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1.  Recurrent Rearrangement during Adaptive Evolution in an Interspecific Yeast Hybrid Suggests a Model for Rapid Introgression 
PLoS Genetics  2013;9(3):e1003366.
Genome rearrangements are associated with eukaryotic evolutionary processes ranging from tumorigenesis to speciation. Rearrangements are especially common following interspecific hybridization, and some of these could be expected to have strong selective value. To test this expectation we created de novo interspecific yeast hybrids between two diverged but largely syntenic Saccharomyces species, S. cerevisiae and S. uvarum, then experimentally evolved them under continuous ammonium limitation. We discovered that a characteristic interspecific genome rearrangement arose multiple times in independently evolved populations. We uncovered nine different breakpoints, all occurring in a narrow ∼1-kb region of chromosome 14, and all producing an “interspecific fusion junction” within the MEP2 gene coding sequence, such that the 5′ portion derives from S. cerevisiae and the 3′ portion derives from S. uvarum. In most cases the rearrangements altered both chromosomes, resulting in what can be considered to be an introgression of a several-kb region of S. uvarum into an otherwise intact S. cerevisiae chromosome 14, while the homeologous S. uvarum chromosome 14 experienced an interspecific reciprocal translocation at the same breakpoint within MEP2, yielding a chimaeric chromosome; these events result in the presence in the cell of two MEP2 fusion genes having identical breakpoints. Given that MEP2 encodes for a high-affinity ammonium permease, that MEP2 fusion genes arise repeatedly under ammonium-limitation, and that three independent evolved isolates carrying MEP2 fusion genes are each more fit than their common ancestor, the novel MEP2 fusion genes are very likely adaptive under ammonium limitation. Our results suggest that, when homoploid hybrids form, the admixture of two genomes enables swift and otherwise unavailable evolutionary innovations. Furthermore, the architecture of the MEP2 rearrangement suggests a model for rapid introgression, a phenomenon seen in numerous eukaryotic phyla, that does not require repeated backcrossing to one of the parental species.
Author Summary
Interspecific hybridization occurs when two different species mate and produce viable offspring. While hybrid offspring are usually sterile, like the mule, which results from a horse–donkey mating, sometimes they are fertile, creating new species. Indeed, many plant and animal species have arisen via this mechanism. Because interspecific hybridization occurs between different yeast species, and because they are such tractable models, yeast are ideally suited for experimentally investigating the genomic consequences of interspecific hybridization. We created an interspecific yeast hybrid by crossing S. cerevisiae and S. uvarum, and then studied genomic changes that occurred as it adaptively evolved in a stressful nitrogen-limiting environment. We discovered that a characteristic rearrangement between the parental species' chromosomes evolved independently many times, and always within a particular gene encoding a protein that imports nitrogen into the cell. Evolved hybrids carrying this rearrangement grew faster under nitrogen-limitation than ancestral hybrids, suggesting that the rearrangement is beneficial in nitrogen-poor environments. Our results suggest that having the genomes of two different species within a cell provides novel sources of variation for evolution to act upon, leading to adaptations that could not occur in either parental species.
doi:10.1371/journal.pgen.1003366
PMCID: PMC3605161  PMID: 23555283
2.  Combining in silico prediction and ribosome profiling in a genome-wide search for novel putatively coding sORFs 
BMC Genomics  2013;14:648.
Background
It was long assumed that proteins are at least 100 amino acids (AAs) long. Moreover, the detection of short translation products (e.g. coded from small Open Reading Frames, sORFs) is very difficult as the short length makes it hard to distinguish true coding ORFs from ORFs occurring by chance. Nevertheless, over the past few years many such non-canonical genes (with ORFs < 100 AAs) have been discovered in different organisms like Arabidopsis thaliana, Saccharomyces cerevisiae, and Drosophila melanogaster. Thanks to advances in sequencing, bioinformatics and computing power, it is now possible to scan the genome in unprecedented scrutiny, for example in a search of this type of small ORFs.
Results
Using bioinformatics methods, we performed a systematic search for putatively functional sORFs in the Mus musculus genome. A genome-wide scan detected all sORFs which were subsequently analyzed for their coding potential, based on evolutionary conservation at the AA level, and ranked using a Support Vector Machine (SVM) learning model. The ranked sORFs are finally overlapped with ribosome profiling data, hinting to sORF translation. All candidates are visually inspected using an in-house developed genome browser. In this way dozens of highly conserved sORFs, targeted by ribosomes were identified in the mouse genome, putatively encoding micropeptides.
Conclusion
Our combined genome-wide approach leads to the prediction of a comprehensive but manageable set of putatively coding sORFs, a very important first step towards the identification of a new class of bioactive peptides, called micropeptides.
doi:10.1186/1471-2164-14-648
PMCID: PMC3852105  PMID: 24059539
Micropeptide; Small open reading frame; Mus musculus; Genome-wide; Ribosome profiling; LincRNA; sORF; ncRNA; Bioactive peptide
3.  Systematic discovery of regulatory motifs in Fusarium graminearum by comparing four Fusarium genomes 
BMC Genomics  2010;11:208.
Background
Fusarium graminearum (Fg), a major fungal pathogen of cultivated cereals, is responsible for billions of dollars in agriculture losses. There is a growing interest in understanding the transcriptional regulation of this organism, especially the regulation of genes underlying its pathogenicity. The generation of whole genome sequence assemblies for Fg and three closely related Fusarium species provides a unique opportunity for such a study.
