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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., (Copyright © 2017 Wong et al.)

Lichen-forming fungi and extracts derived from them have been used as alternative medicine sources for millennia and recently there has been a renewed interest in their known bioactive properties for anticancer agents, cosmetics and antibiotics. Although lichen-forming fungus-derived compounds are biologically and commercially valuable, few studies have been performed to determine their modes of action. This study used chemical-genetic and chemogenomic high-throughput analyses to gain insight into the modes of action of Caloplaca flavoruscens extracts. High-throughput screening of 575 lichen extracts was performed and 39 extracts were identified which inhibited yeast growth. A C. flavoruscens extract was selected as a promising antifungal and was subjected to genome-wide haploinsufficiency profiling and homozygous profiling assays. These screens revealed that yeast deletion strains lacking Rsc8, Pro1 and Toa2 were sensitive to three concentrations (IC25.5, IC25 and IC50, respectively) of C. flavoruscens extract. Gene-enrichment analysis of the data showed that C. flavoruscens extracts appear to perturb transcription and chromatin remodeling., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

While the pace of discovery of human genetic variants in tumors, patients, and diverse populations has rapidly accelerated, deciphering their functional consequence has become rate-limiting. Using cross-species complementation, model organisms like the budding yeast, Saccharomyces cerevisiae, can be utilized to fill this gap and serve as a platform for testing human genetic variants. To this end, we performed two parallel screens, a one-to-one complementation screen for essential yeast genes implicated in chromosome instability and a pool-to-pool screen that queried all possible essential yeast genes for rescue of lethality by all possible human homologs. Our work identified 65 human cDNAs that can replace the null allele of essential yeast genes, including the nonorthologous pair yRFT1/hSEC61A1. We chose four human cDNAs (hLIG1, hSSRP1, hPPP1CA, and hPPP1CC) for which their yeast gene counterparts function in chromosome stability and assayed in yeast 35 tumor-specific missense mutations for growth defects and sensitivity to DNA-damaging agents. This resulted in a set of human-yeast gene complementation pairs that allow human genetic variants to be readily characterized in yeast, and a prioritized list of somatic mutations that could contribute to chromosome instability in human tumors. These data establish the utility of this cross-species experimental approach., (Copyright © 2015 by the Genetics Society of America.)

Systematic analyses of essential gene function using mutant collections in Saccharomyces cerevisiae have been conducted using collections of heterozygous diploids, promoter shut-off alleles, through alleles with destabilized mRNA, destabilized protein, or bearing mutations that lead to a temperature-sensitive (ts) phenotype. We previously described a method for construction of barcoded ts alleles in a systematic fashion. Here we report the completion of this collection of alleles covering 600 essential yeast genes. This resource covers a larger gene repertoire than previous collections and provides a complementary set of strains suitable for single gene and genomic analyses. We use deep sequencing to characterize the amino acid changes leading to the ts phenotype in half of the alleles. We also use high-throughput approaches to describe the relative ts behavior of the alleles. Finally, we demonstrate the experimental usefulness of the collection in a high-content, functional genomic screen for ts alleles that increase spontaneous P-body formation. By increasing the number of alleles and improving the annotation, this ts collection will serve as a community resource for probing new aspects of biology for essential yeast genes., (Copyright © 2015 Kofoed et al.)