Introduction of Plasmid DNA into the Trypanosomatid Protozoan Crithidia Fasciculata

Crithidia fasciculata cells were treated with a plasmid (pDK96) containing pBR322 sequences, a Leishmania tarentolae maxicircle autonomously replicating sequence, and the bacterial gene for aminoglycoside 3' phosphotransferase I inserted between the yeast alcohol dehydrogenase 1 promotor and terminator sequences. Resistant colonies were selected on agar plates containing paromomycin and screened for vector DNA by hybridization. Approximately 1% of the resistant colonies contained detectable vector DNA, which was present as extrachromosomal closed circular molecules ranging in copy number from 1 to 160 per cell. The plasmids could be recovered from Escherichia coli transformed to ampicillin resistance with Crithidia total cell DNA. Most of the recovered plasmids were a deleted product of pDK96, which lacked the maxicircle autonomously replicating sequence and contained a unique fragment of Crithidia nuclear DNA present at a low copy number in the wild-type genome. The plasmid DNA in resistant Crithidia was unstable even under selective conditions and was lost within 30 cell divisions.