Liebscher D H, Coutelle C, Rapoport T A, Hahn V, Rosenthal S, Prehn S, Williamson R
Gene. 1980 May;9(3-4):233-46. doi: 10.1016/0378-1119(90)90325-l.
The successful cloning of recombinants between cDNA from fractionated poly(A)+-RNA of Brockmann bodies of the carp and the plasmid pBR322 in Escherichia coli chi 1776 is reported. One of the recombinant clones has been identified as a preproinsulin-cDNA recombinant by the hybrid-arrest translation assay. Recombination was at the PstI site of pBR322; reconstitution of this site was by 3'-tailing of the vector with dGn. The transformants were screened by in situ hybridization with kinase-labeled poly(A)+-RNA sedimenting at 9S from Brockmann bodies. Restriction analysis was performed on 26 of the strongly hybridizing clones to estimate the size of the inserted cDNA. Six of the recombinants studied contain inserts of a size approximating to full length 9S preproinsulin mRNA. The hybrid-arrest translation assay on selected clones identified one as a recombinant containing the preproinsulin cDNA sequence.
报道了鲤鱼布罗克曼小体分级分离的聚腺苷酸加尾RNA（poly(A)+-RNA）的cDNA与质粒pBR322在大肠杆菌chi 1776中成功克隆重组体。通过杂交抑制翻译试验，其中一个重组克隆被鉴定为前胰岛素原cDNA重组体。重组发生在pBR322的PstI位点；该位点的重建是通过用dGn对载体进行3'端加尾。用来自布罗克曼小体的沉降系数为9S的激酶标记聚腺苷酸加尾RNA进行原位杂交筛选转化体。对26个强杂交克隆进行限制性分析以估计插入的cDNA的大小。所研究的六个重组体含有大小近似全长9S前胰岛素原mRNA的插入片段。对选定克隆的杂交抑制翻译试验鉴定出一个含有前胰岛素原cDNA序列的重组体。
