High-copy-number transformants and co-transformation in Dictyostelium

Abstract

We have recently established a DNA-mediated transformation system for Dictyostelium. The vector (pB10) contains the promoter from the Dictyostelium actin 6 gene fused to the Nm R gene from Tn5 which confers resistance to antibiotic G418. Dictyostelium cells can be stably transformed and express kanamycin phosphotransferase (APHII). There is an average of three to five copies of vector DNA in transformed populations. We have fused an A + T-rich region containing the 3 ' end of the Dictyostelium actin (Act) 8 gene to the end of the Act6-Nm R fusion. Though the fragment is inserted in reverse orientation, this adds a transcription termination and/or 3′ processing site and results in the formation of a discretely sized mRNA from the Act6-Nm R gene fusion. Using this vector, the number of transformants increases by approx. 5–10-fold. We also describe conditions that allow for the isolation of transformants having a high copy number of vector DNA per cell (approx. 150 copies/cell). In addition, we show that cells can be co-transformed with the transformation vector and other pBR322 derivatives. Both plasmid DNAs are present in transformed Dictyostelium cells in high- M r DNA. When cells are grown under selective conditions in the presence of the antibiotic G418, both DNAs are present in high copy number and Dictyostelium genes present on both vectors are transcribed and are properly regulated under the conditions examined. These modifications of the original transformation system should facilitate the introduction of modified genes into Dictyostelium to study gene regulation during development and allow one to examine the effects of high gene dosage.