Kondo K, Inokuchi H, Ozeki H
Department of Biophysics, Faculty of Science, Kyoto University, Japan.
Jpn J Genet. 1989 Dec;64(6):417-34. doi: 10.1266/jjg.64.417.
Transposon Tn3 was inserted into a tRNA operon of the amber suppressor Su+2 on a transducing phage (lambda hcI857nin5pSu+2) by selecting phages with ampicillin resistance and Su- phenotypes. In a strain thus obtained, Tn3 was inserted between the promoter and the first tRNA gene of the operon, which was determined by DNA sequencing. The Su+2 tRNA operon on the transducing phage consisted of two tRNA genes for tRNA(Met) and Su+2 tRNA(2Gln), which was a deletion derivative of the supB-E tRNA operon of E. coli containing seven tRNA genes in the order of promoter-Met-Leu-Gln1-Gln1-Met-Gln2-Gln2. Proliferating the lambda hcI857nin5pSu+2::Tn3 in E. coli cells, a number of phages which had lost Tn3 were isolated, and their tRNA gene compositions as well as the DNA structures of the tRNA operon were analyzed. In many cases the tRNA genes which had been deleted from the original transducing phage were regained from the chromosomal supB-E operon. Thus the loss of Tn3 from the phages was not due to excision of the transposon but due to the replacement of a portion of the tRNA operon, including Tn3, with the host homologous region that did not contain Tn3. This type of replacement takes place rather efficiently as a consequence of Tn3 insertion, owing to the general recombination occurring between homologous tRNA genes of phage and host chromosomes in the presence of either host recA or phage red. No such enhanced recombination in a similar cross between phage and host chromosomes was observed with the Tn3 present in the trans position on an independent plasmid. We conclude that inserting Tn3 in cis promotes general recombination in the neighboring regions. Possible mechanisms for this new type of genetic effect of Tn3 are discussed. During the course of this study, a natural defective mutation (T11) was also detected in one of the duplicated tRNA(2Gln) genes in an E. coli K12 strain we used.
通过筛选具有氨苄青霉素抗性和琥珀抑制缺陷型(Su-)表型的噬菌体,转座子Tn3被插入到转导噬菌体(λhcI857nin5pSu+2)上琥珀抑制因子Su+2的一个tRNA操纵子中。在这样获得的菌株中,通过DNA测序确定Tn3插入到了操纵子的启动子和第一个tRNA基因之间。转导噬菌体上的Su+2 tRNA操纵子由两个tRNA基因组成,分别是tRNA(Met)和Su+2 tRNA(2Gln),它是大肠杆菌supB-E tRNA操纵子的缺失衍生物,该操纵子包含七个tRNA基因,顺序为启动子-Met-Leu-Gln1-Gln1-Met-Gln2-Gln2。在大肠杆菌细胞中增殖λhcI857nin5pSu+2::Tn3,分离出许多丢失了Tn3的噬菌体,并分析了它们的tRNA基因组成以及tRNA操纵子的DNA结构。在许多情况下,从原始转导噬菌体中缺失的tRNA基因从染色体supB-E操纵子中重新获得。因此,噬菌体中Tn3的丢失不是由于转座子的切除,而是由于包含Tn3的部分tRNA操纵子被不含Tn3的宿主同源区域所取代。由于在宿主recA或噬菌体red存在的情况下,噬菌体和宿主染色体的同源tRNA基因之间发生了普遍重组,这种类型的取代作为Tn3插入的结果相当有效地发生。在独立质粒上处于转座位置的Tn3与宿主染色体进行类似杂交时,未观察到这种增强的重组。我们得出结论,顺式插入Tn3促进了邻近区域的普遍重组。讨论了Tn3这种新型遗传效应的可能机制。在这项研究过程中,在我们使用的大肠杆菌K12菌株的一个重复的tRNA(2Gln)基因中也检测到了一个自然缺陷突变(T11)。