Results
Applying comparative genomics approaches, we developed a computational pipeline to systematically discover evolutionarily conserved regulatory motifs in the promoter, downstream and the intronic regions of Fg genes, based on the multiple alignments of sequenced Fusarium genomes. Using this method, we discovered 73 candidate regulatory motifs in the promoter regions. Nearly 30% of these motifs are highly enriched in promoter regions of Fg genes that are associated with a specific functional category. Through comparison to Saccharomyces cerevisiae (Sc) and Schizosaccharomyces pombe (Sp), we observed conservation of transcription factors (TFs), their binding sites and the target genes regulated by these TFs related to pathways known to respond to stress conditions or phosphate metabolism. In addition, this study revealed 69 and 39 conserved motifs in the downstream regions and the intronic regions, respectively, of Fg genes. The top intronic motif is the splice donor site. For the downstream regions, we noticed an intriguing absence of the mammalian and Sc poly-adenylation signals among the list of conserved motifs.
Conclusion
This study provides the first comprehensive list of candidate regulatory motifs in Fg, and underscores the power of comparative genomics in revealing functional elements among related genomes. The conservation of regulatory pathways among the Fusarium genomes and the two yeast species reveals their functional significance, and provides new insights in their evolutionary importance among Ascomycete fungi.
doi:10.1186/1471-2164-11-208
PMCID: PMC2853525  PMID: 20346147
4.  Molecular analysis of the Trichosporon cutaneum DSM 70698 argA gene and its use for DNA-mediated transformations. 
Journal of Bacteriology  1994;176(10):3021-3032.
Genomic clones capable of complementing a previously isolated arginine auxotrophic mutant strain of the filamentous yeast Trichosporon cutaneum DSM 70698 have been identified by DNA-mediated transformation, and a complementing 4,082-bp subfragment was sequenced. This analysis revealed an intact gene (arg4) showing a high degree of homology with the Saccharomyces cerevisiae CPA2 gene encoding the large subunit of carbamoyl-phosphate synthetase (CPS-A). The inferred amino acid sequence of the T. cutaneum argA-encoded protein contains 1,168 residues showing 62% identity with the sequence of the S. cerevisiae CPA2 protein, and the comparison of the two sequences uncovered a putative intron sequence of 81 nucleotides close to the 5' end of the coding region of the T. cutaneum argA gene. The presence of this intron was confirmed by nuclease protection studies and by direct DNA sequence analysis of a cDNA fragment which had been obtained by PCR amplification. The T. cutaneum intron shares the general characteristics of introns found in yeasts and filamentous fungi. A major transcript of around 4 kb was found in Northern (RNA) blots. The T. cutaneum argA coding region was expressed in Escherichia coli under the control of the regulatable tac promoter. A roughly 130-kDa protein which was found to cross-react with an anti-rat CPS antibody in Western blots (immunoblots) was observed. Two putative ATP-binding domains were identified, one in the amino-terminal half of the argA-encoded protein and the other in the carboxy-terminal half. These domains are highly conserved among the known CPS-A sequences from S. cerevisiae, E. coli, and the rat. From these results we conclude that the T. cutaneum argA gene encodes the large subunit of CPS. This is the first gene to be identified and analyzed in the T. cutaneum DSM 70698 strain.
Images
PMCID: PMC205460  PMID: 8188603
5.  Global Mapping of Transposon Location 
PLoS Genetics  2006;2(12):e212.
Transposable genetic elements are ubiquitous, yet their presence or absence at any given position within a genome can vary between individual cells, tissues, or strains. Transposable elements have profound impacts on host genomes by altering gene expression, assisting in genomic rearrangements, causing insertional mutations, and serving as sources of phenotypic variation. Characterizing a genome's full complement of transposons requires whole genome sequencing, precluding simple studies of the impact of transposition on interindividual variation. Here, we describe a global mapping approach for identifying transposon locations in any genome, using a combination of transposon-specific DNA extraction and microarray-based comparative hybridization analysis. We use this approach to map the repertoire of endogenous transposons in different laboratory strains of Saccharomyces cerevisiae and demonstrate that transposons are a source of extensive genomic variation. We also apply this method to mapping bacterial transposon insertion sites in a yeast genomic library. This unique whole genome view of transposon location will facilitate our exploration of transposon dynamics, as well as defining bases for individual differences and adaptive potential.
Synopsis
Transposons, or mobile DNA sequences—first described by Barbara McClintock—are interesting and important residents of all genomes. They are involved in gene creation and regulation, chromosome evolution, and generation of mutations, events that can occur on hugely varying time scales, from millions of years to mere days in the lab. Some transposons have even been “tamed” by geneticists for use as tools for marking genes and making mutations. In yeast, genome sequencing has given us a snapshot of transposons present in one strain at one particular time. The authors developed a method to easily, accurately, and globally track transposons in order to study how their locations change in different strains or during an experiment. The method involves finding pieces of DNA that contain the ends of transposons along with neighboring DNA and attaching these segments to magnetic beads. A magnet is then used to separate the selected DNAs away from the rest of the genome. The transposon-associated DNA is labeled with dyes and applied to a microarray, a glass slide with over 40,000 unique sequence features of yeast DNA attached. Each feature that lights up with the dye marks a transposon location. This new technique allows investigators to easily identify specific strains, to accurately monitor mobile portions of the genome, and to determine the role of transposons in phenotypic differences.
doi:10.1371/journal.pgen.0020212
PMCID: PMC1698948  PMID: 17173485
6.  Global Mapping of Transposon Location 
PLoS Genetics  2006;2(12):e212.
