Potter P M, Wilkinson M C, Fitton J, Carr F J, Brennand J, Cooper D P, Margison G P
Department of Carcinogenesis, Paterson Institute for Cancer Research, Christie Hospital and Holt Radium Institute, Manchester, UK.
Nucleic Acids Res. 1987 Nov 25;15(22):9177-93. doi: 10.1093/nar/15.22.9177.
The plasmid pO61 that was isolated from an E. coli genomic DNA library and codes for O6-alkylguanine (O6AG) DNA alkyltransferase (ATase) activity (1) has been further characterised. Subclones of the 9 Kb insert of pO61 showed that the ATase activity was encoded in a 2Kb Pst1 fragment but a partial restriction endonuclease map of this was different to that of the E. coli ada gene that codes for O6-AG and alkylphosphotriester dual ATase protein. Fluorographic analyses confirmed that the molecular weight of the pO61-encoded ATase was 19KDa i.e. similar to that of the O6AG ATase function that is cleaved from the 39KDa ada protein but rabbit polyclonal antibodies to the latter reacted only very weakly with the pO61-encoded protein. A different set of hybridisation signals was produced when E. coli DNA, which had been digested with a variety of restriction endonucleases was probed with 2Kb Pst 1 fragment or the ada gene. These results provided evidence for the existence of a second ATase gene in E. coli. The 2Kb Pst-1 fragment of pO61 was therefore sequenced and an open reading frame (ORF) that would give rise to a 19KDa protein was identified. The derived amino acid sequence of this showed a 93 residue region with 49% homology with the O6AG ATase region of the ada protein and had a pentamer and a heptamer of identical sequence separated by 34 amino acids in both proteins. The pentamer included the alkyl accepting cysteine residue of the ada O6AG ATase. The hydrophobic domains were similarly distributed in both proteins. Shine-Dalgarno, -10 and -35 sequences were identified and the origin of transcription was located by primer extension and S1 nuclease mapping. The amino-terminal amino acid sequence of the protein was as predicted from the ORF.
从大肠杆菌基因组DNA文库中分离得到的编码O6-烷基鸟嘌呤（O6AG）DNA烷基转移酶（ATase）活性的质粒pO61（1）已得到进一步鉴定。pO61的9 Kb插入片段的亚克隆表明，ATase活性由一个2Kb的Pst1片段编码，但该片段的部分限制性内切酶图谱与编码O6-AG和烷基磷酸三酯双功能ATase蛋白的大肠杆菌ada基因的图谱不同。荧光自显影分析证实，pO61编码的ATase的分子量为19KDa，即与从39KDa的ada蛋白上切割下来的O6AG ATase功能蛋白的分子量相似，但针对后者的兔多克隆抗体与pO61编码的蛋白反应非常微弱。当用2Kb Pst 1片段或ada基因探测经多种限制性内切酶消化的大肠杆菌DNA时，产生了不同的杂交信号集。这些结果为大肠杆菌中存在第二个ATase基因提供了证据。因此，对pO61的2Kb Pst-1片段进行了测序，并鉴定出一个可产生19KDa蛋白的开放阅读框（ORF）。其推导的氨基酸序列显示，有一个93个残基的区域与ada蛋白的O6AG ATase区域有49%的同源性，并且在两种蛋白中都有一个相同序列的五聚体和七聚体，中间相隔34个氨基酸。五聚体包括ada O6AG ATase的烷基接受半胱氨酸残基。两种蛋白中的疏水结构域分布相似。鉴定出了Shine-Dalgarno、-10和-35序列，并通过引物延伸和S1核酸酶作图确定了转录起始位点。该蛋白的氨基末端氨基酸序列与从ORF预测的一致。
