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. 2011 Jul 29;6(7):e22209. doi: 10.1371/journal.pone.0022209

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Wang et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) Western blot analysis of whole-cell extracts using anti-Flag antibody shows the level of H2B (Flag-H2B) and its ubiquitylated form (Flag-H2Bub1) in the indicated strains (derived from Y131) expressing Flag-HTB1 or Flag-htb1 mutants. (B) Telomere silencing analysis of the indicated strains (derived from UCC6389) expressing HTB1 or htb1 mutants. Cells were 10-fold serially diluted and spotted on YC with or without uracil (-Ura), or YC containing FOA (FOA). The strain sir4Δ was used as a control for the defect of telomere silencing. (C) The mRNA level of an endogenous gene near a telomere, YFR057W. Total RNA was extracted from the yeast strains, reverse transcribed and then quantified by real-time PCR using the primer pairs against YFR057W sequences. The obtained signals were normalized with the signal from ACT1, and then the value of WT was taken as 1 before log transformation. (D) Transcript array analysis was performed using strains (derived from Y131) expressing htb1-T122E, S125E, S125E/S126E, and S126E/S127E. Transcripts were isolated and analyzed by Phalanx Yeast OneArray®. Numbers of genes affected in the htb1 mutants compared to WT plotted by their positions from telomeres. The numbers of up-regulated genes are represented by the black bar; down-regulated genes are represented by the white bar.