Characterization of promoters and stable transfection by homologous and nonhomologous recombination in Plasmodium falciparum.
Genetic studies of the protozoan parasite Plasmodium falciparum have been severely limited by the inability to introduce or modify genes. In this paper we describe a system of stable transfection of P. falciparum using a Toxoplasma gondii dihydrofolate reductase-thymidylate synthase gene, modified to confer resistance to pyrimethamine, as a selectable marker. This gene was placed under the transcriptional control of the P. falciparum calmodulin gene flanking sequences. Transfected parasites generally maintained plasmids episomally while under selection; however, parasite clones containing integrated forms of the plasmid were obtained. Integration occurred by both homologous and nonhomologous recombination. In addition to the flanking sequence of the P. falciparum calmodulin gene, the 5' sequences of the P. falciparum and P. chabaudi dihydrofolate reductase-thymidylate synthase genes were also shown to be transcriptionally active in P. falciparum. The minimal 5' sequence that possessed significant transcriptional activity was determined for each gene and short sequences containing important transcriptional control elements were identified. These sequences will provide considerable flexibility in the future construction of plasmid vectors to be used for the expression of foreign genes or for the deletion or modification of P. falciparum genes of interest.
Crabb
B S
BS
The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia.
Cowman
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AF
D000818:Animals; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D017931:DNA Primers; D016054:DNA, Protozoan / Q000032:analysis; D017125:Genes, Protozoan; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011995:Recombination, Genetic; D015183:Restriction Mapping; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis* / Q000235:genetics; D013940:Thymidylate Synthase / Q000096:biosynthesis* / Q000235:genetics; D014122:Toxoplasma / Q000201:enzymology / Q000235:genetics; D014162:Transfection
D017931:DNA Primers; D016054:DNA, Protozoan; D011993:Recombinant Fusion Proteins; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase; D015500:Chloramphenicol O-Acetyltransferase