EP0804573A1 - Methods and compositions for modulating morphogenic protein expression - Google Patents

Abstract

Disclosed are methods and compositions for screening compounds for their ability to modulate expression of a tissue morphogenetic protein, particularly OP-1, OP-1 homologs and closely related proteins, using one or more OP-1-specific, noncoding nucleotide sequences and a suitable reporter gene.

Description

Methods and Compositions for Modulating Morphogenic Protein Expression

Reference to Related Applications

This application is a continuation-in-part of USSN 08/255,250, filed June 7, 1994 which is a continuation-in-part of USSN 07/938,021, filed August 28, 1992 which is a continuation-in- part of USSN 07/752,861, filed August 30, 1991 which is a continuation-in-part of USSN 07/667,274, filed March 11, 1991, the disclosures of which are incorporated herein by reference.

Field of the Invention The invention relates generally to the field of drug screening assays. More particularly, the invention relates to methods and compositions for identifying molecules that modulate production of true tissue morphogenic proteins.

Background of the Invention

A class of proteins recently has been identified, the members of which are true tissue morphogenic proteins. The members of this class of proteins are characterized as competent for inducing the developmental cascade of cellular and molecular events that culminate in the formation of new organ-specific tissue, including any vascular and connective tissue formation as required by the naturally occurring tissue. Specifically, the morphogenε are competent for inducing all of the following biological functions in a morphogenically permissive environment: (1) stimulating proliferation of progenitor cells; (2) stimulating differentiation of progenitor cells; (3) stimulating the proliferation of differentiated cells and (4) supporting the growth and maintenance of differentiated cells. For example, the morphogenic proteins can induce the full developmental cascade of bone tissue morphogenesis, including the migration and proliferation of mesenchymal cells, proliferation and differentiation of chondrocytes, cartilage matrix formation and calcification, vascular invasion, osteoblast proliferation, bone formation, bone remodeling, and hematopoietic bone marrow differentiation. These proteins also have been shown to induce true tissue morphogenesis of non-chondrogenic tissue, including dentin, liver, and nerve tissue.

A particularly useful tissue morphogenic protein is human OP-1 (Osteogenic Protein-1) , described in U.S. 5,011,691; US Pat. No. 5,266,683 and Ozkaynak et al. (1990) EMBO J. 9 : 2085-2093. Species homologues identified to date include mouse OP-1 (see US Pat. 5,266,683) and the Drosophila homologue 60A, described in Wharton et al. (1991) PNAS 88:9214-9218) . Other closely related proteins include OP-2 (Ozkaynak (1992) J. Biol. Chem. 267:25220- 25227 and US Pat. No. 5,266,683); BMP5, BMP6 (Celeste et al. (1991) PNAS 87:9843-9847) and Vgr-1 (Lyons et al. (1989) . These disclosures are incorporated herein by reference.

It previously has been contemplated that these tissue morphogens can be administered to an animal to regenerate lost or damaged tissue. Alternatively, one can envision administering a molecule capable of modulating expression of the endogenous tissue morphogen as a means for providing morphogen to a site in vi vo.

It is an object of this invention to provide compositions and methods of screening compounds which can modulate expression of an endogenous tissue morphogen, particularly OP-1 and closely related genes. The compounds thus identified have utility both in vi tro and in vi vo . Useful compounds contemplated include those capable of stimulating transcription and/or translation of the OP-1 gene, as well as compounds capable of inhibiting transcription and/or translation of the OP-1 gene.

These and other objects and features of the invention will be apparent from the description, drawings and claims which follow.

Summary of the Invention

The invention features compositions and methods for screening candidate compounds for the ability to modulate the effective local or systemic quantity of endogenous OP-1 in an organism, and methods for producing the compounds identified. In one aspect, the method is practiced by: (1) incubating one or more candidate compounds with cells transfected with a DNA sequence encoding, in operative association with reporter gene, a portion of an OP-1 non-coding DNA sequence that is competent to act on and affect expression of the associated receptor gene; (2) measuring the level of reporter gene expression in the transfected cell, and (3) comparing the level of reporter gene expressed in the presence of the candidate compound with the level of reporter gene expressed in the absence of the candidate compound. In a related aspect, the invention features the compound that is identified by use of the method of the invention.

The screening method of the invention provides a simple method of determining a change in the level of a reporter gene product expressed by a cell following exposure to one or more compound(ε) . The level of an expressed reporter gene product in a given cell culture, or a change in that level resulting from exposure to one or more compound(s) indicates that application of the compound can modulate the level of the morphogen expressed and normally associated with the non-coding sequence. Specifically, an increase in the level of reporter gene expression is indicative of a candidate compound's ability to increase OP-1 expression in vivo . Similarly, a decrease in the level of reporter gene expression is indicative of a candidate compound's ability to decrease or otherwise interfere with OP-1 expression in vi vo .

^'The methods and compositions of the invention can be used to identify compounds showing promise as therapeutics for various in vivo and ex vi vo mammalian applications, as well as to identify compounds having numerous utilities. For example, morphogen expression inducing compounds can be used in vi vo to correct or alleviate a diseased condition, to regenerate lost or damaged tissue, to induce cell proliferation and differentiation, and/or to maintain cell and tissue viability and/or a differentiated phenotype in vivo or ex vivo. The compounds also can be used to maintain the viability of, and the differentiated phenotype of, cells in culture. The various in vivo, ex vivo, and in vi tro utilities and applications of the morphogenic proteins described herein are well documented in the art. See, for example, US 92/01968 (WO 94/03200), filed March 11, 1992; US 92/07358 (WO

93/04692), filed August 28; PCT US 92/0743 (WO 93/05751), filed August 28, 1992; US 93/07321 (WO 94/03200), filed July 29, 1993; US 93/08808 (WO 94/06449), filed September 16, 1993; US93/08885 (WO94/06420) , filed September 15, 1993, and US Pat. No. 5,266,683.

Morphogen expression inhibiting compounds identified by the methods, kits and compositions described herein can be used to modulate the degree and/or timing of morphogen expression in a cell. Such compounds can be used both in vi tro and in vi vo to more closely regulate the production and/or available concentration of morphogen.

List of useful terms and Definitions

As used herein, "gene expression" is understood to refer to the production of the protein product encoded by a DNA sequence of interest, including the transcription of the DNA sequence and translation of the mRNA transcript.

As used herein, "operative association" is a fusion of the described DNA sequences with a reporter gene in such a reading frame as to be co-transcribed, or at such a relative positioning as to be competent to modulate expression of the reporter gene. As used herein, "vector" is understood to mean any nucleic acid comprising a nucleotide sequence of interest and competent to be incorporated into a host cell and recombining with and integrating into the host cell genome. Such vectors include linear nucleic acids, plasmids, phagemids, cosmids, YAC'S (yeast artificial chromosomes) and the like.

As used herein, "non-coding sequence" or "non-coding DNA" includes DNA sequences that are not transcribed into RNA sequence, and/or RNA sequences that are not translated into protein. This category of "non-coding sequence" has been defined for ease of reference in the application, and includes sequences occurring 5' to the ATG site which indicates the start codon and sequences 3' to the stop codon, as well as intervening intron sequences that occur within the coding region of the gene. As used herein, an "OPl-specific" non-coding sequence is understood to define a non- coding sequence that lies contiguous to OP1 specific coding sequence at an OP-1 gene locus under naturally-occurring conditionε. The sequences may include 5', 3' and intron sequences.

As used herein, "allelic, species and other sequence variants thereof" includes point mutations, insertions and deletions such as would be naturally occurring or which can genetically engineered into an OP-1 non-coding DNA sequence and which do not affect substantially the regulation of a reporter gene by the OP-1 non-coding sequence. For example, one of ordinary skill in the art can use site directed mutagenesis to modify , as by deletion, for example, one or more of the OP-1 non-coding sequences described herein without substantially affecting the regulation of OP-1 or a reporter gene by the modification. Such modifications are considered to be within the scope of the disclosure provided herein. As used herein, a "Wt-l/Egr-1 consensus binding sequence" or Kt-l/Egr-1 consensus binding element" is a nine base sequence which has been shown to be bound by the DNA binding proteins Wt-1 and Egr-1. The consensus sequence of the Wt-l/Egr-1 binding site has been determined by homology to be GN3NGGGNG, Seq. ID No. 4 (Rauscher et al. , Science 250:1259-1262 (1990), incorporated herein by reference) .

As used herein, a "TCC binding sequence" or "TCC binding element" is an approximately 15 to 20 base sequence of DNA which contains at least three contiguous or non-contiguous repeats of the DNA sequence TCC. The TCC binding sequence identified in human OP-1 genomic DNA is shown in Seq. ID No. 5, and the TCC binding sequence identified in murine OP-1 genomic DNA is shown in Seq. ID No. 6. The TCC binding sequence has also been shown to be bound by the DNA binding proteins Wt-1 and Egr-1 (Wang et al., Proc. Natl. Acad. Sci. 90:8896-8900 (1993)'; Wang et al. , Biochem Biophys Res. Comm. , 188:433-439 (1992)) .

As used herein, a "FTZ binding sequence" or "FTZ binding element" is a Fushi-tarazu DNA sequence (FTZ) that has been shown to be bound by the DNA binding protein Fushi-tarazu (FTZ-F1) . The FTZ binding sequence identified in human OP-1 genomic DNA is shown in Seq. ID No. 7. The FTZ consensus sequence, a consensus sequence for the nuclear hormone receptor εuperfamily, is YCAAGGYCR. Aε used herein, a "εteroid binding sequence" or "steroid binding element" is a DNA sequence that has been shown to be bound by one or more elements, in response to activating signal molecules. Exampleε of such "activating signal molecules" include retinoids, Vitamin D, and also include steroids such as estrogen and progesterone. Useful elements are anticipated to include the FTZ-F1 protein, WT-1 and Egr-1. Activating signal molecules of the nuclear receptor family have recently been εhown to bind to DNA aε homodimerε, heterodimers or as monomers (Parker, M.G., Curr. Op. Cell Biol., 1993, 5:499-504). The formation of heterodimers among the nuclear receptor family moleculeε may significantly increase the diversity of binding elements which are recognized by these nuclear receptors, and provide for differential regulation of genes containing the specific binding sites. In addition, the nuclear receptors have been shown to interact with other accessory factors, such as transcription factors, to stimulate or repress transcription. These interactions, between the nuclear receptors and the nuclear receptors and acceεεory factors, indicate that there could be significant number of nuclear receptor/accessory factor interactionε which have widely different tranεcriptional activitieε.

While the method of the invention is described with reference to a single cell, as will be appreciated by those having ordinary skill in the art, this is only for ease of description, and the method is most efficiently carried out using a plurality of cells.

With respect to transfection of DNA sequenceε in the cell and the method of the invention, all means for introducing nucleic acids into a cell are contemplated including, without limitation, CaP0₄ co-precipitation, electroporation, DEAE-dextran mediated uptake, protoplast fusion, microinjection and lipofusion. A key to the invention is the DNA sequences with which the cell is transfected, rather than the mechanical or chemical process by which the DNA incorporation is accomplished. Uεeful reporter genes are characterized as being easy to transfect into a suitable host cell, easy to detect using an established assay protocol, and genes whose expression can be tightly regulated. Other reporter genes contemplated to have utility include, without limitation, the luciferase gene, the Green Fluorescent Protein (GFP) gene, the chloramphenicol Acetyl Transferase gene (CAT) , human growth hormone, and beta- galactosidase. Additional useful reporter genes are any well characterized genes the expression of which is readily asεayed, and examples of such reporter genes can be found in, for example, F.A. Ausubel et al., Eds., Current Protocols in Molecular Biology, John Wiley Sons, New York, (1989). As will be appreciated by those having ordinary skill in the art, the listed reporter genes are only a few of the possible reporter genes, and it is only for eaεe of description that all available reporter genes are not listed.

While the method, vectors, and cells described recite the use of a reporter gene in operative association with an OP-1 non- coding DNA sequence, it will be apparent to those of ordinary skill in the art that the DNA sequence OP-1, including human OP1, shown in Seq. ID No. 1 or murine OP-1, disclosed in U.S. Patent No. 5,266,683, is also within the scope of a suitable reporter gene. Other suitable reporter genes can be used for ease in assaying for the presence of the reporter mRNA or reporter gene product .

Where a cell line is to be establiεhed, particularly where the transfected DNA iε to be incorporated into the cell'ε genome, lines that can be immortalized are eεpecially desirable. As used herein, "immortalized" cell lines are viable for multiple .pasεages (e.g., greater than 50 generations) without significant reduction in growth rate or protein production.

While the selected non-coding DNA sequences disclosed herein are described using defined baεeε, as will be appreciated by those having ordinary skill in the art, to some degree the lengths of the selected DNA sequences recited are arbitrary and are defined for convenience. As will be understood by those of ordinary skill in the art, shorter sequences of OP-1 non-coding DNA sequence and other fusion DNA's can be used in a vector according to the invention, and can be transfected into a cell, or uεed in the method of the invention for screening a candidate compound for its ability to modulate OP-1 expreεεion. Specifically, it iε standard procedure for molecular biologists to first identify useful - 8 -

regulatory sequences, and then to determine the minimum sequence required, by systematic digestion and mutagenesis e.g., by exonuclease or endonucleaεe digestion, site directed mutagenesis and the like. Accordingly, subsequent, standard routine experimentation iε anticipated to identify minimum sequences and these, shorter sequenceε are contemplated by the invention disclosed herein.

Useful cell types for the method and compositions according to the invention include any eukaryotic cell. Currently preferred are cell types known to express OP-1. Such cells include epithelial cells and cells of uro-genital cell origin, including renal (kidney or bladder) cells, as well as liver, bone, nerve, ovary, cardiac muscle and the like. The cells may be derived from tissue or cultured from an established cell line. See, for example Ozkaynak et al. (1991) Bioche . BioPhys . Res. Com . 179 :116-123 for a detailed deεcription of tissues known to express OP-1. Other useful cells include those known to exhibit a steroid receptor, including cells having an estrogen receptor and cells responsive to the FTZ-F1 protein. Currently preferred cells also have simple media component requirements. Other uεeful representative cells include, but are not limited to, Chinese hamster ovary (CHO) ; canine kidney (MDCK) ; or rat bladder (NBT-2) , and the like. Useful cell types can be obtained from the American Type Culture Collection (ATCC) , Rockville, MD or from the European Collection of Animal Cell Cultures, Portion Down, Salisbury

SP40JG, U.K. As used herein, "derived" means the cells are from the cultured tissue itself, or are a cell line whose parent cells are of the tissue itself.

Aspects and Embodiments of the Invention

In one aspect, the invention features a vector having a reporter gene operatively associated with a portion of one or more OP-1 non-coding sequences. The OP-1 non-coding sequence chosen is independently selected from the 5' (or "upstream") non-coding human or murine OP-1 sequence shown in Seq. ID Nos. 1 and 2, respectively, the 3' (or "downstream") non-coding human or murine OP-1 sequence shown in Seq. ID Nos. 1 or 3, and the human intron non-coding OP-1 sequences shown in Seq. ID No. 1. Also - 9 -

anticipated to be useful are the non-coding sequences (e.g., 5', 3' and intron) of other species homologs of OP-1 and proteins closely related to OP-1. In addition, the portion of OP-1 sequence included in the vector can be a combination of two or more 5' non-coding, 3" non-coding and/or intron OP-1 sequences.

In one embodiment, the vector can include a non-coding 0P1- specific sequence selected from at least one of the following sequence segments of Seq. ID No. 1 presented below, and which define human genomic OP-1 sequence comprising approximately 3.3 Kb of 5' non-coding sequence. In Seq. ID No. 1, the start codon begins at poεition 3318, and the upεtream εequence (bases 1 to 3317) is composed of untranscribed (1 to 2790) and untranslated (2791 to 3317) OPl-εpecific DNA; approximately 1 Kb of which is presented in Fig. 1 (bottom strand) . Useful sequence segments include bases 2548-3317, representing 750 baseε sharing significant (greater than 70% identity) between the mouse and human OP-i homologs (See Fig. 1), and baseε 3170- 3317; 3020-3317; 2790-3317; 2548-2790 of Seq. ID No. 1, all shorter fragments of this region of the DNA. As base 2790 is the mRNA start site, other useful sequences include 2790-3317, representing transcribed but not translated 5' coding sequence and shorter fragments of this DNA region as noted above; upstream fragments of OPl-specific DNA, bases 2548-2790; 1549-2790; 1-2790 of Seq. ID No. 1. Also uεeful εequence εegmentε include the approximately 750 baεes that have homology between the human and mouεe OP-1 sequences with additional upstream sequenceε, 2300 to 3317,; 1300 to 3317; 1-3317; all fragments of the discloεed upstream OPl-specific DNA sequenceε of Seq. ID No. 1.

In another embodiment, the sequences are defined by the non- coding sequences of the mouse OP-1 homolog, including the following 5¹ non-coding sequenceε (Seq. ID No. 2): 2150-2296, 2000-2296, 1788-2296, and 1549-2296 all of which define the 750 baseε εharing high sequence identity with the human homolog (See, Fig. 1); 800-2296; 1-2296; 1549-1788, 800-1788 and 1-1788. Within thiε region alεo exiεt a number Egr/Wt-1 sites (8 in hOP-1; 7 in mOP-1), known in the art to bind the regulatory elements Egr and Wt-1. Accordingly, in another aspect, the invention contemplates a screening material for identifying co pounds which modulate OP-1 expression, the asεay comprising the step of identifying compounds which bind Egr/Wt-1 site. At least oneWt/Egr-1 element, preferably between 1-6 elements, or at least 6 Wt/Egr-1 elementε are included in a sequence. The relative locations of these elements are indicated in Fig. 1 and at positions 3192-3200; 3143-3151; 3027-3035; 2956-2964; 2732-2740; 2697-2704 of Seq. ID No. 1, and positions 2003-2011; 1913-1922; 1818-1826; 1765-1776; 1757-1765; 1731-1739; 1699-1707; 1417-1425 of Seq. ID No. 2 of Seq. ID Nos. 1, 2 subεtantially the same Seq. alignment. The lengthε of baεeε within theεe 5' non-coding εequenceε iε selected to include portions of the sequence of DNA which was determined to be homologous between murine and human genomic OP-1, separately and as a part of a larger sequence including non-homologous DNA. Additionally, the portion of OP-1 sequence selected can be a portion of the region of homology between murine and human OP-1 DNA sequences, bases 2548-2790 or 2548-3317 of Seq. ID No. 1, or bases 1549 to 1788 or 1549 to 2296 of Seq. ID No. 2, and/or at least one of an Wt-l/Egr-1 consensus binding sequence. In still another aspect the portion of OP-1 sequence selected can include a TCC binding sequence, a FTZ binding sequence, a steroid binding sequence, or part or all of an OP-1 intron sequence. The relative positions of the TCC and FTZ elements are indicated in Fig. 1 and at positions 2758-2778 (TCC); 2432-2441 (FTZ) of Seq. ID No. 1 and 1755-1769 (TCC) of Seq. ID No. 2.

