Copy number control by a yeast centromere

Tschumper, Gary; Carbon, John

doi:10.1016/0378-1119(83)90054-9

Cited by 112 publications

(57 citation statements)

References 26 publications

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“…As a control, we purified minichromosomes that cannot assemble kinetochores due to mutations in the conserved centromere domain CDEIII (Ortiz et al 1999). Because these mutant minichromosomes lack kinetochores, the intracellular copy number is ;20-fold higher than the wild-type (WT) centromeric minichromosome (Tschumper and Carbon 1983;data not shown). Despite this copy number difference, the centromeric histone H3 variant (Cse4) and Ndc80 outer kinetochore component specifically copurified with minichromosomes containing functional centromeres (Fig.…”

Section: Yeast Kinetochores Can Be Purified By Isolating Centromeric mentioning

confidence: 99%

Quantitative proteomic analysis of purified yeast kinetochores identifies a PP1 regulatory subunit

Akiyoshi¹,

Nelson²,

Ranish³

et al. 2009

Genes Dev.

103

154

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The kinetochore is a macromolecular complex that controls chromosome segregation and cell cycle progression. When sister kinetochores make bioriented attachments to microtubules from opposite poles, the spindle checkpoint is silenced. Biorientation and the spindle checkpoint are regulated by a balance between the Ipl1/Aurora B protein kinase and the opposing activity of protein phosphatase I (PP1). However, little is known about the regulation of PP1 localization and activity at the kinetochore. Here, we developed a method to purify centromere-bound kinetochores and used quantitative proteomics to identify the Fin1 protein as a PP1 regulatory subunit. The Fin1/PP1 complex is regulated by phosphorylation and 14–3–3 protein binding. When Fin1 is mislocalized, bipolar spindles fail to assemble but the spindle checkpoint is inappropriately silenced due to PP1 activity. These data suggest that Fin1 is a PP1 regulatory subunit whose spatial and temporal activity must be precisely controlled to ensure genomic stability.

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Section: Yeast Kinetochores Can Be Purified By Isolating Centromeric mentioning

confidence: 99%

Quantitative proteomic analysis of purified yeast kinetochores identifies a PP1 regulatory subunit

Akiyoshi¹,

Nelson²,

Ranish³

et al. 2009

Genes Dev.

103

154

View full text Add to dashboard Cite

show abstract

“…Despite the fact that the 2,um circle apparently has the ability to amplify its copy number to high levels, these CEN-2,um circlecomposite plasmids have copy numbers of about one and do not have any obvious toxic effects on the host cells. Perhaps the effects are too small to be noticed; alternatively, the CEN may prevent 2,um circle amplification directly, possibly by blocking replication through the centromere until late in mitosis (34). A replication block at the centromere is one way of explaining the observation that sister chromatids are held together at the centromere until anaphase (8).…”

Section: Discussionmentioning

confidence: 99%

“…Evidence for this comes from Southern hybridization analysis of DNA from CEN plasmid transformants (3,34) and also from tetrad analysis; CEN plasmids segregate 2:2 in about half the tetrads dissected, indicating that most parental diploids have one plasmid copy (6,12). In contrast, ARS plasmids without centromeres have copy numbers of 50 to 100 per plasmid-bearing cell (18).…”

mentioning

confidence: 99%

Toxic effects of excess cloned centromeres.

Futcher¹,

Carbon²

1986

Mol. Cell. Biol.

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“…First, because our systematic libraries were designed to be functional tools, it was important that the inserts did not contain elements that interfered with function. One element that can interfere with both CEN and 2 µ vectors is the centromere itself, as dicentric plasmids are unstable (Mann and Davis, 1983), and centromeres reduce the copy number of 2 µ plasmids (Tschumper and Carbon, 1983). For this reason, each of the 2 µ library inserts that contained an endogenous centromere was subcloned to separate the centromere-proximal genes from their respective centromere.…”

Section: Resultsmentioning

confidence: 99%