Search

We have constructed a new vector for transformation that carries a fusion of the Dictyostelium discoideum actin 6 promoter gene and 5' flanking region with the bacterial Tn5 NeoR (KanR) gene which can confer resistance to the aminoglycoside G418. This vector can be used to transform D. discoideum cells. Approximately 200 to 2,000 transformants were obtained per 10(7) cells. Transformed cell populations carried vector DNA at an average copy number of ca. 5 per cell, and the DNA was stable for more than 40 generations in the absence of selection. We have shown that transformed cells synthesize functional kanamycin phosphotransferase and that initiation of transcription of the actin 6-NeoR gene fusion occurs at the actin 6 cap site. Moreover, analysis of RNA isolated from transformed and untransformed cells during vegetative growth and during development indicated that the actin 6-NeoR gene fusion was regulated in parallel with the endogenous actin 6 gene, suggesting that the upstream flanking regions of actin 6 contain the cis-acting regulatory sequences sufficient for differential regulation of this gene during D. discoideum development. These results indicate that this system can be used to examine control of gene expression during D. discoideum development.

We cloned a 12.3-kilobase (kb) endogenous plasmid, Ddp1, found in several wild-type and laboratory strains of Dictyostelium discoideum into pBR322. The cloned plasmids have been used to cotransform D. discoideum cells with B10S, a transformation vector carrying a gene fusion conferring resistance to G418. Whereas B10S DNA alone appears to integrate in a tandem array, the cloned Ddp1 plasmids replicate extrachromosomally and are stably maintained in the absence of selection with an average copy number of 50 to 100 copies per cell. The Ddp1-derived plasmids can be directly recovered by transforming Escherichia coli with bulk nuclear DNA from these cells. Preliminary deletion analysis indicates that not all regions of Ddp1 are necessary for stable replication in D. discoideum. Several recombinant vectors which replicate extrachromosomally in D. discoideum were also isolated. One contains the Act6-neor gene fusion from B10S recombined into one of the cloned derivatives of Ddp1 and can be used to directly transform D. discoideum amoebae, selecting for G418 resistance. Another recombinant is only 5.6 kb and resulted from a deletion of a 16.6-kb cloned Ddp1 hybrid plasmid. An analysis of the vector DNAs present in clones derived from single D. discoideum transformants is also described.