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. 2005 Jan;25(2):637–651. doi: 10.1128/MCB.25.2.637-651.2005

FIG. 1.

FIG. 1.

The Paf1 elongation complex regulates H2B ubiquitylation. (A) RAD6 (YZS279), SET2 (YTX023), or the indicated Paf1 complex gene (YTX018-YTX022), was deleted from a yeast strain bearing a Flag-tagged copy of histone H2B (YZS276). Cell pellets derived from these strains were loaded directly on an SDS-polyacrylamide gel, and the levels of H2B ubiquitylation were determined by immunoblot analysis using an anti-Flag antibody. The presence of H2B and the monoubiquitylated form of H2B (ubH2B) are indicated. Independently, nuclei were prepared from these strains and analyzed by immunoblot analysis for the presence of H3 lysines 4 (K4), 36 (K36), and 79 (K79) dimethylation. Asterisks indicate N-terminal breakdown products of H3 that are typically observed. (B) The loss of H2B ubiquitylation in the rtf1Δ and paf1Δ strains can be restored. Cell pellets isolated from the WT (YZS276), rft1Δ (YTX018), or paf1Δ (YTX019) strain containing either the vector control plasmid or the appropriate Rtf1-Flag or Paf1-Flag expression plasmid were probed with the anti-Flag antibody. The locations of Rtf1 and Paf1 are indicated. It is noteworthy that Rtf1 shows a variety of N-terminal breakdown products that naturally occur during logarithmic growth.