Olasz F, Stalder R, Arber W
Institute of Molecular Genetics, Agricultural Biotechnological Center, Gödöllö, Hungary.
Mol Gen Genet. 1993 May;239(1-2):177-87. doi: 10.1007/BF00281616.
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.
像复合转座子Tn2706那样携带两个同向IS30元件的质粒，在大肠杆菌中结构不稳定。主要的分离产物是通过两个IS30元件之间序列的位点特异性缺失形成的。产生的共价闭合复制子携带两个作为串联重复的IS30元件，它们之间仅相隔2个碱基对。这种（IS30）2结构极其不稳定，但仍可在其载体质粒上分离出来，纯化后可通过转化重新导入宿主细胞。在recA - 细菌的转化子后代中，导入的（IS30）2结构的复制拷贝仍然存在，同时还有各种分离产物，这些产物为DNA重排的有效发生提供了证据。最丰富的产物是所涉及的IS30元件两个相同末端之间另一次位点特异性重组的结果，这导致质粒上仅存在一个完整的IS30。除此之外，根据是否存在适合IS30转座的靶标，还可见到（IS30）2的各种转座产物。分子内反应导致DNA倒位和缺失，其断点位于IS30末端以外。在分子间反应中，反向转座高频发生，还可得到简单转座和共整合。一项突变研究表明，不仅（IS30）2的形成，而且其进一步的重排反应，包括位点特异性缺失的有效形成，都需要一个完整的IS30转座酶基因以及相关两个IS30元件的两个近端在顺式条件下存在。提出了一个模型，该模型假定（IS30）2中间体在IS30转座途径中起关键作用，其中（IS30）2的形成可能是限速步骤。一旦形成这种结构，它会在携带（IS30）2的亚克隆中引发一阵转座重排。讨论了这些发现的进化意义。
