Sakiyama S, Okiba N, Fujimura S
Biochim Biophys Acta. 1980 Mar 28;607(1):81-91. doi: 10.1016/0005-2787(80)90222-1.
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.
从大鼠腹水肝癌AH 7974的多核糖体中提取信使核糖核酸（mRNA），该肝癌细胞同时含有β-肌动蛋白和γ-肌动蛋白，并在经核酸酶处理的网织红细胞裂解物中进行翻译。通过以下方法对体外合成的β和γ两种同工肌动蛋白进行了鉴定：（1）对脱氧核糖核酸酶I-琼脂糖具有高亲和力；（2）与从牛脑中纯化的肌动蛋白聚合；（3）在二维凝胶上与牛脑肌动蛋白进行共电泳；（4）用木瓜蛋白酶（EC 3.4.22.2）部分消化各同工肌动蛋白，随后进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳，对各同工肌动蛋白进行肽图谱分析。合成β-和γ-同工肌动蛋白时，RNA、Mg2+和K+浓度的最佳条件相同，分别为300微克/毫升、1.5毫摩尔和100毫摩尔。金精三羧酸和7-甲基鸟苷酸（7MeGMP）对β-和γ-肌动蛋白合成的抑制程度相同。这些结果有力地表明，每个同工肌动蛋白基因存在差异翻译控制的可能性极小。当通过蔗糖梯度离心分离多核糖体RNA时，β-和γ-肌动蛋白mRNA均在略重于18 S RNA的区域出现一个尖锐峰。
