Ranes M G, Rauzier J, Lagranderie M, Gheorghiu M, Gicquel B
Unité de Génie Microbiologique, Institut Pasteur, Paris, France.
J Bacteriol. 1990 May;172(5):2793-7. doi: 10.1128/jb.172.5.2793-2797.1990.
Functional domains of pAL5000 were determined by gene disruption and deletion analysis. Of the five plasmid open reading frames (ORFs), ORF1 to ORF5, and a putative origin of replication previously identified (J. Rauzier, J. Moniz-Pereira, and B. Gicquel-Sanzey, Gene 71:315-321), two of the ORFs (ORF3 and ORF4) were deemed dispensable for plasmid replication. A "mini" mycobacterium-Escherichia coli shuttle plasmid applicable for general recombinant DNA studies in mycobacteria was constructed by using the gene for Kanr (Tn903) as a selective marker. Heterologous expression of the gene for Kanr was confirmed by Western blotting (immunoblotting) analysis.
通过基因破坏和缺失分析确定了pAL5000的功能结构域。在五个质粒开放阅读框（ORF），即ORF1至ORF5，以及先前鉴定的一个假定复制起点（J. Rauzier、J. Moniz-Pereira和B. Gicquel-Sanzey，《基因》71:315 - 321）中，其中两个ORF（ORF3和ORF4）被认为对于质粒复制是可有可无的。通过使用卡那霉素抗性基因（Tn903）作为选择标记，构建了一种适用于分枝杆菌一般重组DNA研究的“微型”分枝杆菌 - 大肠杆菌穿梭质粒。通过蛋白质免疫印迹（免疫印迹）分析证实了卡那霉素抗性基因的异源表达。
