Ishiura M, Hazumi N, Koide T, Uchida T, Okada Y
Department of Cell Biology, National Institute for Basic Biology, Okazaki, Japan.
J Bacteriol. 1989 Feb;171(2):1068-74. doi: 10.1128/jb.171.2.1068-1074.1989.
Segments of DNA are deleted from recombinant cosmid DNAs with high frequency during propagation in standard recA Escherichia coli hosts. An attempt has been made to derive an appropriate strain of E. coli, suitable for cosmid cloning, in which such deletions do not occur. We examined the effects of a series of host recombinational mutations on the deletion process, using six independent recombinant cosmids that carry inserts of mouse, Chinese hamster, or human DNA. Various E. coli host cells carrying the recombinant cosmids were cultured serially in liquid medium, and the recombinant cosmid DNAs were extracted from the host cells and analyzed by agarose gel electrophoresis and by gene transfer of the DNAs into cultured mammalian cells. Of the mutations examined, only a recB recC sbcB recJ (or recN) quadruple combination of host mutations prevented the deletion of DNA segments. The recombinant cosmid DNAs propagated in E. coli hosts that carried this combination of mutations were functionally as well as structurally intact. We propose that the recJ (and/or recN) gene is involved in some aspect of the events that lead to deletions of cosmid DNA in a recB recC sbcB genetic background.
在标准recA大肠杆菌宿主中繁殖期间，重组黏粒DNA的DNA片段会高频缺失。人们已尝试获得一种适合黏粒克隆的大肠杆菌菌株，在该菌株中不会发生此类缺失。我们使用六种携带小鼠、中国仓鼠或人类DNA插入片段的独立重组黏粒，研究了一系列宿主重组突变对缺失过程的影响。携带重组黏粒的各种大肠杆菌宿主细胞在液体培养基中连续培养，从宿主细胞中提取重组黏粒DNA，并通过琼脂糖凝胶电泳以及将DNA基因转移到培养的哺乳动物细胞中进行分析。在所检测的突变中，只有recB recC sbcB recJ（或recN）宿主突变的四重组合可防止DNA片段的缺失。在携带这种突变组合的大肠杆菌宿主中繁殖的重组黏粒DNA在功能和结构上均保持完整。我们提出，recJ（和/或recN）基因在recB recC sbcB遗传背景下导致黏粒DNA缺失的某些事件中发挥作用。
