CA2191583C - 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, usin g one or more OP-1-specific, noncoding nucleotide sequences and a suitable reporter gene.

Description

Methods and Compositions for Modulating morphoQenic Protein Expression 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 morphogens are competent for inducing all of the following biological functions in a morphogenically permissive environment: (11 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 mesechymal 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 morphoganesis o! non-chondrogenic tissue, including dentin, liver, and nerve tissuo.
A particularly useful tissue morphogenic protein is human OP-1 (Ostooganic Protein-1), described in U.S. 5,011,691; US Pat. No.
5.266,683 and Ozkaynak et al. (1990) EMHO J. 9: 2085-2093.
Species homologues identified to date include mouse OP-1 (set US
Pat. 5.266,683) and the Drosophila homologue 60A, described in Wharton Qt 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); HMPS, BMP6 (Celeste et al.
(1991) PNAS 87:9843-9847) and Vgr-1 (Lyons et al. (1989).
I: previously has been contemplate,: that these tissue morphogens can be administered to an animal to regenerate lost or daa~age~ tissue. Alternativel~~, one ca.~. e.~.v~ision ad.;~inistering a molecule capable of modulating e:cpression of the endogenous tissue morphogen as a means for pro~~id:.~.g morphogen to a site .r, vivo.
It is an object of this invention to provide compositions and 2~ methods of screening co~:pounds v:r:ch ca-: modulate erpression of an endogenous tissue morphogen, particular l~~ 0: -1 ar.~ closel}~ related genes. The compounds thus identified have utilit~~ both ir, vitro and .r. vivo. Useful compounds contemplated include those capable of stimulating transcription and/or translation of the OP-1 gene, as well as compounds capable o'_ inhibiting transcription and/or translation of the 0P-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 Dt~~ sequence encoding, in operative association v:ith reporter gene, a portion of an OP-1 ~~~~ i8:3 WO 95/33831 PCTlt1S95107349 non-coding DflA sequence that is competent to acC 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 1~ of determining a change in the level of a reporter gene product expressed by a cell follo:,ing e>:posure to one or more compoundts).
The level of an expressed reporter gene product in a given cell culture, or a change in that level resulting frcm exposure to one or more cempound(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 t;:e level of reporter gene.expression is indicative of a candidate compound's ability to increase CF-1 expression _i r.
viva. similarly, a decrease in the level of reporter gene 2(1 expression a indicative of a candidate compound's ability to decrease or otherwise interfere :,~ith CP-1 expression :n v:vo.
The methods and compositions of the invention can be used to identify compounds showing promise as therapeutics for various ir.
v=ve and e;: v_vc mamma2ian applications, as well as to identify coc~pounds having numerous utilities. For example, morphogen expression inducing compounds can be used .viva to correct o:
alleviate a diseased condition, to regenerate lost or damaged tissue, to induce cell proliferation and differentiation, andlor to maintain cell and tissue viability and/or a differentiated phenotype in viva or ex viva. The compounds also can be used to maintain the viability of, and the differentiated phenotype of.
cells in culture. The various in viva, ex viva, and in vitro utilities and applications of the morphogenic proteins described herein are well documented in the art. see, for example, US
92/01968 (4:0 94/03200), filed March 11, 1992; US 92/07358 (Wo 93/04692), filed August 2&; PCT US 92/0743 (470 93/05751), filed August 28, 1992; US 93/07321 (v:C 94/03200?, filed July 29, 1993;
SUBSTITUTE SHEET {RULE 26) US 93/08808 (WO 94/06449), filed September 16, 1993; US93/08885 (W094/06420), 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 vitro and in vivo to more closely regulate the production and/or available concentration of morphogen.
An embodiment of the invention is an isolated nucleic acid comprising a reporter gene in operative association with a single nucleic acid fragment of an OP-1 specific upstream non-coding sequence, wherein said nucleic acid fragment consists of nucleotides 3170 to 3317, 3020 to 3317, 2790 to 3317, 2548 to 3317, 2300 to 3317, 1300 to 1S 3317, 2548 to 2790, 1549 to 2790, or 1 to 2790 of SEQ ID NO:1, wherein said isolated nucleic acid comprises not more than one nucleic acid fragment of said OP-1 upstream sequence, and, wherein said nucleic acid fragment is operative to regulate expression of said reporter gene.
A further embodiment of the invention is a cell transfected with an isolated nucleic acid, said nucleic acid comprising a reporter gene in operative association with a first DNA sequence, wherein said first DNA sequence is: (a) a single nucleic acid fragment of an OP-1 specific upstream non-coding sequence, wherein said nucleic acid fragment 2$ consists of nucleotides 2548 to 3317 or 2548 to 2790 of SEQ ID NO:1; or (b) a nucleic acid fragment of an OP-1 specific upstream non-coding sequence, wherein said nucleic acid fragment consists of nucleotides 1549 to 2297, or 1549 to 1788 of SEQ ID N0:2; or (c) a variant of a nucleic acid fragment of (b) which hybridizes with a nucleic acid complementary to the nucleic acid fragment of (b) under conditions of hybridization in 40~ formamide, 5 x SSPE, 5 x Denhardt's solution, and 0.1~ SDS at 37°C, followed by washing in 0.1 x SSPE, and 0.1~ SDS at 50°C; and a second DNA sequence comprising a sequence which interacts with a DNA binding molecule and affects expression of said reporter gene, wherein said isolated nucleic acid comprises not more than one nucleic acid fragment of (a).

-4a-A further embodiment of the invention provides a method for screening a candidate compound for the ability to modulate expression of OP-1, said method comprising the steps of: (a) incubating a said candidate compound with a cell transfected with an isolated nucleic $ acid as noted above; (b) measuring the level of said reporter gene expressed in said 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's ability to increase OP-1 expression in vivo, and a decrease in reporter gene expression level is indicative of the candidate's ability to inhibit OP-1 expression in vivo.
A further embodiment of the invention provides a method for screening a candidate compound for the ability to modulate expression of OP-1, said method comprising the steps of: (a) incubating a said candidate compound with a cell as noted above; (b) measuring the level of reporter gene expressed in said 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's ability to increase OP-1 expression in vivo, and a decrease in reporter gene expression level is indicative of the candidate's ability to inhibit OP-1 expression in vivo.
A further embodiment of the invention provides a method for producing a candidate compound having the ability to modulate OP-1 expression in a cell, the method comprising the steps of (a) obtaining by the method noted above, a candidate compound, and (b) producing either said candidate compound, or a derivative thereof, having substantially the same OP-1 expression modulating ability as said candidate.
A further embodiment of the invention provides a kit for identifying a candidate molecule capable of modulating OP-1 expression in a cell, the kit comprising: (a) a receptacle containing an isolated nucleic acid as noted above; and (b) means for detecting expression of -4b-said reporter gene following exposure of a said candidate molecule to a cell containing said nucleic acid.
A further embodiment of the invention provides an isolated nucleic acid comprising a reporter gene in operative association with:
(a) a nucleic acid fragment of an OP-1 specific upstream non-coding sequence, wherein said nucleic acid fragment consists of nucleotides 2151 to 2297, 2001 to 2297, 1788 to 2297, 1549 to 2297, 800 to 2297, 1 to 2297, 1549 to 1788, 800 to 1788, or 1 to 1788 of SEQ ID N0:2; or (b) a variant of a nucleic acid fragment of (b) which hybridizes with a nucleic acid complementary to the nucleic acid fragment of (b) under conditions of hybridization in 40~ formamide, 5 x SSPE, 5 x Denhardt's solution, and 0.1~ SDS at 37°C, followed by washing in 0.1 x SSPE, and 0.1~ SDS at 50°C, wherein the nucleic acid fragment of (a) or (b) is operative to regulate expression of said reporter gene.
A further embodiment of the invention provides a kit for identifying a candidate molecule capable of modulating OP-1 expression in a cell, the kit comprising: (a) a receptacle containing an isolated nucleic acid as noted above; and (b) means for detecting expression of said reporter gene following exposure of a said candidate molecule to a cell containing said nucleic acid.
A further embodiment of the invention provides a method for screening a candidate compound for the ability to modulate expression of OP-1, said method comprising the steps of: (a) incubating a said candidate compound with a cell transfected with an isolated nucleic acid as noted above; (b) measuring the level of said reporter gene expressed in said 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's ability to increase OP-1 expression in vivo, and a decrease in reporter gene expression level is indicative of the candidate's ability to inhibit OP-1 expression in vivo.
List of Useful Terms and Definitions -4c-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, 1$ 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, 20 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 25 the coding region of the gene. As used herein, an "OP1-specfic" non-coding sequence is understood to define a non-coding sequence that lies contigous to OP1 specifically coding sequence at an OP-1 gene locus under naturally-occurring CA 02191583 1999-06-09 _ . - 5-conditions. 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 o! 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 r:ithin the scope of the disclosure provided herein.
As used herein, a 'tit-1/~Egr-1 consensus binding sequence' or V:t-1/Egr-1 consensus binding element- is a nine base sequence which has been shown to be bound b~~ the Dt1=. bin3ing proteins 4a-1 and Egr-1. The consensus sequence of the c~:t-1!Egr-1 bindinc S1LE
has been determined by homolooy to ba GIt:.N3GGNG, Seq. ID No. <
(Rauscher et al., Science 250:1259-1262 (1990).
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 o-- non-contiguous repeats of the DNA sequence TCC. The TCC binding sequence identified in human OP-1 genomic DN:, is shown in Seq. ID No. 5, and the TCC
binding sequence identified in a~urine OP-1 genomic Dtdh is shown in Seq. ID No. 6. The TCC binding sequence has also been shokn to be bound by the DNA binding proteins kt-1 and Egr-1 (A~ang et al., Proc. Natl. Acad. Sci. 90:8896-8900 (1993); Hang et al., Biochem Biophys Res. Com~n., 188:633-439 (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 superfamily, is YCAAGGYCR.

W095/33R3t ~ PCTIUS95/~17349 As used herein, a "steroid binding sequence" or "steroid binding element" is a DNA sequence that has been shown to be bound by one or more elements, in response toactivating signal molecules. Examples 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, R"f-1 and Egr-1. Activating signal molecules of the nuclear receptor family have recently been shown to bind to DCdA as homadimers, heterodimers or as monomers (Parker, M.G., 1~ Curr. Op. Cell Biol.., 1993, 5:499-504y. The formation of heterodimers among the nuclear receptor family molecules 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 IS sites. 1.n 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 ttue nuclear receptors and the nuclear receptors and accessory factors, indicate that there could be 2U significant number of nuclear raceptarlaccessory factor interactions o:hich have widely different transcriptional activities.
Y;hi.l.e the method of the invention is described with reference to a single cell, as will be apprec~.aced by those having ordinary ZS skill in the art, this is only for ease of das~~:-iption, and the method is melt efficiently carried out using a plurality of cells.
4.ith respect to transfection of DtIA sequences in the call and the method of Lhe invention, all means for introducing nucleic acids into a cell are contemplated including, without limitation, ~ CaPO4 co-precipitation, electroporation, DEAE-dextran mediated uptake, protoplast fusion, microinjection and lipofusian. A key to the invention is the DC;A sequences with which the cell is transfected, rather than the mechanical or chemical process by which the DNA incorporation is accomplished.
3J Useful reporter genes are characterized as being easy Le transtect 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 SUBSTITUTE SHEET (RULE 26) 2~g~.~8:3 1~ W095133831 PCT/US951Q7349 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 assayed, 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 1~ are only a few of the possible reporter genes, and it is only for ease of description that aI1 available reporter genes are not listed.
While the method, vectors, and cells described recite the use of a reporter gene in operati~.~e association with an CP-1 non-coding DNA sequence, it will be apparent to those of ordinary skill in the art that the Dtls sequence OP-1, including human OPI, shown in Seq. ID No. 1 or murine OP-1, disclosed in ti. 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 2(.l assaying for the presence of the reporter mt2tdA or reporter gene product.
47here a cell line is to be established, particularly where the transfected DNA is to be incorporated into the cell s genome, lines that can be immortalized are especially desirable. As used 2S herein, "immortalized" cell lines are viable for multiple passages (e. g., greater than 50 generations) without significant reduction in growth rate ar protein production.
Y:hile the selected non-coding DNA sequences disclosed herein are described using defined bases, as will be appreciated by those 3() 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 CP-1 non-coding DtdA sequence and other fusion DNA's can be used in a vector according to the 35 invention, and car. be transfected into a cell, or used in the method of the invention for screening a candidate compound for its ability to modulate OP-1 expression. Specifically, it is standard procedure far molecular biologists to first identify useful SUBSTITUTESHEET(RULE28) ~~~~83 WO 95/33831 PCT/US95l07349 _ g_ regulatory sequences, and then to determine the minimum sequence required, by systematic digestion and mutagenesis e.g., by exonuclease or endonuclease digestion, sitedirected mutagenesis and the like. Accordingly, sutsaquant, standard routine S experimentation is anticipated to identify~minimum sequences and these, shorter sequences are contamplztad by the invention disclosed herein.
Useful cell types for the method and compositions according to the invention include any eukaryctic cell. Currently preferred lfl are cell types known to express OP-1. Such cells include epithelial cells and cells of uro-genital cell origin., including renal (kidney an bladder) calls, as well as liver, bone, nerve, ovary, cardiac muscles and the like. The calls may be derived from tissue or cultured from an established call line. sae, for example 15 Ozkaynak et al. (1951) Biochem. BioPhvs. Res. Cetrz~. 179:116-123 for a detailed dasrription of tissues known to express OP-i.
Other useful cells include chose known to exhibit a steroid receptor, including cells having an estrogen receptor and cells responsive to the F'TZ-F1 protein. Currently preferred cells also 2U have simple media component requirements. Other useful representative cells include, but are not limited to, Chinese hamster ovary (CHO:; canine kidney (hfDCK); or rat bladder (NBT-2), and the like. Useful cell types can be obtained from the American 'P.~pe Culture Collection (ATCC), Rockville, N.D or from the European 25 Collection of Anirtuzl Call Cultures, Portion Down, Salisbury SP40JG, U.F;. As used herein, "derived" means the calls are from the cultured tissue itself, or are a cell line whose parent cells zre ef the tissue itself.
30 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. Tk:e OP-1 non-coding sequence chosen is indapendentlp~ selected from the 5' (or "upstream") non-coding 3~ humor. or marine OP-1 sequence shown in Seq. ID Nos. 1 and 2, respectively, the 3' (or "dc,.mstream") non-coding human or marine 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 SUBSTITUTE SHEET (RULE 28) 21~~.58 W~ 95133831 PCTIUS95107349 anticipated to be useful are the non-coding sequences (e.g., 5', 3' and intron) of other species hamologs of OP-1 and proteins closely related to OP-1. In addition, the portion of OP-1 sequence included 1r. 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 OPI-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 Xb I~ of 5' non-coding sequence. In Seq. ID No. 1, the start codon begins at position 3318, and the upstream sequence (bases 1 to 3317) is composed of untranscribed (1 to 2790) and untranslated 12791 to 3317) OP1-specific DfI:,; appra>:imately 1 F;b of which is presented in Fig. 1 (bottom strand).
IS Useful sequence segments include bases 2548-3317, representing 750 bases sharing significant (greater than 70~ identity) between the mouse and human OP-.I hamalags (See Fig. I), and bases 3170-3317; 3020-3317; 2796-3317; 2548-27,0 of Seq. ID Na. 1, all shorter fragments of this region o. tha DL1.'-... As base 2790 is the 2L) m.NA start site, other useful sequences include 2790-3317, representing transcribed but not translated 5' coding sequence znd shorter fragments of this DNA region as noted above; upstream fragments of OP1-specific DL;A, bases 2548-2790; 1549-2790; I-2790 of Seq. ID Ldo. 1. Also useful sequence segments include the 25 approximately 750 bases that have homology between the human and mouse OP-I sequences with additional upstream sequences, 2300 to 3317,; 1300 to 3317; 1-3317; all fragments of the disclosed upstream OP1-specific DLdA sequences of Seq. ID LIO. 1.
In another embodiment, the sequences are defined by the non-3~ coding sequences of the mouse OP-1 homolog, including the following 5' non-coding sequences (Seq. ID No. 2): 215C-2296, 2000-2296, 1788-2296, and 1549-2296 all of which define the 750 bases sharing high sequence identity with the human homolog (See, Fig. 1); 800-2296; 1-2296; 1549-1788, 800-1788 and 1-1788.
3$ tiithin this region also exist a number Egr/wt-1 sites (8 in hOP-1; r in mOP-1), known in the art to bind tt;e regulatory elements Egr and tat-1. Accordingly, in another aspect, the invention contemplates a screening material for identifying SUBSTITUTE SHEET (RULE 26) W09g133831 ~ ~ ~ ~ -~ ~ ~ PCTlUS951073a9 compounds which modulate OP-I expression, the assay comprising the step of identifying compounds which bind EgrlN~t-1 site. At least.
onek'tJEgr-1 element, preferably between 1-6 elements, or at least 6 tdt/Egr-1 elements are included in a sequence. The relative S locations of these elements are indicated'in Fig. 1 and at positions 3192-32DD; 3143-3151; 3027-3035; 2956-2964; 2732-2740;
2697-2704 of Seq. ID No. 1. and positions 2063-2D11; 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 substantially the same Seq.
alignment. 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 marine and humor.
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 marine and human OP-1 DNA sequences, bases 2548-2790 or 2548-3311 of Seq. ID Na,. I, or bases 1549 t.a 1788 or 1549 to 2296 of Seq. SD No. 2, and/or at least one of an tit-I!Egr-1 consensus binding sequence. In still another aspect the potion of OF-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 intrcn sequence. The relative positions of the TCC and FTZ
elements are indicated in Fig. 1 and at positions 2758-277& ifiCC);
2432-2441 (FTZ) of Seq. ID No. 1 and 1755-1769 (TCC) of Saq. ID
2S C:o. 2.
In another aspect, the invention features a cell that has been transfected urith a reporter gene in operative association with a portion of OP-1 non-coding DC,'=. secuence. The portion of OP-1 nan-coding sequence is independently selected Pram the 5' (or 3U upstream) non-coding human or marine GP-i sequence shown in Seq.
' ID Nos. 1 and 2, the 3' (or downstream) non-coding marine OP-1 sequence shaven in Seq. ID No. 3, and the human intron non-coding GP-1 sequence shau:n in Seq. ID No. 1. The six human intron non-coding OP-1 sequences are at bases 3736 to 10760; bases 10897 to IID63; bases 11217 to 11424; uses II623 to 13358; bases 13440 to 10548; bases 15166 to 17250; all of Seq. ID No. 1. In addition the portion of OP-i sequence selected can be a combination of 5' non-coding, 3' non-coding and/or intron OF-1 sequence. Thus, the cell can have been transfected with a reporter gene in operative SUBSTITUTE SHEET (RULE 2fi) 21~1~83 association with a portion cf 5' non-coding OP-1 genomic sequence that is independently selected from bases 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 bases 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 cf Seq. ID No. 2. The lengths of bases within these 5' non-coding sequences is selected to include portions of the sequence of DtdA which was determined to be homologous between marine and human genomic OP-1, separately and as a parC of a larger sequence including non-homologous DCdA. Additionally, the portion of OP-1 sequence selected can be a portion of the region of homology between marine and human OP-1 DNA sequences, such as bases 2548-2790 or 2548-3317 cfSeq. ID No. 1, or bases 1549 to 1.788 or 1549 to 2296 of Seq. 1D No. 2, and at least one of an nt-1lEgr-1 consensus binding sequence, a TCC binding sequence, a FTZ
binding sequence, a steroid binding sequence, and an intron. Thus the portion of OP-1 sequence selected can be a portion of the 5' non-coding human or marine OP-1 genomic DNA sequences, as stated 2~ above, and at least one ia-1lEgr-1 consensus binding sequence alone or in combination with at least one of a TCC binding sequence, a FTZ binding sequence, a steroid binding sequence, and a human OP-1 intron DN?. sequence. In another embodiment more than one 4a-lJEgr-1 element is use3, for example, between 1-6, or at least six. Thess cells are suitable for use in the method of the invention.
In one embodiment, part of the OP-1 coding region is anticipated to have an expression regulatcry function and also can be added to a vector for use in the screening assay described herein. OP-1 protein is translated as a precursor polypeptide having an N-terminal signal peptide sequence (the "pre pro"
region) which is typicallp° less than about 30 amino acid residues, 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 tc yield the mature protein sequence.
The mature sequence comprises both a conserved C-terminal seven cysteine domain and an N-terminal sequence ~.:hich varies significantly in sequence between the various morphogens. The SUBSTITUTE SHEET (RULE 26) mature 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 associates noncovalently with the mature dimeric protein, presumably to enhance solubility and/or targeting properties of the mature species. See, for example, Canadian Patent Application Serial Number 2,141,555. 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 structure (e.g., as part of the mRNA transcript) which itself may modulate OP-1 expression.
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 sequences for transfection are described below, as well as suitable cells containing transfected DNA sequences.
In another aspect, the invention provides molecules, vectors, methods and kits useful in the design and/or identification of OP-1 expression modulating compounds. As used herein a "kit" comprises a cell transfected with a DNA sequence comprising a reporter gene in operative association with a portion of OP-1 upstream DNA sequence and the reagents necessary 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-based or can be composed in part or whole of non-proteinaceous synthetic organic molecules.

