E-Book Overview
Collection of protocols is designed to analyze the relationship between chromatin structure and function, and to elucidate molecular mechanisms that control vital cellular functions such as transcription, replication, recombination, and DNA repair. Methods cover topics from cell-free systems for the constitution of chromatin heterogeneity in vitro, to techniques for in vivo analysis of protein-DNA interactions. Methods include instructions to ensure successful replication and to avoid pitfalls.
E-Book Content
Methods in Molecular Biology
TM
VOLUME 119
Chromatin Protocols Edited by
Peter B. Becker
HUMANA PRESS
Nucleosome Reconstitution
1
1 Expression and Purification of Recombinant Histones and Nucleosome Reconstitution Karolin Luger, Thomas J. Rechsteiner, and Timothy J. Richmond 1. Introduction In vitro studies on nucleosome core particles (NCPs) and nucleosomes have generally been limited to the use of histone proteins isolated from chromatin. Numerous reliable and well-established methods have been described of obtaining single histone proteins in significant quantity (e.g., refs. 1 and 2, and references therein). Briefly, the histone complexes (histone octamer, or histone tetramer and histone dimer) are isolated from “long chromatin,” which is extracted from nuclei. The histone complexes can be further fractionated into individual histone proteins. This approach suffers from several disadvantages. First, the procedure is time-consuming and depends on the availability of fresh tissue or blood from the organism of choice. Second, histone proteins isolated from natural sources are often degraded by contaminating proteases (3). Third, histone isotypes and posttranslational modifications of histone proteins give rise to heterogeneity. The extent of heterogeneity and modification strongly depend on the ty