Transposable genetic elements are ubiquitous, yet their presence or absence at any given position within a genome can vary between individual cells, tissues, or strains. Transposable elements have profound impacts on host genomes by altering gene expression, assisting in genomic rearrangements, causing insertional mutations, and serving as sources of phenotypic variation. Characterizing a genome's full complement of transposons requires whole genome sequencing, precluding simple studies of the impact of transposition on interindividual variation. Here, we describe a global mapping approach for identifying transposon locations in any genome, using a combination of transposon-specific DNA extraction and microarray-based comparative hybridization analysis. We use this approach to map the repertoire of endogenous transposons in different laboratory strains of Saccharomyces cerevisiae and demonstrate that transposons are a source of extensive genomic variation. We also apply this method to mapping bacterial transposon insertion sites in a yeast genomic library. This unique whole genome view of transposon location will facilitate our exploration of transposon dynamics, as well as defining bases for individual differences and adaptive potential.
Synopsis
Transposons, or mobile DNA sequences—first described by Barbara McClintock—are interesting and important residents of all genomes. They are involved in gene creation and regulation, chromosome evolution, and generation of mutations, events that can occur on hugely varying time scales, from millions of years to mere days in the lab. Some transposons have even been “tamed” by geneticists for use as tools for marking genes and making mutations. In yeast, genome sequencing has given us a snapshot of transposons present in one strain at one particular time. The authors developed a method to easily, accurately, and globally track transposons in order to study how their locations change in different strains or during an experiment. The method involves finding pieces of DNA that contain the ends of transposons along with neighboring DNA and attaching these segments to magnetic beads. A magnet is then used to separate the selected DNAs away from the rest of the genome. The transposon-associated DNA is labeled with dyes and applied to a microarray, a glass slide with over 40,000 unique sequence features of yeast DNA attached. Each feature that lights up with the dye marks a transposon location. This new technique allows investigators to easily identify specific strains, to accurately monitor mobile portions of the genome, and to determine the role of transposons in phenotypic differences.
doi:10.1371/journal.pgen.0020212
PMCID: PMC1698948  PMID: 17173485
7.  Whole-Genome Comparison Reveals Novel Genetic Elements That Characterize the Genome of Industrial Strains of Saccharomyces cerevisiae 
PLoS Genetics  2011;7(2):e1001287.
Human intervention has subjected the yeast Saccharomyces cerevisiae to multiple rounds of independent domestication and thousands of generations of artificial selection. As a result, this species comprises a genetically diverse collection of natural isolates as well as domesticated strains that are used in specific industrial applications. However the scope of genetic diversity that was captured during the domesticated evolution of the industrial representatives of this important organism remains to be determined. To begin to address this, we have produced whole-genome assemblies of six commercial strains of S. cerevisiae (four wine and two brewing strains). These represent the first genome assemblies produced from S. cerevisiae strains in their industrially-used forms and the first high-quality assemblies for S. cerevisiae strains used in brewing. By comparing these sequences to six existing high-coverage S. cerevisiae genome assemblies, clear signatures were found that defined each industrial class of yeast. This genetic variation was comprised of both single nucleotide polymorphisms and large-scale insertions and deletions, with the latter often being associated with ORF heterogeneity between strains. This included the discovery of more than twenty probable genes that had not been identified previously in the S. cerevisiae genome. Comparison of this large number of S. cerevisiae strains also enabled the characterization of a cluster of five ORFs that have integrated into the genomes of the wine and bioethanol strains on multiple occasions and at diverse genomic locations via what appears to involve the resolution of a circular DNA intermediate. This work suggests that, despite the scrutiny that has been directed at the yeast genome, there remains a significant reservoir of ORFs and novel modes of genetic transmission that may have significant phenotypic impact in this important model and industrial species.
Author Summary
The yeast S. cerevisiae has been associated with human activity for thousands of years in industries such as baking, brewing, and winemaking. During this time, humans have effectively domesticated this microorganism, with different industries selecting for specific desirable phenotypic traits. This has resulted in the species S. cerevisiae comprising a genetically diverse collection of individual strains that are often suited to very specific roles (e.g. wine strains produce wine but not beer and vice versa). In order to understand the genetic differences that underpin these diverse industrial characteristics, we have sequenced the genomes of six industrial strains of S. cerevisiae that comprise four strains used in commercial wine production and two strains used in beer brewing. By comparing these genome sequences to existing S. cerevisiae genome sequences from laboratory, pathogenic, bioethanol, and “natural” isolates, we were able to identify numerous genetic differences among these strains including the presence of novel open reading frames and genomic rearrangements, which may provide the basis for the phenotypic differences observed among these strains.
doi:10.1371/journal.pgen.1001287
PMCID: PMC3033381  PMID: 21304888
8.  The new modern era of yeast genomics: community sequencing and the resulting annotation of multiple Saccharomyces cerevisiae strains at the Saccharomyces Genome Database 
The first completed eukaryotic genome sequence was that of the yeast Saccharomyces cerevisiae, and the Saccharomyces Genome Database (SGD; http://www.yeastgenome.org/) is the original model organism database. SGD remains the authoritative community resource for the S. cerevisiae reference genome sequence and its annotation, and continues to provide comprehensive biological information correlated with S. cerevisiae genes and their products. A diverse set of yeast strains have been sequenced to explore commercial and laboratory applications, and a brief history of those strains is provided. The publication of these new genomes has motivated the creation of new tools, and SGD will annotate and provide comparative analyses of these sequences, correlating changes with variations in strain phenotypes and protein function. We are entering a new era at SGD, as we incorporate these new sequences and make them accessible to the scientific community, all in an effort to continue in our mission of educating researchers and facilitating discovery.