The OP-1 expression modulating compounds thus identified then can be produced in reasonable quantities using standard recombinant expression or chemical synthesis technology well known and characterized in the art and/or as described herein. For example, automated means for the chemical synthesis 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 production of OP-1 in a kidney cell line is a candidate for systemic administration. The candidate can be assayed in an animal model to determine the candidate molecule's efficacy in vivo. For example, the ability of a compound to upregulate levels of circulating OP-1 in vivo can be used to correct bone metabolism diseases such as osteoporosis (See, for example, Canadian Patent Application Serial Number 2,116,559). Useful in vivo animal models for systemic administration are disclosed in the art and below.
As demonstrated herein below, OP-1 is differentially expressed in different cell types. Accordingly, it further is anticipated that a candidate compound will have utility as an inducer of OP-1 expression in one cell type but not in another. Thus, the invention further contemplates testing a candidate compound for its utility in modulating expression of OP-1 in different cells in vivo, including different cells known to express OP-1 under native physiological conditions.
Thus, 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 assay for compounds which modulate the expression of OP-1. These identified compounds can be used to modulate OP-1 production and its available concentrations in both in vivo and in vitro contexts.
Brief Description of the Drawings WO 95f33831 ~ PCTlUS95107349 Fig. 1 shows the alignment of upstream sequences of the murine and human OP-1 gene. The murine sequence is present in the upper sequence lines and the hu.~nan sequence is the lawer sequence on all lines. The murine sequence is numbered backwards, counting back S from the first ATG of the translated sequence which is shown highlighted. For purposes of alignment, dashes are introduced into the DNA sequence, and three portions of human DNA sequence have been cut from the sequence and placed underneath a gap, belaw a solid triangle;
la Fig. 2 shows a time course of murine uterus CP-1 mRNA
regulation by estrcgen; and Fig. 3a shawl a schematic of the 2 kb and s kb OP-1 mRNAS, the hybridisation locations of pzobes 1 through 7 (indicated by the bars under the schematic;. The solid line indicates OP-1 mRN:,, 1S the " indicate potential poly A signals, the boxes indicate the translated portion of CP-1 mFtP7:-, with the hatched box shoi,~ing the TuF-(3 -like domain. The dashsd lines indicate genomic DNA
sequences. The arrows mark the locations ef the cleavage site for GP-1 maturation.
20 Fig. ?b. shows a Northern blot hyb_idizac cn analysts e( 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 era indicated by the 4- and 2-en the left side of Fig. 3b, and a 4.24 to 9.a9 kb RtdA size ladder is -indicated bv_ 25 dashes to the right cf the figure.
Detailed Description As will be more fully described below, we have identified regions in the OP1 genetic sequence useful in identifying 3~ 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 status of the cell in a tissue. Specifically, as described herein below, OP-1 protein expression is differentially regulated in uteriae tissue 35 depending on th.e status of the uterine tissue. For example, OP-1 erpression is dramatically down-regulated in uterine manse tissue during pregnancy, whereas it is normally expressed in this tissue in virgin mice. Moreover, OP-1 expression in other tissues such SUBSTITUTE SHEET (RULE 26) as renal tissue apparently is unaffected during pregnancy.
Administration 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 nor.-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 1~ having utility in a method for the screening of compounds for their ability to modulate GP-1 expression.
In view of this disclosure and the examples provided below, a method for identifying molecules which can affect OP-1 e~;pression LS in a particular cell type in vivo nc~.r is provided.
Cloning of Hunan and Nouse GP-1 Gene Non-coding Seauences In the Northern blot analysis of murine organs multiple OP-1 transcripts, are detected namely, three species of 1.8, 2.2, 2.4 20 kb and a prominent 4.0 kb RNA species (OZkaynak et ai., 1992, _J.
Biol. Chem., 267:25220-25227; Ozkaynak et al; Biochsm. Biophvs Res. Comm., _179:116-123). The pattern is similar in rats with only the 1.8 kb species absent. The estrogen-mediated dow:lregulation of OP-1 mRtlA affects all of these species. In 25 order to prove that the 4.0 kb mRfl.~, is in fact a transcript from the same OP-1 locus, cCNF. clones were isolated from a mouse teratocarcinoma cDtdA library.
Four independent clones were obtained that added sequence information to the publishe3 mouse cDNA sequence. Two of these 3~ cDNA clones have longer 5'-untranslated sequences (G.4 and 0.3 kb) than previously reported i0.1 kb). Three of the murine clones contain additional 1.4 kb at the 3'-end. The combined sequences add up to a total OP-1 cDNA size of 3.5 kb, about 0.5 kb shorter than the 4.0 kb mRNA observed on Northern blots. cDNA clones that 35 represent the 2 kb and 4 kb messages are shown schematically in Figure 3a. Since the polyA-tail is lacking in those cDNA clones that extend the 3'-information, it was anticipated that missing 0.5 kb sequence occurs at the 3'-end.
SUBSTITUTE SHEET (RULE 26) WO 95133831 ~ ~ ~ ~ ~ ~ ~ PCT/U595I07349 In order to obtain the sequence immediately adjacent to the 3'-end of the 3.5 kb cD2aA sequence, a mouse genomic library, ML1039J (Clontech), was screened with a.:.3'-end cDNA specific probe (0.45 kb, 3'-end XmnI-EcoRI fragment-of marine DP-1 cDNA) S according to the parameters described~beiow for the cloning of upstream non-coding sequences. This screen yielded four lambda clones which were analyzed by southern blotting. All clones yielded a I.5 kb XmnI fragment which was subcloned from lambda X71 into a Hluescript vector and sequenced. Three polyadenylation signals (AATAAA) (Proudfoot et al, (1976) Nature, 263:211-214) were found in this genomic fragment, at 3.52-, 3.58-, and 3.59 kb (shown schematically in Fig. 3a by the ',). The 3'-end ct7NA and the genomic DNA sequences in the 1.5 kb Xmn2 fragment overlap by 0.4 kb in a region that immediately precedes the second 1S polyadenylation signal located at 3.5 kb (Figure 3a, region indicated by probe 6j and are in complete agreement within this stretch.
Humor. upstream non-ceding sequence and additional mouse upstream non-coding sequence were obtained b,= screening hu:aan and 2~ mouse genomic libraries, HL1G67J and h.:.1G30J respectively (Clontech). All libraries u:ere screened by an initial plating of 750,000 plaques (approximately 50,000 ;~laques/pl.ate).
Hybridizations were Bane in 408 farma>nide, 5 x SSPE, 5 x Denhardt's solution, and 0.1% SDS at 37°C. Nonspecific courts 25 were removed in 0.1 x SSPE, G.1 % SDS by shaking at 50°C. Human and mouse upstream genomic D2~7A sequences were obtained from clones lambda 03 and lambda 033, respectively (Clontech, HLIt~~'J and i;L1030J). These lambda clones were isolated using a '"?-labeled probe made from a human 0.47 kb EcoRI OP-1 cDfQA fragment (obtained 3~ from p0115) containing mainly 5' non-coding and exon 1 sequences.
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 fcr marine was obtained by subcloning a 1.1 kb PstI fragment from the genomia phage clone 35 lambda 1533. This fragment overlaps »~ith the 5'-end of the longest marine eDt9A clone by 0.3 kb in the 5' non-coding region and provided 0.8 kb additional sequence information. A schematic diagram of the 2- and 4 kb OP-i messages is shown in Figure 3a SU6STITUTE SHEET (RULE 26) ~~.~~. i83 ~ W095/33831 PC't/US9i107349 with dashed lines indicating supplementing information derived from murine upstream and docanstream genomic DNA.
-All sequencing was done according to Sanger et al. (1977) Proc. Natl. Acad. Sci. _74:5463-5467, using exonuclease III-mediated unidirectional deletion (bzkaynak et al., (1987) BioTechniques, 5:770-773), subcloning of restriction fragments, and synthetic primers. Compressians were resolved by performing the reactions at 70°C with Taq polymerase and using 7-deaz>-GTP
(U. S. Biochemical Corp., Cleveland, OH).
Verification of OP-1 mRtQA Seavences by Northern Blotting To verify the structures of the short and long mRP7A species observed, Northern blot hybridizations 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 prctein coding sequence, and genomic regions upstream or downstream of the predicted mRN."-.s, respectively.
Hybridization of these probes to individual Northern blot strips containing mouse kidney mRDi.=: is consistent with the 2~ predicted 4 kb mRNA structure. As shas:r: in Fig. 3a, and Fig. 3b, the genomic DN=. probes 1 and 2 did not hybridize to any message.
Probe 2 is specific to the upstream sequences 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. Frobe 6, specific to sequences between the first and second polyadenylation signals, hybridized only to the 4 kb message. Finally, probe ' which is specific to sequences further dou:nstream of the fourth (last) palyadenylation signal, did not hybridize to any message. The results obtained with these probes confirm the two OP-1 mRNA.structures and the approximate 6'- and 3'-end boundaries of OP-1 transcripts shown in Figure 3a.
This demonstrates that the 2 kb and 4 kb mF.NA's are from the same OP-1 genomic locus rather than from multiple genes.
The extensive 3' sequence included in the 4 kb mRNA transcript suggests that the 3' untranslated sequence may' play a role in OP-1 gene expression particularly as it has been detected across species namely, in mouse, rat, dog, human and chicken. Multiple SUBSTITUTE SHEET (RULE 26) - 18' stop codons in all three possible translation reading frames rule out the likelihood that this sequence encodes a peptide. The untranslated sequence itself may act therefoze to influence mRNA
stability.. For example, the sequence may interact with another S 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., 4:2157-2168).
Alternatively, the 3'-end sequences may be interacting 1~ with the 5'-en d sequences thereby affecting initiation of protein synthesis or, the 3'-end sequences may be servin g as a binding site for other RNAS which can interfere with the binding of an expression in mod ulating molecule, including repressor molecule. (Kla usner et al., 1989, Science, 246:870-872; Kozak, 1992, Ann. Rev. Cell Biol., 8:197-225.
Comparison o' 5' >'on-codino Seauences o° Hu~en and Mouse OP-1 DNn The clo.~.ing of the 5' non-coding aeno.-..ic mur ine and human OP-1 2~ Dt:n sequences de-onstrated that a high degree of sequence homology exists between the human and murine ~' non-coding DN=. sa~uences.
The homology extends from the basE im:~eWatel:~ upstream of the translation start site for the OP-1 morphogen protein to approximately 750 bases upstream of the translation start site, as 25 is shown in the shaded regio:~s of Fig. 1, kith the marine sequences being the upper lines and the human sequences being the lower lines. The 5' nucleotide of the region of homolog}~ for the human OP-1 5' non-codinc seauence is base 2548 of Seq. ID No. 1 and for the r"urine OP-1 5' non-coding sequence is base 1549 of seq. ID No. 2. The significant homology between the human and marine 5' non-coding sequences of OP-1 suggest that this region may be important in the regulation of OP-1 expression. As will be discussed in more detail below, this region contains several conserved DN:, sequences which have been identified as the DN?, 35 binding sequences for two DN~ binding proteins, Wt-1 and Egr-l, which both recognize these DNA sequences. The DNA binding sequences for 4:t-1/Egr-1 present in human and marine are marked in Fig. 1 with a single line. also, the TCC binding sequence, a DNA
bindi:~g sequence for Y:t-1 and Egr-1, is marked in Fig. 1 b;' the double line. ~n'T-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, 86:8737-87x1; 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-1/ht-1 binding sequence (5'-GNG NGG GNG-3') (Rupprecht et al., 1994, J. Biol. Chem., 269:
0198-6202; Herner et al., 1994, : Biol. Chem., 269: 12940-12996 are located before and after the presumed transcriptional initiation site (Fig. 1, shown by solid single lines!. The presence o: these has significance in lig.',t o; the elevated 1e:~21s of ~:t-1 mRNA in the rat uterus decidua during pregnancy (Zhou e~
al., 1°93, Differentiation, 59:10-1141.
The analysis also revealed, in the human upstream region, a pattern o~ seven TCC repeats, present at -561, im.T~ediately 3' of two Egr/k't-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 upstrea.~r, region contains a similar, albeit less ob~~ious sequence at -356 and at position 1755-1769 of Seq. ID No. 2. This TCC-repeat pattern is found in the promoters of PDGF-A and several other growth-related genes, and 4;t-1 has been found to activate transcription when either of the sequences are present and to suppress it when both sequences are present. (~:ang et al., 1992, Biochem. Hiophys Res. Comm., _188:433-439; kang et e1. 1993, PNAS, ~ 90:8896-8900). Accordingly, estrog en receptor may exert its effect on OP-1 expression in uterus by upregulating Wt-1, either directly or indirectly. Alternatively or, in addition other regulatory elements, located further upstream of th a 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 is marked by carats below: the human DN?. sequence. A FTZ binding sequence is ~1~1a~~
Vf0 95133831 PCTlUS95tt17349 bound by the Fushi-tarazu protein (FTG-Fl), which is a member of the superfamily of nuclear receptors (Parker. (1993) Current opinion in Cell Biology. S:S99-504....j. The superfamily of nuclear receptor proteins include steroid hormones, retinaids, thyroid hormone, nerve growth factor and Fushi-tarazu, and are structurally related. FTZ-F1 is likely to belong to a subfamily of nuclear receptors that bind DNA as monomers.
The FTZ-F1 protein is a positive regulator at the fushi-tarazu gene in blastoderm stage embryos of Drasophila . FTZ-F1 is clasely~ related in the silkworm (Bombyx) B:nFTZ-F1 protein and the mouse embr~;~onal long terminal repeat binding protein (ELF) and all of them are members of the nuclear hormone receptor superfamily, which recognizes the same 9 base pair sequence, 5'-PyC~RGGPyCPU-3'. The FTZ binding sequence does not apparently have a direct or inverted repeat. In contrast, other members of the nuclear hormcne receptor superfamil}~ usually bind to repeated sequences.
Nevertheless, the FTZ-F1, Bm~zTZ-F1 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 tha nuclear hormone receptor superfamily.
(riitachi et al., 1992, hfcl, a.~.d Celi Bioloav December, pp. 6667-5672.).
The mR.fdA transcription initiation site for human oP-1 is markzd an Fig. 1 by the upward arrow, and the 0P-1 protein translation initiation site is marked on Fig. 1 by the solid triangles just prior to the highlighted ATG. The transcription initiation site for the human. OF-1 gene is at bass 2790 of Seq. ID
Na. 1 and the analogous site for murine is at base 1788 of Seq. ID
No. 2. The translation initiation site for the human OP-1 gene is ~ at base 3318 of Seq. ID No. 1 and for murine it is at base 2296 of Seq. ID No. 2. The high degree of identity that the murine and human DNA sequences share in the region between the transcription initiation site and the translation initiation site, suggests that this region. likely plays a role in the modulation of the 3S expression of the OP-1 gene product.
Rnalvsis of OP-1 Gene Excressio~, in Mouse Tissues SUBSTfTUTE SHEET (RULE 26}

