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note
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Single-stranded biotinylated 5S DNA template (top strand between the positions -113 and +218 relative to the transcription start site) was synthesized with a 5′-biotinylated primer (-113....-96, 5′-ATTTCACACAGGAAACAG-3′) and plasmid pXP10 (12), linearized by Hind III. Probes were synthesized as described (29), with the following modifications: the 5′-biotinylated template strand of 5S DNA was immobilized on streptavidin-agarose before primer extensions, and the ligation of nicks was omitted (Fig. 1).
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28
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10244276982
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note
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Nucleosome cores were reconstituted onto radiolabeled DNA fragments by salt-dilution exchange with chicken erythrocyte core particles (12). The original 1 M NaCI 60-μl exchange reaction contained donor chromatin (5 μg), naked nonspecific DNA (1 μg), and photoactive labeled 5S fragment (50 fmol). Reconstitution was monitored by electrophoresis in 0.7% agarose gel in 0.5 × TBE [1× tris-borate EDTA (TBE) consists of 90 mM tris base, 90 mM boric acid, 2.5 mM EDTA]. After electrophoresis, the gels were dried and autoradiographed. Approximately 25 ng of reconstituted nucleosomes, including ∼0.25 fmol of a photoprobe-containing nucleosome, were incubated with or without various amounts of linker histones in 10 μl of binding buffer [10 mM tris-HCl (pH 8.0), 50 mM NaCl, 0.1 mM EDTA, 5% (v/v) glycerol, 0.05% Triton X-100]. Samples were incubated at room temperature for 15 min and loaded onto 0.7% agarose gels in 0.5X TBE, as described above. To avoid potential complications with nonspecific linker histone binding, we subsequently used linker histones (H1°, H5)/nucleosome ratios of about 0.5, as confirmed by the titration experiment described above. For the deletion mutant H1°Δ126-C and for the globular domain of histone H5, it was not possible to follow their binding to nucleosomes by gel mobility-shift (13), so we used a linker histone amount corresponding to 25% of the amount sufficient to cause extensive DNA aggregation in the samples, based on the observation that the full-length linker histones cause DNA aggregation at ∼2:1 ratio to the nucleosomes.
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29
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10244247883
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note
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2 and digested with 2 ng of micrococcal nuclease (MNase) for 5 to 60 min at room temperature. Digestion was terminated by the addition of EDTA (5 mM), SDS (0.25% w/v), and DNA was deproteinized by proteinase K (1 μg/ml) (37°C, 30 min), phenol-extracted, and etnanol-precipitated. Digestion products were resolved on 9% acrylamide gel in 1X TBE, and core particle and chromatosome-sized bands were excised and electroeluted (90 V, 25 min) and loaded on a sequencing gel.
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30
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10244237824
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note
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The samples were adjusted to 15 mM NaCl and photocross-linked by mild ultraviolet irradiation. Cross-linked DNA was chemically digested by 70% formic acid, 2% diphenylamine (70°C, 20 min), so that only 5′,3′-phosphorylated oligopyrimidine blocks remained covalently bound to the histones. The reactants were extracted by 10 volumes of ether (twice). The samples were freeze-dried, loaded to 15% SDS gel, stained with Coomassie blue to reveal unlabeled protein positions, dried, and autoradiographed.
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preparation of the manuscript
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We thank the referees for useful comments and T. Vo for preparation of the manuscript.
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Vo, T.1
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