Article Figures & Data
Figures
- FIG. 1.
HU Synchronization of procyclic T. brucei. (A) Cells were treated with 0.2 mM HU for 12 h (0 h to 12 h), stained with propidium diiodide, and analyzed by flow cytometry. (B) Cells were treated the same as described for panel A, but the results are from the period after HU washout (12 h to 24 h). Panels A and B are from different but identical experiments. (C) Growth of T. brucei in medium supplemented with 0.2 mM HU (•), 1 mM HU (▴), or no HU (⧫). Extrapolation of the growth curve, after washout of 0.2 mM HU, indicated a lag of ∼6 h before the resumption of normal growth. Values on the y axis were calculated from measured values of cells/ml multiplied by the dilution factor.
- FIG. 2.
Kinetics of kDNA replication in the presence of HU and after HU washout. The TdT-labeling pattern reveals the extent of replication. TdT-positive (TdT+) cells are undergoing replication, whereas TdT-negative (TdT-) cells include those that have not initiated replication and those that have completed postreplication gap repair. Based on the results of previous studies (10), kinetoplasts with labeling in antipodal sites and network poles are defined as “early” replicative stage, those with most or all of the network uniformly labeled are in “late” stages, and those with two TdT-positive networks are in the “post”-replicative stage. Cells were incubated with 0.2 mM HU for 12 h (0 h to 12 h), washed, and further incubated without HU (12 h to 24 h). Samples were examined by fluorescence microscopy after DAPI staining and TdT labeling. About 100 cells were categorized at each time point. (A) Examples of the different stages of kDNA replication. Cells are categorized by their TdT status and by the number of nuclei and kinetoplasts. Scale bar, 5 μm. (B) Kinetics of the appearance of the different stages during HU treatment. (C) The results of a different experiment, after HU washout, are shown.
