Fig. 1. Southern blot of genomic DNAs from the parental RH strain, CTK11 and CAT-GFP [21]. BglII-digested DNA was electrophoresed, transferred to nitrocellulose and probed using the PstI/NotI fragment from pCAT-GFP that contains the common 3′ DHFR untranslated sequence [21]. The hybridization band in RH genomic DNA (lane 1) demonstrates the signal intensity for the single copy DHFR gene. In CTK11 genomic DNA (lane 2), the endogenous DHFR signal is masked by a more intense band at ≈9 Kb that represents approximately five copies of the CAT-HSTK fusion sequence. A second site at ≈6.5 Kb is likely a single-copy insertion based on the hybridization signal. For comparison, genomic DNA from CAT-GFP tachyzoites (lane 3) containing ≥20 copies of the transgene is shown [21].

Fig. 2. (A) The growth of CTK11 tachyzoites is sensitive to thymidine (-•-), ganciclovir (-○-) and BrdU (-♦-). Drug sensitivity is assessed by the calculated number of parasite doublings based on the average number of parasites per vacuole after 36 h growth (-▪-, controls). IC₅₀s for thymidine, ganciclovir and BrdU are 3.0, 2.3 and 1.2 μM, respectively. (B) CTK11 growth inhibition by low doses of thymidine is reversible. Tachyzoites were incubated for 6, 12, 18 or 24 h with 10 μM thymidine (-•-), 50 μM thymidine (--), 100 μM thymidine (-▪-) and 10 μM ganciclovir (-○-), the drug was removed and plaques allowed to grow for 10 days. Percent plaque survival is based on a control with no drug. Note that 10 μM thymidine (-•-) for 6 h is largely reversible while the same treatment with 10 μM ganciclovir (-○-) is lethal.

Fig. 3. (A) Density plot for an asynchronous, exponentially growing population of CTK11 tachyzoites fixed in 70% ethanol and stained using PI. Distributions were generated by flow cytometry using a Becton-Dickinson FACSCaliber and plotted forward scatter (FSC-H) verses fluorescence measured in FL2-H. G1 (1 N) and S populations are readily evident in this distribution. (B) Histogram plot for the distribution in (A). The mode fluorescence for G1 and S are FL2=401 and FL2=720, respectively. (C,D) Density and histogram plots for a population of freshly excysted VEG sporozoites, fixed as described. A single G1 distribution (FL2=399) is evident while S and G2+M are conspicuously absent. (E,F) The density and histogram plots for a population of CTK11 tachyzoites treated 4 h in 10 μM thymidine. A primary 1 N distribution is evident (FL2=394) indicating that growth of these parasites has been arrested in late G1.

Fig. 4. CTK11 tachyzoite population blocked 4 h using 10 μM thymidine and released. Histogram data was collected each hour for 7 h following release and plotted as in Fig. 3. (R1) CTK11 tachyzoites released 1 h. Note the population is uni-modal and has begun to move into S phase (compare to the blocked population in Fig. 3F, and R5). (R2) Histogram distribution 2 h post-release. The population has increased fluorescence and is primarily in S phase, but a small percentage with a 2 N content (FL2=800) is evident (15%, G2+M). In R3, the histogram is bi-modal with the largest population distributed in G2+M (43%) and G1 parasites have re-emerged (30%, FL2=401). (R4) Note the increased proportion of G1 parasites as the G2+M population continues to undergo cytokinesis. By R5, the population is again G1 and remains as such through R6 (histogram not shown). A total of 7 h after release from the thymidine block (R7), parasites have synchronously transited S, G2+M and G1 to re-enter S phase of the subsequent cell cycle.