Benoff S, Nadal-Ginard B N
Biochemistry. 1979 Feb 6;18(3):494-500. doi: 10.1021/bi00570a019.
An mRNA-dependent reticulocyte cell-free protein synthesizing system very efficient in the translation of myosin heavy-chain mRNA from a rat myogenic cell line is described. This system exhibits a high degree of fidelity with regard to the spectrum and relative proportion of the different proteins synthesized from a sample of cytoplasmic RNA as compared to the proteins synthesized in vivo by the cells from which the RNA is prepared. The main feature of this system is the use of a K+ and Cl- concentration similar to those of the reticulocyte cytoplasm. Using this system, myosin heavy chain, identified by low-salt precipitation, electrophoretic mobility, and partial peptide analysis, represents 17% of the total protein synthesis when cytoplasmic RNA from well-fused L6E9 cells is used. Furthermore, when RNA preparations from growing myoblasts, that when analyzed in other cell-free translational systems seem not to contain any myosin heavy-chain mRNA, are tested in the system reported here, they are proven to contain high amounts of translatable myosin heavy-chain mRNA.
本文描述了一种依赖mRNA的无细胞网织红细胞蛋白质合成系统，该系统在翻译来自大鼠成肌细胞系的肌球蛋白重链mRNA方面非常高效。与从制备RNA的细胞体内合成的蛋白质相比，该系统在从细胞质RNA样本合成的不同蛋白质的种类和相对比例方面表现出高度的保真度。该系统的主要特点是使用了与网织红细胞细胞质相似的K+和Cl-浓度。使用该系统时，通过低盐沉淀、电泳迁移率和部分肽分析鉴定的肌球蛋白重链，当使用来自融合良好的L6E9细胞的细胞质RNA时，占总蛋白质合成的17%。此外，当在本文报道的系统中测试来自生长中的成肌细胞的RNA制剂时（在其他无细胞翻译系统中分析时似乎不含有任何肌球蛋白重链mRNA），结果证明它们含有大量可翻译的肌球蛋白重链mRNA。
