Guntaka R V, Mitsialis S A
Gene. 1980 Dec;12(1-2):113-21. doi: 10.1016/0378-1119(80)90021-9.
Two avian tumor virus DNA fragments of 4.2 and 3.2 kb were inserted into pBR322 in the two possible orientations for each fragment. In the Escherichia coli host cells, RNA polymerase initiates transcription of large quantities (up to 0.5 to 1% of total E. coli RNA) of virus-specific RNA in the recombinant plasmids carrying the 4.2-kb fragment (pATV-6) but not in pATV-2 which contains the 3.2-kb fragment. Two SacI cleavage sites flank the putative promoter in the 4.2-kb viral insert. Deletion in the 1.2-kb SacI fragment obliterated the ability of pATV-6 to synthesize viral RNA. Digestion of the 1.2-kb SacI fragment with PvuI generates two fragments of 0.63 and 0.57 kb. Deletion of the 0.57-kb but not the 0.63-kb PvuI-SacI fragment completely eliminated the ability of the recombinant to synthesize viral RNA. These results strongly suggest that viral RNA in E. coli transcription is indeed initiated at a size present in the viral genome and that this site is localized in the 0.57-kb PvuI-SacI fragment.
将两个大小分别为4.2 kb和3.2 kb的禽肿瘤病毒DNA片段以每种片段的两种可能方向插入pBR322中。在大肠杆菌宿主细胞中，RNA聚合酶在携带4.2 kb片段的重组质粒（pATV - 6）中启动大量（高达大肠杆菌总RNA的0.5%至1%）病毒特异性RNA的转录，但在含有3.2 kb片段的pATV - 2中则不会启动。在4.2 kb病毒插入片段中，假定的启动子两侧有两个SacI切割位点。1.2 kb SacI片段的缺失消除了pATV - 6合成病毒RNA的能力。用PvuI消化1.2 kb SacI片段产生两个大小分别为0.63 kb和0.57 kb的片段。缺失0.57 kb而不是0.63 kb的PvuI - SacI片段完全消除了重组体合成病毒RNA的能力。这些结果有力地表明，大肠杆菌中转录的病毒RNA确实在病毒基因组中存在的一个大小处起始，并且该位点位于0.57 kb的PvuI - SacI片段中。
