Toxic effects of excess cloned centromeres.

Futcher, Bruce; Carbon, John

doi:10.1128/mcb.6.6.2213

Cited by 86 publications

(81 citation statements)

References 21 publications

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“…It has been found that transcription directed into a yeast centromere is deleterious to CEN function in vivo (7,13,16,31). In this report, it is demonstrated that transcription directed into an ARS sequence impairs ARS function in vivo.…”

mentioning

confidence: 64%

Transcription interferes with elements important for chromosome maintenance in Saccharomyces cerevisiae.

Snyder¹,

Sapolsky²,

Davis³

1988

Mol. Cell. Biol.

138

119

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Transcription directed into a Saccharomyces cerevisiae autonomously replicating sequence (ARS) causes high-frequency loss of minichromosomes. Conditionally stable artificial yeast chromosomes were constructed that contain an inducible GAL promoter upstream of ARS). Under growth conditions in which the promoter was inactive, these chromosomes were mitotically stable; however, when the GAL promoter was induced, the chromosomes became extremely unstable as a result of transcriptional impairment of ARS function. This interference by the GAL promoter occurred only in cis but can occur from either side of ARSI. Transcriptional interference ofARS function can be monitored readily by using a visual colony-color assay (P. Hieter, C. Mann, M. Snyder, and R. W. Davis, Cell 40:381-392, 1985), which was further developed as a sensitive in vivo assay for sequences which rescue ARS from transcription. DNA fragments from the 3' ends of genes, inserted downstream of the GAL promoter, protected ARS function from transcriptional interference. This assay is expected to be independent of both RNA transcript stability and processing. Philippsen et al. have shown that transcription into a yeast centromere inhibits CEN function in vivo

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mentioning

confidence: 64%

Transcription interferes with elements important for chromosome maintenance in Saccharomyces cerevisiae.

Snyder¹,

Sapolsky²,

Davis³

1988

Mol. Cell. Biol.

138

119

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“…1). The fact that introduction of multiple CEN plasmids slows cell proliferation (Futcher and Carbon 1986) could contribute to this phenotype. Based on these control experiments, we restricted our analysis to no more than two CEN plasmids.…”

Section: Resultsmentioning

confidence: 99%

Aneuploid proliferation defects in yeast are not driven by copy number changes of a few dosage-sensitive genes

2015

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Aneuploidy—the gain or loss of one or more whole chromosome—typically has an adverse impact on organismal fitness, manifest in conditions such as Down syndrome. A central question is whether aneuploid phenotypes are the consequence of copy number changes of a few especially harmful genes that may be present on the extra chromosome or are caused by copy number alterations of many genes that confer no observable phenotype when varied individually. We used the proliferation defect exhibited by budding yeast strains carrying single additional chromosomes (disomes) to distinguish between the “few critical genes” hypothesis and the “mass action of genes” hypothesis. Our results indicate that subtle changes in gene dosage across a chromosome can have significant phenotypic consequences. We conclude that phenotypic thresholds can be crossed by mass action of copy number changes that, on their own, are benign.

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“…For example, when overexpression generates high levels of free ␤-tubulin, cells containing a plasmid copy of RBL2 accumulate four to seven RBL2 plasmids in order to survive. Although CEN plasmids are normally present at a relatively low copy number (1 to 2 copies per cell), selective pressure can increase the copy number of CEN plasmids to ϳ12 copies per cell (6,13). The increase in Rbl2p levels in pCEN-RBL2 GAL-TUB2 cells due to the accumulation of multiple RBL2 plasmids defines the minimum amount of Rbl2p needed for cells to survive ␤-tubulin overexpression.…”

Section: Discussionmentioning

confidence: 99%

Protection from Free β-Tubulin by the β-Tubulin Binding Protein Rbl2p

Abruzzi

Smith

Chen

et al. 2002

Molecular and Cellular Biology

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Free ␤-tubulin not in heterodimers with ␣-tubulin can be toxic, disrupting microtubule assembly and function. We are interested in the mechanisms by which cells protect themselves from free ␤-tubulin. This study focused specifically on the function of Rbl2p, which, like ␣-tubulin, can rescue cells from free ␤-tubulin. In vitro studies of the mammalian homolog of Rbl2p, cofactor A, have suggested that Rbl2p/cofactor A may be involved in tubulin folding. Here we show that Rbl2p becomes essential in cells containing a modest excess of ␤-tubulin relative to ␣-tubulin. However, this essential activity of Rbl2p/cofactorA does not depend upon the reactions described by the in vitro assay. Rescue of ␤-tubulin toxicity requires a minimal but substoichiometric ratio of Rbl2p to ␤-tubulin. The data suggest that Rbl2p binds transiently to free ␤-tubulin, which then passes into an aggregated form that is not toxic.Studies of cellular control of microtubule assembly have focused primarily on the assembly reaction from ␣/␤-tubulin heterodimers to microtubule polymers and on the identification of protein cofactors and structures that modulate this polymerization (8-10). Results obtained by several approaches suggest that cells may also regulate microtubule morphogenesis at stages preceding the polymerization reaction. Of particular interest are proteins that appear to interact with the ␣-and ␤-tubulin polypeptides and modulate their activities. We are studying these proteins in the yeast Saccharomyces cerevisiae in order to understand their in vivo functions.One of these yeast proteins is Rbl2p. Identified in a search for proteins that, when overexpressed, rescue cells from the toxicity of free ␤-tubulin (5), Rbl2p binds monomeric ␤-tubulin to form a heterodimer that excludes ␣-tubulin, both in vivo and in vitro (5). Pulse-labeling experiments demonstrate that Rbl2p can bind both newly synthesized ␤-tubulin before it is incorporated into ␣/␤-tubulin heterodimers and ␤-tubulin released by dissociation of heterodimers (4). However, the precise function of Rbl2p in vivo is not known.Biochemical experiments with the vertebrate homolog of Rbl2p, cofactor A, suggest one possible function. Cofactor A was purified from extracts based on its activity in an in vitro tubulin-folding assay that monitors the exchange of tubulin polypeptides released from the cytosolic chaperonin Tri-C into preexisting ␣/␤-tubulin heterodimers (14, 30). Five cofactors facilitate this reaction. Three of them-cofactors C, D, and E-are necessary for the reaction. The functions of the other two-cofactors A and B-are a subset of the functions of cofactors D and E, respectively, and are not essential in the assay. However, their presence substantially stimulates the reaction (approximately fourfold for cofactor A [21]).These experiments also suggest a pathway for the exchange reaction between unfolded tubulin polypeptides and heterodimers. When ␤-tubulin polypeptides are released from the cytosolic chaperonin, they are able initially to bind either cofactor A or c...

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Toxic effects of excess cloned centromeres.

Cited by 86 publications

References 21 publications

Transcription interferes with elements important for chromosome maintenance in Saccharomyces cerevisiae.

Transcription interferes with elements important for chromosome maintenance in Saccharomyces cerevisiae.

Aneuploid proliferation defects in yeast are not driven by copy number changes of a few dosage-sensitive genes

Protection from Free β-Tubulin by the β-Tubulin Binding Protein Rbl2p

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