A cell cycle model for the tachyzoite of Toxoplasma gondii using the Herpes simplex virus thymidine kinase.
Toxoplasma gondii (RH strain) tachyzoites were transfected with a plasmid containing a fusion of the chloramphenicol acetyl transferase and the Herpes simplex virus-2 thymidine kinase coding regions and transgenic parasites obtained by chloramphenicol selection. CTK11, a single high expressing clone was isolated based on immunofluorescence and contained approximately five integrated copies of the fusion sequence. Lysates prepared from this clone displayed thymidine kinase activity of 2.9 pmol min(-1) microg(-1) protein, whereas thymidine kinase activity was not detected in lysates from the parental RH strain. Growth of CTK11 tachyzoites was fully inhibited in 5 microM ganciclovir and thymidine and in 2.5 microM 5-bromo-2'-deoxyuridine. While the inhibitory effects of ganciclovir were lethal, low concentrations of thymidine (10 microM) were largely reversible. Asynchronously growing CTK11 tachyzoites were found to contain major G1 (1 N) and S phase (1 N+) distributions as determined by relative propidium iodide fluorescence and with reference to the haploid (1 N) DNA content of a T. gondii sporozoite population. CTK11 tachyzoites blocked 4 h in 10 microM thymidine exhibited mean fluorescence consistent with a 1 N complement of DNA indicating growth was arrested in G1. Following the removal of excess thymidine, parasites immediately entered S phase, thus confirming the late G1 block. Parasites with a 2 N complement of DNA (G2 + M) first appear at 2 h post-release, while 1 N (G1) parasites re-appear at 3 h suggesting the length of S phase is < or = 2 h and that of G2 + M is < or = 1 h. Within 7 h, parasites had transited G2 + M and much of G1 and re-entered S of the subsequent cell cycle--a time consistent with the doubling of these parasites in culture. Thus, the CTK11 tachyzoite cell cycle is similar to those of higher eukaryotic cells and is characterized by major G1 and S phases and a relatively short G2 + M.
Radke
J R
JR
Department of Veterinary Molecular Biology, Montana State University, Bozeman 59717-3610, USA.
White
M W
MW
D000818:Animals; D000963:Antimetabolites / Q000494:pharmacology; D001976:Bromouracil / Q000031:analogs & derivatives; D002453:Cell Cycle / Q000187:drug effects; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D016054:DNA, Protozoan / Q000378:metabolism; D018274:Electroporation; D005434:Flow Cytometry; D016193:G1 Phase; D016195:G2 Phase; D015774:Ganciclovir / Q000494:pharmacology; D005822:Genetic Vectors; D018258:Herpesvirus 2, Human / Q000201:enzymology / Q000235:genetics; D008938:Mitosis; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016196:S Phase; D012641:Selection, Genetic; D013936:Thymidine / Q000494:pharmacology; D013937:Thymidine Kinase / Q000235:genetics* / Q000378:metabolism; D014122:Toxoplasma / Q000166:cytology / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development*; D014529:Uridine / Q000031:analogs & derivatives / Q000494:pharmacology
D000963:Antimetabolites; D016054:DNA, Protozoan; D011993:Recombinant Fusion Proteins; D001976:Bromouracil; D015500:Chloramphenicol O-Acetyltransferase; D013937:Thymidine Kinase; C006824:5-bromouridine; D015774:Ganciclovir; D013936:Thymidine; D014529:Uridine