Rtt109 Prevents Hyper-Amplification of Ribosomal RNA Genes through Histone Modification in Budding Yeast (original) (raw)
Figure 8
Less ERC is generated and multimeric ERC integrates into rDNA during hyper-amplification.
A) 2D gel electrophoresis to detect ERC before (growth on raffinose) and after (growth on galactose) FOB1 induction. The wild type (RTT109) and rtt109 mutant with YCplac22-GAL-FOB1 in the fob1 defective background were incubated as in the experiment to monitor hyper-amplification and were sampled for DNA at time zero (Raffinose) and 9 hr after galactose mediated FOB1 induction (Galactose). The scheme is shown in the right side of the figure. B) Quantification of ERC before and after FOB1 induction. Quantification is described in Materials and Methods. C) Integration of plasmid with rDNA into chromosome XII during hyper-amplification; EtBr staining (left) and hybridization with URA3 as probe (right). Asterisks indicate the positions of the plasmid with and without rDNA (See Figure S4 to confirm the band with asterisks derived from the plasmid). The arrowhead indicates the position of the hyper-amplified band of chromosome XII [ChrXII(hyp.)]. The rtt109 mutant with an empty vector (lanes 1–4) or with a plasmid containing an rDNA unit (ERC-like plasmid, lanes 5–8) were incubated after FOB1 induction. As the control, the wild type strain with the ERC-like plasmid was incubated (lanes 9–10, RTT109). At the indicated time after Fob1 induction, DNA was prepared for CHEF analysis. Southern hybridization was performed with a URA3 probe. D) 2D gel electrophoresis to detect the repeats amplified by rolling circle replication. (Upper) A scheme for rolling circle amplification with the IGS plasmid. The amplification forms a cluster of plasmid sequences in the rDNA. Copy number of a cluster is estimated by _Bgl_II digestion. In this case, five copies were amplified. (Lower) Plasmid sequences were detected by a specific probe (amp). The scheme is shown on the right side of the figure. The numbers below the linear position are copy numbers generated by rolling circle amplification.