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

摘要

A burgeoning interest in the role of chromatin structure in a wide variety of chromosome functions has established a need for methods to obtain chromatin in its native form. Here we describe a simple and efficient method for biochemical isolation of selected chromatin fragments from yeast chromosomes. The approach involves three steps. First, site-specific recombination in 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 cultures in which recombination has been induced. Third, differential centrifugation is used to separate excised chromatin rings from chromosomes and other cellular debris. Using this methodology, we show that rings containing the transcriptionally repressed HMR mating-type locus can be formed and isolated in high yield. Furthermore, we show that the isolation procedure results in significant enrichment of recombinant rings. Finally, we show that the nucleosomal organization of the recombined material is not altered during isolation. Copyright 1999 Academic Press.