Minshull J, Hunt T
Nucleic Acids Res. 1986 Aug 26;14(16):6433-51. doi: 10.1093/nar/14.16.6433.
Single-stranded cDNA clones complementary to the 5' end of TMV RNA have been used to explore the conditions necessary for efficient 'hybrid arrest of translation' in the reticulocyte lysate. It is shown that incubations of 20 minutes at 60 degrees in 0.1 M KCl are sufficient to give almost complete arrest of translation using a clone complementary to the 5'-non-coding region and first 171 coding nucleotides of TMV RNA. However, hybrids with DNA complementary to regions of the mRNA downstream of the first AUG gave variable and in some cases almost no arrest of translation in the reticulocyte lysate unless they were first digested with RNase H. A simple and rapid method for giving complete and highly specific arrest of translation of particular mRNAs in complex mixtures has been developed using both cDNA clones and synthetic oligodeoxynucleotides in conjunction with RNase H digestion. Evidence is presented that suggests that 'hybrid arrest of translation' in the wheat-germ cell-free system is primarily due to the action of RNase H. When a reticulocyte lysate was doped with 20 U/ml of RNase H, its ability to translate unannealed mRNA was unaffected but it translated DNA/RNA hybrids extremely poorly.
与烟草花叶病毒（TMV）RNA 5'端互补的单链 cDNA 克隆已被用于探索在网织红细胞裂解物中实现高效“翻译杂交阻断”所需的条件。结果表明，在 0.1 M KCl 中于 60 摄氏度孵育 20 分钟，使用与 TMV RNA 5'-非编码区及前 171 个编码核苷酸互补的克隆足以几乎完全阻断翻译。然而，与 mRNA 中第一个 AUG 下游区域互补的 DNA 形成的杂交体，在网织红细胞裂解物中导致的翻译阻断情况不一，在某些情况下几乎没有阻断，除非先用 RNase H 消化。利用 cDNA 克隆和合成寡脱氧核苷酸并结合 RNase H 消化，已开发出一种简单快速的方法，可在复杂混合物中完全且高度特异性地阻断特定 mRNA 的翻译。有证据表明，小麦胚芽无细胞系统中的“翻译杂交阻断”主要是由于 RNase H 的作用。当向网织红细胞裂解物中加入 20 U/ml 的 RNase H 时，其翻译未退火 mRNA 的能力未受影响，但翻译 DNA/RNA 杂交体的能力极差。
