Fig. 1.
Isolation of mononucleosomes from yeast. (A) Chromatin fractions from wild-type, H3 K4R, htz1Δ or htz1Δ strains expressing HA–Htz1p. Histones in chromatin were analyzed by immunoblots probed with anti-HA antibodies (top), and then stripped and reprobed with anti-H3 antibodies (bottom). (B) Mononucleosome preparation. DNA extracted from nuclear extracts from 1×108 cell equivalents of yeast expressing HA–Htz1p were digested with 0 or 2.0 U MNase/mg of solid and analyzed by agarose gel electrophoresis. (C–E) Analyses of antibody specificity and genotype of yeast used for single molecule studies. Immunoblots of whole cell extracts from the indicated yeast strains were probed with anti-HA antibodies (top panels) to evaluate HA–Hzt1p expression, then stripped and reprobed with anti-H4 K12ac (C), anti-H3 K4me3 (D) or anti-H3 K36me3 (E) antibodies (middle panels) to evaluate histone modifications. Blots were then restripped and reprobed with anti-H3 antibodies because H3 served as a loading control (bottom panels).