Database URL: http://www.yeastgenome.org/
doi:10.1093/database/bat012
PMCID: PMC3595989  PMID: 23487186
9.  Yeast genome analysis identifies chromosomal translocation, gene conversion events and several sites of Ty element insertion 
Nucleic Acids Research  2009;37(19):6454-6465.
Paired end mapping of chromosomal fragments has been used in human cells to identify numerous structural variations in chromosomes of individuals and of cancer cell lines; however, the molecular, biological and bioinformatics methods for this technology are still in development. Here, we present a parallel bioinformatics approach to analyze chromosomal paired-end tag (ChromPET) sequence data and demonstrate its application in identifying gene rearrangements in the model organism Saccharomyces cerevisiae. We detected several expected events, including a chromosomal rearrangement of the nonessential arm of chromosome V induced by selective pressure, rearrangements introduced during strain construction and gene conversion at the MAT locus. In addition, we discovered several unannotated Ty element insertions that are present in the reference yeast strain, but not in the reference genome sequence, suggesting a few revisions are necessary in the latter. These data demonstrate that application of the chromPET technique to a genetically tractable organism like yeast provides an easy screen for studying the mechanisms of chromosomal rearrangements during the propagation of a species.
doi:10.1093/nar/gkp650
PMCID: PMC2770650  PMID: 19710036
10.  Genome sequencing and genetic breeding of a bioethanol Saccharomyces cerevisiae strain YJS329 
BMC Genomics  2012;13:479.
Background
Environmental stresses and inhibitors encountered by Saccharomyces cerevisiae strains are the main limiting factors in bioethanol fermentation. Strains with different genetic backgrounds usually show diverse stress tolerance responses. An understanding of the mechanisms underlying these phenotypic diversities within S. cerevisiae populations could guide the construction of strains with desired traits.
Results
We explored the genetic characteristics of the bioethanol S. cerevisiae strain YJS329 and elucidated how genetic variations in its genome were correlated with specified traits compared to similar traits in the S288c-derived strain, BYZ1. Karyotypic electrophoresis combined with array-comparative genomic hybridization indicated that YJS329 was a diploid strain with a relatively constant genome as a result of the fewer Ty elements and lack of structural polymorphisms between homologous chromosomes that it contained. By comparing the sequence with the S288c genome, a total of 64,998 SNPs, 7,093 indels and 11 unique genes were identified in the genome of YJS329-derived haploid strain YJSH1 through whole-genome sequencing. Transcription comparison using RNA-Seq identified which of the differentially expressed genes were the main contributors to the phenotypic differences between YJS329 and BYZ1. By combining the results obtained from the genome sequences and the transcriptions, we predicted how the SNPs, indels and chromosomal copy number variations may affect the mRNA expression profiles and phenotypes of the yeast strains. Furthermore, some genetic breeding strategies to improve the adaptabilities of YJS329 were designed and experimentally verified.
Conclusions
Through comparative functional genomic analysis, we have provided some insights into the mechanisms underlying the specific traits of the bioenthanol strain YJS329. The work reported here has not only enriched the available genetic resources of yeast but has also indicated how functional genomic studies can be used to improve genetic breeding in yeast.
doi:10.1186/1471-2164-13-479
PMCID: PMC3484046  PMID: 22978491
Bioethanol; Saccharomyces cerevisiae; Stress; Genome; RNA-Seq
11.  Whole-genome sequencing of a laboratory-evolved yeast strain 
BMC Genomics  2010;11:88.
Background
Experimental evolution of microbial populations provides a unique opportunity to study evolutionary adaptation in response to controlled selective pressures. However, until recently it has been difficult to identify the precise genetic changes underlying adaptation at a genome-wide scale. New DNA sequencing technologies now allow the genome of parental and evolved strains of microorganisms to be rapidly determined.
Results
We sequenced >93.5% of the genome of a laboratory-evolved strain of the yeast Saccharomyces cerevisiae and its ancestor at >28× depth. Both single nucleotide polymorphisms and copy number amplifications were found, with specific gains over array-based methodologies previously used to analyze these genomes. Applying a segmentation algorithm to quantify structural changes, we determined the approximate genomic boundaries of a 5× gene amplification. These boundaries guided the recovery of breakpoint sequences, which provide insights into the nature of a complex genomic rearrangement.
Conclusions
This study suggests that whole-genome sequencing can provide a rapid approach to uncover the genetic basis of evolutionary adaptations, with further applications in the study of laboratory selections and mutagenesis screens. In addition, we show how single-end, short read sequencing data can provide detailed information about structural rearrangements, and generate predictions about the genomic features and processes that underlie genome plasticity.
doi:10.1186/1471-2164-11-88
PMCID: PMC2829512  PMID: 20128923
12.  Deciphering the Hybridisation History Leading to the Lager Lineage Based on the Mosaic Genomes of Saccharomyces bayanus Strains NBRC1948 and CBS380T 
PLoS ONE  2011;6(10):e25821.
