1S911-mediated intramolecular transposition is naturally temperature sensitive
1S911-mediated intramolecular transposition is naturally temperature sensitive
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DOI：
10.1046/j.1365-2958.1997.4951854.x
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发表时间：
1997-08-01
影响因子：
3.6
通讯作者：
Chandler, M
中科院分区：
文献类型：
--
作者：
Haren, L;Betermier, M;Chandler, M
这里显示，细菌插入序列IS 911表现出温度敏感的转座表型。先前的结果已经证明，升高水平的IS 911转座酶OrfAB产生显著量的8字形形式，其通过转座子链之一的切割和环化产生，以及切除的环状形式，其中两个转座子链都已环化。我们在这里表明，与37 ℃相比，在42 ℃下体内观察到的两种类型的分子水平大大降低。另一方面，将温度降低到30摄氏度导致产量显著增加。在此温度下的转座活性足够高，允许在体内检测一个有缺陷的单一IS 911染色体拷贝的切除的环状形式时，OrfAB提供反式。一个类似的温度-活性曲线观察到的无细胞反应，使用部分纯化的OrfAB和生成的数字8形式独特。此外，获得了OrfAB的两个点突变体，这使得反应在体内和体外都具有部分温度抗性。这些结果表明转座酶本身的某些特性对升高的温度敏感。
It is shown here that the bacterial insertion sequence IS911 exhibits a temperature-sensitive transposition phenotype. Previous results have demonstrated that elevated levels of the IS911 transposase OrfAB generate significant quantities of a figure-eight form, created by cleavage and circularization of one of the transposon strands, and of an excised circular form, in which both transposon strands have been circularized. We show here that the level of both types of molecule observed in vivo was greatly reduced at 42 degrees C compared with 37 degrees C. On the other hand, reducing the temperature to 30 degrees C resulted in a significant increase in production. Transposition activity at this temperature was sufficiently high to permit detection in vivo of an excised circular form of a defective single IS911 chromosomal copy when OrfAB is supplied in trans. A similar temperature-activity profile is observed for a cell-free reaction that uses partially purified OrfAB and generates the figure-eight form uniquely. Moreover, two point mutants of OrfAB were obtained, which render the reactions partially temperature resistant both in vivo and in vitro. These results suggest that some property of transposase itself is sensitive to elevated temperatures.