Machida Y, Machida C, Ohtsubo H, Ohtsubo E
Proc Natl Acad Sci U S A. 1982 Jan;79(2):277-81. doi: 10.1073/pnas.79.2.277.
We demonstrate that mutants with deletions at either end of the insertion sequence IS1 lose the ability to mediate cointegration of two plasmids, whereas mutants with deletions or an insertion within IS1 can mediate cointegration at a reduced frequency. These results, together with the nucleotide sequence analysis of the IS1 mutants, indicate that the two ends of IS1 (insL and insR) and two genes (insA and insB) that are encoded by IS1 are required for cointegration. Using a plasmid carrying two copies of IS1, we found that the individual IS1s mediate cointegration at different characteristic frequencies, and that each of two parts of plasmid DNA segments flanked by the two IS1s is a transposon, mediating plasmid cointegration at a unique frequency. When one IS1 was replaced with a mutant IS1, the remaining wild-type IS1 complemented the cointegration ability of the mutant IS1 as well as a resulting mutant transposon that was then flanked by a wild-type IS1 and a mutant IS1. The efficiency of this complementation reflected the characteristic ability of an individual IS1 present on the plasmid to promote cointegration. The results suggest that the IS1-encoded proteins are produced in different amounts, depending on the location of IS1 in the plasmid, and that these amounts determine the efficiency of complementation of the cointegration ability of a mutant IS1 as well as a mutant transposon. However, the location of an individual IS1 itself can also determine the frequency of cointegration in the presence of a given amount of the IS1 proteins. On the basis of the observation that the cointegration ability of a mutant IS1 is less efficiently complemented than is the ability of a mutant transposon, we also suggest that the IS1-encoded proteins can function in trans, but act preferentially on the IS1 or transposon sequence from which they are produced in promoting cointegration.
我们证明，插入序列IS1两端缺失的突变体失去了介导两个质粒共整合的能力，而IS1内部有缺失或插入的突变体能够以较低频率介导共整合。这些结果，连同对IS1突变体的核苷酸序列分析，表明IS1的两端（insL和insR）以及由IS1编码的两个基因（insA和insB）是共整合所必需的。使用携带两个IS1拷贝的质粒，我们发现单个IS1以不同的特征频率介导共整合，并且由两个IS1侧翼的质粒DNA片段的两部分中的每一部分都是一个转座子，以独特的频率介导质粒共整合。当一个IS1被一个突变的IS1取代时，剩余的野生型IS1补充了突变IS1的共整合能力以及随后由野生型IS1和突变IS1侧翼的所得突变转座子的共整合能力。这种互补的效率反映了质粒上存在的单个IS1促进共整合的特征能力。结果表明，IS1编码的蛋白质产生的量不同，这取决于IS1在质粒中的位置，并且这些量决定了突变IS1以及突变转座子共整合能力的互补效率。然而，单个IS1本身的位置在存在给定数量的IS1蛋白质时也可以决定共整合的频率。基于观察到突变IS1的共整合能力比突变转座子的能力更难被有效互补，我们还表明IS1编码的蛋白质可以反式作用，但在促进共整合时优先作用于产生它们的IS1或转座子序列。
