Defining the cell cycle for the tachyzoite stage of Toxoplasma gondii.
Tachyzoite endodyogeny is characterized by a three phase cell cycle comprised of major G1 and S phases with mitosis following immediately upon the conclusion 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. In all strains, parasites with a 2 N DNA content are a relatively small subpopulation and when tachyzoites expressing a fluorescent nuclear marker (green-fluorescent-protein fused to proliferating-cell-nuclear-antigen) were observed by time-lapse microscopy, there appeared to be little delay between S phase and mitosis. Measurements of the DNA content of RH parasites by flow cytometry demonstrated that the G1 and S periods were approximately 60 and approximately 30% of a single division cycle, although these phases were longer in strains that display a slower growth rate. The overall length of S phase was determined by [3H]-thymidine autoradiography using transgenic parasites expressing herpes simplex thymidine kinase and validated by Northern analysis of S phase specific genes during synchronous growth. The fraction of S phase parasites by flow cytometry paralleled autoradiography, however, within S phase, the distribution of parasites was bimodal in all strains 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 near-diploid ( approximately 1.8 N) complement, suggesting parasites in late S phase have a slower rate of DNA replication. In lieu of a short or missing G2, where checkpoints are thought to operate in other eukaryotes, the bimodal replication of tachyzoite chromosomes may represent a distinct premitotic checkpoint associated with endodyogeny.
Radke
J R
JR
Department of Veterinary Molecular Biology, Marsh Laboratory, Montana State University, 59717-3610, Bozeman, MT, USA.
Striepen
B
B
Guerini
M N
MN
Jerome
M E
ME
Roos
D S
DS
White
M W
MW
D000818:Animals; D002453:Cell Cycle / Q000502:physiology*; D002455:Cell Division; D016054:DNA, Protozoan / Q000032:analysis; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D016193:G1 Phase; D016195:G2 Phase; D015870:Gene Expression; D008938:Mitosis; D018809:Proliferating Cell Nuclear Antigen / Q000032:analysis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016196:S Phase; D014122:Toxoplasma / Q000166:cytology / Q000235:genetics / Q000254:growth & development*
D016054:DNA, Protozoan; D018809:Proliferating Cell Nuclear Antigen; D012333:RNA, Messenger