Manis J J, Kline B C
Mol Gen Genet. 1977 Apr 29;152(3):175-82. doi: 10.1007/BF00268815.
The plasmids pSC138 and pML31 each contain the EcoRI-generated f5 replicator fragment of the conjugative plasmid F in addition to an EcoRI fragment encoding antibiotic resistance: ampicillin resistance derived from Staphylococcus aureus in pSC138 and kanamycin resistance from Escherichia coli in pML31. We have mapped one HindIII and two BamHI restriction sites in the f5 region of these plasmids and one HindIII site in the antibiotic resistance region of each plasmid. The HindIII site in the Km region of pML31 occurs in the kan gene whereas the HindIII site in the Ap region of pSC138 appears to occur in an area important for the regulation of beta-lactamase production. By means of in vitro recombinant DNA manipulation of plasmids pML31 and pSC138, we have shown that approximately 1.9 X 10(6) daltons of the 6.0 X 10(6) dalton f5 fragment can be deleted without disrupting plasmid stability. In addition, we have used these same techniques to isolate a novel F-controlled Ap plasmid cloning vehicle which contains a single restriction site for each of the enzymes EcoRI, HindIII, and BamHI. This cloning vehicle has been linked via either its EcoRI or HindIII site to a ColE1 plasmid replicon to yield stable recombinants.
质粒pSC138和pML31除了含有一个编码抗生素抗性的EcoRI片段外，还各自含有由EcoRI产生的接合质粒F的f5复制子片段：pSC138中来自金黄色葡萄球菌的氨苄青霉素抗性以及pML31中来自大肠杆菌的卡那霉素抗性。我们已在这些质粒的f5区域定位了一个HindIII和两个BamHI限制性酶切位点，并在每个质粒的抗生素抗性区域定位了一个HindIII位点。pML31的Km区域中的HindIII位点位于卡那基因中，而pSC138的Ap区域中的HindIII位点似乎位于对β-内酰胺酶产生调控很重要的区域。通过对质粒pML31和pSC138进行体外重组DNA操作，我们已表明，在不破坏质粒稳定性的情况下，可以缺失6.0×10⁶道尔顿f5片段中的约1.9×10⁶道尔顿。此外，我们已使用这些相同技术分离出一种新型的F控制的Ap质粒克隆载体，该载体对EcoRI、HindIII和BamHI每种酶都含有一个单一限制性酶切位点。该克隆载体已通过其EcoRI或HindIII位点与ColE1质粒复制子连接以产生稳定的重组体。
