Analysis of mRNA populations by cDNA.mRNA hybrid-mediated inhibition of cell-free protein synthesis.
Analysis of mRNA populations by cDNA.mRNA hybrid-mediated inhibition of cell-free protein synthesis.
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通过 cDNA.mRNA 杂交介导的无细胞蛋白质合成抑制来分析 mRNA 群体。
DOI：
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发表时间：
1978
影响因子：
11.1
通讯作者：
W. Held
中科院分区：
文献类型：
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作者：
N. Hastie;W. Held
将mRNA与相应的cDNA杂交可特异性抑制mRNA在体外的翻译。利用珠蛋白cDNA与珠蛋白mRNA的杂交作为模型系统，我们发现mRNA杂交的饱和和珠蛋白合成的完全抑制都需要等量的cDNA。翻译失活速率与杂交速率一致，符合预测的动力学形式。该方法已应用于小鼠肝脏中一组丰富的mrna的分析。肝脏mRNA与肝脏总cDNA的杂交略微过量至低C(0)t值，特异性地抑制了几种主要多肽的翻译。在翻译之前将杂交种熔化，可以恢复这些多肽的合成。此外，我们发现不同的肝脏mrna以不同的动力学失活；结果表明，主要尿多肽和白蛋白mrna在小鼠肝脏中分别是最丰富和第二丰富的。讨论了该技术的一般应用。
Hybridization of mRNA to its corresponding cDNA was found to specifically inhibit translation of the mRNA in vitro. Using hybridization of globin cDNA to globin mRNA as a model system, we found that equivalent amounts of cDNA were required both for the saturation of the mRNA hybridization and for complete inhibition of globin synthesis. Also, the rate of inactivation of translation was identical to the rate of hybridization and followed the predicted kinetic form. This assay has been applied to the analysis of a set of abundant mRNAs in mouse liver. Hybridization of liver mRNA with total liver cDNA in slight excess to a low C(0)t value specifically inhibited translation of several major polypeptides. Melting of the hybrids prior to translation restored synthesis of these polypeptides. Moreover, we found that different liver mRNAs are inactivated with different kinetics; the results suggest that the mRNAs for the major urinary polypeptide and for albumin are the most abundant and second most abundant, respectively, in mouse liver. The general applications of this technique are discussed.