Mendiola M V, Bernales I, de la Cruz F
Departmento de Biología Molecular, Universidad de Cantabria, Santander, Spain.
Proc Natl Acad Sci U S A. 1994 Mar 1;91(5):1922-6. doi: 10.1073/pnas.91.5.1922.
Insertion sequence 91 (IS91) inserts specifically at GTTC or CTTG target sequences without duplication of the target. After insertion, the right inverted repeat (IRR) lies adjacent to the 3' end of the target sequences (or 5' to the complementary sequence CAAG or GAAC). We have analyzed the effects of alteration of each terminus of IS91 on transposition activity in Escherichia coli. IRR is absolutely required for transposition. Deletion analysis indicates that a 14-bp segment is not sufficient, but an 81-bp sequence within the IRR region is sufficient. Furthermore, the GTTC/CTTG target site is also required. The left inverted repeat (IRL) of IS91 is dispensable. Plasmid fusions originated by one-ended transposition of IS91 derivatives lacking IRL occur at about the same frequency as cointegrate formation observed for the wild-type element. In the one-ended-type fusions, the inserted fragment of donor DNA is flanked at one end (constant end) by IRR and at the other end by a GTTC or CTTG sequence present in the donor (variable end) in a way that usually results in multiple tandem insertions of the donor plasmid in the target site. These results are easily accommodated by a rolling-circle replicative transposition mechanism. This model also draws support from the finding that the IS91 transposase is related in sequence to the superfamily of rolling-circle replication proteins and the observation that IRR shows some conservation in sequence and secondary structure with the origins of replication of some rolling-circle replication plasmids.
插入序列91（IS91）特异性地插入GTTC或CTTG靶序列，且不重复靶序列。插入后，右侧反向重复序列（IRR）与靶序列的3'端相邻（或与互补序列CAAG或GAAC的5'端相邻）。我们分析了IS91每个末端的改变对大肠杆菌中转座活性的影响。转座绝对需要IRR。缺失分析表明，一个14bp的片段是不够的，但IRR区域内的一个81bp序列是足够的。此外，GTTC/CTTG靶位点也是必需的。IS91的左侧反向重复序列（IRL）是可有可无的。缺乏IRL的IS91衍生物通过单端转座产生的质粒融合，其发生频率与野生型元件观察到的共整合形成频率大致相同。在单端型融合中，供体DNA的插入片段一端（恒定端）侧翼为IRR，另一端侧翼为供体中存在的GTTC或CTTG序列（可变端），这种方式通常导致供体质粒在靶位点的多个串联插入。这些结果很容易用滚环复制转座机制来解释。该模型还得到以下发现的支持：IS91转座酶在序列上与滚环复制蛋白超家族相关，以及观察到IRR在序列和二级结构上与一些滚环复制质粒的复制起点有一定的保守性。
