(Buckingham, 1977). Experiments using actinomycin D suggested that mRNA coding for muscle protein accumulates just prior to cell fusion (e.g. Yaffe and Dym, 1973 (Glover, 1977). We have employed this technique to isolate muscle coding sequences from heterogeneous fractions of mouse muscle mRNA. The protocol is summarized in figure 2. Poly A + RNA was separated on low salt/sucrose gradients to obtain partially enriched fractions for the myosin light chains, the actins, and the myosin heavy chain mRNAs. Although the proteins are very abundant in muscle (20-60 p. 100 total protein), the level of protein synthesis and of the corresponding mRNA is very much lower (1-5 p. 100) which makes purification of the mRNAs difficult (c/f globin mRNA 50-90 p. 100). Once the mixture of double-stranded cDNAs has been integrated into plasmids ( fig. 2) and bacterial clones selected with the expected antibiotic resistance, the major problem is identification of the clones. A preliminary screening was carried out in situ by the technique of Grunstein and Hogness (1975), using 32 P-labelled C DNA from the original RNA fraction and from muscle and nonmuscle RNA to obtain as much information as possible. Clones were then selected and grown in bulk culture, and the plasmid DNA was purified. This DNA was used in hybridization/translation assays with muscle mRNA, either in the hybrid-arrested translation technique (HART) (Paterson et al.,1977) (Minty et al., 1981). The DBM filter technique indicated that this clone cross-hybridizes with muscle and non-muscle actin mRNA. After hybrid-arrested translation and analysis of the products on two-dimensional gels, it appears to affect the translation of (1.-and y-actin mRNAs more than that of (3-actin mRNA. In order to determine which sequence is incorporated into the plasmid, the inserted C DNA segment was isolated and its DNA sequence analyzed by the method of Maxam and Gilbert (1977). The result is shown in figure 3 where it is clear that the clone contains a sequence corresponding to oe-actin. Analysis of this sequence by restriction mapping and partial DNA sequencing indicates that it contains 1 350 nucleotides, including part of the non-coding 3' terminal of the mRNA and lacking a short sequence at the 5' coding end of the messenger (fig. 4)
A recombinant plasmid with a cDNA sequence transcribed from mouse skeletal muscle RNA is shown to hybridize with mRNAs for myosin light chains LC1F and LC3F. The inserted fragment corresponds exclusively to the 3'-noncoding region ofthe mRNA. It hybridizes almost exclusively with the two light chain messengers from fast skeletal muscle RNA of adult mouse. Slight hybridization is seen with RNA from heart muscle and embryonic skeletal muscle. The implications ofthe conservation ofthe 3'-noncoding regions between the two mRNAs are discussed.
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