Nurk A, Tamm A, Hôrak R, Kivisaar M
Institute of Molecular and Cell Biology, Estonian Biocentre, Tartu.
Gene. 1993 May 15;127(1):23-9. doi: 10.1016/0378-1119(93)90612-7.
Plasmid pEST1463 carrying the promoterless pheBA operon was cloned into Pseudomonas putida PaW85, and phenol-utilizing colonies were isolated on minimal plates containing phenol as the only carbon and energy source. In these clones, chromosomally located Tn4652 was transposed upstream from the coding sequencing of pheA (encoding phenol monooxygenase). Sequence analysis together with mapping of the transcription start point of the pheBA operon in the recombinant plasmids revealed that fusions of the -10 sequences present in the pheBA operon and -35 sequence located in the terminal inverted repeats of Tn4652 had generated functional promoters under selective pressure in P. putida cells. These promoter sequences show similarity to the Escherichia coli RNA polymerase sigma 70 promoter consensus sequence. In three of the six fusion promoters studied, the generation combined two distinct events: transposition of Tn4652 into DNA containing potential -10 sequences and point mutations in these sequences. These mutations made the -10 sequences more like the sigma 70 promoter consensus sequences.
携带无启动子pheBA操纵子的质粒pEST1463被克隆到恶臭假单胞菌PaW85中，利用苯酚的菌落被分离于以苯酚作为唯一碳源和能源的基本培养基平板上。在这些克隆中，位于染色体上的Tn4652转座到pheA（编码苯酚单加氧酶）编码序列的上游。序列分析以及重组质粒中pheBA操纵子转录起始点的定位显示，pheBA操纵子中存在的-10序列与位于Tn4652末端反向重复序列中的-35序列的融合在恶臭假单胞菌细胞中的选择压力下产生了功能性启动子。这些启动子序列与大肠杆菌RNA聚合酶σ70启动子共有序列相似。在所研究的六个融合启动子中的三个中，这种产生结合了两个不同事件：Tn4652转座到含有潜在-10序列的DNA中以及这些序列中的点突变。这些突变使-10序列更类似于σ70启动子共有序列。
