FORMATION OF THE TANDEM REPEAT (IS30)2 AND ITS ROLE IN IS30-MEDIATED TRANSPOSITIONAL DNA REARRANGEMENTS
FORMATION OF THE TANDEM REPEAT (IS30)2 AND ITS ROLE IN IS30-MEDIATED TRANSPOSITIONAL DNA REARRANGEMENTS
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DOI：
10.1007/bf00281616
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发表时间：
1993-05-01
期刊：
影响因子：
--
通讯作者：
ARBER, W
中科院分区：
文献类型：
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作者：
OLASZ, F;STALDER, R;ARBER, W
质粒携带两个IS 30元件在相同的方向，如在复合转座子Tn 2706，在大肠杆菌中的结构不稳定。一级分离产物是通过两个IS 30元件之间携带的序列的位点特异性缺失形成的。得到的共价闭合复制子携带两个IS 30元件作为仅间隔2bp的串联重复。这种（IS 30）2结构极不稳定，但仍可在其载体质粒上分离，纯化后可通过转化重新导入宿主细胞。在recA-细菌的转化体的后代中，引入的（IS 30）2结构的复制拷贝仍然存在，以及各种分离产物，其为DNA重排的有效产生提供了证据。最丰富的是所涉及的IS 30元件的两个相同末端之间的另一个位点特异性重组的产物，这导致质粒上仅存在一个完整的IS 30。除此之外，根据IS 30转座的适当靶点的存在，还可以看到（IS 30）2的各种转座产物。分子内反应导致DNA倒位和缺失，断裂点不是IS 30末端。在分子间反应中，逆转座发生的频率很高，也可以得到简单转座和协整。一项突变研究揭示了一个完整的IS 30转座酶基因和两个IS 30元件的两个近端的顺式要求，这两个元件不仅涉及（IS 30）2的形成，而且还涉及其进一步的重排反应，包括有效形成位点特异性缺失。提出了一个模型，假设（IS 30）2中间体在IS 30转座途径中发挥关键作用，其中（IS 30）2的形成可能是限速的。一旦这种结构形成，它会在携带（IS 30）2的亚克隆中引起一系列的易位重排。这些研究结果的进化意义进行了讨论。
Plasmids carrying two IS30 elements in the same orientation, as in the composite transposon Tn2706, are structurally unstable in Escherichia coli. A primary segregation product is formed by site-specific deletion of the sequences carried between the two IS30 elements. The resulting covalently closed replicon carries the two IS30 elements as tandem repeats separated by only 2 bp. This (IS30)2 Structure is extremely unstable, but it can nevertheless be isolated on its vector plasmid and, after purification, can be reintroduced into host cells by transformation. Among the descendants of transformants of recA- bacteria, replicated copies of the introduced (IS30)2 structure are still present, together with various kinds of segregation products which provide evidence for the efficient generation of DNA rearrangements. Most abundant is the product of another site-specific recombination between two identical ends of the IS30 elements involved, which results in the presence of just one intact IS30 on the plasmid. Apart from this, and depending on the presence of appropriate targets for IS30 transposition, various transposition products of (IS30)2 are also seen. Intramolecular reactions lead to DNA inversions and deletions with breakpoints other than IS30 ends. In intermolecular reactions inverse transposition occurs at high frequency and one also obtains simple transposition and cointegration. A mutational study revealed the requirement in cis of one intact IS30 transposase gene and of both proximal ends of the two IS30 elements concerned not only for the formation of (IS30)2, but also for its further rearrangement reactions, including the efficient formation of site-specific deletions. A model is proposed, which postulates that (IS30)2 intermediates play a key role in IS30 transposition pathways in which the formation of (IS30)2 may be rate-limiting. Once this structure is formed, it gives rise to a burst of transpositional rearrangements in the subclone carrying (IS30)2. Evolutionary implications of these findings are discussed.