Selective Excision of the Centromere Chromatin Complex from Saccharomyces revisiae

Abstract. We have taken advantage of the known structural parameters associated with centromere DNA in vivo to construct a CEN fragment that can be selec-tively excised from the chromatin DNA with restric-tion endonucleases. CEN3 DNA is organized in chro-matin such that a 220-250-bp region encompassing the elements of centromere homology is resistant to nuclease digestion. Restriction enzyme linkers encod-ing the Bam HI-recognition site were ligated to a 289 base pair DNA segment that spans the 220-250-bp protected core (Bloom et al., 1984). Replacement of this CEN3-Bam HI linker cassette into a chromosome or plasmid results in formation of a complete struc-tural and functional centromeric unit. A centromere core complex that retains its protected chromatin con-formation can be selectively excised from intact nuclei by restriction with the enzyme Barn HI. The centro-meric protein-DNA complex is therefore not depen-dent upon the intact torsional constraints on linear chromosomes for its structural integrity. Isolation of this complex provides a novel approach to characteriz-ing authentic entromeric proteins bound to DNA in their native state. T HE centromere region functions as a cis-acting ele-ment for chromosomal segregation i both mitotic and meiotic ell divisions in the yeast Saccharomyces cerevisiae. Specific centromeric DNA sequences (CEN) are required in cis for proper chromosomal transmission during cell growth. The sequence requirements have been inves-tigated by altering DNA in vitro and determining CEN func-tion in an in vivo assay for chromosomal stability (Carbo