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. 2014 Mar 25;70(Pt 4):444–449. doi: 10.1107/S2053230X14004695

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© Urahama et al. 2014

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

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Reconstitution of the human testis-specific TSH2B nucleosome. (a) Alignment of the human H2B and TSH2B amino-acid sequences. The secondary structure of TSH2B in the nucleosome is shown at the top of the panel. Amino-acid residues that differ between H2B and TSH2B are represented with a white background. (b) Expression of His₆-tagged TSH2B. Lane 1 contains molecular-mass markers. Lane 2 contains the whole-cell extract of the E. coli cells producing His₆-tagged TSH2B. The proteins were analyzed by 16% SDS–PAGE with Coomassie Brilliant Blue staining. (c) The Ni–NTA agarose column fraction. Lane 1 contains molecular-mass markers. His₆-tagged TSH2B eluted from the Ni–NTA agarose column was analyzed by 16% SDS–PAGE with Coomassie Brilliant Blue staining (lane 2). The TSH2B sample after His₆-tag removal by thrombin protease was analyzed by 16% SDS–PAGE with Coomassie Brilliant Blue staining (lane 3). (d) The purified H2A (lane 2), H2B (lane 3) and TSH2B (lane 4) were analyzed by 18% SDS–PAGE with Coomassie Brilliant Blue staining. Lane 1 contains molecular-mass markers. (e) Analysis of the histone octamers by gel-filtration chromatography. H2A, H3.1, H4 and TSH2B or H2B were incubated without DNA in the presence of 2 M NaCl. The samples were then subjected to HiLoad 16/60 Superdex 200 gel-filtration column chromatography. The gel-filtration profile of the histone octamer reconstituted with H2A, TSH2B, H3.1 and H4 is shown in the upper panel, and that of the histone octamer reconstituted with H2A, H2B, H3.1 and H4 is shown in the lower panel. (f) Purified canonical H2B (lane 2) and TSH2B (lane 3) nucleosomes were analyzed by 6% nondenaturing polyacrylamide gel electrophoresis (PAGE). DNA was visualized by ethidium bromide staining. Lane 1 indicates naked DNA. (g) The histone compositions of the purified H2B (lane 2) and TSH2B (lanes 3) nucleosomes were analyzed by 18% SDS–PAGE. Histones were visualized by Coomassie Brilliant Blue staining. A trace amount of degradation products was incorporated into the TSH2B nucleosome, as indicated by an asterisk. Lane 1 contains molecular-mass markers.