West R W, Neve R L, Rodriguez R L
Gene. 1979 Nov;7(3-4):271-88. doi: 10.1016/0378-1119(79)90048-9.
Derivatives of the Escherichia coli drug-resistance plasmid pBR316 have been constructed which act as molecular probes for promoter-containing DNA restriction fragments from various prokaryotic genomes. The plasmids, designated pBRH1 and pBRH3B, contain a unique EcoRI restriction site located within the promoter for the tetracycline resistance (Tcr) gene. This site was created by the insertion of a chemically synthesized octanucleotide, containing the EcoRI cleavage sequence, into the HindIII site of pBR316. Base-pair alterations within the Tc promoter produced by this insertion resulted in a substantial reduction (pBRH3B) or elimination (pBRH1) in ability of these plasmids to confer Tc resistance to the host strain. Cloning of EcoRI-cleaved foreign DNA fragments into the EcoRI site of these plasmids allows for the isolation of recombinant transformants with Tcr levels greater than that of the plasmid vector. Further characterization of these recombinant plasmids demonstrates that the Tcr phenotype is dependent upon the orientation of the inserted fragment, but not on the molecular weight. We have concluded that these fragments carry promoters which, in the proper orientation, allow for the transcription of the Tcr gene. The utility of these "promoter-probe" plasmids lies in the ability to select for promoter-containing DNA fragments by insertional activation of the Tcr gene.
已构建出大肠杆菌耐药质粒pBR316的衍生物，它们可作为分子探针，用于探测来自各种原核生物基因组的含启动子DNA限制片段。这些被命名为pBRH1和pBRH3B的质粒，在四环素抗性（Tcr）基因的启动子区域内含有一个独特的EcoRI限制位点。该位点是通过将一个含有EcoRI切割序列的化学合成八核苷酸插入pBR316的HindIII位点而产生的。这种插入导致Tc启动子内的碱基对改变，从而使这些质粒赋予宿主菌株Tc抗性的能力大幅降低（pBRH3B）或完全丧失（pBRH1）。将EcoRI切割的外源DNA片段克隆到这些质粒的EcoRI位点，可分离出Tcr水平高于质粒载体的重组转化体。对这些重组质粒的进一步表征表明，Tcr表型取决于插入片段的方向，而不取决于分子量。我们得出结论，这些片段携带启动子，在适当的方向上，这些启动子可使Tcr基因转录。这些“启动子探针”质粒的实用性在于能够通过Tcr基因的插入激活来选择含启动子的DNA片段。