WO 95133831 ~ PCTII1S95/07349 A detailed analysis of the uro-genital tract of rats has revealed OP-1 mRNA expression in the renal (kidney), and bladder tissues, as well as at other sites of the urogenital organ system.
The most abundant levels are present in renal and uterine tissue $ (6 week old mica), 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 polyiA)+ RNA (5mg), were loaded into each lane.
1~ Preparation of RNA and Northern blot hybridization analysis was conducted as follows. 8-week-old female mice, strain CD-1, were obtaine3 from Charles River Laboratories, Wilmington, MA.
Total RNA, from the various organs of mice was prepared using the acid-guanidine thiocyarate-phenol-chloroform method (Chomczynski IS et al., (19871 Anal. Biochem. _162:156-159). The Rt7A was dissolved in TES buffer (10 ml-; Tris-HC1, 1 m: tJa~-EDTA, 0.18 SDS, pH7.5) containing Proteinase P; (Stratagene, La Jolla, CA; approx. 1 mg prcteinase /ml TES) and incubated at 37°C for 1 hr. Poly (A)+ RNA
~has selected in a batch procedure cn oligc(dT')-cellulose 2~ (Stratagene, La Jolla, CA) in 0.5 td NaCl, 10 mM Tris-HC1, 1 mM
tta -EDT:, pY. 7.4 (1 x binding buffer; . For the selection of poly (A)+ RNA, total RtdA obtained from 1 g of tissue was mixed with approximately O.lg of oligo(dT)-cellulose (in 11 ml TES containing 0.5 M NaCl). The tubes containing the RtaA and oligo(dT)-cellulose 2$ ware gently shaken for approx. 2 hrs. Thereafter, the oligo(dT)-cellulose was washed twice in lx binding butter and once in O.Sx binding buffer (0.25 M NaCl, 10 m~ht Tris-HC1, 1 mtu Na -EDTA, pH

7.4) and poly (A)+ RNA o:as eluted with c,~atezand precipitated with ethanol.
3~ Poly(A)+ RNA (5 mg per lane) was electrophoresed on 1.28 agarose-formaldehyde gels with 1 mg of 400 )tg/m1 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 3$ et al., eds., (1990) Current Protocols in Malecular Bioloov, John tdiley & Sons, taew York). Follawing eleccrcphoresis, the gels were photographed, rinsed briefly in water, and blotted overnight onto t7ytran (SChleicher & Schuell Inc., Keene, NH) or Duralon-W
SUBSTITUTE SHk'~T (RULE 26) ~191~~~
WO 95!33831 PCTIUS95107349 _ 2~ _ (Stratagene) membranes in 10 x SSC. The membranes were dried aL
80° for 30 min. and irradiated with C1V light (1 mw=/cmj for 2S
sec.).
The 3zP-labeled probe was made from a murine OF-1 cDNA
$ fragment (0.68 kb BstXI-BG1I frg.) by random hexanucleotide priming (Feinberg et al., (1984) Anal':~'Eiochem.. 137:266-267).
The hybridizations were dons in 40%~formamide, Sx SSPE, Sx Denhardt's, 0.1% SDS, pH 7..5 at 37°C overnight. The non-specific counts were washed off by shaking in 0.1x SSPE, 0.1% SDS at SO°C.
In For re-use, filters were stripped in 1 m": Tris-HC1, 1 mM Cda~-EDTA, 0.1% SDS, pH 7.5 at 80° C for .0 min.
Analysis ef OF~-1 Expression During Preananc~,~ in Mice An examination of the effect o_ pracnar:~.~t ur:cn GF-1 ex_~rass:cn 15 was undercakea by measuring OP-1 mRllF; levels in kidney, Gvary and uterus, before, during, and after pregnanc:~ (virgins, 2-day pcst~-coital (pp), 4-day pc, 6-day pc, 8-day pc, 13"~ day pc, 17-day pc, 3-day lactating, and retired breeders) bNar.thern blot hybridization of poly(A)+ Rt:,. These measurements demonstrated 2~ Chat, while kidneys show nc pregnancy-related changes in OP-1 mRC7A
levels, the uterine levels beca~r:e nearly undetectable by 6-day pc, However, no changes were observed in the cvaries. A dramatic and rapid decline in GP-1 message in uterine cissu~ becween d.a_: 3 and 4 of pregnancy is apparent in the comparison a~ith virgin animals.
25 The levels of GP-1 mRNA in the embryo and maternal levels in uterus of 8 week old mice at day 13 and 16 of the pregnanc;= were also compared. D,'hile the GP-1 expression in the pregnant uterus is dramatically reduced, high levels cf GP-1 message are found in the mouse embryo at 13- and 16-days. Thus, at a stage of 3n pregnancy when OP-1 mRNA expression in the maternal uterus is almost undetectable, embryonal. OP-1 expression is high. The high embryonal OP-2 expression also is detected consistent with the relatively high levels of OF-1 mRS7A, found in human placenta. The level of OP-1 mRL(A measured in the embryo is in the same range as ~5 that measured in adult kidney or virgin uterus 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 vary specific stages of tissue and organ morphogensis. 4dhile not being SUBSTITUTE SHEET {RULE 26) WO 95f33831 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 expression 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 sustained 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 subcutaneausly administered 17~i-estradiol, or progesterone, or a combination of both, IS In the first experiment the rapid increase in estrogen and progesterone levels during pregnancy was simulated. Plan-pregnant mice were injecte_ subcutaneously on four consecutive days with increasing doses, starting with 20 mg 17p-estradiel, 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 m.°taA was isolated~from uteri and kidneys. A striking negative effect of 17(i-estradiol on the uterine OF-1 mRNA expression was observed, but no effect by progesterone was seen. In the kidneys, however, mRPdA levels did not change after 17p-estradiol or progesterone treatment.
Another experiment addressed the time course: 17(i-estradiol was administered to virgin female mice ac a constant dose of 200 mg (SO ml of 4 mg/ml 17(i-estradiol per day, subcutaneously in DhfSO
[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 173-estradiol and almost undetectable levels by 48 hours, as shown in Fig. 2. In the figure, the lanes correspond as follows: frcr,~ left to right, 0-day (negative control), 0-day= (negative control), 0.5-, 1-, 2-, SUBSTITUTE SHEET (RULE 26) ~I~~. i~~
WO 95133531 PCTlUS95107349 3-, 4-, 6-, 6-, 7-, and~8=days.~~ The arrowheads mark the two major GP-1 mRNA species.. A modest amount of message reappears a few days later (Figure 2).
The uterus has been identified as a major site of OP-I
expression.. The level of OP-I expression in uterine tissue is comparable to that observed in renal tissue. However, during pregnancy, by day four, the uterine OP-1 mRNA levels are reduced to the limit of detection.. The loss of OP-1 expression corresponds withalso is rising levels of estrogen during this same IO time frame. The same dramatic loss of uterine OP-1 message also is observed in estrogen-treated animals, suggesting that estrogen is involved i.n negative regulation of OP-1 expression in uterine tissue, The effect of estrogen is rapid, with most of the message disappearing after L2 hours of 17(S-sstradicl administration. Tk:e IS reappearance of some OP-1 message at later days may be due to a counter-regulatory mechanism. In contrast to the modulated O?-1 mR2:~ levels in the uterus, no substantial changes occur in renal tissue during pregnancy or in response to estrogen treatment.
Therefore, OP-1 mRNA er;pression in these different organs is 2~ regulated independentl;=. The differential expression may be due, for ex~ple, to a Sack of estrogen receptors in renal tissue.
Alternatively. co-regulation by means of one or mere accessory molecules chat interact with estrogen or a related nuclear receptor moleculefs) may allow for the independent. regulation.
2$ For example, each of 4dt-. protein ;which binds to the wt-ifEgr-1 eles~ent) and oP-1 protein are required for norsal kidney development, and each are expressed at high levels durina_ kidney tissue development. As described above the OP-1 promoter region contains kt-1 consensus binding elements. Wt-1 protein also has 30 been shown to negatively regulate the transcription of the insulin grown: 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 ba that Ya-1 protein, either alone or in combination with one or more molecules is 3$ 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.
SUBSTITUTE SKEET (RULE 26) ~~9~.~8'3 WO 95133831 PCTIUS95t07349 Implications of Tissue Specific Differential Regulation of OP-1 Expression Estrogen also has been shown to inhibit the uterine expression of calbindin-DzBH, a vitamin D dependent calcium binding protein, the a-subunit expression of the glycoprotein hormones, and other proteins involved in bone formation. Estrogen also has been shown to cause dramatic decreases in the steady state mRNA levels of the bone matrix proteins osteocalcin, prepro a2(Iy chain type I
collagen, osteonectin, osteopontin, and alkaline phosphatase in an 1~ ovariectomized rat, which is a rat model for osteoporosis, Estrogen agpears to mediate its beneficial effect on bone metabolism in the osteoporotic model through inhibition of osteoclasts. Estrogen does not reverse osteoporosis. By contrast, OP-1, which is e>:pressed in uterine, renal and bane tissues, is able to induce an increase in bore mass in the esteoporotic model. Thus, the negative effect of estrogen on OP-1 expression in uterine tissue may seem unexpected in view of estrogen's effect on bone metabolism.
In addition to the 5' non-coding D2dA sequences of OP-1, the other non-coding sequences such as introns and 3° non-coding sequences may be involved in the modulation. of OP-1 protein expression. This invention presents 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 expression will be identified by culturing cells transfected with the non-coding sequences, in operative association with a reporter gene, with one or more compound(s), measuring the level of reporter gene expression, and comparing this Ievel of expression 3d to the level of reporter gene expression in the absence of the compound(s).
EXEMPLARY CELLS, VECTORS, REPORTER GENES AP:D ASSAYS FOR USE IN
SCREENING COMPOUDIDS A'HICH MODULATE OP-1 REGULATORY SEQUENCES
I. Useful Cells Any eukaryotic cell, including an immortalized cell line suitable for long term culturing conditions is contemplated to be useful for the method an3 cell of the invention. Useful cells SUBSTITUTE SHEET (RULE 26) 21~~~8J
wa 9sr33am PCTlUS951073d9 should be easy to transfect, are capable of stably maintaining foreign DNA with an unrearranged sequence, and have the necessary cellular components for efficient transcription and translaticr, of the protein. including any elements required for post-$ translational modification and secretion, 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 aisa has simple media composition requirements, and rapid generation times.
1U Particularly useful cell lines are mammalian cell lines, including myeloma. HeLa, fibroblast, embryonic and various tissue cell lines, e.g., ki.dney, liver, lung and the like. A large number of cell lines now are available through the American T,~pe Culture Collection (Rockville, MD) or through the curopean Collection of 1$ Animal Cell Cultures (Porton Down, Salisbury, S?d OJO, U.Y,.) 4:here, as here, the expression of a reporter gene that is controlled by non-coding se:7~aences of the morphog~en OP-1 is to be analyzed, particularly useful cells and cell lines are envisioned to include eukaryotic, preferably mammalian cells of a tissue and 2Q cell type known to express OP-1 andlor closely related~proteins.
Such cells, inciude, without limitation, cells of uro-genital cell origin, including kidnes~, bladder and ovary cel:.s, lung. liver, mammary gland and cardiac cells, cells of gonadal origin. cells of gaserointestinal origin. glial cells and other cell lines known to 25 erpress endogenous genes encoding morphagenic proteins. Preferred cell lines are of epithelial origin.
II. ~xemp.lary VectorslVector Construction Considerations Useful vectors for use in the invention include, but are not 3U limited to cosmids, phagemids, yeast artificial chromosomes or other large vectors. Vectors that can be maintained c.~ithin the nucleus or integrated into the genome by homologous recombination are also useful. For example a vector such as PSV2CAT would be useful.
$S Selected porticns of non-coding OP-1 sequence 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 possihle, and can be engineered into the sequence when needed. If restriction SUBSTITUTE SHEET (RULE 26) 2:~~158~

_ 27 _ endonuclease sites are needed to be engineered iiao 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 S 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 bases upstream 1~ of the translation initiation site as shown by the shading in Fig.
1. This region is positions 2548-3317 of Seq. TD No. 1 and positions 1549-2296 of Seq. ID tdo. 2. The mRNA transcription initiation site lies within this region at position 2790 of Seq.
ID No. 1 and by analogy at position 1788 of Seq. ID No. 2, shown 15 in Fig. 1 by the upward arrow. This suggesCS that positions 2548-2790 of Seq. ID tde. 1 and 1549-i788 of Seq. ID t7o. 2 contain conserved promoter elements for the expression of OP-1 lnRtdA, and approximately 500 bases at positions 2791-3327 of Seq. ID No. 1 and positions 1790-2290' of Seq. ID No. 2 contain conserved 2~ elements of the transcribed, but not translated, sequences all or part of which may be involved in the regulation of OP-1 expression. Additionall_: sequen.es upstream of the homology region may also be involved in the regulation of OP-1 expression.
Thus a range of upstream sequences" including sequences upstream 25 of the transcription initiation site and not including the approximately 500 bases of transcribed sequence, can be fused in operative association with a reporter gene to modulate er;pression of the gene.
3' non-coding sequences and intron sequences also car, be fused in operative association 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 2190-331.7; 2548-2790 or 2548-3317 of Seq. ID No. 1, and either/both of 3' sequences or intron sequences in operative 35 association with a reporter gene. The positions of the six introns are shown in Seq. ID No. 1 as bases 3736 to 10700; bases 10897 to 11063; bases 11217 to 11424; bases 11623 to 13358; bases 13440 to 10548; bases 15166 to 17250;
SUBSTITUTE SHEET (RULE 26) - 28.
Also envisioned is a nucleic acid construct comprising a small fragment of 5' non-coding oP-1 sequence in combination with additional conserved elements such as one or more in't-1/Egr-1 binding sequences; a TCC binding sequence and/or a FT2 binding sequence in operative association with a reporter gene. such a nucleic acid construct also could include intron sequences andlor 3' non-coding sequences.
A range of useful 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 also are useful. Such smaller fragments can be identified to deleting bases from one o_- both ends of the provided 5' non-coding fragments, using tech..~.iques that are well known in the art and testing the truncated constructs for their ability to modulate repozter gene expression.
In this way, the shortest modulating sequences can be identified.
III. Transfection Considerations hnl method for incorporating nucleic acids into cells of ..merest is contemplated in the method of the invention. Calcium 2~ phosphate (CaPO;), followed by glycerol shock is a standard means use3 in the art for introducing vectors, particularly plasmid DN.'-.
into mammalian cells. A representative method is disclosed in Cockett et al., (1990) Hiotechnolog y 8: 662-667). Othe r methods that m ay be used include electroporation, protoplast fusion, particularly useful in myeloma transfections, microinjections, lip ofections and DEAE-dextran mediated uptake. Methods for these procedures a re described in F.M. Ausubel, ed., Cur rent Protocols in Molecular Biology, John Wiley & Sons, New York (1989).
As will be appreciated by those having skill in the art, optimal DNA concentrations per transfection will vary according to the transfection protocol. For calcium phosphate transfection, for exa.~~.ple, preferably 5-10 pg plasmid DNA per plasmid type is transfected. In addition, the DNA to be transfected preferably is essentially free of contaminants that may interfere with DNA
incorporation. A standard means used in the art for purifying DNA
is b1 ethidium bromide banding.

