DNase I footprinting of the nucleosome in whole nuclei
Section snippets
Materials and methods
Nuclei. Chicken blood was collected in PBS (0.15 M NaCl, 0.015 M phosphate, pH 7.6) containing 50 mg/l heparin. Cells were never exposed to EDTA. The cells were lysed in 50 mM Tris pH 7.5, 6 mM MgCl2, 0.25 M sucrose and 1% Triton X-100, followed by several washes in the same buffer without Triton as in Ref. [3].
Nucleosome footprints. Nucleosome footprints were obtained as previously described in Ref. [12], but the nuclei were digested with DNase I before isolation of nucleosomes. Briefly, nuclei at
Results and discussion
To obtain a direct footprint of the additional protection imposed by the higher-order chromatin structure on the nucleosome: (i) whole nuclei were digested with DNase I, (ii) nucleosomes were isolated by micrococcal nuclease digestion and sucrose gradient fractionation, (iii) histone tails were removed by partial trypsin digestion, (iv) nucleosomal DNA was trimmed to core particle size and (v) gel purified and 5’-end-labelled DNA fragments were examined on a denaturing polyacrylamide gel. A
Acknowledgments
I thank Dr. Yana Proykova for discussions and critical reading of the manuscript. This work was supported by The Wellcome Trust Grant No. 037008.
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