Hughes J E, Ashtorab H, Welker D L
Dept. of Biology, Utah State University, Logan 84322-5500.
Dev Genet. 1988;9(4-5):495-504. doi: 10.1002/dvg.1020090426.
Cellular slime molds are one of only three types of eukaryotes known to contain circular nuclear plasmids. Unlike the 2-microns circle in Saccharomyces, different strains of Dictyostelium can carry different, nonhomologous plasmids. Covalently closed, circular DNA plasmids have been identified in D. discoideum, D. mucoroides, D. giganteum, and D. purpureum. These plasmids range in size from 1.3-27 kb and in copy number from 50-300 molecules per cell. Plasmids have been identified in approximately one-fifth of all isolates examined. The organization of their DNA in nucleosomes establishes their presence in the nucleus. We have successfully cotransformed endogenous Dictyostelium plasmids into D. discoideum using the G418 resistance shuttle vector B10S. Transformants carrying D. discoideum plasmids are recovered at much higher frequency than those carrying plasmids from the other Dictyostelium species. We have constructed recombinant plasmids based on the D. discoideum plasmid Ddp2 and the G418 resistance gene. With these extrachromosomal vectors, transformed cells are recovered at frequencies of up to 10(-4) per input cell, the vectors are stably maintained at high copy number in the absence of selection, and the vectors can be used to introduce foreign DNA sequences into D. discoideum cells.
细胞黏菌是已知仅有的三种含有环状核质粒的真核生物类型之一。与酿酒酵母中的2微米环状质粒不同，不同的盘基网柄菌菌株可携带不同的、非同源的质粒。在盘基网柄菌、黏液网柄菌、巨大网柄菌和紫色网柄菌中已鉴定出共价闭合的环状DNA质粒。这些质粒的大小在1.3 - 27 kb之间，拷贝数为每个细胞50 - 300个分子。在所检测的所有分离物中，约五分之一鉴定出了质粒。它们的DNA在核小体中的组织方式证实了其存在于细胞核中。我们已使用G418抗性穿梭载体B10S成功地将内源性盘基网柄菌质粒共转化到盘基网柄菌中。携带盘基网柄菌质粒的转化体的回收频率远高于携带其他盘基网柄菌物种质粒的转化体。我们基于盘基网柄菌质粒Ddp2和G418抗性基因构建了重组质粒。利用这些染色体外载体，转化细胞的回收频率高达每输入细胞10(-4)，这些载体在无选择条件下能以高拷贝数稳定维持，并且可用于将外源DNA序列导入盘基网柄菌细胞。