In another aspect, the invention features a cell that has been transfected with a reporter gene in operative aεεociation with a portion of OP-1 non-coding DNA εequence. The portion of OP-1 non¬ coding εequence is independently selected from the 5' (or upstream) non-coding human or murine OP-1 sequence shown in Seq. ID Nos. 1 and 2, the 3' (or downstream) non-coding murine OP-1 sequence shown in Seq. ID No. 3, and the human intron non-coding OP-1 sequence shown in Seq. ID No. 1. The six human intron non¬ coding OP-1 sequences are at baseε 3736 to 10700; bases 10897 to 11063; bases 11217 to 11424; feeseε 11623 to 13358; bases 13440 to 10548; baseε 15166 to 17250; all of Seq. ID No. 1. In addition the portion of OP-1 εequence εelected can be a combination of 5' non-coding, 3' non-coding and/or intron OP-1 εequence. Thus, the cell can have been transfected with a reporter gene in operative association with a portion of 5' non-coding OP-1 genomic sequence that is independently selected from baseε 3170 to 3317; 3020 to 3317; 2790 to 3317; 2548 to 3317; 2300 to 3317; 1300 to 3317; 1 to 3317; 2548 to 2790; 1549 to 2790; and 1 to 2790; all of Seq. ID No. 1 or baεeε 2150 to 2296; 2000 to 2296; 1788 to 2296; 1549 to 2296; 800 to 2296; 1 to 2296; 1549 to 1788; 800 to 1788; 1 to 1788; all of Seq. ID No. 2. The lengths of bases within these 5' non-coding sequences is selected to include portions of the sequence of DNA which was determined to be homologous between murine and human genomic OP-1, separately and as a part of a larger sequence including non-homologous DNA. Additionally, the portion of OP-1 sequence selected can be a portion of the region of homology between murine and human OP-1 DNA sequenceε, such as bases 2548-2790 or 2548-3317 of Seq. ID No. 1, or bases 1549 to 1788 or 1549 to 2296 of Seq. ID No. 2, and at least one of an Wt- 1/Egr-l consensus binding sequence, a TCC binding sequence, a FTZ binding εequence, a steroid binding sequence, and an intron. Thus the portion of OP-1 εequence εelected can be a portion of the 5' non-coding human or murine OP-1 genomic DNA sequences, as stated above, and at least one Wt-l/Egr-1 consensus binding sequence alone or in combination with at least one of a TCC binding sequence, a FTZ binding sequence, a steroid binding εequence, and a human OP-1 intron DNA εequence. In another embodiment more than one wt-l/Egr-1 element iε used, for example, between 1-6, or at least six. These cells are suitable for use in the method of the invention.

In one embodiment, part of the OP-1 coding region iε anticipated to have an expression regulatory function and also can be added to a vector for use in the screening assay described herein. OP-1 protein iε translated as a precursor polypeptide having an N-terminal signal peptide εequence (the "pre pro" region) which iε typically leεε than about 30 amino acid reεidues, followed by a "pro" region which is about 260 amino acid residues, followed by the additional amino acid residues which comprise the mature protein. The pre pro and pro regions are cleaved from the primary translation sequence to yield the mature protein sequence. The mature sequence comprises both a conserved C-terminal seven cysteine domain and an N-terminal sequence which varies significantly in sequence between the variouε morphogens . The ature polypeptide chains dimerize and these dimers typically are stabilized by at least one interchain disulfide bond linking the two polypeptide chain subunits. After the pro domain is cleaved from the OP-1 protein it associateε noncovalently with the mature dimeric protein, preεumably to enhance solubility and/or targeting properties of the mature species. See, for example, PCT/US93/07189, filed July 29, 1993. The pro region represents the nucleotide sequence occurring approximately 87 bases downstream of the ATG start codon, and continues for about 980 bases. The nucleotide sequence encoding the pro region is highly enriched in a "GC" sequence, which well may be competent to form a secondary εtructure (e.g., as part of the mRNA transcript) which itself may modulate OP-1 expresεion. Accordingly, part or all of the nucleotide sequence encoding an OP-1 pro region, particularly that portion corresponding to a GC rich region, may be used, preferably in combination with one or more OP-1 non coding sequences, in the compositions and methods of the invention.

In another embodiment, the method can be practiced using a cell known to express the OP-1 gene. Suitable DNA sequenceε for tranεfection are deεcribed below, as well as suitable cells containing transfected DNA sequences.

In another aspect, the invention provides molecules, vectors, methods and kits uεeful in the design and/or identification of OP-1 expression modulating compounds. As used herein a "kit" comprises a cell transfected with a DNA εequence compriεing a reporter gene in operative association with a portion of OP-1 upstream DNA sequence and the reagents neceεεary for detecting expression of the reporter gene. The portion of OP-1 upstream DNA chosen can be any of the various portions which have been described herein.

Following this disclosure, medium flux screen assays, and kits therefore, for identifying OP-1 expression modulating compounds are available. These compounds can be naturally occurring molecules, or they can be designed and biosynthetically created using a rational drug design and an established structure/function analysis methodology. The compounds can be amino acid-baεed or can be compoεed in part or whole of non-proteinaceouε εynthetic organic molecules. The OP-1 expression modulating compounds thus identified then can be produced in reasonable quantities using standard recombinant expresεion or chemical synthesiε technology well known and characterized in the art and/or as described herein. For example, automated means for the chemical εynthesis of nucleic and amino acid sequences are commercially available. Alternatively, promising candidates can be modified using standard biological or chemical methodologies to, for example, enhance the binding affinity of the compound for a DNA element and the preferred candidate derivative then can be produced in quantity.

Once a candidate compound has been identified it can be tested for its effect on OP-1 expression. For example, a compound which upregulates (increases) the pro__ction of OP-1 in a kidney cell line is a candidate for systemic administration. The candidate can be asεayed in an animal model to determine the candidate molecule'ε efficacy in vi vo . For example, the ability of a compound to upregulate levelε of circulating OP-1 in vi vo can be uεed to correct bone metabolism diseaεeε εuch aε oεteoporoεiε (See, for example, PCT/US92/07932, supra). Useful in vi vo animal models for systemic administration are disclosed in the art and below.

As demonεtrated herein below, OP-1 iε differentially expresεed in different cell types. Accordingly, it further is anticipated that a candidate compound will have utility aε an inducer of OP-1 expreεεion in one cell type but not in another. Thuε, the invention further contemplates testing a candidate compound for itε utility in modulating expression of OP-1 in different cells in vivo, including different cells known to express OP-1 under native physiological conditions. Thuε, in view of this disclosure, one of ordinary skill in recombinant DNA techniques can design and construct appropriate DNA vectors and transfect cells with appropriate DNA sequences for use in the method according to the invention to asεay for compounds which modulate the expreεεion of OP-1. Theεe identified compoundε can be uεed to modulate OP-1 production and itε available concentrations in both in vi vo and in vi tro contexts.

Brief Description of the Drawings Fig. 1 shows the alignment of upstream sequences of the murine and human OP-1 gene. The murine sequence iε present in the upper sequence lines and the human sequence is the lower sequence on all lines. The murine sequence is numbered backwards, counting back from the first ATG of the translated sequence which is shown highlighted. For purposeε of alignment, daεheε are introduced into the DNA sequence, and three portions of human DNA sequence have been cut from the sequence and placed underneath a gap, below a solid triangle; Fig. 2 shows a time course of murine uterus OP-1 mRNA regulation by estrogen; and

Fig. 3a shows a schematic of the 2 kb and 4 kb OP-1 mRNAε, the hybridization locations of probes 1 through 7 (indicated by the bars under the schematic) . The solid line indicates OP-1 mRNA, the * indicate potential poly A signals, the boxes indicate the translated portion of OP-1 mRNA. with the hatched box showing the TGF-β -like domain. The dashed lines indicate genomic DNA sequences. The arrows mark the locations of the cleavage site for OP-1 maturation. Fig. 3b. showε a Northern blot hybridization analyεiε of OP-1 specific 2 kb and 4 kb mRNAs in murine uterine tissue. Lanes 1 through 7 correspond to probes 1 through 7 respectively. The 2 kb and 4 kb mRNAs are indicated by the 4- and 2-on the left side of Fig. 3b, and a 0.24 to 9.49 kb RNA εize ladder is indicated by dashes to the right of the figure.

Detailed Description

As will be more fully described below, we have identified regions in the 0P1 genetic sequence useful in identifying molecules capable of modulating OP-1 expression in vivo. Also as described herein, we have determined that OP-1 expression in vivo can be dependent both on cell type and on the statuε of the cell in a tiεsue. Specifically, as described herein below, OP-1 protein expresεion iε differentially regulated in uterine tiεsue depending on the status of the uterine tiεεue. For example, OP-1 expression is dramatically down-regulated in uterine mouse tissue during pregnancy, whereas it is normally expresεed in this tiεsue in virgin mice. Moreover, OP-1 expression in other tissues such as renal tisεue apparently iε unaffected during pregnancy. Adminiεtration of estrogen to a virgin mouse is capable of duplicating this down-regulation of OP-1 gene expression.

We investigated the DNA sequences responsible for the regulation of OP-1 gene expression by cloning non-coding sequences for the human and mouse OP-1 gene. The tissue specific modulation of OP-1 gene expression, and the significant homology which was found between an approximately 750 base region of human and murine 5' non-coding OP-1 genomic sequence, implicate these sequences as having utility in a method for the screening of compoundε for their ability to modulate OP-1 expresεion.

In view of this disclosure and the examples provided below, a method for identifying molecules which can affect OP-1 expression in a particular cell type in vivo now is provided.

Cloning of Human and Mouse OP-1 Gene Non-coding Sequenceε

In the Northern blot analysis of murine organs multiple OP-1 transcripts, are detected namely, three species of 1.8, 2.2, 2.4 kb and a prominent 4.0 kb RNA species (Ozkaynak et al. , 1992, J. Biol. Chem., 267:25220-25227; Ozkaynak et al; Biochem. Biophys. Res. Comm. , 179:116-123) . The pattern is similar in rats with only the 1.8 kb species absent. The eεtrogen-mediated downregulation of OP-1 mRNA affectε all of these species. In order to prove that the 4.0 kb mRNA is in fact a transcript from the same OP-1 locus, cDNA clones were isolated from a mouse teratocarcinoma cDNA library.

Four independent clones were obtained that added sequence information to the published mouse cDNA sequence. Two of these cDNA clones have longer 5' -untranslated εequenceε (0.4 and 0.3 kb) than previously reported (0.1 kb) . Three of the murine clones contain additional 1.4 kb at the 3 '-end. The combined sequenceε add up to a total OP-1 cDNA εize of 3.5 kb, about 0.5 kb shorter than the 4.0 kb mRNA observed on Northern blots. cDNA clones that represent the 2 kb and 4 kb messages are shown schematically in Figure 3a. Since the polyA-tail iε lacking in thoεe cDNA cloneε that extend the 3 ' -information, it waε anticipated that missing 0.5 kb sequence occurs at the 3' -end. In order to obtain the εequence immediately adjacent to the 3 ' -end of the 3.5 kb cDNA εequence, a mouse genomic library, ML1039J (Clontech), was screened with a 3 ' -end cDNA specific probe (0.45 kb, 3 ' -end Xmnl-EcoRI fragment of murine DP-1 cDNA) according to the parameters described below for the cloning of upεtream non-coding εequenceε. Thiε εcreen yielded four lambda clones which were analyzed by Southern blotting. All clones yielded a 1.5 kb XmnI fragment which was subcloned from lambda 071 into a Bluescript vector and sequenced. Three polyadenylation signalε (AATAAA) (Proudfoot et al, (1976) Nature, 263:211-214) were found in thiε genomic fragment, at 3.52-, 3.58-, and 3.59 kb (shown schematically in Fig. 3a by the *) . The 3 ' -end cDNA and the genomic DNA sequences in the 1.5 kb XmnI fragment overlap by 0.4 kb in a region that immediately precedes the second polyadenylation signal located at 3.5 kb (Figure 3a, region indicated by probe 6) and are in complete agreement within this stretch.

Human upstream non-coding sequence and additional mouse upstream non-coding εequence were obtained by εcreemng human and mouεe genomic libraries, HL1067J and ML1030J respectively

(Clontech) . All libraries were screened by an initial plating of 750,000 plaques (approximately 50,000 olaques/plate) . Hybridizations were done in 40% formamide, 5 x SSPE, 5 x Denhardt 's solution, and 0.1% SDS at 37°C. Nonspecific counts were removed in 0.1 x SSPE, 0.1 % SDS by εhaking at 50°C. Human and mouεe upstream genomic DNA sequences were obtained from clones lambda 03 and lambda 033, respectively (Clontech, HL1067J and ML1030J) . Theεe lambda clones were isolated using a ^3<!P-labeled probe made from a human 0.47 kb EcoRI OP-1 cDNA fragment (obtained from p0115) containing mainly 5' non-coding and exon 1 sequenceε.

A 7 kb EcoRI fragment from the human genomic clone, lambda 03, was isolated which contains 5 kb of upstream non-coding sequence. Additional upstream sequence information for murine was obtained by subcloning a 1.1 kb PstI fragment from the genomic phage clone lambda 033. This fragment overlaps with the 5 ' -end of the longest murine cDNA clone by 0.3 kb in tne 5' non-coding region and provided 0.8 kb additional sequence information. A schematic diagram of the 2- and 4 kb OP-1 mesεages is εhown in Figure 3a with dashed lines indicating supplementing information derived from murine upstream and downstream genomic DNA.

All εequencing was done according to Sanger et al. (1977) Proc. Natl. Acad. Sci. 7_4:5463-5467, using exonuclease III- mediated unidirectional deletion (Ozkaynak et al. , (1987)

BioTechniques, _5:770-773), subcloning of restriction fragments, and synthetic primers. Compressions were resolved by performing the reactions at 70°C with Taq polymerase and using 7-deaza-GTP (U.S. Biochemical Corp., Cleveland, OH).

Verification of OP-1 mRNA Sequences by Northern Blotting

To verify the structures of the short and long mRNA species observed, Northern blot hybridizationε were performed with probes made from seven non-overlapping DNA fragments (Fig. 3a; probes 1 through 7) specific to the 5' and 3' non-coding region, the protein coding sequence, and genomic regions upstream or downstream of the predicted mRNAs, respectively.

Hybridization of these probes to individual Northern blot stripε containing mouεe kidney mRNA iε consistent with the predicted 4 kb mRNA structure. As shown in Fig. 3a, and Fig. 3b, the genomic DNA probes 1 and 2 did not hybridize to any message. Probe 2 is specific to the upstream εequenceε immediately adjacent to the cDNA. Probes 3, 4, and 5, specific to 5' non-coding, coding, and 3" non-coding regions, respectively, hybridized to both the 2 kb and 4 kb messages, hence these sequences are present in both messages. Probe 6, specific to sequences between the first and second polyadenylation signals, hybridized only to the 4 kb mesεage. Finally, probe 7 which is specific to sequenceε further downεtream of the fourth (last) polyadenylation signal, did not hybridize to any message. The resultε obtained with theεe probes confirm the two OP-1 mRNA structures and the approximate 5'- and 3 ^■-end boundaries of OP-1 transcriptε εhown in Figure 3a. Thiε demonstrates that the 2 kb and 4 kb mRNA's are from the same OP-1 genomic locuε rather than from multiple geneε. The extensive 3' sequence included in the 4 kb mRNA transcript εuggeεtε that the 3' untranslated sequence may play a role in OP-1 gene expresεion particularly as it has been detected across species namely, in mouse, rat, dog, human and chicken. Multiple εtop codonε in all three posεible translation reading frames rule out the likelihood that this sequence encodes a peptide. The untranslated sequence itself may act therefore to influence mRNA stability.. For example, the sequence may interact with another protein as has been described for transferrin receptor mRNA.

Here, IRE-binding protein (IRE; iron response element) stabilizes the transferrin receptor mRNA by binding to the 3 ' -end of the mRNA (Standard et al. , 1990, Genes Dev. , l:2157-2168, incorporated herein by reference). Alternatively, the 3 ' -end sequences may be interacting with the 5' -end sequenceε thereby affecting initiation of protein εyntheεiε or, the 3 ' -end sequenceε may be εerving aε a binding εite for other RNAε which can interfere with the binding of an expression in modulating molecule, including repressor molecule. (Klausner et al . , 1989, Science, 246:870-872; Kozak, 1992, Ann. Rev. Cell Biol., 8 :197-225, incorporated herein by reference) .

Compariεon of 5' Non-coding Sequences of Human and Mouse OP-1 DNA

The cloning of the 5' non-coding genomic murine and human OP-1 DNA sequences demonstrated that a high degree of sequence homology exists between the human and murine 5' non-coding DNA sequences. The homology extends from the base immediately upstream of the translation start site for the OP-1 morphogen protein to approximately 750 bases upstream of the translation start site, aε is shown in the shaded regions of Fig. 1, with the murine sequences being the upper lines and the human sequences being the lower lines. The 5' nucleotide of the region of homology for the human OP-1 5' non-coding εequence iε base 2548 of Seq. ID No. 1 and for the murine OP-1 5' non-coding sequence is base 1549 of Seq. ID No. 2. The significant homology between the human and murine 5' non-coding sequences of OP-1 εuggeεt that thiε region may be important in the regulation of OP-1 expression. As will be discusεed in more detail below, thiε region containε εeveral conserved DNA sequences which have been identified as the DNA binding sequences for two DNA binding proteins, Wt-1 and Egr-1, which both recognize these DNA sequences. The DNA binding sequences for Wt-l/Egr-1 present in human and murine are marked in Fig. 1 with a single line. Also, the TCC binding sequence, a DNA binding sequence for Wt-1 and Egr-1, is marked in Fig. 1 by the - 19 -

double line. WT-1 and Egr-1 proteins have also been implicated in the regulation of expression of several genes which are unrelated to OP-1.

Alignments of mouse and OP-1 human genomic sequences reveals a conserved stretch of 0.75 kb just upstream of the first ATG that contains several patterns with marked similarity to the zinc- finger protein binding sequence (5'-GCG GGG GCG-3 ' ) specific for Egr-1 and Wt-1 (Christy et al. , 1989, PNAS, ^6:8737-8741; Rauscher et al., 1990, Science, 250:1259-1262; Drummond et al. , 1992, Science, 257 :664-678) . In mouse, a total of 8, and in human 7, patterns, conforming to the degenerate Egr-l/Wt-1 binding εequence (5'-GNG NGG GNG-3 ' ) (Rupprecht et al . , 1994, J. Biol. Chem., 269: 6198-6202; Werner et al. , 1994, J. Biol. Chem., 269: 12940-12946 are located before and after the preεu ed transcriptional initiation site (Fig. 1, shown by εolid εingle lines). The presence of theεe has significance in light of the elevated levels of Wt-1 mRNA in the rat uterus decidua during pregnancy (Zhou et al., 1993, Differentiation, 54:109-114) .

