Borovok I A
Mol Biol (Mosk). 1990 Sep-Oct;24(5):1419-28.
Using thermoelimination (at 42 degrees C) of the thermoinducible coliphage P1tsCmr omega::TnV (TnV is a Tn5 derivative which contains the replication origin (Rep) of plasmid pSC101), more than 110 KmrCms Escherichia coli K12 clones were selected. It was supposed that the KmrCms phenotype could result only from insertion of TnV (Kmr) into E. coli chromosome and the loss of phage (Cms). It was found that the majority of KmrCms clones (35-90%) contained miniplasmids. Their molecular sizes did not exceed the TnV size (6.1 kb). The formation of miniplasmids called pTnV was observed both in RecA+ cells (C600) and in RecA- (HB101), more often in the latters. Interestingly, that miniplasmids of only several molecular sizes were detected: from 6.1 kb (pTnV60) to 4.35 kb (pTnV43). A restriction analysis showed that DNA of the majority of pTnV plasmids had varying deletions (0.3-1.3 kb) of mainly IS50L element which together with IS50R flank TnV. Very low transposition frequencies (approx. 10(-8) Kmr transconjugants per transferred R388) of all pTnV types (including pTnV60 plasmids containing probably microdeletions of the joining "outside" IS50's ends) suggest that pTnV plasmids are not intermediates in TnV transposition. Possibly the circularized TnV derivatives (pTnV's) are side products of the transposition resulting from the abortive attempts of an excised and autonomous transposon molecule to insert into itself. In the present paper the possible mechanisms of the origin of limited pTnV type numbers are also discussed.
利用热诱导噬菌体P1tsCmr ω::TnV(TnV是一种Tn5衍生物,含有质粒pSC101的复制起点(Rep))在42℃下进行热消除,筛选出了110多个KmrCms大肠杆菌K12克隆。据推测,KmrCms表型可能仅由TnV(Kmr)插入大肠杆菌染色体以及噬菌体(Cms)丢失所致。结果发现,大多数KmrCms克隆(35 - 90%)含有微型质粒。它们的分子大小不超过TnV的大小(6.1 kb)。在RecA +细胞(C600)和RecA -细胞(HB101)中均观察到了称为pTnV的微型质粒的形成,在后者中更为常见。有趣的是,仅检测到几种分子大小的微型质粒:从6.1 kb(pTnV60)到4.35 kb(pTnV43)。限制性分析表明,大多数pTnV质粒的DNA主要是IS50L元件有不同程度的缺失(0.3 - 1.3 kb),IS50L元件与IS50R一起位于TnV两侧。所有pTnV类型(包括可能在连接“外侧”IS50末端有微缺失的pTnV60质粒)的转座频率都非常低(约每转移一个R388产生10^(-8)个Kmr转接合子),这表明pTnV质粒不是TnV转座的中间体。可能环化的TnV衍生物(pTnV)是转座的副产物,是由切除并自主的转座子分子试图自身插入时的失败尝试导致的。本文还讨论了有限数量的pTnV类型产生的可能机制。