Saccharomyces bayanus is a yeast species described as one of the two parents of the hybrid brewing yeast S. pastorianus. Strains CBS380T and NBRC1948 have been retained successively as pure-line representatives of S. bayanus. In the present study, sequence analyses confirmed and upgraded our previous finding: S. bayanus type strain CBS380T harbours a mosaic genome. The genome of strain NBRC1948 was also revealed to be mosaic. Both genomes were characterized by amplification and sequencing of different markers, including genes involved in maltotriose utilization or genes detected by array-CGH mapping. Sequence comparisons with public Saccharomyces spp. nucleotide sequences revealed that the CBS380T and NBRC1948 genomes are composed of: a predominant non-cerevisiae genetic background belonging to S. uvarum, a second unidentified species provisionally named S. lagerae, and several introgressed S. cerevisiae fragments. The largest cerevisiae-introgressed DNA common to both genomes totals 70kb in length and is distributed in three contigs, cA, cB and cC. These vary in terms of length and presence of MAL31 or MTY1 (maltotriose-transporter gene). In NBRC1948, two additional cerevisiae-contigs, cD and cE, totaling 12kb in length, as well as several smaller cerevisiae fragments were identified. All of these contigs were partially detected in the genomes of S. pastorianus lager strains CBS1503 (S. monacensis) and CBS1513 (S. carlsbergensis) explaining the noticeable common ability of S. bayanus and S. pastorianus to metabolize maltotriose. NBRC1948 was shown to be inter-fertile with S. uvarum CBS7001. The cross involving these two strains produced F1 segregants resembling the strains CBS380T or NRRLY-1551. This demonstrates that these S. bayanus strains were the offspring of a cross between S. uvarum and a strain similar to NBRC1948. Phylogenies established with selected cerevisiae and non-cerevisiae genes allowed us to decipher the complex hybridisation events linking S. lagerae/S. uvarum/S. cerevisiae with their hybrid species, S. bayanus/pastorianus.
doi:10.1371/journal.pone.0025821
PMCID: PMC3187814  PMID: 21998701
13.  Proteome-wide evidence for enhanced positive Darwinian selection within intrinsically disordered regions in proteins 
Genome Biology  2011;12(7):R65.
Background
Understanding the adaptive changes that alter the function of proteins during evolution is an important question for biology and medicine. The increasing number of completely sequenced genomes from closely related organisms, as well as individuals within species, facilitates systematic detection of recent selection events by means of comparative genomics.
Results
We have used genome-wide strain-specific single nucleotide polymorphism data from 64 strains of budding yeast (Saccharomyces cerevisiae or Saccharomyces paradoxus) to determine whether adaptive positive selection is correlated with protein regions showing propensity for different classes of structure conformation. Data from phylogenetic and population genetic analysis of 3,746 gene alignments consistently shows a significantly higher degree of positive Darwinian selection in intrinsically disordered regions of proteins compared to regions of alpha helix, beta sheet or tertiary structure. Evidence of positive selection is significantly enriched in classes of proteins whose functions and molecular mechanisms can be coupled to adaptive processes and these classes tend to have a higher average content of intrinsically unstructured protein regions.
Conclusions
We suggest that intrinsically disordered protein regions may be important for the production and maintenance of genetic variation with adaptive potential and that they may thus be of central significance for the evolvability of the organism or cell in which they occur.
doi:10.1186/gb-2011-12-7-r65
PMCID: PMC3218827  PMID: 21771306
14.  Genomic Sequence Diversity and Population Structure of Saccharomyces cerevisiae Assessed by RAD-seq 
G3: Genes|Genomes|Genetics  2013;3(12):2163-2171.
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.
doi:10.1534/g3.113.007492
PMCID: PMC3852379  PMID: 24122055
RAD-seq; yeast; phylogenetics; population structure; genetic diversity
15.  Saccharomyces cerevisiae Σ1278b Has Novel Genes of the N-Acetyltransferase Gene Superfamily Required for l-Proline Analogue Resistance 
Journal of Bacteriology  2000;182(15):4249-4256.
We discovered on the chromosome of Saccharomyces cerevisiae Σ1278b novel genes involved in l-proline analogue l-azetidine-2-carboxylic acid resistance which are not present in the standard laboratory strains. The 5.4 kb-DNA fragment was cloned from the genomic library of the l-azetidine-2-carboxylic acid-resistant mutant derived from a cross between S. cerevisiae strains S288C and Σ1278b. The nucleotide sequence of a 4.5-kb segment exhibited no identity with the sequence in the genome project involving strain S288C. Deletion analysis indicated that one open reading frame encoding a predicted protein of 229 amino acids is indispensable for l-azetidine-2-carboxylic acid resistance. The protein sequence was found to be a member of the N-acetyltransferase superfamily. Genomic Southern analysis and gene disruption showed that two copies of the novel gene with one amino acid change at position 85 required for l-azetidine-2-carboxylic acid resistance were present on chromosomes X and XIV of Σ1278b background strains. When this novel MPR1 or MPR2 gene (sigma 1278b gene for l-proline analogue resistance) was introduced into the other S. cerevisiae strains, all of the recombinants were resistant to l-azetidine-2-carboxylic acid, indicating that both MPR1 and MPR2 are expressed and have a global function in S. cerevisiae.