The analysis also revealed, in the human upstream region, a pattern of seven TCC repeats, present at -561, immediately 3' of two Egr/Wt-1 sequences (at -624 and -587) (Figure 1, shown by double solid lines and at position 2758-2778 of Seq. ID No. 1) . The mouse upstream region contains a similar, albeit lesε obviouε εequence at -356 and at position 1755-1769 of Seq. ID No. 2. Thiε TCC-repeat pattern is found in the promoters of PDGF-A and several other growth-related genes, and Wt-1 has been found to activate transcription when either of the εequences are present and to suppress it when both sequences are present. (Wang et al . , 1992, Biochem. Biophys Reε. Comm. , 188:433-439; Wang et al. 1993, PNAS, _9_0:8896-8900 incorporated herein by reference) . Accordingly, estrogen receptor may exert its effect on OP-1 expresεion in uteruε by upregulating Wt-1, either directly or indirectly. Alternatively or, in addition other regulatory elementε, located further upεtream of the OP-1 gene may be involved in estrogen regulation.

Also on Fig. 1, the human 5' non-coding DNA sequence is shown to contain a Fushi-tarazu (FTZ) binding sequence which iε marked by caratε below the human DNA εequence. A FTZ binding sequence iε bound by the Fushi-tarazu protein (FTZ-F1), which iε a member of the superfamily of nuclear receptors (Parker, (1993) Current opinion in Cell Biology, _5:499-504, ) . The superfamily of nuclear receptor proteins include steroid hormones, retinoids, thyroid hormone, nerve growth factor and Fuεhi-tarazu, and are structurally related. FTZ-F1 is likely to belong to a εubfamily of nuclear receptorε that bind DNA as monomers.

The FTZ-F1 protein is a positive regulator at the fuεhi-tarazu gene in blastoderm stage embryos of Drosophila . FTZ-F1 is closely related in the silkworm (Bombyx) BmFTZ-Fl protein and the mouse embryonal long terminal repeat binding protein (ELP) and all of them are members of the nuclear hormone receptor superfamily, which recognizes the same 9 base pair sequence, 5 ' -PyCAAGGPyCPu- 3'. The FTZ binding sequence doeε not apparently have a direct or inverted repeat. In contrast, other members of the nuclear hormone receptor superfamily usually bind to repeated sequences. Neverthelesε, the FTZ-F1, BmFTZ-Fl and ELP proteins have high affinities for the FTZ binding site DNA, indicating that the mechanism that the binding is somewhat different from that of other members of the nuclear hormone receptor superfamily.

(Hitachi et al . , 1992, Mol. and Cell Biology December, pp. 5667- 5672.) .

The mRNA transcription initiation εite for human OP-1 iε marked on Fig. 1 by the upward arrow, and the OP-1 protein tranεlation initiation site iε marked on Fig. 1 by the εolid triangleε juεt prior to the highlighted ATG. The transcription initiation site for the human OP-1 gene is at baεe 2790 of Seq. ID No. 1 and the analogouε εite for murine iε at base 1788 of Seq. ID No. 2. The tranεlation initiation site for the human OP-1 gene is at base 3318 of Seq. ID No. 1 and for murine it iε at baεe 2296 of Seq. ID No. 2. The high degree of identity that the murine and human DNA εequenceε share in the region between the tranεcription initiation εite and the tranεlation initiation εite, εuggeεtε that this region likely plays a role in the modulation of the expresεion of the OP-1 gene product.

Analysis of OP-1 Gene Expression in Mouse Tiεεues A detailed analysis of the uro-genital tract of rats has revealed OP-1 mRNA expreεsion in the renal (kidney) , and bladder tisεues, as well as at other εites of the urogenital organ system. The most abundant levels are present in renal and uterine tissue (8 week old mice), while much lower levels were found in ovaries. The mRNA level of G3DPH, a "housekeeping function" molecule, was used as an internal control for recovery and quality of mRNA preparations and equal amounts of poly(A)+ RNA (5mg) , were loaded into each lane. Preparation of RNA and Northern blot hybridization analysis was conducted as follows. 8-week-old female mice, strain CD-I, were obtained from Charles River Laboratories, Wilmington, MA. Total RNA, from the various organε of mice waε prepared uεing the acid-guanidine thiocyanate-phenol-chloroform method (Chomczynski et al., (1987) Anal. Biochem. 162:156-159) . The RNA was diεεolved in TES buffer (10 ml. Tris-HCl, 1 mM Na_:-EDTA, 0.1% SDS, pH7.5) containing Proteinase K (Stratagene, La Jolla, CA; approx. 1 mg proteinaεe /ml TES) and incubated at 37°C for 1 hr. Poly (A)⁺ RNA waε εelected in a batch procedure on oligo(dT) -cellulose (Stratagene, La Jolla, CA) in 0.5 M NaCl, 10 mM Triε-HCl, 1 mM

Na.-EDTA, pH 7.4 (1 x binding buffer) . For the selection of poly

(A)+ RNA, total RNA obtained from 1 g of tiεεue waε mixed with approximately 0.Ig of oligo(dT) -celluloεe (in 11 ml TES containing 0.5 M NaCl) . The tubeε containing the RNA and oligo(dT) -cellulose were gently shaken for approx. 2 hrs. Thereafter, the oligo(dT)- cellulose was waεhed twice in lx binding buffer and once in 0.5x binding buffer (0.25 M NaCl, 10 mM Triε-HCl, 1 mM Na -EDTA, pH 7.4) and poly (A)+ RNA waε eluted with water and precipitated with ethanol. Poly(A)+ RNA (5 mg per lane) waε electrophoresed on 1.2% agarose-formaldehyde gels with 1 mg of 400 μg/ml ethidium bromide added to each sample prior to heat denaturation (Rosen et al. , (1990) Focus, _12:23-24). Electrophoresis was performed at 100 Volts with continuous circulation of the 1 x MOPS buffer (Ausubel et al. , eds., (1990) Current Protocols in Molecular Biology, John Wiley _. Sons, New York). Following electrophoresiε, the gels were photographed, rinsed briefly in water, and blotted overnight onto Nytran (Schleicher _. Schuell Inc., Keene, NH) or Duralon-UV

SUBSTITUTE SHEET {RULE 26) (Stratagene) membranes in 10 x SSC . The membranes were dried at

8 B00°° ffιor 30 min. and irradiated with UV light (1 mW /cm for 25 sec. )

The ³²P-labeled probe was made from a murine OP-1 cDNA fragment (0.68 kb BstXI-BGlI frg.) by random hexanucleotide priming (Feinberg et al., (1984) Anal. Biochem. , 137:266-267) . The hybridizationε were done in 40% formamide, 5x SSPE, 5x Denhardt's, 0.1% SDS, pH 7.5 at 37°C overnight. The non-specific counts were washed off by shaking in O.lx SSPE, 0.1% SDS at 50°C. For re-use, filters were stripped in 1 mM Tris-HCl, 1 mM Na₂-EDTA, 0.1% SDS, pH 7.5 at 80° C for 10 min.

Analysis of OP-1 Expression During Pregnancy in Mice

An examination of the effect of pregnancy upon OP-i expression was undertaken by measuring OP-1 mRNA levels in kidney, ovary and uterus, before, during, and after pregnancy (virgins, 2-day post- coital (pc) , 4-day pc, 6-day pc, 8-day pc, 13 day pc, 17-day pc, 3-day lactating, and retired breeders) by Northern blot hybridization of poly (A) + RNA. These measurements demonεtrated that, while kidneys show no pregnancy-related changes in OP-1 mRNA levels, the uterine levels became nearly undetectable by 6-day pc. However, no changeε were obεerved in the ovaries. A dramatic and rapid decline in OP-1 message in uterine tiεεue between day 3 and 4 of pregnancy iε apparent in the comparison with virgin animals. The levels of OP-1 mRNA in the embryo and maternal levels in uterus of 8 week old mice at day 13 and 16 of the pregnancy were also compared. While the OP-1 expression in the pregnant uterus is dramatically reduced, high levels of OP-1 message are found in the mouse embryo at 13- and 16-days. Thus, at a stage of pregnancy when OP-1 mRNA expreεεion in the maternal uterus is almost undetectable, embryonal OP-1 expresεion iε high. The high embryonal OP-1 expreεεion also is detected consistent with the relatively high levels of OP-1 mRNA, found in human placenta. The level of OP-1 mRNA measured in the embryo is in the same range as that meaεured in adult kidney or virgin uteruε tissue. Hence, it is likely that OP-1 plays a critical role in the development of the embryo which may require appropriate amounts of OP-1 at very specific stageε of tiεεue and organ morphogensis. While not being limited to any given theory, it is possible that OP-1 expression in uterine tissue during pregnancy potentially could interfere with the level of OP-1 produced by the developing embryo, and thereby interfere with proper development of the embryo. Therefore, a shut-down or inhibition of uterine OP-1 expresεion during pregnancy might be for the benefit of the fetus.

Effect of Estrogen and Progesterone on OP-1 Expression

During pregnancy the estrogen and progesterone levels increase many fold and high levels are εustained until birth. To determine whether these hormonal changes are responsible for the altered OP- 1 transcription in pregnant uterine tissue, non-pregnant female mice were εubcutaneously adminiεtered 17β-eεtradiol, or progesterone, or a combination of both. In the first experiment the rapid increase in estrogen and progesterone levels during pregnancy was εimulated. Non-pregnant mice were injected εubcutaneouεly on four conεecutive days with increasing doseε, εtarting with 20 mg 17β-eεtradiol, or 100 mg progesterone or the combination of both and doubling the dose on each following day. On the fourth day the animals were sacrificed and mRNA was iεolated from uteri and kidneyε. A εtriking negative effect of 17β-estradiol on the uterine OP-1 mRNA expression waε obεerved, but no effect by progesterone was seen. In the kidneys, however, mRNA levels did not change after 17β-estradiol or progesterone treatment.

Another experiment addreεεed the time course: 17β-eεtradiol was administered to virgin female mice at a conεtant doεe of 200 mg (50 ml of 4 mg/ml 17β-estradiol per day, subcutaneously in DMSO [dimethyl sulfoxide] + 150 ml 150 mM NaCl) (Figure 2). Following this, their uteri were extracted, poly (A) + RNA was prepared, equal amounts of poly(A)+ RNA (5 mg) was loaded into each lane of a 1.2% agarose-formaldehyde gel and analyzed by Northern blot hybridization. The effect was rapid, with considerable decrease of OP-1 mRNA 12 hours after administration of 17β-estradiol and almost undetectable levels by 48 hours, as shown in Fig. 2. In the figure, the lanes correspond as followε: from left to right, 0-day (negative control), 0-day (negative control), 0.5-, 1-, 2-, 3-, 4-, 5-, 6-, 7-, and 8-days. The arrowheads mark the two major OP-1 mRNA species. A modest amount of meεsage reappears a few days later (Figure 2) .

The uterus has been identified as a major site of OP-1 expression. The level of OP-1 expresεion in uterine tiεsue is comparable to that observed in renal tisεue. However, during pregnancy, by day four, the uterine OP-1 mRNA levels are reduced to the limit of detection. The loss of OP-1 expresεion correεpondε withalso is rising levels of estrogen during this εame time frame. The εame dramatic loεs of uterine OP-1 mesεage alεo iε observed in estrogen-treated animals, suggeεting that estrogen iε involved in negative regulation of OP-1 expression in uterine tissue. The effect of estrogen is rapid, with most of the mesεage disappearing after 12 hours of 17β-estradiol administration. The reappearance of εome OP-1 mesεage at later dayε may be due to a counter-regulatory mechaniεm. In contraεt to the modulated OP-1 mRNA levelε in the uteruε, no εubεtantial changes occur in renal tissue during pregnancy or in response to estrogen treatment. Therefore, OP-1 mRNA expresεion in these different organε iε regulated independently. The differential expreεεion may be due, for example, to a lack of estrogen receptors in renal tissue. Alternatively, co-regulation by means of one or more accessory molecules that interact with estrogen or a related nuclear receptor molecule(ε) may allow for the independent regulation. For example, each of Wt-1 protein (which binds to the wt-l/Egr-1 element) and OP-1 protein are required for normal kidney development, and each are expresεed at high levelε during kidney tiεsue development. As described above the OP-1 promoter region contains Wt-1 conεensuε binding elementε. Wt-1 protein alεo has been εhown to negatively regulate the tranεcription of the insulin growth factor II gene and the platelet-derived growth factor A chain gene. Kreidberg et al . , Cell, 1993, 74:679-691. Without being limited to a given theory, it may be that wt-1 protein, either alone or in combination with one or more molecules is involved in the expression of OP-1. For example, Wt-1 protein may act in concert with a nuclear hormone receptor element, including, for example,the estrogen receptor element. Implications of Tissue Specific Differential Regulation of 0P- 1 Expression

Estrogen also has been shown to inhibit the uterine expression of calbindin-D_28k, a vitamin D dependent calcium binding protein, the α-subunit expression of the glycoprotein hormones, and other proteinε involved in bone formation. Estrogen also haε been shown to cause dramatic decreases in the steady state mRNA levels of the bone matrix proteins osteocalcin, prepro α2(I) chain type I collagen, osteonectin, oεteopontin, and alkaline phoεphataεe in an ovariectomized rat, which iε a rat model for oεteoporosis.

Eεtrogen appears to mediate its beneficial effect on bone metaboliεm in the oεteoporotic model through inhibition of oεteoclasts. Eεtrogen doeε not reverse oεteoporosis. By contrast, OP-1, which is expreεεed in uterine, renal and bone tiεεues, is able to induce an increaεe in bone mass in the osteoporotic model. Thuε, the negative effect of eεtrogen on OP-1 expreεsion in uterine tissue may seem unexpected in view of estrogen's effect on bone metabolism.

In addition to the 5' non-coding DNA sequences of OP-1, the other non-coding sequences such aε intronε and 3 ' non-coding εequenceε may be involved in the modulation of OP-1 protein expression. This invention preεentε a method in which these non¬ coding sequences are assayed while in operative association with a reporter gene for their influence on the expression of OP-1. Non- coding sequences which are involved in the modulation of OP-1 expresεion will be identified by culturing cells transfected with the non-coding sequences, in operative association with a reporter gene, with one or more compound(ε), measuring the level of reporter gene expression, and comparing this level of expreεsion to the level of reporter gene expression in the absence of the compound(s) .

EXEMPLARY CELLS, VECTORS, REPORTER GENES AND ASSAYS FOR USE IN SCREENING COMPOUNDS WHICH MODULATE OP-1 REGULATORY SEQUENCES

I. Useful Cells

Any eukaryotic cell, including an immortalized cell line suitable for long term culturing conditions iε contemplated to be uεeful for the method and cell of the invention. Useful cells εhould be eaεy to tranεfect, are capable of εtably maintaining foreign DNA with an unrearranged εequence, and have the neceεεary cellular componentε for efficient tranεcription and tranεlation of the protein, including any elementε required for post- tranεlational modification and εecretion, if necessary. Where the cell is to be transfected with a non-dominating selection gene, the cell genotype preferably is deficient for the endogenous selection gene. Preferably, the cell line also haε simple media composition requirements, and rapid generation times. Particularly useful cell lines are mammalian cell lineε, including myeloma, HeLa, fibroblast, embryonic and variouε tisεue cell lineε, e.g., kidney, liver, lung and the like. A large number of cell lineε now are available through the American Type Culture Collection (Rockville, MD) or through the European Collection of Animal Cell Cultures (Porton Down, Salisbury, SP4 OJG, U.K.)

Where, aε here, the expression of a reporter gene that is controlled by non-coding sequences of the morphogen OP-1 is to be analyzed, particularly uεeful cells and cell _.lines are envisioned to include eukaryotic, preferably mammalian cells of a tissue and cell type known to expresε OP-1 and/or closely related proteinε.

Such cellε, include, without limitation, cells of uro-genital cell origin, including kidney, bladder and ovary cel.'.s, lung, liver, mammary gland and cardiac cells, cells of gonadal origin, cells of gaεtrointestinal origin, glial cellε and other cell lineε known to express endogenous genes encoding morphogenic proteinε. Preferred cell lines are of epithelial origin.

II. Exemplary Vectors/Vector Construction Considerationε

Useful vectors for use in the invention include, but are not limited to cosmids, phagemids, yeast artificial chromosomes or other large vectors. Vectors that can be maintained within the nucleus or integrated into the genome by homologous recombination are also useful. For example a vector such aε PSV2CAT would be uεeful. Selected portions of non-coding OP-1 εequence can be cloned into a useful vector using standard molecular cloning techniques, as will be apparent to one of ordinary skill in the art. Restriction endonuclease sites will be utilized when possible, and can be engineered into the sequence when needed. If restriction endonuclease sites are needed to be engineered into the sequence, eight base recognition sites are preferable because they generally occur infrequently in DNA and will enhance a practitioners ability to obtain the sequence of interest. Restriction endonuclease sites can be engineered into the non-coding sequence using the common techniques such as site directed mutagenesis and PCR with primers including the desired restriction endonuclease site.

As discussed above, murine and human OP-1 sequences share a region of high homology covering approximately 750 baseε upstream of the translation initiation site as shown by the shading in Fig. 1. This region is positions 2548-3317 of Seq. ID No. 1 and positions 1549-2296 of Seq. ID No. 2. The mRNA transcription initiation site lieε within this region at position 2790 of Seq. ID No. 1 and by analogy at poεition 1788 of Seq. ID No. 2, εhown in Fig. 1 by the upward arrow. Thiε suggests that positions 2548- 2790 of Seq. ID No. 1 and 1549-1788 of Seq. ID No. 2 contain conserved promoter elements for the expresεion of OP-1 mRNA, and approximately 500 baεes at poεitionε 2791-3317 of Seq. ID No. 1 and positions 1790-2296 of Seq. ID No. 2 contain conserved elements of the transcribed, but not translated, εequeήceε all or part of which may be involved in the regulation of OP-1 expression. Additionally sequences upstream of the homology region may also be involved in the regulation of OP-1 expression. Thuε a range of upstream sequences, including sequences upstream of the transcription initiation εite and not including the approximately 500 baεeε of transcribed sequence, can be fused in operative association with a reporter gene to modulate expresεion of the gene.

3 ' non-coding εequenceε and intron εequenceε also can be fused in operative asεociation with a reporter gene, either separately or in combination with each other or with 5' non-coding sequences. For example, one can place the 5' sequences defined by positions 2790-3317; 2548-2790 or 2548-3317 of Seq. ID No. 1, and either/both of 3" sequenceε or intron sequences in operative association with a reporter gene. The positions of the six introns are shown in Seq. ID No. 1 aε baseε 3736 to 10700; baεeε 10897 to 11063; bases 11217 to 11424; baseε 11623 to 13358; baεeε 13440 to 10548; baεeε 15166 to 17250; Alεo enviεioned iε a nucleic acid construct compriεing a small fragment of 5' non-coding OP-1 sequence in combination with additional conεerved elementε εuch aε one or more Wt-l/Egr-1 binding εequences; a TCC binding sequence and/or a FTZ binding sequence in operative association with a reporter gene. Such a nucleic acid construct alεo could include intron εequenceε and/or 3" non-coding εequences.

A range of uεeful 5' non-coding fragments has been provided, and as will be apparent to those of ordinary skill in the art, smaller fragments of OP-1 sequence alεo are useful. Such smaller fragmentε can be identified to deleting bases from one or both ends of the provided 5' non-coding fragments, using techniques that are well known in the art and testing the truncated constructs for their ability to modulate reporter gene expreεsion. In thiε way, the shorteεt modulating εequences can be identified.

