Precise Gene-Dose Alleles for Chemical Genetics

Yan, Zhun; Berbenetz, Nicolas M.; Giaever, Guri; Nislow, Corey

doi:10.1534/genetics.109.103036

Cited by 14 publications

(12 citation statements)

References 18 publications

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“…To further support this conclusion, we also found that the greatest degree of MTX sensitivity was achieved when two copies of wild-type SEC21 were expressed in cells ( Figure 5 ). These are consistent with SEC21 representing an indirect target of MTX that nonetheless can confer robust drug resistance in a gene dosage-dependent manner, similarly to DFR1 target ( Yan et al 2009 ).…”

Section: Resultssupporting

confidence: 82%

Genome-Wide Screen Reveals sec21 Mutants of Saccharomyces cerevisiae Are Methotrexate-Resistant

Wong

Flibotte

Sinha

et al. 2017

G3 Genes|Genomes|Genetics

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Drug resistance is a consequence of how most modern medicines work. Drugs exert pressure on cells that causes death or the evolution of resistance. Indeed, highly specific drugs are rendered ineffective by a single DNA mutation. In this study, we apply the drug methotrexate, which is widely used in cancer and rheumatoid arthritis, and perform evolution experiments on Baker’s yeast to ask the different ways in which cells become drug resistant. Because of the conserved nature of biological pathways between yeast and man, our results can inform how the same mechanism may operate to render human cells resistant to treatment. Exposure of cells to small molecules and drug therapies imposes a strong selective pressure. As a result, cells rapidly acquire mutations in order to survive. These include resistant variants of the drug target as well as those that modulate drug transport and detoxification. To systematically explore how cells acquire drug resistance in an unbiased manner, rapid cost-effective approaches are required. Methotrexate, as one of the first rationally designed anticancer drugs, has served as a prototypic example of such acquired resistance. Known methotrexate resistance mechanisms include mutations that increase expression of the dihydrofolate reductase (DHFR) target as well as those that maintain function yet reduce the drug’s binding affinity. Recent evidence suggests that target-independent, epistatic mutations can also result in resistance to methotrexate. Currently, however, the relative contribution of such unlinked resistance mutations is not well understood. To address this issue, we took advantage of Saccharomyces cerevisiae as a model eukaryotic system that combined with whole-genome sequencing and a rapid screening methodology, allowed the identification of causative mutations that modulate resistance to methotrexate. We found a recurrent missense mutation in SEC21 (orthologous to human COPG1), which we confirmed in 10 de novo methotrexate-resistant strains. This sec21 allele (S96L) behaves as a recessive, gain-of-function allele, conferring methotrexate resistance that is abrogated by the presence of a wild-type copy of SEC21. These observations indicate that the Sec21p/COPI transport complex has previously uncharacterized roles in modulating methotrexate stress.

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Section: Resultssupporting

confidence: 82%

Genome-Wide Screen Reveals sec21 Mutants of Saccharomyces cerevisiae Are Methotrexate-Resistant

Wong

Flibotte

Sinha

et al. 2017

G3 Genes|Genomes|Genetics

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“…A more inclusive collection of bar-coded yeast strains containing both essential and nonessential genes with decreased abundance of mRNA perturbation (DAmP) is also available to study chemical genetics [ 66 ]. Using this approach, it was shown that the protein product of IPP1 genes, a cytoplasmic inorganic pyrophosphatase is specifically inhibited by NaF [ 67 ].…”

Section: Discussionmentioning

confidence: 99%

Lithium Chloride Sensitivity in Yeast and Regulation of Translation

Hajikarimlou

Hunt

Kirby

et al. 2020

IJMS

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For decades, lithium chloride (LiCl) has been used as a treatment option for those living with bipolar disorder (BD). As a result, many studies have been conducted to examine its mode of action, toxicity, and downstream cellular responses. We know that LiCl is able to affect cell signaling and signaling transduction pathways through protein kinase C and glycogen synthase kinase-3, which are considered to be important in regulating gene expression at the translational level. However, additional downstream effects require further investigation, especially in translation pathway. In yeast, LiCl treatment affects the expression, and thus the activity, of PGM2, a phosphoglucomutase involved in sugar metabolism. Inhibition of PGM2 leads to the accumulation of intermediate metabolites of galactose metabolism causing cell toxicity. However, it is not fully understood how LiCl affects gene expression in this matter. In this study, we identified three genes, NAM7, PUS2, and RPL27B, which increase yeast LiCl sensitivity when deleted. We further demonstrate that NAM7, PUS2, and RPL27B influence translation and exert their activity through the 5′-Untranslated region (5′-UTR) of PGM2 mRNA in yeast.

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“…Use of heterologous constructions where one could adjust the promoter strength would provide the most conclusive data. A possible strategy would be to build upon a recently published protocol [54] which combines diverse loss-of-function alleles, to systematically modulate gene dosage in budding yeast. The use of this protocol on four enzyme-coding genes supports that [0 0 0] is a default sign motif [54] .…”

Section: Discussionmentioning

confidence: 99%

Allele Interaction – Single Locus Genetics Meets Regulatory Biology

et al. 2010

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BackgroundSince the dawn of genetics, additive and dominant gene action in diploids have been defined by comparison of heterozygote and homozygote phenotypes. However, these definitions provide little insight into the underlying intralocus allelic functional dependency and thus cannot serve directly as a mediator between genetics theory and regulatory biology, a link that is sorely needed.Methodology/Principal FindingsWe provide such a link by distinguishing between positive, negative and zero allele interaction at the genotype level. First, these distinctions disclose that a biallelic locus can display 18 qualitatively different allele interaction sign motifs (triplets of +, – and 0). Second, we show that for a single locus, Mendelian dominance is not related to heterozygote allele interaction alone, but is actually a function of the degrees of allele interaction in all the three genotypes. Third, we demonstrate how the allele interaction in each genotype is directly quantifiable in gene regulatory models, and that there is a unique, one-to-one correspondence between the sign of autoregulatory feedback loops and the sign of the allele interactions.Conclusion/SignificanceThe concept of allele interaction refines single locus genetics substantially, and it provides a direct link between classical models of gene action and gene regulatory biology. Together with available empirical data, our results indicate that allele interaction can be exploited experimentally to identify and explain intricate intra- and inter-locus feedback relationships in eukaryotes.

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Precise Gene-Dose Alleles for Chemical Genetics

Cited by 14 publications

References 18 publications

Genome-Wide Screen Reveals sec21 Mutants of Saccharomyces cerevisiae Are Methotrexate-Resistant

Genome-Wide Screen Reveals sec21 Mutants of Saccharomyces cerevisiae Are Methotrexate-Resistant

Lithium Chloride Sensitivity in Yeast and Regulation of Translation

Allele Interaction – Single Locus Genetics Meets Regulatory Biology

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