Pledger David W, Coates Craig J
Department of Biology (MSC-158), Texas A&M University, Kingsville, TX 78363, USA.
Insect Biochem Mol Biol. 2005 Oct;35(10):1199-207. doi: 10.1016/j.ibmb.2005.06.002.
The development of genetic strategies to control the spread of mosquito-borne diseases through the use of class II transposons has been hampered by suboptimal rates of transformation and the absence of post-integration mobility for all transposons evaluated to date. Two Mos1 mariner transposase mutants were produced by the site-directed mutagenesis of amino acids, E137 and E264, to K and R, respectively. The effects of these mutations on the transpositional activities of Mos1-derived transposon constructs were evaluated by interplasmid transposition assays in Escherichia coli and Aedes aegypti. The transpositional activities of two Mos1 transposons, one with imperfect wild type inverted terminal repeats (ITRs) and another that contained two perfectly matched 3' ITRs, were increased when the mutant transposases were supplied in trans in E. coli. The use of the perfect repeat transposon with wild type transposase did not result in an increase in transposition frequency in Ae. aegypti. However, an improvement in the integrity of the transposition process did occur, as evidenced by a lower rate of recombination events in which the transgene was transferred. An increase in the transpositional activity of the perfect repeat transposon was observed in the mosquito in the presence of either mutant transposase, and in the case of the E264R transposase, the observed increase in transposition frequency was also accompanied by a further improvement in the integrity of transposition. We discuss the possible contributions of these mutant residues to the transposition of the perfect repeat Mos1 transposon, the implications of these results with respect to the molecular evolution of Mos1, and the potential uses of the perfect repeat transposon and mutant transposases for the improvement of Mos1 mediated germ line transformation of Ae. aegypti.
通过使用II类转座子来控制蚊媒疾病传播的遗传策略的发展，一直受到转化效率不理想以及迄今为止评估的所有转座子整合后缺乏移动性的阻碍。通过将氨基酸E137和E264分别定点突变为K和R，产生了两个Mos1水手转座酶突变体。通过在大肠杆菌和埃及伊蚊中进行质粒间转座试验，评估了这些突变对源自Mos1的转座子构建体转座活性的影响。当在大肠杆菌中反式提供突变转座酶时，两个Mos1转座子的转座活性增加，其中一个具有不完美的野生型反向末端重复序列（ITR），另一个包含两个完美匹配的3' ITR。使用具有野生型转座酶的完美重复转座子在埃及伊蚊中并未导致转座频率增加。然而，转座过程的完整性确实得到了改善，这表现为转基因转移的重组事件发生率较低。在存在任何一种突变转座酶的情况下，在蚊子中观察到完美重复转座子的转座活性增加，并且在E264R转座酶的情况下，观察到的转座频率增加还伴随着转座完整性的进一步改善。我们讨论了这些突变残基对完美重复Mos1转座子转座的可能贡献、这些结果对Mos1分子进化的影响，以及完美重复转座子和突变转座酶在改善Mos1介导的埃及伊蚊种系转化方面的潜在用途。
