DNA-mediated transformation in Dictyostelium discoideum: regulated expression of an actin gene fusion.

Abstract

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.