Stochastic galactokinase expression underlies <scp>GAL</scp> gene induction in a <scp>GAL</scp>3 mutant of Saccharomyces cerevisiae

Kar, Rajesh Kumar; Qureshi, Mohd. Tanvir; DasAdhikari, Akshay Kumar; Zahir, Taiyeb; Venkatesh, K. V.; Bhat, Paike Jayadeva

doi:10.1111/febs.12741

Cited by 14 publications

(22 citation statements)

References 77 publications

(86 reference statements)

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“…In contrast, we observed a 7–18% survival rate on selection of the isogenic progeny ( Table 4 and Table S2 ). These results support the involvement of a nongenetic event in the galactose-tolerant phenotype of the two original euploid strains, possibly deriving from cell-to-cell differences in intracellular protein or transporter levels ( Acar et al 2005 ; Kar et al 2014 ). We further found that single cells from clonal populations of galactose-tolerant progeny also demonstrated low survival rates when grown on galactose medium, suggesting that this nongenetic event could affect mitotically dividing cells as well as germinating spores ( Table S2 ).…”

Section: Resultssupporting

confidence: 77%

Allelic Variation, Aneuploidy, and Nongenetic Mechanisms Suppress a Monogenic Trait in Yeast

Sirr¹,

Cromie²,

Jeffery³

et al. 2014

Genetics

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Clinically relevant features of monogenic diseases, including severity of symptoms and age of onset, can vary widely in response to environmental differences as well as to the presence of genetic modifiers affecting the trait’s penetrance and expressivity. While a better understanding of modifier loci could lead to treatments for Mendelian diseases, the rarity of individuals harboring both a disease-causing allele and a modifying genotype hinders their study in human populations. We examined the genetic architecture of monogenic trait modifiers using a well-characterized yeast model of the human Mendelian disease classic galactosemia. Yeast strains with loss-of-function mutations in the yeast ortholog (GAL7) of the human disease gene (GALT) fail to grow in the presence of even small amounts of galactose due to accumulation of the same toxic intermediates that poison human cells. To isolate and individually genotype large numbers of the very rare (∼0.1%) galactose-tolerant recombinant progeny from a cross between two gal7Δ parents, we developed a new method, called “FACS-QTL.” FACS-QTL improves upon the currently used approaches of bulk segregant analysis and extreme QTL mapping by requiring less genome engineering and strain manipulation as well as maintaining individual genotype information. Our results identified multiple distinct solutions by which the monogenic trait could be suppressed, including genetic and nongenetic mechanisms as well as frequent aneuploidy. Taken together, our results imply that the modifiers of monogenic traits are likely to be genetically complex and heterogeneous.

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Section: Resultssupporting

confidence: 77%

Allelic Variation, Aneuploidy, and Nongenetic Mechanisms Suppress a Monogenic Trait in Yeast

Sirr¹,

Cromie²,

Jeffery³

et al. 2014

Genetics

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“…In a wild type yeast strain (W303) bearing a GFP reporter gene expressed from the GAL1 promoter we find that ~80% of cells induce significant GFP expression within an hour of galactose addition, and greater than 95% become fully induced within 4 hours (Figure 1A). In contrast, consistent with previous observations (33), only a minor subset of gal3 yeast induce GFP expression even at 24 hours, and after 96 hours only ~10% of the cells induce significant expression (Figure 1B). Importantly however, individual gal3 cells are capable of inducing GFP expression comparable to levels similar to wild type.…”

Section: A Minor Subset Of Gal3 Yeast Induce Full Gal Expression In Rsupporting

confidence: 93%

TOR signaling modulates Cdk8-dependentGALgene expression inSaccharomyces cerevisiae

