Colasanti J, Denhardt D T
Department of Microbiology and Immunology, University of Western Ontario, London, Canada.
Mol Gen Genet. 1987 Sep;209(2):382-90. doi: 10.1007/BF00329669.
Recombinant DNA techniques were used to study various aspects of rep gene function in Escherichia coli. In order to enhance expression of the Rep protein, the rep gene was cloned into the vector pKC30 under the control of the lambda pL promoter. By trimming away a portion of the DNA sequence immediately upstream of the translational start site of rep, we were able to obtain very high levels of Rep protein upon induction. Cells carrying such plasmids showed no ill effects from the high concentration of the protein. To ascertain the consequence of the absence of Rep protein on the cell, the chromosomal copy of the gene was deleted using a homologous recombination technique. The viability of E. coli strains completely lacking the rep gene proves that the Rep function is not essential, at least in wild-type cells under laboratory conditions. We confirmed that in the absence of Rep function there is an increase in the average number of growing forks in exponentially growing cells; augmentation of Rep protein levels above normal, however, did not detectably decrease the number of growing forks.
利用重组DNA技术研究了大肠杆菌中rep基因功能的各个方面。为了增强Rep蛋白的表达，将rep基因克隆到受λ pL启动子控制的载体pKC30中。通过切除rep翻译起始位点上游紧邻的一部分DNA序列，我们能够在诱导后获得非常高水平的Rep蛋白。携带此类质粒的细胞未表现出因高浓度该蛋白而产生的不良影响。为了确定细胞中缺乏Rep蛋白的后果，使用同源重组技术删除了该基因的染色体拷贝。完全缺乏rep基因的大肠杆菌菌株的活力证明，至少在实验室条件下的野生型细胞中，Rep功能并非必不可少。我们证实，在缺乏Rep功能的情况下，指数生长细胞中生长叉的平均数量会增加；然而，将Rep蛋白水平提高到正常水平以上并未检测到生长叉数量的明显减少。
