Isolation of a collagenase cDNA clone and measurement of changing collagenase mRNA levels during induction in rabbit synovial fibroblasts.
To facilitate our studies on the mechanisms controlling collagenase production at a molecular level in rabbit synovial fibroblasts, we have constructed a cDNA library using mRNAs isolated from cells induced with crystals of monosodium urate monohydrate. We have screened this library with cDNA probes made from induced and control mRNA populations. From among 30 clones that hybridized preferentially to the induced-cell probe, 4 contained collagenase sequences. The largest, a clone of 650 base pairs, was identified by its ability to hybrid select a mRNA that could be translated in a cell-free system into a product that was precipitable with monospecific antibody to collagenase. Using this clone to probe blots of RNA from induced cells, we detected the appearance of a collagenase mRNA of 2.7 kilobases within 5 hr of addition of urate. The level of collagenase mRNA continued to increase for 35-40 hr, when it was 60 to 90 times more abundant in induced cells than in control cells. The increase in mRNA levels correlated with an increase in immunoreactive collagenase protein that was detectable in culture medium by 10 hr.
D000818:Animals; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification*; D004262:DNA Restriction Enzymes; D004790:Enzyme Induction; D005347:Fibroblasts / Q000201:enzymology; D007700:Kinetics; D003012:Microbial Collagenase / Q000096:biosynthesis / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D010944:Plants / Q000378:metabolism; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics*; D011817:Rabbits; D013582:Synovial Fluid / Q000201:enzymology*; D014908:Triticum / Q000378:metabolism
D012333:RNA, Messenger; D004247:DNA; D004262:DNA Restriction Enzymes; D003012:Microbial Collagenase