CONSTRUCTION AND CHARACTERIZATION OF NEW CLONING VEHICLES .1. AMPICILLIN-RESISTANT DERIVATIVES OF PLASMID PMB9
CONSTRUCTION AND CHARACTERIZATION OF NEW CLONING VEHICLES .1. AMPICILLIN-RESISTANT DERIVATIVES OF PLASMID PMB9
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DOI：
10.1016/0378-1119(77)90074-9
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发表时间：
1977-01-01
期刊：
影响因子：
3.5
通讯作者：
BOYER, HW
中科院分区：
文献类型：
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作者：
BOLIVAR, F;RODRIGUEZ, RL;BOYER, HW
通过限制性内切酶的体外重组和Ap [氨苄青霉素]抗性（Apr）基因的体内遗传易位，构建了一种新的克隆载体，即质粒pBR313。该载体来源于ColE1样质粒，虽然它不产生大肠杆菌素E1，但它仍然保留了大肠杆菌素E1免疫性。pBR313中携带的Apr和四环素抗性（Tcr）标记分别来自pRSF2124和pSC101的氨苄青霉素转座子（TnA）。在构建pBR 313的过程中，TnA组分发生了改变，使pBR 313中的Apr基因不能再转位。该质粒的MW为5.8 Mdalton，用13种限制性内切酶表征，其中6种（EcoRI、SmaI、HpaI、HindIII、BamHI和SalI）在独特的限制性位点切割质粒。这允许对由这6种酶产生的DNA片段进行分子克隆。后3种酶HindIII、BamHI和SalI的限制性位点位于Tcr基因中。将DNA片段克隆到这些位点改变了Tcr机制的表达，从而为携带重组质粒分子的[大肠杆菌]细胞提供了选择。描述了一种富集携带重组质粒分子的AprTcs细胞的方法。
In vitro recombination via restriction endonucleases and the in vivo genetic translocation of the Ap [ampicillin] resistance (Apr) gene resulted in the construction of a new cloning vehicle, the plasmid pBR313. This vector was derived from a ColE1-like plasmid and, while it does not produce colicin E1, it still retains colicin E1 immunity. The Apr and tetracycline resistance (Tcr) markers carried in pBR313 were derived from the ampicillin transposon (TnA) of pRSF2124 and pSC101, respectively. During the construction of pBR313, the TnA component was altered and the Apr gene in pBR313 can no longer be translocated. This plasmid has a MW of 5.8 Mdalton and was characterized using 13 restriction enzymes, 6 of which (EcoRI, SmaI, HpaI, HindIII, BamHI and SalI) cleave the plasmid at unique restriction sites. This allows the molecular cloning of DNA fragments generated by these 6 enzymes. The restriction sites for the latter 3 enzymes, HindIII, BamHI and SalI, are located in the Tcr gene(s). Cloning DNA fragments into these sites alters the expression of the Tcr mechanisms thus providing a selection for [Escherichia coli] cells carrying recombinant plasmid molecules. An enrichment method for AprTcs cells carrying recombinant plasmid molecules is described.