IL'. Exemplary Reporter Genes There are numerous reporter systems commercially available.
which include, without limitation, the chlozamphenicol acetyltransferase (CAT), luciferase, GAL4, and the human growth S hormone (hGH) assay systems.
CAT is a well charactezized and frequently used reporter system and a major advantage of this system is that it is an extensively validated and widely accepted measure of promoter activity. See, for example, Gorman, C.M., Moffat, L.F., and Howard, B.H. (1982) Mol. Cell. Hiel., 2:1046-1051 for a description of the reporter gene and general methodology. In this system cells are harvested 2-3 days after transfection with CAT
e>:pression vectors and extracts prepared. The extracts are incubate with acetyl Co~ and zadioactive chloramphenicol.
Following the incubation acetylated chloramphenicol is sepa:ated f:o~; nonacetylated form by thir: la..~e= c::_or,.a:o?raphy . In this assay the degree of acetylatio.~, re;leccs the C::T gene acti:mty with the particula: promoter.
Another well-recognized zeporte: systea is the firefly 0 luciferase reporter syste"~. See, for exa.Tple Gould, S.J., and Subramani, S. (198E) Anal. bioche.-.,.,., 7:COC-408 for a description c-' the reporter gene ana general n~sthodolog~~. The luciferase assay is fast and has increases sensitivity. The system also is particularly useful in bulk tra.~.s:ections or i: the pro.~"oter of interest is weak. In this assay trans:ected cells arE grown under standard conditions, and when cultured under assay co;,ditions both ~':? and the substrate lucife:in is added to the cell lysate. The enzyme luciferase catal~~zes a rapid, ATP dependent oxidation of the substrate which then emits light. The total light output is measured using a luninometer according to manufacturer's instructions (e.g., Cromega) and is proportional to the amount of luciferase present over a wide range of enzyme concentrations.
A third reporter system is based on immunologic detection of h GH, it is quick and a asy to use. (Selden, R., 3S Burke-Howie, K. Rowe, M.E., Goodman, H.M., and Moore, D.D.
(1986), Mol. C ell. Biol., 6:3173-3179). hGH is assayed in the media, rather than in cell a xtracts. This allows 2~~(: i8~~
WO 95133831 PCT/L1995l073~19 direct monitoring over by a single population of transfected cells over time.
As indicated above and as will be appreciated by these having ordinary skill in the art, particular details of the conventional S means for transfection, expressicn, and.: assay of recombinant genes are well documented in the art arid aYeunderstood by those having ordinary skill in the art. The instant invention enables and discloses vectors, cells and a method for screening compounds to determine the capability of compounds to modulate the expression ~Q 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 steps used in recombinant production of foreign genes in mammalian expression systems car. be found in a number of texts and laboratory manuals in the art, such as, for example, F.P:. ;,usubel 1~ et al., ~d., Current Protocols in Molecular Sia_l~ov, John G:iley &
Sons, Ne~.J Yerk, ;19891.
VII2. Exemplary HOtinOIOgOUSit~~Gn-HOuTsOlOgOU5 ReCOmbin3tiOn One approach to scraen for inducers of (organ-specific) oP-1 2~ expression in a particular cell line derived from a particular tissue such as renal a.r uterine tissue, is through gene cargeting by homologous recombination (Sedivy= et al., S~:.H. Freer,~an E. Co., idew York (1992); A.S. 4ialdman, Crit. Rev. Cncoi. Hematol. 12, 49 (1992)). In one strategy the endogenous (genomic) OP-1 gene is 25 replaced by another reporter gene which is optimally suited far screening assays, such as the firefly luciferase gena. 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 elements can be assembled.
3Q Genomic OP-1 upstream and promoter sequences preferably 3000 to 5000 nucleotides in length, and which mediate the homologous recombination, are attached to the lu.ciferase gene. The OP-1 upstream sequences down to the first coding ATG can be attached at the start codan A~.~; of the lucif~rase coding sequence, using a 35 restriction site such as 27co1, which can be introduced by site directed mutagenesis into both the promoter and the luciferase sequences.
SUBSTITUTE SHEET (RULE 26) Also included is a selective marker, preferably the neo gene, without its o~.~n promoter. Preferably, selectable marker ineo) is placed downstream of the reporter gene (luciferase), after an intercistronic sequence derived from the poliovirus 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 85:8583 (1988): Sedivy et al., PNAS USA 86, 227 (1989); Dorin et al., Science 243:1357 (1989). As de scribed therein, the endogen s OP-1 gene is replaced by th a lucif erase and neo coding sequences and the expression of these sequences then assayed in a standard A
screening protocol.
A genetic arrangement of OP-1 promoter (as much genomic OP-1 upstream sequence as possible, up to 10,000 bp) and reporter gene (without its original promoter but joined directly to th a OP-1 ATG or in its vicinity) can also be introduced into cells on standard a ukaryotic expression vectors. These vectors carry selectable markers (neo, d hfr, etc-) and will typically be integrated into the host gen ome with variable copy number ranging from one to several co pies without efforts at amplification. Also, if desired, the vector or gene copy number can be a nhanced using a well characterized amplifiable gene, suc h as dhfr in conjunction with methotrex ate. Commercial vect ors designed for auto nomous replicatia~ without integration are raa~ily available. Ona sour:E
vectcr is the Episomal Expression Epstelr. Barr Virus Vector (PREP, Invitrogen Corp., San Diego CA).
Introns also can be tested for regulatory sequences 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. Albertsor., H.M. et al. PNAS USA, 87:4256, 1990), or other vectors adapted to allow transfer of large sequences, e.g..
up to 1 megabases. As for the OP-1 S' or 3' noncoding sequences described above, the intron sequence or a portion thereof is inccrporated in operative association H'ith a reporter gene and the ability of the sequence to modulate reporter gene expressions then associated.
X. Exemplary Screening Assay for Compounds which Alter OP-1 Gene or S Reporter Gene Levels.
Candidate compounds (s) which may be administered to affect the level of a given endogenous morphogen, such as OP-1, or a reporter gene that is fused to OP-1 non-coding sequence may be found using the followng screening assay, in which the level of reporter gene production by a cell type which produces measurable levels of the reporter gene expression 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 expression, a detailed description also may be found in Canadian Patent Application Serial No. 2,111,656.
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 plasmid in the cell nucleus or the vector can be integrated into the host cell genome, preferably at the OP-1 genomic locus.
Cell samples for testing the level of reporter gene expression are collected periodically and evaluated for reporter gene expression using the appropriate assay for the given reporter gene as indicated in the section describing reporter gene assays, or, alternatively, a portion of the cell culture itself can be collected periodically and used to prepare polyA(+) RNA for mRNA analysis.
Once candidate compounds are identified, they can be produced in reasonable, useful quantities using standard methodologies known in the art. Amino acid-based molecules can be encoded by synthetic nucleic acid molecules, and expressed in a recombinant expression system as described herein above or in the art. Alternatively, such molecules can be chemically synthesized, e.g., by means of an automated peptide synthesizer, for example.

