RABBIT GENOMIC CLONES FOR SYNOVIAL FIBROBLAST COLLAGENASE: TOOLS FOR UNDERSTANDING CONNECTIVE TISSUE DISEASE

The collagenases are central to normal connective tissue remodeling and to both pathological disorders of matrix metabolism and proliferative diseases requiring matrix destruction. Our goal is to understand molecular mechanisms controlling collagenase activity and synthesis. We feel that these questions can best be approached using recombinant DNA technology. Using a partial collagenase cDNA clone, it was previously shown by our lab that induction of collagenase synthesis in primary cultures of rabbit synovial fibroblasts is accompanied by a large increase in intracellular collagenase mRNA. I now report characterization of the genes for synovial cell collagenase of rabbits. Titration hybridizations suggest that there are about 2 copies of the gene per haploid genome. The gene copy number is not increased in cells induced by phorbol esters to synthesize collagenase, indicating that gene amplification is not required for induction. For further studies, I isolated genomic clones for synovial fibroblast collagenase of rabbits from a Charon 4A library using the cDNA clone. Restriction mapping and crossed-contact hybridizations demonstrate that these clones contain DNA inserts with overlapping homology. Polymorphisms in the restriction map suggest that these clones may represent either different genetic loci or allelic forms of the same locus. Each clone contains a 3.95 kb Eco RI fragment which hybridizes to the partial cDNA clone for synovial cell collagenase. Using in vitro-labelled mRNA from induced synovial cells as a probe, I localized the remainder of the collagenase gene found on the clones and demonstrated its mosaic structure. The direction of gene transcription was determined by S1 nuclease protection studies. Repetitive sequences around the gene were also localized and characterized. I have begun to sequence the collagenase cDNA to enable a rapid determination of the primary structure of collagenase. This information may provide insight into the molecular mechanisms of collagenase activity and its control.