Olsen J C, Watson K F
Nucleic Acids Res. 1982 Feb 11;10(3):1009-27. doi: 10.1093/nar/10.3.1009.
The early DNa products of reverse transcription have been analyzed from reconstructed reactions containing avian myeloblastosis virus 35S RNA . tRNAtrp complex and highly purified reverse transcriptase. We describe conditions for the synthesis of genome-length complementary DNA and two discrete species of plus strand DNA (the same chemical polarity as the viral RNA genome) about 300 and 400 nucleotides in length. Plus DNA400 and plus DNA300 were detected by molecular hybridization with DNA probes complementary to sequences from both the 3'- and 5'-ends of the viral RNA. Both species appear to be copied from the 5'-end of minus strand DNA by their hybridization properties and their early synthesis when only the 5'-end of minus strand DNA is available as template. Restriction endonuclease mapping of plus DNA400 and plus DNA300 rules out a precursor-product relationship between the two. Rather the results suggest a unique initiation site for both species, with plus DNA400 containing internal sequences not present in plus DNA300. Plus DNA400 and plus DNA300 appear to be analogous to early plus DNA species detected in cells early after retrovirus infection. Thus, purified reverse transcriptase appears to be enzymatically sufficient for synthesis of genome-length complementary DNA and initiation and synthesis of early plus strand DNA as observed in infected cells.
已从含有禽成髓细胞瘤病毒35S RNA、色氨酸tRNA复合物和高度纯化逆转录酶的重建反应中分析了逆转录早期的DNA产物。我们描述了合成基因组长度互补DNA以及两种长度分别约为300和400个核苷酸的离散正链DNA（与病毒RNA基因组具有相同化学极性）的条件。通过与与病毒RNA 3'-端和5'-端序列互补的DNA探针进行分子杂交检测到正链DNA400和正链DNA300。从它们的杂交特性以及当只有负链DNA的5'-端可作为模板时它们的早期合成情况来看，这两种类型的正链DNA似乎都是从负链DNA的5'-端复制而来。对正链DNA400和正链DNA300进行限制性内切酶图谱分析排除了两者之间存在前体-产物关系。相反，结果表明这两种类型的正链DNA都有一个独特的起始位点，正链DNA400含有正链DNA300中不存在的内部序列。正链DNA400和正链DNA300似乎类似于在逆转录病毒感染后早期在细胞中检测到的早期正链DNA类型。因此，纯化的逆转录酶在酶促作用下似乎足以合成基因组长度的互补DNA，并启动和合成如在受感染细胞中观察到的早期正链DNA。