Z~~1~~

Nan-amino acid-based molecules can be produced by standard organic chemical synthesis procedures.
Provided below is an exemplary protccel for carrying cut the method of the invention, using the CAT gene as the reporter gene and one or more mammalian cell lines known to express 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 Far Transfections A DNA fragment containing the OP-1 promoter can be joined to a I~ 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 fer OP-1 mRt7.'-.). Using this technology we have found se~~eral cell lines which make high levels of OP-'1 mRivr., and some IS o-' these lines are the kidney line IMCD, the bladder line NBT II.
An approximately 5 Kb EcoRI, BamHi genomic fragment containing approximately 4 Rb of upstrear,~ OF-1 sequences as well as part of the first intran is blurt-ende;.' with T4 DN=., polymerise and cloned into a pclylinker of a pUC vector (p0146-i?. An appro:cimately 3.5 2~ kb DtdA fragment containing human OP-1 upstream sequences is obtained by del~>ting a portion of coding sequences and the first intron from p0146-1 with the restriction enzyme EheI. The -3.Skb fragment has blunt ends and contains mostly 5' non-coding sequences and also includes a short stretch of 30 bases into the 25 O?-1 gene. This upstream fragment is of -3.Skb ligated to a 1.6 kb HindIII-BamHI fragment from the CAT gene obtained from the vector SV2CAT by 5~ HindiII end blunted ligation. The l.6kb CAT
gene fragment contains about 7G bases of upstream sequences.
These ligated fragments are cloned into Bluescript RS(-) vector 3~ (Stratgene, La Jalla, CA). This construct in turn is subjected to site specific mutagenesis to delete the extra sequences (approximately 30 basest from the 3' end of the OP-1 upstream sequences and the adjacent 5' non-coding sequences (approximately i0 bases) from the CAT gene. This mutagenesis results in the 35 elimination of any OP-1 coding sequences from the promoter fragment as well as any non-coding sequences upstream of the CAT
gene. Thus the resulting construct is a fusion of OP-1 upstream sequences with the CRT gene sequences which encode the CAT
SUBSTITUTE SHEET (RULE 26) 21~1~$x W0 95133fi3d PCT/U89SJ07349 protein. This approximatell~ 5 kb ~ragmenc is they, excised from Bluescript using HindIII ard.EaamHIand ligated into a HindIII-BamHI cut and gel purified bank-bone of the pSV2CAT vector, for transfectian into suitable cell lines.
Suitable cell lines include cell lines that have been shown to contain high levels of OP-1 mRP7A, indicating that the OP-1 promoter is active in the cells. Two of these cell lines are mouse inner medullary collecting duct (IMCD) cells, and the rat bladder carcinoma line (NBT II). However other cell lines of the 1~ uro-genital system that produce high levels of the OP-1 message car. be used in addition tc the many previously mentioned cell types and cell lines.
The transfection ef this vector into an OP-1 producing cell line is accomplished fcllawing standard techniques, i.e., i$ transfeation using calcium phosphate, liposome mediated transfection, eleatropcration, er Dt..ri.-dextran transfection.
The transfected cells are harvested 48-'2 hours a.fcer transfeation with the C:.T expression vector and extracts are made by successive freeze-thawing. 2 u1 of 200 uCi/ml 14C-2~ choramphenicol (35 to 55 mCi/mmoli. 2G ~tl cf 4 :o-:1 aaet.;.,.~1 CoA, 32,5 u.1 of 1 M Tris-HCl, pH %.5, and 75.5 ~1 of water is added Lc 20 ml ef call extract, and incubated for 1 hour at 37 degrees Celsius.
Upon completion of incubation, 1 ml ethyl acetate is added to the reaction, microcentrifuged far 1 minute and the top layer is 25 removed. This top layer is dried down in a Speedvac far 45 rminutes, and each sample is resuspended in 30 ml of ethyl acetate.
The samples are spotted onto a plastic-backed Tr,C sheet for chromatography. The thin layer is then devalaped in a tank containing 200 ml of 19:1 chloroform/methanol. The chromatography 3d is run for 2 hours and placed under film for autoradiography. The activity of the C14 in the monoacetylated chlcramphenicol series is calculated as described in Current Protocols in Molecular Biology, 1993 {Ausubel et al., eds. John u:iley & Sons, New York).
Upon determination of CAT activity, the main construct can be 3$ deleted in sections to determine the regions that are responsible for the observed CAT activity. Alternatively, the upstream sequences can be deletes unidireccionally, using an exonuclease SUBSTITUTE SHEET (RULE 26) ~~~Ia83 W0 95133931 P(.°1'IUS95107349 such as Ha131, and the deletion product can be analy2ed in the CAT
activity assay. This system car. also be used in the method of the invention to screen compounds for their ability to modulate OP-1 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-1 reporter gene is preferred in the method disclosed herein, as will Id be appreciated by those having ordinary skill in the art, OP-1 coding sequence also may be used in the screening method of the invention. The OP-1 expression preferably is determined by an immunoassay or by 2aorthern or dct blot or other means for measuring mRL7=, transcript. See, for example, h0 95J11983, published May 4, 1995 fcr a detailed description on assaying changes in OP-1 levels in a cell or fluid.
XI. Exemplary Screening Assay for Compounds which Alter OP-1 Gene Expression in Endogenous Cell Type Models.
2a OP-I is expressed in a variety of different cell types, including renal, bane, lung, heart, uterine, cardiac and neural tissue. Candidate compounds can be identified which have a modulating effect on cells of one tissue type but not another, and/or wherein the effect is modulated in the different cells.
The assay describe3 bolo.; can be used to evaluate the effect of a candidate compounds) in a parCicular cell type known to express OF-1 under physiological conditions.
Cell cultures of kidney, adrenals, urinary bladder, brain, or other organs, may be prepared as described widely in the 3~ 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 sliced ~(1-4 mm) tissues. Priman~ tissue cultures and established cell lines, also derived from kidney, adrenals, urinary, bladder, brain, mammary, or other tissues 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 i1-7 days).
Cells may be cultured, for example, in Dulbecco's Modified Eagle medium (Gibco, Long Island, L7Y) containing serum (e. g., fetal calf SUBSTITUTE SHEET (RULE 26) ~i9~.~~:~
W 0 95!33R31 PCTlU5951073~19 serum at 1~-10~, Gi.bco) or in serum-deprived medium, as desired, or in defined medium (e. g., containing insulin, transferrin, glucose, albumin, or other growth factors).
Samples far testing the level of OP-1 production includes culture supernatants or cell lysates, collected periodically and.
evaluated for OP-1 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 fcr R2JA
IQ analysis. To monitor _de nova OP-1 synthesis, same cultures are labeled according to conventional procedures with an r5S-methicninei~SS-cysteine mixture for 6-24 hours and then evaluated to OP-1 synthesis by conventional immunoprecipitation methods.
I$ XII. Exemplary In viva Primal hfodel far Testir;g Effica:.y of Compounds to Modulate OF-I E>:pressian It previously has been demonstrated that OF1 can effect csteoporosis on the standard ovariectomized rat model, as indicated by the dose-response increase in alkaline phosphate and 2~ osteocalcin levels following i.njecticn with OF-I. The osteoporotic rat model provides an ::n v3vo model for evaluating the efficacy of a candidate modulating compound. In order to determine tire effect of a candidate morphogen stimulating agent en OP-I production and, thereby, nn bona production _Sn _viva, alkaline 25 phosphate and cosaFOCalcin le~=els are measured under co;:ditions which promote osteoporosis, e.g., wherein ostecporosis is induced by ovary removal in rats and in the presence and absence of a candidate modulating ccmpeund. .. compound competent to enhznce or induce endogenous OP-1 expression should result in increased 3~ osteocalcin and alkaline phosphate levels.
Forty Long-Evans rats (Charles River Laboratories, 4.'ilmington) weighing about 200g each are ovariectomized (OVX) using standard surgical procedures, and ten rats are sham operated. The ovariectomization of the rats produces an osteaporotic condition 35 within the rats as a result of decreased estrogen production.
Food and water are provided ad libitum. Eight days after ovariectomy, the rats, prepared as described above, are divided into three groups: (A) sham-operated rats; (B) ovariectamized rats receiving 1 ml of phosphate-buffered saline (PBS) i.v. in the SUBSTITUTE SHEET (RULE 26) tail vein: and (C) cvariectomized rats receiving vnrious dose ranges of the candiate stimulating agent either by intravenous injection through the tail vein or direct administration to kidney tissue.
S The effect of the candidate compound on in v~vo bone lozmation ' can be determined by preparing sections of bone tissue from the ovariectomized rats. Each rat is 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. Hone sections are prepared and the distaance separating each tetracycline straining is measured to determine the amount of new bone growth. The levels of OP-1 in serum following injection of the candidate agent also can be manitered on a periodic basis using, for example, the immunoassay described in sections V and V:I above.
V. Exemplazy Determination of o?-1 Protein Pzoduction 2~ 4:here OP-1 acts as the repcztez gene, detection fo the gene pzosuct readily can be assayed using antibodies specific to the pzotein and standard immunoassay testings. For example, OP-1 may be detected using a polyclonal antibod~~ specific for OP-1 in an ELIS:,, as follows .
lug/100 u1 of affinity-purified polyclonal rabbit IgG
specific for OP-1 is added to each well of a ?6-well plate and incubated at 37°C for an houz. The wells are washed four times with 0.167M sodium borate buffer ;,ith 0.15 M NaCl (HSH), pH 8.2, containing O.li Tween*20. To minimize non-specific binding, the wells are blocked by filling completely with 19 bovine serum albumin (BSA) in HSB and incubating for 1 hour at 37°C. The wells aze then washed four times with HSH containing 0.1~ Tween 20. A
100 u1 aliquot of an appropriate dilution of each of the test samples of cell culture supernatant is added to each well in tziplicate and incubated at 37'C for 30 min. After incubation, 100 u1 biotinylated rabbit anti-OP-1 serum (stock solution is about 1 mg/ml and diluted 1:400 in BSB containing It HSA before use) is added to each well and incubated at 37'C foz 30 min. The wells are then washed fouz times with BSB containing 0.1; Tweeri *Trade mark 20. 100 u1 streptavidin-alkaline tSouthern Hiotachnology associates, Inc. Birmingham, Alabama, diluted 1:2000 in BSH
containing 0.1~ Tween*20 before use) is added to each well and incubated at 37°C for 30 min. The plates are washed four times S with 0.5M Tris buffered Saline (TBS), pH 7,2. 50u1 substrate (ELISA Amplification System Kit, Life Technologies, Inc., Hethesda, MD) is added to each well incubated at room temperature for 15 min. Then, 50 p1 amplifier (from the same amplification system kit) is added and incubated for another 15 min at room temperature. The reaction is stopped by the addition of 50 ~:1 0.3 M sulphuric acid. The OD at 490 nm of the solution in each well is recorded. To quantitate OP-1 in culture media, a OP-1 standard curve is performed in parallel with the test samples.
Eae::plar)~ Pro~uction of O?-1 Polyclo:,al and Monoclona:
1S Fztibody Pol)~clonal antibody for 0~-1 prcte:.~. may be p=epared a=
fellows. Each rabbit is given a primary im,:":nization of 100 erg; 500 u1 E. col i produced OP-? mo:~ome= (a.~nino acids 328-431 of OP-1) in O.lo SDS mixed with X00 u1 Complete Freund's Adjuvant. The antigen is injected subcutaneously at multiple sites or. the back and flanks of the anima'. The rabbit is boasted after a month in the same manner usi.~.g incomplete Freund's Adjuvant. Test bleeds are taken fro:.- the ear vein seven days later. T~:o additional boosts and test b'_eeds are performed at r..onthl)' intervals until antibody against Or-1 is detected in the serum using an ELIS~, assa)~. Then, the rabbit is boosted monthly with 100 y~g of antigen and bled (15 r.,l per bleed) a: days seven and ten after boosting.
Monoclonal antibody specific for OP-1 protein may be prepared as follows. F, mouse is given two injections of E. coil produced OP-1 monomer. The first injection contains 100ug of OP-1 in complete Freund's adjuvant and is given subcutaneously. The second injection contains 50 ug of OP-1 in incomplete adjuvant and is given intraperitoneally. The mouse then receives a total of 230 ug of OP-1 (amino acids 307-431 of OP-1) in four intraperitoneal injections at various times over an eight month period. One week prior to fusion, both mice are boosted intraperitoneally with 100 ug of OP-1 (307-431) and 30 ug of the N-terminal peptide (Serzs,-Asn~oa-Cys) conjugated through the added * Trade mark 2~~~.58 W0 95)3383 ( PCT/US95/07344 cysteine to bovine serum albumin with SMCC crosslinking agent.
This boost was repeated five days (IP), four days (SP), three days (IP) and one day (IV) prior to fusion. The mouse spleen cells are then fused 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-1 (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.
1~ VIi. Exemplary Process far Detecting OP-1 in Serum Presente3 below is a sample protocol far identifying OP-1 in serum. Following this general methodology OP-1 may he detected in body fluids, including serum, and can be used in a protocol for evaluating the efficacy of an OP-1 modulating compound in viva.
15 A monoclonal antibody raised against mammalian, reaombinantly produced OP-1 using 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-GelT'", from Bio-Rad Laboratories, 2fl Richmond, CA, prepared falloo:ing manufacturer's instructions) and used to purify OP-1 from serum. Human serum then was passed aver the column and eluted with 3M P:-thiocyanate. K-thiocyanante fractions then were dialyzed in 6tt urea, 20mM PO;, pH 7.0, applied to a CS HPLC columr.~ and eluted with a 20 r,~inute, 25-50$
25 acetonitrile/0.18 TFA gradient. Mature, recombinantly produced OF-1 homodimers elute between 2G-22 minutes, and are used as a positive control. Fractions the;: were collected and tesce3 for the presence of OF-1 b)~ standard immunoblot using an OP-1 specific antibody. Using this method OP-1 readily was detected in human 3~ serum. See also, PCT/US92/07432 for a detailed description of the assay.
IX. Considerations for Formulations and Methods for Administering Therapeutic Agents Where the OF-1-modulating agent identified herein comprises 35 part of a tissue or organ preservation solution, any commercially available preservation solution may be used to advantage. For example, useful solutions known in the art include Collins SUBSTITUTE SHEET (RULE 26) ~1158a solution, Kisconsin solution, Helzer solution, ~urocallins solution and lactated Ringer's solution. Generally, an organ preservation solution usually ppssesses one or more of the following pzoperties: (a) an osmotic pressure substantially equal.
to that of the inside of a mammalian cell, (solutions typically are hyperosmolar and have K+ and/or Mg++ ions present in an. amount sufficient to produce an osmotic pressure slightly higher than the inside of a mammalian cell); (b) the solution typically is capable of maintaining substantially normal ATP levels in the cells; and (c) the solution usually allows optimum maintenance of glucose metabolism in the cells. Organ preservation solutions also may contain anticoagulants, energy sources such as glucose, fructose and other sugars, metabolites, hea~,ry metal chelators, glycerol and other materials of high viscosity to enhance survival at low temperatures, free aa:ygen radical inhibiting agents and a pH
indicator. A detailed description of preser~.~ation solutions and useful compenent.s may be fauna, fcr example, in US Patent No, 5,002,95.
Where the OP-1-modulating agent is to be provided to an indi~.idual, 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 bs~ in,ectian to the tissue or organ locus) or systemically (e. g., parenterally or orally).
Useful solutions for parenteral admin:stratien may be prepared by any of the methods well known in the pharmaceutical art, described, for example, in Reminaton~s Pharmaceutical Sciences (Gennaro, A., ea.}, Mack Pub., 1990. Formulations may incl.vde, for example, polyalkylene glycols such as polyethylene 3~ glycol, oils of vegetable original, hydrogenated naphthalenes, and the like. Formulations for direct administration, in particular, may include glycerol and other compositions of high viscosity to help maintain the agent at the desired locus. Biocoaipatible, preferably bioresorbable, polymers, including, for example, hyaluronic acid, collagen, tricalciurn phosphate, polybutyrate, lactide and glycolide polymers and lactidelglycolide copolymers, may be useful excipients to control the release of the agent =n Vi VO.
SUBSTITUTE SHEET {RUL~ 26) ~19~.~8a3 i WO 95133831 PCT/US9SI07349 As will be appreciated by chose skilled in the art, the concentration of the compounds described in a therapeutic composition will vary depending upon a number of factors, including the dosage of the drug to be administered, the chemical $ characteristics (e. g., hydrophobicityy of the compounds employed, and the route of administration. Where the morphogen-stimulating agent is 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 storage!, the frequency with Which the preservation solution is changed, and the type of storage anticipated, e.g., low temperature. In general terms, preferred ranges include a concentration range between about 0.1 ng to 100 Ng!kg per tissue or organ weight per day.
6:here the therapeutic agent a to be administered to a donor or recipient, the preferred dosage of drug to be administered also is 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 pro~.~ided in an aqueous physiological buffer solution containing about. 0.0018 to 102 w/v compound for parenteral administration. 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 abouC 0.1 u9/kg to 100 mglkg of body 4:eight per day.
The invention may he 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 claims are therefore intended to be embraced therein..
SUBSTITUTE SHEET (RULE 26) WQ 95133831 ~ ~, ,~ ~ ~ ~ ~ -42- PCT/US95107349 SEQUENCE LISTING
{1) GENERAL INFORMATION:
(I) APPLICAPIT: OZF.AYNAK, ENGiN;
OPPERMANN, H~RMANN
(ii) TITLE OF INVENTION: METHODS AND CCMPOSiTIOtdS FOR MODULATING
MORPHOGEPIIC PROTEIt: EXPRESSION
(iii) NUMBER OF SEQUENCES: 7 (iv) CORR~SPOfIDEtICE ADDRESS:
(A) ADDRESSEE: PATENT ADMINISTRATOR, CREATIVE BICP~LOLECULBS
INC.
(B) STREET: 45 SOUTH STR~ET
(C) LIT:: HOPF:INTON
(D) STATE: M~
(E) COUNTRY: USn (F) ZIP: 07148 (v) CCM?UTE.R READABLE FORtd (:-.) M~DiUtd TYPE: Floppy disk (B) COMPUTER: IBM PC Compatible (C) OPERATIPd~~ SYSTEtd: PC-DOSIMS-DOS
(D) SOF'TrdARE: Patentin Release =1.0, Version =1.25 (vi) CURREPIT APPLICATION D.'-.T:-.:
(A) APPLICATION ivITMBER: US 07!938.021 (B) FILIN~~ DATE: 28-AUG-1992 (C) CLASSIFICATION:
(Viii) ATTORtdE:'iAGEfIT ItdFOR1~'LTION:
(Aj NAME: KELLEY. ROBIfI D
(B) REGISTRATION NUMBER: 34.x37 (C) REFEREIGCElDCCF;ET fTJhiBER: CRP-091 (ix) TELECOMMUfIICATION INFOP,fIATI0P7:
(A.) TELEPHONE: {5D8)-435-9001 (B) TELEFAX: (508)-435-4992 (2) INFOP,N'.ATIOtI FOR SEQ ID NO:I:
(I) SEQUEPICE CHARACTERISTICS:
(A) LECIGTH: 17415 base pairs {B) TYPE: nucleic acid (C) STRAtIDEDf7ESS: single (D) TOPOLOGY: linear {ii) MOLECULE TYPE: DNP. (genomic) SUBSTITUTE SHEET (RULE 26) ~11~8~3 ~ WO 95133831 PCTlUS95l07349 (vi) ORSGItdAL SOURCE:
(A) ORGANISM: homo Sapiens (xi) SEQUENCE
DESCRIPTIOC7:
SEQ ID
NO:1:

AGACCTGCTGCCCCTGCCCCCAGCCTCACCTGCTTGTG:vj.GATCCCTCCAAAAGATTTGA24p GAGTAGATAF:AFAGCAGAGACTACTACTGAAG~_'-.CAGGGCTGCTTTGGCTCCTT?.TTATT300 TCAGACTTTGGAF.GkAAATGACCTCCTTTTTCTCTACTGGCACTGAGTGCATAGCTGACC360 TAGCAAGCCAGGCCTGGAGGGCGTGiGCAGGGCTGGGGACCGAGCCTGGTTTCTGTTCCC42G

TGCTCTGCAGCTCAAGCACTTGCTGTTCCTCCACCTGGGATGCCTTTCC~CTGGArl'.1:-,GCC480 TGTCTCTTTCTTGTCTTTCAGGACTCAGGTCAGTGGCATCTCCTCCAi,iaAACTCCCCTTC54G

GTGGCACTCCTGCAATTTCCCCAGTCTCCCTGGGG~tAGGATTCCTGCTTGCCAGGATGCC720 CACCTTTCCTTCTCCCTCCTGCATGTCCTCCTCTGCCTGGCTTCTGAATTGTTTCCAGAG7$D

AGAGTGATAGACAAGATCTGCCTCTCCTTCAGTCCCTGAATCTTATTTnAGGCTCTTGCT840 ACTGTCCAGGTTGCAGGCCCCACAGCTCGCCTCCTGATATCCTGTGCTCCATGCT'IGTCT114Q

SUBSTITUTE SHEET (RULE 26) WO 95!33831 ~ ~ ~ ~ ~ ~ '~~ PCTIU595I07349 _ qø _ ATAATTCTCTTCCCCAG~~TCTCCTGCTCCAGGAAGGACAGCCCCA.4P.GTGAGGCTTAGCA1500 TTTTACCCCGCTGCCCTCACTGCCACTCTGGGTGCAGTAesTTCCCTTGAGATCCCACACC1680 GGCAGAGGGACCGG'IGGGTTCTGAGTGGTCTvGGGACTCCCTGTGACAGCGTu"CAT(iGCT1740 CGGTATTGATTGAGGGATGAATGGATGAGGAGAGACAGGAGAGGAGCCCGATGCGGAGGT1$00 ACTGCCACGAGGAGGGCTGATGGGAAGCCCTAGTGGTGGGGCTGGCa3TGTCTCCTCTCAG1920 GCTGAGGGGTGGCTGGAAAGAT:-.CAGGGCCCCGAAGAGGAGGAGGTGGGAAGAACCCCCC1980 CAGCTCACAC GCAGTTCACT TATTCACTCA ACAP~TCGTG ACTGCGCACG TACAGTGGCT 2046 GGCCCCAGTG CTCTGGGTGT CTAGCGGGGG TnAGiv,GGCA ATAnAGAAGG CACGGAGTAA 2160 CTCAA.'~.CAGC AATTCCAGP.C AGCaAGRG~i ACTnCAGG:L~. AGAA.~'.ACAAA CGTGCGAGGG

GCGAGGCGAG GAP,ACAACCT CAGCTTGGCA GGTCTTGGAG GTCTCTGGGA GGAGAAnGCA 22F.,C~
GCGTCTGATG GGGGCGGGAG GTGGTGAGTG GGGAGAGGTC C:.GGCGGAGG GP=.TGGCGAG 2340 i,ACJ'ATACATGGGACTA:~'GGGCAAGACCGGCTCAAGGTCACCGCTTCCAGGACCTTCTAT2460 TTCCGCGCCACCTCCGCGCTCCCCCAACTTTTCCCACCGCGGTCCGCAGCCCP.CCCGTCC2520 TG4TCGGGCC GCCTTCCTGG TCCGGACCGC GAGTGCCGAG AGGGwAGGGC CGGCTCCGAT 2580 TCCTCCAGCC GCATCCCCGC GACGTCCCGC CAGGCTCTAG GCACCCCGTG GGCACTCAGT 2fi4G

GGP.AGP:.AGGA GCGCTCGCCC GCCCGCCTGC CTCCTCGCTG CCTCCCCGGC GTTGGCTCTC 3006 SUBSTITUTE SHEET (RULE 26) 2~~~.~83 W0 9513383I PCT/US95l07349 GGCCGCCTGCCCCCTCTGCCACC'IGGGGCGGTGCGGGCCCGGAGCCCGGAGCCCGGGTAG 3346 AGCGCGAGATCCTCTCCATTTTGGGCT'I'GCCCCACCGCCCGCGCCCGCACCTCCAGGGC?.3540 GCGGCGGGCCCGGCGGCCAGGGCTTCTCCTACCCCTACT-.AGGCCGTCTTCAGTACCCAGG 3660 GCTTCGTCAACCTCGGTGAGT~,.=.GGGC?.GGCG.=,GGGTACGCCGTCTCCTTTCGGGGGCi,C3180 TTTGAGACTGGG:,GGGAGGGAGCCGCTTCTTCTATGCAGCCCGCCCAGCTTTCCGCTCCT 3$40 GGCTGAAATCGCAGTGCCTGCCCGAGGGTCTCCC.=.CCCACAGCCCTATGACTCCCAAGCT 39GG

GT~TGCGCCCCCAGGTCGGGCCGCTGGGTCGGTGAGCCTGTAGGGGTTACTGGGn.AGGAG3966 GGGCGTTCia=.AGCGCGGGGCTCGGTCATGTG:,GCTGTCCCGGGCCGGCGCCG.~,TCCCT'~.'-.4680 CCTGGATGTAAAGGGCCCTTCCCGGCG.'-.GGCTGCCTTGCCGCCCTTCCTGGGCCCCTCTC 4140 AGCCCTGCCTGGCTCTGGCATCGCGGCCGTCGCACCCCCTTACCCTCCCTGTCA.sGCCCT42GG

GTGCCTCAGCCTCCCGAGTAGCTGGGATTACAGGCATGCGCACCATGCCTGGCTnATTTT 4560 .