III. Transfection Considerations

Any method for incorporating nucleic acids into cells of interest is contemplated in the method of the invention. Calcium phosphate (CaPO , followed by glycerol shock iε a standard means used in the art for introducing vectors, particularly plasmid DNA into mammalian cells. A repreεentative method iε disclosed in Cockett et al . , (1990) Biotechnology 8 : 662-667, incorporated^" herein by reference. Other methods that may be used include electroporation, protoplast fusion, particularly useful in myeloma tranεfections, microinjections, lipofections and DEAE-dextran mediated uptake. Methodε for these procedures are described in F.M. Ausubel, ed., Current Protocolε in Molecular Biology, John Wiley _. Sonε, New York (1989). Aε will be appreciated by thoεe having εkill in the art, optimal DNA concentrations per transfection will vary according to the transfection protocol. For calcium phoεphate transfection, for example, preferably 5-10 μg plasmid DNA per plasmid type is transfected. In addition, the DNA to be transfected preferably is esεentially free of contaminantε that may interfere with DNA incorporation. A εtandard means used in the art for purifying DNA is by ethidium bromide banding.

SUBSTITUTE SHEET (RULE 26. IV. Exemplary Reporter Geneε

There are numerous reporter systemε commercially available, which include, without limitation, the chloramphenicol acetyltransferase (CAT), luciferaεe, GAL4, and the human growth hormone (hGH) assay systems.

CAT is a well characterized and frequently used reporter system and a' major advantage of this εystem is that it is an extensively validated and widely accepted measure of promoter activity. See, for example, Gorman, CM., Moffat, L.F., and Howard, B.H. (1982) Mol. Cell. Biol., 2:1044-1051 for a description of the reporter gene and general methodology. In this system cells are harvested 2-3 days after tranεfection with CAT expression vectors and extracts prepared. The extracts are incubated with acetyl CoA and radioactive chloramphenicol. Following the incubation acetylated chloramphenicol is εeparated from nonacetylated form by thin layer chro atography. In thiε assay the degree of acetylation reflects the CAT gene activity with the particular promoter.

Another well-recognized reporter system is the firefly luciferase reporter system. See, for example Gould, S.J., and

Subramani, S. (1988) Anal. Biochem. , ^:4°4-408 ^{for a} description of the reporter gene and general methodology. The luciferase aεεay is fast and has increased εenεitivity. The εyεtem also is particularly useful in bulk transfections or if the promoter of interest is weak. In thiε assay transfected cells are grown under standard conditions, and when cultured under assay conditions both ATP and the substrate luciferin is added to the cell lysate. The enzyme luciferase catalyzes a rapid, ATP dependent oxidation of the subεtrate which then emits light. The total light output is meaεured using a luminometer according to manufacturer'ε inεtructions (e.g., Cromega) and is proportional to the amount of luciferaεe preεent over a wide range of enzyme concentrations.

A third reporter system is baεed on immunologic detection of hGH, it is quick and easy to use. (Selden, R., Burke-Howie, K. Rowe, M.E., Goodman, H.M., and Moore, D.D. (1986), Mol. Cell. Biol. , _6:3173-3179 incorporated herein by reference) . hGH iε assayed in the media, rather than in cell extracts. This allows direct monitoring over by a single population of transfected cells over time.

As indicated above and as will be appreciated by those having ordinary skill in the art, particular details of the conventional meanε for tranεfection, expression, and assay of recombinant genes are well documented in the art and are understood by those having ordinary skill in the art. The instant invention enables and discloses vectorε, cellε and a method for screening co poundε to determine the capability of compoundε to modulate the expreεεion of OP-1 via the non-coding sequences of the OP-1 genomic DNA.

Further details on the various technical aspects of each of the stepε used in recombinant production of foreign genes in mammalian expresεion εystems can be found in a number of texts and laboratory manuals in the art, such as, for example, F.M. Ausubel et al. , Ed., Current Protocols in Molecular Biology, John Wiley _ Sons, New York, (1989) .

VIII. Exemplary Homologouε/Non-Homologous Recombination

One approach to screen for inducerε of (organ-εpecific) OP-1 expression in a particular cell line derived from a particular tisεue εuch aε renal or uterine tisεue, iε through gene targeting by homologous recombination (Sedivy et al . , W.H. Freeman Co., New York (1992); A.S. Waldman, Crit. Rev. Oncol. He atol . 12, 49 (1992)) . In one strategy the endogenous (genomic) OP-1 gene is replaced by another reporter gene which iε optimally εuited for εcreening aεεays, εuch aε the firefly luciferaεe gene. To target the OP-1 gene in an appropriate cell line, e.g., a kidney cell line or NBT-2, the following arrangement of genetic elementε can be aεεembled. Genomic OP-1 upεtream and promoter εequenceε preferably 3000 to 5000 nucleotides in length, and which mediate the homologous recombination, are attached to the luciferase gene. The OP-1 upstream sequenceε down to the firεt coding ATG can be attached at the εtart codon ATG of the luciferaεe coding εequence, uεing a restriction εite such as Ncol, which can be introduced by site directed mutagenesis into both the promoter and the luciferaεe sequenceε. Alεo included is a selective marker, preferably the neo gene, without its own promoter. Preferably, selectable marker (neo) is placed downstream of the reporter gene (luciferase), after an intercistronic sequence derived from the polibviruε genome and which allows translation of the sequence marker on the same transcript as the reporter gene transcripts. Details of this approach, including specific intercistronic sequences and the detailed steps of homologous recombination, are described in the art, including (Jasin et al. , PNAS USA 5:8583 (1988); Sedivy et al. , PNAS USA 86, 227 (1989) ; Dorin et al. , Science 243 :1357 (1989) the disclosures of which are incorporated herein by reference. As described therein, the endogenes OP-1 gene is replaced by the luciferase and neo coding sequences and the expression of these sequenceε then aεayed in a εtandard A screening protocol.

A genetic arrangement of OP-1 promoter (as much genomic OP-1 upεtream sequence as possible, up to 10,000 bp) and reporter gene (without its original promoter but joined directly to the OP-1 ATG or in its vicinity) can alεo be introduced into cellε on standard eukaryotic expression vectors. These vectors carry selectable markers (neo, dhfr, etc.) and will typically be integrated into the hoεt genome with variable copy number ranging from one to εeveral copieε without effortε at amplification. Alεo, if desired, the vector or gene copy number can be enhanced uεing a well characterized amplifiable gene, εuch as dhfr in conjunction with methotrexate. Commercial vectorε deεigned for autonomous replication without integration are readily available. One source vector is the Episomal Expreεεion Epstein Barr Virus Vector (pREP, Invitrogen Corp., San Diego CA) . Introns also can be tested for regulatory εequences as described hereinabove using the methods described herein. One or more intron sequences derived from a genomic OP-1 locus preferably is introduced into proper mammalian cells using, for example, a yeast artificial chromosome (pYACneo, Clontech, Inc. Palo Alto, CA) (Ref. Albertson, H.M. et al. PNAS USA, 87:4256, 1990), or other vectors adapted to allow transfer of large sequenceε, e.g., up to 1 megabaεeε. As for the OP-1 5' or 3' noncoding sequenceε deεcribed above, the intron εequence or a portion thereof iε incorporated in operative asεociation with a reporter gene and the ability of the sequence to modulate reporter gene expressions then associated.

X. Exemplary Screening Asεay for Compounds which Alter OP-1 Gene or Reporter Gene Levels

Candidate compound(s) which may be adminiεtered to affect the level of a given endogenouε morphogen, εuch as OP-1, or a reporter gene that is fused to OP-1 non-coding sequence may be found using the following screening assay, in which the level of reporter gene production by a cell type which produces measurable levels of the reporter gene expreεsion product by incubating the cell in culture with and without the candidate compound, in order to assess the effects of the compound on the cell. This can be accomplished by detection of the reporter expression product either at the protein or RNA level. The protocol is based on a procedure for identifying compounds which alter endogenous levels of morphogen expresεion, a detailed description also may be found in PCT US 92/07359.

Cultured cells are transfected with portions of OP-1 non- coding sequences in operative association with a reporter gene, and such transfected cells are maintained with the vector remaining as a plaεmid in the cell nucleus or the vector can be integrated into the host cell genome, preferably at the OP-1 genomic locus. Cell εampleε for teεting the level of reporter gene expreεεion are collected periodically and evaluated for reporter gene expreεεion uεing the appropriate aεsay for the given reporter gene as indicated in the section describing reporter gene asεays, or, alternatively, a portion of the cell culture itself can be collected periodically and uεed to prepare polyA(+) RNA for mRNA analysis.

Once candidate compounds are identified, they can be produced in reasonable, uεeful quantitieε uεing εtandard methodologies known in the art. Amino acid-based molecules can be encoded by synthetic nucleic acid molecules, and expreεsed in a recombinant expresεion system as described herein above or in the art. Alternatively, such moleculeε can be chemically synthesized, e.g., by means of an automated peptide εyntheεizer, for example. Non-amino acid-based moleculeε can be produced by standard organic chemical syntheεis procedures.

Provided below is an exemplary protocol for carrying out the method of the invention, using the CAT gene as the reporter gene and one or more mammalian cell lineε known to expreεε OP-1. The example is non limiting, and other cells, reporter genes and OP-1 non-coding sequences are envisioned.

Exemplary Construction Of Representative Vectors For Transfections

A DNA fragment containing the OP-1 promoter can be joined to a reporter gene for transfection into a cell line that expresses endogenous OP-1. Suitable cell lines are selected by Northern blot hybridization to an OP-1 specific probe (by analyzing the cell extracts for OP-1 mRNA) . Using this technology we have found several cell lines which make high levels of OP-1 mRNA, and some of these lines are the kidney line IMCD, the bladder line NBT II.

An approximately 5 Kb EcoRI, BamHI genomic fragment containing approximately 4 Kb of upstream OP-1 εequences as well as part of the first intron is blunt-ended with T4 DNA polymerase and cloned into a polylinker of a pUC vector (p0146-l). An approximately 3.5 kb DNA fragment containing human OP-l upstream εequenceε iε obtained by deleting a portion of coding εequences and the firεt intron from p0146-l with the reεtriction enzyme Ehel. The -3.5kb fragment has blunt ends and contains moεtly 5' non-coding εequences and alεo includeε a εhort εtretch of 30 baεes into the 0?-l gene. This upstream fragment is of -3.5kb ligated to a 1.6 kb Hindlll-BamHI fragment from the CAT gene obtained from the vector SV2CAT by 5' Hindlll end blunted ligation. The 1.6kb CAT gene fragment contains about 70 baseε of upstream sequenceε. Theεe ligated fragments are cloned into Bluescript KS(-) vector (Stratgene, La Jolla, CA) . Thiε construct in turn is subjected to εite specific mutagenesis to delete the extra εequenceε (approximately 30 baseε) from the 3' end of the OP-l upεtream sequences and the adjacent 5' non-coding sequences (approximately 70 bases) from the CAT gene. This mutageneεiε reεultε in the elimination of any OP-l coding εequences from the promoter fragment as well as any non-coding sequenceε upstream of the CAT gene. Thus the resulting conεtruct is a fusion of OP-l upεtream εequences with the CAT gene sequences which encode the CAT protein. Thiε approximately 5 kb fragment iε then excised from Bluescript using Hindlll and BamHI and ligated into a Hindlll- BamHI cut and gel purified back-bone of the pΞV2CAT vector, for transfection into suitable cell lines. Suitable cell lines include cell lines that have been shown to contain high levels of OP-l mRNA, indicating that the OP-l promoter is active in the cellε. Two of these cell lines are mouεe inner medullary collecting duct (IMCD) cellε, and the rat bladder carcinoma line (NBT II) . However other cell lineε of the uro-genital system that produce high levels of the OP-l mesεage can be uεed in addition to the many previously mentioned cell types and cell lineε.

The tranεfection of thiε vector into an OP-l producing cell line is accomplished following standard techniques, i.e., transfection using calcium phosphate, lipoεome mediated tranεfection, electroporation, or DEA.E-dextran tranεfection.

The tranεfected cells are harvested 48-72 hours after tranεfection with the CAT expreεεion vector and extracts are made by succeεεive freeze-thawing. 2 μl of 200 μci/ml 14C- choramphenicol (35 to 55 mCi/mmol), 20 μl of 4 mM acetyl CoA, 32.5 μl of 1 M Triε-HCl, pH 7.5, and 75.5 μl of water is added to 20 ml of cell extract, and incubated for 1 hour at 37 degreeε Celεius. Upon completion of incubation, 1 ml ethyl acetate is added to the reaction, microcentrifuged for 1 minute and the top layer iε removed. Thiε top layer iε dried down in a SpeedVac for 45 minutes, and each sample is resuεpended in 30 ml of ethyl acetate. The samples are spotted onto a plastic-backed TLC εheet for chromatography. The thin layer is then developed in a tank containing 200 ml of 19:1 chloroform/methanol . The chromatography is run for 2 hours and placed under film for autoradiography. The activity of the C¹⁴ in the monoacetylated chloramphenicol εerieε iε calculated as described in Current Protocols in Molecular Biology, 1993 (Ausubel et al. , eds. John Wiley Sons, New York).

Upon determination of CAT activity, the main conεtruct can be deleted in sections to determine the regions that are responsible for the observed CAT activity. Alternatively, the upstream sequences can be deleted unidirectionally, using an exonuclease such as Bal31, and the deletion product can be analyzed in the CAT activity assay. This system can also be used in the method of the invention to screen compounds for their ability to modulate OP-l expression by dividing the cells into several groups, and culturing one group in the absence of any added compounds, and culturing the other groups with one or more candidate compound, and comparing the resulting levels of CAT activity.

While a readily assayable, well characterized, non OP-l reporter gene is preferred in the method disclosed herein, as will be appreciated by thoεe having ordinary skill in the art, OP-l coding sequence alεo may be used in the screening method of the invention. The OP-l expression preferably is determined by an immunoasεay or by Northern or dot blot or other means for measuring mRNA transcript. See, for example, WO 95/11983, published May 4, 1995 for a detailed description on asεaying changes in OP-l levels in a cell or fluid.

XI. Exemplary Screening Asεay for Compoundε which Alter OP-l Gene Expression in

Endogenous Cell Type Models. OP-l is expresεed in a variety of different cell typeε, including renal, bone, lung, heart, uterine, cardiac and neural tissue. Candidate compounds can be identified which have a modulating effect on cells of one tiεεue type but not another, and/or wherein the effect iε modulated in the different cells. The asεay described belov; can be used to evaluate the effect of a candidate compound(ε) in a particular cell type known to express OP-l under physiological conditions.

Cell cultures of kidney, adrenals, urinary bladder, brain, or other organs, may be prepared as described widely in the literature. For example, kidneys may be explanted from neonatal or new born or young or adult rodents (mouse or rat) and used in organ culture as whole or εliced (1-4 mm) tissueε. Primary tiεεue cultures and established cell lines, also derived from kidney, adrenals, urinary, bladder, brain, mammary, or other tiεεueε may be established in multiwell plates (6 well or 24 well) according to conventional cell culture techniques, and are cultured in the absence or presence of serum for a period of time (1-7 days) . Cells may be cultured, for example, in Dulbecco's Modified Eagle medium (Gibco, Long Island, NY) containing serum (e.g., fetal calf serum at 1%-10%, Gibco) or in serum-deprived medium, as desired, or in defined medium (e.g., containing insulin, transferrin, glucose, albumin, or other growth factors) .

Samples for testing the level of OP-l production includes culture supernatants or cell lyεateε, collected periodically and evaluated for OP-l production by immunoblot analysis (Sambrook et al., eds., 1989, Molecular Cloning, Cold Spring Harbor Press, Cold Spring Harbor, NY), or a portion of the cell culture itself, collected periodically and used to prepare polyA+ RNA for RNA analysiε. To monitor de novo OP-l εyntheεiε, some cultures are labeled according to conventional procedures with an ³⁵S- methionine/³⁵S-cyεteine mixture for 6-24 hourε and then evaluated to OP-l εynthesiε by conventional im unoprecipitation methodε.

XII. Exemplary In vi vo Animal Model for Testing Efficacy of Compounds to Modulate OP-l Expresεion

It previously haε been demonstrated that OP1 can effect osteoporoεiε on the εtandard ovariectomized rat model, aε indicated by the doεe-response increase in alkaline phosphate and osteocalcin levelε following injection with OP-l. The osteoporotic rat model provides an in vivo model for evaluating the efficacy of a candidate modulating compound. In order to determine the effect of a candidate morphogen stimulating agent on OP-l production and, thereby, on bone production in vi vo, alkaline phosphate and oεteocalcin levels are meaεured under conditions which promote oεteoporoεiε, e.g., wherein oεteoporoεis is induced by ovary removal in rats and in the presence and absence of a candidate modulating compound. A compound competent to enhance or induce endogenous OP-l expression should result in increased osteocalcin and alkaline phosphate levels.

Forty Long-Evans rats (Charles River Laboratorieε, Wilmington) weighing about 200g each are ovariectomized (OVX) uεing εtandard εurgical procedureε, and ten ratε are sham operated. The ovariectomization of the rats produces an osteoporotic condition within the rats as a result of decreased estrogen production. Food and water are provided ad libitum. Eight dayε after ovariectomy, the ratε, prepared as described above, are divided into three groups: (A) sham-operated rats; (B) ovariectomized rats receiving 1 ml of phosphate-buffered saline (PBS) i.v. in the tail vein; and (C) t ariectomized rats receiving various dose ranges of the candiate stimulating agent either by intravenous injection through the tail vein or direct administration to kidney tissue. The effect of the candidate compound on in vivo bone formation can be determined by preparing sections of bone tissue from the ovariectomized rats. Each rat iε injected with 5 mg of tetracycline, which will stain the new bone (visualized as a yellow color by fluorescence) , on the 15th and 21st day of the study, and on day 22 the rats are sacrificed. The body weights, uterine weights, serum alkaline phosphate levels, serum calcium levels and serum osteocalcin levels then were determined for each rat. Bone sectionε are prepared and the diεtaance εeparating^" each tetracycline straining iε meaεured to determine the amount of new bone growth. The levelε of OP-l in εerum following injection of the candidate agent alεo can be monitered on a periodic baεis using, for example, the immunoassay described in sectionε V and VII above.

