Characterization of neonatal cardiac myosin heavy chain mRNA and synthesis of complementary DNA

Abstract

A procedure for the extraction of intact RNA from whole heart tissue using guanidine hydrochloride has been accomplished. Subsequent chromatography on oligo(dT)-cellulose of the total RNA and translation of the poly (A)⁺ RNA in a cell-free reticulocyte translation system has shown that the myosin heavy chain mRNA from rat heart tissue was present. The myosin heavy chain mRNA was identified by several criteria: (i) sizing by sucrose density gradient centrifugation, (ii) migration of a polypeptide translation product coincided with a myosin heavy chain marker in one-dimensional sodium dodecyl sulfate – polyacrylamide gel electrophoresis, and (iii) immunoprecipitation of the mRNA translation product with a myosin heavy chain specific antibody. A myosin heavy chain mRNA enriched fraction was used to direct cDNA synthesis in an in vitro system. An optimal condition has been elucidated in which large molecular weight cDNA strands are produced. Evidence is presented here for the production of cDNA strands in excess of 4000 nucleotides which includes the heavy chain myosin in a high potassium – low sodium transcription assay, as revealed by dot blot hybridization with a cDNA clone of the myosin heavy chain.