotide according to Claim 18 or 19 comprising a sequence of nucleotides encoding an amino acid sequence substantially as set forth in SEQ ID NO:4 or an amino acid sequence having at least 50% similarity thereto.

24. An isolated polynucleotide according to Claim 23 comprising the nucleotide sequence set forth in SEQ ID NO: 1.

25. An isolated polynucleotide according to Claim 24 comprising the nucleotide sequence set forth in SEQ ID NO:3.

26. A method for producing a uterine estrogen agonist-mediating polypeptide, said method comprising introducing into a cell a nucleic acid molecule comprising a nucleotide sequence corresponding to a uterine estrogen agonist-inducible genetic sequence and subjecting said cell to conditions sufficient to permit expression of the nucleotide sequence and then recovering the polypeptide or membrane portions comprising same.

27. A method according to Claim 26 wherein the uterine estrogen agonist- inducible genetic sequence is expressed in estrogen agonist treated ovariectomized uterus cells or tissue but substantially not or in very low amounts in non-estrogen agonist treated ovariectomized uterine tissue.

28. A method according to Claim 25 or 26 comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:l or a nucleotide sequence having at least 50% similarity thereto or a nucleotide sequence capable of hybridizing to SEQ ID NO:l or a complementary form thereof under low stringency conditions.

29. A method according to Claim 25 or 26 comprising a sequence of nucleotides encoding an amino acid sequence substantially as set forth in SEQ ID NO:2 or an amino acid sequence having at least 50% similarity thereto.

30. A method according to Claim 25 or 26 comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:3 or a nucleotide sequence having at least 50% similarity thereto or a nucleotide sequence capable of hybridizing to SEQ ID NO: 3 or a complementary form thereof under low stringency conditions.

31. A method according to Claim 25 or 26 comprising a sequence of nucleotides encoding an amino acid sequence substantially as set forth in SEQ H) NO:4 or an amino acid sequence having at least 50% similarity thereto.

32. A method according to Claim 31 comprising the nucleotide sequence set forth in SEQ J-D NO: 1.

33. A method according to Claim 32 comprising the nucleotide sequence set forth in SEQ ID NO:3.

34. Use of a nucleic acid molecule according to any one of Claims 1 to 8 in the manufacture of a medicament for the treatment of a condition in a mammal.

35. Use according to Claim 34 wherein the nucleic acid molecule encodes a protein comprising the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:4 or an amino acid sequence having at least about 50% similarity thereto.

36. A regulatory element associated with a genomic nucleotide sequence corresponding to the nucleic acid molecule according to any one of Claims 1 to 8.

37. A regulatory element according to Claim 36 wherein the element is a promoter.

38. A regulatory element according to Claim 36 wherein the element is a terminator.

39. A regulatory element according to Claim 36 wherein the element is a