PMCID: PMC101931  PMID: 10894734
16.  Conservation of Expression and Sequence of Metabolic Genes Is Reflected by Activity Across Metabolic States 
PLoS Computational Biology  2006;2(8):e106.
Variation in gene expression levels on a genomic scale has been detected among different strains, among closely related species, and within populations of genetically identical cells. What are the driving forces that lead to expression divergence in some genes and conserved expression in others? Here we employ flux balance analysis to address this question for metabolic genes. We consider the genome-scale metabolic model of Saccharomyces cerevisiae, and its entire space of optimal and near-optimal flux distributions. We show that this space reveals underlying evolutionary constraints on expression regulation, as well as on the conservation of the underlying gene sequences. Genes that have a high range of optimal flux levels tend to display divergent expression levels among different yeast strains and species. This suggests that gene regulation has diverged in those parts of the metabolic network that are less constrained. In addition, we show that genes that are active in a large fraction of the space of optimal solutions tend to have conserved sequences. This supports the possibility that there is less selective pressure to maintain genes that are relevant for only a small number of metabolic states.
Synopsis
The regulation of gene product activity allows cells to efficiently cope with various tasks under varying conditions. Given that, one may have expected that striving for efficiency would cause genetically similar cells to have similar regulation. However, in reality, high variations in gene expression levels are detected between different strains and even between genetically identical cells taken from the same culture. What are the driving forces that lead to expression divergence in some genes and conserved expression in others?
To address this question, the authors study the conservation of regulation in yeast metabolism, using a computational model. They find that genes coding for reactions whose flux rates are narrowly constrained by the cellular need to maximize growth rate tend to have strictly conserved regulation and expression. However, when a wide range of flux rates is compatible with high cellular growth rates, the corresponding regulation and expression patterns are free to diverge. Furthermore, enzymes that participate in a large number of alternative metabolic behaviors tend to be encoded by genes with a highly conserved sequence. Taken together, these findings support the hypothesis that maintaining large variability in the overall expression and metabolic repertoire of the cell is under marked evolutionary selection.
doi:10.1371/journal.pcbi.0020106
PMCID: PMC1550272  PMID: 16933982
17.  Conservation of Expression and Sequence of Metabolic Genes Is Reflected by Activity Across Metabolic States 
PLoS Computational Biology  2006;2(8):e106.
Variation in gene expression levels on a genomic scale has been detected among different strains, among closely related species, and within populations of genetically identical cells. What are the driving forces that lead to expression divergence in some genes and conserved expression in others? Here we employ flux balance analysis to address this question for metabolic genes. We consider the genome-scale metabolic model of Saccharomyces cerevisiae, and its entire space of optimal and near-optimal flux distributions. We show that this space reveals underlying evolutionary constraints on expression regulation, as well as on the conservation of the underlying gene sequences. Genes that have a high range of optimal flux levels tend to display divergent expression levels among different yeast strains and species. This suggests that gene regulation has diverged in those parts of the metabolic network that are less constrained. In addition, we show that genes that are active in a large fraction of the space of optimal solutions tend to have conserved sequences. This supports the possibility that there is less selective pressure to maintain genes that are relevant for only a small number of metabolic states.
Synopsis
The regulation of gene product activity allows cells to efficiently cope with various tasks under varying conditions. Given that, one may have expected that striving for efficiency would cause genetically similar cells to have similar regulation. However, in reality, high variations in gene expression levels are detected between different strains and even between genetically identical cells taken from the same culture. What are the driving forces that lead to expression divergence in some genes and conserved expression in others?
To address this question, the authors study the conservation of regulation in yeast metabolism, using a computational model. They find that genes coding for reactions whose flux rates are narrowly constrained by the cellular need to maximize growth rate tend to have strictly conserved regulation and expression. However, when a wide range of flux rates is compatible with high cellular growth rates, the corresponding regulation and expression patterns are free to diverge. Furthermore, enzymes that participate in a large number of alternative metabolic behaviors tend to be encoded by genes with a highly conserved sequence. Taken together, these findings support the hypothesis that maintaining large variability in the overall expression and metabolic repertoire of the cell is under marked evolutionary selection.
doi:10.1371/journal.pcbi.0020106
PMCID: PMC1550272  PMID: 16933982
18.  Correlating Gene Expression Variation with cis-Regulatory Polymorphism in Saccharomyces cerevisiae 
Identifying the nucleotides that cause gene expression variation is a critical step in dissecting the genetic basis of complex traits. Here, we focus on polymorphisms that are predicted to alter transcription factor binding sites (TFBSs) in the yeast, Saccharomyces cerevisiae. We assembled a confident set of transcription factor motifs using recent protein binding microarray and ChIP-chip data and used our collection of motifs to predict a comprehensive set of TFBSs across the S. cerevisiae genome. We used a population genomics analysis to show that our predictions are accurate and significantly improve on our previous annotation. Although predicting gene expression from sequence is thought to be difficult in general, we identified a subset of genes for which changes in predicted TFBSs correlate well with expression divergence between yeast strains. Our analysis thus demonstrates both the accuracy of our new TFBS predictions and the feasibility of using simple models of gene regulation to causally link differences in gene expression to variation at individual nucleotides.
doi:10.1093/gbe/evq054
PMCID: PMC2953268  PMID: 20829281
Saccharomyces cerevisiae; transcription factors; transcription factor binding sites; population genetics; gene expression; SNP; eQTL
19.  Novel genes exhibit distinct patterns of function acquisition and network integration 
Genome Biology  2010;11(12):R127.
