Gautier F, Bonewald R
Mol Gen Genet. 1980;178(2):375-80. doi: 10.1007/BF00270487.
A physical map for plasmid R1162 (Sm, Su, IncP4) was constructed. Neither EcoRI, PstI nor EcaI cut within a region essential for replication, molbilization or streptomycin resistence. Plasmid R1162 can replicate in E. coli as well as in Pseudomonas species and shows a strong dependence for DNA polymerase I in E. coli. By RP4 induced mobilization, R1162 can be transferred from E. coli to Pseudomonas AM1. A hybrid plasmid pFG7 (MW=8.4 x 10(6), Sm, Su, Ap, Tc) was constructed between pBR322 and R1162, which allows the selection of hybrid plasmids by insertional inactivation with the restriction enzymes HindIII, BamHI, SalI, ClaI. Transformation of E. coli SK1592 with Ecal-cut and ligated R1162-DNA and Pseudomonas AMI-DNA and subsequent mobilization of the hybrid plasmids into Pseudomonas AM1/M15a (methanol dehydrogenase-) led to the isolation of Pseudomonas AM1/M15a colonies, which could grow on methanol again. Back-conjugation into E. coli SK1592, subsequent mobilization studies and plasmid analysis suggests that the gene for Pseudomonas methanol dehydrogenase has been cloned in this vector.
构建了质粒R1162（链霉素敏感、磺胺敏感、IncP4）的物理图谱。EcoRI、PstI和EcaI都不会在复制、转移或链霉素抗性所必需的区域内切割。质粒R1162既能在大肠杆菌中复制，也能在假单胞菌属中复制，并且在大肠杆菌中对DNA聚合酶I有很强的依赖性。通过RP4诱导转移，R1162可以从大肠杆菌转移到假单胞菌AM1。在pBR322和R1162之间构建了一个杂种质粒pFG7（分子量 = 8.4×10⁶，链霉素敏感、磺胺敏感、氨苄青霉素抗性、四环素抗性），它允许通过用限制性内切酶HindIII、BamHI、SalI、ClaI进行插入失活来筛选杂种质粒。用EcaI切割并连接的R1162 - DNA和假单胞菌AMI - DNA转化大肠杆菌SK1592，随后将杂种质粒转移到假单胞菌AM1/M15a（甲醇脱氢酶缺陷型）中，从而分离出了能再次在甲醇上生长的假单胞菌AM1/M15a菌落。回交至大肠杆菌SK1592、随后的转移研究和质粒分析表明，假单胞菌甲醇脱氢酶基因已克隆到该载体中。