V. Exemplary Determination of OP-l Protein Production Where OP-l actε as the reporter gene, detection fo the gene product readily can be aεεayed uεing antibodies specific to the protein and standard immunoassay testingε. For example, OP-l may be detected uεing a polyclonal antibody specific for OP-l in an ELISA, as follows. lμg/100 μl of affinity-purified polyclonal rabbit IgG specific for OP-l is added to each well of a 96-well plate and incubated at 37°C for an hour. The wells are washed four timeε with 0.167M εodium borate buffer with 0.15 M NaCl (BSB) , pH 8.2, containing 0.1% Tween 20. To minimize non-εpecific binding, the wells are blocked by filling completely with 1% bovine serum albumin (BSA) in BSB and incubating for 1 hour at 37°C. The wells are then waεhed four times with BSB containing 0.1% Tween 20. A 100 μl aliquot of an appropriate dilution of each of the test samples of cell culture supernatant iε added to each well in triplicate and incubated at 37°C for 30 min. After incubation, 100 μl biotinylated rabbit anti-OP-1 εerum (εtock solution is about 1 mg/ml and diluted 1:400 in BSB containing 1% BSA before use) iε added to each well and incubated at 37°C for 30 min. The wellε are then waεhed four timeε with BSB containing 0.1% Tween 20. 100 μl streptavidin-alkaline (Southern Biotechnology Aεεociateε, Inc. Birmingham, Alabama, diluted 1:2000 in BSB containing 0.1% Tween 20 before uεe) iε added to each well and incubated at 37^CC for 30 min. The plates are washed four times with 0.5M Tris buffered Saline (TBS), pH 7.2. 50μl substrate (ELISA Amplification System Kit, Life Technologies, Inc., Bethesda, MD) is added to each well incubated at room temperature for 15 min. Then, 50 μl amplifier (from the same amplification system kit) iε added and incubated for another 15 min at room temperature. The reaction is stopped by the addition of 50 μl 0.3 M sulphuric acid. The OD at 490 nm of the solution in each well is recorded. To quantitate OP-l in culture media, a OP-l standard curve is performed in parallel with the test samples.

VI. Exemplary Production of OP-l Polyclonal and Monoclonal Antibody

Polyclonal antibody for OP-l protein may be prepared as follows. Each rabbit is given a primary immunization of 100 μg/500 μl E. coli produced OP-l monomer (amino acids 328-431 in SEQ ID NO:5) in 0.1% SDS mixed with 500 μl Complete Freund's Adjuvant. The antigen iε injected εubcutaneously at multiple sites on the back and flanks of the animal. The rabbit is booεted after a month in the same manner using incomplete Freund's Adjuvant. Test bleeds are taken from the ear vein seven days later. Two additional booεtε and test bleeds are performed at monthly intervalε until antibody against OP-l is detected in the serum using an ELISA asεay. Then, the rabbit iε booεted monthly with 100 μg of antigen and bled (15 ml per bleed) at days εeven and ten after boosting.

Monoclonal antibody specific for OP-l protein may be prepared as follows. A mouse is given two injectionε of E. coli produced OP-l monomer. The firεt injection containε lOOμg of OP-l in complete Freund'ε adjuvant and iε given εubcutaneouεly. The εecond injection containε 50 μg of OP-l in incomplete adjuvant and is given intraperitoneally. The mouse then receives a total of 230 μg of OP-l (amino acids 307-431 in SEQ ID NO:5) in four intraperitoneal injections at various times over an eight month period. One week prior to fusion, both mice are boosted intraperitoneally with 100 μg of OP-l (307-431) and 30 μg of the N-terminal peptide (Ser_2.3-Asn₃o₉-Cys) conjugated through the added cyεteine to bovine serum albumin with SMCC crosslinking agent. This booεt waε repeated five dayε (IP) , four days (IP) , three days (IP) and one day (IV) prior to fusion. The mouεe spleen cells are then fuεed to myeloma (e.g., 653) cells at a ratio of 1:1 using PEG 1500 (Boeringer Mannheim) , and the cell fusion is plated and screened for OP-1-specific antibodies using OP-l (307-431) as antigen. The cell fusion and monoclonal screening then are according to standard procedures well described in standard texts widely available in the art. VII. Exemplary Process for Detecting OP-l in Serum

Presented below is a sample protocol for identifying OP-l in serum. Following thiε general methodology OP-l may be detected in body fluids, including serum, and can be used in a protocol for evaluating the efficacy of an OP-l modulating compound in vi vo . A monoclonal antibody raised against mammalian, recombinantly produced OP-l uεing standard immunology techniques well described in the art and described generally in example VI., above, was immobilized by passing the antibody over an agarose- activated gel (e.g., Affi-Gel™, from Bio-Rad Laboratories, Richmond, CA, prepared following manufacturer'ε instructions) and used to purify OP-l from serum. Human serum then waε paεεed over the column and eluted with 3M K-thiocyanate. K-thiocyanante fractionε then were dialyzed in 6M urea, 20mM P0₄, pH 7.0, applied to a C8 HPLC column, and eluted with a 20 minute, 25-50% acetonitrile/0.1% TFA gradient. Mature, recombinantly produced OP-l homodimerε elute between 20-22 minuteε, and are uεed aε a poεitive control. Fractions then were collected and teεted for the preεence of OP-l by standard immunoblot uεing an OP-l εpecific antibody. Uεing thiε method OP-l readily waε detected in human serum. See also, PCT/US92/07432 for a detailed description of the assay.

IX. Considerationε for Formulationε and Methodε for Adminiεtering Therapeutic Agentε

Where the OP-1-modulating agent identified herein comprises part of a tisεue or organ preservation solution, any commercially available preservation solution may be uεed to advantage. For example, uεeful εolutions known in the art include Collins εolution, Wisconsin solution, Belzer solution, Eurocollins solution and lactated Ringer's εolution. Generally, an organ preεervation εolution uεually poεεeεεes one or more of the following properties: (a) an osmotic pressure subεtantially equal to that of the inεide of a mammalian cell, (solutions typically are hyperosmolar and have K+ and/or Mg++ ions present in an amount sufficient to produce an osmotic presεure slightly higher than the inside of a mammalian cell); (b) the εolution typically is capable of maintaining subεtantially normal ATP levelε in the cells; and (c) the solution usually allows optimum maintenance of glucose metabolism in the cellε. Organ preεervation εolutionε alεo may contain anticoagulantε, energy εources such as glucose, fructose and other εugarε, metaboliteε, heavy metal chelatorε, glycerol and other materialε of high viscosity to enhance survival at low temperatures, free oxygen radical inhibiting agents and a pH indicator. A detailed description of preservation solutions and useful components may be found, for example, in US Patent No. 5,002,965.

Where the OP-1-modulating agent iε to be provided to an individual, e.g., the donor prior to harvest, or the recipient prior to or concomitant with transplantation, the therapeutic agent may be provided by any suitable means, preferably directly (e.g., locally, as by injection to the tissue or organ locus) or εyεtemically (e.g., parenterally or orally). Uεeful εolutions for parenteral administration may be prepared by any of the methods well known in the pharmaceutical art, described, for example, in Remington's Pharmaceutical Sciences (Gennaro, A., ed.), Mack Pub., 1990. Formulations may include, for example, polyalkylene glycols such as polyethylene glycol, oils of vegetable original, hydrogenated naphthaleneε, and the like. Formulationε for direct administration, in particular, may include glycerol and other compoεitionε of high viεcoεity to help maintain the agent at the deεired locus. Biocompatible, preferably bioresorbable, polymerε, including, for example, hyaluronic acid, collagen, tricalcium phosphate, polybutyrate, lactide and glycolide polymerε and lactide/glycolide copolymerε, may be uεeful excipients to control the release of the agent in vivo. As will be appreciated by thoεe εkilled in the art, the concentration of the compoundε deεcribed in a therapeutic compoεition will vary depending upon a number of factorε, including the dosage of the drug to be administered, the chemical characteristics (e.g., hydrophobicity) of the compounds employed, and the route of administration. Where the morphogen-stimulating agent iε part of a preservation solution, the dosage likely will depend for example, on the size of the tissue or organ to be transplanted, the overall health status of the organ or tissue itself, the length of time between harvest and transplantation (e.g., the duration in εtorage) , the frequency with which the preservation solution is changed, and the type of εtorage anticipated, e.g., low temperature. In general terms, preferred ranges include a concentration range between about 0.1 ng to 100 μg/kg per tisεue or organ weight per day.

Where the therapeutic agent iε to be ad iniεtered to a donor or recipient, the preferred dosage of drug to be administered alεo iε likely to depend on such variables as the type and extent of progression of the disease, the overall health status of the particular patient, the relative biological efficacy of the compound selected, the formulation of the compound excipients, and its route of administration. In general terms, a suitable compound of this invention may be provided in an aqueous physiological buffer solution containing about 0.001% to 10% w/v compound for parenteral adminiεtration. Typical dose ranges are from about 10 ng/kg to about 1 g/kg of body weight per day; and preferred dose range is from about 0.1 μg/kg to 100 mg/kg of body weight per day.

The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claimε are therefore intended to be embraced therein. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: OZKAYNAK, ENGIN

OPPERMANN, HERMANN

(ii) TITLE OF INVENTION: METHODS AND COMPOSITIONS FOR MODULATING MORPHOGENIC PROTEIN EXPRESSION

(iii) NUMBER OF SEQUENCES: 7

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: PATENT ADMINISTRATOR, CREATIVE BIOMOLECULES

INC.

(B) STREET: 45 SOUTH STREET

(C) CITY: HOPKINTON

(D) STATE: MA

(E) COUNTRY: USA

(F) ZIP: 07148

(V) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy diεk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: PatentIn Releaεe =1.0, Verεion .1.25

(Vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US 07/938,021

(B) FILING DATE: 28-AUG-1992

(C) CLASSIFICATION:

(Viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: KELLEY, ROBIN D

(B) REGISTRATION NUMBER: 34,637

. (C) REFERENCE/DOCKET NUMBER: CRP-091

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (508) -435-9001

(B) TELEFAX: (508) -435-0992

(2) INFORMATION FOR SEQ ID Nθ:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17415 base pairε

(B) TYPE: nucleic acid

(C) STRANDEDNESS: εingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE:

(A) ORGANISM: homo sapiens

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:1 :

TCAACCGGTC TCTTTAGGTT TTGGCTGTGC TTATTACTAT TCATTCAACA GGTACTAATT 60

GAGCACCTGC TGTGTGCCAG GCTCAGAATA GGCTCAGGTG AGATGCACAA AGAAGGGTAA 120

ACTAGAATCC TTGCTTAGAC ACTGACGGAT CAGTTGTTTC ATATGTAAAT TGTAGCACCA 180

AGACCTGCTG CCCCTGCCCC CAGCCTCACC TGCTTGTGAA GATCCCTCCA AAAGATTTGA 240

GAGTAGATAA AAAGCAGAGA CTACTACTGA AGAACAGGGC TGCTTTGGCT CCTTATTATT 300

TCAGACTTTG GAAGAAAATG ACCTCCTTTT TCTCTACTGG CACTGAGTGC ATAGCTGACC 360

TAGCAAGCCA GGCCTGGAGG GCGTGTGCAG GGCTGGGGAC CGAGCCTGGT TTCTGTTCCC 420

TGCTCTGCAG CTCAGCACT TGCTGTTCCT CCACCTGGGA TGCCTTTCCC TGGAAAAGCC 480

TGTCTCTTTC TTGTCTTTCA GGACTCAGGT CAGTGGCATC TCCTCCAAAA ACTCCCCTTC 540

CCACCCTCCA TCACCTCACC CTGTTTATCT GCGCCCCCGC CCCCACTGCC TGTCACTTAT 600

TGCAGGCTGA AGTGACCCAG GCTCTCCAGT TGTACACTCT CAGATGGACC CTGGACGACT 660

GTGGCACTCC TGCAATTTCC CCAGTCTCCC TGGGGTAGGA TTCCTGCTTG CCAGGATGCC 720

CACCTTTCCT TCTCCCTCCT GCATGTCCTC CTCTGCCTGG CTTCTGAATT GTTTCCAGAG 780

AGAGTGATAG ACAAGATCTG CCTCTCCTTC AGTCCCTGAA TCTTATTTAA GGCTCTTGCT 840

TTGCTTCCCT GGCCTGGAGG CGGCTCCTTG ATGGAGTCTG CCATGTGGGT TCGCTCATGG 900

CCATGTCTTC CTGCCCAGCA TGGTGCTTGG CCCTGGGACT GGCCA AA TATCTGGGCC 960

AGGTGCAAAA TTAGTACGGG GCAGGGGGTA CTTTGTTCAT AGGTGATTCA GAACCACATA 1020

TGGTGACCTC AGAGTAGGAA ACCAAGTGTG GGGCCCTTAA GAGCTGGGGG GCCCTGTACG 1080

ACTGTCCAGG TTGCAGGCCC CACAGCTCGC CTCCTGATAT CCTGTGCTCC ATGCTTGTCT 1140

GTTGAAGGAA GGAGTGAATG GATGAAGAGC AGGTGGTGGG GGGTGGTTTG AGGGCCTTGC 1200

TGGTGGGTGG GTAGAGGCCC CTCCCTGGCA TGGGGCTCAA GACCTGTTCC ATCCCACAGC 1260

CTGGGGCTGT GTGTAAATGG CCAGGACCTG CAGGCTGGCA TTTTTCTGCT CCTTGCCTGG 1320

CTCTGGCTCC CCTTTCTCCA CCCATGTGGC CCCTCAGGCT GCCATCTAGT CCAAAAGTCC 1380

CAAGGGAGAC CCAGAGGCCA CTTGGCAAAC TACTTCTGCT CCAGAAAACT GTAGAAGACC 1440 ATAATTCTCT TCCCCAGCTC TCCTGCTCCA GGAAGGACAG CCCCAAAGTG AGGCTTAGCA 1500

GAGCCCCTCC CAGACAAGCG CCCCCGCTTC CCCAACCTCA GCCCTTCCCA GTTCATCCCA 1560

AAGGCCCTCT GGGGACCCAC TCTCTCACCC AGCCCCAGGA GGGAAGGAGA CAGGATGAAC 1620

TTTTACCCCG CTGCCCTCAC TGCCACTCTG GGTGCAGTAA TTCCCTTGAG ATCCCACACC 1680

GGCAGAGGGA CCGGTGGGTT CTGAGTGGTC TGGGGACTCC CTGTGACAGC GTGCATGGCT 1740

CGGTATTGAT TGAGGGATGA ATGGATGAGG AGAGACAGGA GAGGAGGCCG ATGGGGAGGT 1800

CTCAGGCACA GACCCTTGGA GGGGAAGAGG ATGTGAAGAC CAGCGGCTGG CTCCCCAGGC 1860

ACTGCCACGA GGAGGGCTGA TGGGAAGCCC TAGTGGTGGG GCTGGGGTGT CTGGTCTCAG 1920

GCTGAGGGGT GGCTGGAAAG ATACAGGGCC CCGAAGAGGA GGAGGTGGGA AGAACCCCCC 1980

CAGCTCACAC GCAGTTCACT TATTCACTC ACAAATCGTG ACTGCGCACG TACAGTGGCT 2040

ACCAGGCGCT GGGTTCAAGG CACTGCGGGT ACCAGAGGTG CGGAGAAGAT CGCTGATCCG 2100

GGCCCCAGTG CTCTGGGTGT CTAGCGGGGG TAAGAAGGCA ATAAAGAAGG CACGGAGTAA 2160

CTCAAACAGC AATTCCAGAC AGCAAGAGAA ACTACAGGAA AGAAAACAAA CGTGCGAGGG 2220

GCGAGGCGAG GAAACAACCT CAGCTTGGCA GGTCTTGGAG GTCTCTGGGA GGAGAAAGCA 2280

GCGTCTGATG GGGGCGGGAG GTGGTGAGTG GGGAGAGGTC CAGGCGGAGG GAATGGCGAG 2340

CGAGAGACAG GCTGGCAACG GCTTCAGGGA GGCGCGGAGG GGTCAGCGTG GCTGGCTTAA 2400

AAGGATACAT GGGACTAGGG GCAAGACCGG CTCAAGGTCA CCGCTTCCAG GACCTTCTAT 2460

TTCCGCGCCA CCTCCGCGCT CCCCCAACTT TTCCCACCGC GGTCCGCAGC CCACCCGTCC 2520

TGCTCGGGCC GCCTTCCTGG TCCGGACCGC GAGTGCCGAG AGGGCAGGGC CGGCTCCGAT 2580

TCCTCCAGCC GCATCCCCGC GACGTCCCGC CAGGCTCTAG GCACCCCGTG GGCACTCAGT 2640

AAACATTTGT CGAGCGCTCT AGAGGGAATG AATGAACCCA CTGGGCACAG CTGGGGGGAG 2700

GGCGGGGCCG AGGGCAGGTG GGAGGCCGCC GGCGCGGGAG GGGCCCCTCG AAGCCCGTCC 2760

TCCTCCTCCT CCTCCTCCGC CCAGGCCCCA GCGCGTACCA CTCTGGCGCT CCCGAGGCGG 2820

CCTCTTGTGC GATCCAGGGC GCACAAGGCT GGGAGAGCGC CCCGGGGCCC CTGCTATCCG 2880

CGCCGGAGGT TGGAAGAGGG TGGGTTGCCG CCGCCCGAGG GCGAGAGCGC CAGAGGAGCG 2940

GGAAGAAGGA GCGCTCGCCC GCCCGCCTGC CTCCTCGCTG CCTCCCCGGC GTTGGCTCTC 3000

TGGACTCCTA GGCTTGCTGG CTGCTCCTCC CACCCGCGCC CGCCTCCTCA CTCGCCTTTT 3060 CGTTCGCCGG GGCTGCTTTC CAAGCCCTGC GGTGCGCCCG GGCGAGTGCG GGGCGAGGGG 3120

