Ivanov V A, Melnikov A A, Siunov A V, Fodor I I, Ilyin Y V
Institute of Cell Biophysics, Academy of Sciences, Poustchino, Moscow Region, USSR.
EMBO J. 1991 Sep;10(9):2489-95. doi: 10.1002/j.1460-2075.1991.tb07788.x.
The mobile element jockey is similar in structural organization and coding potential to the LINEs of various organisms. It is transcribed at different stages of Drosophila ontogenesis. The Drosophila LINE family includes active transposable elements. Current models for the mechanism of transposition involve reverse transcription of an RNA intermediate and utilization of element-encoded proteins. As demonstrated here, a 2.23 kb DNA fragment from the region of jockey encoding the putative reverse transcriptase was stably introduced into an expression system under inducible control of the Escherichia coli lac regulatory elements. We describe the expression of the 92 kDa protein and identify this polypeptide alone as the authentic jockey reverse transcriptase based on some of its physical and enzymic properties. The jockey polymerase demonstrates RNA and DNA-directed DNA polymerase activities but lacks detectable RNase H, has a temperature optimum at 26 degrees C, requires Mg2+ or Mn2+ as a cofactor and is inactivated by sulphydryl reagent. The enzyme prefers poly(rC) and poly(rA) as template and 'activated' DNA is not effective.
移动元件“赛马”在结构组织和编码潜能方面与各种生物体的长散在核元件(LINEs)相似。它在果蝇个体发育的不同阶段转录。果蝇LINE家族包括活跃的转座元件。目前的转座机制模型涉及RNA中间体的逆转录以及元件编码蛋白的利用。如本文所示,来自“赛马”区域编码假定逆转录酶的一个2.23 kb DNA片段被稳定地引入到在大肠杆菌乳糖调节元件诱导控制下的表达系统中。我们描述了92 kDa蛋白的表达,并根据其一些物理和酶学特性将该多肽鉴定为真正的“赛马”逆转录酶。“赛马”聚合酶表现出RNA和DNA指导的DNA聚合酶活性,但缺乏可检测到的核糖核酸酶H,最适温度为26℃,需要Mg2+或Mn2+作为辅因子,并且会被巯基试剂灭活。该酶更喜欢以聚(rC)和聚(rA)作为模板,而“活化”的DNA无效。