CAGGTGATCCTCCCGCCTCAGCCTCCC:-~AGTGGTGCTGGGA"TraCAGGCGTGAnGCTGT 4680 SUBSTfTUTE SHEET (RULE 26) 21~~~8~
W09S133831 ~ PCTIUS95l(173,t9 ATTTTCAGCATTTACCACGTGTGGCGCGCAAACCACAGGTTTTGGCGATTGGGTTGCGCG 4$00 GGATCTCAGAGCTGACGACCGCGGGGGCCTGGGGGTCCCGGTTTCCGACTGGAGCCGCGA 4$60 GTGCGCGCACATTCTCCAGACT'TGCTCAAACTAACCCCCCGGAGCAGCGCACGGGCTGGG 49$0 CGCTTTCATTCTACTTGTGTAACTTGCTGCGAAF?.CCCGAACCAAGTCAAGACAGCAAAC 5100 TCACGCCCACGGGCC'SGTGTCAACATGGAAATAATC:,ATACTGAAGCCCCACGCTGGGCAC 5160 CTGGGGCGTGGACTGGGC.GCGCGGGGGPhGCGCAGATCCGCCTTCATGCTTCCCCTCCTC 5220 CTGAT.=,AG3TCCCTGGAGTTCCCGGGA~vCCAT.TGTCTGT.=.CTTAATAnTA::CT?..~.ATCCn52"d0 nCTAGTGA.'-1CCAAGCTTCAGCGAGGCAAGGG~.GGG:-,GGTT'I'AGATGCCc'~Ai,ATi?.CCTTS.i40 G

C~,.GTT TAAATTATACTAAGCAGCCAGTTAAGAAGGA.nGCAGCAATATATGACCTG 5406 ATTTAGAi,C'CATCTCC4'~.Gi~TG~.~aTGnGGTGG:..=.sGA~,GCivAG
GTGCAG?.TGAG'IGGGCT5460 GCATGTGTGCTTGTATATCATCGTGTCCTCCTG.i:GG:~.?GACACCAGG:"-iCTGGAGAGAG 552'D

ATTT'TACTGGAGGGGTATATGGCGGGGGCATAG~.~.T~vGGGCTTr.CGGAGTGGG:.GGTGGGG5580 TCTGATTTTTCGTCGTCTGCACTTCTGTATTTGTGATTTTTTTTiIAACAATGTGTATTTA 5650 TTA.nCTATACCt';F.A~AATAAAGGAAAATTCC .=..=,TnCAT:,C::T~.TP.A?,TAATGAeICCGC::5'r GAGCTCTGTCGCCCTCCTGAAGCCTGGGGTTi~GCCAGG'~CCCTTTCTCTGGTGGGGGATT 5760 TrTAGCATCTTCCCTTCiGTTGGGTnCCCCGGnCTCCCnCTG.~,=.TGTGCAGGTCCCAGTG 5826 GCTGCCTTCAGAGCCTGGCTGGAATCATTAn~H~THTT TGT:..TCTCTGGCTTCTGCA 5$$0 GAAGGCCC'IGCAAACCAAGAGCAAAAAAGCCCCCAGTGCTTATGGGCCGGCAGTGTGGGC 5940 nACTTTGGTGCGGGCTATTTGCTCCCCCCATGGCGGCAGGAGCAAGCTGGGACTTGTTTG 6060 GGAAGGCCACAGCTGGGTGG?":TTCCTCCTCTGGCTGTACATACACCTTTCAATCCATTT 61.20 CT'TTCATCTTGAAAGGACAAAGACCGGCTTGTCTGAGCCTCTTAATCAGTCAGGCTGGCT 61$0 TTGGGCTTTuGGGACCC~GACTTTCTCAGGTCTAGCTTTCTGC~ACATCACTCC.~"L=hTTA6240 GA'lGGCAGAGTGGCTTT'TF.ACAGAGCGCACTGACCTTGTTTTCTTTCTCTCTCTGTCCCT 30U

SUBSTITUTE SHEET (RULE 26) 2~.~~.58'~
~ WO 95!33831 PCT/US95/07349 AGATGCTTCTAATGTTGGCCTTTAitTTTCTGCTAAGCAGCAGCACACAiviTAAATGGCCT 6420 TGAACTGGATTGAGAGGAT~GAACAATAGAAGGAGGATATGGCTCAGGACAGTCAAGTAC 6660 TGGARGAGGGAAAGGTACAAAGAGGTGTTGGCACTG.AATGACCCTGAACAGGGCTGCCCT 6720 -GGAAATATCAGAGGTGAGTGACA.'1AGAGAACT'CTAGTCGAGGTCTGGA."-..GTCF.ATTATT6780 A

TCCAGGAGCGCTGAGGTTCTGGGAATTCCCAGTGCTGGCTACCATGCCATTCTT'TTCTCA6906 TTCACTCAAGAGCGTATTGGGATATGCGTGCATGisAisGCAATGTAATTATGGGCACARCC 6960 TC:vISACCTGCTCTAATTTTTTTTTTTTTTGGAGATGGAGTCTCGC1CCTCACCCAGGC 7620 TGGAGTGCri.,TGGCGCGF.TCTCAGCTCACTGCF.nGCTCi,GACCTCCAGGGTTC.=.CACCAT7086 CCTGACCTCGTGATCCACCCGCCTCGGCCTCCCAA.~aCTTCTGGGATTACAGGCGTGACAG 7260 CCGTGCCCGGAATCTGCTCTA.'-~TTTTTTFLnAGATATCATTTGCAAACTTTGGGCACTTGA 7320 GTCACTCAGTAAGATATTATTTACAACCCCACCATAG CAAACCTCTGTCCTAGAATG 73$0 ATT

TTGTCGAGTTAGGCATCTGGCTTGCAGCiv.CAGCTGGCTTTCCTGTCTATGCTGTCTCCT I44G

TCCAGGGA:~ATGTTTCACC'CTTCATATTGAGGe,.AATGGGCACAGAGAACCCATTTCTCT 7500 TGACCTCAGTCTCCCAAGTAGCTGGGACCATAGGCAT'GCACCACTF"-.TGCCTGGCTkATTT 7746 AACTCCTGGACTCAAGCA.ATCTTCCCACCTTTGCCTACCAGAGTGCCGGGATTACAGGTG 7860 TGAGCCATCATGCTAGTTGCGCACAGTTGGGCG:,suACTGACAGATGAGAAAGCAGAACCT 7920 SUBSTITUTE SHEET (RULE 26) ~~~~i~3 R'O 9if33$31 PCTIUS~SIQ7344 , - 4$ -CGTGAGTCCA CfiCAGTAAGA GACTCCCTAC TTTCTTTCTG AGTCTTTGTT TCTCATCAAT 7980 TGAATGGCAA TAAACAACTT GGTGGCCCAA GAGTTGATGA CAACAGTCCT ATAAGATTAT. 8040 ACATGTA~ GAAACAGAGT ATTCTAC:-.AA TATCAGTTAT TGATAGTTCA ATAGGCAACC SIQO
TGACATTACC TTTTCTTGGA ACTTGATGAA.CAACTCAGAA ACfiCATTAAT ATCAAACCCA 8I6D
ATGGTGAGCA CTTGGT~TTT ATTTATGGCT GTAAGAGAAG AAATTGAATT AACTCTATGT 822D

AAGCTTAGAJ, GTGAATTCTA CTTACTTATT ACTTAAAAGT GGTTCTCAAA CTTCAAGGTG 8400 A.ATCR3,F,ATC ATCTGTAGAG CTTGTT1A.=.A CACAGGTTGC TGGTCCACCC CAAGAG2'G'TC 8460 TTGAGTCAGT AGGTCTCAAG TAGGGCTCip.i Giv,IATGCAT TTCT?.FtTGAG CTCCAGGTGA 8520 GT~,".2'ArIGTGT TAGTCGTCGG TCTTGGGACC ACAACTTTGG Giv:CAATTGA TTTAGr'.AGAA 8580 CTC:v=sGATC AGAAfiGGGGT G'v'?.nTrlT':TT Ti:w,.aTTGTG GT~~a:o.4TACG -CnTkA?,CAGA 864D
i,Ar",GCTACeI'~. TTTTAACCAC TTAG AGAGAG GTGGGATCTA AGAACAG~ fi'A'GTTATGCC 870D
ATCAAAGGTGAGTTCAGATAAGC.TTATTAAnTGGTATCTATGGATAAF,CTTCAGGGGCC$760 CTGTGGAGCACCC."~ATG~TGGGATGGGGTCCAGGTGTGCTATGGTTTG,~>ATGTGGTTTG8820 A

TCCCTACAA'y.AACTCATGTTGA.=u~.TTTAATTGCC:,GTGTAACATT:,TTGnGAGGTT:,TGG8880 ACTTTTAAGAGGCATTTGGGTCATGAGGGATCCACCTTCR.GGG."-.TTAG1'~CAGTCTCCAG89A0 GGAGTGAG'iGAGTTCCCATTCT."-nGTGGGACTGGATT."-.GTTACCAT?.CAGTGGT7C;TTATA9000 AAGTGnG-~CT GCTTCTGGTG TTTTa.TCTGT TTGCnGGCAC iTCCTTCCCC TTCCnCTTCT 9060 CTGCCAGGTT AGGATGCAGC ATGAGGCCCT CACCAGiuIGC TGACCAGATG TGGC'TGCCTG 9120 ATCTTT.,P.?.CT TCCCAGTCCC CAGAACCATG AGCTAAATAA ACCTTTT'TTC TCTATAAATT 9180 GAACACTACT GACTTC4'CCC ATACTCTGGC CTATGGACAA GAGTGACAGA CAGACAAGAG 93D0 CCAGTGAC'IG CCAGCTAATG CTATCATAGA CCCCACCTTT CCCCTGACTT GATTGGACCA 9420 GAAGCACCCT CCTGA2'CCAT GGCC'AACAA~.' CAGATTCACT TTCAAGAATT TG.--v'~CTAAGA 94&0 GACACTAGGA AGATGGCCCT TGAGCTGTGA GTCCTACACT TGA?,AGTTCT TAGCATCTTG 9540 SUBSTITUTE SH~~T (RULE 26) '~ WO 95133831 PCT/US951073~t9 CCTCTTCRGC AATGGCCCRC TCRCCTTCRA kRRGGCTGAA GAGCAGACTG GCTGGGTTCT 10020 TCRTGGTGGA GGGGCAGTCT GGGAGGTTI~T AAGGTTGRAG i-.TGRAAACTT TCRCTTTTGG 10080 CTCAATGGTC TGAAAAAGAG AAGGRCCAGC nRGTGA.4CTG RAGCCTCCTG GAnAGCATCT 10140 TGRT?_?CAGG GGCAGRGTTT CRRGATGRGA AGCTGTGGCA CTTRCTCTGG CTTTGGRP.AT 10200 ACARTTTTCR ATRACATRGT CTACCCTCCC' CTCCTTCCCC CRCCTTCACC TCTTCTTTCR 10320 TCACRGGCTT ACAGGGCACC TCTTRGAGCC AGGCACGGTG TTGGGATCRG G.ARCRRGGCC 10380 RCTGCTCRCA TCCAGAGCCT GTGCT:=.CTTn AG.:,'nGCTTCC i,GGACCTCTT GGnTGGCTGT 10440 GGTTAGTGCC CTRCTTTTCC CAGCAGGTTG GATGCAGRnT CATGCTCTTG TCGTTCRGGA 10500 TG.=.CCRTGGG GACCATGGGT CTGAG~CCTGT GACCCTCCAG TCTRCAGTGT GTTGGTGAGG 10560 AnGGAGCAGT TGTCACTGGG GTCRCTGGCA ATGGGCRTGC CTCCATCTAG CTTAGGCAAG ~ 10620 RTGCTTAGAC TCRGAGCCAG AGnGTGRi,.~C CCAGACi:CTn RTGRGCTGTC GGTGTTGCTG 16580 ATCGAGAGTT CCGGTTTGRT CTTTCCAAGA TCCCAGAAGG GGRAGCTGTC ACGGCAGCCG 10$00 A=.TTCCCGAT CTACAAGGRC TACATCCGGG RRCGCTTCGA CAATGAGACG TTCCGGATCA 10860 AGGTGCTGAG TTTCCTCTGG GGGCAGAGGA AGAAGG2C,GT GRGGGTTTCC CTCCCCTCCC 10980 CTCTGGGCCT CGGAGGAGGG CZVGCTGGTG ?'TTGACATCA CAGCCACCRG CAACCACTGG 11160 SUBSTITUTE SHEET {RULE 26) Wfl 95133831. ~ ~ ~ ~ ~ ~ '~ P4TIUS95107349 TCCCCCGCCA CTGCCAGTCC TAATGCAGCC TGTGCTCCTG GACTTCAGGA GGGTCTCAGC 112$0 GTGCACTTGG CiGCTTCCTG TCCAGGGCAG ACGATCAACC CCAAGTTGGC GGGCCTGATT 11460 GTCCACTTCC GCAGCATCCG GTCCACGGGG AGCAAACAGC GCAGCCAGAA CCGCTCCAAG 115$C
ACGCCCAAGA ACCAGGAAGC CCTCGGATGG CCAACGTGGC P.GGGTATCTT AGGTGGGAGG 11640 GATCACAGAC CCACCACAGG P-"yCCCAGCAG GCCCCCCCGA CCGCAGGAGA CTGACTAAAA 11700 TCATTCAGTG CTCACCFAGA TGCTCTGAGC TCTCTTCGAT TTTAGCAAAC CAGGAGTCC~11?57 AAGATCTAAG GAGAGCTGGG GGTTTG=.CTC CGAGAGCT~~G :,GCAGTCCCC .'-=.GaCCTGGT 11&2G
CTTGACTCAC GAGTTAGACT CCACTCAGAG GCTGACTG~-'C TCCAGGGTCT AC~,CCTCTi~,h 11$$0 GGGCGACACT GGGCTCAAGC AGACTGCCGT TT'TCTATATG GGATGAGCCT TCACAGGGCA 11940 GCCAGTTGGG ATGGGTTGAG GTTTGGCTGT r.~..."~~CATCi,G.n ?,.~:CCCA.AGTC RnATGCGCTT

