Mouse actin messenger RNAs. Construction and characterization of a recombinant plasmid molecule containing a complementary DNA transcript of mouse alpha-actin mRNA.

Abstract

An RNA fraction enriched in a-actin mRNA (isolated by sucrose-gradient fractionation of mouse skeletal muscle poly(A+) RNA) was used to synthesize doublestranded complementary DNA.Transcripts more than 900 nucleotides in length were inserted into the Pst I site of plasmid pBR 322 using poly(dC).poly(dG)tailing, and these molecules were used to transform Escherichia coli C 600. Transformant clones were hybridized in situ with complementary DNA transcribed from the actin-enriched muscle RNA, and those containing DNA sequences complementary to RNAs abundant in this population were selected.DNA was extracted from these clones, fixed to diazobenzyloxymethyl paper, and used to select the complementary RNA sequence.In this way, clone 91 was shown to contain an inserted sequence complementary to muscle and nonmuscle actin mRNAs.Comparison of the nucleotide sequence of part of the inserted DNA with the published amino acid sequences for mammalian actins showed that in four positions (278,286,296, and 298) the sequenced region encodes amino acids specific to a-actin.Sizing on agarose gels of the Pst I digest of this clone showed that it contains an inserted sequence of approximately 1350 base-pairs.This represents approximately 90% of the coding sequence for a-actin plus about 300 nucleotides of 3' noncoding sequence.Hybridization of the labeled plasmid DNA to RNA, separated on agarose gels and transferred to diazobenzyloxymethyl-paper, showed that mouse skeletal muscle and nonmuscle actin mRNAs have lengths of 1650 and 2100 nucleotides, respectively.A 200-nucleotide fragment of the plasmid, corresponding to the 3' noncoding region of the mRNA, hybridized specifically to the muscle actin mRNA.Proliferating cultures of a mouse myogenic cell line contained, in addition to the nonmuscle actin mRNAs, an actin mRNA slightly smaller than 1650 nucleotides which did not hybridize to the 200-nucleotide fragment of the plasmid.This