Genetic rescue of a Toxoplasma gondii conditional cell cycle mutant.
Growth rate is a major pathogenesis factor in the parasite Toxoplasma gondii; however, how cell division is controlled in this protozoan is poorly understood. Herein, we show that centrosomal duplication is an indicator of S phase entry while centrosome migration marks mitotic entry. Using the pattern of centrosomal replication, we confirmed that mutant ts11C9 undergoes a bimodal cell cycle arrest that is characterized by two subpopulations containing either single or duplicated centrosomes which correlate with the bipartite genome distribution observed at the non-permissive temperature. Genetic rescue of ts11C9 was performed using a parental RH strain cDNA library, and the cDNA responsible for conferring temperature resistance (growth at 40 degrees C) was recovered by recombination cloning. A single T. gondii gene encoding the protein homologue of XPMC2 was responsible for genetic rescue of the temperature-sensitive defect in ts11C9 parasites. This protein is a known suppressor of mitotic defects, and in tachyzoites, TgXPMC2-YFP localized to the parasite nucleus and nucleolus which is consistent with the expected subcellular localization of critical mitotic factors. Altogether, these results demonstrate that ts11C9 is a conditional mitotic mutant containing a single defect which influences two distinct control points in the T. gondii tachyzoite cell cycle.
White
Michael W
MW
Department of Veterinary Molecular Biology, Montana State University, Bozeman, MT 59717-3610, USA. uvsmw@montana.edu
Jerome
Maria E
ME
Vaishnava
Shipra
S
Guerini
Michael
M
Behnke
Michael
M
Striepen
Boris
B
D000595:Amino Acid Sequence; D000818:Animals; D002453:Cell Cycle / Q000235:genetics*; D018385:Centrosome / Q000502:physiology / Q000648:ultrastructure; D017124:Conserved Sequence; D004261:DNA Replication; D016054:DNA, Protozoan / Q000235:genetics; D005092:Exonucleases / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D018087:Plastids / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014122:Toxoplasma / Q000235:genetics* / Q000254:growth & development
D016054:DNA, Protozoan; D005092:Exonucleases