Smith M D, Abrahamson R, Minton K W
Department of Pathology, F. E. Hebert School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814.
Plasmid. 1989 Sep;22(2):132-42. doi: 10.1016/0147-619x(89)90022-x.
An Escherichia coli plasmid that confers kanamycin resistance (Kmr) was inserted into the large Deinococcus radiodurans cryptic plasmids pUE10 and pUE11, yielding pS28 and pS19. The method of insertion involved both in vitro splicing and the natural transformation of D. radiodurans and yielded full-length clones in E. coli of pUE10 and pUE11. Both pS28 and pS19 replicated and expressed Kmr in E. coli and D. radiodurans. In both pS28 and pS19, D. radiodurans plasmid sequences were immediately upstream from the Kmr determinant. Transformation experiments suggested that Kmr expression in D. radiodurans was initiated in upstream D. radiodurans sequences. Restriction maps of pS28 and pS19 showed that each plasmid contained three MraI sites. Both pS28 and pS19 transformed the MraI-producing D. radiodurans strain R1 at low frequencies. D. radiodurans strain Sark, which naturally contains pUE10 and pUE11, was transformed by pS28 and pS19 at much higher frequencies. A Sark derivative that was cured for pUE10 was isolated by screening Sark/pS28 subisolates for loss of kanamycin resistance.
一个携带卡那霉素抗性（Kmr）的大肠杆菌质粒被插入到耐辐射球菌的隐蔽质粒pUE10和pUE11中，产生了pS28和pS19。插入方法涉及体外剪接和耐辐射球菌的自然转化，并在大肠杆菌中获得了pUE10和pUE11的全长克隆。pS28和pS19在大肠杆菌和耐辐射球菌中都能复制并表达Kmr。在pS28和pS19中，耐辐射球菌的质粒序列都位于Kmr决定簇的上游。转化实验表明，耐辐射球菌中Kmr的表达是由上游的耐辐射球菌序列启动的。pS28和pS19的限制性图谱显示，每个质粒都含有三个MraI位点。pS28和pS19都能以低频率转化产生MraI的耐辐射球菌菌株R1。天然含有pUE10和pUE11的耐辐射球菌菌株Sark被pS28和pS19以高得多的频率转化。通过筛选Sark/pS28亚分离物中卡那霉素抗性的丧失，分离出了一个缺失pUE10的Sark衍生物。
