Defining the cell cycle for the tachyzoite stage of Toxoplasma gondii

Tachyzoite endodyogeny is characterized by a three phase cell cycle compris ed of major G1 and S phases with mitosis Following immediately upon the con clusion of DNA replication, Cytokinesis, which begins with the formation of daughter apical complexes, initiates in late S phase and overlaps mitosis. There is no evidence to support an extended G2 period in these parasites. Tn all strains, parasites with a 2 N DNA content are a relatively small sub population and when tachyzoites expressing a fluorescent nuclear marker (gr een-fluorescent-protein fused to proliferating-cell-nuclear-antigen) were o bserved by time-lapse microscopy. there appeared to be little delay between S phase and mitosis. Measurements of the DNA content of RH parasites by fl ow cytometry demonstrated that the G1 and S periods were approximate to 60 and approximate to 30% of a single division cycle, although these phases we re longer in strains that display a slower growth rate. The overall length of S phase was determined by [H-3]-thymidine autoradiography using transgen ic parasites expressing herpes simplex thymidine kinase and validated by No rthern analysis of S phase specific genes during synchronous growth. The fr action of S phase parasites by flow cytometry paralleled autoradiography, h owever. within S phase, the distribution of parasites was bimodal in all st rains examined, Parasites containing a 1-1.7 N DNA complement were a small fraction when compared to the major S phase population which contained a ne ar-diploid (approximate to 1.8 N) complement. suggesting parasites in late S phase have a slower rate of DNA replication. In lieu of a short or missin g G2, where checkpoints are thought to operate in other eukaryotes, the bim odal replication of tachyzoite chromosomes may represent a distinct premito tic checkpoint associated with endodyogeny. (C) 2001 Elsevier Science B.V. All rights reserved.