Translation of chick calvarial procollagen messenger RNA'S by a messenger RNA dependent reticulocyte lysate.
A method was developed for the extraction of RNA from chick embryo calvaria which should be generally applicable to other connective tissues. Total RNA prepared by this method was translated by a mRNA-dependent reticulocyte lysate into discrete pro alpha chains. Several criteria were used to identify these translation products, including (1) preferential labeling with [3H]proline, (2) appropriate migration on sodium dodecyl sulfate-polyacrylamide gels, (3) selective sensitivity to collagenase digestion, and (4) specific precipitability by two different antisera against procollagen. Data from the immunoprecipitation experiments indicated that the majority of the pro alpha chains contained the carboxy-terminal antigenic determinants. These results demonstrate that this translation system can be used as an assay for intact procollagen mRNAs and as a source of in vitro synthesized pro alpha chains for future structural analysis.
D000818:Animals; D001842:Bone and Bones / Q000378:metabolism; D002642:Chick Embryo; D008274:Magnesium / Q000494:pharmacology; D008715:Methionine / Q000378:metabolism; D003012:Microbial Collagenase / Q000378:metabolism; D010446:Peptide Fragments / Q000096:biosynthesis / Q000276:immunology; D011188:Potassium / Q000494:pharmacology; D011347:Procollagen / Q000096:biosynthesis* / Q000276:immunology; D011392:Proline / Q000378:metabolism; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism*; D012156:Reticulocytes / Q000378:metabolism*
D010446:Peptide Fragments; D011347:Procollagen; D012333:RNA, Messenger; D011392:Proline; D008715:Methionine; D003012:Microbial Collagenase; D008274:Magnesium; D011188:Potassium