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首页> 外文期刊>Plasmid: An International Journal Devoted to Extrachromosomal Gene Systems >Co-insertional replication is responsible for tandem multimer formation during plasmid integration into the Dictyostelium genome.
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Co-insertional replication is responsible for tandem multimer formation during plasmid integration into the Dictyostelium genome.

机译：共插入复制负责将质粒整合到双歧杆菌基因组中的串联多聚体形成。

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摘要

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.
机译：我们研究了盘基网柄菌基因组中整合转化载体的建立，以深入了解质粒插入物的形成，并研究确定存在于此类插入物中的质粒拷贝数的条件。将赋予对新霉素和/或杀稻瘟菌素的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的整合是基于质粒的重组整合或限制性酶介导的整合。通过脉冲场凝胶的Southern印迹分析和通过最近公开的直接电穿孔入大肠杆菌的方法，检查了所得转化体的基因组DNA。发现插入位点的数目取决于所用的转化方法，而抗性所需的每个插入位点的最小质粒拷贝数取决于选择性药物的类型和浓度。共转化研究揭示了来自任何给定插入位点的载体多聚体的严格均质组成。这表明多聚体通过单个质粒单体的共插入复制而不是通过随后的涉及同源重组的额外插入事件产生。仅当通过同源重组建立插入时，才发生整合载体的多聚化。而且，质粒拷贝的数目似乎是随机的，是在转化时确定的，并且不随选择方案的后续改变而改变。

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