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Additional file 1: Table S1. Strains used in this study. Table S2. Sedimentation scores in the presence of Nicotinamide (NAM). Sedimentation rates were determined by resting culture tubes and measuring the time needed for the clearance of the upper 50% of the culture (TS50). Values less than 1 indicate shorter TS50 relative to the non-treated sample. Table S3. List of PCR primers. Figure S1. All strains were simultaneously grown in YPD medium in a Thermo-Scientific Multiskan shaker-spectrophotometer. Time-course OD600 values are plotted. One of two independent experiments with all strains analysed in the same 96 well tray is shown. Figure S2. Exponentially growing cultures were harvested at OD600 = 1, stained with Propidium Iodine and analysed by flow cytometry. Figure S3. Genomic DNA was isolated from saturated liquid cultures and amplified by PCR with primers flanking the FLO1, FLO5, FLO9 and FLO11 genes. The PCR products were analysed on 1% agarose gels. Figure S4. Canavanine resistance in select strains. Four independent cultures of 107 cells were spread on plates containing 60 μg/mL canavanine, the CanR colonies were counted and plotted using “stock” graph by MS Excel©. The actual numbers of CanR colonies on each plate are listed in the table below. The assay was performed only with the strains, which do not harbor the can1-100 mutation. Figure S5. MMS sensitivity of the analysed strains. Exponentially growing cultures (OD600 = 1) of the strains shown on top were serially diluted and 5 microliter aliquots were spotted on YPD plates containing 0, 0.005, 0.01 and 0.02% MMS (shown on the right). One of two independent experiments is shown. Figure S6. Mating efficiency in double deletion mutants. Exponentially growing cultures (OD600 = 1) of the strains shown on the horizontal axis were serially diluted, mixed with 105 W303 cells of the opposing mating type in 0.25 mL of YPD medium and incubated for 4 h at 30 °C with gentle shaking. Five microliter aliquots were then spotted on SC dropout plates selecting for diploid cells and on plates selecting for both diploids and the tested haploids. SD dropout media were different for the different strains. The efficiency of mating was calculated as per cent of the number of diploids divided by the number of diploids/haploids. Figure S7. Sensitivity of chromatin to MNase digestion. 100 mL of exponentially growing cultures (OD600 = 1.6) of the strains shown on top of each panel were harvested and washed and cells were crushed by bead beating in Lysis buffer (140 mM NaCl, 50 mM Tris.HCl pH 7.6, 2 mM EDTA plus Protease Inhibitors). The extract was spun for 10 min at 13,000g, the chromatin pellet was resuspended in 1.5 mL MNase buffer plus Protease Inhibitors containing 6000 gels units of Microccocal nuclease (NEB) and incubated at 37 °C. Aliquots were removed at the times indicated and mixed with 1/10th volume STOP solution (10% SDS, 25 mM EDTA, 100 mM EGTA), DNA was purified and analysed on 1.2% agarose gels. The right-hand and the left-hand panel are from different experiments. At lease four experiments with each mutant strain in parallel with W303 were performed. |
