cDNA clone analysis of six co-regulated mRNAs encoding skeletal muscle contractile proteins.

Abstract

Ac[complementary]DNA cloning approach was used to investigate muscle gene regulation during differentiation of cultured embryonic quail myoblasts. A cNDA clone library of cultured myofiber poly(A)+RNA was constructed and screened by colony hybridization with cDNA probes of myoblast and myofiber RNA. Myofiber-specific cDNA clones (28) were identified and, by cross-hybridization analysis, these clones were found to represent, at most, 18 different myofiber-specific RNA. Of these RNA, 6 were identified by sequence analysis of the cDNA clones. These 6 RNA encode the contractile proteins .alpha.-actin, .alpha.-tropomyosin, myosin heavy chain, myosin light chain 2, troponin C and troponin I. The embryonic muscle contractile protein sequences are identical with, or closely match, those of adult skeletal muscle proteins and include both fast fiber (myosin light chain 2 and troponin I) and slow fiber (troponin C) isotypes. RNA gel transfer hybridization analysis showed that the cellular abundances of these contractile protein mRNA increase 20- to 30-fold or more during myoblast differentiation. Coordinate activation of contractile protein synthesis during myogenesis is controlled by mechanisms that direct the accumulation of contractile protein mRNA rather than their translational utilization. With the possible exception of myosin heavy chain, the contractile protein genes expressed by cultured embryonic muscle encode adult muscle proteins of both fast and slow fiber types, consistent with a co-activation-selective repression model of gene regulation during fiber type differentiation in developing skeletal muscle.