Co-insertional replication is responsible for tandem multimer formation during plasmid integration into the Dictyostelium genome
Co-insertional replication is responsible for tandem multimer formation during plasmid integration into the Dictyostelium genome
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DOI：
10.1006/plas.1997.1326
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发表时间：
1998-01-01
期刊：
影响因子：
2.6
通讯作者：
Fisher, PR
中科院分区：
文献类型：
--
作者：
Barth, C;Fraser, DJ;Fisher, PR
我们研究了在盘基网柄藻基因组中整合转化载体的建立，以深入了解质粒插入的形成，并研究确定这种插入中存在的质粒拷贝数的条件。将赋予对新霉素和/或杀稻瘟菌素抗性的转化载体作为磷酸钙共沉淀物或通过电穿孔引入细胞中。质粒DNA的整合基于质粒的重组整合或限制性酶介导的整合。通过脉冲场凝胶Southern印迹分析和最近发表的直接电穿孔到大肠杆菌中的方法检查所得转化体的基因组DNA。发现插入位点的数目取决于所使用的转化方法，并且抗性所需的每个插入位点的质粒拷贝的最小数目取决于选择性药物的类型和浓度。共转化研究揭示了一个严格的同质组合物的载体多聚体从任何给定的插入位点。这表明多聚体是通过单个质粒单体的共插入复制而产生的，而不是通过随后涉及同源重组的额外插入事件而产生的。整合载体的多聚化仅在通过同源重组建立插入时发生。此外，质粒拷贝数似乎是随机的，在转化时建立，并且不随随后选择方案的改变而改变。(C)北京：科学出版社.
We investigated the establishment of integrating transformation vectors in the genome of Dictyostelium discoideum to gain insight into the formation of the plasmid insertions and to investigate the conditions that determine the number of plasmid copies present in such insertions. Transformation vectors conferring resistance to neomycin and/or blasticidin were introduced into the cell as a calcium phosphate coprecipitate or by electroporation. The integration of the plasmid DNA was based on either recombinational integration of plasmids or restriction enzyme-mediated integration. The genomic DNA of the resulting transformants was examined by Southern blot analysis of pulsed-field gels and by the recently published method of direct electroporation into Escherichia coli The number of insertion sites was found to be dependent on the transformation method used, and the minimum number of plasmid copies per insertion site required for resistance depended on the type and the concentration of the selective drug. Cotransformation studies revealed a strictly homogeneous composition of vector multimers from any given insertion site. This suggests that multimers arise by co-insertional replication of a single plasmid monomer, rather than by subsequent additional insertion events involving homologous recombination. The multimerization of the integrated vector only occurred when the insertion was established by homologous recombination. Moreover, the number of plasmid copies appeared to be random, was established at the time of the transformation, and did not change with subsequent alterations to the selection regime. (C) 1998 Academic Press.