Ahern K G, Howard P K, Firtel R A
Department of Biology, University of California, San Diego, La Jolla 92093.
Nucleic Acids Res. 1988 Jul 25;16(14B):6825-37. doi: 10.1093/nar/16.14.6825.
Initial experiments with the endogenous 12.3 kb Dictyostelium discoideum plasmid Ddp1 led to the generation of a large shuttle vector, Ddp1-20. In addition to Ddp1, this vector contains pBR322 and a gene fusion that confers G418 resistance in Dictyostelium cells. We have shown that Ddp1-20 replicates extrachromosomally in Dictyostelium cells and can be grown in Escherichia coli cells (1). We have now examined deletions within this vector to identify the elements essential for extrachromosomal replication and stable maintenance of the plasmid. We find that a 2.2 kb fragment is sufficient to confer stable, extrachromosomal replication with a reduction in copy number from about 40 to approximately 10-15 copies per cell. Vectors containing additional Ddp1 sequences have a higher copy number. The 2.2 kb region contains none of the complete, previously identified transcription units on Ddp1 expressed during vegetative growth or development. These results suggest that gene products expressed by Ddp1 are not essential for replication, stability, or partitioning of the plasmid between daughter cells. Vectors carrying only the 2.2 kb fragment plus the gene fusion conferring G418 resistance transform Dictyostelium cells with high efficiency using either calcium phosphate mediated transformation or electroporation. Finally, we have examined the relative levels of expression of actin promoters driving neoR genes when in extrachromosomal or integrating vectors.
对内生的12.3 kb盘基网柄菌质粒Ddp1进行的初步实验产生了一个大型穿梭载体Ddp1 - 20。除了Ddp1，该载体还包含pBR322和一个在盘基网柄菌细胞中赋予G418抗性的基因融合体。我们已经表明，Ddp1 - 20在盘基网柄菌细胞中进行染色体外复制，并且可以在大肠杆菌细胞中生长（1）。我们现在研究了该载体内的缺失情况，以确定染色体外复制和质粒稳定维持所必需的元件。我们发现一个2.2 kb的片段足以赋予稳定的染色体外复制能力，每个细胞的拷贝数从约40个减少到大约10 - 15个。含有额外Ddp1序列的载体具有更高的拷贝数。2.2 kb区域不包含在营养生长或发育过程中表达的Ddp1上任何完整的、先前已鉴定的转录单元。这些结果表明，Ddp1表达的基因产物对于质粒在子细胞之间的复制、稳定性或分配并非必需。仅携带2.2 kb片段加上赋予G418抗性的基因融合体的载体，使用磷酸钙介导的转化或电穿孔法都能高效转化盘基网柄菌细胞。最后，我们研究了在染色体外或整合载体中驱动neoR基因的肌动蛋白启动子的相对表达水平。
