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. 2013 Aug 15;9(8):e1003677. doi: 10.1371/journal.pgen.1003677

Figure 1. Mediator is required for heterochromatic silencing at the pericentromere.

Figure 1

(A) Schematic of fission yeast centromere 1. Locations of ura4 and ade6 reporter genes inserted within the pericentromeric region are shown (imr1L::ura4+ and otr1R::ade6). Black bars indicate the location of primers or probes used for ChIP, RT-PCR and northern analysis. (B) Silencing assay at the pericentromere. Shown are the results of serial dilutions of the indicated strains spotted onto non-selective media (N/S), medium with 5-fluoroorotic acid (5-FOA), and medium with a limited amount of adenine (Low Ade) to assay ura4+ and ade6+ expression. (C) Spots on Low Ade medium using Mediator mutants (med18Δ, med20Δ, med8-K9 and med31-H1), which are defective in heterochromatic silencing at the pericentromere. (D) Silencing assay at the pericentromere. Shown are the results of serial dilutions of the indicated strains spotted onto N/S, 5-FOA and Low Ade media to assay ura4+ and ade6+ expression. w indicates white epiclones (med18Δ-w and med20Δ-w), and p indicates pink epiclones (med18Δ-p and med20Δ-p). (E) Northern Analysis of dh, otr1R::ade6 and imr1L::ura4 forward strand transcripts in wild-type (wt) and mutant cells using oligonucleotide probes. rRNA was used as a loading control. (F) Quantitative RT-PCR analysis of cen dh forward transcript levels relative to a control act1+, normalized to the wild type in the indicated strains. Error bars show the standard error of the mean (n = 3).