FIG. 4.
Complementation shows that PUS1 is indeed involved in the Tbd phenotype. (A) Clones of TBD8 parasites complemented with the genomic PUS1 locus, TBD8comp, or a control vector, TBD8pHANA were created. These parasites were induced to switch with differentiation medium for 4 days and labeled with the bradyzoite-specific cyst wall marker Dolichos bifluorescein and with the tachyzoite surface marker anti-SAG1. Fifteen fields were analyzed for tachyzoite vacuoles and bradyzoite cysts based on immunofluorescence. Three biological replicates with at least two technical replicates were performed and the data graphed with standard deviations. *, P < 0.005 relative to WT. (B) Quantitative PCR was performed on cDNA from each strain to verify the restoration of PUS1 transcription in TBD8comp. The graph represents two independent experiments with technical triplicates and the standard deviation of the cycle threshold.