The Journal of Cell Biology, Vol 107, 9-15, Copyright © 1988 by The Rockefeller University Press

ARTICLES

Selective excision of the centromere chromatin complex from Saccharomyces cerevisiae

M Kenna, E Amaya and K Bloom
Department of Biology, University of North Carolina, Chapel Hill 27599- 3280.

We have taken advantage of the known structural parameters associated with centromere DNA in vivo to construct a CEN fragment that can be selectively excised from the chromatin DNA with restriction endonucleases. CEN3 DNA is organized in chromatin such that a 220-250- bp region encompassing the elements of centromere homology is resistant to nuclease digestion. Restriction enzyme linkers encoding 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 structural and functional centromeric unit. A centromere core complex that retains its protected chromatin conformation can be selectively excised from intact nuclei by restriction with the enzyme Bam HI. The centromeric protein-DNA complex is therefore not dependent upon the intact torsional constrains on linear chromosomes for its structural integrity. Isolation of this complex provides a novel approach to characterizing authentic centromeric proteins bound to DNA in their native state.
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This article has been cited by other articles:

• Wright, J H, Gottschling, D E, Zakian, V A (1992). Saccharomyces telomeres assume a non-nucleosomal chromatin structure.. Genes Dev. 6: 197-210 [Abstract]  

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