A polyploid admixed origin of beer yeasts derived from European and Asian wine populations.

Strains of Saccharomyces cerevisiae used to make beer, bread, and wine are genetically and phenotypically distinct from wild populations associated with trees. The origins of these domesticated populations are not always clear; human-associated migration and admixture with wild populations have had a strong impact on S. cerevisiae population structure. We examined the population genetic history of beer strains and found that ale strains and the S. cerevisiae portion of allotetraploid lager strains were derived from admixture between populations closely related to European grape wine strains and Asian rice wine strains. Similar to both lager and baking strains, ale strains are polyploid, providing them with a passive means of remaining isolated from other populations and providing us with a living relic of their ancestral hybridization. To reconstruct their polyploid origin, we phased the genomes of two ale strains and found ale haplotypes to both be recombinants between European and Asian alleles and to also contain novel alleles derived from extinct or as yet uncharacterized populations. We conclude that modern beer strains are the product of a historical melting pot of fermentation technology.

Natural Variation in SER1 and ENA6 Underlie Condition-Specific Growth Defects in Saccharomyces cerevisiae.

Despite their ubiquitous use in laboratory strains, naturally occurring loss-of-function mutations in genes encoding core metabolic enzymes are relatively rare in wild isolates of Saccharomyces cerevisiae Here, we identify a naturally occurring serine auxotrophy in a sake brewing strain from Japan. Through a cross with a honey wine (white tecc) brewing strain from Ethiopia, we map the minimal medium growth defect to SER1, which encodes 3-phosphoserine aminotransferase and is orthologous to the human disease gene, PSAT1 To investigate the impact of this polymorphism under conditions of abundant external nutrients, we examine growth in rich medium alone or with additional stresses, including the drugs caffeine and rapamycin and relatively high concentrations of copper, salt, and ethanol. Consistent with studies that found widespread effects of different auxotrophies on RNA expression patterns in rich media, we find that the SER1 loss-of-function allele dominates the quantitative trait locus (QTL) landscape under many of these conditions, with a notable exacerbation of the effect in the presence of rapamycin and caffeine. We also identify a major-effect QTL associated with growth on salt that maps to the gene encoding the sodium exporter, ENA6 We demonstrate that the salt phenotype is largely driven by variation in the ENA6 promoter, which harbors a deletion that removes binding sites for the Mig1 and Nrg1 transcriptional repressors. Thus, our results identify natural variation associated with both coding and regulatory regions of the genome that underlie strong growth phenotypes.

Independent Origins of Yeast Associated with Coffee and Cacao Fermentation.

Modern transportation networks have facilitated the migration and mingling of previously isolated populations of plants, animals, and insects. Human activities can also influence the global distribution of microorganisms. The best-understood example is yeasts associated with winemaking. Humans began making wine in the Middle East over 9,000 years ago [1, 2]. Selecting favorable fermentation products created specialized strains of Saccharomyces cerevisiae [3, 4] that were transported along with grapevines. Today, S. cerevisiae strains residing in vineyards around the world are genetically similar, and their population structure suggests a common origin that followed the path of human migration [3-7]. Like wine, coffee and cacao depend on microbial fermentation [8, 9] and have been globally dispersed by humans. Theobroma cacao originated in the Amazon and Orinoco basins of Colombia and Venezuela [10], was cultivated in Central America by Mesoamerican peoples, and was introduced to Europeans by Hernán Cortés in 1530 [11]. Coffea, native to Ethiopia, was disseminated by Arab traders throughout the Middle East and North Africa in the 6(th) century and was introduced to European consumers in the 17(th) century [12]. Here, we tested whether the yeasts associated with coffee and cacao are genetically similar, crop-specific populations or genetically diverse, geography-specific populations. Our results uncovered populations that, while defined by niche and geography, also bear signatures of admixture between major populations in events independent of the transport of the plants. Thus, human-associated fermentation and migration may have affected the distribution of yeast involved in the production of coffee and chocolate.

Allelic variation, aneuploidy, and nongenetic mechanisms suppress a monogenic trait in yeast.

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.

BEST: barcode enabled sequencing of tetrads.

Tetrad analysis is a valuable tool for yeast genetics, but the laborious manual nature of the process has hindered its application on large scales. Barcode Enabled Sequencing of Tetrads (BEST)1 replaces the manual processes of isolating, disrupting and spacing tetrads. BEST isolates tetrads by virtue of a sporulation-specific GFP fusion protein that permits fluorescence-activated cell sorting of tetrads directly onto agar plates, where the ascus is enzymatically digested and the spores are disrupted and randomly arrayed by glass bead plating. The haploid colonies are then assigned sister spore relationships, i.e. information about which spores originated from the same tetrad, using molecular barcodes read during genotyping. By removing the bottleneck of manual dissection, hundreds or even thousands of tetrads can be isolated in minutes. Here we present a detailed description of the experimental procedures required to perform BEST in the yeast Saccharomyces cerevisiae, starting with a heterozygous diploid strain through the isolation of colonies derived from the haploid meiotic progeny.

