FIG 1 .

Temperature-sensitive (ts) mutants defective in chromosome replication. (A) The growth of chemical ts mutants 13-64D5 and 11-51A1 was inhibited by high temperatures leading to lethal arrest. At the nonpermissive temperature of 40°C, the ts mutants arrested within one or two divisions, whereas at the permissive temperature of 34°C, the ts mutants replicated similar to the parent strain. Note that all of the strains, including the parent strain, grew more slowly at 34°C compared to the parent strain at 40°C. The vacuole size shown on the y axis is the number of parasites per vacuole. RHKO, RHΔku80Δhxgprt knockout strain. (B) Flow cytometric analysis of ethanol (EtOH)-fixed ts mutant parasites stained with propidium iodide after RNase treatment showed similar sub-1N aneuploidy that is consistent with chromosome loss at 40°C. The DNA content of the ts mutants grown at 34°C exhibits the 1N and 1.8N peaks representative of an asynchronous growing tachyzoite population (6). For each sample, 10,000 parasites were analyzed (y axis) using an FL-2 linear scale; 1N and 2N DNA fluorescent values are indicated on the x axis and by the vertical dashed lines. (C) IFA analysis of ts mutants 13-64D5 and 11-51A1 compared to the parent strain grown at 34°C (image series 1 to 5) and 40°C (image series 2 to 6). The parasites were stained for DNA with DAPI and internal daughter budding with anti-IMC1. The IMC1 Guide column show noncolored/inverted images in order to better differentiate individual mother parasites and daughter buds. The circles in the IMC1 Guide images indicate the number of vacuoles and the approximate size base of differential interference contrast (DIC) images (results not shown). Mutant images at 40°C (series 4 and 6) show a similar phenotype of unequal masses of chromosomal DNA accumulated free of abnormal daughter parasites. Note that the small DAPI focus in the buds of series 6 is plastid DNA. The scale bar in the series 1 fluorescent image applies to all images.