H2A2-GFP was analyzed by wide-field fluorescence microscopy as reported before15 (link). Briefly, series of z-focal plane images (15–20 planes, 0.15–0.3 μm depth) were collected on a Leica DMI6000, using a 63×/1.30 immersion objective and an ultrasensitive DFC 350 digital camera, and processed with the AF6000 software (Leica). Scale bars in micrographs depict 5 μm; BF stands for bright field.
DNA content by flow cytometry analysis (FACS) was done as previously described using a BD FACScalibur equipment7 (link). An asynchronous culture of each strain growing at permissive temperature was used to calibrate the 1C and 2C peaks before reading the samples. Strains carrying the cdc15-2 and cdc14-1 alleles rendered a minor 2C peak at the G1 arrest. This peak corresponded to 10–20% of G1 samples and comprised cells responsive to alpha-factor but that remain attached as pairs (FigureS7 ). This phenotype likely stems from delayed septation at permissive temperatures. This minor 2C transposes into a 4C peak when cells are released from the G1 arrest, giving the false impression of re-replication.
DNA content by flow cytometry analysis (FACS) was done as previously described using a BD FACScalibur equipment7 (link). An asynchronous culture of each strain growing at permissive temperature was used to calibrate the 1C and 2C peaks before reading the samples. Strains carrying the cdc15-2 and cdc14-1 alleles rendered a minor 2C peak at the G1 arrest. This peak corresponded to 10–20% of G1 samples and comprised cells responsive to alpha-factor but that remain attached as pairs (Figure
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