Double cross-over gene replacement within the sec 7 domain of a GDP-GTP exchange factor from Plasmodium falciparum allows the generation of a transgenic brefeldin A-resistant parasite line.
High molecular weight ADP ribosylation factor GDP-GTP exchange factors (ARF-GEF) play an essential role in the formation of COP I coated transport vesicles and are characterized by a structurally and functionally conserved sec 7 domain. The genome of the malaria parasite Plasmodium falciparum encodes a single ARF-GEF that contains an unusual sec 7 domain. In comparison to the sec 7 domain of other eukaryotes, the plasmodial sec 7 domain is characterized by an insertion sequence of 146 amino acids that disrupt helices essential for the GDP-GTP exchange activity of the protein. In a previous study we have shown a correlation between a methionine to isoleucine exchange in helix H of the sec 7 domain and resistance to brefeldin A in a parasite line generated by drug selection. Here we have transfected brefeldin A sensitive parasites with plasmid constructs containing the sec 7 domain of the resistant line either with or without the insertion sequence. Transfection with sec 7 sequences including the insertion resulted in brefeldin A resistant parasites in which double cross-over recombination had replaced the endogenous sec 7 sequences with the transgenic sequences. Thus, the point mutation in helix H is sufficient to confer brefeldin A resistance in P. falciparum. Transfections using constructs lacking the insertion did not result in resistant parasites. Gene replacement by targeted double cross-over recombination is a rare event in P. falciparum. This approach has taken advantage of the fact that the successful integration of the transgene results in a drug selectable phenotype. We anticipate that the strategy described here will be useful for the identification of mutations within target genes that have the potential to confer increased drug resistance.
Wiek
Sabine
S
FB Biology, Philipps-University, 35032 Marburg, Germany.
Cowman
Alan F
AF
Lingelbach
Klaus
K
D020727:ADP-Ribosylation Factors / Q000737:chemistry / Q000235:genetics; D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology*; D003434:Crossing Over, Genetic; D004351:Drug Resistance; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry* / Q000235:genetics*; D021261:Parasitic Sensitivity Tests; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000235:genetics; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D019076:Transgenes
D020662:Guanine Nucleotide Exchange Factors; D011500:Protein Synthesis Inhibitors; D020126:Brefeldin A; D020727:ADP-Ribosylation Factors