van den Hondel C A, Verbeek S, van der Ende A, Weisbeek P J, Borrias W E, van Arkel G A
Proc Natl Acad Sci U S A. 1980 Mar;77(3):1570-4. doi: 10.1073/pnas.77.3.1570.
We have used the TEM beta-lactamase transposon Tn901, located on Escherichia coli plasmid pRI46, to introduce in vivo a genetic marker into plasmid pUH24, present in the cyanobacterial strain Anacystis nidulans R-2. Restriction enzyme analysis and heteroduplex studies of the 8.3 x 10(6)-dalton plasmids pCH1-pCH5, present in the ampicillin-resistant A. nidulans R-2 colonies obtained after transformation with pRI46, demonstrated that these plasmids consist of the complete sequence of Tn901 inserted at different places into plasmid pUH24. The pUH24::Tn901 recombinant plasmids transform A. nidulans R-2 with a frequency of 10(-4)--10(-5) per microgram of plasmid DNA and contain a single cleavage site for the restriction enzyme Xho I. From pCH1 a plasmid of 5.5 x 10(6) daltons,pUC1, was constructed with only a part of the Tn901 sequence and an additional single cleavage site for the restriction enzyme BamHI. This plasmid, as well as plasmids pCH1-pCH5, are potentially useful as vectors for cloning genes in cyanobacteria and for studying cyanobacterial plasmid biology.
我们使用了位于大肠杆菌质粒pRI46上的TEMβ-内酰胺酶转座子Tn901，将一个遗传标记体内导入到蓝藻菌株集胞藻6803 R-2中存在的质粒pUH24中。对用pRI46转化后获得的氨苄青霉素抗性集胞藻6803 R-2菌落中存在的8.3×10⁶道尔顿质粒pCH1 - pCH5进行的限制性内切酶分析和异源双链研究表明，这些质粒由插入到质粒pUH24不同位置的Tn901完整序列组成。pUH24::Tn901重组质粒以每微克质粒DNA 10⁻⁴ - 10⁻⁵的频率转化集胞藻6803 R-2，并含有限制性内切酶Xho I的单个切割位点。从pCH1构建了一个5.5×10⁶道尔顿的质粒pUC1，它只含有Tn901序列的一部分以及限制性内切酶BamHI的另一个单个切割位点。该质粒以及质粒pCH1 - pCH5在作为蓝藻基因克隆载体和研究蓝藻质粒生物学方面具有潜在用途。
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