Isolation of selected chromatin fragments from yeast by site-specific recombination in vivo

A burgeoning interest in the role of chromatin structure in it wide variety of chromosome functions has established a need for methods to obtain chrom atin in its native form. Here we describe a simple and efficient method for biochemical isolation of selected chromatin fragments from yeast chromosom es. The approach involves three steps. First, site-specific recombination i n vivo is used to excise a chromosomal domain of interest In the form of a small extrachromosomal ring Second, whole cell lysate is prepared from cult ures in which recombination has been induced. Third, differential centrifug ation is used to separate excised chromatin rings from chromosomes and othe r cellular debris. Using this methodology, we show that rings containing th e transcriptionally repressed HMR mating-type locus can be formed and isola ted in high yield. Furthermore, we show that the isolation procedure result s in significant enrichment of recombinant rings. Finally, we show that the nucleosomal organization of the recombined material is not altered during isolation. (C) 1999 Academic Press.