Bachrach Gilad, Colston M Joseph, Bercovier Herve, Bar-Nir Dror, Anderson Colin, Papavinasasundaram K G
Division of Mycobacterial Research, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK1.
Department of Clinical Microbiology, The Hebrew University-Hadassah Medical School, Jerusalem, PO Box 12272, Israel2.
Microbiology (Reading). 2000 Feb;146 ( Pt 2):297-303. doi: 10.1099/00221287-146-2-297.
A 9.2 kb cryptic Mycobacterium fortuitum plasmid, pMF1, was isolated from strain 110 and its restriction map constructed. A 4.2 kb HindIII fragment of pMF1 was found to support replication in mycobacteria and this fragment was cloned and sequenced to characterize the replication elements of the plasmid. Computer analysis identified a putative Rep protein (362 amino acids) with high homology to the putative Rep protein of the Mycobacterium celatum plasmid pCLP and limited homology, mostly in the N-terminal region, to the Rep proteins of Mycobacterium avium pLR7, M. fortuitum pJAZ38 and Mycobacterium scrofulaceum pMSC262. A region containing a putative ori site was located upstream of the rep gene; this region displayed high homology at the nucleotide level with the predicted ori of pCLP and pJAZ38. A plasmid carrying the 4.2 kb HindIII fragment and a kanamycin resistance marker, designated pBP4, was maintained as a single-copy plasmid in Mycobacterium smegmatis and was stably inherited in the absence of antibiotic selection. Plasmid pBP4 was incompatible with the pJAZ38 replicon but was compatible with the widely used pAL5000 replicon, indicating that among the mycobacterial vectors now available there are two incompatibility groups. Significantly, the plasmid was able to replicate in the pathogen Mycobacterium tuberculosis, making it a useful tool for gene expression studies. To provide a choice of restriction sites and easy manipulation, a 2.1 kb fragment containing the minimal replication region was cloned to make the mycobacterial shuttle vector pBP10, which showed similar stability to pBP4.
从110菌株中分离出一个9.2 kb的隐秘偶然分枝杆菌质粒pMF1，并构建了其限制性图谱。发现pMF1的一个4.2 kb HindIII片段可支持在分枝杆菌中复制，对该片段进行克隆和测序以表征质粒的复制元件。计算机分析鉴定出一个推定的Rep蛋白（362个氨基酸），它与塞拉分枝杆菌质粒pCLP的推定Rep蛋白具有高度同源性，并且与鸟分枝杆菌pLR7、偶然分枝杆菌pJAZ38和瘰疬分枝杆菌pMSC262的Rep蛋白主要在N端区域具有有限的同源性。一个包含推定ori位点的区域位于rep基因上游；该区域在核苷酸水平上与pCLP和pJAZ38的预测ori具有高度同源性。携带4.2 kb HindIII片段和卡那霉素抗性标记的质粒，命名为pBP4，在耻垢分枝杆菌中作为单拷贝质粒维持，并在无抗生素选择的情况下稳定遗传。质粒pBP4与pJAZ38复制子不兼容，但与广泛使用的pAL5000复制子兼容，这表明在现有的分枝杆菌载体中有两个不兼容组。重要的是，该质粒能够在病原体结核分枝杆菌中复制，使其成为基因表达研究的有用工具。为了提供限制酶切位点的选择并便于操作，克隆了一个包含最小复制区域的2.1 kb片段，构建了分枝杆菌穿梭载体pBP10，其稳定性与pBP4相似。