CCCGGGGCCA GCACCGAGCA GGGGGCGGGG GTCCGGGCAG AGCGCGGCCG GCCGGGGAGG 3180

GGCCATGTCT GGCGCGGGCG CAGCGGGGCC CGTCTGCAGC AAGTGACCGA GCGGCGCGAC 3240

GGCCGCCTGC CCCCTCTGCC ACCTGGGGCG GTGCGGGCCC GGAGCCCGGA GCCCGGGTAG 3300

CGCGTAGAGC CGGCGCGATG CACGTGCGCT CACTGCGAGC TGCGGCGCCG CACAGCTTCG 3360

TGGCGCTCTG GGCACCCCTG TTCCTGCTGC GCTCCGCCCT GGCCGACTTC AGCCTGGACA 3420

ACGAGGTGCA CTCGAGCTTC ATCCACCGGC GCCTCCGCAG CCAGGAGCGG CGGGAGATGC 3480

AGCGCGAGAT CCTCTCCATT TTGGGCTTGC CCCACCGCCC GCGCCCGCAC CTCCAGGGCA 3540

AGCACAACTC GGCACCCATG TTCATGCTGG ACCTGTACAA CGCCATGGCG GTGGAGGAGG 3600

GCGGCGGGCC CGGCGGCCAG GGCTTCTCCT ACCCCTACAA GGCCGTCTTC AGTACCCAGG 3660

GCCCCCCTCT GGCCAGCCTG CAAGATAGCC ATTTCCTCAC CGACGCCGAC ATGGTCATGA 3720

GCTTCGTCAA CCTCGGTGAG TAAGGGCAGG CGAGGGTACG CCGTCTCCTT •TCGGGGGCAC 3780

TTTGAGACTG GGAGGGAGGG AGCCGCTTCT TCTATGCAGC CCGCCCAGCT TTCCGCTCCT 3840

GGCTGAAATC GCAGTGCCTG CCCGAGGGTC TCCCACCCAC AGCCCTATGA CTCCCAAGCT 3900

GTGTGCGCCC CCAGGTCGGG CCGCTGGGTC GGTGAGCCTG TAGGGGTTAC TGGGAAGGAG 3960

GGATCCTCCG AAGTCCCCTC CATGTTACGC CGCCGGCCGC ATCTCTGGGG CTGGAGGCAA 4020

GGGCGTTCAA AGCGCGGGGC TCGGTCATGT GAGCTGTCCC GGGCCGGCGC CGGTCCGTGA 4080

CCTGGATGTA AAGGGCCCTT CCCGGCGAGG CTGCCTTGCC GCCCTTCCTG GGCCCCTCTC 4140

AGCCCTGCCT GGCTCTGGCA TCGCGGCCGT CGCACCCCCT TACCCTCCCT GTCAAGCCCT 4200

ACCTGTCCCC TCGTGGTGCG CCCGCCTTAG GCTACCGGCC GCTCCGAGCC TTGGGGCCCC 4260

TCTCCGGGCG CCGATGCCCC ATTCTCTCTT GGCTGGAGCT GGGGAAGAAA CGGTGCCATT 4320

GCTAATTTTC TTTGTTTTCT TTCTTTGTTT ATTTTTTTCT TTTTTCTTTT TTTTTCTTTT 4380

CTTTTCTTTT CTTTTTTTTT TTTTTTGAGA CGGAGTTTCA CTCTTGCTCG CCCAGACTGG 4440

AGTGCAATGG CGCGATCTCT GCTCACCGCA ACCTCTGCCT CCCGGGTTCA AGCGATTCTC 4500

GTGCCTCAGC CTCCCGAGTA GCTGGGATTA CAGGCATGCG CACCATGCCT GGCTAATTTT 4560

GTATTTTAGT AGAGACAGGG TTTCTCCATG TTAGGCAGGC TGGTCTCGAA CTCCCGATCT 4620

CAGGTGATCC TCCCGCCTCA GCCTCCCAAA GTGGTGCTGG GATTACAGGC GTGAAGCTGT 4680 GCCCTGCCGC TAGTCTTCTA TTTTAAGTAT TTAGTGGTAG GTCCCGGGCC GGCAGAATCT 4740

ATTTTCAGCA TTTACCACGT GTGGCGCGCA AACCACAGGT TTTGGCGATT GGGTTGCGCG 4800

GGATCTCAGA GCTGACGACC GCGGGGGCCT GGGGGTCCCG GTTTCCGACT GGAGCCGCGA 4860

CGACCCCGGC GACGGCAGCC TGGGGCTGCA GCCGAGGGCC GGGGAGCTCC CCCTCCATAT 4920

GTGCGCGCAC ATTCTCCAGA CTTGCTCAAA CTAACCCCCC GGAGCAGCGC ACGGGCTGGG 4980

ACTGATGATC AAATATTTGG TTTCCGAGAT AACACACCCC GATAGCGCTG TTTCCTGAGC 5040

CGCTTTCATT CTACTTGTGT AACTTGCTGC GAAAACCCGA ACCAAGTCAA GACAGCAAAC 5100

TCACGCCCAC GGGCCTGTGT CAACATGGAA ATAATGATAC TGAAGCCCCA CGCTGGGCAC 5160

CTGGGGCGTG GACTGGGGGC GCGGGGGAAG CGCAGATCCG CCTTCATGCT TCCCCTCCTC 5220

CTGATAAGGT CCCTGGAGTT CCCGGGAGCC ATTGTCTGTA CTTAATAATA ACTAAATCCA 5280

ACTAGTGAAC CAAGCTTCAG CGAGGCAAGG GGAGGGAGGT TTAGATGCCA AAATTACCTT 5340

CAAAAAAGTT TAAATTATAC TAAGCAGCC GTTAAGAAGG AAGCAGCAAT ATATGACCTG 5400

ATTTAGAACC ATCTCCAAGA TGTATGAGGT GGΛAAGAAGC AAGGTGCAGA TGAGTGGGCT 5460

GCATGTGTGC TTGTATATCA TCGTGTCCTC CTGGAGGAAG ACACCAGGAA CTGGAGAGAG 5520

ATTTTACTGG AGGGGTATAT GGCGGGGGCA TAGCTGGGGC TTACGGAGTG GGAGGTGGGG 5580

TCTGATTTTT CGTCGTCTGC ACTTCTGTAT TTGTGATTTT TTTAAAACAA TGTGTATTTA 5640

TTAACTATAC CAAAAAATAA AGGAAAATTC CAAATACATA CATATAAATA ATG ACCGCA 5700

GAGCTCTGTC GCCCTCCTGA AGCCTGGGGT TAGCCAGGGC CCTTTCTCTG GTGGGGGATT 5760

TATAGCATCT TCCCTTCTGT TGGGTACCCC GGACTCCCAC TGAATGTGCA GGTCCCAGTG 5820

GCTGCCTTCA GAGCCTGGCT GGAATCATTA AAAAGGTATT TGTAATCTCT GGCTTCTGCA 5380

GAAGGCCCTG CAAACCAAGA GCAAAAAAGC CCCCAGTGCT TATGGGCCGG CAGTGTGGGC 5940

TAGGCCCGGG GCTCCCTGTC CCCAAGAGAA AGACCAGGTT GCTCGGAGGG TGCCTCTGGG 6000

AACTTTGGTG CGGGCTATTT GCTCCCCCCA TGGCGGCAGG AGCAAGCTGG GACTTGTTTG 6060

GGAAGGCCAC AGCTGGGTGG TTTTCCTCCT CTGGCTGTAC ATACACCTTT CAATCCATTT 6120

CTTTCATCTT GAAAGGACAA AGACCGGCTT GTCTGAGCCT CTTAATCAGT CAGGCTGGCT 6180

TTGGGCTTTG GGGACCCTGA CTTTCTCAGG TCTAGCTTTC TGGGACATCA CTCCAAATTA 6240

GATGGCAGAG TGGCTTTTAA CAGAGCGCAC TGACCTTGTT TTCTTTCTCT CTCTGTCCCT 6300 AAACTCGAGG TCATTAGTTA GGTGAAGACC TGGGCTGCAG TTTGGCGAGA CACTTCCTGT 6360

AGATGCTTCT AATGTTGGCC TTTAATTTCT GCTAAGCAGC AGCACACAAA TAAATGGCCT 6420

GTCCCTTCTA TCCTGTTGTA GCTTGGAATT TCTCCATAGG AGGGACTTGG GGGTGGCAGT 6480

AGGGTTGGAG AGGGTTGGGG GGAGGTGTAG GAGACTTGTC TGGCCACTGA GTTTGCTGAG 6540

AAAGTACTGC TATAGTGTTT TTCCTTGGAT TGCAAATCAT GTTGATCTGA ACTGCTGATT 6600

TGAAGTGGAT TGAGAGGATG GAACAATAGA AGGAGGATAT GGCTCAGGAC AGTCAAGTAC 6660

TGGAAGAGGG AAAGGTACAA AGAGGTGTTG GCACTGAATG ACCCTGAACA GGGCTGCCCT ^■ 6720

GGAAATATCA GAGGTGAGTG ACAAAGAGAA CTCTAGTCGA AGGTCTGGAA GTCAATTATT 6780

GTCTCCAGCT TTTGTCCCAC CCTAAGGGAT GGAGCATGAA CTTCATGCAT GTAACATCCC 6840

TCCAGGAGCG CTGAGGTTCT GGGAATTCCC AGTGCTGGCT ACCATGCCAT TCTTTTCTCA 6900

TTCACTCAAG AGCGTATTGG GATATGCGTG CATGAAAGCA ATGTAATTAT GGGCACAACC 6960

TCAAAACCTG CTCTAATTTT TTTTTTTTTT GGAGATGGAG TCTCGCTCCA ^'TCACCCAGGC 7020

TGGAGTGC.AA TGGCGCGATC TCAGCTCACT GCAAGCTCAG ACCTCCAGGG TTCACACCAT 7080

TCTCCTGCCT CAGCCTCCCG AGTAGCTGGG AATACAGGCG CCCGCACCAT GCGCGGCTAA 7140

TTTTTTTGTA TTTTTAGTAG AGACGGGGTT TCACTGTGTT AGCCAGGATG GTCTCGATCT 7200

CCTGACCTCG TGATCCACCC GCCTCGGCCT CCCAAAGTTC TGGGATTACA GGCGTGACAG 7260

CCGTGCCCGG AATCTGCTCT AATTTTTTAA AGATATCATT TGCAAACTTT GGGCACTTGA 7320

GTCACTCAGT AAGATATTAT TTACAACCCC ACCATAGATT CAAACCTCTG TCCTAGAATG 7380

TTGTCGAGTT AGGCATCTGG CTTGCAGCAA CAGCTGGCTT TCCTGTCTAT GCTGTCTCCT 7440

TCCAGGGAGG ATGTTTCACC CTTCATATTG AGGAAATGGG CACAGAGAAC CCATTTCTCT 7500

TACTCATCAT GTAACTTCAG TGGGATGGTC AGATCTATCT TTAACCTGGC CACTCTTCCA 7560

CAAGCTCACA CTGACTCCAG CAAGATCTTA AACTAGAAGG CAGGAGTTCA AATCCTAGCT 7620

GGTGCAGTGG CCAAATCTCG GCTCACAGCA CCTTCTGCCT CCTGGGCTCA AGCGATCCTC 7680

TGACCTCAGT CTCCCAAGTA GCTGGGACCA TAGGCATGCA CCACTATGCC TGGCTAATTT 7740

TTGTATTTTT GTAATTTTTT GTAGAGACAG AGTTTCACCA TGTTGCCCAG CCCAGTCTTG 7800

AACTCCTGGA CTCAAGCAAT CTTCCCACCT TTGCCTACCA GAGTGCCGGG ATTACAGGTG 7860

TGAGCCATCA TGCTAGTTGC GCACAGTTGG GCGAAACTGA CAGATGAGAA AGCAGAACCT 7920 CGTGAGTCCA CTCAGTAAGA GACTCCCTAC TTTCTTTCTG AGTCTTTGTT TCTCATCAAT 7980

TGAATGGCAA TAAACAACTT GGTGGCCCAA GAGTTGATGA CAACAGTCCT ATAAGATTAT 8040

ACATGTAAAA GAAACAGAGT ATTCTACAAA TATCAGTTAT TGATAGTTCA ATAGGCAACC 8100

TGACATTACC TTTTCTTGGA ACTTGATGAA CAACTCAGAA ACTCATTAAT ATCAAACCCA 8160

ATGGTGAGCA CTTGGTCTTT ATTTATGGCT GTAAGAGAAG AAATTGAATT AACTCTATGT 8220

AAATGCCAAC TAAGAACATC GAAGTCTGAA ATCAACAGTT TTCCTCGCTC ATACGACACA 8280

CCCAAACTCA AGCAGTGGTT CCAAGCCCCT TTGGAAAATA CCATGGGCTA ACGACTTTAA 8340

AAGCTTAGAA GTGAATTCTA CTTACTTATT ACTTAAAAGT GGTTCTCAAA CTTCAAGGTG 8400

AATCAAAATC ATCTGTAGAG CTTGTTAAAA CACAGGTTGC TGGTCCACCC CAAGAGTGTC 8460

TTGAGTCAGT AGGTCTCAAG TAGGGCTCAA GAATATGCAT TTCTAATGAG CTCCAGGTGA 8520

GTCTAAGTGT TAGTCGTCGG TCTTGGGACC ACAACTTTGG GAACAATTGA TTTAGAAGAA 8580

CTCAAAGATC AGAAAGGGGT GGAATATTTT TAAAATTGTG GTAAAATACG CA AAACAGA 8640

AAAGGTACAA TTTTAACCAC TTAGAGAGAG GTGGGATCTA AGAACAGAAA TTGTTATGCC 8700

ATCAAAGGTG AGTTCAGATA AGCATTATTA AATGGTATCT ATGGATAAAC TTCAGGGGCC 8760

CTGTGGAGCA ACCCAATGCT GGGATGGGGT CCAGGTGTGC TATGGTTTGG ATGTGGTTTG 8820

TCCCTACAAA AACTCATGTT GAAATTTAAT TGCCAGTGTA ACATTATTGA GAGGTTATGG 8880

ACTTTTAAGA GGCATTTGGG TCATGAGGGA TCCACCTTCA GGGATTAGTG CAGTCTCCAG 8940

GGAGTGAGTG AGTTCCCATT CTAGTGGGAC TGGATTAGTT ACCATACAGT GGTTGTTATA 9000

AAGTGAGGCT GCTTCTGGTG TTTTATCTGT TTGCAGGCAC TTCCTTCCCC TTCCACTTCT 9060

CTGCCAGGTT AGGATGCAGC ATGAGGCCCT CACCAGAAGC TGACCAGATG TGGCTGCCTG 9120

ATCTTGAACT TCCCAGTCCC CAGAACCATG AGCTAAATAA ACCTTTTTTC TCTATAAATT 9180

ACGCAGTCTA GAGTATTCTA TTATAGCAAC ACAAGACAGA CTAAGACACA GTGGTAGAAA 9240

GAACACTACT GACTTCTCCC ATACTCTGGC CTATGGACAA GAGTGACAGA CAGACAAGAG 9300

TGAATATCAG GGCCCTCAGG CACATTCCTC TCTGCCCCTT CCTCCCTTCT TGCAGAGTCT 9360

CCAGTGACTG CCAGCTAATG CTATCATAGA CCCCACCTTT CCCCTGACTT GATTGGACCA 9420

GAAGCAGCCT CCTGATCCAT GGCCAACAAT CAGATTCACT TTCAAGAATT TGAACTAAGA 9480

GACACTAGGA AGATGGCCCT TGAGCTGTGA GTCCTACACT TGAAAGTTCT TAGCATCTTG 9540 - 49 -

GTCAGGTACC CACCAGGGCC ATGTGCAAAC TGAGATAATG GGGACATGGA ACAAGGGTAA 9600

GTGGAGAGGG CTGGCTGGAG AGAGACGGGC AGAGGAAAGC CCTGCCAAGA GGAGCAGAGA 9660

TGAGAGACCT TGGAGGGAGA GGTAATAAAA GGAGGCAAAG ATGATTTTCC ATGCTTACAA 9720

CTCACAGCTG AGGCCTAACT ATCTTTATGT CCATAAGAGG CATCCTTGTG TCGAACCTCT 9780

CCTCTTTCTT GGGTCAATGG GGGATGGTTG CAAGGGACCA TCAGTAGGAA GGCATAGTAC 9840

ACTAACCCAG TCTGGGGTGG GCTTTTAGAC TAGTCTTCCT CCCATGCTCC TCCTCCCATT 9900

GGAACCCCGG ACTTTCAAGA CTGCTACCTA GCACACCAGT GCACCAGATG TCACTCAAAA 9960

CCTCTTCAGC AATGGCCCAC TCACCTTCAA AAAGGCTGAA GAGCAGACTG GCTGGGTTCT 10020

TCATGGTGGA GGGGCAGTCT GGGAGGTTTT AAGGTTGAAG ATGAAAACTT TCACTTTTGG 10080

CTCAATGGTC TGAAAAAGAG AAGGACCAGC AAGTGAACTG AAGCCTCCTG GAAAGCATCT 10140

TGATAACAGG GGCAGAGTTT CAAGATGAGA AGCTGTGGCA CTTACTCTGG CTTTGGAAAT 10200

GACCTCTAAG TATCTCAGTT AATTAAAGGA GTCAAACTCT AGACTCGAAG GAGAAGATCT 10260

ACAATTTTCA ATAACATAGT CTACCCTCCC CTCCTTCCCC CACCTTCACC TCTTCTTTCA 10320

TCACAGGCTT ACAGGGCACC TCTTAGAGCC AGGCACGGTG TTGGGATCAG GAACAAGGCC 10380

ACTGCTCACA TCCAGAGCCT GTGCTACTTA AGAAGCTTCC AGGACCTCTT GGATGGCTGT 10440

GGTTAGTGCC CTACTTTTCC CAGCAGGTTG GATGCAGAAT CATGCTCTTG TCGTTCAGGA 10500

TGACCATGGG GACCATGGGT CTGAGCCTGT GACCCTCCAG TCTACAGTGT GTTGGTGAGG 10560

AAGGAGCAGT TGTCACTGGG GTCACTGGCA ATGGGCATGC CTCCATCTAG CTTAGGCAAG 10620

ATGCTTAGAC TCAGAGCCAG AGAGTGAAAC CCAGACACTA ATGAGCTGTC GGTGTTGGTG 10680

TGTGTTCTCT TCCTCTTCCA GTGGAACATG ACAAGGAATT CTTCCACCCA CGCTACCACC 10740

ATCGAGAGTT CCGGTTTGAT CTTTCCAAGA TCCCAGAAGG GGAAGCTGTC ACGGCAGCCG 10800

AATTCCGGAT CTACAAGGAC TACATCCGGG AACGCTTCGA CAATGAGACG TTCCGGATCA 10860

GCGTTTATCA GGTGCTCCAG GAGCACTTGG GCAGGTGGGT GCTATACGGG TATCTGGGAG 10920

AGGTGCTGAG TTTCCTCTGG GGGCAGAGGA AGAAGGTGGT GAGGGTTTCC CTCCCCTCCC 10980

ACCCCATGAG CTCTGCTTCC CATCTGTTGG GGTAGTGGAG CTGTGACCTG CTAACGCGAA 11040

GCCCGTGTCT CTCCTCCTCT CTCGCAGGGA ATCGGATCTC TTCCTGCTCG ACAGCCGTAC 11100

CTCTGGGCCT CGGAGGAGGG CTGGCTGGTG TTTGACATCA CAGCCACCAG CAACCACTGG 11160 GTGGTCAATC CGCGGCACAA CCTGGGCCTG CAGCTCTCGG TGGAGACGCT GGATGGTGAG 11220 TCCCCCGCCA CTGCCAGTCC TAATGCAGCC TGTGCTCCTG GACTTCAGGA GGGTCTCAGC 11280 AGTGCTCATG CTTGCTTCAC TACAAACAGG CTTCCCCGCC CCTCCCAACC AGTACTCCAT 11340

