Toxoplasma gondii: Characterization of temperature-sensitive tachyzoite cell cycle mutants

We mutagenized RH Delta hxgprt strain tachyzoites of Toxoplasma gondii usin g N-nitroso-N-ethylurea and analyzed 40 clonal isolates (of 3680 ENU mutant s) that were unable to grow in cell culture at 40 degreesC. These isolates grew normally at 34 degreesC, but showed variable growth at temperatures be tween 34 and 39 degreesC. The inability to grow at 40 degreesC was also cor related with a loss of virulence in mice for those mutants examined. We fur ther characterized the temperature-sensitive (rs) isolates using flow cytom etry and propidium iodide staining and identified three types of cell cycle -related mutations. Regardless of temperature, in the isolates ts1C12, ts7B 4, and ts7B10, the distribution of parasites with a haploid DNA content was substantially higher (approximate to 85%) than that observed for RH Delta hxgprt (approximate to 60%). Four other isolates, ts4F6, ts6C11, ts8G10, an d ts11F5, contained G1-related mutations, and in each case, the DNA distrib ution among parasites at the permissive temperature was similar to that of the parental strain, but at 40 degreesC only a single population containing a IN nuclear DNA complement was evident. Furthermore, there was no evidenc e of nuclear division or cytokinesis at 40 degreesC, and these parasites de monstrated a distended cytoplasm typical of G1 arrest in other cell types. Finally, parasites of the ts11C9 mutant arrested in two near-equal populati ons with either 1N or 2N complements of nuclear DNA. All arrested ts11C9 pa rasites contained a single nucleus, and a major subfraction of the 2N popul ation contained abnormal and incompletely formed daughters-indicating that the initiation of daughter formation can occur in the absence of nuclear di vision. (C) 2000 Academic Press.