Tracking of Single and Multiple Genomic Loci in Living Yeast Cells

Abstract

Nuclear organization is involved in numerous aspects of cellular function. In yeast, analysis of the nuclear position and dynamics of the silent and active mating-type loci has allowed to gain insight into the mechanisms involved in directing mating-type switching. The fluorescent repressor operator systems (FROS) have proven to be a powerful technique to tag DNA sequences to investigate chromosome position and dynamics in living cells. FROS rely on the transgenic expression of a bacterial repressor fused to a fluorescent protein which can bind to its respective operator DNA sequence integrated as multicopy tandem arrays at a specific genomic site. Different FROS exist which facilitate the tagging of up to three different loci simultaneously. This chapter describes detailed protocols for FROS usage and analysis in the yeast Saccharomyces cerevisiae.