cDNA cloning and sequence of a new type of actin in mouse B16 melanoma

Parent B16 melanoma and B16-F1 cell lines express a third actin (Ax) in addition to beta- and gamma-actin. It has the same molecular mass (43,000 daltons) and a more acidic isoelectric point (pI = 5.2) than the latter two actins (pI = 5.3) (Taniguchi, S., Kawano, T., Kakunaga, T., and Baba, T. (1986) J. Biol. Chem. 261, 6100-6106). We constructed a cDNA library from poly(A)+ RNA of B16-F1 and then isolated Ax actin candidate clones. According to the nucleotide sequencing analysis for one of the candidate clones, pMA 30, the predicted amino acid sequence was composed of 375 amino acids and was similar to that of beta-actin, but differed at the 28th amino acid in that leucine replaced the arginine of beta-actin. When RNA synthesized from the clone pMA 30 with the SP6 transcription system was translated in vitro using reticulocyte lysate, we identified a polypeptide which had the same isoelectric point and molecular weight as Ax actin; the polypeptide had binding activity to DNase I, a common characteristic of native actin. These observations provide evidence that the clone pMA 30 encodes the mRNA for Ax actin. In the nucleotide sequence of the Ax cDNA, there are: 1) one base change in the coding region which causes a loss of the SmaI site and an amino acid exchange, as mentioned above; 2) four deletion sites in the 3'-noncoding region; 3) one insertion site in the 3'-noncoding region; and 4) one base change in the 5'-noncoding region, as compared with hitherto known mouse beta-actin cDNA. These differences between Ax and beta-actin cDNA indicate that the Ax actin is encoded by an unique gene set, independent of beta-actin.