Colony size measurement of the yeast gene deletion strains for functional genomics

Memarian, Negar; Jessulat, Matthew; Alirezaie, Javad; Mir-Rashed, Nadereh; Xu, Jianhua; Zareie, Mehri; Smith, Myron L.; Golshani, Ashkan

doi:10.1186/1471-2105-8-117

“…YPD agar plates without (control), and with a subinhibitory concentration of eugenol (0.18 mg/ml, experimental), were inoculated by hand-pinning sets of 384 mutant strains per plate using a floating pin replicator as previously described [18]. After 1–2 days incubation at 30°C, digital images of the plates were captured and analyzed using Growth Detector software [19]. The relative size of colonies was used as a measure for growth differences under experimental and control conditions.…”

Section: Methodsmentioning

confidence: 99%

The Antifungal Eugenol Perturbs Dual Aromatic and Branched-Chain Amino Acid Permeases in the Cytoplasmic Membrane of Yeast

Darvishi

¹

,

Omidi

²

,

Bushehri

³

et al. 2013

Self Cite

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Eugenol is an aromatic component of clove oil that has therapeutic potential as an antifungal drug, although its mode of action and precise cellular target(s) remain ambiguous. To address this knowledge gap, a chemical-genetic profile analysis of eugenol was done using ∼4700 haploid Saccharomyces cerevisiae gene deletion mutants to reveal 21 deletion mutants with the greatest degree of susceptibility. Cellular roles of deleted genes in the most susceptible mutants indicate that the main targets for eugenol include pathways involved in biosynthesis and transport of aromatic and branched-chain amino acids. Follow-up analyses showed inhibitory effects of eugenol on amino acid permeases in the yeast cytoplasmic membrane. Furthermore, phenotypic suppression analysis revealed that eugenol interferes with two permeases, Tat1p and Gap1p, which are both involved in dual transport of aromatic and branched-chain amino acids through the yeast cytoplasmic membrane. Perturbation of cytoplasmic permeases represents a novel antifungal target and may explain previous observations that exposure to eugenol results in leakage of cell contents. Eugenol exposure may also contribute to amino acid starvation and thus holds promise as an anticancer therapeutic drug. Finally, this study provides further evidence of the usefulness of the yeast Gene Deletion Array approach in uncovering the mode of action of natural health products.

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“…S. cerevisiae strains used in this study are listed in Supplemental Material, Table S1. All strains have been previously described (Deutschbauer et al 2005; Memarian et al 2007). …”

Section: Methodsmentioning

confidence: 99%

“…Common cell spotting assays range from colony pinning assays, in which a pin is used to deliver a patch of cells onto the surface of solid agar media to serial dilution spotting analysis, wherein single colonies are obtained (Memarian et al 2007; Shah et al 2007; Lawless et al 2010). While these methods are universally accepted, there are significant caveats to their use.…”

mentioning

confidence: 99%

One-Cell Doubling Evaluation by Living Arrays of Yeast, ODELAY!

Herricks

¹

,

Dilworth

²

,

Mast

³

et al. 2017

G3 Genes|Genomes|Genetics

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Cell growth is a complex phenotype widely used in systems biology to gauge the impact of genetic and environmental perturbations. Due to the magnitude of genome-wide studies, resolution is often sacrificed in favor of throughput, creating a demand for scalable, time-resolved, quantitative methods of growth assessment. We present ODELAY (One-cell Doubling Evaluation by Living Arrays of Yeast), an automated and scalable growth analysis platform. High measurement density and single-cell resolution provide a powerful tool for large-scale multiparameter growth analysis based on the modeling of microcolony expansion on solid media. Pioneered in yeast but applicable to other colony forming organisms, ODELAY extracts the three key growth parameters (lag time, doubling time, and carrying capacity) that define microcolony expansion from single cells, simultaneously permitting the assessment of population heterogeneity. The utility of ODELAY is illustrated using yeast mutants, revealing a spectrum of phenotypes arising from single and combinatorial growth parameter perturbations.

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“…Common cell spotting assays range from colony pinning assays, in which a pin is used to deliver a patch of cells onto the surface of solid agar media to serial dilution spotting analysis, wherein single colonies are obtained (Memarian et al 2007;Shah et al 2007;Lawless et al 2010). While these methods are universally accepted, there are significant caveats to their use.…”

mentioning

confidence: 99%

One-cell Doubling Evaluation by Living Arrays of Yeast, ODELAY!

Herricks

¹

,

Dilworth

²

,

Mast

³

et al. 2016

Preprint

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Cell growth is a complex phenotype widely used in systems biology to gauge the impact of genetic and environmental perturbations. Due to the magnitude of genome-wide studies, resolution is often sacrificed in favor of throughput, creating a demand for scalable, time-resolved, quantitative methods of growth assessment. We present ODELAY (One-cell Doubling Evaluation by Living Arrays of Yeast), an automated and scalable growth analysis platform. High measurement density and single-cell resolution provide a powerful tool for large-scale multiparameter growth analysis based on the modeling of microcolony expansion on solid media. Pioneered in yeast but applicable to other colony forming organisms, ODELAY extracts the three key growth parameters (lag time, doubling time, and carrying capacity) that define microcolony expansion from single cells, simultaneously permitting the assessment of population heterogeneity. The utility of ODELAY is illustrated using yeast mutants, revealing a spectrum of phenotypes arising from single and combinatorial growth parameter perturbations.

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Colony size measurement of the yeast gene deletion strains for functional genomics

Cited by 66 publications

References 28 publications

The Antifungal Eugenol Perturbs Dual Aromatic and Branched-Chain Amino Acid Permeases in the Cytoplasmic Membrane of Yeast

The Antifungal Eugenol Perturbs Dual Aromatic and Branched-Chain Amino Acid Permeases in the Cytoplasmic Membrane of Yeast

One-Cell Doubling Evaluation by Living Arrays of Yeast, ODELAY!

One-cell Doubling Evaluation by Living Arrays of Yeast, ODELAY!

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