Hizi A, Barber A, Hughes S H
Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Israel.
Virology. 1989 May;170(1):326-9. doi: 10.1016/0042-6822(89)90389-9.
We have described a strain of Escherichia coli that expresses high levels of enzymatically active, soluble, HIV-1 reverse transcriptase (A. Hizi, C. McGill, and S. H. Hughes, Proc. Natl. Acad. Sci. USA, 85, 1218-1222, 1988). The clone can be used as a source of the enzyme and to generate and characterize mutations in the reverse transcriptase. We have made a series of small in-frame insertions in the region that encodes the reverse transcriptase. When the mutant plasmids are reintroduced into E. coli, they induce the synthesis of mutant forms of the enzyme. With one interesting exception, the reduction in RNA-dependent DNA polymerizing activity seen in the mutants correlates well with the degree of sequence conservation among the various reverse transcriptases. Insertions into regions that are evolutionarily conserved have a more profound effect on RNA-dependent DNA polymerase activity than do insertions into regions that are less conserved. The exception to this simple correlation is that a small insertion into the region encoding RNase H gives rise to a protein with essentially no RNA-dependent DNA polymerase activity. We suggest that this mutation may affect the ability of the reverse transcriptase to fold properly, which might explain our previous observation that small carboxyl terminal deletions profoundly affect RNA-dependent NAD polymerase activity.
我们已经描述了一种大肠杆菌菌株，它能高水平表达具有酶活性的可溶性HIV-1逆转录酶（A. 希齐、C. 麦吉尔和S. H. 休斯，《美国国家科学院院刊》，85卷，1218 - 1222页，1988年）。该克隆可作为这种酶的来源，并用于产生和鉴定逆转录酶中的突变。我们在编码逆转录酶的区域进行了一系列小的读框内插入。当将突变质粒重新导入大肠杆菌时，它们会诱导合成该酶的突变形式。除了一个有趣的例外，在突变体中观察到的RNA依赖性DNA聚合活性的降低与各种逆转录酶之间的序列保守程度密切相关。插入进化上保守的区域对RNA依赖性DNA聚合酶活性的影响比插入保守性较低的区域更为深远。这种简单相关性的例外情况是，在编码核糖核酸酶H的区域进行一个小的插入会产生一种基本上没有RNA依赖性DNA聚合酶活性的蛋白质。我们认为这种突变可能会影响逆转录酶正确折叠的能力，这或许可以解释我们之前的观察结果，即小的羧基末端缺失会深刻影响RNA依赖性NAD聚合酶活性。
