Restriction endonuclease mapping and mutagenesis of the F sex factor replication region
Restriction endonuclease mapping and mutagenesis of the F sex factor replication region
复制标题
F性因子复制区的限制性核酸内切酶作图和诱变
DOI：
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发表时间：
1977
期刊：
影响因子：
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通讯作者：
B. Kline
中科院分区：
文献类型：
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作者：
J. Manis;B. Kline
质粒pSC 138和pML 31除了编码抗生素抗性的EcoRI片段外，还各自含有接合质粒F的EcoRI产生的f5复制子片段：pSC 138中的氨苄青霉素抗性来源于金黄色葡萄球菌，pML 31中的卡那霉素抗性来源于大肠杆菌。我们在这些质粒的f5区域绘制了一个HindIII和两个BamH I限制性位点，并在每个质粒的抗生素耐药性区域绘制了一个HindIII位点。pML 31 Km区的Hind Ⅲ位点位于kan基因中，而pSC 138 Ap区的Hind Ⅲ位点似乎位于对β-内酰胺酶产生的调节重要的区域。我们已经表明，6.0 × 106道尔顿道尔顿的F5片段中的约1.9 × 106道尔顿可以被缺失而不破坏质粒稳定性。此外，我们已经使用这些相同的技术来分离一种新的F-控制的Ap质粒克隆载体，该载体含有EcoRI、HindIII和BamHI酶中每一种的单一限制性位点。该克隆载体通过其EcoRI或HindIII位点与ColE 1质粒复制子连接，以产生稳定的重组体。
SummaryThe 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 β-lactamase production.By means of in vitro recombinant DNA manipulation of plasmids pML31 and pSC138, we have shown that ∼ 1.9x106 daltons of the 6.0x106 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.