By Wu L.A., Allis C.D.
DNA within the nucleus of plant and animal cells is kept within the type of chromatin. Chromatin and the Chromatin remodelling enzymes play an immense function in gene transcription.
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Extra info for Chromatin and Chromatin Remodeling Enzymes
Production of Recombinant Histones The expression of histones in bacterial culture overcomes some problems with isolating material from higher organisms, related to animal welfare regulations and histone homogeneity. Recombinant material will not have N- or C-terminal tail modifications that can cause heterogeneity in isolated vertebrate material. However, unless grown by large-scale fermentation, the yields from recombinant histone production are most often less than can be derived from chicken erythrocyte preparations.
3. 1 M; optional). 4. Analyze by native PAGE as described below, and autoradiograph the gel. 12 J. M. Gottesfeld, C. Melander, R. K. Suto, H. Raviol, K. Luger, and P. B. Dervan, J. Mol. Biol. 309, 625 (2001). 13 J. M. Gottesfeld and K. Luger, Biochemistry 40, 10927 (2001). 40 biochemistry of histones, nucleosomes, and chromatin  Small-Scale Reconstitution of NCP Small-scale reconstitution works well for amounts of NCP 25 and 500 g. Multiple setups can be dialyzed in one vessel. , full-length histones, globular histones, and histone variants) into NCPs can vary, mainly because of inaccuracies in the concentration of histone octamer.
4. Analyze the products by using the high-resolution gel-shift assay described earlier1 and later in the chapter. Sample that has not been heat treated is run as a control. For subsequent large-scale reconstitutions, choose conditions that (1) contain only a small (if any) excess of free DNA and (1) are completely shifted to a single position. Large-Scale Reconstitution Large-scale reconstitution and purification of up to 4 mg of nucleosomes require about 5 days and involve the following steps.