CP.ACCAGTAG AAAATTCACC AGCC~CGCi:G:~. GCT:..GGTTG GGTG >ACP-.TT AGGGTTGGTT

GATCCAGGAG CTCAACAGTG TCCTCTGAGC CCCAGCTCCT TCTGCCCCAC CCCACCATCT 1212'0 TCACTGCTGC TTCCTCTCAA GGCCACAGCT GTAGTTGGCC AGGGGGGCTT CATTA'rT'TT1' 121°C.~

TGGGA.nCTTT TTCCAG»nGT CTCTATGTCT TTTAGTTT'vT GTTGGGTCAC TTGCCCTTCC 1230D
TGAACCACTT CCTGACTCCT GG.=:C.'wGGnT'G TGCACTG.iTV i~'GCTTAGCTT TGGGGi,TCTn ATAGTGACTT TACAAAGCCT CTTTGAGn?.G GTGACATTGG AACCn.AGGCT TGAGCAGACA 12420 CAACAAAGAT TGCAGGGAGG GGCATTGCAG GTGGAGGAAA CGGCACATGC AAGAGCCCTG 124$0 CGTGGGAGTG AGCTTGGTGT TTGGTCAATC AGTIuTCAGA GCACACCGGG CCC'IK;TCAGC 12546 AGGCACAGCC TGGGCCTGCT Ci'GAGTATGA CAGAGAGCCC CTGGGAAGTT GTAGGTGGAG 12600 GCAGTGTGTG TGAGAGAGAG ACAAGACAGA CAGACAGACA CTTCTCAA'fl:.. TTTACAACTG 12720 CTCAGGCCCT GACCCGAAiC, CTTCCAi,ATT TACGTAGTTC TGGiSAAACCC CCTGTATCAT 127B6 SU~STfTUTE SHEET (RULE 26) - 51~

TTCGATTCCC TTATGGAATC CAGGCAGATG TAGCATTTRP. ACAACACACG TGTATRAAkG 13080 CAGGTTAGP.C CCTCGRACTG GCTTTTGRRT CGGGA.'yt-.TTT ACCCCCCRGC CGTTCTGTGC 13260 TTCATTGCTG TTCACRTCAC TGCCTAAGAT GG:4GGA.ACTT TGATGTGTGT GTGTTTCTTT 13320 CTCCTCACTG GGCTCTGCTT CTTCACTTCC TTGTCAnTGC ACACAnCAGC AGCRGGCACC 13380 RGAGGCAGGC CTTGTRRGAA GCACGAGCiG TATGTCRGCT TCCGAGRCCT GGGCTGGCAG 13440 GCCTCGCTAG GGAAGGGGAC TTGGGCTnhG G=.T1C.'-.GGGn GGCCTCnTG~:~ F,.:aTUCGAG,vG

CAGAnATGTG GTTGAGRCTT G?P,CTCG:~s.>C CP:.;GAACCCA AACA~~TTTGG nCT~~TGAACC

TTCCTGRACC AGTCAACTGT TTCRGGGGTG GGGTCATGTT P.CTGGCACAT GGCTGCCCCC 13926 TCTGGAGCCR TTTGCRTGGA GTGAGGCAAA AGGCAG~vGG.'-a TGRATCTnGG AGAGGRGTGA 13980 CRGGGCAACT GGTvGTRTTC TRCCCTCAGG GRGCTTGTAG TTCAGTGGGA GAGTCCAGAA 14226 GCCCTCCTGT GAGCCTGCCC TCAGGACTGA TG.ACCACATC TACCTGCRGC TGGGACRG.=.A 14346 CCCP.AACTCC AGGGGCCTCT GCTGGr.AGAT TCCATGTGCT TAAGCATCAC TGRGGRGTRT 14400 SUBSTITUTE SHEET (RULE 26) ~~.~1 >8 W 0 95133831 PCTlLI5951073.19 GGGCAACATT

CTCTCTGGCAGGTGGGCAAACCGAGGCATGGAGGTT'Z'GTTTAAGGTGAACTGCCAGTGTG145$0 TGACCACCTAGTGGGGGTAGAGCTGATGATTC~CTCACACCGCAGGCTCCTTCCTGTGCC14640 GCTTTCCTTCATTTTTATTTTATGTTTiTTTAGAAATGGGGTC'Pc'GCTCTGTCACCCAGG14760 CTGGGGTGCAGTGGTGTCATCATACGTCACCGCAGCT'I"i'GAGCCGTCTTCCCACTCAGTC14820 TF.CTAAGCTTGGACTATAGGCCAAGACTATAGAGTGGTCCTTCTTTCCATTCTTTTGGGA148$0 CCATGAGAGGCCACCCATGTTTCCTGCCCCTGCTGGGCCCTGCTGCTCAGAAGGCATGG2'14940 CTGAGGCTTTCRCCTTGGTCGTGAGCCTTCGTGGTGGTTTCTTTCAGCATGGGGT'I'GGGA15000 ATCGCGCCTGAAGGCTACGCGCGCTACTACTGTGACGGGGAGTGTGCCTTCCCTCTG:,itC15120 TCCTACATGAACGCCRCCAACCACGCCATCGTGCAGF.CGCiGGTGGGTGTCACGCCATCT151.$0 TGGGGTGTGGTCACCTGGGCCGGGCAGGCTGCGCf'.,GCCACCAGATCC'IVCTGCCTCCAAG15240 CTGGGGCCTGAGTAGATGTCAGCCCF.TTG.~CATGTCnTGnCTTTTGGGGGCCCCTTGCGC15300 CGTTAAF,FF.A"~r'".TCA:v'-vAAT'IGTACTTTaTG='.CTGGTTTGGTJnT?.A:,G:,GGAGTnTA.BT:5366 CTTCGACCCTGGAGTTCATTTATTTCTCCTAATTTTT:vvT,GTAACTAAA:.GTTGTATGGi:15420 CTCCTTTGAGGATGCTTGTAGTA~TGTGGGTGCTGGTTACGGTGCCTAAGAGCACTGGGC154$6 CCCTGCTTCATTTTCCAGTAGAGGAAACAGGTAAACAGATGAGA.naTTTCAGTGAGGGGC15540 ACAGTGRTCAGAAGC:'i<sGCCAGCAGGATiu,TGGGATGGAGAGATGAG3GGGGACCCATGG15600 TGGTTATGTAGCTCCC'IGTCATGAACATCATCTGAGCCTTTCCTGCCTACTGATCATCCA15$40 ATCTCATCTATCCCTTTCATTTGATTCTGCTCTTTGAGGGCi~GGCGTTTTTGTTTCTTTG1.6020 SU$STITUTE SHEET (RULE 26) '~~~~.5$

knGCCCTCAT TGGGATTGAA GGTCCTT."-.GG CTGGnAACAG AAGAGTCCTC CCCAACCTGT 16140 GGGCAACTGT CTTCTAGAGA GTCCTCATGA A.4TGCTTCCA GAGGAAATGA GCTGATGGCT ' 16440 GGAATTTGCT TTAAAATCAT TCi,AGGTGGA GOAGGTGGGG AAGGGTATGG ATGTGTAAGA 165C0 GTTTG.AF.ATT GTCCATCATA AAATGTGTAn A.°vlGCA~1GCT GGCCTATGTC

GOCTGGAGGT GGTAACAG.-"eG TGCCAGTCA.('.. TGATGCTCAA GCCTGGCACC TACAGTTGCT 16620 G:,.'-..=.ACCCAG AAGTTTCACG TTG.AA:i?,~4AA CAGGRCAGTG GA."-,TCTCTGG CCCTGTCTTG

e'.AOACGTGGC AGATCTGCTA ACACTG~AT~:.T TGGTTGGCTG CCGTCAGCTT AGGTTGAGTG 16740 TTGCTTATOA GTvGACCTCA CGAGGCACTO ATAGGOATTT GAGTOTATGT GTOCCTGTCC 16866 CAOATCCTOT GTAAGGTGOn GnGAAGT~~Cn TGAGCAAGAT GG.=,GCnCTTC TAOTGGGTCC 16920 A=.GTCAGGGA CnCTATTCAG CAATCTACAG TGCACAGGGC .~'eGTTCCCCAA CAGAGAATTA 16980 CCTGGTCCTG AATGTCGGAT CTGGCCOCTT CCTTCCCCAC TGTATP.ATGT GA:,.=:?.CCTCT 17040 ATGCTTTGTT CCOCTTGTCT GCAAAACAGG GRT:-.ATCCCn G?.?.CTG.AGTT GTCCATGT:-~. 17070 AGTOCTTAGA ACAGGGAGTG CTTGGCTTGG GGAGTGTCAC CTGCA.GTCAT TCATTATG::C 17160 CAGACAGGAT GTTTCTTTAT AGnP.ACGTGG AGGCCAGTTA G.=,?.CGACTCA CCGCTTCTCA 17220 CCnCTGCCCA TGTT2"IhGTG TGTGTTTCAG GTCCACTTCA TCAACCCGGA AACGGTGCCC 172$0 (2) INFORMATION FOR SEQ Ip N0:2:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2298 base pairs (E) TYPE: nucleic acid (C) STRAf4DEpNESS: single SUBSTfTUTE SHEET (RULE 26) WO 95133831 ' ~ ~ ~ ~ ~ ~ ~ PCTlU595107349 (D) TOPOLOGY: linear (ii) MOL~CULE TYPE: DNA (genom'ic).
(ix) FEATURE:
(A) NAhIEtKEY: misc_feature (B) LOCATION: 1..229$
(D) OTHER INFORMATION: mote= "MOP1 UPSTREAM SEpUENCE"
(x1) SEQUEtQCE
DESCRIPTION:
SEQ
ID-N0:2:

TGCATAGGTCACACATCCCTCCTCTACCCAAGGCTAGCC.AGGTGCCCTATCTCTCCCTTC60 TTGCTACCCTTCTGTTCCGGACCTTACTGF.AGGGCCTTAGTGTTTCCAGGGGC:.'Ci,i;GAn1$U

CCAGGTAGCCGTGGAGG CCATGv~1TGCCTGCCCACTCn.C.C:.ii-~GT'L'CTi;i;TGvCT~'':~4C
1~TG

GCCTGTGTGTGGCACCCATGCAGGCCACAGAAGGCCAC.zCA,C'r.GCCTTCAGGATGAGGCA~U(;'~

G3GCCCCTTTGTTTATTCAnTATCAAGAACTGTF.ACGTGGTCACCGGAGGTCATGTCTCC36U

A-GCTCGCAGCCTGCTTGGCCTCF.GATCACCCACCACAGCAGGTCCAGGGAGGGGCCTCTC420 F.GGTCTGCACTGGGCCAGGGACTCAGTACTGGTGGGCF.TCCAAGGCCTGGGCTAAGACCT98U

GCF~r.GTTTCTTTTAGCCCCTCAGACAGTCACATC.'7CCTA.=..AATTCCTACCF;PiGGAGCCCT560 GAGAGACCTAGGTAGTTATCTCTGTTCC.--.GGAAGCCTG:.inF,Gz,CCAGGC'I'TCCC'ATCTCn60U

CCCTAGGACTTCAAGAGGGACCCCCTACTCA:SGGCCCTTCCCCF.GCCCCTF.CTTL:CCF.TT66u TTACCACCCC'TGAAkCGCTTGCTTGTCG~;CCACCTTCAGCA:Y.GCRGG.=s.=.GCCTGGCTCA72C

CCATCCCCACTCACTCACTGCCATTCTGGGTGAAGGCTGCT~TGCTCCCATTTTTCAGAT7$D

TAGGAAACGGAGGCTCCAFr'.GAGCAGCP.nTCC'ACTGAGAGACCCAGTATCTGTCTGGGAC$4C

GTTTCCTCCTGGGAGGAGAGGGAGGCTAGTCCTTTG.AGACAGGAAAATCGAGTCGGGAGC900 GGGGTTATTAGACAGiGAGGTACCAAAGGACTCATGTCAGGACCCCGCCCCCCCAAGAGA1D20 GGAGGGGGTCGGAACATTCTCTAGTCCCAGATTTCACTTATGTACTCTGTAGAGCTGCAG10$0 CTGGGCCCGGATCCCTTGGGTGCTGGACTTGAGGGGAGGTGTGTGTGTGTGTG:'GTGTGA1200 GTGTGTGTGTATGTATGTGTGTGTTGGGGGAGTGAAGTGTAGAAAGAACTTTATC'ILCAC1260 SUBSTITUTE SHEET (RULE 26) 2~ ~~.58:~

GGGAGAACGT TCCCCGAGGAACAAAAGCCAGGATAGCAGAGGGGCFaAGCG GTGGGGGTAC13$0 GGACACCCGG TGGGCACTCGGTAA.ATATTTGTAGAGCGCCCTGGGAGGAA TGAATGAAGC1680 GGGGCGGGGA RGTCAGTCCTCCCGCTCCTCCCCCGCTCCCCGGCCCCAGC GCGCCCF-$CT1$00 CCGGGGCTCC CGAGGCGGCGGGCGGGCGATCCGGGCGCGCAGGGCCCTTG TATTGGGCAC1$60 GCGGGAGATC GGAiviGGGGTTTGTTGCTGGTGCCCGCGGGCCTGAGCGCG ATCAGAGCGG192D

GAGGAGGGAG CTAGGGTTCGCTCRGCGCCCAGCTGCCTCTCCGGCACTCG CTCTCCGGAC19$0 CGGGCCAGA.n. CTGAGTAAAGGACRGGGGCGTCCCGGGCAAAGCGCAGCCG GCCGGGGAGT216D

GGCCATGTGT GGCGAGGCCGCCTTGRRGCTCGCCTGCAGCP~GTGRCCTC CGGTCGTGGA2220 (2) INFORMATION
FOR SEQ ID N0:3:

(i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 2997 base pairs (B) TYPE: nu cleic acid (C) STRANDEDNESS: e singl (D) TOPOLOGY : linear (ii) MOLECULE TYPE: DC7R
(genomic) (ix) FEATURE:
(R) NAMEJKEy: misc,_feature (B) LOCATION: 1..2997 iD) OTHER If7FORhuF,TIOf;: Jnote= "MOP1 TERMI2~1J,L SEQUENCE"
SUBSTITUTE SHEET (RULE 26j '~~~.~~~
WO 95f33831 PGTIU595I07349 (xi9 SEQUENCE
DESCRIPTION:
SEQ ID
N0:.3:

TRGCTCTTCCTGAGACCCTGACCTTTGCGGGGCCACRCCTTTCC~TCT TCGhTGTCTC 6C

CCTTCCCCTCACCTCCCCAACCGGAAGCAT~GTAAGGGTTCCAGAAACCTGAGCGTGCAGG 1$0 CAGCTGATGAGCGCCCTTTCCTTCTv~GCACGTGACGGACAAGATCCTACCAGCTACCACA 240 CTGGCGCTCTGRGTCTTTGAGGAGTAATCGCAAGCCTCGTTCAGCTGCAGCAGF,AGGAAG36U

AATGATATGTCACAATAAARCCCATGARTGRA'TGGTTAGGATACAGATATATTTTCCT 480 Fu',e',CA1TTTATCCCCGTTTCTTGGTTThT'ICTGACTTTGTI-w,'~r'aGAAR.'iGCCGGGGCTG 540 CCCAA'-1TCCRGCCTGTAGGGAGG:aGGRGGhGGhTGTCTGCTCRGRAG.-;GGCC~,GTGAy..G660 ATGTGGCCTCAARGGGTGTTGGGiaTG~.'--.GnTGGAGGGAGGTF.2T:CRTGChChCACACACR 720 CACACACACACRCACACACAC:~TGCATG=.TACACRCRCACACAC:~CRCACACACACACG:~780 TGCACRCACRChCACACACACRCACRCACACACRCACGC?.CGCACGCACGCRCAC'.GCACG840 CATGCRTGCRCACACACACGCACRCnCAC.'-.TCTG1GCG~ATGTAGACT'PTGGARTGGCT 9G0 CTGCCAGTCCCTCAGCCCCAATTCCTGCCCChiVGT:,~~G1,AA.TCChTGAGn:.is.'-~GC'ritlisG9n0 CThACAAGCRCAGCGGACCCTACCTGAGGAAGCRCAGGGGATGCRGGCTCTTChGGACAC li;~U

TGTCCTCCkAACAAGCCCCCTCTGGCACCTCTGTGGCCGRGCTCCGGAGCCAGGTCC~GG 10$0 CCTTCRCAGCTGCCTCTCTTCACTCTCAACCCTAACAGAAGGTTCTGCGACAGA'I"I'GGTT114U

