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 plas mid containing a fusion of the chloramphenicol acetyl transferase and the Herpes simplex virus-2 thymidine kinase coding regions and transge nic parasites obtained by chloramphenicol selection. CTK11, a single h igh expressing clone was isolated based on immunofluorescence and cont ained approximately five integrated copies of the fusion sequence. Lys ates prepared from this clone displayed thymidine kinase activity of 2 .9 pmol min(-1) mu g(-1) protein, whereas thymidine kinase activity wa s not detected in lysates from the parental RH strain. Growth of CTK11 tachyzoites was fully inhibited in 5 mu M ganciclovir and thymidine a nd in 2.5 mu M 5-bromo-2'-deoxyuridine. While the inhibitory effects o f ganciclovir were lethal, low concentrations of thymidine (10 mu M) w ere largely reversible. Asynchronously growing CTK11 tachyzoites were found to contain major G1 (1 N) and S phase (1 N +) distributions as d etermined 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 mu M thymidine exhibited mean fl uorescence consistent with a 1 N complement of DNA indicating growth w as arrested in G1. Following the removal of excess thymidine, parasite s immediately entered S phase, thus confirming the late G1 block. Para sites with a 2 N complement of DNA (G2 + M) first appear at 2 h post-r elease, while 1 N (G1) parasites re-appear at 3 h suggesting the lengt h of S phase is less than or equal to 2 h and that of G2 + M is less t han or equal to 1 h. Within 7 h, parasites had transited G2 + M and mu ch of G1 and re-entered S of the subsequent cell cycle-a lime consiste nt with the doubling of these parasites in culture. Thus, the CTK11 ta chyzoite 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. (C) 1998 Elsevier Science B.V. All rights reserved.