Chromatin structure mapping in Saccharomyces cerevisiae in vivo with DNaseI

Most methods for assessment of chromatin structure involve chemical or nucl ease damage to DNA followed by analysis of distribution and susceptibility of cutting sites. The agents used generally do not permeate cells, making n uclear isolation mandatory. In vivo mapping strategies might allow detectio n of labile constituents and/or structures that are Post when chromatin is swollen in isolated nuclei at low ionic strengths. DNase I has been the mos t widely used enzyme to detect chromatin sites where DNA is active in trans cription, replication or recombination. We have introduced the bovine DNase I gene into yeast under control of a galactose-responsive promoter. Expres sion of the nuclease leads to DNA degradation and cell death. Shorter expos ure to the active enzyme allows mapping of chromatin structure in whole cel ls without isolation of nuclei. The validity and efficacy of the strategy a re demonstrated by footprinting a labile repressor bound to its operator. I nvestigation of the inter-nucleosome linker regions in several types of rep ressed domains has revealed different degrees of protection in cells, relat ive to isolated nuclei.