GTTCAGCCTT TTGATCCTGC AGCCCTGTCC CGCTCGTGGC CCTCCTGTAA CTGCTCTTCT 11400

GTGCACTTGG CTGCTTCCTG TCCAGGGCAG ACGATCAACC CCAAGTTGGC GGGCCTGATT 11460

GGGCGGCACG GGCCCCAGAA CAAGCAGCCC TTCATGGTGG CTTTCTTCAA GGCCACGGAG 11520

GTCCACTTCC GCAGCATCCG GTCCACGGGG AGCAAACAGC GCAGCCAGAA CCGCTCCAAG 11580

ACGCCCAAGA ACCAGGAAGC CCTCGGATGG CCAACGTGGC AGGGTATCTT AGGTGGGAGG 11640

GATCACAGAC CCACCACAGG AACCCAGCAG GCCCCGGCGA CCGCAGGAGA CTGACTAAAA 11700

TCATTCAGTG CTCACCAAGA TGCTCTGAGC TCTCTTCGAT TTTAGCAAAC CAGGAGTCCG 11757

AAGATCTAAG GAGAGCTGGG GGTTTGACTC CGAGAGCTCG AGCAGTCCCC AAGACCTGGT 11820

CTTGACTCAC GAGTTAGACT CCACTCAGAG GCTGACTGTC TCCAGGGTCT ACACCTCTAA 11880

GGGCGACACT GGGCTCAAGC AGACTGCCGT TTTCTATATG GGATGAGCCT TCACAGGGCA 11940

GCCAGTTGGG ATGGGTTGAG GTTTGGCTGT AGACATCAGA AACCCAAGTC AAATGCGCTT 12000

CAACCAGTAG AAAATTCACC AGCCCGCAGA GCTAAGGTTG GGTGGACATT AGGGTTGGTT 12060

GATCCAGGAG CTCAACAGTG TCCTCTGAGC CCCAGCTCCT TCTGCCCCAC CCCACCATCT 12120

TCAGTGCTGC TTCCTCTCAA GGCCACAGCT GTAGTTGGCC AGGGGGGCTT CATTATTTTT 12180

TGCTCCTGGG CAGTAGGAGG AAGAGAATGA ATGTCTCTCC ATGGGTCTTT CTTAGGAATG 12240

TGGGAACTTT TTCCAGAAGT CTCTATGTCT TTTAGTTTGT GTTGGGTCAC TTGCCCTTCC 12300

TGAACCACTT CCTGACTCCT GGACAGGATG TGCACTGATG AGCTTAGCTT TGGGGATCTA 12360

ATAGTGACTT TACAAAGCCT CTTTGAGAAG GTGACATTGG AACCAAGGCT TGAGCAGACA 12420

CAACAAAGAT TGCAGGGAGG GGCATTGCAG GTGGAGGAAA CGGCACATGC AAGAGCCCTG 12480

CGTGGGAGTG AGCTTGGTGT TTGGTCAATC AGTTGTCAGA GCACACCGGG CCCTGTCAGC 12540

AGGCACAGCC TGGGCCTGCT CTGAGTATGA CAGAGAGCCC CTGGGAAGTT GTAGGTGGAG 12600

GAAAGACAGG TCATGACTAG GAAAAAAGCA ATCCCTCTGT TGTGGGGTGG AAGGAAGGTT 12660

GCAGTGTGTG TGAGAGAGAG ACAAGACAGA CAGACAGACA CTTCTCAATG TTTACAAGTG 12720

CTCAGGCCCT GACCCGAATG CTTCCAAATT TACGTAGTTC TGGAAAACCC CCTGTATCAT 12780 TTTCACTACT CAAAGAAACC TCGGGAGTGT TTTCTTCTGA AAGGTCATCA GGTTTTGACT 12840

CTCTGCTGTC TCATTTCTTC TTGCTGGTGG TGGTGATGGT TGCTTGTCCC AGGCCCTGTC 12900

CCGCATCCTC TTGCCCCTGC AGAGGGATGA GTGTGTTGGG GCCTCACGAG TTGAGGTTGT 12960

TCATAAGCAG ATCTCTTTGA GCAGGGCGCC TGCAGTGGCC TTGTGTGAGG CTGGAGGGGT 13020

TTCGATTCCC TTATGGAATC CAGGCAGATG TAGCATTTAA ACAACACACG TGTATAAAAG 13080

AAACCAGTGT CCGCAGAAGG TTCCAGAAAG TATTATGGGA TAAGACTACA TGAGAGAGGA 13140

ATGGGGCATT GGCACCTCCC TTAGTAGGGC CTTTGCTGGG GGTAGAAATG AGTTTTAAGG 13200

CAGGTTAGAC CCTCGAACTG GCTTTTGAAT CGGGAAATTT ACCCCCCAGC CGTTCTGTGC 13260

TTCATTGCTG TTCACATCAC TGCCTAAGAT GGAGGAACTT TGATGTGTGT GTGTTTCTTT 13320

CTCCTCACTG GGCTCTGCTT CTTCACTTCC TTGTCAATGC AGAGAACAGC AGCAGGCACC 13380

AGAGGCAGGC CTTGTAAGAA GCACGAGCTG TATGTCAGCT TCCGAGACCT GGGCTGGCAG 13440

GTAAGGGGCT GGCTGGGTCT GTCTTGGGTG TGGGCCCTCT GGCGTGGGCT CCCACAGGCA 13500

GCGGGTGCTG TGCTCAGTCT TGTTTCTCAT CTCTGCCAGT TAAGACTCCA GTATCAAGTG 13560

GCCTCGCTAG GGAAGGGGAC TTGGGCTAAG GATACAGGGA GGCCTCATGA AATCCGAGAG 13620

CAGAAATGTG GTTGAGACTT GAACTCG.AAC CAGGAACCCA AACACTTTGG ACTCTGAACC 13680

CCATTCTCTG CATGCACCTC ATTCCCATCC CTTGGCTGGC TGCTTCTCAA GATGATGCCG 13740

GGCCGTGTGT TTGAATGTAG ATACCTGGGG AGCCATCTCC CCCTCTGCCC TCTGACTTCA 13800

TTTACCCCAT TCCCATTCCC ACGGGAGGGA CGGATCTCCC CAGCTTGGTT CAGGCGCTTG 13860

TTCCTGAACC AGTCAACTGT TTCAGGGGTG GGGTCATGTT ACTGGCACAT GGCTGCCCCC 13920

TCTGGAGCCA TTTGCATGGA GTGAGGCAAA AGGCAGGGGA TGAATCTAGG AGAGGAGTGA 13980

GGGTCATGTG ATCCACCTGC CGTGAGCTCT GGATCGTGAT TCTCATTCAG CAGTCACGAG 14040

CATCTCGAGC GTTCTGGGCC CTGTTCTAGG TACTGGATTG GAGATGCAGC GATGAACACT 1 100

GCAATGTGTC TGCCCTGTGG GGCTCAAATA TCCCTGGAGA GGGTATTGTC ATGAGGTCAT 14160

CAGGGCAACT GGTGGTATTC TACCCTCAGG GAGCTTGTAG TTCAGTGGGA GAGTCCAGAA 14220

TCTTCCCTGG GGATTATGCC CAGACACACT CAGGGCGTAC GTGCACACAG CCAGCTCTGA 14280

GCCCTCCTGT GAGCCTGCCC TCAGGACTGA TGACCACATC TACCTGCAGC TGGGACAGAA 14340

CCCAAACTCC AGGGGCCTCT GCTGGAAGAT TCCATGTGCT TAAGCATCAC TGAGGAGTAT 14400 ATTGATTATT GGGCAACATT TCTGTGCCAC CCAGACCCTA GAGGCAAGGA TGGCACATGG 14460

ATCCCTTACT GACCAGTGCA CCCGGAGCCA GCATGGGTGA TGCCATTATG AGTTATTAGC 14520

CTCTCTGGCA GGTGGGCAAA CCGAGGCATG GAGGTTTGTT TAAGGTGAAC TGCCAGTGTG 14580

TGACCACCTA GTGGGGGTAG AGCTGATGAT TGCCTCACAC CGGAGGCTCC TTCCTGTGCC 14640

GCGTTCTGTC CAGAAGACAC AGCCATGGAT GTCCATTTTA GGATCAGCCA AGCCCGTGGG 14700

GCTTTCCTTC ATTTTTATTT TATGTTTTTT TAGAAATGGG GTCTTGCTCT GTCACCCAGG 14760

CTGGGGTGCA GTGGTGTGAT CATACGTCAC CGCAGCTTTG AGCCGTCTTC CCACTCAGTC 14820

TACTAAGCTT GGACTATAGG_ CCAAGACTAT AGAGTGGTCC TTCTTTCCAT TCTTTTGGGA 14880

CCATGAGAGG CCACCCATGT TTCCTGCCCC TGCTGGGCCC TGCTGCTCAG AAGGCATGGT 14940

CTGAGGCTTT CACCTTGGTC GTGAGCCTTC GTGGTGGTTT CTTTCAGCAT GGGGTTGGGA 15000

TGCTGTGCTC AGGCTTCTGC ATGGTTTCCC ACACTCTCTT CTCCTCCTCA GGACTGGATC 15060

ATCGCGCCTG AAGGCTACGC GCGCTACTAC TGTGAGGGGG AGTGTGCCTT ^'CCCTCTGAAC 15120

TCCTACATGA ACGCCACCAA CCACGCCATC GTGCAGACGC TGGTGGGTGT CACGCCATCT 15180

TGGGGTGTGG TCACCTGGGC CGGGCAGGCT GCGGGGCCAC CAGATCCTGC TGCCTCCAAG 15240

CTGGGGCCTG AGTAGATGTC AGCCCATTGC CATGTCATGA CTTTTGGGGG CCCCTTGCGC 15300

CGTTAAAAAA AAATCAAAAA TTGTACTTTA TGACTGGTTT GGTATAAAGA GGAGTATAAT 15360

CTTCGACCCT GGAGTTCATT TATTTCTCCT AATTTTTAAA GTAACTAAAA GTTGTATGGG 15420

CTCCTTTGAG GATGCTTGTA GTATTGTGGG TGCTGGTTAC GGTGCCTAAG AGCACTGGGC 15480

CCCTGCTTCA TTTTCCAGTA GAGGAAACAG GTAAACAGAT GAGAAATTTC AGTGAGGGGC 15540

ACAGTGATCA GAAGCGGGCC AGCAGGATAA TGGGATGGAG AGATGAGTGG GGACCCATGG 15600

GCCATTTCAA GTTAAATTTC AGTCGGGTCA CCAGGAAGAT TCCATGTGAT AATGAGATTA 15660

ACGTGCCCAG TCACGGCGAC ACTCAGTAGG TGTTATTCCT GCTCTGCCAA CAGCAACCAT 15720

AGTTGATAAG AGCTGTTAGG GATTTTGTCC TTTTGCTTAG AATCCAAGGT TCAAGGACCT 15780

TGGTTATGTA GCTCCCTGTC ATGAACATCA TCTGAGCCTT TCCTGCCTAC TGATCATCCA 15840

CCCTGCCTTG AATGCTTCTA GTGACAGAGA GCTCACTACC AGGACTACTC CCTCCTTTCA 15900

TTTAGTAATC TGCCTCCTTC TTTTCTTGTC CCTGTCCTGT GTGTTAAGTC CTGGAGAAAA 15960

ATCTCATCTA TCCCTTTCAT TTGATTCTGC TCTTTGAGGG CAGGGGTTTT TGTTTCTTTG 16020 TTTGTTTTTT TAAGTGTTGG TTTTCCAAAG CCCTTGCTCC CCTCCTCAAT TGAAACTTCA 16080

AAGCCCTCAT TGGGATTGAA GGTCCTTAGG CTGGAAACAG AAGAGTCCTC CCCAACCTGT 16140

TCCCTGGCCT GGATGTGCTG TGCTGTGCCA GTATCCCCTG GAAGGTGCCA GGCATGTCTC 16200

CCCGGCTGCC AGGGGACACA TCTCTATCCT TCTCCAACCC CTGCCTTCAT GGCCCATGGA 16260

ACAGGAGTGC CATCGCCCTG TGTGCACCTA CTTCCATCAG TATTTCACCA GAGATCTGCA 16320

GGATCAAAGT GAATTCTCCA GGGATTGTGA AATGATGCGA TTGTGGTCAT GTTTAAAAGG 16380

GGOCAACTGT CTTCTAGAGA GTCCTGATGA AATGCTTCCA GAGGAAATGA GCTGATGGCT 16440

GGAATTTGCT TTAAAATCAT TCAAGGTGGA GCAGGTGGGG AAGGGTATGG ATGTGTAAGA 16500

GTTTGAAATT GTCCATCATA AAATGTGTAA AAAGCATGCT GGCCTATGTC AGCAGTCACA 16560

GCCTGGAGGT GGTAACAGAG TGCCAGTCAC TGATGCTCAA GCCTGGCACC TACAGTTGCT 16620

GGAAACCCAG AAGTTTCACG TTGAAAACAA CAGGACAGTG GAATCTCTGG CCCTGTCTTG 16680

AACACGTGGC AGATCTGCTA ACACTGATCT TGGTTGGCTG CCGTCAGCTT AGGTTGAGTG 16740

GCGGTCTTCC CTTAGTTTGC TTAGTCCCCG CTATTCCCTA TTGTCTTACC TCGGTCTATT 16800

TTGCTTATCA GTGGACCTCA CGAGGCACTC ATAGGCATTT GAGTCTATGT GTCCCTGTCC 16860

CACATCCTCT GTAAGGTGCA GAGAAGTCCA TGAGCAAGAT GGAGCACTTC TAGTGGGTCC 16920

AAGTCAGGGA CACTATTCAG CAATCTACAG TGCACAGGGC AGTTCCCCAA CAGAGAATTA 16980

CCTGGTCCTG AATGTCGGAT CTGGCCCCTT CCTTCCCCAC TGTATAATGT GAAAACCTCT 17040

ATGCTTTGTT CCCCTTGTCT GCAAAACAGG GATAATCCCA GAACTGAGTT GTCCATGTAA 17070

AGTGCTTAGA ACAGGGAGTG CTTGGCTTGG GGAGTGTCAC CTGCAGTCAT TCATTATGCC 17160

CAGACAGGAT GTTTCTTTAT AGAAACGTGG AGGCCAGTTA GAACGACTCA CCGCTTCTCA 17220

CCACTGCCCA TGTTTTGGTG TGTGTTTCAG GTCCACTTCA TCAACCCGGA AACGGTGCCC 17280

AAGCCCTGCT GTGCGCCCAC GCAGCTCAAT GCCATCTCCG TCCTCTACTT CGATGACAGC 17340

TCCAACGTCA TCCTGAAGAA ATACAGAAAC ATGGTGGTCC GGGCCTGTGG CTGCCACTAG 17400

CTCCTCCGAG AATTC 17415 (2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2298 base pairε

(B) TYPE: nucleic acid

(C) STRANDEDNESS: sing1e (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..2298

(D) OTHER INFORMATION: /note= "MOP1 UPSTREAM SEQUENCE"

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

TGCATAGGTC ACACATCCCT CCTCTACCCA AGGCTAGCCA GGTGCCCTAT CTCTCCCTTC 60

TTCGGTGCCT CCTCCGACTG GGCTCTGACG TTCTCAGAGA GAACGAAAGG GAAAGACTGC 120

TTGCTACCCT TCTGTTCCGG ACCTTACTGA AGGGCCTTAG TGTTTCCAGG GGCCCAAGAA 180

CCAGGTAGCC GTGGAGGTTG CCATGCTTGC CTGCCCACTC ACCCAACGTT CTCCTGCCTG 2 C

GCCTGTGTGT GGCACCCATG CAGGCCACAG AAGGCCACAC ACAGCCTTCA GGATGAGGCA 300

GGGCCCCTTT GTTTATTCAA TATCAAGAAC TGTAACGTGG TCACCGGAGG TCATGTCTCC 360

AGCTCGCAGC CTGCTTGGCC TCAGATCACC CACCACAGCA GGTCCAGGGA GGGGCCTCTC 420

AGGTCTGCAC TGGGCCAGGG ACTCAGTACT GGTGGGCATC CAAGGCCTGG GCTAAGACCT 480

GCAAGTTTCT TTTAGCCCCT CAGACAGTCA CATCACCTAA AATTCCTACC AAGGAGCCCT 540

GAGAGACCTA GGTAGTTATC TCTGTTCCAG GAAGCCTGAA AGACCAGGCT TCCCATCTCA 600

CCCTAGGACT TCAAGAGGGA CCCCCTACTC AAGGCCCTTC CCCAGCCCCT ACTTGCCATT 660

TTACCACCCC TGAAACGCTT GCTTGTCGCC CACCTTCAGC AAAGCAGGAA GCCTGGCTCA 720

CCATCCCCAC TCACTCACTG CCATTCTGGG TGAAGGCTGC TTTGCTCCCA TTTTTCAGAT 780

TAGGAAACGG AGGCTCCAAA GAGCAGCAAT CCACTGAGAG ACCCAGTATC TGTCTGGGAC 840

GTTTCCTCCT GGGAGGAGAG GGAGGCTAGT CCTTTGAGAC AGGAAAATCG AGTCGGGAGC 900

TCTTCTGAAC TTGGGTACCA ACTGCCTACT CCTCAGGCCC CTGACCTGGG GCTAGGGGTA 960

GGGGTTATTA GACAGTGAGG TACCAAAGGA CTCATGTCAG GACCCCGCCC CCCCAAGAGA 1020

GGAGGGGGTG GGAACATTCT CTAGTCCCAG ATTTCACTTA TGTACTCTGT AGAGCTGCAG 1080

CATCTGGGGT TTGAAGGCTT TGGGTTAAAA GATACTTGGG AAGGAAAAGC CGAGAAGTAC 1140

CTGGGCCCGG ATCCCTTGGG TGCTGGACTT GAGGGGAGGT GTGTGTGTGT GTGTGTGTGA 1200

GTGTGTGTGT ATGTATGTGT GTGTTGGGGG AGTGAAGTGT AGAAAGAACT TTATCTCCAC 1260 ATTATCTCTG CCCGTCCTGG AAGGTTCCCA GAGGAAGTGG CACCCGAGGG GGGAGGGGCA 1320

GGGAGAACGT TCCCCGAGGA ACAAAAGCCA GGATAGCAGA GGGGCAAGCG GTGGGGGTAC 1380

CGAGGGGGTT TTGCATGACT GGAGCAAATG GAGTGTTGGG GGGGGCGGTT CGAAAGATGA 1440

GCCAGGTCCA AGAGTGGCCA CCTCCGAGGA GCCTTCTCGG ATTCCTGCGC TCCCTCCTGG 1500

ATGCTTTCCT AGCACAGCCC TTAGTTGCTA CACTTTGGCC ACTTCCAAGT GCGAGTCCCG 1560

AGAGAGCTGG GCAGATTGGG ATTCTTCTCT CTGGGTCCCT GCGGCGTCTG TCCCAGTGCC 1620

GGACACCCGG TGGGCACTCG GTAAATATTT GTAGAGCGCC CTGGGAGGAA TGAATGAAGC 1680

CATTGGGCCA GGCTTGGGGA GGGCGGGGAC AGGCGCAGGT GGGAGGCAGC GGGAGCGGGA 1740

GGGGCGGGGA AGTCAGTCCT CCCGCTCCTC CCCCGCTCCC CGGCCCCAGC GCGCCCAACT 1800

CCGGGGCTCC CGAGGCGGCG GGCGGGCGAT CCGGGCGCGC AGGGCCCTTG TATTGGGCAC 1860

GCGGGAGATC GGAAAGGGGT TTGTTGCTGG TGCCCGCGGG CCTGAGCGCG ATCAGAGCGG 1920

GAGGAGGGAG CTAGGGTTCG CTCAGCGCCC AGCTGCCTCT CCGGCACTCG CTCTCCGGAC 1980

TGTAGGTCTG CAAGCTGCTG CTCCTCCCAC CCCGGCCCGC CTCCTCGCTC TCTTGCTCGC 2040

TCTCTGGAGT TGCTGTGCTA GCCTTGCCGT GCGTCCTGGC GAGTGCGGGC CGAGGGGCCC 2100

CGGGCCAGAA CTGAGTAAAG GACAGGGGCG TCCCGGGCAA AGCGCAGCCG GCCGGGGAGT 2160

GGCCATGTGT GGCGAGGCCG CCTTGAAGCT CGCCTGCAGC AAGTGACCTC GGGTCGTGGA 2220

CCGCTGCCCT GCCCCCTCCG CTGCCACCTG GGGCGGCGCG GGCCCGGTGC CCCGGATCGC 2280

GCGTAGAGCC GGCGCGATG 2299 (2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2997 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..2997

