Ohtsubo H, Zenilman M, Ohtsubo E
J Bacteriol. 1980 Oct;144(1):131-40. doi: 10.1128/jb.144.1.131-140.1980.
In vivo recombination was found to occur between plasmid pHS1, a temperature-sensitive replication mutant of pSC101 carrying tetracycline resistance, and plasmid ColE1 after selection for tetracycline resistance at the restrictive temperature, 42 degrees C. Extensive analysis of the physical structures of three of these recombinant plasmids, using restriction endonucleases and the electron microscope heteroduplex method, revealed that the plasmid pHS1 was integrated into different sites on ColE1. The recombinant plasmids contained a duplication of a unique 1-kilobase (kb) sequence of pHS1 in a direct orientation at the junctions between the two parental plasmid sequences. This was confirmed by comparing the nucleotide sequence of the recombinants and their parental plasmids. Nucleotide sequence analysis further revealed that nine nucleotides at the site of recombination of ColE1 were duplicated at the junction of each of the 1-kb sequences. The formation of recombinants was independent of RecA function. Based on our previous finding that a plasmid containing a deoxyribonucleic acid insertion (IS) element can recombine with a second plasmid to generate a duplication of the IS element, we conclude that the 1-kb sequence is an insertion sequence, which we named IS102. For convenience, we have also denoted the IS102 sequence as eta theta to assign the orientation of the sequence. Eighteen nucleotides at one end (eta end) were found to be repeated in an inverted orientation at the other end (theta end) of IS102. The nucleotide sequence of the eta end of the sequence was found to be identical to the sequence at the ends of the transposon Tn903, which is responsible for transposition of the kanamycin resistance gene.
在42℃的限制温度下选择四环素抗性后,发现携带四环素抗性的pSC101温度敏感复制突变体质粒pHS1与质粒ColE1之间在体内发生重组。使用限制性内切酶和电子显微镜异源双链法对其中三个重组质粒的物理结构进行了广泛分析,结果表明质粒pHS1整合到了ColE1的不同位点。重组质粒在两个亲本质粒序列的连接处包含一段以直接方向排列的pHS1独特1千碱基(kb)序列的重复。通过比较重组体及其亲本质粒的核苷酸序列证实了这一点。核苷酸序列分析进一步表明,ColE1重组位点的九个核苷酸在每个1-kb序列的连接处重复。重组体的形成与RecA功能无关。基于我们之前的发现,即含有脱氧核糖核酸插入(IS)元件的质粒可与第二个质粒重组以产生IS元件的重复,我们得出结论,该1-kb序列是一个插入序列,我们将其命名为IS102。为方便起见,我们还将IS102序列表示为ηθ以确定该序列的方向。发现IS102一端(η端)的18个核苷酸在另一端(θ端)以反向重复。发现该序列η端的核苷酸序列与转座子Tn903末端的序列相同,Tn903负责卡那霉素抗性基因的转座。