Visualization of large-scale chromatin structure and dynamics using the lac operator/lac repressor reporter system.

Publisher Summary In situ hybridization methods have been pivotal in allowing the exploration of chromosome and gene arrangements within interphase nuclei. However, the requirement of DNA denaturation for in situ hybridization is incompatible with direct in vivo observations. Moreover, the harsh conditions required for DNA denaturation are antagonistic to ultrastructural preservation. This has led to a large gap between primarily light microscopy studies, using in situ hybridization to examine the locations of specific chromosomal regions or loci, and ultrastructural analysis of higher order, “generic” chromatin structure, primarily by electron microscopy. In an attempt to bridge these two structural approaches, this chapter explores the use of protein–DNA recognition as a tag for localizing specific chromosome regions to provide an alternative to in situ hybridization. The chapter uses the lac operator/repressor system for an initial test case for several reasons. The lac repressor had been demonstrated as having the capability of binding in vitro to an operator sequence packaged within nucleosomes with only a small decrease in binding affinity. Later in vivo experiments confirmed the specific binding of lac repressor to lac operator within eukaryotic cells.