(D) OTHER INFORMATION: /note= "MOP1 TERMINAL SEQUENCE" (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:3 :

TAGCTCTTCC TGAGACCCTG ACCTTTGCGG GGCCACACCT TTCCAAATCT TCGATGTCTC 60

ACCATCTAAG TCTCTCACTG CCCACCTTGG CGAGGAGCCA ACAGACCAAC CTCTCCTGAG 120

CCTTCCCCTC ACCTCCCCAA CCGGAAGCAT GTAAGGGTTC CAGAAACCTG AGCGTGCAGG 180

CAGCTGATGA GCGCCCTTTC CTTCTGGCAC GTGACGGACA AGATCCTACC AGCTACCACA 240

GCAAACGCCT AAGAGCAGGA AAAATGTCTG CCAGGAAAGT GTCCATTGGC CACATGGCCC 300

CTGGCGCTCT GAGTCTTTGA GGAGTAATCG CAAGCCTCGT TCAGCTGCAG CAGAAGGAAG 360

GGCTTAGCCA GGGTGGGCGC TGGCGTCTGT GTTGAAGGAA AACCAAGCAG AAGCCACTGT 420

AATGATATGT CACAATAAAA CCCATGAATG AAAATGGTTA GGATACAGAT ATATTTTCCT 480

AAACAATTTA TCCCCGTTTC TTGGTTTATT CTGACTTTGT AAACAGAAAA GCCGGGGCTG 540

TGGAGGATGG AGAGGCCCCT CCTTTCCGTC TCGTCTCGTT GTGTGTGTTT ACCAGACCTG 600

CCCAAATCCA GCCTGTAGGG AGGAGGAGGA GGATGTCTGC TCAGAAGAGG CCAGTGAGGG 660

ATGTGGCCTC AAAGGGTGTT GGGATG AGA TGGAGGG.AGG TATGCATGCA CACACACACA 720

CACACACACA CACACACACA CATGCATGAT ACACACACAC ACACACACAC ACACACACGA 780

TGCACACACA CACACACACA CACACACACA CACACACGCA CGCACGCACG CACACGCACG 840

CATGCATGCA CACACACACG CACACACACA TCTGAAGCGC ATGTAGACTT TGGAATGGCT 900

CTGCCAGTCC CTCAGCCCCA ATTCCTGCCC CATGGTAGGA AATCCATGAG AAAAGCAAAG 960

CTAACAAGCA CAGCGGACCC TACCTGAGGA AGCACAGGGG ATGCAGGCTC TTCAGGACAC 1020

TGTCCTCCAA ACAAGGCCCC TCTGGCACCT CTGTGGCCGA GCTCCGGAGC CAGGTCCTGG 1080

CCTTCACAGC TGCCTCTCTT CACTCTCAAC CCTAACAGAA GGTTCTGCGA CAGATTGGTT 1.140

TCTGGATCTG AGGGAGATGG CAGAACAGGG TTGTACTGGC TTAGAAGGTT CAACCATGCT 1200

TCCTGCTTCA GAGGTGGGAT GTTGGTTATG GCTCAAACAA GGCCTCTCTG CCTGAGTTTG 1260

CAGAGCCCCA GCTGCCCCAA TGGTTCCTAG CTTCAAATGC AGAGGGTTAA ACTGGCTGCC 1320

AGTGTTTCCT GCATCCACAC AAAGAATGAG GTTAGCCAGG CAGGACCTAT GGCCATGTCG 1380

CATCTGGTCA GGTGGGGAAC CAATTCTTCA TGTCTGTGTC CCTGGAAACA CTGGGCTCTC 1440

TTCTGTTCTG TTTTAGTTTT TCTTCTTCAG TAGCTTGGGC TGCAGCTTCT ACTCTGCCCA 1500

TTCGATGTGG GGGAAGGCCA TTTCTTTTTG TAATTTGTTC TGTGTGTTTG CAGATCTGGG 1560 GCTTTTTGTG TGACTCCCCT GTGGTGCACA TTTTACTTTA GAGCCCTAGT CTGCCTGCAG 1620

TCGGTGTCTC TTATACGTTT AAATGTGTAA ATAGTTGTGA CAAGACAAAG AAATTATTTA 1680

TTTCCATCTG AAGCTCTTTC CAAAGGCTCC TCACAGAGAA CAATGAGGCC GACTTCCTTC 1740

AGTCTGTTTG TTTTCTTATT TAAGACTATT TATTAACAGT TGGACCGATG TACCCATAGC 1800

TGTCGAATAA AGTGGTCCTT AGTGAAAATT CTGTATAAAT AGAGTAAGAA GGGGTTTGAC 1860

TTTGCAATAA AAGGAGACAT TTGGTTCTGG TTGTCCGACC CATGTGTGTA TTTGTGTCTT 1920

TCCCCCTGAA CTCCTGGACA CTGGAGTCTC ATCGGCTGAG AACCCTCGAC CTTGATCTCG 1980

ACTGTTAACG GGATGTTTAT CATCCAGGCC CAGGGGAAGT CGGGCGCTCC TCAATATTTG 2040

GTGCAGCTGT GTGGGGCTCC CTGGGCGGGA GAGACGGAAC CAAACAACAA ATGTGAGTTT 2100

GGTAAGGCTG GATGGCAAAG AGTGCCTTTG ATTGAACTAC AGCCCAGCTG TCAGCAGCTG 2160

CTTCAAAGAG GCAGGGGGTA AATTAGCTGT GTTTACTGCT AACATAGTCG AAAGATTTAG 2220

TCATCCCAAT AAAATAGAGG CACAAGAGAG AAGAGGGGGG GGTGTATACC CCAAACTTGA 2280

AAGCCATGCT GGCCTCACAG CTGGCGTCAT TCAGTGCCCG TCACACCCGG GCAGTTGGGG 2340

GCTGCCCTCG CAGGCCAAGC TGTGGAGGTG GGCAGCCCAC CGCAGGCTGG AGAAGGGAGT 2400

GCCCCCCACC TCCCCGGCAA GCTCAGGGCA GTGCTCATCT GGCTACATCG GTCTTTGAAG 2460

TGCGCACGAA GGTCACCTGA CGGATGTTTC TAGAATCCCA GGCGATGCTT GGGACAGGCT 2520

GCTCTCTCTT CCCCTGTTGA CTCAGACCCA GCAACCCAGC CGTCCTAACA CATTCCAGCC 2580

CCTGCGATTT CTAAACCTTT CCTGTCACTG TCCCGACAAC TCAGCTTTTG TTCTGTTTTC 2640

CAGGCTGAAG CCCAGAGCCA CAAGCCGGAG GGTCCAGATG TGGCCTCTCA GATGTGTGCC 2700

TTAGCCTCTC AACCCCACCC CCACCCCCAA CCCCAGTGAT GTTTACACAT CTTAAAAAAC 2760

ACTAATCTGT TGCCAATATG TTTTTGCAAA TAAGGAGTTT GGGCTTCTCT TGAGCGGGCC 2820

ACCTGGTTCC TCCCTGTGTG CTGCTCCTAA CTGAACAGAG GTGCCAGGGC CGTTGTCACA 2880

CATACACACA CCCCCGCCAT GGCCTCATCC ACAAACGGTC GAGGTCAGCT GACATCTTCA 2940

AAATGGCTGA CGGATGTCTA CTTGTGCCCA CGACCCAAAA GGAATAGGAA AATGGAA 2997 (2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: CDNA

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..9

(D) OTHER INFORMATION: /note= "WT1/EGR CONSENSUS SEQUENCE"

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: GNGNGGGNG (2) INFORMATION FOR SEQ ID NO: 5 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..21

(D) OTHER INFORMATION: /note= "WT1/EGR HUMAN TCC BINDING SITE"

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5 : TCCTCCTCCT CCTCCTCCTC C 21

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..15

(D) OTHER INFORMATION: /note= "WT1/EGR MOUSE TCC BINDING SITE"

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: - 59 -

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..9

(D) OTHER INFORMATION: /note= "HUMAN FTZ BINDING SITE"

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: TCAAGGTCA

Claims

What is claimed is:

1. A vector comprising a DNA sequence defining a reporter gene in operative association with at least one OPl-specific non¬ coding sequence lying contiguous to the OP1 gene under naturally-occurring conditions and competent to affect expression of said reporter gene on said vector.

2. The vector of claim 1 wherein said non-coding sequence is capable of being acted on by a nucleic acid binding molecule, thereby to affect expresεion of εaid reporter gene.

3. The vector of claim 1 wherein εaid non-coding εequence iε selected from the group of DNA εequences defined by baεes 3170 to 3317 (Seq. ID No. 1); 3020 to 3317 (Seq. ID No. 1) 2790 to 3317 (Seq. ID No. 1); 2548 to 3317 (Seq. ID No. 1) 2150 to 2296 (Seq. ID No. 2); 2000 to 2296 (Seq. ID No. 2) 1788 to 2296 (Seq. ID No. 2); 1549 to 2296 (Seq. ID No. 2), including allelic, species and other sequence variants thereof.

4. The vector of claim 1 wherein said non-coding εequence iε εelected from the group of DNA sequences defined by baseε 2300 to 3317 (Seq. ID No. 1); 1300 to 3317 (Seq. ID No. 1); 1 to 3317 (Seq. ID No. 1); 2548 to 2790 (Seq. ID No. 1); 1549 to 2790 (Seq. ID No. 1), 1 to 2790 (Seq. ID No. 1); 800 to 2296 (Seq. ID No. 2); 1 to 2296 (Seq. ID No. 2); 1549 to 1788 (Seq. ID No. 2); 800 to 1788 (Seq. ID No. 2); 1 to 1788 (Seq. ID No. 2), including allelic, species and other sequence variantε thereof .

5. The vector of claim 1 wherein said non-coding sequence iε defined by part or all of Seq. ID No. 3 including allelic, species and other sequence variant thereof.

6. The vector of claim 1 wherein said non-coding sequence comprises part or all of an OP1 intron sequence.

7. The vector of claim 6 wherein said sequence defining part or all of an OPl intron is selected from the group of sequences consiεting of baεeε 3736 to 10700; bases 10897 to 11063; bases 11217 to 11424; bases 11623 to 13358; bases 13440 to 10548; bases 15166 to 17250; all of Seq. ID No. 1, including allelic, specieε and other εequence variantε thereof.

8. The vector of claim 1 wherein said vector comprises at least a second said non-coding sequence.

9. The vector of claim 8 wherein said second non-coding sequence is independently εelected from the group of εequences defined in claims 2, 3, 4, 5 or 7.

10. The vector of claim 1 wherein εaid non-coding εequence defineε at leaεt one Wt-l/Egr conεenεus binding element.

11. The vector of claim 1 wherein said non-coding sequence defines between one and six Wt-l/Egr binding elements.

12. The vector of claim 1 wherein said non-coding sequence defines at least part of an FTZ binding element.

13. The vector of claim 1 wherein said non-coding εequence defines a steroid binding element.

14. A cell tranεfected with a vector of any of claims 1, 3, 4, 5, 6, 10, 11 or 12.

15. The transfected cell of claim 14 wherein at least part of the DNA of said vector is operatively integrated into the cellular genome.

16. The transfected cell of claim 15 wherein said cell's genome has an OP-l gene locus and at least part of said transfected DNA is operatively integrated into εaid genome at εaid OP-l locuε .

17. The tranεfected cell of claim 14 wherein said cell expresεeε OPl under naturally-occurring conditionε.

18. The transfected cell of claim 17 wherein said cell is an epithelial cell.

19. The tranεfected cell of claim 17 wherein εaid cell iε of kidney, renal, urogenital, liver, bone, cardiac, lung, or nerve cell origin.

20. A cell comprising a transfected vector, said vector defining a reporter gene in operative association with at least two DNA sequenceε, the firεt said sequence comprising part or all of a sequence selected from the group consisting of bases 2548 to 2790 (Seq. ID No. 1); bases 2548 to 3317 (Seq. ID No. 1); baseε 1549 to 1788 (Seq. ID No. 2); baεes 1549 to 2296 (Seq. ID No. 2) , including allelic, species and other sequence variants thereof, and the second εaid εequence defining a sequence capable of being acted on by a DNA binding molecule and competent to affect expression of said reporter gene.

21. The cell of claim 20 wherein said second DNA sequence compriseε at least one Wt-l/Egr-1 consenεuε element (Seq. ID No. 4) .

22.. The cell of claim 21 wherein said second DNA sequence compriεeε between one and six v.'t-1/Egr-l consensuε elementε (Seq. ID No. 4) .

23. The cell of claim 21 wherein εaid second DNA sequence comprises at least six Wt-l/Egr-1 consensus elements (Seq. ID No. 4) .

24. The cell of claim 20 wherein said second DNA sequence is selected from the group of εequenceε consisting of a TCC element, an FTZ binding element and a steroid binding element.

25. The cell of claim 20 further comprising a third DNA sequence in operative association with said reporter gene and competent to affect expression of εaid gene, εaid third DNA εequence being independently selected from the group of sequences consisting of a TCC element, an FTZ binding element and a steroid binding element.

26. The cell of claim 20 further comprising a third DNA sequence in operative asεociation with said reporter gene and competent to affect expression of said gene, said third DNA sequence being independently selected from the group of sequenceε conεiεting of bases 3736 to 10700 (Seq. ID No. 1) ; bases 10897 to 11063 (Seq. ID No. 1) ; baseε 11217 to 11424 (Seq. ID No. 1) ; bases 11623 to 13358 (Seq. ID No. 1); bases 13440 to 10548 (Seq. ID No. 1) ; bases 15166 to 17250 (Seq. ID No. 1) , including allelic, specieε and other εequence variantε thereof .

27. A method for εcreening a candidate compound for the ability to modulate expreεsion of OP-l, said method comprising the stepε of :

(a) incubating a said candidate compound with a cell transfected with a vector comprising a DNA sequence defining a reporter gene in operative association with at least one OPl-specific non-coding εequence lying contiguous to the OPl gene under naturally-occurring conditions and competent to affect expresεion of εaid reporter gene on said vector;

(b) measuring the level of reporter gene expressed in said cell; and

(c) comparing said level with that of said reporter gene expresεed in said cell in the absence of said candidate compound, wherein an increase in reporter gene expresεion level iε indicative of said candidate's ability to increase OP-l expreεsion in vi vo, and a decreaεe in reporter gene expression level is indicative of the candidate'ε ability to inhibit OP-l expreεεion in vivo .

28. The method of claim 27 wherein εaid non-coding εequence iε capable of being acted on by a nucleic acid binding molecule, thereby to affect expreεsion of said reporter gene.

29. The method of claim 27 wherein said non-coding sequence is selected from the group of DNA sequenceε defined by bases 3170 to 3317 (Seq. ID No. 1); 3020 to 3317 (Seq. ID No. 1) 2790 .to 3317 (Seq. ID No. 1); 2548 to 3317 (Seq. ID No. 1) 2150 to 2296 (Seq. ID No. 2); 2000 to 2296 (Seq. ID No. 2) 1788 to 2296 (Seq. ID No. 2); 1549 to 2296 (Seq. ID No. 2), including allelic, species and other sequence variantε thereof .

30. The method of claim 27 wherein said non-coding sequence is selected from the group of DNA εequences defined by baεeε 230C to 3317 (Seq. ID No. 1) ; 1300 to 3317 (Seq. ID No. 1); 1 to 3317 (Seq. ID No. 1) ; 2548 to 2790 (Seq. ID No. 1) ; 1549 to 2790 (Seq. ID No. 1) , 1 to 2790 (Seq. ID No. 1); 800 to 2296 (Seq. ID No. 2) ; 1 to 2296 (Seq. ID No. 2) ; 1549 to 1788 (Seq. ID No. 2) ; 800 to 1788 (Seq. ID No. 2) ; 1 to 1788 (Seq. ID No. 2) , including allelic, εpecies and other εequence variantε thereof.

31.^' The method of claim 27 wherein said non-coding εequence iε εelected from the group of εequences defined by claims 5, 6 or 7.

32. A method for εcreening a candidate compound for the ability to modulate expression of OP-l, said method compriεing the εteps of :

(a) incubating a εaid candidate compound with a cell according to claim 20, 21, 24 or 25;

(b) measuring the level of reporter gene expressed in εaid cell; and

(c) comparing said level with that of said reporter gene expressed in said cell in the absence of said candidate compound, wherein an increase in reporter gene expression level is indicative of said candidate'ε ability to increase OP-lexpression in vi vo, and a decrease in reporter gene expresεion level is indicative of the candidate'ε ability to inhibit OP-l expreεεion in vi vo .

33. A compound that iε identified by the method of claim 27 or 32.

34. A εubstantially pure nucleic acid comprising a DNA sequence defined by bases 1 to 1871 of Seq. ID No. 2, including allelic, species and other sequence variantε thereof.

35. A subεtantially pure nucleic acid comprising a DNA εequence defined by baεeε 1 to 2997 of Seq. ID No. 3, including allelic, εpecieε and other εequence variants thereof.

36. The vector of claim 1, 3, 4, 5 or 7 further comprising part or all of a nucleotide εequence encoding an OPl pro protein in operative aεεociation with εaid reporter gene.

37. The method of claim 27 wherein εaid vector further compriεes part or all of a nucleotide εequence encoding an OPl pro protein in operative aεsociation with said reporter gene.

38. A method for producing a candidate compound having the ability to modulate OP-l expresεion in a cell, the method compriεing the εteps of:

(a) obtaining, by the method of claim 27, a candidate compound, and

(b) producing either said candidate compound, or a derivative thereof having substantially the same OP-l expression modulating ability as said candidate.

39. The method of claim 38 wherein said candidate compound, or derivative thereof, produced in step (b) is by recombinant DNA techniques, or by nonbiological peptide synthesiε.

40. A kit for identifying a candidate molecule capable of modulating OP-l expreεεion in a cell, the kit compriεing: (a) a receptacle adapted to receive a sample, εaid sample containing a vector encoding a DNA sequence compriεing a reporter gene in operative association with at least one OP-1-specific non-coding sequence lying contiguous to the OP-l gene under naturally-occuring conditions and competent to affect expression of εaid reporter gene, wherein said vector is carried in a cell,

(b) means for detecting expression of said reporter gene following exposure of a said candidate compound to said sample.

41. The kit of claim 40 wherein εaid reporter gene comprises an OP-l DNA sequence.