Résumé
DNA content after elutriation. A FACS analyses and determination of DNA content. The first cell collection was made 55 min after T0, because we previously determined that this was the amount of time required for C. glabrata cells to re-enter the cell cycle after elutriation. Subsequent collections were made at 5 min intervals. DNA was labeled with Sytox Green [56] and analyzed by FACS (MACS Quant, Miltenyi Biotec) and relative amounts of G1 and G2 cells were calculated using FlowJo. Note that at T0, there is already a detectable amount of cells that have started to replicate their genome, suggesting that replication restarts soon after mitosis, at early origins. For each time point, peaks corresponding respectively to G1 and G2 cells are shown, along with percentages of cells in each phase. Dotted lines represent average intensity values of G1 and G2 peaks. B Table summarizing these results. DNA content was calculated using the formula (G1 cells + 2 Ă G2 cells)/(G1 cells + G2 cells) [13]. The curve plots DNA content according to each time point. Note that time points are spaced at 5 min, except between T0 and T1 (55 min). Genomic DNA from time points shaded in gray were extracted and sequenced. C FACS profiles of the first elutriated fraction of two independent experiments (T0). The small peak corresponds to cells that have already started DNA replication, although no bud is visible by visual examination. (PDF 230 kb)