Selective excision of the centromere chromatin complex from Saccharomyces cerevisiae.
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.
Kenna
M
M
Department of Biology, University of North Carolina, Chapel Hill 27599-3280.
Amaya
E
E
Bloom
K
K
D002467:Cell Nucleus / Q000378:metabolism; D002503:Centromere / Q000032:analysis / Q000378:metabolism*; D002843:Chromatin / Q000032:analysis / Q000378:metabolism*; D002875:Chromosomes / Q000378:metabolism*; D004262:DNA Restriction Enzymes; D004271:DNA, Fungal / Q000032:analysis / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000032:analysis / Q000302:isolation & purification*; D015245:Deoxyribonuclease BamHI; D015252:Deoxyribonucleases, Type II Site-Specific; D004587:Electrophoresis, Agar Gel; D009693:Nucleic Acid Hybridization; D009698:Nucleoproteins / Q000032:analysis / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000648:ultrastructure
D002843:Chromatin; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D009698:Nucleoproteins; D004262:DNA Restriction Enzymes; D015245:Deoxyribonuclease BamHI; D015252:Deoxyribonucleases, Type II Site-Specific; C035594:TTTAAA -specific type II deoxyribonucleases