We identified the chromosomal addresses of a cohesin subunit, Mcd1p, in vivo by chromatin immunoprecipitation coupled with high resolution PCR-based chromosomal walking. The mapping of new Mcd1p-binding sites (cohesin-associated regions [CARs]) in single-copy sequences of several chromosomes establish their spacing (∼9 kb), their sequestration to intergenic regions, and their association with AT-rich sequences as general genomic properties of CARs. We show that cohesins are not excluded from telomere proximal regions, and the enrichment of cohesins at the centromere at mitosis reflects de novo loading. The average size of a CAR is 0.8–1.0 kb. They lie at the boundaries of transcriptionally silenced regions, suggesting they play a direct role in defining the silent chromatin domain. Finally, we identify CARs in tandem (rDNA) and interspersed repetitive DNA (Ty2 and subtelomeric repeats). Each 9-kb rDNA repeat has a single CAR proximal to the 5S gene. Thus, the periodicity of CARs in single-copy regions and the rDNA repeats is conserved. The presence and spacing of CARs in repetitive DNA has important implications for genomic stability and chromosome packaging/condensation.
The 70-kDa heat shock proteins (hsp7Os) function as molecular chaperones in a wide variety of cellular processes through cycles of binding and release from substrate protens coupled to cycles ofATP hydrolysis. In the prokaryote Esckerichia coi, the hsp7O DnaK functions with two other proteins, DnaJ and GrpE, which modulate the activity of DnaK. While numerous hsp7Os and DnaJ-related proteins have been identified in' eukaryotes, to our knowledge no GrpErelated proteins have been reported. We report the isolation and characterization ofa eukaryotic grpE-related gene, MfGE.
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