Abstract
Transcriptional inactivation of the budding yeast centromere has been a widely used tool in studies of chromosome segregation and aneuploidy. In haploid cells when an essential chromosome contains a single conditionally inactivated centromere (GAL-CEN), cell growth rate is slowed and segregation fidelity is reduced; but colony formation is nearly 100%. Pedigree analysis revealed that only 30% of the time both mother and daughter cell inherit the GAL-CEN chromosome. The reduced segregation capacity of the GAL-CEN chromosome is further compromised upon reduction of pericentric cohesin (mcm21∆), as reflected in a further diminishment of the Mif2 kinetochore protein at GAL-CEN. By redistributing cohesin from the nucleolus to the pericentromere (by deleting SIR2), there is increased presence of the kinetochore protein Mif2 at GAL-CEN and restoration of cell viability. These studies identify the ability of cohesin to promote chromosome segregation via kinetochore assembly, in a situation where the centromere has been severely compromised.
Highlights
Proper microtubule attachment is required for accurate chromosome segregation
The most commonly used conditionally inactivated centromere tool is the insertion of a galactose inducible promoter upstream of the centromeric sequence, termed GAL-CEN
When pericentromeric cohesin recruitment is impaired by deleting MCM21, viability is reduced to 23%
Summary
Attachment to the mitotic spindle requires the formation of a multiprotein kinetochore at the specialized chromosomal locus, the centromere. Termed GAL-CEN, this conditional centromere is functional when cells are grown on glucose but its function is inhibited when cells are grown on galactose [2]. Both chromosomes and autonomous mini-chromosomes harboring the GAL-CEN3 construct show severe defects in chromosome segregation upon kinetochore inactivation on galactose. In haploid cells carrying a nonessential GAL-CEN3 plasmid, or diploids carrying a single GAL-CEN3 chromosome, the percentage of GAL-CEN3 containing cells dropped to less than 5–10% within 10 generations following centromere inactivation [2]. The transcriptional inactivation of a centromere has been widely used to study consequences of aneuploidy [4,5,6]
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