Smith D F, Searle P F, Williams J G
Nucleic Acids Res. 1979 Feb;6(2):487-506. doi: 10.1093/nar/6.2.487.
A 1700 nucleotide DNA sequence derived from Xenopus vitellogenin mRNA has been cloned in the bacterial plasmid pBR322. The identity of the cloned sequence was verified in two ways. Firstly, the plasmid DNA was shown to hybridise to an RNA of the correct size (6,700 nucleotides). This was shown by in situ hybridisation to electrophoretically separated RNA and also by the formation of "R-loops" with purified vitellogenin mRNA. Then, using a novel procedure in which plasmid DNA covalently bound to diazotised paper is used to select complementary mRNA sequences, the cloned sequence was shown to hybridise to an mRNA which directed the synthesis of vitellogenin when translated in a reticulocyte lysate cell-free system.
一段源自非洲爪蟾卵黄原蛋白mRNA的1700个核苷酸的DNA序列已被克隆到细菌质粒pBR322中。通过两种方法验证了克隆序列的身份。首先,显示质粒DNA与正确大小(6700个核苷酸)的RNA杂交。这通过对电泳分离的RNA进行原位杂交以及与纯化的卵黄原蛋白mRNA形成“R环”得以证明。然后,使用一种新方法,即利用共价结合到重氮化纸上的质粒DNA来选择互补的mRNA序列,结果显示克隆序列与一种mRNA杂交,该mRNA在无细胞的网织红细胞裂解物系统中翻译时指导卵黄原蛋白的合成。