Characterisation and nucleotide sequence of ogt, the O6-alkylguanine-DNA-alkyltransferase gene of E. coli.
Characterisation and nucleotide sequence of ogt, the O6-alkylguanine-DNA-alkyltransferase gene of E. coli.
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大肠杆菌O6-烷基鸟嘌呤-DNA-烷基转移酶基因ogt 的特征和核苷酸序列。
DOI：
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发表时间：
1987
影响因子：
14.9
通讯作者：
G. P. Margison
中科院分区：
文献类型：
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作者：
Philip M. Potter;M. Wilkinson;J. Fitton;Francis J Carr;John Brennand;Donald P. Cooper;G. P. Margison
从大肠杆菌基因组DNA文库中分离到编码O6-烷基鸟嘌呤(O6AG)DNA烷基转移酶(ATase)活性(1)的pO61，并对其进行了进一步鉴定。PO61的9kb插入片段的亚克隆表明，该AT酶活性编码在2kb的Pst1片段中，但其部分限制性内切酶图谱与编码O6-AG和烷基磷酸三酯双ATase蛋白的E.Coliada基因的部分限制性内切酶图谱不同。荧光光谱分析证实，pO61编码的ATase的相对分子质量为19 KDa，与39 KDa蛋白裂解的O6AG ATase功能相似，但后者的兔多克隆抗体仅与pO61编码的蛋白反应很弱。用2kb的PST-1片段或ada基因探测用各种限制性内切酶消化的大肠杆菌DNA时，产生了一组不同的杂交信号。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，并鉴定了一个可产生19 KDa蛋白的开放阅读框(ORF)。其推导的氨基酸序列与ADA蛋白的O6AG ATase区有一个93个残基区，同源性为49%，两个蛋白中都有一个五聚体和一个七聚体序列相同，由34个氨基酸隔开。该五聚体包括接受ADAO6AG AT酶半胱氨酸残基的烷基。疏水结构域在两种蛋白质中的分布相似。通过扩增和S1核酸酶定位，确定了该基因的转录起始点。蛋白质的氨基末端氨基酸序列与开放阅读框架预测的一致。
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.