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. 2014 Oct 9;42(20):12650–12667. doi: 10.1093/nar/gku915

Figure 3.

Figure 3.

Rad9 and Aft1 exhibit synthetic effect and may affect transcriptional elongation. Cultures of the strains were grown in rich medium (YPD) to an OD600 = 1.0. Seven serial dilutions of the cells were spotted (A) on YPD plates, (B) on YPD plates which contained 300 μg/ml 6-AU and (C) on YPD plates which contained 50 μM FeCl3. Plates were incubated for 2 days at 30°C. Microscopy studies showed that the examined mutants have similar cell size in YPD rich medium (data not shown). The OD600 of exponentially growing cells (starting from an OD600 = 0.25) was measured every 20 min (D) inYPD, (E) in YPD plus 300 μg/ml 6-AU and (F) in YPD plus 50 μM FeCl3, until they reached stationary phase, using the BioLector technology. Values of each growth curve were normalized over the value of the first measurement in each case (wt, rad9Δ, aft1Δ, rad9Δaft1Δ). The relative growth rate of the strains in each condition was calculated and the values are shown in parentheses. (G) ChIP analysis that shows Rad9–13Myc and Aft1–9Myc enrichment to yeast centromeres. Normalization was performed firstly over INPUT chromatin and secondly by measuring the binding of Rad9–13Myc on FRE2 coding region (where Rad9 and Aft1 binding is minimal in the used growth conditions) and then dividing Rad9–13Myc or Aft1–9Myc enrichment by this ‘non-specific’ enrichment. Experiments were performed at least in duplicate. The same binding pattern was also obtained after normalizing over PHO5 coding region with similar results (data not shown). Due to high content in AT nucleotides in centromeric areas, some of the primers were not functional in real-time PCR (chr IX and XI). (H) Aft1-dependency of Rad9 localization to centromeres. The centromeric localization of Rad9–13Myc in wt and aft1Δ cells, as well as the centromeric localization of Aft1–9Myc in wt and rad9Δ cells was examined by ChIP in CENIII, CENXII and CENXV. All strains were grown under inducing conditions (SC BCS BPS). Normalization was performed firstly over INPUT chromatin and secondly by measuring the enrichment of Rad9–13Myc on PHO5, as described in the legend to Figure 2.