High-throughput and sensitive assay to measure yeast cell growth: a bench protocol for testing genotoxic agents

Toussaint, Martin; Conconi, Antonio

doi:10.1038/nprot.2006.304

Cited by 60 publications

(55 citation statements)

References 25 publications

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“…Growth of each treatment was measured by optical density (OD 600 ) every 15 min for 23 h using an automated microplate reader (for culture details see electronic supplementary material). We used the R package grofit [35] to estimate two parameters used to compare population growth rates: the length of the lag phase (l) and the cell doubling time [36].…”

Section: (D) Experimental Designmentioning

confidence: 99%

Phylogenetic relatedness predicts priority effects in nectar yeast communities

2011

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Priority effects, in which the outcome of species interactions depends on the order of their arrival, are a key component of many models of community assembly. Yet, much remains unknown about how priority effects vary in strength among species in a community and what factors explain this variation. We experimented with a model natural community in laboratory microcosms that allowed us to quantify the strength of priority effects for most of the yeast species found in the floral nectar of a hummingbirdpollinated shrub at a biological preserve in northern California. We found that priority effects were widespread, with late-arriving species experiencing strong negative effects from early-arriving species. However, the magnitude of priority effects varied across species pairs. This variation was phylogenetically non-random, with priority effects stronger between closer relatives. Analysis of carbon and amino acid consumption profiles indicated that competition between closer relatives was more intense owing to higher ecological similarity, consistent with Darwin's naturalization hypothesis. These results suggest that phylogenetic relatedness between potential colonists may explain the strength of priority effects and, as a consequence, the degree to which community assembly is historically contingent.

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Section: (D) Experimental Designmentioning

confidence: 99%

Phylogenetic relatedness predicts priority effects in nectar yeast communities

2011

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“…Haploid double mutants (yfegts-URA3 bud23Δ::KanMX) were then assayed for genetic interaction by comparing the growth of the double mutant with the parental single mutants. The doubling times and growth curves for each strain were generated as described (Toussaint and Conconi 2006). We scored neutral genetic interactions as having the fitness predicted by the product of the fitness of individual mutants.…”

Section: Genetic Interactions Of Bud23mentioning

confidence: 99%

“…For quantification of genetic interactions, cells were cultured in 100 µL of YPD containing 75 µg/mL Ampicillin in 96-well plates by shaking continuously for 36 h in a microplate reader (Powerwave; Biotek). The doubling times were calculated from growth curves as described (Toussaint and Conconi 2006). The fitness of each strain was calculated as the ratio of the doubling time of the wild type to that of the mutant (Dixon et al 2009).…”

Section: Construction Of Strains For the Genetic Screenmentioning

confidence: 99%

The rRNA methyltransferase Bud23 shows functional interaction with components of the SSU processome and RNase MRP

Sardana

White

Johnson

2013

RNA

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Bud23 is responsible for the conserved methylation of G1575 of 18S rRNA, in the P-site of the small subunit of the ribosome. bud23Δ mutants have severely reduced small subunit levels and show a general failure in cleavage at site A2 during rRNA processing. Site A2 is the primary cleavage site for separating the precursors of 18S and 25S rRNAs. Here, we have taken a genetic approach to identify the functional environment of BUD23. We found mutations in UTP2 and UTP14, encoding components of the SSU processome, as spontaneous suppressors of a bud23Δ mutant. The suppressors improved growth and subunit balance and restored cleavage at site A2. In a directed screen of 50 ribosomal trans-acting factors, we identified strong positive and negative genetic interactions with components of the SSU processome and strong negative interactions with components of RNase MRP. RNase MRP is responsible for cleavage at site A3 in pre-rRNA, an alternative cleavage site for separating the precursor rRNAs. The strong negative genetic interaction between RNase MRP mutants and bud23Δ is likely due to the combined defects in cleavage at A2 and A3. Our results suggest that Bud23 plays a role at the time of A2 cleavage, earlier than previously thought. The genetic interaction with the SSU processome suggests that Bud23 could be involved in triggering disassembly of the SSU processome, or of particular subcomplexes of the processome.

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“…For expediency, we screened compounds on 96-well plates using optical density as an index of growth recovery, as described for yeasts (27). Growth of Vpr-expressing yeast in the presence or absence of small molecules was measured spectrophotometrically and plotted as percentage growth (with the growth recovery of yeast in the presence of 1, our positive control in the screen, expressed as 100%).…”

Section: Resultsmentioning

confidence: 99%

Vipirinin, a Coumarin-based HIV-1 Vpr Inhibitor, Interacts with a Hydrophobic Region of VPR

Ong

Watanabe

Saito

et al. 2011

Journal of Biological Chemistry

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The human immunodeficiency virus 1 (HIV-1) viral protein R (Vpr) is an accessory protein that has been shown to have multiple roles in HIV-1 pathogenesis. By screening chemical libraries in the RIKEN Natural Products Depository, we identified a 3-phenyl coumarin-based compound that inhibited the cell cycle arrest activity of Vpr in yeast and Vpr-dependent viral infection of human macrophages. We determined its minimal pharmacophore through a structure-activity relationship study and produced more potent derivatives. We detected direct binding, and by assaying a panel of Vpr mutants, we found the hydrophobic region about residues Glu-25 and Gln-65 to be potentially involved in the binding of the inhibitor. Our findings exposed a targeting site on Vpr and delineated a convenient approach to explore other targeting sites on the protein using small molecule inhibitors as bioprobes.

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High-throughput and sensitive assay to measure yeast cell growth: a bench protocol for testing genotoxic agents

Cited by 60 publications

References 25 publications

Phylogenetic relatedness predicts priority effects in nectar yeast communities

Phylogenetic relatedness predicts priority effects in nectar yeast communities

The rRNA methyltransferase Bud23 shows functional interaction with components of the SSU processome and RNase MRP

Vipirinin, a Coumarin-based HIV-1 Vpr Inhibitor, Interacts with a Hydrophobic Region of VPR

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