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. 2008 Feb 16;36(7):2208–2218. doi: 10.1093/nar/gkn060

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© 2008 The Author(s)

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 1. — Preparation and nucleosome formation ability of H3t/H4. (A) SDS–PAGE analysis of proteins from each preparation step for H3t/H4. Lanes 2 and 3: the whole cell lysates of the E. coli cells expressing His₆-tagged H3t and His₆-tagged H4, respectively. Lanes 4 and 5: the peak Ni-NTA agarose fractions of His₆-tagged H3t and His₆-tagged H4, respectively. Lanes 6 and 7: His₆-tagged H3t/H4 before and after the removal of the hexahistidine tag, respectively. The positions of the size markers (lane1) are indicated on the left. (B) SDS–PAGE analysis of purified histones used in this study. Lane 1: size markers. Lanes 2–4: histones; H2A/H2B (lane 2), H3t/H4 (lane 3) and H3.1/H4 (lane 4). (C) Nucleosome reconstitution analyzed by non-denaturing 6% PAGE. Lane 1: naked DNA. Lane 2: nucleosomes reconstituted with H2A/H2B and H3t/H4 by the salt-dialysis method. Nucleosomes with different translational positions are indicated. (D) MNase assay analyzed by non-denaturing 10% PAGE. Naked DNA (lanes 2–5) and H3t-containing nucleosomes reconstituted by the salt-dialysis method (lanes 6–9) were treated with MNase, and the resulting DNA fragments were analyzed. Lane 1: the molecular mass markers (10 bp DNA ladder). Amounts of MNase (unit/μl) were 0.18 (lanes 2 and 6), 0.13 (lanes 3 and 7), 0.08 (lanes 4 and 8) and 0 (lanes 5 and 9). In H3t-containing nucleosomes, 146 bp DNA, which is tightly wrapped around the nucleosomes and resistant to MNase, is detected (lanes 6–8).