In vitro synthesis of beta- and gamma-isoactins by messenger RNA of rat ascites hepatoma.

Messenger RNA was extracted from polysomes of rat ascites hepatoma, AH 7974, which contains both beta- and gamma-actins, and was translated in nuclease-treated reticulocyte lysate. The isoactins, beta and gamma, synthesized in vitro were characterized by (1) a high affinity to DNAase I-agarose; (2) polymerization with actin purified from bovine brain; (3) coelectrophoresis with bovine brain actin on two-dimensional gel, and (4) peptide mapping of each isoactin by partial digestion with papain (EC 3.4.22.2) followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimum conditions with respect to the concentration of RNA, Mg2+, and K+ for the synthesis of beta- and gamma-isoactins were identical: 300 micrograms/ml, 1.5 mM, and 100 mM, respectively. Aurintricarboxylic acid and 7-methyl-GMP (7MeGMP) inhibited the synthesis of beta- and gamma-actins to the same extent. These results strongly suggest that there is very little possibility of differential translational control of each isoactin gene. When polysomal RNA was separated by sucrose gradient centrifugation, both beta- and gamma-actin mRNAs appeared as a sharp peak at the region slightly heavier than 18 S RNA.