Cell-free translation of mammalian myosin heavy-chain messenger ribonucleic acid from growing and fused-L6E9 myoblasts

Abstract

An mRNA-dependent reticulocyte cell-free protein synthesizing system very efficient in the translation of myosin heavy-chain mRNA from a rat myogenic cell line was described. This system exhibited a high degree of fidelity with regard to the spectrum and relative proportion of the different proteins synthesized from a sample of cytoplasmic RNA as compared to the proteins synthesized in vivo by the cells from which the RNA is prepared. The main feature was the use of a K+ and Cl- concentration similar to those of the reticulocyte cytoplasm. Using this system, myosin heavy chain, identified by low-salt precipitation, electrophoretic mobility and partial peptide analysis, represents 17% of the total protein synthesis when cytoplasmic RNA from well-fused L6 E9 [subclone of L6-fetal rat skeletal muscle myoblast] cells was used. When RNA preparations from growing myoblasts, that when analyzed in other cell-free translational systems seem not to contain any myosin heavy-chain mRNA, were tested in the system, they were proven to contain high amounts of translatable myosin heavy-chain mRNA.