Mitochondrial RNA polymerase is an essential enzyme in erythrocytic stages of Plasmodium falciparum.
We have shown that transgenic Plasmodium falciparum parasites expressing the yeast DHODH (dihydroorotate dehydrogenase) are independent of the mtETC (mitochondrial electron transport chain), suggesting that they might not need the mitochondrial genome (mtDNA), since it only encodes three protein subunits belonging to the mtETC and fragmentary ribosomal RNA molecules. Disrupting the mitochondrial RNA polymerase (mtRNAP), which is critical for mtDNA replication and transcription, might then cause the generation of a ρ(0) parasite line lacking mtDNA. We made multiple attempts to disrupt the mtRNAP gene by double crossover recombination methods in parasite lines expressing yDHODH either episomally or integrated in the genome, but were unable to produce the desired knockout. We verified that the mtRNAP gene was accessible to recombination by successfully integrating a triple HA tag at the 3' end via single cross-over recombination. These studies suggest that mtRNAP is essential even in mtETC-independent P. falciparum parasites.
Center for Molecular Parasitology and Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, PA 19129, USA.
Hangjun
H
Ke
Joanne M
JM
Morrisey
Suresh M
SM
Ganesan
Michael W
MW
Mather
Akhil B
AB
Vaidya
D000818:Animals; D003434:Crossing Over, Genetic; D004272:DNA, Mitochondrial / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D000090722:Dihydroorotate Dehydrogenase; D004579:Electron Transport; D004912:Erythrocytes / Q000469:parasitology*; D055786:Gene Knockout Techniques / Q000379:methods; D050259:Genes, Mitochondrial; D017125:Genes, Protozoan; D008018:Life Cycle Stages; D008928:Mitochondria / Q000201:enzymology*; D024101:Mitochondrial Proteins / Q000378:metabolism; D030781:Organisms, Genetically Modified / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity; D015800:Protozoan Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D014162:Transfection
D004272:DNA, Mitochondrial; D016054:DNA, Protozoan; D000090722:Dihydroorotate Dehydrogenase; D024101:Mitochondrial Proteins; D015800:Protozoan Proteins; D044925:Oxidoreductases Acting on CH-CH Group Donors; D012321:DNA-Directed RNA Polymerases