TCCTGCTTCAGAGGTGGGATGTTGGTTATGGCTCAAACAAGGCCTCTCTGCCTGAGiTTG 1260 CAGAGCCCCAGCTGCCCCAATGGTTCCTRGCTTCAAATGCAGAGGGTTAARCTGGCTG~:C1326 AGTGTTTCCTGCATCCACACAAAGAATGAGGTTAGCCAGGCRGGACCTATG;a"CCATGTCG13$U

TTCTGTTCTGTTTTAGTTTTTCTTCTTCAGTAGCTTGGGCTGCAGCTTCTACTCTGCCCA ~

TTCGATGTGGGGGnAGGCCATTTCTTTTTGTAATTTGT'ICTGTGTGTTTGCACATCTGGG 1560 SUBSTITUTE SHEET (RULE 26) ~I~:~58~
W 0 95f33831 PCT/US95/07349 GCTTTTTvTG TGACTCCCCT 1620 GTGGTGCACA TTTTACTTTA
GAGCCCTAGT CTGCCTGCAG

ATAGTTGTGA
CAAGACAAAG

CAATGAGGCC

TGGACCGATG

TGTCGAATAA AGTGGTCCTTAGTGAFAATTCTGTATAAATAGAGTAAGAAGGGGTT'IGAC1860 TTTGCkATAA AAGGAGACATTTGGTTCTGGTTGTCCGACCCATGTGTGTATTTGTGTCTT 1920 ACTGTTAACG GGATGTTTATCATCCAGGCC'CAGGGGAAGTCGGGCGCTCCTCAATATTZi~.2040 GTGCAGCTGT GTGGGGCTCCCTGGGCGGGAGAGACGGAACCAAACAACA.AATGTGAGTTT 2106 CTTCFAAGAG GCAGGGGGTAA.4TTAGCTGTGTTTACTGCTAACATAGTCGAAi,GATTTAG2220 TCATCCCnnT AA:,ATAGAGGCACi-AG AAGAGGGGGGGGTGTATACCCCAAACTTGA 2280 :GAG

AivGCCATGCT GGCCTCACAGCTGGCGTCATTCAGTGCCCGTCACACCCGGGCAGTTGGGG 2346 iV~CGCACG."-,A CGGATGTTTC.TAGA.j.TCCCAGGCGATGCTTGGGACAGGCT 2520 GGTCACCTGA

GCTCTCTCTT CCCCTGTTGFp,CTCAGACCCAGCAACCCF.GCCGTCCTrI:oCnCATTCCAGCC 2580 CAGGCTGi,nG CCCAGAGCCACFAGCCGGAGGGTCCAGATGTGGCCTCTCAGATGTGTGCC 2700 TTAGCCTCTC AACCCCACCCCCACCCCC.P.CCCCAGTGATGTTTACACATCT Tii.rW,F,C2760 (2) INFORMATION
FOR SEQ ID N0:4:

(i) SEQUENCE CHARACTERISTICS:
(A) LEt7GTFi: 9 base pairs (B) TYPE: nucleic acid (C) STRANDEDt~7ESS: single SUBSTITUTE SHEET (RULE 26) (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
( ix ) FEATUF:E
(A) NAME/KEY: misc_feature (B) LOCATION: 1..9 (D) OTHER INFORMATION: /note= "WT1/EGR CONSENSUS SEQUENCE"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:

(2) INFORMATION FOR SEQ ID N0:5:
(1) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) 7.'YPE: 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) cJTHER INFORMATION: /note= "WT1/EGR HUMAN TCC BINDING SITE"
(xi) SEQUE:NCE DESCRIPTION: SEQ ID N0:5:

(2) INFORMATION FOR SEQ ID N0:6:
(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 INFORM;TIOI:: /note= "WT1/EGR MOUSE TCC BINDING SITE"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:

(2) INFORMATION FOR S:EQ ID N0:7:
(i) SEQU:ENCE CHARACTERISTICS:
(Al LENGTH; 9 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TS'PE: DNA (genomic) (ix) FEATURE:
(;4) NAME/KEY: misc_feature (B) LOCATION: 1..9 (D) OTHER INFORMATION; /note= INHUMAN FTZ BINDING SITE"
(xi) SEQLTENCE DESCRIPTION: SEQ ID N0:7:

Claims (44)

1. An isolated nucleic acid comprising a reporter gene in operative association with a single nucleic acid fragment of an osteogenic protein-1 (OP-1) specific upstream non-coding sequence, wherein said nucleic acid fragment consists of nucleotides 3170 to 3317, 3020 to 3317, 2790 to 3317, 2548 to 3317, 2300 to 3317, 1300 to 3317, 2548 to 2790, 1549 to 2790, or 1 to 2790 of SEQ ID NO:
1, wherein said isolated nucleic acid comprises not more than one nucleic acid fragment of said OP-1 upstream sequence, and, wherein said nucleic acid fragment is operative to regulate expression of said reporter gene.

2. The nucleic acid of claim 1, wherein said nucleic acid fragment consists of nucleotides 3170 to 3317, 3020 to 3317, 2790 to 3317, or 2548 to 3317 of SEQ
ID NO:1.

3. The nucleic acid of claim 1, wherein said nucleic acid fragment consists of nucleotides 2300 to 3317, 1300 to 3317, 2548 to 2790, 1549 to 2790, or 1 to 2790 of SEQ ID NO:l.

4. The nucleic acid of claim 1, wherein said nucleic acid fragment comprises at least one Wt-1/Egr consensus binding element.

5. The nucleic acid of claim 1, wherein said nucleic acid fragment comprises between one and six Wt-1/Egr binding elements.

6. The nucleic acid of claim 1, wherein said nucleic acid fragment comprises at least part of a Fushi-tarazu protein binding element.

7. The nucleic acid of claim 1, wherein said nucleic acid fragment comprises a steroid responsive element.

8. A cell transfected with the nucleic acid of any one of claims 1 to 6.

9. The transfected cell of claim 8, wherein at least part of said isolated nucleic acid is operatively integrated into the cellular genome.

10. The transfected cell of claim 9, wherein the genome of said cell has an osteogenic protein-1 (OP-1) gene locus and at least part of said transfected nucleic acid is operatively integrated into said genome at said OP-1 locus.

11. The transfected cell of claim 8, wherein said cell expresses endogenous osteogenic protein-1 (OP-1).

12. The transfected cell of claim 11, wherein said cell is an epithelial cell.

13. The transfected cell of claim 11, wherein said cell is of urogenital, liver, bone, cardiac, lung, or nerve cell origin.

14. A cell transfected with an isolated nucleic acid, said nucleic acid comprising a reporter gene in operative association with a first DNA sequence, wherein said first DNA sequence is:
(a) a single nucleic acid fragment of an osteogenic protein-1 (OP-1) specific upstream non-coding sequence, wherein said nucleic acid fragment consists of nucleotides 2548 to 3317 or 2548 to 2790 of SEQ ID NO:1; or (b) a nucleic acid fragment of an OP-1 specific upstream non-coding sequence, wherein said nucleic acid fragment consists of nucleotides 1549 to 2297, or 1549 to 1788 of SEQ ID NO:2; or (c) a variant of a nucleic acid fragment of (b) which has the same function as the nucleic acid fragment of (b) and which hybridizes with a nucleic acid complementary to the nucleic acid fragment of (b) under conditions of hybridization in 40% formamide, 5 × SSPE, 5 ×
Denhardt's solution, and 0.1% SDS at 37°C, followed by washing in 0.1 × SSPE, and 0.1% SDS at 50°C;

and a second DNA sequence comprising a sequence which interacts with a DNA binding molecule and affects expression of said reporter gene, wherein said isolated nucleic acid comprises not more than one nucleic acid fragment of (a).

15. The cell of claim 14, wherein said second DNA sequence comprises at least one Wt-1/Egr-1 consensus element SEQ ID NO:4

16. The cell of claim 15, wherein said second DNA sequence comprises between one and six Wt-1/Egr-1 consensus elements SEQ ID NO:4.

17. The cell of claim 15, wherein said second DNA sequence comprises at least six Wt-1/Egr-1 consensus elements SEQ ID NO:4.

18. The cell of claim 14, wherein said second DNA sequence is selected from the group of sequences consisting of a TCC element, a Fushi-tarazu protein binding element and a steroid responsive element.

19. The cell of claim 14, further comprising a third DNA sequence in operative association with said reporter gene, said third DNA sequence being independently selected from the group of sequences consisting of a TCC
element, a Fushi-tarazu protein binding element and a steroid responsive element.

20. A method for screening a candidate compound for the ability to modulate expression of osteogenic protein-1 (OP-1), said method comprising the steps of (a) incubating a candidate compound with a cell transfected with an isolated nucleic acid of claim 1;
(b) measuring the level of said reporter gene expressed in said 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's ability to increase OP-1 expression in vivo, and a decrease in reporter gene expression level is indicative of the candidate's ability to inhibit OP-1 expression in vivo.

21. The method of claim 20, wherein said reporter gene is in operative association with a single nucleic acid fragment consisting of nucleotides 3170 to 3317, 3020 to 3317, 2?90 to 3317, or 2548 to 3317 of SEQ ID NO:1.

22. The method of claim 20, wherein said reporter gene is in operative association with a single nucleic acid fragment consisting of nucleotides 2300 to 3317, 1300 to 3317, 2548 to 2790, 1549 to 2790, or 1 to 2790 of SEQ ID NO:1.

23. A method for screening a candidate compound for the ability to modulate expression of osteogenic protein-1 (OP-1), said method comprising the steps of (a) incubating a said candidate compound with a cell according to any one of claim 14, 15, 18, or 19;
(b) measuring the level of reporter gene expressed in said 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's ability to increase OP-1 expression in vivo, and a decrease in reporter gene expression level is indicative of the candidate's ability to inhibit OP-1 expression in vivo.

24. An isolated nucleic acid having a nucleotide sequence comprising nucleotides 1 to 1871 of SEQ ID NO:2.

25. An isolated nucleic acid having a nucleotide sequence comprising nucleotides 1 to 3317 of SEQ ID NO:1.

26. The method of claim 20, wherein said isolated nucleic acid further comprises part or all of a nucleotide sequence encoding an osteogenic protein-1 (OP-1) pro protein in operative association with said reporter gene.

27. A kit for identifying a candidate molecule capable of modulating osteogenic protein-1 (OP-1) expression in a cell, the kit comprising:
(a) a receptacle containing an isolated nucleic acid of claim 1; and (b) means for detecting expression of said reporter gene following exposure of a said candidate molecule to a cell containing said isolated nucleic acid.

28. The kit of claim 27, wherein said reporter gene comprises a nucleic acid comprising an osteogenic protein-1 (OP-1) coding region sequence.

29. The method of claim 26, wherein said cell is an epithelial cell.

30. The method of claim 20 or 23, wherein said cell is an epithelial cell.

31. The method of claim 26, wherein said cell is of urogenital, liver, bone, cardiac, lung, or nerve cell origin.

32. The method of claim 20 or 23, wherein said cell is of urogenital, liver, bone, cardiac, lung, or nerve cell origin.

33. The kit of claim 27, wherein said reporter gene is in operative association with a single nucleic acid fragment consisting of nucleotides 3170 to 3317, 3020 to 3317, 2790 to 3317, or 2548 to 3317 of SEQ ID NO:1.

34. The kit of claim 27, wherein said reporter gene is in operative association a single nucleic acid fragment consisting of nucleotides 2300 to 3317, 1300 to 3317, 2548 to 2790, 1549 to 2790, or 1 to 2790 of SEQ ID NO:1.

35. An isolated nucleic acid comprising a reporter gene in operative association with:
(a) a nucleic acid fragment of an osteogenic protein-1 (OP-1) specific upstream non-coding sequence, wherein said nucleic acid fragment consists of nucleotides 2151 to 2297, 2001 to 2297, 1788 to 2297, 1549 to 2297, 800 to 2297, 1 to 2297, 1549 to 1788, 800 to 1788, or 1 to 1788 of SEQ ID NO:2; or (b) a variant of a nucleic acid fragment of (a) which has the same function as the nucleic acid fragment of (a) and which hybridizes with a nucleic acid complementary to the nucleic acid fragment of (a) under conditions of hybridization in 40% formamide, 5 × SSPE, 5 ×
Denhardt's solution, and 0.1 % SDS at 37°C, followed by washing in 0.1 × SSPE, and 0.1 % SDS at 50°C;
wherein the nucleic acid fragment of (a) or (b) is operative to regulate expression of said reporter gene.

36. The nucleic acid of claim 35, wherein said reporter gene is in operative association with:
(a) a nucleic acid fragment consisting of nucleotides 2151 to 2297, 2001 to 2297, 1788 to 2297, or 1549 to 2297 of SEQ ID NO:2; or (b) a variant of a nucleic acid fragment of (a) which has the same function as the nucleic acid fragment of (a) and which hybridizes with a nucleic acid complementary to the nucleic acid fragment of (a) under conditions of hybridization in 40% formamide, 5 × SSPE, 5 ×
Denhardt's solution, and 0.1 % SDS at 37°C, followed by washing in 0.1 × SSPE, and 0.1 % SDS at 50°C.

37. The nucleic acid of claim 35, wherein said reporter gene is in operative association with:

(a) a nucleic acid fragment consisting of nucleotides 800 to 2297, 1 to 2297, 1549 to 1788, 800 to 1788, or 1 to 1788 of SEQ ID NO:2; or (b) a variant of a nucleic acid fragment of (a) which has the same function as the nucleic acid fragment of (a) and which hybridizes with a nucleic acid complementary to the nucleic acid fragment of (a) under conditions of hybridization in 40% formamide, 5 × SSPE, 5 ×
Denhardt's solution, and 0.1% SDS at 37°C, followed by washing in 0.1 × SSPE, and 0.1 % SDS at 50°C.

38. The nucleic acid of any one of claim 1, 2, 3, 35, 36, or 37 further comprising part or all of a nucleotide sequence encoding an osteogenic protein-1 (OP-1) pro protein in operative association with said reporter gene.

39. A vector comprising the isolated nucleic acid of any one of claim 1, 2, 3, 35, 36, or 37.

40. A kit for identifying a candidate molecule capable of modulating osteogenic protein-1 (OP-1) expression in a cell, the kit comprising:
(a) a receptacle containing an isolated nucleic acid of claim 35; and (b) means for detecting expression of said reporter gene following exposure of a said candidate molecule to a cell containing said isolated nucleic acid.

41. The kit of claim 40, wherein said reporter gene comprises a nucleic acid comprising an osteogenic protein-1 (OP-1) coding region sequence.

42. The kit of claim 40, wherein said reporter gene is in operative association with:
(a) a nucleic acid fragment consisting of nucleotides 2151 to 2297, 2001 to 2297, 1788 to 2297, or 1549 to 2297 of SEQ ID NO:2; or (b) a variant of a nucleic acid fragment of (a) which has the same function as the nucleic acid fragment of (a) and which hybridizes with a nucleic acid complementary to the nucleic acid fragment of (a) under conditions of hybridization in 40% formamide, 5 × SSPE, 5 ×
Denhardt's solution, and 0.1% SDS at 37°C, followed by washing in 0.1 × SSPE, and 0.1% SDS at 50°C.

43. The kit of claim 40, wherein said reporter gene is in operative association with:
(a) a nucleic acid fragment consisting of nucleotides 800 to 2297, 1 to 2297, 1549 to 1788, 800 to 1788, on to 1788 of SEQ ID NO:2; or (b) a variant of a nucleic acid fragment of (a) which has the same function as the nucleic acid fragment of (a) and which hybridizes with a nucleic acid complementary to the nucleic acid fragment of (a) under conditions of hybridization in 40% formamide, 5 × SSPE, 5 ×
Denhardt's solution, and 0.1% SDS at 37°C, followed by washing in 0.1 × SSPE, and 0.1% SDS at 50°C.

44. A method for screening a candidate compound for the ability to modulate expression of osteogenic protein-1 (OP-1), said method comprising the steps of:
(a) incubating a said candidate compound with a cell transfected with an isolated nucleic acid of claim 35;
(b) measuring the level of said reporter gene expressed in said 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's ability to increase OP-1 expression in vivo, and a decrease in reporter gene expression level is indicative of the candidate's ability to inhibit OP-1 expression in vivo.