MOLECULAR STUDIES OF CONTRACTILE PROTEIN GENE REGULATION DURING MUSCLE DIFFERENTIATION AND THE STRUCTURE OF THE FAST MUSCLE MYOSIN LIGHT CHAIN 1/3 GENE
GARFINKEL, LEONARD ISAAC
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A cDNA clone library was constructed, using as starting material mRNA from adult rat skeletal muscle. From this library, clones corresponding to alpha actin, myosin light chain 2, troponin C, troponin T, alpha tropomyosin, and beta tropomyosin were selected. In addition, a clone homologous to both myosin light chain 1 and 3 mRNAs was selected.;Studies using these clones in filter hybridizations and RNA blot hybridizations lead to the conclusion that steady state levels of contractile protein mRNAs in adult rat skeletal muscle are maintained at strict stoichiometric levels. Levels of these mRNAs in L(,6)E(,9) myotubes, while not strictly stoichiometric, can be divided into two groups of higher and lower abundance. Synthetic rates of these mRNAs are not strictly coordinated. However, they can be divided into high and low synthetic rate groups with the same membership as the comparable abundance groups. All of the contractile protein mRNAs, except that encoding alpha actin, have long half lives.;The accumulation of the contractile protein mRNAs during muscle differentiation can be achieved by increases in synthetic rate and mRNA stability. One coordinately regulated set of mRNAs, including those encoding myosin heavy chain, myosin light chain 2, alpha tropomyosin, and beta tropomyosin appear to accumulate by virtue of both of these mechanisms. A second set, consisting of troponin C and troponin T mRNAs, appears to be regulated solely by large increases in mRNA stability. Actin mRNA does not appear to belong to either of these sets.;The cDNA clone homologous to myosin light chain 1 and 3 mRNAs was used to determine the chromosomal arrangement of the gene(s) encoding these mRNAs. The two mRNAs are identical along a length of 700 bases, as determined by S1 mapping, and differ from each other only in the length and sequence of their 5' ends, as shown by RNase H mapping. Southern blot analysis showed that both mRNAs are encoded by a single gene, with a repetitive element in the region corresponding to the 3' non-translated region of the mRNAs. The two different mRNAs must arise either by differential transcription using two different promoters at the 5' end of the gene, or by differential splicing of a single primary transcript.