Developmentally regulated expression of chimeric genes containing muscle actin DNA sequences in transfected myogenic cells.

AUTOR(ES)

Melloul, D

RESUMO

A recombinant plasmid containing 2/3 of the rat skeletal muscle actin structural gene plus 730 bp of its 5' flanking region, spliced to the 3' end of the human epsilon-globin gene, was introduced into cells of the rat myogenic line L8. Myogenic clones carrying the actin/globin chimeric gene were isolated. In many of these clones, the expression of the gene greatly increased during differentiation (up to greater than 50-fold) and, in some clones, the amount of the chimeric gene transcripts in the differentiated cultures exceeded that of the native muscle actin gene transcripts. Furthermore, the temporal relation between differentiation of the cultures and the accumulation of the transcripts from the transferred genes was very similar to that of the native skeletal muscle actin gene, suggesting a similar mechanism of regulation. Endonuclease S1 analysis indicated a correct initiation and termination of the mRNA but suggested that a fraction of the chimeric actin/globin transcripts was not properly processed. To test whether the increased expression of the transferred gene which occurred during differentiation was determined by DNA sequences in the 5' region of the muscle actin gene, a plasmid (p alpha-CAT) containing 730 bp of the 5' flanking region of the rat skeletal muscle actin gene (plus the exon of the 5' untranslated region, and 25 bp of the first intron), spliced to the bacterial structural gene coding for chloramphenicol acetyl transferase (CAT), was constructed and introduced into L8 cells. In the majority of the isolated clones containing this plasmid, CAT activity increased many-fold during differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)

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