Hawe

Mestnikov

Horvath

et al. 2020

Preprint

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1Cdk8 of the RNA Polymerase II mediator complex regulates genes by 2 phosphorylating sequence specific transcription factors. Despite conserved importance 3 for eukaryotic transcriptional regulation, the signals regulating Cdk8 are unknown. Full 4 induction of the yeast GAL genes requires phosphorylation of Gal4 by Cdk8, and we 5 exploited this requirement for growth of gal3 yeast on galactose to identify mutants 6 affecting Cdk8 activity. Several mutants from the screen produced defects in TOR 7 signaling. A mutant designated gal four throttle (gft) 1, gft1, was identified as an allele of 8 hom3, encoding aspartokinase. Defects in gft1/ hom3 caused hypersensitivity to 9 rapamycin, and constitutive nuclear localization of Gat1. Furthermore, mutations of tor1 10 or tco89, encoding TORC1 components, also prevented GAL expression in gal3 yeast, 11and tco89 was determined to be allelic to gft7. Disruption of cdc55, encoding a subunit 12 of PP2A regulated by TOR signaling, suppressed the effect of gft1/ hom3, gft7/ tco89, 13 and tor1 mutations on GAL expression in gal3 yeast, but not of cdk8/ srb10 disruptions or 14 Gal4 S699A mutation. Mutations of gft1/ hom3 and tor1 did not affect kinase activity of 15Cdk8 in vitro, but caused loss of Gal4 phosphorylation in vivo. These observations 16 demonstrate that TOR signaling regulates GAL induction through the activity of PP2A/ 17 Cdc55, and are consistent with the contention that Cdk8-dependent phosphorylation of 18 Gal4 S699 is opposed by PP2A/ Cdc55 dephosphorylation. These results provide insight 19 into how induction of transcription by a specific inducer can be modulated by global 20 nutritional signals through regulation of Cdk8-dependent phosphorylation. 21 on EB-gal. Because less than 10% of otherwise wild type gal3 yeast produce a colony on 128 EB-gal (Figure 2), we were unable to implement a conventional synthetic genetic array 129 (SGA) screen using the non-essential gene deletion collection for this purpose. 130Consequently, we employed u.v.-irradiation of a gal3 W303 strain and replica plating, to 131 identify mutants that were incapable of growth on EB-gal, but which produced robust 132 colonies on media containing 3-carbon molecules as the sole carbon source. Initial 133 mutants recovered from this process were transformed with a plasmid bearing genomic 134 GAL3 and re-assayed for growth on EB-gal; mutants capable of growth when expressing 135Gal3 were designated the gal four throttle (gft) mutants, a collection which is comprised 136 of several groups with distinct phenotypes that will be detailed in a separate report. The 137

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“…In non-inducing conditions, GAL3 is basally expressed, whereas GAL1 transcription is repressed, with the result that Gal3p is the main galactose sensor. Importantly, in cells where Gal3p is absent or in cells with previous exposure to galactose, Gal1p becomes the primary sensor of galactose (Abramczyk et al, 2012; Kar et al, 2014). …”

Section: Resultsmentioning

confidence: 99%

“…Stains with a GAL3 deletion exhibit a partial penetrance phenotype in which growth on galactose is dependent on GAL1 leakage (Kar et al, 2014). We find that after GAL10 ncRNA inhibition, gal3 Δ strains grow faster on galactose containing plates (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…5E) due to increased GAL1 leakage. To quantify this observation, cells were grown on plates containing glucose and galactose + ethidium bromide, where growth on galactose reflects activation of the GAL pathway (Kar et al, 2014). In wt cells, CRISPRi of GAL10 ncRNA has no effect on the percentage of colonies formed on galactose plates, but in gal3 Δ cells the percentage increased from 0.56% ± 0.13 to 3.73% ± 0.34 colonies, a 6.7 fold increase (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Single-Molecule Imaging Reveals a Switch between Spurious and Functional ncRNA Transcription

et al. 2015

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Summary Eukaryotic transcription is pervasive, and many of the resulting RNAs are non-coding. It is unknown if ubiquitous transcription is functional or simply reflects stochastic transcriptional noise. By single-molecule visualization of the dynamic interplay between coding and non-coding transcription at the GAL locus in living yeast cells, we show that antisense GAL10 ncRNA transcription can switch between functional and spurious under different conditions. During galactose induction, GAL10 sense transcription occurs in short stochastic bursts which are unaffected by transcription of antisense GAL10 ncRNA, even when both are present simultaneously at the same locus. In contrast, when GAL10 is not induced, ncRNA transcription is critical to prevent transcriptional leakage of GAL1 and GAL10. Suppression of ncRNA transcription by strand-specific CRISPR/dCas9 results in transcriptional leakage of the inducer GAL1, leading to a more sensitive transcription activation threshold, an alteration of metabolic switching, and a fitness defect in competition experiments.

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Stochastic galactokinase expression underlies GAL gene induction in a GAL3 mutant of Saccharomyces cerevisiae

Cited by 14 publications

References 77 publications

Allelic Variation, Aneuploidy, and Nongenetic Mechanisms Suppress a Monogenic Trait in Yeast

Allelic Variation, Aneuploidy, and Nongenetic Mechanisms Suppress a Monogenic Trait in Yeast

TOR signaling modulates Cdk8-dependentGALgene expression inSaccharomyces cerevisiae

Single-Molecule Imaging Reveals a Switch between Spurious and Functional ncRNA Transcription

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