CHROMATIN ASSEMBLY EXTRACTS FROM DROSOPHILA EMBRYOS

This chapter describes the protocols for the preparation of chromatin assembly extracts from Drosophila embryos and procedures for the assembly of regularly spaced nucleosomes on plasmid DNA and for the analysis of the resulting chromatin. The in vitro chromatin assembly system from Drosophila embryos enables the reconstitution of cloned genes in chromatin with regularly spaced nucleosomes. As the reconstituted chromatin is transcriptionally repressed, this template approaches the in vivo structure of inert chromatin that is the substrate for interaction with transcription factors, RNA polymerase, and other sequence-specific-binding proteins. Thus, the ability to reconstitute transcriptionally repressed chromatin provides a starting point for investigations on the mechanism of action of these proteins in a near-physiological context. The assembly system should also be useful for the analysis of the pathway of histone deposition, for an analysis of the higher orders of chromatin folding, and more generally for mechanisms of DNA replication and recombination in a chromatin context. © 1994, Elsevier Science Publishers, B.V.