CHARACTERIZATION OF PROMOTERS AND STABLE TRANSFECTION BY HOMOLOGOUS AND NONHOMOLOGOUS RECOMBINATION IN PLASMODIUM-FALCIPARUM

Genetic studies of the protozoan parasite Plasmodium falciparum have b een severely limited by the inability to introduce or modify genes. In this paper we describe a system of stable transfection of P. falcipar um using a Toxoplasma gondii dihydrofolate reductase-thymidylate synth ase gene, modified to confer resistance to pyrimethamine, as a selecta ble marker. This gene was placed under the transcriptional control of the P. falciparum calmodulin gene flanking sequences, Transfected para sites generally main rained plasmids episomally while under selection; however, parasite clones containing integrated forms of the plasmid w ere obtained, Integration occurred by both homologous and nonhomologou s recombination, In addition to the flanking sequence of the P. falcip arum calmodulin gene, the 5' sequences of the P. falciparum and P. cha baudi dihydrofolate reductase-thymidylate synthase genes were also sho wn to be transcriptionally active in P. falciparum. The minimal 5' seq uence that possessed significant transcriptional activity was determin ed for each gene and short sequences containing important transcriptio nal control elements were identified. These sequences will provide con siderable flexibility in the future construction of plasmid vectors to be used for the expression of foreign genes or for the deletion or mo dification of P. falciparum genes of interest.