Identification and characterization of a drug-sensitive strain enables puromycin-based translational assays in Saccharomyces cerevisiae.

Puromycin is an aminonucleoside antibiotic with structural similarity to aminoacyl tRNA. This structure allows the drug to bind the ribosomal A site and incorporate into nascent polypeptides, causing chain termination, ribosomal subunit dissociation and widespread translational arrest at high concentrations. In contrast, at sufficiently low concentrations, puromycin incorporates primarily at the C-terminus of proteins. While a number of techniques utilize puromycin incorporation as a tool for probing translational activity in vivo, these methods cannot be applied in yeasts that are insensitive to puromycin. Here, we describe a mutant strain of the yeast Saccharomyces cerevisiae that is sensitive to puromycin and characterize the cellular response to the drug. Puromycin inhibits the growth of yeast cells mutant for erg6∆, pdr1∆ and pdr3∆ (EPP) on both solid and liquid media. Puromycin also induces the aggregation of the cytoplasmic processing body component Edc3 in the mutant strain. We establish that puromycin is rapidly incorporated into yeast proteins and test the effects of puromycin on translation in vivo. This study establishes the EPP strain as a valuable tool for implementing puromycin-based assays in yeast, which will enable new avenues of inquiry into protein production and maturation.

Genomic sequence diversity and population structure of Saccharomyces cerevisiae assessed by RAD-seq.

The budding yeast Saccharomyces cerevisiae is important for human food production and as a model organism for biological research. The genetic diversity contained in the global population of yeast strains represents a valuable resource for a number of fields, including genetics, bioengineering, and studies of evolution and population structure. Here, we apply a multiplexed, reduced genome sequencing strategy (restriction site-associated sequencing or RAD-seq) to genotype a large collection of S. cerevisiae strains isolated from a wide range of geographical locations and environmental niches. The method permits the sequencing of the same 1% of all genomes, producing a multiple sequence alignment of 116,880 bases across 262 strains. We find diversity among these strains is principally organized by geography, with European, North American, Asian, and African/S. E. Asian populations defining the major axes of genetic variation. At a finer scale, small groups of strains from cacao, olives, and sake are defined by unique variants not present in other strains. One population, containing strains from a variety of fermentations, exhibits high levels of heterozygosity and a mixture of alleles from European and Asian populations, indicating an admixed origin for this group. We propose a model of geographic differentiation followed by human-associated admixture, primarily between European and Asian populations and more recently between European and North American populations. The large collection of genotyped yeast strains characterized here will provide a useful resource for the broad community of yeast researchers.

High-throughput tetrad analysis.

Tetrad analysis has been a gold-standard genetic technique for several decades. Unfortunately, the need to manually isolate, disrupt and space tetrads has relegated its application to small-scale studies and limited its integration with high-throughput DNA sequencing technologies. We have developed a rapid, high-throughput method, called barcode-enabled sequencing of tetrads (BEST), that uses (i) a meiosis-specific GFP fusion protein to isolate tetrads by FACS and (ii) molecular barcodes that are read during genotyping to identify spores derived from the same tetrad. Maintaining tetrad information allows accurate inference of missing genetic markers and full genotypes of missing (and presumably nonviable) individuals. An individual researcher was able to isolate over 3,000 yeast tetrads in 3 h, an output equivalent to that of almost 1 month of manual dissection. BEST is transferable to other microorganisms for which meiotic mapping is significantly more laborious.

Cell-based assays for identification of novel double-strand break-inducing agents.

BACKGROUND: We are developing cell-based assays to identify anticancer agents that are selectively toxic to cells with defined mutations. As a test, we used a three-stage strategy to screen compounds from the National Cancer Institute's repository for agents that are selectively toxic to double-strand break repair-deficient yeast cells. METHODS: Compounds identified in the screen were further analyzed by use of yeast and vertebrate cell-based and in vitroassays to distinguish between topoisomerase I and II poisons. RESULTS: Of the more than 85 000 compounds screened, 126 were selectively toxic to yeast deficient in DNA double-strand break repair. Eighty-seven of these 126 compounds were structurally related to known topoisomerase poisons, and 39 were not. Twenty-eight of the 39 were characterized, and we present data for eight of the compounds. Among these eight compounds, we identified two novel topoisomerase II poisons (NSC 327929 and NSC 638432) that were equipotent to etoposide in biochemical tests and in cells, five (NSC 63599, NSC 65601, NSC 380271, NSC 651646, and NSC 668370) with topoisomerase I-dependent toxicity in yeast that induced DNA damage and toxicity in mammalian cells, and one (NSC 610898) that directly bound to DNA and induced strand breaks. CONCLUSIONS: Cell-based assays can be used to identify molecules that are selectively toxic to cells with a predetermined genetic background, including mutations in genes involved in the cell cycle and its checkpoints, for which there are currently no selectively toxic compounds.