Background
Genes are created by a variety of evolutionary processes, some of which generate duplicate copies of an entire gene, while others rearrange pre-existing genetic elements or co-opt previously non-coding sequence to create genes with 'novel' sequences. These novel genes are thought to contribute to distinct phenotypes that distinguish organisms. The creation, evolution, and function of duplicated genes are well-studied; however, the genesis and early evolution of novel genes are not well-characterized. We developed a computational approach to investigate these issues by integrating genome-wide comparative phylogenetic analysis with functional and interaction data derived from small-scale and high-throughput experiments.
Results
We examine the function and evolution of new genes in the yeast Saccharomyces cerevisiae. We observed significant differences in the functional attributes and interactions of genes created at different times and by different mechanisms. Novel genes are initially less integrated into cellular networks than duplicate genes, but they appear to gain functions and interactions more quickly than duplicates. Recently created duplicated genes show evidence of adapting existing functions to environmental changes, while young novel genes do not exhibit enrichment for any particular functions. Finally, we found a significant preference for genes to interact with other genes of similar age and origin.
Conclusions
Our results suggest a strong relationship between how and when genes are created and the roles they play in the cell. Overall, genes tend to become more integrated into the functional networks of the cell with time, but the dynamics of this process differ significantly between duplicate and novel genes.
doi:10.1186/gb-2010-11-12-r127
PMCID: PMC3046487  PMID: 21187012
20.  Saturation of the Human Phenome 
Current Genomics  2010;11(7):482-499.
The phenome is the complete set of phenotypes resulting from genetic variation in populations of an organism. Saturation of a phenome implies the identification and phenotypic description of mutations in all genes in an organism, potentially constrained to those encoding proteins. The human genome is believed to contain 20-25,000 protein coding genes, but only a small fraction of these have documented mutant phenotypes, thus the human phenome is far from complete. In model organisms, genetic saturation entails the identification of multiple mutant alleles of a gene or locus, allowing a consistent description of mutational phenotypes for that gene. Saturation of several model organisms has been attempted, usually by targeting annotated coding genes with insertional transposons (Drosophila melanogaster, Mus musculus) or by sequence directed deletion (Saccharomyces cerevisiae) or using libraries of antisense oligonucleotide probes injected directly into animals (Caenorhabditis elegans, Danio rerio). This paper reviews the general state of the human phenome, and discusses theoretical and practical considerations toward a saturation analysis in humans. Throughout, emphasis is placed on high penetrance genetic variation, of the kind typically asociated with monogenic versus complex traits.
doi:10.2174/138920210793175886
PMCID: PMC3048311  PMID: 21532833
Human genome; phenome; genetics; saturation mutagenesis.
21.  Widespread remodeling of mid-coding sequence nucleosomes by Isw1 
Genome Biology  2010;11(5):R49.
In yeast, the chromatin remodeler Isw1 shifts nucleosomes from mid-coding, to more 5’ regions of genes and may regulate transcriptional elongation.
Background
The positions of nucleosomes along eukaryotic DNA are defined by the local DNA sequence and are further tuned by the activity of chromatin remodelers. While the genome-wide effect of most remodelers has not been described, recent studies in Saccharomyces cerevisiae have shown that Isw2 prevents ectopic expression of anti-sense and suppressed transcripts at gene ends.
Results
We examined the genome-wide function of the Isw2 homologue, Isw1, by mapping nucleosome positioning in S. cerevisiae and Saccharomyces paradoxus strains deleted of ISW1. We found that Isw1 functions primarily within coding regions of genes, consistent with its putative role in transcription elongation. Upon deletion of ISW1, mid-coding nucleosomes were shifted upstream (towards the 5' ends) in about half of the genes. Isw1-dependent shifts were correlated with trimethylation of H3K79 and were enriched at genes with internal cryptic initiation sites.
Conclusions
Our results suggest a division of labor between Isw1 and Isw2, whereby Isw2 maintains repressive chromatin structure at gene ends while Isw1 has a similar function at mid-coding regions. The differential specificity of the two remodelers may be specified through interactions with particular histone marks.
doi:10.1186/gb-2010-11-5-r49
PMCID: PMC2898075  PMID: 20459718
22.  Characterization of a novel CDC gene (ORC1) partly homologous to CDC6 of Saccharomyces cerevisiae. 
Molecular Biology of the Cell  1996;7(3):409-418.
A novel cell cycle gene was identified by a computer search for genes partly homologous to known CDC genes, CDC6 of Saccharomyces cerevisiae and CDC18 of Schizosaccharomyces pombe, using the nucleotide sequence data base for S. cerevisiae produced by the Yeast Sequencing Project. The protein sequence coded by the cloned gene was found to be identical to that of purified ORC1 protein. Disruption of the gene and subsequent tetrad analysis revealed that the gene was essential for growth. The function of the gene product was analyzed by depleting the protein from the cell using a mutant haploid strain containing the disrupted ORC1 gene on the chromosome and a galactose-inducible gene coding for HA-tagged ORC1 protein on a single copy plasmid. The HA-tagged protein was expressed during growth in the presence of galactose but began to decrease rapidly upon depletion of galactose. Analysis of the cell cycle progression of the mutant cells by FACS after the removal of galactose from the medium, and microscope observations of cells and their nuclei revealed that the normal progression of 2N cells was immediately impeded as the ORC1 protein started to decrease. This was blocked completely in the cells that had progressed to the S phase under conditions deficient in ORC1 protein followed by cell death. Two-dimensional gel analysis of the replication intermediates after the galactose removal revealed that the depletion of ORC1 protein caused a decrease in the frequency of initiation of chromosomal replication, eventually resulting in the inhibition of replication as a whole. The function of the ORC1 protein in the cell cycle progression of S. cerevisiae is discussed in light of current information on ORC.
