Barth C, Wilczynska Z, Pontes L, Fraser D J, Fisher P R
School of Microbiology, La Trobe University, Melbourne, Australia.
Plasmid. 1996 Sep;36(2):86-94. doi: 10.1006/plas.1996.0036.
Transformation of Escherichia coli with Dictyostelium discoideum genomic DNA containing integrated shuttle vectors in multicopy, tandemly duplicated format resulted in the establishment of the linear plasmid molecules as circular monomeric replicons. The transformation efficiencies were comparable to those obtained with circular plasmid DNA and the recovered plasmids were free of deletions and rearrangements. Digestion of the genomic DNA prior to the transformation using restriction enzymes that cut within the inserted plasmids reduced the transformation efficiency dramatically and a high proportion of the recovered plasmids carried deletions. Our results provide evidence that the linear plasmid multimers cyclize in E. coli by homologous recombination in order to be established as autonomously replicated plasmids. The efficiency of recircularization was found to be independent of the recA gene product but dramatically reduced in the absence of recB recC or sbcB gene products. However, the paradoxically high efficiency of transformation with plasmid multimers of a recB recC sbcB mutant indicated the presence of an additional pathway for recombinational recircularization independent of these gene products. Unlike previous studies using as a DNA source linearized plasmid monomers and dimers that were created in vitro, the use of linear plasmid multimers integrated into the D. discoideum genome ensured that none of the E. coli transformants we obtained could be attributed to low levels of uncut circular plasmid molecules. The efficient recovery of the plasmid monomers faithfully reflects the structure of the insertion and thus provides a useful tool in the characterization of such plasmid insertions in the genome of D. discoideum.
用含有多拷贝、串联重复形式的整合穿梭载体的盘基网柄菌基因组DNA转化大肠杆菌，导致线性质粒分子以环状单体复制子的形式建立。转化效率与用环状质粒DNA获得的效率相当，回收的质粒没有缺失和重排。在转化前使用能在插入质粒内切割的限制性酶消化基因组DNA，会显著降低转化效率，并且回收的质粒中有很大比例带有缺失。我们的结果提供了证据，表明线性质粒多聚体在大肠杆菌中通过同源重组环化，以便作为自主复制的质粒建立起来。发现环化效率与recA基因产物无关，但在没有recB、recC或sbcB基因产物时会显著降低。然而，recB recC sbcB突变体的质粒多聚体转化效率异常高，这表明存在一条独立于这些基因产物的重组环化额外途径。与之前使用体外产生的线性化质粒单体和二聚体作为DNA来源的研究不同，使用整合到盘基网柄菌基因组中的线性质粒多聚体确保了我们获得的大肠杆菌转化体中没有一个可归因于未切割的环状质粒分子的低水平。质粒单体的高效回收忠实地反映了插入结构，因此为盘基网柄菌基因组中此类质粒插入的表征提供了一个有用的工具。
