Modulators of morphogen expression and methods of identifying the same

Abstract

Disclosed are methods and compositions for identifying compounds having an ability to modulate expression of a morphogen, particularly OP-1, OP-1 homologues and closely related proteins, using one or more OP-1-specific, non-coding sequences and a suitable reporter gene. In preferred embodiments, the OP-1-specific non-coding DNA sequence comprises a Pax-responsive OP-1-modulating element.

1 . A vector comprising a DNA sequence encoding a reporter gene in operative association with at least one OP-1-specific non-coding DNA sequence capable of modulating expression of said reporter gene, wherein said non-coding sequence comprises at least a first Pax-responsive OP-1 modulating element which is responsive to a first Pax gene expression product.  
     
     
         2 . The vector of  claim 1  wherein said non-coding sequence is selected from the group of DNA sequences corresponding to bases 108-121, 139-154, 157-167, 365-378, 497-511, 598-613, 1123-1140, 1144-1161, 1285-1297, 1750-1762, 2001-2023, 2365-2378, 2931-2944 of SEQ. ID No. 1, including allelic, species and other sequence variants thereof.  
     
     
         3 . The vector of  claim 1  wherein said non-coding sequence corresponds to part or all of SEQ. ID No. 2 including allelic, species and other sequence variants thereof.  
     
     
         4 . The vector of  claim 1  wherein said non-coding sequence comprises between one and twelve first Pax-responsive OP-1 modulating elements.  
     
     
         5 . The vector of  claim 1  wherein said non-coding sequence comprises at least part of a first Pax-responsive OP-1 modulating element.  
     
     
         6 . The vector of  claim 1  further comprising at least a second non-coding sequence, wherein said second non-coding sequence comprises at least a second Pax-responsive OP-1 modulating element which is responsive to a second Pax gene expression product, said second Pax gene expression product differing from said first Pax gene expression product.  
     
     
         7 . The vector of  claim 6  wherein said second non-coding sequence is selected from the group of DNA sequences corresponding to bases 491-503, 737-747, 891-903, 994-1006 of SEQ. ID. No. 1, including allelic, species and other sequence variants thereof.  
     
     
         8 . The vector of  claim 6  wherein said second non-coding sequence corresponds to part or all of SEQ. ID No. 3 including allelic, species and other sequence variant thereof.  
     
     
         9 . The vector of  claim 6  wherein said second non-coding sequence comprises four or more second Pax-responsive OP-1 modulating elements.  
     
     
         10 . The vector of  claim 6  wherein said second non-coding sequence defines at least part of a second Pax-responsive OP-1 modulating element.  
     
     
         11 . (canceled)  
     
     
         12 . (canceled)  
     
     
         13 . (canceled)  
     
     
         14 . (canceled)  
     
     
         15 . (canceled)  
     
     
         16 . (canceled)  
     
     
         17 . (canceled)  
     
     
         18 . (canceled)  
     
     
         19 . (canceled)  
     
     
         20 . (canceled)  
     
     
         21 . A substantially pure nucleic acid comprising a DNA sequence corresponding to bases 108-121, 139-154, 157-167, 365-378, 497-511, 598-613, 1123-1140, 1144-1161, 1285 1297, 1750-1762, 2001-2023, 2365-2378, 2931-2944 of SEQ. ID No. 1, or corresponding to bases 491-503, 737-747, 891-903, 994-1006 of SEQ. ID. No. 1, including allelic, species and other sequence variants of any of the foregoing.  
     
     
         22 . (canceled)  
     
     
         23 . (canceled)  
     
     
         24 . (canceled)  
     
     
         25 . A nucleic acid comprising nucleotides 2606-2690 of SEQ ID NO: 1.  
     
     
         26 . A method for increasing OP-1 gene expression comprising the step of deleting a gene expression silencer contained within the AatII-PvuII upstream fragment (residues 2606 2690 of SEQ ID NO: 1).  
     
     
         27 . A recombinant OP-1 gene lacking a gene expression silencer contained within the AatII PvuII upstream fragment (residues 2606-2690 of SEQ ID NO: 1).  
     
     
         28 . A recombinant cell containing the OP-1 gene of  claim 27.