Wood D O, Hollinger M F, Tindol M B
J Bacteriol. 1981 Mar;145(3):1448-51. doi: 10.1128/jb.145.3.1448-1451.1981.
A pBR322:RSF1010 composite plasmid, constructed in vitro, was used as a cloning vector in Pseudomonas aeruginosa. This nonamplifiable plasmid, pMW79, has a molecular weight of 8.4 X 10(6) and exists as a multicopy plasmid in both P. aeruginosa and Escherichia coli. In P. aeruginosa strain PAO2003, pMW79 conferred resistance to carbenicillin and tetracycline. Characterization of pMW79 with restriction enzymes revealed that four enzymes (BamHI, SalI, HindIII, and HpaI) cleaved the plasmid at unique restriction sites. Cloning P. aeruginosa chromosomal deoxyribonucleic acid fragments into the BamHI or SalI site of pMW79 inactivated the tetracycline resistance gene. Thus, cells carrying recombinant plasmids could be identified by their carbenicillin resistance, tetracycline sensitivity phenotype. Deoxyribonucleic acid fragments of approximately 0.5 to 7.0 megadaltons were inserted into pMW79, and the recombinant plasmids were stably maintained in a recombination-deficient (recA) P. aeruginosa host.
一种体外构建的pBR322:RSF1010复合质粒被用作铜绿假单胞菌的克隆载体。这种不可扩增的质粒pMW79，分子量为8.4×10⁶，在铜绿假单胞菌和大肠杆菌中均以多拷贝质粒形式存在。在铜绿假单胞菌菌株PAO2003中，pMW79赋予对羧苄青霉素和四环素的抗性。用限制酶对pMW79进行表征显示，四种酶（BamHI、SalI、HindIII和HpaI）在独特的限制位点切割该质粒。将铜绿假单胞菌染色体脱氧核糖核酸片段克隆到pMW79的BamHI或SalI位点会使四环素抗性基因失活。因此，携带重组质粒的细胞可通过其羧苄青霉素抗性、四环素敏感性表型来鉴定。大约0.5至7.0兆道尔顿的脱氧核糖核酸片段被插入到pMW79中，并且重组质粒在重组缺陷型（recA）铜绿假单胞菌宿主中得以稳定维持。