Images
PMCID: PMC275893  PMID: 8868469
23.  Genome-wide Fitness Profiles Reveal a Requirement for Autophagy During Yeast Fermentation 
G3: Genes|Genomes|Genetics  2011;1(5):353-367.
The ability of cells to respond to environmental changes and adapt their metabolism enables cell survival under stressful conditions. The budding yeast Saccharomyces cerevisiae (S. cerevisiae) is particularly well adapted to the harsh conditions of anaerobic wine fermentation. However, S. cerevisiae gene function has not been previously systematically interrogated under conditions of industrial fermentation. We performed a genome-wide study of essential and nonessential S. cerevisiae gene requirements during grape juice fermentation to identify deletion strains that are either depleted or enriched within the viable fermentative population. Genes that function in autophagy and ubiquitin-proteasome degradation are required for optimal survival during fermentation, whereas genes that function in ribosome assembly and peroxisome biogenesis impair fitness during fermentation. We also uncover fermentation phenotypes for 139 uncharacterized genes with no previously known cellular function. We demonstrate that autophagy is induced early in wine fermentation in a nitrogen-replete environment, suggesting that autophagy may be triggered by other forms of stress that arise during fermentation. These results provide insights into the complex fermentation process and suggest possible means for improvement of industrial fermentation strains.
doi:10.1534/g3.111.000836
PMCID: PMC3276155  PMID: 22384346
S. cerevisiae; fermentation; fitness profiling; environmental stress; autophagy
24.  Biogenesis of mitochondria: a mutation in the 5'-untranslated region of yeast mitochondrial oli1 mRNA leading to impairment in translation of subunit 9 of the mitochondrial ATPase complex. 
Nucleic Acids Research  1987;15(5):1965-1977.
A temperature-conditional mit- mutant of Saccharomyces cerevisiae has been characterized; the mutant strain h45 cannot grow at 36 degrees C on nonfermentable substrates yet appears to be normal at 28 degrees C. The mutation in strain h45 maps genetically to the oli1 region of the mitochondrial DNA (mtDNA) genome, and prevents the synthesis at 36 degrees C of the oli1 gene product, subunit 9 of the mitochondrial ATPase complex. Since the level of oli1 mRNA in mutant h45 is close to normal at 36 degrees C, it is concluded that there is a specific block in translation of this mRNA at the non-permissive temperature. DNA sequence analysis of mtDNA from strain h45 reveals an additional T residue inserted 88 bp upstream of the oli1 coding region, in the A,T-rich sequence that is transcribed into the 5'-untranslated region of the oli1 mRNA. Sequence data on two revertants show that one returns to wild-type parental (J69-1B) mtDNA sequence, whilst the other contains an inserted A residue adjacent to the T inserted in the original h45 mutant. The results are discussed in terms of the stability of folds in RNA upstream of putative ribosome-binding sites in mitochondrial mRNA, and the potential action of nuclear-coded proteins that might be activators of the translation of specific mitochondrial mRNAs in yeast mitochondria.
Images
PMCID: PMC340611  PMID: 2951651
25.  The Awesome Power of Yeast Evolutionary Genetics: New Genome Sequences and Strain Resources for the Saccharomyces sensu stricto Genus 
G3: Genes|Genomes|Genetics  2011;1(1):11-25.
High-quality, well-annotated genome sequences and standardized laboratory strains fuel experimental and evolutionary research. We present improved genome sequences of three species of Saccharomyces sensu stricto yeasts: S. bayanus var. uvarum (CBS 7001), S. kudriavzevii (IFO 1802T and ZP 591), and S. mikatae (IFO 1815T), and describe their comparison to the genomes of S. cerevisiae and S. paradoxus. The new sequences, derived by assembling millions of short DNA sequence reads together with previously published Sanger shotgun reads, have vastly greater long-range continuity and far fewer gaps than the previously available genome sequences. New gene predictions defined a set of 5261 protein-coding orthologs across the five most commonly studied Saccharomyces yeasts, enabling a re-examination of the tempo and mode of yeast gene evolution and improved inferences of species-specific gains and losses. To facilitate experimental investigations, we generated genetically marked, stable haploid strains for all three of these Saccharomyces species. These nearly complete genome sequences and the collection of genetically marked strains provide a valuable toolset for comparative studies of gene function, metabolism, and evolution, and render Saccharomyces sensu stricto the most experimentally tractable model genus. These resources are freely available and accessible through www.SaccharomycesSensuStricto.org.
doi:10.1534/g3.111.000273
PMCID: PMC3276118  PMID: 22384314
Saccharomyces genome; genome assembly; evolutionary genetics; sensu stricto; genomics;  yeast species

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