Mutated beta-actin gene: coexpression with an unmutated allele in a chemically transformed human fibroblast cell line.

Abstract

By in vitro translation, we have identified the mRNA species that codes for a novel actin polypeptide (Ax-actin) in the chemically transformed human fibroblast line HuT-14. The relatedness of the coding sequences of the Ax- and beta-actin genes is indicated by our finding that pcDd actin ITL-I DNA, a recombinant plasmid DNA that contains a DNA sequence complementary to actin mRNA of Dictyostelium discoideum, hybridizes both the Ax-actin mRNA and the beta-actin mRNA but not the gamma-actin mRNA. In contrast, pcHa-1 DNA, a recombinant plasmid constructed by cloning a DNA sequence complementary to human actin mRNA from HuT-14 cells into pBR322, hybridized to all three mRNA species. In addition, no difference was observed between Ax- and beta-actin mRNAs when their molecular size was determined either by sucrose density gradient sedimentation or by methyl mercury agarose gel electrophoresis. Southern blot transfer of radioactive pcDd actin DNA to restriction endonuclease-digested Hut-14 DNA produced only a single hybrid band (a 6-kilobase fragment); the pcHa-1 DNA probe detected one additional band (a 3-kilobase fragment). These results suggest that HuT-14 cells contain only one copy per haploid genome for Ax- or beta-actin. When considered together with recent determination of the entire amino acid sequences of Ax- and beta-actin, our findings indicate that Ax-actin is the product of a mutated beta-actin gene and are evidence for the occurrence of a mutation in a chemically transformed cell.